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Regions of the genome that have been the target of positive selection specifically along the human lineage are of special importance in human biology. We used high throughput sequencing combined with methods to enrich human genomic samples for particular targets to obtain the sequence of 22 chromosomal samples at high depth in 40 kb neighborhoods of 49 previously identified 100–400 bp elements that show evidence for human accelerated evolution. In addition to selection, the pattern of nucleotide substitutions in several of these elements suggested an historical bias favoring the conversion of weak (A or T) alleles into strong (G or C) alleles. Here we found strong evidence in the derived allele frequency spectra of many of these 40 kb regions for ongoing weak-to-strong fixation bias. Comparison of the nucleotide composition at polymorphic loci to the composition at sites of fixed substitutions additionally reveals the signature of historical weak-to-strong fixation bias in a subset of these regions. Most of the regions with evidence for historical bias do not also have signatures of ongoing bias, suggesting that the evolutionary forces generating weak-to-strong bias are not constant over time. To investigate the role of selection in shaping these regions, we analyzed the spatial pattern of polymorphism in our samples. We found no significant evidence for selective sweeps, possibly because the signal of such sweeps has decayed beyond the power of our tests to detect them. Together, these results do not rule out functional roles for the observed changes in these regions—indeed there is good evidence that the first two are functional elements in humans—but they suggest that a fixation process (such as biased gene conversion) that is biased at the nucleotide level, but is otherwise selectively neutral, could be an important evolutionary force at play in them, both historically and at present. Understanding the forces that have shaped the evolution of the human genome is one of the most exciting problems in modern genomics. Two approaches to this problem are focused on identification and characterization of those genomic regions that have evolved the slowest and fastest along the human lineage [1]–[5]. The slowest evolving regions may contain elements that cannot be disturbed without disrupting essential function. The fastest evolving regions may harbor elements whose function is unique to our species lineage. To eliminate non-functional regions, both of these complementary approaches begin with a search for regions that are conserved throughout mammalian history or longer. The ultra conserved elements [1] maintain this conservation along the human lineage, and have been shown to be under purifying (negative) selection [6], strongly suggesting that they are functionally important to our species, although in ways that are still largely unknown. By contrast, several groups have searched for positive selection along the human lineage by focusing on those previously slowly evolving regions of the genome that have evolved most quickly along the human lineage [4], [5], [7]. These regions, such as those in the set of Human Accelerated Regions (HARs) [7], may include some of the genetic changes that make our species biologically unique. Indeed, biological characterization of the topmost elements on this list of candidates has proven fruitful: HAR1 is part of a novel RNA gene (HAR1F) that is expressed during neocortical development [3]; HAR2 (or HACNS1) is a conserved non-coding sequence that has been shown to function as an enhancer in the developing limb bud with the human-specific sequence enhancing expression in the presumptive anterior wrist and proximal thumb [8]. Since the HARs were identified based on an excess of fixed differences between the human reference genome and sequences that are highly conserved among chimp, mouse and rat, such differences could have arisen at any time within the 5 million years that have elapsed since our common ancestor with the chimpanzee. As such it is important to recognize that even if such differences resulted from positive selection for advantageous mutations, they may have occurred so long ago that we have little power to find evidence for such selection using only the present day sequences available to us. Furthermore, as previously noted [7], positive selection might not be the sole explanation for the rapid evolution that is evident in the HARs. Biased gene conversion (BGC) may also hasten the fixation of mutations in a local manner independent of any fitness benefits [9], [10]. BGC arises as a byproduct of recombination between homologous chromosomal regions. In this process DNA double stranded breaks are repaired and the alleles from one chromosome are copied to the other, with a bias for conversion of A or T (weak hydrogen bonding) alleles to G or C (strong hydrogen bonding) alleles [11]–[14]. A neutral locus can thus mimic the rapid evolution of loci under positive selection [9], [10], and furthermore, BGC may in fact drive fixation of deleterious alleles [15], the precise opposite of a positive, adaptive evolutionary effect. One of the most powerful tools for identifying those regions that have been subjected to directional selection comes from examining the distribution of allele frequencies segregating within a species. For example, analysis of this distribution, known as the site frequency spectrum (SFS), allows for the identification of loci that have been involved in selective sweeps in the last few hundred thousand years. Analysis of the SFS has been used to identify targets of natural selection that may be responsible for genetic traits that are uniquely human, such as language [16] or cognition [17]. In the current work we investigate the top 49 HARs that were identified as having a 5% false discovery rate [3]. But rather than restricting our attention to the core elements, which are 100–400bp in length, we consider the polymorphism in a set of 22 chromosomal human samples in a 40kb neighborhood of each of these HAR elements, with an eye to capturing perturbations in the SFS at linked sites, and/or regionally biased patterns of allele fixation. Our samples are drawn from a single population, the Yoruba from Ibidan, Nigeria in order to avoid confounding issues of population admixture as well as to take advantage of a greater degree of variation in this population. We use an adaptation of several techniques previously developed [18]–[21] to enrich genomic DNA from our sample individuals for the target genomic neighborhoods. The enriched DNA is then subject to high throughput sequencing followed by genome-wide mapping of many overlapping sequences to determine genotypes at sites in the target regions, and hence derive the site frequency spectra. With these spectra in hand, it is possible to test for the hallmarks of BGC. We employed an approach that compares the separate site frequency spectra for the weak-to-strong (i. e. A or T to G or C) (W2S) and strong-to-weak (S2W) mutations to determine if any shift towards high frequency, normally characteristic of a selective sweep, is biased towards one of the two sets of mutations. This signal would indicate an ongoing process in the current human population. Similarly, one can compare the proportion of W2S changes among already fixed substitutions on the human or chimp lineage to that among the still segregating sites. A W2S bias in fixed differences relative to polymorphisms would indicate that the regions have historically been subject to a BGC-like biased process. On the other hand, the spectra may contain evidence of positive selection. Various techniques have emerged in recent years to search for signatures of positive selection using population genetic data [22]–[24], but many are based on the phenomenon of genetic hitchhiking [25], [26] in which fixation of beneficial mutations results in a skew in the site frequency spectrum. One such approach [27] is based on the composite likelihood of allele frequencies wherein the probability of the observed allele frequency at each polymorphic position is calculated based on its distance from a site under putative positive selection. This probability explicitly takes into account the strength of recombination and selection. A variant of this approach has been implemented [28] in the SweepFinder program that we use herein. It has been previously used [29] in a genome-wide search for sweeps at the scale of 500kb, since that study' s data was restricted to loci with common polymorphisms. The power of that approach is probably limited to finding sweeps not much older than 200,000 years but has the attractive property that it is robust to demographic history [29]. Unlike other approaches that have been used [22], [23], [30], [31] it also does not require that the sweep be ongoing or differentially concluded in separate populations. Since we have discovered many novel polymorphisms by resequencing our samples, we use this method to take a more focused look at our 40kb HAR neighborhoods in search of adaptive evolutionary forces. When the HARs were first described, a strong W2S substitution bias was noted in the human-specific substitutions in these elements [3], [7]. This bias was extremely pronounced in HARs 1,2, 3, and 5, but also noticeable as a general trend in the entire set 1–49. This evidence suggested that BGC could have had a historical role in the evolution of the HARs. Here we analyze our list of segregating sites in the 40kb HAR neighborhoods to determine if such bias is still ongoing in the human population. In each region, we separately computed the derived allele frequency spectra for the W2S mutations and the S2W mutations. We then tested for an offset in the spectra between the two categories with a two-sided Mann Whitney U (MWU) test (see Materials and Methods). This test has been shown to have good power to detect fixation bias [34]. We found a significant (p 0. 05) difference in 11 out of 49 harseq regions (Table 1). In all 11 significant harseq regions, the offset was for the W2S mutations to be segregating at higher derived allele frequencies than S2W. This implies that regardless of the rate of introduction of W2S or S2W mutations, it is the W2S mutations that are more likely to reach high frequency and eventually fix in the human population. This is certainly consistent with a mechanism of gene conversion that favors selection of G or C alleles from a heterozygote or some other selective force generally favoring higher GC content. The novel features of this result are that it indicates that this process is ongoing and not confined to the core 100–400bp HAR elements. This ongoing W2S fixation bias distinguishes the harseq regions from ctlreg regions and Seattle SNPs regions. Significant MWU tests were observed at none of ctrlreg50-62 (Supplementary Table 2 in Text S1) and five out of 62 Seattle SNPs regions (Supplementary Table 4 in Text S1), one of which has higher derived allele frequencies in S2W compared to W2S mutations. The distribution of the MWU test statistic in the test regions is also biased towards W2S mutations compared to the 62 Seattle SNPs regions (Supplementary Figure 6 in Text S1). Applying the MWU test to simulations of a neutral coalescent model (see Materials and Methods) showed that the p-values from this test accurately reflect the fraction expected by chance from a neutrally evolving locus (Supplementary Figure 2 in Text S1). The two harseq regions with the greatest offset were harseq21 and harseq34 (Figure 1). We note that across these two 40kb regions, the ratio of W2S to S2W segregating sites is not extreme; it is the W2S shift towards higher frequency in the population that is significant. These ratios are consistent with the smoothed ratios reported in the top several thousand conserved candidate HAR elements [7] even though our 40kb regions do not comprise largely conserved regions. It is natural to theorize that BGC will be a stronger driving force in areas of high recombination and studies have shown there to be a good correlation with the male recombination rate in particular [35]. We examined the recombination rates in the enclosing 1Mb windows as determined by the deCODE project [36]. Harseq21 is an outlier in that it is contained in a genomic region of extremely high recombination rate (male 4. 29 cM/Mb, sex-averaged 3. 43 cm/Mb, in contrast to genome-wide averages of 0. 93 cM/Mb and 1. 29 cm/Mb respectively). But this is not true of harseq34 (0 male and 1. 42 cm/Mb sex-averaged). For the remainder of the regions with a significant p-value on the MWU test, the rates vary. An additional (not unrelated) factor that has been strongly correlated with biased substitutions is chromosomal position near telomeres [35]. With the exception of harseq1 this is not the case for the regions with significantly shifted W2S spectra (Table 1). We also performed the MWU test for the shift in W2S sites toward higher frequencies after pooling all the segregating sites in our 49 harseq regions, and found a p-value. By contrast, the test was not significant (p = 0. 26) for the pooled data in our 13 control regions, thereby controlling for possible systematic bias in our sequencing and genotyping techniques. We thus conclude that in many of the neighborhoods of the top 49 HARs there is an ongoing force driving W2S mutations to higher frequency in the human population. Since the HARs were essentially defined based on fixed differences between the human and chimp reference genomes in otherwise strongly conserved elements [7], we compared such human/chimp fixed differences to the segregating sites in our human samples. Our set of fixed differences was based on high quality base calls from the reciprocal best alignments [37] of human and chimp genomes. We further restricted this list to the locations within our regions for which we had the above-mentioned 35-fold coverage for an individual in our sample. Finally, we removed from this initial fixed difference list those positions that we found to be segregating in our samples, or that appeared in the dbSNP129 database [38] (see Materials and Methods). The latter two filters removed 6. 7% of the fixed differences at high coverage positions. Since we do not have information on sites that are segregating in the chimp population, we could not remove those, but would expect the number to be similarly small. We separated the mutations in our segregating site set into the categories: W2S, S2W, and neither. We similarly divided the set of fixed differences, regardless of whether the substitution occurred on the chimp or human lineage. As in reference [35], we performed a variant of the McDonald-Kreitman (MK) test to compare the W2S∶S2W ratios in the sets of mutations and the sets of substitutions (see Materials and Methods). We found a significant (p 0. 05) difference between the substitution patterns of segregating versus fixed sites in 11 of the 49 harseq regions (Table 2). In all but one (harseq39) of these 11, the fixed substitutions had relatively more W2S mutations (compared to S2W) from the ancestral form than did the segregating sites. Four of the 11 fell in the 40kb neighborhoods of the top 11 HARs, and indeed the strongest result (p = 0. 00015) was for harseq1. This is not surprising, since the HAR1 element has 18 fixed differences, all W2S [3]. By contrast, none of the ctlreg regions and nine out of 62 Seattle SNPs regions had a significant p-value on this test (Supplementary Tables 2 and 4 in Text S1). All of the nine significant Seattle SNPs regions had a higher W2S∶S2W ratio in fixed differences than in segregating sites. Thus, the harseq regions have a much stronger signal for historical fixation bias than our control regions and a somewhat stronger signal than the genic Seattle SNPs regions. Applying the MK test to simulations of a neutrally evolving primate phylogeny (see Materials and Methods) showed that the p-values from this test accurately reflect the fraction expected by chance from a neutrally evolving locus (Supplementary Figure 3 in Text S1). We also recapitulate and expand the finding [7] that this bias towards W2S fixation is associated with telomeres, since 7 of the 11 significant regions under the MK test were found either in the karyotype band containing a telomere or the one immediately adjacent. (This count includes the two cases of harseq19 and harseq36 from chromosome 2 that fall adjacent to the ancestral telomeric fusion event at 2q14. 1.) Although many of the 11 regions (including ones near telomeres) have elevated recombination rates, it is also worth noting that 5 of the 11 are in regions with much lower than average male recombination rates (including the two near the chromosome 2 fusion site, and harseq11 on chrX). One noteworthy negative result from the MK test, (and the MWU test as well) is harseq2. It had been noted [7] that the core HAR2 element showed a strong bias towards W2S fixations, and that this extended to a region of 1kb. Here we find no significant signal for either the MK or MWU test in our 40kb neighborhood of that element (Supplementary Table 2 in Text S1). Another noteworthy negative result on these tests is the harseq6 region, which has an extremely elevated rate of mutation as estimated either by nucleotide diversity [39] or by the number of segregating sites [40] (Supplementary Table 1 in Text S1), but apparently no strong bias towards weak-to-strong fixation (Supplementary Table 2 in Text S1). Although the MK test, like the MWU test, is consistent with the mechanism of BGC favoring fixation of G or C alleles, in fact only one of our regions (harseq1) had significant results in both tests. The complementary evidence from the MWU test (a total of 20 of our 49 test regions are W2S-significant on one test or the other) indicates that the ongoing bias in favor of W2S mutations has also probably led to human specific substitutions in otherwise conserved elements. Because BGC is posited to operate on a scale much smaller than the 40kb of our target sequencing regions, perhaps operating at localized recombination hotspots, and because the (100–400bp) HAR elements at the core of our target regions were suspected of arising in part due to BGC [7], we wanted to test whether the MWU and MK signals depended on these core elements. We therefore performed the same tests after masking out the central 500bp, 1kb, 5kb or 10kb of each region. We found that signals of both ongoing and historical fixation bias are fairly robust to removing sequences including and flanking the core HAR element. For the MWU test, all but two (harseq1, harseq18) of the 11 regions that were significant at the 5% level for the MWU test were still significant at that level with the central 5kb omitted. Seven of the 11 remained significant even with 10kb omitted (Table 1). For the MK test, the results were slightly more sensitive to masking. Considering the 11 regions with a significant result on that test (including the one favoring S2W fixation), 9 (7) of these were still significant with the central 1kb (5kb) masked out (Table 2). We conclude that the evolutionary forces behind these results is not confined to the small HAR elements themselves, but rather that any bias in the substitutions found in the HARs is likely a byproduct of the forces acting at a larger scale. To test whether there might be other localized elements within the 40kb regions driving these results we performed the tests under a set of fifteen overlapping 5kb masks (centered at a 2. 5kb spacing along each region). Among the 11 MWU significant regions, 5 were still significant at 10% under this regime, while 3 completely lost significance (p 20%) for at least one such mask (Table 1). Of the 11 MK significant regions, 5 were still significant at 10% (including harseq1) under this regime, while 1 completely lost significance (p 20%) under at least one mask (Table 2). It is worth noting that the harseq1 result reflects the fact that of the 105 segregating sites we found in that 40kb neighborhood, 71 were S2W and only 19 W2S (Supplementary Table 2 in Text S1). We conclude from this set of tests that the evolutionary forces behind W2S fixation bias are not necessarily highly local. If fixation bias relies on recombination hotspots and BGC, we have to posit that such hotspots extend over a long range of bases, or are somehow temporally and spatially variable (cf. [41]). To determine if the results for the MWU and MK tests on the HAR neighborhoods are consistent with a model of GC-biased evolution, we performed simulations under a model of BGC (see Materials and Methods). Of 499 simulations, for the MK test 130 were significant (p) with W2S bias (none significant with S2W bias), and for the MWU test 51 were significant with W2S bias (one significant with S2W bias). The union of the significant W2S simulations on the two tests comprised 169 cases while the intersection comprised 12 cases. Compared to the simulations, the 49 HAR regions had significantly more than the expected number of MWU W2S cases (p = 0. 003 for binomial probability of at least 11/49 cases using the simulation rate of 51/499), but the MK test does not (p = 0. 77 binomially comparing 10/49 to 130/499). On the other hand, the small (one case) intersection of MWU and MK tests in the HAR regions is not unexpected based on the simulations. That is, using the fraction 12/499 (= 0. 024) of the MWU and MK intersection in the simulations as the expected rate, the small fraction 1/49 (= 0. 020) in the HAR regions is not statistically significant using either a binomial (p = 0. 67) or Poisson (p = 0. 67) test. Finally, for neither the simulations (Fisher' s Exact test p = 0. 74) nor the HAR regions (p = 0. 42) is there a significantly greater correlation between the MWU and MK results than expected by chance. Together, these analyses indicate that the MWU and MK results for the 49 HAR regions are consistent with a model of GC-biased evolution in terms of the overlap between the tests, although the number of MWU cases is enriched compared to the simulation model. For each of the studied regions, we used the SFS to calculate two population genetic statistics that can sometimes indicate positive selection: Tajima' s D [42], which is based on the folded SFS, and Fay and Wu' s H [43]. Neither of these statistics exceeded the value of in any region (Supplementary Table 1 in Text S1). We next compared the distributions of these two statistics in the 49 harseq regions and the 13 ctlreg regions, to those for the same population (YRI) in 104 genic regions resequenced by Seattle SNPs (see Materials and Methods). We found the ctlreg regions to be indistinguishable from the Seattle SNPs for these statistics, while the harseq regions were only mildly more negative for Tajima' s D (Wilcoxon rank sum p = 0. 08) and not significantly different for H (Supplementary Figure 1 in Text S1). These results strongly suggest that the site frequency spectrum in harseq regions is indistinguishable from that found in either our control regions (ctlreg), or in the Seattle SNPs data set. Thus we have no reason to believe that harseqs represent some kind of genomic outlier with respect to recent selective events. Examination of these statistics calculated separately for the W2S and S2W segregating sites (Supplementary Figure 7 in Text S1) shows that the W2S subset in the harseq regions has a significantly more negative value of H than in the Seattle SNPs, which is consistent with the shift to higher derived allele frequencies for this subset noted above using the MWU test. To test for evidence of a selective sweep, we analyzed the spatial variation of derived allele frequencies at the segregating sites from our 22 chromosomal samples in each of the target 40kb regions using the SweepFinder program. This software determines a composite likelihood ratio (CLR) statistic comparing the hypothesis of a complete selective sweep at the location to the null hypothesis of no sweep using Test 2 from [28]. We tested along a grid of 1000 points in each target region (see Materials and Methods). This test has been shown to be robust to demographic deviations from the standard neutral model in its ability to use an arbitrary background site frequency spectrum [29]. We tested with two such backgrounds: the first from the pooled set of all the data in the 49 harseq regions plus 13 ctlreg regions, the second from the same population (YRI) as our samples but with frequencies taken from the Seattle SNPs resequencing data [33] for a large set (104) of genic regions. It should be noted that using the SFS from our data as the background to define the neutral model should be particularly conservative in that we are testing any given region for deviations from that neutral model. To determine the significance of the maximum CLR values, we performed coalescent simulations of each target region and ran the SweepFinder program on each simulated set of segregating sites (see Materials and Methods). We report as a p-value the fraction of simulations of each target that had a CLR greater than or equal to the actual maximum CLR for that target (Supplementary Table 2 in Text S1) The harseq regions with the most significant five SweepFinder p-values are listed in Table 3. These are nearly all at the 95% confidence level for either of the two background distributions used, but we note that none are individually significant after a conservative Bonferroni correction, given the 49 harseq regions that were tested. Since these may nevertheless harbor mutations that were selected for in the human lineage, here we briefly note some of their characteristics that can be seen in tracks from the UC Santa Cruz Genome Browser (Supplementary Figure 4 in Text S1). Unlike the other four SweepFinder hits, which all contain introns or exons of coding genes, harseq25 is in a gene “desert”. The nearest known gene, approximately 1Mb away on chromosome 4, is ODZ3, which is a transmembrane signaling protein most highly expressed in brain. Note that harseq25 also has a significant result on the MWU test discussed above. Evidence for a sweep in the harseq9 region is intriguing because it encompasses the 42-codon long, second exon of the PTPRT gene, a phosphatase with possible roles in the central nervous system. However, the human amino acid sequence of this exon matches the other primates chimp, gorilla, and orangutan, except where chimp has an obviously non-ancestral ThrAla substitution. The human sequence does have a single GA substitution near the 3′ splice site just upstream of this exon, but it falls in a position between the polypyrimidine (Py) tract and the AG acceptor site, for which the consensus sequence across many splice sites is evenly divided among the 4 nucleotides. The location of harseq11 on chromosome X places its evidence for a sweep in the first intron of the 2. 4Mb long dystrophin gene DMD. The evidence for a sweep in harseq16 is offset to one end of its region, about 20kb from an apparent pseudogene comprising a single coding exon with a 270-codon open reading frame (ORF) that is probably derived from the Poly-A binding protein PABPC1. The harseq24 region encompasses the second through fourth exons of the SKAP2 gene with the strongest evidence for a sweep about 10kb from the closest exon, but closer to a LINE transposable element that is present also in chimp, orangutan, and rhesus macaque (but lost in gorilla). Although the above evidence for selective sweeps is not statistically significant, and none of it seems to point directly to a mutation in a core HAR element based on the position of the SweepFinder peak CLR values, it is important to note that while having the advantage of robustness to demography and recombination rate, our tests would not likely have power to detect sweeps that occurred beyond the last 200,000 years [29]. Under an assumption that substitutions in the HAR elements occurred uniformly over the last 5 million years and that most of these substitutions were adaptive, we estimate (see Materials and Methods) that we would be able to detect fewer than 8 with our tests. Therefore this negative result should not be interpreted as ruling out a role for adaptive evolution in the HARs. In the era of comparative genomics, strong signals of conservation across multiple species serve as signposts that can indicate regions where evolutionary forces may be preserving functional elements that are subject to purifying selection (e. g. [6]). By contrast, signals of positive selection pointing to adaptive changes in one lineage are harder to find, often employing sets of polymorphic sequences from multiple individuals of the same species. We exploited the two recently developed techniques of genomic enrichment and high throughput sequencing to characterize the polymorphism in a single human population across 40kb neighborhoods of the 49 HARs (harseq regions). We investigated the harseq regions because the HARs were defined based on a presumption that the human lineage specific fixed differences therein might have arisen due to adaptive evolutionary forces. On the other hand, it has been emphasized by some that the presumably evolutionarily neutral mechanism of BGC can influence the frequency spectra at polymorphic positions, or cause fixation of alleles in a way that partially mimics the action of adaptive evolution. Indeed, fixation bias was noted in connection with the limited set of human specific alleles for some of the HARs when they were first described [7]. With the extensive novel polymorphism in our samples, we were able to carefully characterize fixation bias — both historical and ongoing — in the harseq regions and to conduct tests for recent selective sweeps across these regions. Our deep resequencing data is noteworthy because it eliminates issues of SNP ascertainment bias that could have skewed previous investigations of polymorphism near HARs. We applied several established population genetic tests, as well as an application of the MWU test, to identify differences in the fixation patterns of W2S and S2W mutations. Consistent with published reports [7], [9], [35], we find evidence of historical W2S fixation bias in harseq regions. Using a MK test, we compared the proportion of W2S mutations among already fixed substitutions on the human or chimp lineage to that among the still segregating sites in our samples. We found that 11 of our 49 regions show statistically significant evidence of historical bias in allele fixation, with all but one favoring W2S fixation. These results strengthen and expand previous findings by identifying signals for W2S bias in much larger regions flanking the core HAR regions in an ascertainment-free population sample. This study goes beyond previous approaches by also looking at ongoing W2S fixation bias in the segregating site frequency spectrum. We performed a MWU test using only sites that are still segregating in the human population, separating out W2S from S2W mutations. This second test is designed to detect a phenomenon of bias that is currently driving W2S mutations to higher frequency in the population than S2W mutations. We found statistically significant evidence for this bias (and none in the opposite direction) in the regions flanking 11 of 49 HARs. For both of our tests, we showed that the core HAR element is generally not the main source of the signal that we detected, since the signal usually remains strong even when we mask out the central 1kb or even 5kb of the region. This is not consistent with BGC due to a recombination hot spot that has remained in the same location for millions of years, because the length scale of the effect of BGC is set by the length of the heteroduplex tract formed during recombination that needs to be repaired, which is thought to be 500bp (e. g. [44]–[46]). However, it is consistent with a model in which the location of recombination hotspots drift fairly rapidly over evolutionary time scales, but may be denser in some regions [41], [47]–[50]. It is noteworthy that there was little overlap in the regions identified by these two tests, one for older W2S fixations and the other for present day forces toward fixation, with a total of 20 found in one or the other. Although this is consistent with the hypothesis that the regional focus of BGC, which may be recombination hot spots, drifts significantly on a time scale of many hundreds of thousands or millions of years, we also found from simulations of GC-biased evolution over these time scales that the relatively minimal overlap between the tests is not unexpected. Another explanation for W2S fixation bias near HARs is selection for increased GC-content or individual fitness-improving GC alleles. To investigate these hypotheses and to attempt to disentangle the possible roles of BGC and positive selection in shaping the HARs, we applied a recently developed powerful method for detecting selective sweeps. Selection was previously investigated in much larger (500kb) regions using more sparse polymorphic loci [29]. That study found 101 regions with strong evidence for a selective sweep within 100kb of a known gene. Here, we found only 5 possible candidates for such sweeps among our 49 target regions (and none that were significant after correction for multiple hypothesis testing). Three of these candidates overlap regions with significant evidence of historical (2) or ongoing (1) W2S bias. As we are dealing with a lineage-specific evolutionary period of about 5 million years, and these tests can only see back a few hundred thousand years, it is quite possible that the original signal for selective sweeps in these regions has already decayed beyond our ability to recognize it in human population genetic data. That is, the lack of evidence for recent sweeps does not rule out the possibility that some of the excess substitutions in HARs were fixed by older selection. Similarly, the evidence for GC-biased evolution based on current population genetic data may not fully reflect patterns of polymorphism in the past. Consistent with the idea that HAR regions may have experienced positive selection too long ago to be detected with population genetic methods, very few positively selected regions in the human lineage have been identified to date, despite the existence of numerous public databases. Selective sweeps that have been identified have typically been the product of very recent events in human history, such as dairy farming affecting the lactase gene [51] or climate differences influencing a salt sensitivity variant [52]. Such environmental or cultural changes result in differences in the genetic makeup of disparate human populations, and such differences can be exploited to find evidence of recent, possibly still ongoing, selective sweeps. An alternative hypothesis that deserves consideration is that HARs may have an unusually high level of recent substitution due to a recent relaxation in purifying selection along the human lineage (e. g. [53]). Using previously described methods [7], we compared estimates of the rates of substitution in the 49 HAR elements to the neutral rate. We find that the human substitution rate exceeds the expected neutral rate in all 49 HARs, while this is true for the chimp substitution rate in only 10 HARs. Furthermore, in 33 HARs the human substitution rate significantly exceeds the neutral rate (Poisson p-value) while none of the chimp substitution rates significantly exceed the neutral rate. This evidence argues against the hypothesis that these HAR elements are the product of relaxed selection. We have focused in our study on 40kb neighborhoods of 49 HAR elements (and 13 similar control regions) because of their intrinsic interest but also because the scope of our study was appropriate to the state of the art of recently emerged enrichment and sequencing technologies. As larger data sets become available we will be able to apply our analysis on a genome-wide scale. Such analysis should give us insights into the properties associated with genomic regions that display this ongoing W2S fixation bias and their potential biological consequences. Despite the evidence that the unusually high level of recent substitution in the more extreme HAR elements, such as HAR1 and HAR2, could be due to the process of BGC, there is ample evidence that these genomic elements remain functional, and thus the effect of BGC was to mutationally stress but not destroy these elements. HAR1 shows a very strong pattern of compensatory substitutions within its RNA helix structures, indicating a selective force to maintain these helix structures. The W2S substitutions all strengthen the RNA helices of HAR1, and in one case, a substitution appears to extend one of them. Human HAR1 and HAR2 both show evidence of specific function, the former by its highly specific expression pattern during neurodevelopment and the latter by its ability to enhance gene expression during limb development. Whether the human-specific evolutionary changes to these elements reflect a process that was essentially like swimming upstream against an onslaught of non-selective BGC just to keep in place on the fitness landscape, or whether the mutational stress pushed these elements into a configuration that enabled some positive selection for higher fitness in humans, remains to be seen. Genomic DNA for our samples was obtained from the NHGRI Sample Repository for Human Genetic Research distributed by the Coriell Institute for Medical Research [Camden NJ] [54]. All of the 11 samples were chosen from the Yoruba from Ibidan Nigeria (YRI) HapMap population. In particular, the samples were chosen as a subset of the Seattle SNPs P2 panel [33]. The Seattle SNPs PGA-VDR (and Coriell repository numbers were): DY01 (NA18502), DY03 (NA19223), DY04 (NA19201), DY17 (NA19143), DY18 (NA18517), DY19 (NA18856), DY20 (NA19239), DY21 (NA18871), DY22 (NA19209), DY23 (NA19152), DY24 (NA19210). Sample preparation as described below was similar for all samples, except that prior to processing, the DY01, DY03, DY04 samples were subject to whole genome amplification (WGA) using the Repli-G Kit (Qiagen N. V, The Netherlands) according to manufacturer' s specifications. It was found that WGA caused a loss of coverage in some isolated target regions, most notably in the 28kb section of the harseq1 region with coordinates hg18: chr20: 61,183,966–61,212,244, except for the core HAR1 element at chr20: 61,203,919–61,204,081. The primary target regions consisted of 20kb extensions in both directions from the 49 most statistically significant Human Accelerated Regions (HARs) identified as having a 5% false discovery rate [3]. Additional 40kb control regions were chosen in neighborhoods of 13 of the set of 34,498 vertebrate conserved elements that had extremely low LRT scores in the test used to define the HARs. The enrichment arrays were obtained from Nimblegen Systems (Madison WI) who designed the probes on their 385K array based on our specifications of the coordinates of the 62 target genomic regions (Supplementary Table 1 in Text S1). Probes were chosen to tile the target regions from both DNA strands. The design process avoided probes in highly repetitive sequences as described previously [19], [21]. The fraction of bases in the target regions covered by the probes ranged from 98% (harseq12) to 65% (harseq4) with a median of 88%. Details of probed bases are available upon request. The library preparation of the samples for SOLiD (Applied Biosystems, Foster City, CA) sequencing generally followed the manufacturer' s protocols for barcoded SOLiD System 2. 0 Fragment Library Preparation (samples DY01, DY03, DY04) and SOLiD System 3. 0 Barcoded Fragment Library Preparation (remaining samples), with changes as necessary for enrichment on the Nimblegen arrays as noted in the following: Samples were sheared to approximately 100bp using the Covaris S2 System Program B (Covaris Inc, Woburn, MA). End repair was performed with End-It DNA End-Repair Kit (Epicentre Biotechnologies, Madison, WI) per manufacturer' s instructions. Single stranded oligos for the P1 and P2-barcoded SOLiD adaptors were ordered from Invitrogen Corporation and annealed per the SOLiD protocol to form double stranded adaptors, which were ligated to the end-repaired DNA fragments using the Quick Ligation Kit (New England BioLabs, Ipswich, MA) per manufacturer' s directions, leaving a nick at the 3′ end of each genomic DNA strand where the 5′ end of the adaptor was not phosphorylated. Samples DY01, DY03, DY04 were size selected to 150250bp from a 6% polyacrylamide gel and purified via ethanol precipitation. This was followed by nick translation and ligation mediated PCR (LMPCR) amplification (6 cycles) in a combined reaction per the SOLiD protocols using Takara ExTaq polymerase (Takara Bio, Madison, WI). After dividing into 10 aliquots, an additional 10 cycles of ExTaq LMPCR amplification were performed on these samples in preparation for array hybridization. Samples DY17–DY20 were first nick translated without amplification using Pfu polymerase (Stratagene, La Jolla, CA). Samples DY21–DY24 were nick translated and ExTaq LMPCR amplified (6 cycles). Quantitation using a DNA 2100 BioAnalyzer (Agilent Technologies, Waldbronn, Germany) showed that the PCR associated with nick translation prior to size selection mainly served to amplify the nick translated adaptors. After the nick translation and any initial LMPCR, all of samples DY17–DY24 were size selected on an E-Gel SizeSelect 2% agarose gel (Invitrogen) per manufacturer' s instructions. These size selected samples were then ExTaq LMPCR amplified (6,9 or 10 cycles) in preparation for array hybridization. Array hybridization to the Nimblegen arrays was performed on the Nimblegen Hybridization System 4 station under Mix Mode “B” for 64 to 70 hours using the Nimblegen Sequence Capture Kit per manufacturer' s instructions. Prior to hybridization, samples DY21–24 were pooled to a total of 5. 7g. All the other samples were hybridized individually, in amounts ranging from 1. 9g to 8. 0g per sample. For competitive hybridization to probes on the array that might nonselectively bind repetitive DNA, Human Cot-1 DNA (Invitrogen) that had been Covaris sheared to approximately 100bp was added to the hybridization mix in a 5∶1 ratio by weight. Additionally, to block the adaptor ends of the denatured, single stranded DNA fragments from binding to each other, a 10∶1 molar excess of adaptor oligos (P2 with an unused barcode sequence and P1) was also added. At the completion of hybridization, the slide was washed with the Nimblegen Sequence Capture Wash and Elution kit per manufacturer' s directions, and the enriched DNA was eluted with 350L of C purified water using an affixed SA200 SecureSeal Hybridization Chamber (Grace Bio-Labs, Bend OR). A secondary elution with an additional 350L was also taken. Quantitative real-time PCR (qPCR) was performed on the eluted material to determine the rough fraction of target DNA and to compare the primary and secondary elutions. For this purpose qPCR amplicons within the target were compared to amplicons not in the target regions. It was generally found that the primary elution captured more than 95% of the target DNA (data not shown). By normalizing to pre-enrichment material, and taking into account the fact that the 2. 1Mbp target region comprised approximately 0. 1% of the entire human genome, it was estimated that more than 35% of the eluted material fell in the target regions (data not shown). The eluted material was ExTaq LMPCR amplified (samples DY01 for 19 cycles, samples DY03–04 for 15 cycles, pooled samples DY21–24 for 10 cycles, samples DY17–20 for 12 cycles,) in preparation for the emulsion PCR step of SOLiD sequencing that was performed in the UC Santa Cruz Genome Sequencing Center. Samples DY01, DY03, DY04 were processed with the SOLiD Version 2 system, producing 35 bases of sequence information for each read. The remaining samples were processed with the SOLiD Version 3 system, producing 50 bases of sequencing information for each read. The 35mer (DY01, DY03, DY04) or 50mer (remaining samples) sequencing reads were mapped to the whole human genome using the bwa program [55] which generates mappings and associated quality scores in the sam format [56] that can be processed with the samtools suite. The bwa program is aware of the colorspace nature of the SOLiD sequencing reads, and uses a dynamic programming algorithm to infer the best nucleotide sequence for the read [55]. All reads were also mapped to the DNA of the Epstein-Barr Virus, which was used to transform the Coriell cell lines from which the supplied genomic DNA was extracted. For the female samples, the Y chromosome was excluded from the mapping. For the male samples, the pseudo-autosomal region of the Y chromosome was excluded from the mapping. The set of mappings for each sample was then filtered to the regions covered by the probes on the Nimblegen enrichment array described above. To eliminate spurious pileups caused by overamplification of particular molecules in the library preparation process, the mapped reads were further filtered to select at most 4 reads from each strand at a given genomic starting position. Where there were more than 4, the 4 with the highest total read quality (not the best mapping quality, which would bias against reads containing non-reference alleles) from the SOLiD instrument were selected. Between 40% and 60% of the reads for a given sample were successfully mapped, and of those reads, between 33% and 48% mapped to bases covered by the probes on the enrichment array (Supplementary Table 3 in Text S1). For samples DY01, DY03, DY04 about 50% of the latter reads were lost in the “maximum 4 per strand” pileup elimination step, while only 11% to 23% were lost in this step in the remaining samples (Supplementary Table 3 in Text S1). This was likely due to the difference in LMPCR cycles used for the different samples as noted above. To determine the coverage at each position in the target region and the consensus genotypes for each sample, the command “samtools pileup -v” was used with default parameters for its consensus calling model. Possible confounding of the genotypes due to contamination by paralogous sequences was avoided in two ways. First, as noted, only genotypes at positions delimited by the Nimblegen probes were used in the analysis and these probes were designed to avoid repetitive sequences. Second, the bwa mapping algorithm assigns low mapping quality to reads that are not genome-wide unique, and the samtools consensus caller requires high mapping quality. To filter SNPs from among the not homozygous reference genotypes, “samtools. pl varFilter” was run with default parameters, except that the maximum read depth was set to 425, because with up to 4 reads of length 50 on each strand, it was possible to get coverage of 400. This command filters out potential SNPs when more than 2 fall within a 10bp window, on the grounds that there might be an insertion/deletion event rather than separate SNPs, and also filters out reads with RMS mapping quality value less than 25. A similar quality filter was applied to the genotype calls that were homozygous reference. Because of stochastic variation in the composition of reads from the two chromosomes of each diploid individual, low coverage might cause an erroneous homozygous call in a true heterozygote. Therefore a further filter restricted the subsequent analysis to the SNP or homozygous reference calls made for a sample only at positions for which the coverage was 35 or greater. For each target region, the count of the union of such positions across all samples is listed in Supplementary Table 1 in Text S1. As shown in Supplementary Figure 5 in Text S1, the vast majority of the segregating sites that remain after the application of our 35× coverage filter are in Hardy-Weinberg Equilibrium, with only 0. 3% having a p-value less than 0. 05. For purposes of all subsequent analysis, an ancestral allele at each position in the target regions was determined from the Enredo-Pecan-Ortheus (EPO) pipeline [57], [58] as published on the 1000 Genomes website [59]. This pipeline determines the common ancestor of human and chimp at a locus by considering alignments of the human, chimp, orangutan, and rhesus macaque genomes. From the sets of filtered genotype calls in the 11 diploid samples as described above all the segregating sites were selected. A set of filters was applied to this list to produce the final set of segregating site derived (i. e. non-ancestral) allele frequencies (DAFs) for all downstream analyses. To avoid skewing the DAFs towards higher frequencies, segregating sites with less than 8 chromosomal samples were eliminated. Also eliminated were any positions with more than 2 alleles among the reference, ancestral, or sample alleles, or where the ancestral allele was not determined by the EPO pipeline. Lowercase values of the EPO ancestral allele, which result from various cases without complete evidence in all species were not eliminated. The SweepFinder program [28] was applied to the allele frequencies for the final list of segregating sites to determine the composite likelihood ratio (CLR) of a selective sweep at each one of a grid of 1000 positions across each target region. The model used in this program requires a background derived allele frequency spectrum. Two such backgrounds were used. First, all of the DAFs from all filtered segregating sites in our sample were aggregated and used as input to the command “SweepFinder -f”, which accounts for missing data using a Broyden-Fletcher-Goldfarb-Shanno (BFGS) algorithm. We refer to this as the “harseq” background. A second, presumably more neutral background was obtained from the African-Derived (AD) YRI subset of 24 individuals in the Seattle SNPs P2 panel. The DAFs for all segregating sites in all 104 genes resequenced for this panel by Seattle SNPs [33] were included. As for the harseq background, the “seasnp” background was obtained with the command “SweepFinder -f” applied to these DAFs. The resulting “seasnp” frequency spectrum ran from 1 to 47 and was reduced to a spectrum running from 1 to 21, as needed by SweepFinder with our data, by hypergeometric weighting the relevant components of the input allele frequencies at each target allele frequency. (1) Dividing by the sum of the in Eqn 1 produces a valid frequency spectrum that sums to 1. A similar hypergeometric weighting was also required to reduce the spectrum to a range from 1 to 19 for the harseq1,2 and ctlreg60 regions. In the latter regions missing data reduced the maximum number of samples at the segregating sites to 20. P-values for our SweepFinder results were obtained via coalescent simulation conditional on the observed number of segregating sites in focal region, the observed coverage of this region, and the estimated recombination rate for a given region according to the pedigree data of [36] assuming a human effective population size of. The second point is important here in that our resequencing of both the harseq regions and the control regions was not perfectly complete, but instead was partial owing to an inability to design proper probes for our Nimblegen enrichment procedure (see above) in certain genomic segments. For each region examined we performed coalescent simulations under the standard neutral model which has been shown to be conservative for the SweepFinder procedure [29]. From the set of fixed differences between human and chimp in the 49 core HAR elements we use the EPO determined ancestral allele (see above) to count 206 as the total number of human lineage specific substitutions. Since a small number of substitutions are expected to occur by chance even in constrained elements, we used the number of substitutions on the chimp lineage as an estimate of the minimum number of non-adaptive substitutions in each HAR. These total 16 for all 49 HARs. So, we approximate that at most 190 (= 206-16) substitutions were adaptive in humans. In reality some of the excess nucleotide changes for a given HAR were probably segregating at the same time on the same haplotype. So, 190 is most likely an overestimate of the number of adaptive events in HARs. But suppose there were indeed 190 separate adaptive substitutions and that these occurred uniformly over the last 5 million years. Further assume that any sweep from the last 200,000 years could be detected by SweepFinder. Then, 4% of the 190 adaptive substitutions (i. e., 7. 6 sweeps) should be in the detectable time frame. Since the number 190 and the percentage 4% are both upper bounds, we conclude that at most 8 and probably much fewer than 8 sweeps would be detectable by our SweepFinder analysis even if all 49 HARs were shaped by adaptive evolution. Our finding of no significant sweeps after Bonferroni correction and 5 significant before correction is therefore consistent with expectations. To determine if mutations from an ancestral weak (A or T) basepair to a strong (G or C) basepair (W2S mutations) are more likely to spread in the population represented by our samples, we compared W2S segregating sites to S2W segregating sites for each target region and for the aggregate set of segregating sites. We performed a Mann-Whitney U (MWU) test for a difference between the W2S and S2W derived allele frequency spectra. The test was performed in the R language with the command “wilcox. test (paired = FALSE, alternative = two. sided) ”. The resulting “location” parameter was normalized to 22 samples and is positive if W2S mutations are segregating at higher frequencies than S2W. The resulting p-values are in Supplementary Table 2 in Text S1. To determine if relatively more W2S mutations fixed along the human or chimp lineages than are segregating in the human population represented by our samples, we first determined the high mapping quality chimp reference bases that differ from the human reference using reciprocal best alignments of the chimp and human genomes [37]. This set was then restricted to the positions in our target regions for which we had a genotype call for at least one sample with read depth of coverage of 35 or greater as discussed above. From this set of fixed differences we removed any for which the EPO ancestral allele was not determined as discussed above, or for which we had a segregating site, or which appeared in dbSNP release 129 [38]. The remaining fixed differences as well as the segregating sites were divided into W2S or S2W (or other). A McDonald Kreitman-like (MK) test on the resulting 2×2 contingency table was performed in the R language with the command “fisher. test (alternative = two. sided) ”. The resulting p-values are in Supplementary Table 2 in Text S1. For the significant cases it was easy to determine from the data in the contingency table if the fixed differences favored S2W mutations relatively more than the segregating sites (column “S2W” in Table 2). For the target regions with significant p-values on either the MWU or MK tests, we tested whether the significance was due to a restricted locus within the region, by removing all segregating sites and fixed differences under a mask of a given size at a given position within the region and rerunning the test with the remaining data (Table 1 and Table 2). We downloaded the data for the 104 genes resequenced with the Seattle SNPs P2 panel. The genotypes for our 11 YRI samples (which are a subset of the P2 panel) and the coordinates for the genotyped segregating sites were obtained from the global “prettybase” file mapped to UCSC hg18 coordinates. The total set of positions genotyped was obtained from the individual gene “genbank” files by excluding the features defined as “Region not scanned for variation” and aligning the remaining regions to the hg18 coordinates of the full extent of the genic region sequenced specified in the associated “ucscDataFile”. Given these coordinates and the genotypes at the segregating sites, the same techniques as described above for our resequencing data was applied to derive ancestral alleles and human/chimp fixed differences, and to perform the MWU and MK tests. Our data was derived from (probed) regions of a relatively tight size distribution (Supplementary Table 1 in Text S1). By contrast, the sizes of Seattle SNPs variation-mapped genic regions varied widely. Some were rather small and contained few segregating sites. Therefore, we included only genic regions with a minimum of 10kb variation-mapped and a minimum of 40 segregating sites. Additionally, a small number of the genic regions were excluded because of data missing from the “prettybase” file or because there were no associated high quality reciprocal best human chimp differences as described above, possibly because of paralogous genes in one or the other lineage. The remaining set of results for the MWU and MK tests on 62 genic regions (Supplementary Table 4 in Text S1) were used for comparison to our 49 harseq regions. To determine if the p-values for the MWU and MK tests were accurate, we also conducted the tests on sets of simulated data under a neutral model. For the MWU test, we performed coalescent simulations using Hudson' s ms program [60]. For each simulation we generated 22 samples at 85 segregating sites (the average number of W2S plus S2W segregating sites in the 49 harseq target regions) and then randomly assigned the sites as either W2S or S2W in Bernoulli trials using the W2S∶S2W ratio from the 49 harseq regions of 2057∶2114. After calculating the MWU test p-value for each simulation, the fraction of simulations with p-value less than a given value was computed, as well as the subset of that fraction in which the W2S spectrum was offset towards higher derived allele frequencies (Supplementary Figure 2 in Text S1). For the MK test, for each simulation we separately derived a set of human-chimp fixed differences and a set of segregating sites. The fixed differences were derived using the phyloBoot program from the PHAST package [61]. We used a phylogenetic model and substitution rate matrix derived from 4-fold degenerate amino-acid coding synonymous sites across the genome as an unbiased neutral model. The equilibrium GC-content of this model was adjusted to reflect the genome-wide average GC-content. From the primate sequences so generated, we extracted positions containing a human-chimp difference that could also be unambiguously assigned an ancestral allele based on the macaque allele at that position. Each simulation used 335 such sites, (the average number of fixed differences in the 49 harseq target regions) which were divided based on whether they were W2S or S2W (or other). The segregating sites for each simulation were derived from 101 (the average total number of segregating sites in the 49 harseq target regions) Bernoulli trials, randomly dividing them as W2S or S2W (or other) according the corresponding ratios in the fixed differences from all of the phyloBoot simulations. After calculating the MK test p-value for each simulation, the fraction of simulations with p-value less than a given value was computed, as well as the subset of that fraction for which the ratio of W2S∶S2W was higher for the simulated fixed differences than for the simulated segregating sites (Supplementary Figure 3 in Text S1). Simulations of GC-biased evolution due to BGC were generated using a forward time Wright-Fisher model of a population. Simulations were run at a population size of 10,000, which is approximately comparable to the long term human effective population size. Details of the simulation method can be found in [62] and references therein. Briefly, we model BGC as a selection process in which each W2S (S2W) mutation adds (subtracts) some normal deviate fitness value to the haplotype on which it is found. This model is approximately equal to the normal shift model [63] if we were only to consider the W2S subset of mutations. Simulations were run for 40 * N generations as a burnin period to reach stationarity, at which point we modeled a vicariance event representing the human chimp divergence. After the population split we ran the two populations for 6. 5 units of 4N generations, to approximate the divergence time between humans and chimps. We assume the strength of BGC acting was 4NB = 1. 3 as recently estimated from human data [10], [64]. We also assumed a ratio of 4NB∶4Nu of 1. The MWU and MK tests were performed as above using a single sample from the chimp lineage and 50 samples from the human lineage. Association between MWU and MK tests on simulated (and HAR region) data was assessed using Fisher' s Exact Test on the 2×2 contingency table defined by the counts of significant or not significant tests: {MWU, notMWU}×{MK, notMK}. The numbers of significant tests by either or both MWU and MK were compared between the simulated and HAR region data using binomial and Poisson tests.

The search for functional regions in the human genome, beyond the protein-coding portion, often relies on signals of conservation across species. The Human Accelerated Regions (HARs) are strongly conserved elements, ranging in size from 100-400 bp, that show an unexpected number of human-specific changes. This pattern suggests that HARs may be functional elements that have significantly changed during human evolution. To analyze the evolutionary forces that led these changes, we studied 40 kb neighborhoods of the top 49 HARs. We took advantage of recently developed DNA sequencing technology, coupled with methods to isolate genomic DNA for our target regions only, to determine the genotypes in 22 chromosomal samples. This polymorphism data showed no significant evidence for adaptive selective sweeps in HAR regions. By contrast, we found strong evidence for a nucleotide bias in the fixation of mutations from A or T to G or C basepairs. Our work reveals that this bias in the HAR neighborhoods is not just an historic phenomenon, but is ongoing in the present day human population. This finding adds credence to the possibility that non-selective forces, such as biased gene conversion, could have contributed to the evolution of several of these regions.

lay_plos

Tropical pathogens often cause febrile illnesses in humans and are responsible for considerable morbidity and mortality. The similarities in clinical symptoms provoked by these pathogens make diagnosis difficult. Thus, early, rapid and accurate diagnosis will be crucial in patient management and in the control of these diseases. In this study, a microfluidic lab-on-chip integrating multiplex molecular amplification and DNA microarray hybridization was developed for simultaneous detection and species differentiation of 26 globally important tropical pathogens. The analytical performance of the lab-on-chip for each pathogen ranged from 102 to 103 DNA or RNA copies. Assay performance was further verified with human whole blood spiked with Plasmodium falciparum and Chikungunya virus that yielded a range of detection from 200 to 4×105 parasites, and from 250 to 4×107 PFU respectively. This lab-on-chip was subsequently assessed and evaluated using 170 retrospective patient specimens in Singapore and Thailand. The lab-on-chip had a detection sensitivity of 83. 1% and a specificity of 100% for P. falciparum; a sensitivity of 91. 3% and a specificity of 99. 3% for P. vivax; a positive 90. 0% agreement and a specificity of 100% for Chikungunya virus; and a positive 85. 0% agreement and a specificity of 100% for Dengue virus serotype 3 with reference methods conducted on the samples. Results suggested the practicality of an amplification microarray-based approach in a field setting for high-throughput detection and identification of tropical pathogens. Many infectious diseases are more prevalent in the tropical and subtropical regions where ecological, geographical and socioeconomic factors facilitate their propagation. The high diversity of such tropical pathogens include bacteria, fungi, helminths, parasites, and viruses that mirrors the rich biodiversity in the tropics and sub-tropical regions [1]–[3]. Many of these pathogens are transmissible through an insect vector or an invertebrate host [4]–[7], and transmission is affected by climate that can significantly influence vector behavior and physiology [8], including the extrinsic incubation period of vector-borne pathogens [9], [10]. Furthermore, global changes such as anthropogenic climate change and climate variability, habitat encroachment by the growing human population, volume of international travel, migration, trade and pollution create new opportunities for microbial spread [11]–[13]. The world is subjected to a plethora of tropical pathogens. Table 1 provides an overview of 14 tropical diseases, stratified into protozoan, bacterial, and viral infections that are globally important. However, some of these tropical diseases are often intimately connected to paucity of local and global burden estimates, poverty, geographical isolation and lack of coordinated approaches for disease controls [14]. Firstly, there are protozoan infections: malaria, which remains one of the most devastating and difficult parasitic diseases to be controlled and further threatened by the emergence and spread of resistance to anti-malarial drugs [15]–[17]; Chagas disease which is one of the most neglected tropical disease with a lifelong infection [18]–[20]; and human African trypanosomiasis with 60 million people at risk in Africa [21]–[23]. Next are bacterial infections: leptospirosis, which has been identified as one of the most widespread zoonosis in the world, exemplified by outbreaks in rural and urban environments [24]–[27], and more recently, emerged as a disease of the adventure traveler [28]; meliodosis that has been reported with a global distribution [29], [30]; and salmonellosis, which causes enteric fever and has a high global incidence [31]. Finally, the most prevalent infections are those of viral origins: Chikungunya fever in the Indian Ocean islands, the Indian subcontinent, southeast Asia, Africa, Europe and its emergence in the Americas [32]–[37]; Dengue fever including the emergence of dengue hemorrhagic fever [38]–[43]; West Nile fever in America [44], [45] and the increasing extensive distribution through Africa, Middle East, Europe and Asia [46]; Japanese encephalitis in Australasia [47] and in Asia [48]; yellow fever in West and Central Africa [49]; high incidence rates of hand, food and mouth disease in Asia [50]–[53]; Rift valley fever which has spread to Yemen, Saudi Arabia, northern Egypt and the French island of Mayotte [54]; and Hantavirus hemorrhagic fever which can cause serious diseases in humans with mortality rates of 12% (hemorrhagic fever with renal syndrome) and 60% (Hantavirus pulmonary syndrome) in some outbreaks [4], [55]. Despite being medically important, the incidence rates of some of these diseases are grossly underestimated and this reflects the clinical index of suspicion of the diseases which could have resulted from a lack of access to rapid diagnostics [18], [25], [29]. The global spread of tropical diseases emphasizes the importance of preparedness to address them. The first goal of this preparedness is fast and accurate diagnosis of medically important diseases. Differential diagnosis is based mainly on clinical examination, taking into account which diseases are locally prevalent, potential exposure, and the relevant travel history. However, the similarity and the non-specific nature of the symptoms provoked by many tropical pathogens (Table 1) complicates correct diagnosis by classical clinical observations [25], [56]–[61]. Yet, a correct diagnosis is necessary to institute effective control measures, from timely therapeutic intervention [62], [63], to effective treatment [64] and effective clinical management in deploying appropriate community-wide control measures to improve the patients' clinical outcome, disease mapping, impact monitoring, and post-elimination surveillance. Correct diagnosis can only be determined through reliable laboratory-confirmed detection and identification of tropical pathogens in clinical specimens. Polymerase chain reaction (PCR) has been used in the diagnosis of several infectious diseases [51], [65]–[70] as it is a highly specific and sensitive method for molecular detection [71]–[73]. Moreover, much progress has been made with molecular multiplexing [74]–[78]. With the advent of microarray technology which permits simultaneous detection of a given sequence in a sample by hybridization to thousands of defined probes [79], amplification and microarray integrated assays have been made possible [74], [80]–[82]. In this study, microfluidic technology was combined with reverse transcription (RT), PCR amplification, and microarray hybridization to develop a silicon based micro electro mechanical systems (MEMS) integrated lab-on-chip that can simultaneously detect and differentiate between 26 pathogen species (including bacteria, parasites and viruses) that cause 14 tropical diseases. The detection platform is composed of the disposable lab-on-chip, a temperature control system (TCS) for the accurate control of thermal process and an optical reader for the fluorescence microarray image acquisition. The ability of the lab-on-chip to provide a “blood-to-diagnosis” solution in the detection of known and divergent pathogens was demonstrated on retrospective patient specimens. This system allows the simultaneous identification and discrimination of a large number of candidate tropical pathogens. It is undoubtedly a potential game-changer in the field of molecular diagnostics, as it provides an effective and rapid means to establish the presence of defined potential pathogens. The use of human samples was approved by the National Healthcare Group' s Domain-Specific Ethics Review Board (DSRB reference no. B/08/026), and written informed consent was obtained from all participants. Approval was also obtained for the use of archived samples from The Oxford Tropical Research Ethics Committee (OxTREC) as part of the surveillance routine. Plasma samples from 30 PCR-confirmed Chikungunya virus (CHIKV) patients who were admitted to the Communicable Disease Centre at Tan Tock Seng Hospital (TTSH) during the outbreak from August 1 to September 23,2008 [83], [84] were included. Plasma samples were also collated from 10 healthy donors with informed consent (DSRB reference no. B/08/026) and used as negative controls. RNA samples were extracted using the QIAamp viral RNA mini kit (Qiagen, Hilden, Germany), according to manufacturer' s instructions. One hundred and twenty five archived nuclei acid samples extracted from specimens at the Shoklo Malaria Research Unit (SMRU) clinic on the Thai-Burmese border between 1999 and 2011 as part of two surveillance studies [16], [85] were included. DNA extracts from packed red blood cells obtained from patients (refugees and migrants) with a clear malaria diagnosis (part of the malaria burden observational study) were tested with the lab-on-chip assay. The sensitivity and specificity of the chip assay was then determined against microscopy diagnosis used by the Thailand clinics [16]. Non-malaria specimens collected from patients presenting with undifferentiated febrile illness were also evaluated with the lab-on-chip. Viral RNA extracted from acute plasma specimens that had been stored at −80°C since 2008 were used. These had previously been tested with a range of tests including Dengue RT-PCR [85]. In addition, whole blood samples from 5 native healthy volunteers were extracted using the DNeasy blood and tissue kit and QIAamp viral RNA mini kit (Qiagen) and used as negative controls. Cultures of the 3D7 clone of the NF54 strain of Plasmodium falciparum (P. falciparum) were performed using sealable flasks with RPMI-HEPES medium at pH 7. 4, supplemented with 50 mg/mL hypoxanthine, 25 mM NaHCO3,2. 5 mg/mL gentamicin, and 0. 5% (weight/volume) Albumax II (Gibco, Singapore) in an atmosphere containing 5% CO2, as previously described [86], [87]. The CHIKV isolate used in this study was originally isolated from a French patient returning from Reunion Island during the 2006 CHIKF outbreak [88]. After passages in Vero-E6 cultures, virus stocks were washed, and precleared by centrifugation before storing at −80°C. All virus stocks were titered by plaque assay and quantified by quantitative RT-PCR (qRT-PCR) as previously described [89], [90]. Target gene sequences of each pathogen (Table S1 in Text S1) were first obtained from Genbank database. Sequence alignments were performed using the ClustalW algorithm [91] in MegAlign (DNAStar, Inc., Madison, WI). A consensus sequence representing clinically relevant strains (Table S1 in Text S1) was created for each pathogen. Each target oligonucleotide sequence was designed through multiple, successive steps of evaluation of candidate sequences, based on user-defined criteria, followed by analysis with Basic Local Alignment Tool (BLAST) [92] against the nucleotide sequence database (nr/nt) [93] for non-target genomes potentially present in the specimen that could cause interference. Genus-specific PCR primers were designed for all chosen target genes sequences as previously described [94], [95]. Genus-specific (for Plasmodium, Flaviviruses and Hantaviruses) and species-specific capture probes were selected to target 2 to 4 regions of the targeted gene to confirm specificity and to overcome the problem of poor hybridization within the amplicon as a result of strain-specific gene polymorphisms. Efforts to improve specificity included the design of short length capture probes of 20 to 30 nucleotides in line with other studies which have shown that shorter length probes showed higher specificity [96]. For each pathogen, a PCR product encompassing the targeted region was prepared using the consensus sequence and cloned into the T7 polymerase expression vector pGEMT-easy (Promega, Madison, WI) as described [70]. Serial diluted plasmid DNA or in vitro-transcribed RNA from respective quantified stocks was used as the DNA copy number control for DNA pathogens or RNA copy number control for RNA pathogens. The lab-on-chip was manufactured on a silicon wafer based on MEMS and mounted on a printed circuit board (PCB) support that provides mechanical, thermal, and electrical connection [94], [95], [97] (Figure S1 in Text S1). It encompassed two silicon microreactors (12 µL) connected to a microarray chamber. The microarray chamber (3. 5 mm×9. 0 mm) contains 126 spots consisting of duplicate oligo-probes spotted onto the surface through a piezo-array system [95] to ensure that differential signals do not occur by chance. The enzymatic thermal cycling and hybridization reactions on the lab-on-chip are performed by the electronic TCS. Tropical pathogen detection was split into two chip versions to be subjected to two different multiplex reactions; DNA chip with a customized microarray layout specific for DNA pathogens and RNA chip specialized for RNA pathogen detection. PCR was performed on a DNA chip in a constituted reaction of 200 nM forward and 500 nM Cy5-conjugated reverse primers in 23 µL final volume using the QuantiTect multiplex RT-PCR NoROX kit (Qiagen). Amplification was carried out with initial denaturation at 90°C for 15 min, followed by 45 cycles of 95°C for 15 sec, 60°C for 40 sec, and 72°C for 30 sec, then final extension at 72°C for 60 sec. RT-PCR was carried out on the RNA chip using SuperScript III one-step RT-PCR system with platinum Taq (Life Technologies) in a 23 µL reaction volume containing a concentration of forward and Cy5-conjugated reverse primers in the range of 200 nM to 700 nM. After a 20-min reverse transcription step at 50°C, enzyme activation was initiated at 95°C for 120 sec, followed by denaturation at 95°C for 10 sec. Amplification was performed in a manner of touch down PCR to enhance the specificity of the initial primer-template duplex formation and hence specificity of the final PCR product [98]. The annealing temperature in the initial cycle was initiated at 60°C (5°C above the average melting temperature of the primers for RNA pathogen detection). In the subsequent 10 cycles, the annealing temperature was decreased in steps of 1°C/cycle until a temperature was reached to 50°C, and followed by extension at 72°C for 50 sec. Following these 10 cycles, 40 cycles with a temperature of 95°C for 15 sec, annealing temperature of 56°C for 40 sec, and then a final extension for 50 sec at 72°C completed the program. Upon completion of PCR or RT-PCR, denaturation of amplicons proceeded at 95°C for 3 min, followed by hybridization at 58°C for 30 min. The lab-on-chip was washed and spin dried. The dried chip was scanned in the optical reader [95] (Veredus Laboratories) which has an excitation filter for Cy5. Accompanied software analysis was based on hybridization of amplicons to target-specific capture probes with the highest signals expected from a perfect match. Spot segmentation and intensity calculation of the microarray image was performed by overlaying a virtual grid over the microarray image using the corner features as reference points. For positive detection of Plasmodium parasites, Flaviviruses and Hantavirus, at least 1 out of 2 genus-specific probes must give a positive signal to indicate the presence of the respective genera, and at least 50% of species-specific probes must hybridize for species differentiation (Table S1 in Text S1). For the rest of the pathogens, at least 2 out of 3 pathogen-specific probes must give a positive signal for a positive detection of the pathogen (Table S1 in Text S1). The detection threshold and specificity of the lab-on-chip assay was evaluated by using 4 µL of quantitative standards (to cover a range of 101 to 104 copies per chip for each pathogen) and assessing the signal intensity and presence of cross hybridization at each copy number. Triplicates were run to ensure intra-experimental reproducibility. The lowest titer (DNA or RNA copies per chip) with 2 or more out of 3 chips positive for the assayed pathogen was further expanded to another 21 replicate runs to confirm the LoD which was the indicated titer that would yield more than 95% positive detection, as well as to evaluate inter-assay reproducibility. Sorted P. falciparum parasites were serial diluted in phosphate-buffered saline (PBS) and added to whole blood to obtain spiked samples with final concentrations of 1 to 103 parasites/µL [87]. In parallel, CHIKV virus stock was serial diluted before spiking into aliquots of whole blood to cover 1 to 105 PFU/µL. Spiked experiments were repeated twice for inter-experimental reproducibility. Sensitivity of the chip assay was compared with that of nested PCR [99] or qRT-PCR [70] respectively. The volume of the isolated nuclei acid subsequently used in for all comparison assays was kept constant at 4 µL. All statistical analyses were performed using Prism 6. 03 (GraphPad Software, Inc., La Jolla, CA). Lab-on-chip outcome on previously laboratory-confirmed samples was analyzed using Fisher exact test. P values less than 0. 05 were considered statistically significant. The objective of developing a portable microfluidic integrated lab-on-chip (Figure S1 in Text S1) was to provide a seamless one-time screening test for multiple tropical pathogens that exhibit similar or non-specific symptoms. Twenty-six pathogen species that cause 14 globally important but yet neglected tropical diseases (Table 1 and Table S1 in Text S1) were considered for the panel. A typical workflow for the detection of these pathogens was defined. It comprises of a processing step (blue) that includes the sample extraction and reaction setup. This is then followed by the on-chip identification and differentiation (red) (Figure 1) to ensure accurate implementation of the assay. Microarray spots were simultaneously assessed to calculate differences in signal intensities, thereby identifying unique patterns (Figure S2 in Text S1). Hybridization to a series of target-specific probe sets provided presence/absence information for the tropical pathogen, while also revealing the species of the causative agent (Figures S2, S3 in Text S1). The rationale of the analytical evaluation of the lab-on-chip was to define the LoD of the assay for all the pathogens. LoD of the lab-on-chip was determined as the lowest copy number which, in terms of plasmid copy for DNA or RNA transcript copy for RNA, when added to the chip, led to more than 95% positive pathogen identification outcome. Table S1 in Text S1 shows the lowest detectable dilution for each pathogen. The results revealed an individual sensitivity that ranged from 102 to 103 copies per chip (Figure 2). Target-specific hybridization signal saturation was observed at concentrations as low as 104 copies for all the pathogens (Figure 2). Notably, a highly sensitive detection range of 3 orders of magnitude between LoD and signal saturation was achieved for most of the tropical pathogens, mainly S. enterica, T. brucei and T. cruzi under the DNA pathogen category, together with RNA viruses such as West Nile virus, yellow fever virus, Enterovirus 71 and rift valley virus (Figure 2). Although a narrow detection range of 10 copies was observed for Hantaviruses with LoD at 103 copies, the rest of the pathogens stayed within the broad detection range of approximately 2 orders of magnitude. It should be noted that to date, cases of Hantavirus infections in patients yielded very low or non-detectable viral load levels [55], [100]. Probe specificity evaluation showed no significant cross reactivity (Figures S2 and S3 in Text S1). The efficiency of a detection assay is often dependent on the efficiency of the nuclei acid extraction method from clinical specimens [101], [102]. Some methods may even interfere with the PCR reaction [103], [104]. The purpose of the investigation was to assess the efficiency of the extraction method and the sensitivity of the lab-on-chip using P. falciparum and CHIKV as targets. The read-out for the lab-on-chip and that of nested PCR is illustrated in Table 2 and in Figure 3. The presence of P. falciparum in the extracted spiked samples was demonstrated by the presence of hybridized genus-specific and species-specific probes on the microarray for lab-on-chip, while that by nested PCR relied on the presence a PCR band on agarose gel [99]. Positive detection of P. falciparum by the lab-on-chip was observed at 100 parasites, while positive bands were detected at 5 parasites by nested PCR (Table 2 and Figure 3). Although the nested PCR method [99] is more sensitive with a difference of more than one log when compared to the lab-on-chip (Table 2 and Figure 3), it is more labor intensive. The estimated PFU isolated from CHIKV-spiked samples (in red) compared to the viral load derived from qRT-PCR is shown in Table 3 and in Figure 4. The detection threshold for CHIKV was 50 PFU (Figure 4B, 4C). More importantly, the sensitivity of the detection range of the lab-on-chip and viral load quantification by qRT-PCR are similar, clearly demonstrating the superiority of the lab-on-chip (Figure 4). In order to assess the clinical performance of the assay, the lab-on-chip was evaluated on retrospective clinical specimens to compare its diagnostic capability with reference methods. The screening and order of diagnostic testing of 170 samples received in Singapore and Thailand are illustrated in Figure 5. Sixty-four out of 77 P. falciparum positive samples and 21 out of 23 P. vivax positive samples were concordant with the microscopic diagnosis (Tables 4,5). The sensitivity and the specificity for the detection of P. falciparum was 83. 1% (72. 9% to 90. 7%) and 100% (96. 1% to 100%) (Table 4, Figure 6), and that of P. vivax was 91. 3% (71. 9% to 98. 9%) and 99. 3% (96. 3% to 99. 9%) (Table 5, Figure 6). Fourteen P. falciparum positive samples with low levels of parasitemia did not yield a positive detection for P. falciparum, but 11 out of the 14 were tested positive for Plasmodium. Although species differentiation was not achieved with these 11 samples, the assay did provide a diagnosis for Plasmodium. The validation also yielded a good positive 90. 0% agreement (73. 5% to 97. 9%) and excellent specificity 100% (97. 4% to 100%) for the CHIKV detection (Table 6, Figure 6). Finally, the assay showed an average positive 85% agreement (62. 1% to 96. 8%) (17 out of 20 DENV positive samples) and a specificity of 100% (97. 5% to 100%) for DENV 3 detection (Table 7, Figure 6). The 3 CHIKV samples that were not detected positive by the lab-on-chip were that with low viral load of less than 102 viral copies/µL quantified by qRT-PCR [70]. All healthy donor samples tested were negative. While every disease presents specific diagnostic challenges, clinical needs associated with specificity, sensitivity, total analysis time, and implementation would eventually impact the design and development of the diagnostic method. In this study, an integrated strategy for miniaturizing and simplifying complex laboratory assays for the detection of 14 globally important tropical diseases stood out favorably in terms of seamless implementation and pathogen coverage compared to conventional laboratory diagnostic methodologies. The mainstay to detect protozoan infections such as Chagas disease, human African trypanosomiasis, and malaria infection relies in the conclusive visualization of the parasites in blood [18], [21], [105]. The reliable identification of these infections requires high quality training in specimen preparation and a competency in identifying the parasites when compared to the facile interpretation of the lab-on-chip microarray analysis. Bacteria culture remains as one of the most effective procedures in identifying bacterial infections [106]–[108] and is also crucial in generating pools of clinical strains for pathogenesis studies. However, the process is labor and time intensive, spanning from a few days to several weeks when compared to the lab-on-chip assay that is completed within 4 hours. It is also dependent on stringent transport conditions and well-maintained equipments to maintain specimen viability. While methods based on serological reactivity to pathogen-specific antibodies [109]–[111] have been developed to identify several viral infections and are useful in differentiating viruses within the same family or genus, cross reactivity remains a conflicting issue [100], [112], [113]. In spite of cross reactivity issues, serology is still widely used to confirm diagnosis due to limitations in the detection window of nucleic acids [83], [85], [100]. Here, the analytical performance of the lab-on-chip has highlighted its specificity with no cross reactivity observed between the 5 Plasmodium species, between DENV and the other 3 Flaviviruses, and among the 6 Hantaviruses, achieved in just one test. Future iterations of the lab-on-chip could include protein-based arrays as additional serology screens [114], [115] for some diseases that are clinically warranted as orthogonal diagnosis based on nucleic acid, protein, and other biomarkers will be where the field is heading. Simultaneous laboratory screening of a clinical specimen from a patient with unspecific symptoms for as many tropical agents as possible would either lead to pathogen identification or narrow down the possible causes through elimination. However, combining the various assays for parallel screening of tropical diseases is not a feasible approach given the high diversity of the protocols with many limitations associated with each pathogen. Even though amplification microarray assays [80]–[82] have been developed to circumvent the need for parallel tests, detection in these assays was restricted to one virus family, despite an improvement in pathogen coverage, and thus still considered as low throughput. Moreover, simultaneous detection was achieved only after 3 separate amplification reactions for the 3 respective virus families [80]. Miniaturized total analysis systems [116] have evolved, that has led to miniaturized PCR devices being extensively studied [117]. A few reports have demonstrated rapid on-chip detection of Influenza A virus [118], [119] and human immunodeficiency virus [120], however the development of a miniaturized assay for the detection of multiple tropical diseases pathogens including the validation on patient specimens has yet to be demonstrated. The design and process of the lab-on-chip evaluation was approached systematically. It was first evaluated using quantitative standards. The LoD of the lab-on-chip was shown to range from 102 to 103 copies and signal saturation for target-specific capture probes' hybridization was at 104 copies. This observation was crucial as the efficiency of the chip to detect the relevant pathogen in a clinical sample load on the chip containing 104 or more copies of that pathogen would be 100%. When considering the detection limit of the lab-on-chip of the pathogen in a clinical sample, the target concentration required to get the minimum amount of nuclei acids after sample extraction in the amplification reaction must be investigated. Comparison of the lab-on-chip with nested PCR using spiked P. falciparum samples and with qRT-PCR on spiked CHIKV samples has proven the efficiency of the extraction method and also emphasized a more superior trade-off between the assay' s sensitivity and its utility in the systemic differentiation of P. falciparum and detection of CHIKV. The lab-on-chip assay' s ability to detect CHIKV at 50 PFU/µL demonstrated high clinical relevance as it was shown that the mean CHIKV viral load in patients ranged between 126 to 241 PFU/µL [83]. One of the key objectives of the clinical validation was to investigate the lab-on-chip' s performance and acceptability in field settings and the degree to which the results would determine the quality of the diagnosis for surveillance and patient management to improve health outcomes. The clinical validation of P. vivax offered a sensitivity that was equivalent to microscopy. Although there was a proportion of P. falciparium samples (14 out of 77 samples) with low parasitiamia that were not positively detected for P. falciparum on the lab-on-chip, the assay did manage to give a partial diagnosis (of the samples being Plasmodium positive) for 11 of these samples. Although the lab-on-chip did not positively differentiate samples with extremely low levels of parasitemia, the low parasite burden of these patients could represent the early stages of malaria. Taken together, the analytical performance of the lab-on-chip for P. falciparum and P. vivax in the range of 102 copies, and the demonstration of its diagnostic utility using spiked samples and clinical specimens showed the applicability of the assay for Plasmodium detection. The clinical performance of the lab-on-chip for DENV and CHIKV was comparable to RT-PCR. For DENV, comparisons among the diagnostic tests at SMRU have demonstrated RT-PCR to have the best operating characteristics (sensitivity 89%, specificity 96%, positive predictive value 94%, negative predictive value 92%) [85]. This suggested that the chip would be potentially sufficient to function as a single assay for confirmation of Dengue infection, since it allowed for accurate confirmation. Similarly, the assay sensitivity for CHIKV was on par with that of RT-PCR, and achieved a positive 90% agreement with patients' samples. The cost of the assay compared to that of single assays is high. Advancements in the integration of the lab-on-chip with nuclei extraction capabilities [95] and a higher density microarray with reduced chip cost would provide a more cost-effective comprehensive coverage. While the lab-on-chip assay has showed that miniaturized multiplex PCR could reach the desired clinical sensitivity, future work should attempt to recalibrate the mix of multiplex primers and modify amplification cycling conditions for improved sensitivity. One of the key milestones for lab-on-chip systems would be the direct testing of clinical specimens obtained during the acute infection phase and provide accurate diagnosis to complement clinical assessments.

Tropical diseases consist of a group of debilitating and fatal infections that occur primarily in rural and urban settings of tropical and subtropical countries. While the primary indices of an infection are mostly the presentation of clinical signs and symptoms, outcomes due to an infection with tropical pathogens are often unspecific. Accurate diagnosis is crucial for timely intervention, appropriate and adequate treatments, and patient management to prevent development of sequelae and transmission. Although, multiplex assays are available for the simultaneous detection of tropical pathogens, they are generally of low throughput. Performing parallel assays to cover the detection for a comprehensive scope of tropical infections that include protozoan, bacterial and viral infections is undoubtedly labor-intensive and time consuming. We present an integrated lab-on-chip using microfluidics technology coupled with reverse transcription (RT), PCR amplification, and microarray hybridization for the simultaneous identification and differentiation of 26 tropical pathogens that cause 14 globally important tropical diseases. Such diagnostics capacity would facilitate evidence-based management of patients, improve the specificity of treatment and, in some cases, even allow contact tracing and other disease-control measures.

lay_plos

The unnoticed lives that the pair had hitherto led began, from the day of the suspended wedding onwards, to be observed and discussed by other persons than Arabella. The society of Spring Street and the neighbourhood generally did not understand, and probably could not have been made to understand, Sue and Jude's private minds, emotions, positions, and fears. The curious facts of a child coming to them unexpectedly, who called Jude "Father," and Sue "Mother," and a hitch in a marriage ceremony intended for quietness to be performed at a registrar's office, together with rumours of the undefended cases in the law-courts, bore only one translation to plain minds. Little Time--for though he was formally turned into "Jude," the apt nickname stuck to him--would come home from school in the evening, and repeat inquiries and remarks that had been made to him by the other boys; and cause Sue, and Jude when he heard them, a great deal of pain and sadness. The result was that shortly after the attempt at the registrar's the pair went off--to London it was believed--for several days, hiring somebody to look to the boy. When they came back they let it be understood indirectly, and with total indifference and weariness of mien, that they were legally married at last. Sue, who had previously been called Mrs. Bridehead now openly adopted the name of Mrs. Fawley. Her dull, cowed, and listless manner for days seemed to substantiate all this. But the mistake (as it was called) of their going away so secretly to do the business, kept up much of the mystery of their lives; and they found that they made not such advances with their neighbours as they had expected to do thereby. A living mystery was not much less interesting than a dead scandal. The baker's lad and the grocer's boy, who at first had used to lift their hats gallantly to Sue when they came to execute their errands, in these days no longer took the trouble to render her that homage, and the neighbouring artizans' wives looked straight along the pavement when they encountered her. Nobody molested them, it is true; but an oppressive atmosphere began to encircle their souls, particularly after their excursion to the show, as if that visit had brought some evil influence to bear on them. And their temperaments were precisely of a kind to suffer from this atmosphere, and to be indisposed to lighten it by vigorous and open statements. Their apparent attempt at reparation had come too late to be effective. The headstone and epitaph orders fell off: and two or three months later, when autumn came, Jude perceived that he would have to return to journey-work again, a course all the more unfortunate just now, in that he had not as yet cleared off the debt he had unavoidably incurred in the payment of the law-costs of the previous year. One evening he sat down to share the common meal with Sue and the child as usual. "I am thinking," he said to her, "that I'll hold on here no longer. The life suits us, certainly; but if we could get away to a place where we are unknown, we should be lighter hearted, and have a better chance. And so I am afraid we must break it up here, however awkward for you, poor dear!" Sue was always much affected at a picture of herself as an object of pity, and she saddened. "Well--I am not sorry," said she presently. "I am much depressed by the way they look at me here. And you have been keeping on this house and furniture entirely for me and the boy! You don't want it yourself, and the expense is unnecessary. But whatever we do, wherever we go, you won't take him away from me, Jude dear? I could not let him go now! The cloud upon his young mind makes him so pathetic to me; I do hope to lift it some day! And he loves me so. You won't take him away from me?" "Certainly I won't, dear little girl! We'll get nice lodgings, wherever we go. I shall be moving about probably--getting a job here and a job there." "I shall do something too, of course, till--till-- Well, now I can't be useful in the lettering it behoves me to turn my hand to something else." "Don't hurry about getting employment," he said regretfully. "I don't want you to do that. I wish you wouldn't, Sue. The boy and yourself are enough for you to attend to." There was a knock at the door, and Jude answered it. Sue could hear the conversation: "Is Mr. Fawley at home?... Biles and Willis, the building contractors, sent me to know if you'll undertake the relettering of the ten commandments in a little church they've been restoring lately in the country near here." Jude reflected, and said he could undertake it. "It is not a very artistic job," continued the messenger. "The clergyman is a very old-fashioned chap, and he has refused to let anything more be done to the church than cleaning and repairing." "Excellent old man!" said Sue to herself, who was sentimentally opposed to the horrors of over-restoration. "The Ten Commandments are fixed to the east end," the messenger went on, "and they want doing up with the rest of the wall there, since he won't have them carted off as old materials belonging to the contractor in the usual way of the trade." A bargain as to terms was struck, and Jude came indoors. "There, you see," he said cheerfully. "One more job yet, at any rate, and you can help in it--at least you can try. We shall have all the church to ourselves, as the rest of the work is finished." Next day Jude went out to the church, which was only two miles off. He found that what the contractor's clerk had said was true. The tables of the Jewish law towered sternly over the utensils of Christian grace, as the chief ornament of the chancel end, in the fine dry style of the last century. And as their framework was constructed of ornamental plaster they could not be taken down for repair. A portion, crumbled by damp, required renewal; and when this had been done, and the whole cleansed, he began to renew the lettering. On the second morning Sue came to see what assistance she could render, and also because they liked to be together. The silence and emptiness of the building gave her confidence, and, standing on a safe low platform erected by Jude, which she was nevertheless timid at mounting, she began painting in the letters of the first Table while he set about mending a portion of the second. She was quite pleased at her powers; she had acquired them in the days she painted illumined texts for the church-fitting shop at Christminster. Nobody seemed likely to disturb them; and the pleasant twitter of birds, and rustle of October leafage, came in through an open window, and mingled with their talk. They were not, however, to be left thus snug and peaceful for long. About half-past twelve there came footsteps on the gravel without. The old vicar and his churchwarden entered, and, coming up to see what was being done, seemed surprised to discover that a young woman was assisting. They passed on into an aisle, at which time the door again opened, and another figure entered--a small one, that of little Time, who was crying. Sue had told him where he might find her between school-hours, if he wished. She came down from her perch, and said, "What's the matter, my dear?" "I couldn't stay to eat my dinner in school, because they said--" He described how some boys had taunted him about his nominal mother, and Sue, grieved, expressed her indignation to Jude aloft. The child went into the churchyard, and Sue returned to her work. Meanwhile the door had opened again, and there shuffled in with a businesslike air the white-aproned woman who cleaned the church. Sue recognized her as one who had friends in Spring Street, whom she visited. The church-cleaner looked at Sue, gaped, and lifted her hands; she had evidently recognized Jude's companion as the latter had recognized her. Next came two ladies, and after talking to the charwoman they also moved forward, and as Sue stood reaching upward, watched her hand tracing the letters, and critically regarded her person in relief against the white wall, till she grew so nervous that she trembled visibly. They went back to where the others were standing, talking in undertones: and one said--Sue could not hear which--"She's his wife, I suppose?" "Some say Yes: some say No," was the reply from the charwoman. "Not? Then she ought to be, or somebody's--that's very clear!" "They've only been married a very few weeks, whether or no." "A strange pair to be painting the Two Tables! I wonder Biles and Willis could think of such a thing as hiring those!" The churchwarden supposed that Biles and Willis knew of nothing wrong, and then the other, who had been talking to the old woman, explained what she meant by calling them strange people. The probable drift of the subdued conversation which followed was made plain by the churchwarden breaking into an anecdote, in a voice that everybody in the church could hear, though obviously suggested by the present situation: "Well, now, it is a curious thing, but my grandfather told me a strange tale of a most immoral case that happened at the painting of the Commandments in a church out by Gaymead--which is quite within a walk of this one. In them days Commandments were mostly done in gilt letters on a black ground, and that's how they were out where I say, before the owld church was rebuilded. It must have been somewhere about a hundred years ago that them Commandments wanted doing up just as ours do here, and they had to get men from Aldbrickham to do 'em. Now they wished to get the job finished by a particular Sunday, so the men had to work late Saturday night, against their will, for overtime was not paid then as 'tis now. There was no true religion in the country at that date, neither among pa'sons, clerks, nor people, and to keep the men up to their work the vicar had to let 'em have plenty of drink during the afternoon. As evening drawed on they sent for some more themselves; rum, by all account. It got later and later, and they got more and more fuddled, till at last they went a-putting their rum-bottle and rummers upon the communion table, and drawed up a trestle or two, and sate round comfortable and poured out again right hearty bumpers. No sooner had they tossed off their glasses than, so the story goes, they fell down senseless, one and all. How long they bode so they didn't know, but when they came to themselves there was a terrible thunder-storm a-raging, and they seemed to see in the gloom a dark figure with very thin legs and a curious voot, a-standing on the ladder, and finishing their work. When it got daylight they could see that the work was really finished, and couldn't at all mind finishing it themselves. They went home, and the next thing they heard was that a great scandal had been caused in the church that Sunday morning, for when the people came and service began, all saw that the Ten Commandments wez painted with the 'nots' left out. Decent people wouldn't attend service there for a long time, and the Bishop had to be sent for to reconsecrate the church. That's the tradition as I used to hear it as a child. You must take it for what it is wo'th, but this case to-day has reminded me o't, as I say." The visitors gave one more glance, as if to see whether Jude and Sue had left the "nots" out likewise, and then severally left the church, even the old woman at last. Sue and Jude, who had not stopped working, sent back the child to school, and remained without speaking; till, looking at her narrowly, he found she had been crying silently. "Never mind, comrade!" he said. "I know what it is!" "I can't BEAR that they, and everybody, should think people wicked because they may have chosen to live their own way! It is really these opinions that make the best intentioned people reckless, and actually become immoral!" "Never be cast down! It was only a funny story." "Ah, but we suggested it! I am afraid I have done you mischief, Jude, instead of helping you by coming!" To have suggested such a story was certainly not very exhilarating, in a serious view of their position. However, in a few minutes Sue seemed to see that their position this morning had a ludicrous side, and wiping her eyes she laughed. "It is droll, after all," she said, "that we two, of all people, with our queer history, should happen to be here painting the Ten Commandments! You a reprobate, and I--in my condition... O dear!"... And with her hand over her eyes she laughed again silently and intermittently, till she was quite weak. "That's better," said Jude gaily. "Now we are right again, aren't we, little girl!" "Oh but it is serious, all the same!" she sighed as she took up the brush and righted herself. "But do you see they don't think we are married? They WON'T believe it! It is extraordinary!" "I don't care whether they think so or not," said Jude. "I shan't take any more trouble to make them." They sat down to lunch--which they had brought with them not to hinder time--and having eaten it, were about to set to work anew when a man entered the church, and Jude recognized in him the contractor Willis. He beckoned to Jude, and spoke to him apart. "Here--I've just had a complaint about this," he said, with rather breathless awkwardness. "I don't wish to go into the matter--as of course I didn't know what was going on--but I am afraid I must ask you and her to leave off, and let somebody else finish this! It is best, to avoid all unpleasantness. I'll pay you for the week, all the same." Jude was too independent to make any fuss; and the contractor paid him, and left. Jude picked up his tools, and Sue cleansed her brush. Then their eyes met. "How could we be so simple as to suppose we might do this!" said she, dropping to her tragic note. "Of course we ought not--I ought not--to have come!" "I had no idea that anybody was going to intrude into such a lonely place and see us!" Jude returned. "Well, it can't be helped, dear; and of course I wouldn't wish to injure Willis's trade-connection by staying." They sat down passively for a few minutes, proceeded out of the church, and overtaking the boy pursued their thoughtful way to Aldbrickham. Fawley had still a pretty zeal in the cause of education, and, as was natural with his experiences, he was active in furthering "equality of opportunity" by any humble means open to him. He had joined an Artizans' Mutual Improvement Society established in the town about the time of his arrival there; its members being young men of all creeds and denominations, including Churchmen, Congregationalists, Baptists, Unitarians, Positivists, and others--Agnostics had scarcely been heard of at this time--their one common wish to enlarge their minds forming a sufficiently close bond of union. The subscription was small, and the room homely; and Jude's activity, uncustomary acquirements, and, above all, singular intuition on what to read and how to set about it--begotten of his years of struggle against malignant stars--had led to his being placed on the committee. A few evenings after his dismissal from the church repairs, and before he had obtained any more work to do, he went to attend a meeting of the aforesaid committee. It was late when he arrived: all the others had come, and as he entered they looked dubiously at him, and hardly uttered a word of greeting. He guessed that something bearing on himself had been either discussed or mooted. Some ordinary business was transacted, and it was disclosed that the number of subscriptions had shown a sudden falling off for that quarter. One member--a really well-meaning and upright man--began speaking in enigmas about certain possible causes: that it behoved them to look well into their constitution; for if the committee were not respected, and had not at least, in their differences, a common standard of CONDUCT, they would bring the institution to the ground. Nothing further was said in Jude's presence, but he knew what this meant; and turning to the table wrote a note resigning his office there and then. Thus the supersensitive couple were more and more impelled to go away. And then bills were sent in, and the question arose, what could Jude do with his great-aunt's heavy old furniture, if he left the town to travel he knew not whither? This, and the necessity of ready money, compelled him to decide on an auction, much as he would have preferred to keep the venerable goods. The day of the sale came on; and Sue for the last time cooked her own, the child's, and Jude's breakfast in the little house he had furnished. It chanced to be a wet day; moreover Sue was unwell, and not wishing to desert her poor Jude in such gloomy circumstances, for he was compelled to stay awhile, she acted on the suggestion of the auctioneer's man, and ensconced herself in an upper room, which could be emptied of its effects, and so kept closed to the bidders. Here Jude discovered her; and with the child, and their few trunks, baskets, and bundles, and two chairs and a table that were not in the sale, the two sat in meditative talk. Footsteps began stamping up and down the bare stairs, the comers inspecting the goods, some of which were of so quaint and ancient a make as to acquire an adventitious value as art. Their door was tried once or twice, and to guard themselves against intrusion Jude wrote "Private" on a scrap of paper, and stuck it upon the panel. They soon found that, instead of the furniture, their own personal histories and past conduct began to be discussed to an unexpected and intolerable extent by the intending bidders. It was not till now that they really discovered what a fools' paradise of supposed unrecognition they had been living in of late. Sue silently took her companion's hand, and with eyes on each other they heard these passing remarks--the quaint and mysterious personality of Father Time being a subject which formed a large ingredient in the hints and innuendoes. At length the auction began in the room below, whence they could hear each familiar article knocked down, the highly prized ones cheaply, the unconsidered at an unexpected price. "People don't understand us," he sighed heavily. "I am glad we have decided to go." "The question is, where to?" "It ought to be to London. There one can live as one chooses." "No--not London, dear! I know it well. We should be unhappy there." "Why?" "Can't you think?" "Because Arabella is there?" "That's the chief reason." "But in the country I shall always be uneasy lest there should be some more of our late experience. And I don't care to lessen it by explaining, for one thing, all about the boy's history. To cut him off from his past I have determined to keep silence. I am sickened of ecclesiastical work now; and I shouldn't like to accept it, if offered me!" "You ought to have learnt classic. Gothic is barbaric art, after all. Pugin was wrong, and Wren was right. Remember the interior of Christminster Cathedral--almost the first place in which we looked in each other's faces. Under the picturesqueness of those Norman details one can see the grotesque childishness of uncouth people trying to imitate the vanished Roman forms, remembered by dim tradition only." "Yes--you have half-converted me to that view by what you have said before. But one can work, and despise what one does. I must do something, if not church-gothic." "I wish we could both follow an occupation in which personal circumstances don't count," she said, smiling up wistfully. "I am as disqualified for teaching as you are for ecclesiastical art. You must fall back upon railway stations, bridges, theatres, music-halls, hotels--everything that has no connection with conduct." "I am not skilled in those... I ought to take to bread-baking. I grew up in the baking business with aunt, you know. But even a baker must be conventional, to get customers." "Unless he keeps a cake and gingerbread stall at markets and fairs, where people are gloriously indifferent to everything except the quality of the goods." Their thoughts were diverted by the voice of the auctioneer: "Now this antique oak settle--a unique example of old English furniture, worthy the attention of all collectors!" "That was my great-grandfather's," said Jude. "I wish we could have kept the poor old thing!" One by one the articles went, and the afternoon passed away. Jude and the other two were getting tired and hungry, but after the conversation they had heard they were shy of going out while the purchasers were in their line of retreat. However, the later lots drew on, and it became necessary to emerge into the rain soon, to take on Sue's things to their temporary lodging. "Now the next lot: two pairs of pigeons, all alive and plump--a nice pie for somebody for next Sunday's dinner!" The impending sale of these birds had been the most trying suspense of the whole afternoon. They were Sue's pets, and when it was found that they could not possibly be kept, more sadness was caused than by parting from all the furniture. Sue tried to think away her tears as she heard the trifling sum that her dears were deemed to be worth advanced by small stages to the price at which they were finally knocked down. The purchaser was a neighbouring poulterer, and they were unquestionably doomed to die before the next market day. Noting her dissembled distress Jude kissed her, and said it was time to go and see if the lodgings were ready. He would go on with the boy, and fetch her soon. When she was left alone she waited patiently, but Jude did not come back. At last she started, the coast being clear, and on passing the poulterer's shop, not far off, she saw her pigeons in a hamper by the door. An emotion at sight of them, assisted by the growing dusk of evening, caused her to act on impulse, and first looking around her quickly, she pulled out the peg which fastened down the cover, and went on. The cover was lifted from within, and the pigeons flew away with a clatter that brought the chagrined poulterer cursing and swearing to the door. Sue reached the lodging trembling, and found Jude and the boy making it comfortable for her. "Do the buyers pay before they bring away the things?" she asked breathlessly. "Yes, I think. Why?" "Because, then, I've done such a wicked thing!" And she explained, in bitter contrition. "I shall have to pay the poulterer for them, if he doesn't catch them," said Jude. "But never mind. Don't fret about it, dear." "It was so foolish of me! Oh why should Nature's law be mutual butchery!" "Is it so, Mother?" asked the boy intently. "Yes!" said Sue vehemently. "Well, they must take their chance, now, poor things," said Jude. "As soon as the sale-account is wound up, and our bills paid, we go." "Where do we go to?" asked Time, in suspense. "We must sail under sealed orders, that nobody may trace us... We mustn't go to Alfredston, or to Melchester, or to Shaston, or to Christminster. Apart from those we may go anywhere." "Why mustn't we go there, Father?" "Because of a cloud that has gathered over us; though 'we have wronged no man, corrupted no man, defrauded no man!' Though perhaps we have 'done that which was right in our own eyes.'"

Jude and Sue's postponement of their marriage is noticed by the neighbors at Aldbrickham, who begin to gossip about them. Little Father Time is teased at school. Sue and Jude decide to go off to London for a few days and then return, pretending they have been married there. But the scandal continues, and the neighbors begin to snub them. Jude and Sue are deeply hurt and think of moving away. In the meantime Jude gets a new job at the country church nearby, where he has to restore the lettering of the Ten Commandments. Sue, who is now expecting a baby, goes to help Jude with his work. Unfortunately, the churchwarden and the cleaner recognize them, and they begin to gossip. Eventually, Jude loses his job because of the rumors. Later he is forced to resign from the committee of the local Artisans Mutual Improvement Society, whose members also hear of the scandal. Sue and Jude finally decide to move away from Aldbrickham, and they arrange to auction off their furniture. Although they are not certain where they will go, they decide against going to London. Jude cannot even decide what he will do for a living, since he has already given up on his church work. During the sale Jude and Sue overhear much of the local gossip about their unmarried status and are appalled. Sue is upset at the sale of her pet pigeons to the poulterer, and passing by the shop later, she sets them free. Her remarks on nature's law of "mutual butchery" leave Little Father Time very disturbed.

booksum

Background The federal government has historically established minimum eligibility requirements for Medicaid and CHIP and provided states with considerable flexibility in expanding eligibility to individuals in households with higher incomes. PPACA made numerous changes to existing federal Medicaid and CHIP eligibility requirements and specified eligibility criteria for new types of assistance, such as the premium tax credit. PPACA also provided for a continued focus on certain CHIPRA initiatives; specified additional policies to facilitate eligible children’s enrollment in Medicaid, CHIP, and the premium tax credit; and included provisions to facilitate children’s access to private health insurance. Federal and state implementation of PPACA enrollment and eligibility provisions is under way. Current Eligibility Requirements and Enrollment Policies for Medicaid and CHIP Eligibility for Medicaid and CHIP is limited to U.S. citizens and certain legally residing immigrants and is generally based on household income in relation to the FPL. For Medicaid, the federal government requires that states cover children with household incomes at or below specific eligibility levels, which range from 100 through 133 percent of FPL depending on the age of the child. States have flexibility to increase eligibility levels beyond the federally required levels for children of specific ages. For example, several states have Medicaid eligibility levels of 185 percent of FPL for infants, and a more limited number of states also have eligibility levels higher than the federal requirement for children older than age 1. Because Medicaid eligibility levels vary by children’s age, some members of a given family may qualify for Medicaid, while others do not. With CHIP programs, states cover children whose household incomes are too high for Medicaid eligibility; most states’ CHIP eligibility levels are between 200 and 300 percent of FPL. States use different methods for counting household income; for example, some states disregard portions of certain types of income, such as earned income, and states have varying standards regarding which household members to include when determining family size. Eligibility Requirements for Medicaid, CHIP, and the Premium Tax Credit under PPACA With regard to changes to children’s eligibility for Medicaid and CHIP and eligibility specifications for the new premium tax credit, which are to be fully effective in 2014, PPACA included the following provisions. PPACA expanded Medicaid eligibility to children and adults under age 65 with household incomes at or below 133 percent of FPL. As a result, minimum eligibility levels for Medicaid will generally be the same for all family members. Some children with household incomes higher than 133 percent of FPL will continue to be eligible for Medicaid in states that have established higher eligibility levels for children. These states are not allowed to lower their Medicaid eligibility levels for children until fiscal year 2020. PPACA required a uniform method of counting household income, based on a household’s modified adjusted gross income (MAGI) to determine eligibility for Medicaid, CHIP, and the premium tax credit. As a result, household income for Medicaid and CHIP, as well as for the premium tax credit, will be determined consistently in all states. PPACA defined eligibility criteria for the new premium tax credit, which will apply in all states. Similar to Medicaid and CHIP, eligibility for the premium tax credit will be limited to U.S. citizens and legally residing immigrants. Eligibility will also be limited to individuals with household incomes between 100 and 400 percent of FPL. In addition, to be eligible for the premium tax credit, an individual cannot have access to public insurance such as Medicaid or CHIP or to affordable employer-sponsored health insurance that provides a minimum value. A child’s eligibility for Medicaid, CHIP, and the premium tax credit can change over time under PPACA as his or her household income fluctuates. For example, a child who begins the year eligible for the premium tax credit may become eligible for Medicaid or CHIP if household income declines during the year. Conversely, depending on the state, a child who begins the year eligible for Medicaid or CHIP may lose eligibility for these programs if household income increases. Enrollment Policies for Medicaid, CHIP, and the Premium Tax Credit under PPACA PPACA also contained provisions to facilitate eligible children’s enrollment in Medicaid, CHIP, and private health insurance subsidized by premium tax credits. For example, PPACA extended funding for CHIPRA outreach and enrollment grants through fiscal year 2015, prohibited states from requiring in-person interviews for enrollment beginning in 2014, provided for income to be verified through a federally managed hub of data electronically accessible to states, and specified a coordinated enrollment process, whereby with one federally defined uniform application, states will assess families for eligibility for Medicaid, CHIP, or the premium tax credit. PPACA also made funding available to states to plan and implement exchanges, which will provide eligible individuals and families—including those eligible for premium tax credits—the ability to compare, select, and enroll in participating private health insurance plans with standardized benefit and cost-sharing packages. Under PPACA, exchanges must be established in every state by January 1, 2014, either by the state itself or by the Secretary of HHS. PPACA’s Private Health Insurance Market Provisions Although not the focus of this report, PPACA also contained provisions to facilitate children’s access to private health insurance, apart from the provision of the premium tax credit. For example, as of September 2010, PPACA prohibited health plans and issuers from limiting or denying coverage for children under age 19 because of preexisting health conditions. (See table 1.) Implementing PPACA’s changes to Medicaid and CHIP eligibility determination and enrollment policies and preparing for implementation of the premium tax credit and other provisions of PPACA will require significant state and federal efforts. In August 2011, CMS and IRS separately issued three proposed rules to implement key PPACA provisions related to eligibility and enrollment for Medicaid, CHIP, and the premium tax credit; the CMS rules were finalized in March 2012. According to CMS, more detailed guidance, such as the specific information to be collected in the uniform application or the nature of the data available from the federal hub, will be distributed at a later date. The IRS proposed rule specified how to calculate household MAGI for determining premium tax credit eligibility, and the CMS rules adopted these methods for determining Medicaid and CHIP eligibility, with certain exceptions. IRS finalized its proposed rule in May 2012 with minimal change to these methods. The IRS proposed rule also described the standard for determining whether an individual has access to affordable employer-sponsored insurance for purposes of determining eligibility for the premium tax credit. Under the proposed affordability standard, employer-sponsored insurance is considered affordable if the cost of a self-only plan—meaning a plan that only covers the employee—does not exceed 9.5 percent of household income. Under the proposed standard, if one family member has access to affordable self-only employer- sponsored insurance, all other family members who are eligible to enroll in the employee’s plan are also considered to have access to affordable insurance and are therefore ineligible for the premium tax credit. In this manner, the proposed rule applied the same standard to all family members eligible for the employee’s plan, even if the cost of enrolling the family as a whole exceeds the 9.5 percent threshold. In the preamble to its proposed rule, IRS stated that the PPACA statute specifies using the self-only insurance affordability standard for employees as well as for spouses and dependents of an employee, citing a report issued by the Joint Committee on Taxation that similarly interpreted the law. Some who commented on the proposed rule suggested that it would be more consistent with congressional intent to interpret the statute to require the use of the cost to an employee of insuring all eligible family members in determining access to affordable employer-sponsored insurance. In its final premium tax credit rule, IRS confirmed that the proposed self-only insurance affordability standard would apply to employees, but it deferred a decision on the affordability standard for other eligible family members, such as children, to future rule making. Therefore, because this report focuses on children, the relevant affordability standard remains a proposed standard, and is referred to as such for the remainder of the report. An Estimated Three- Quarters of Uninsured Children Would Be Eligible for Medicaid, CHIP, or the Premium Tax Credit under PPACA, but the Proposed Affordability Standard May Result in Some Children Remaining Uninsured Over three-quarters of uninsured children in January 2009 would be eligible for Medicaid, CHIP, or the premium tax credit under 2014 PPACA eligibility rules, according to our estimates. Applying final CMS and proposed IRS rules for 2014 program eligibility to 2009 SIPP data, we estimate that on the basis of household income and other eligibility criteria, such as citizenship, nearly 68 percent of the approximately 7 million children who were uninsured in January 2009 would be eligible for Medicaid or CHIP—about 48 percent for Medicaid and about 20 percent for CHIP. In addition, 7.5 percent of the uninsured children would be eligible for the premium tax credit. Nearly 13 percent of the uninsured children were noncitizens for whom we did not estimate eligibility because of limitations in the data.approximately 12 percent of uninsured children would be ineligible for We estimate that the final Medicaid, CHIP, or the premium tax credit. Specifically, 5.5 percent would be ineligible because they were in families with a household income that was too high—at greater than 400 percent of FPL. The remaining 6.6 percent would be ineligible because, though their families were considered low-income in that they met the household income requirements for the premium tax credit, they were considered to have access to affordable employer-sponsored insurance based on IRS’s proposed affordability standard. In particular, these children had at least one parent with employer-sponsored insurance that had an estimated cost below 9.5 percent of household income for a self-only plan. (See fig. 1.)These children would not be automatically eligible for the premium tax credit if the affordability standard were instead based on a family plan; their eligibility would depend on the cost of the family plan to which they had access. See appendix I for more information about our estimates. The proposed affordability standard could potentially affect significantly more children than the approximately 460,000 uninsured children we estimated above under certain scenarios. Many children eligible for CHIP have a parent with employer-sponsored insurance. Under PPACA, CHIP is not funded beyond 2015, and, even if federal funding is extended, states may opt to reduce eligibility levels for CHIP or eliminate Without CHIP- CHIP programs altogether beginning in fiscal year 2020.funded Medicaid expansion or separate CHIP programs, we estimate that an additional 1.9 million children who would otherwise be eligible for CHIP would be considered to have access to affordable insurance under this proposed standard and would be ineligible for the premium tax credit.(See fig. 2.) In commenting on IRS’s proposed rule on eligibility for the premium tax credit, some states and other organizations noted that IRS’s proposed interpretation of access to affordable employer-sponsored insurance— defining affordability on the basis of the cost of a self-only plan, and not on the cost of a family plan—could result in some children remaining uninsured. They explained that although a self-only plan for the employee may cost less than the 9.5 percent threshold, a family plan that would also insure the employee’s eligible family members could exceed it. As a result, some employees would not be able to afford the higher premiums to insure their family members, who therefore could remain uninsured. We did not estimate the cost associated with defining the affordability standard based on the cost of a family plan. The cost of such a change would depend on multiple factors, many of which remain uncertain, such as the availability of CHIP funding beyond 2015, the extent to which eligible families avail themselves of the premium tax credit, employer decisions, and the extent to which additional enrollees could affect the aggregate cost of premiums. The Congressional Budget Office has commented on the high degree of uncertainty inherent in projecting the future actions of employees and employers under PPACA as well as other factors that may affect federal costs, such as the number of individuals and families who will have household income in specific eligibility ranges in future years. We did not examine how many of the children estimated to be ineligible for the premium tax credit because of access to affordable employer- sponsored insurance would become eligible if the affordability standard were instead based on the cost of a family plan; the cost of family plans available to employees who chose not to purchase them was not available in the data we analyzed. However, separate data on the cost of family plans among employees who purchased a family plan suggest that some of these uninsured children, particularly those in families facing higher-than-average premium contributions, could become eligible for the premium tax credit if the affordability standard were based on the cost of a family plan. For example, in a 2011 survey, the Kaiser Family Foundation and Health Research & Education Trust found that on average, employees contributed $4,129 annually for a family plan, or 28 percent of the total cost to the employer of an annual family premium, which averaged $15,073. For a family of four with household income equivalent to 250 percent of the FPL, $4,129 represents about 7 percent of household income. However, the percentage of the annual premium paid by employees ranged widely around this average, and 15 percent of employees with family plans paid more than 50 percent of the annual premium. For a family of four with household income equivalent to 250 percent of the FPL, paying 51 percent of the average annual premium (or $7,687) would represent just over 13 percent of household income, exceeding the 9.5 percent threshold. Whether families ultimately choose to purchase insurance for children will depend on many factors, including individual decisions regarding what they can afford for health insurance. An Estimated 14 Percent of Children Eligible for Medicaid, CHIP, or the Premium Tax Credit under PPACA Would Experience a Change in Eligibility within 1 Year Applying final CMS and proposed IRS 2014 PPACA eligibility rules to children in 2009, we estimate that nationally, 9 percent of children eligible for Medicaid, CHIP, or the premium tax credit experienced a change in household income within 6 months that would affect their eligibility for a specific form of assistance, and 14 percent of these children experienced (See table 2.) In addition, some at least one such change within 1 year.children experienced multiple income changes within these time periods that would affect their eligibility for assistance more frequently. We estimate that, nationally, 2 percent of eligible children experienced changes in household income that would affect eligibility two or more times within 6 months, and 6 percent experienced two or more such changes within 1 year. The effect of continuous eligibility policies for Medicaid and CHIP on the frequency of eligibility changes becomes apparent when we consider children in states with versus states without such policies separately. Eligibility changes are higher than the national average in states without continuous eligibility policies in either their Medicaid or CHIP programs, and lower than the national average in states with them. In states with continuous eligibility policies for Medicaid and CHIP, eligibility changes under PPACA would be limited to children who begin the year eligible for the premium tax credit but experience a decrease in household income that would result in eligibility for Medicaid or CHIP instead. Therefore, the percentage of children experiencing changes in eligibility, and at risk of experiencing disruptions in coverage, is lower than the national average among children in the 23 states that have adopted continuous eligibility in both their Medicaid and CHIP programs and greater than the national average among children in the 18 states that do not have continuous eligibility in either program. Specifically, we estimate that about 3 percent of eligible children in states with continuous eligibility for Medicaid and CHIP experienced a change in household income that would affect eligibility under PPACA within 1 year. In contrast, we estimate that about 19 percent of eligible children in states without continuous eligibility experienced a change in household income that would affect program eligibility under PPACA at least once within 6 months, and about 30 percent experienced such a change within 1 year. (See table 3.) Changes in eligibility caused by income fluctuations could deter children’s enrollment in relevant programs if the process for changing enrollment is burdensome for the families and could further complicate other eligibility complexities, such as variation in eligibility within households. Eligibility for specific types of assistance can vary within households because low- to moderate-income adults with household incomes greater than 133 percent of FPL will typically be ineligible for any assistance or will be eligible for the premium tax credit rather than Medicaid or CHIP, while children in some of these households—particularly in states with higher income eligibility levels for Medicaid and CHIP—will be eligible instead for Medicaid or CHIP. We estimate that based on 2009 data, 21 percent of children eligible for Medicaid, CHIP, or the premium tax credit under PPACA would have different eligibility from their parents as of the beginning of the year. However, because of income fluctuations that occurred over the course of the year, we estimate that an additional 9 percent of eligible children would encounter this situation. CMS Has Provided States with Tools to Increase Enrollment, and States Express a Need for Further Guidance and Note Budget Constraints CMS has provided states with incentives and guidance to implement current initiatives to improve enrollment policies and has made progress assisting states in implementing PPACA requirements aimed at further simplifying Medicaid and CHIP enrollment. State officials reported ongoing challenges with regard to enrolling eligible children, including the need for timely guidance to implement PPACA provisions, concerns about enrolling family members who are not eligible for the same program, and state budget constraints. CMS Has Provided States with Financial and Technical Assistance to Facilitate the Enrollment and Retention of Eligible Children in Medicaid and CHIP Through an array of financial incentives and technical assistance, CMS has worked with states to enroll and retain eligible children in Medicaid and CHIP and to set up state exchanges under PPACA. Many of these efforts were initiated with funds appropriated under CHIPRA and continue under PPACA. For example, CHIPRA appropriated $100 million for fiscal years 2009 through 2013 in outreach grants and related efforts to improve the enrollment and retention of underserved populations in Medicaid and CHIP, and by the end of fiscal year 2011, CMS had awarded $80 million in such grants. CMS awarded the first round of outreach grants in fiscal year 2009 to 69 applicants in 43 states, which included state agencies and community-based and other nonprofit groups, and the second round in fiscal year 2011 to 39 applicants in 23 states.officials from selected states, officials noted that the outreach grants had helped the agencies reach eligible children. For example, Oregon Medicaid officials said that the CHIPRA outreach grant their agency received was crucial to reaching the state’s Hispanic population. The grant sought to support outreach by safety net providers, public health departments, and school-based health centers. Since fiscal year 2009, CMS has also awarded performance bonuses annually to states that implemented at least five of the eight enrollment initiatives outlined in CHIPRA and met specific enrollment goals, which are based on the state’s current Medicaid enrollment and population growth. (See table 4.) The number of states receiving these bonuses has more than doubled over the 3 years that bonuses have been awarded, increasing from 10 states in fiscal year 2009 to over 23 states in fiscal year 2011. In 2011, the amount of the performance bonuses ranged from approximately $1.3 million for Idaho to over $28 million for Maryland. (See app. II for a summary of the states that received these performance bonuses and the amounts of the awards.) In addition, among the 23 states that received a performance bonus in 2011, 16 received an enhanced bonus for exceeding their enrollment target by more than 10 percent.bonuses annually through fiscal year 2013. Conclusions A key goal of PPACA was to increase Americans’ access to affordable health insurance. PPACA expanded eligibility for existing federal health programs and private health insurance, offered a new premium tax credit to offset the cost of private health insurance for some low- to moderate- income families whose incomes are too high to qualify for Medicaid or CHIP, and provided means for streamlining enrollment. Although our estimates are based on 2009 data, they illustrate the potential impact of PPACA, when fully implemented in 2014, on children’s access to affordable health insurance, and highlight the importance of many of the policies introduced in CHIPRA and continued in PPACA. For example, our estimates suggest that about 68 percent of children who were uninsured in 2009 would be eligible for Medicaid or CHIP under PPACA, underscoring the continued importance of outreach and simplified enrollment policies to ensure that eligible children are enrolled in the appropriate program. Similarly, significantly higher estimates of changes in eligibility within a year among children in states without continuous eligibility policies compared to states with such policies underscore the importance of a continued emphasis on such policies to minimize changes in eligibility. In addition, a small but significant number of uninsured children from low- to moderate-income families whose incomes are too high to qualify for Medicaid or CHIP would be ineligible for the premium tax credit under IRS’s proposed definition of access to affordable employer-sponsored insurance, which is based on the cost of a self-only plan available to the employee. Yet the cost of insuring other eligible family members could be higher and potentially unaffordable for some families. One implication of this proposal is that some families in which one member has an offer of self-only, employer-sponsored health insurance could be less likely to obtain family insurance than if no employer insurance were offered, because of their ineligibility for the premium tax credit. We recognize that in finalizing the affordability standard for an employee’s eligible family members, IRS must weigh many complex factors, such as costs to the federal government and effects on employers and families, some of which are difficult to predict, as well as the scope of its authority. However, under the proposed standard, an offer of affordable employer-sponsored health insurance to one family member could impede other family members’ access to affordable insurance—an outcome which would not further the broader goals of PPACA. Recommendation for Executive Action In the Department of the Treasury’s future rule making, we recommend that the Secretary of the Treasury, in consultation with the Commissioner of Internal Revenue, consider the impact of the proposed standard for determining affordability of employer-sponsored insurance on children and other family members who are eligible to enroll, and whether it would be consistent with the goals of PPACA to adopt an alternative approach that would consider the cost of insuring eligible family members, or as necessary, seek clarification from Congress regarding its intent with respect to this standard. Agency Comments We provided a draft of this report for comment to HHS and the Department of the Treasury. Neither HHS nor the Department of the Treasury provided general comments on the report or its recommendation. Department of the Treasury officials provided technical comments, which we incorporated as appropriate. As agreed with your offices, unless you publicly announce the contents of this report earlier, we plan no further distribution until 30 days from the report date. At that time, we will send copies to relevant congressional committees, the Secretary of Health and Human Services, the Secretary of the Treasury, and other interested parties. In addition, the report will be available at no charge on the GAO website at http://www.gao.gov. If you or your staff have any questions about this report, please contact me at (202) 512-7114 or iritanik@gao.gov. Contact points for our Offices of Congressional Relations and Public Affairs may be found on the last page of this report. GAO staff who made key contributions to this report are listed in appendix III. Appendix I: Scope and Methodology of the Survey of Income and Program Participation Analysis Our first two objectives were to assess the extent to which uninsured children would be eligible for Medicaid, the State Children’s Health Insurance Program (CHIP), or the premium tax credit available under the Patient Protection and Affordable Care Act (PPACA) and the extent to which they would experience a change in eligibility among these forms of assistance because of changes in household income during a year. We identified the Survey of Income and Program Participation (SIPP), a nationally representative, longitudinal survey conducted by the U.S. Census Bureau, as a useful data set for our purposes because it provides detailed monthly information over a multiyear period about specific types of income, family relationships, and health insurance status of individuals and households representing the civilian, noninstitutionalized population of the United States. We analyzed data from the most recently available SIPP, which began in 2008, and surveyed the occupants of approximately 42,000 households. Our analysis of SIPP data is subject to limitations. The analysis uses 2009 data to illustrate the extent to which uninsured children would be eligible for Medicaid, CHIP, or the premium credit program had proposed and final 2014 PPACA eligibility rules been in effect at that time. To the extent that patterns in household income, insurance status, or other eligibility criteria differ in 2014, eligibility in 2014 will also differ. In addition, the estimates are based on a sample of the population and may differ from estimates that would be obtained if the full population had been surveyed using the same methods, and the estimates are based on self-reported information that may contain errors because of factors such as differing interpretation of survey questions, inability or unwillingness of survey participants to provide correct information, or data processing errors. The Census Bureau reported that quality control and edit procedures were used to reduce such errors.data have shown that the SIPP captures less income compared to other Studies of SIPP income surveys, particularly for higher-income survey participants.analysis focuses on lower-income survey participants, to the extent that the SIPP data underrepresent income in this population as well, our estimates would indicate that more children meet income requirements for Medicaid or CHIP versus the premium tax credit, and for the premium tax credit versus being ineligible for any type of assistance, than other surveys might suggest. Our analysis variables approximate but do not always fully capture key PPACA eligibility criteria, such as household income or citizenship status, as described further below. To determine the reliability of SIPP data, we reviewed related documentation and conducted electronic testing for missing data, outliers, and apparent errors. For example, we tested whether persons who reported being uninsured in January 2009 had reported having health insurance in the prior and following month. We also compared our results to estimates based on data from another Census Bureau survey, the American Community Survey, and to other studies that addressed related research questions. We determined that the SIPP data were sufficiently reliable for the purposes of our engagement. Analysis Variables A child’s eligibility for Medicaid, CHIP, or the premium tax credit under PPACA is based in part on having household income below specified limits relative to the federal poverty level (FPL). The Centers for Medicare & Medicaid Services (CMS) and the Internal Revenue Service (IRS) have specified methods for determining a child’s household income under PPACA in final and proposed eligibility rules, and differences exist in how household income is determined for Medicaid and CHIP versus the premium tax credit. A child’s eligibility for these programs is also based on citizenship or legal residence, and, for the premium tax credit, on whether the child is considered to have access to other affordable insurance. From the available SIPP data, we developed variables for our analysis based on these eligibility rules. Household composition. We created two household composition variables for children on the basis of final CMS and proposed IRS rules for determining household composition for the premium tax credit and for Medicaid or CHIP. The premium tax credit household composition variable defined households as composed of a taxpayer and spouse, if applicable, and tax dependents. Tax dependents were defined as follows: Children under age 19 (or ages 19 through 23 who were full-time students) whose taxable income (together with a spouse’s income, if applicable) was not more than half of household income. Other family members, who (together with a spouse, if applicable), earned less than the IRS threshold and whose total income was not more than half of household income. Taxpayers were those who did not meet the above definition of a tax dependent. This definition of a tax dependent excluded those with significant income, but did not capture tax rules about the amount of financial support a taxpayer must provide for children or other dependents in order to claim them as tax dependents. For example, most children under age 19 were defined as tax dependents. Households of tax-dependent children included the child, the child’s taxpayer parents or guardians, and any other tax dependents of the taxpayers, such as the child’s siblings. When children lived with two unmarried parents, the parent with the higher income was designated as the taxpayer parent. This household composition variable did not account for children who may be claimed as tax dependents by a noncustodial parent or for spouses who choose to file taxes separately. The Medicaid household composition variable was the same as the premium tax credit household composition variable, with certain exceptions. When a tax-dependent child did not live with a taxpayer parent or lived with two parents who were not married to one another, or had household income below tax filing thresholds, the child’s household for purposes of determining Medicaid and CHIP eligibility was composed of the child, the child’s parents, siblings under age 19 (or ages 19 and 20 who were full-time students), and any children of the child. In addition, pregnant women were counted as two household members when determining Medicaid and CHIP eligibility. Pregnancy status is not directly available from the SIPP data; we estimated that women were pregnant in a given month if they had a new infant during one of the following 8 months. Household income. We constructed four household income variables for children based on rules for counting income for Medicaid and CHIP and for the premium tax credit under PPACA. Tax dependent’s income was not included in any household income variable if it was less than the amount that would necessitate filing a tax return. To approximate modified adjusted gross income (MAGI) household income under PPACA, a child’s premium tax credit household income was defined as the sum of all income, less means-tested assistance income; child support or foster care payments; veterans and workers compensation or sickness or accident insurance payments; or gifts from relatives or friends—self-reported by individuals included in the premium tax credit household composition variable defined above, during calendar year 2009. A child’s baseline Medicaid household income was the sum of the same income types in the Medicaid household composition variable defined above, during specific months of 2009. A child’s adjusted Medicaid household income was equal to the baseline Medicaid household income variable, less income deductions applied in specific states in their Medicaid eligibility determination processes, including deductions of certain amounts of earned income and child care expenses. A child’s adjusted CHIP household income was equal to the baseline Medicaid household income variable, less income deductions applied in specific states in their CHIP eligibility determination processes, including deductions of certain amounts of earned income and child care expenses. FPL. We constructed four percentages of FPL variables based on the four household income variables and two household composition variables defined above. A child’s baseline Medicaid percentage of FPL was the Medicaid household income variable divided by the 2009 poverty threshold applicable to the child’s state and family size contained in the Medicaid household composition variable. A child’s adjusted Medicaid percentage of FPL was the adjusted Medicaid household income variable divided by the 2009 poverty threshold applicable to the child’s state and family size contained in the Medicaid household composition variable. A child’s adjusted CHIP percentage of FPL was the adjusted CHIP household income variable divided by the 2009 poverty threshold applicable to the child’s state and family size contained in the Medicaid household composition variable. A child’s premium tax credit percentage of FPL was the premium tax credit household income variable divided by the 2009 poverty threshold applicable to the child’s state and household size contained in the premium tax credit household composition variable. Insurance status. Employer-sponsored insurance was defined as insurance obtained through an individual’s or a family member’s employer, former employer, union, or the military. Individuals were not categorized as having employer-sponsored insurance if they also had Medicaid or CHIP coverage. We used a procedure that the Census Bureau has adopted for the American Community Survey to address under-reporting of Medicaid coverage. Specifically, respondents were recategorized as having Medicaid if they were one of the following: a child under age 19 and the unmarried child of a parent with public a citizen parent with public assistance, a citizen parent married to a citizen with public assistance or a foster child, or a Supplemental Security Income recipient who met one of the following conditions: (1) did not have children or (2) had children but was not working. Individuals were defined as uninsured if they were not categorized as having employer-sponsored or other private insurance, Medicaid, CHIP, or other public insurance. Access to affordable employer-sponsored insurance. Children who had a taxpayer parent as part of their household composition who had employer-sponsored insurance, and children who were taxpayers and had employer-sponsored insurance, were defined as having met the proposed standard for access to affordable employer-sponsored insurance if the average annual employee contribution for a self-only plan, $921, was less than or equal to 9.5 percent of premium tax credit household income. This definition of access to affordable employer- sponsored insurance did not take into account the requirement that employer-sponsored insurance must provide a minimum value in order to be considered affordable, and it assumed that children were eligible to enroll in a parent’s employer-sponsored insurance. Citizenship or legal residence. Citizenship status is available in SIPP data, but the legal status of noncitizens is not directly available from SIPP data. We defined noncitizens as legally residing if they or a parent reported receiving public insurance, such as Medicaid, or other public assistance, which requires documentation of citizenship or legal residence. The remaining noncitizens were defined as potentially ineligible noncitizens. Methodology Based on the variables defined above, we categorized children as eligible or ineligible for Medicaid, CHIP, and the premium tax credit under proposed and final 2014 PPACA eligibility rules. We defined children as eligible for Medicaid under PPACA if they were citizens or legally residing noncitizens whose baseline Medicaid percentage of FPL was less than or equal to 138 percent, or who had an adjusted Medicaid percentage of FPL that was less than or equal to the 2012 state-specific income eligibility level for their age group. Foster children and Supplemental Security Income recipients were also defined as Medicaid eligible. We defined children as eligible for CHIP under PPACA if they were citizens or legally residing noncitizens not estimated to be eligible for Medicaid, with an adjusted CHIP percentage of FPL that was less than or equal to the applicable 2012 CHIP state-specific income eligibility level. CHIP included both CHIP-funded Medicaid expansion programs and separate CHIP programs. Children with employer- sponsored or other private insurance were defined as ineligible for separate CHIP programs. We defined children as eligible for the premium tax credit under PPACA if they were citizens or legally residing non-citizens not estimated to be eligible for Medicaid or CHIP, with premium tax credit percentage of FPL between 100 and 400 percent and without access to affordable employer-sponsored insurance. Our analyses considered three groups of children: uninsured children ages 0 through 18, CHIP-eligible children ages 0 through 18 who were uninsured or publicly insured, and all children ages 0 through 18 eligible for Medicaid, CHIP, or the premium tax credit. We limited our analysis to children who participated in the SIPP for all of calendar year 2009. Among uninsured children, we used January 2009 SIPP data to estimate the percentage who would be eligible for Medicaid, CHIP, and the premium tax credit based on the above definitions of 2014 PPACA eligibility rules, as well as the percentage who would be ineligible. Among uninsured or publicly insured children estimated to be eligible for CHIP, we used January 2009 SIPP data to estimate the percentage who would be eligible for the premium tax credit based on the above definitions of 2014 PPACA eligibility rules, as well as the percentage who would be ineligible, if CHIP were not available. Among all children estimated to be eligible for Medicaid, CHIP, or the premium tax credit in January 2009, we used calendar year 2009 SIPP data to estimate the percentage who would have experienced one or two changes in eligibility for specific types of assistance, including becoming ineligible for any type of assistance, under 2014 PPACA eligibility rules within 6 months and a year. We incorporated state-specific continuous eligibility policies; for children living in states that according to CMS had a continuous eligibility policy in place as of 2012, we did not count them as changing eligibility for the relevant program even if their income eligibility changed. Among all children estimated to be eligible for Medicaid, CHIP, or the premium tax credit in January 2009, we used calendar year SIPP data to estimate the percentage who would be eligible for Medicaid and CHIP under 2014 PPACA eligibility rules with a Medicaid percentage of FPL higher than 138 percent in January 2009 and during 2009 as a whole, in order to examine the percentage of children who could be eligible for different program than their parents. For all estimated percentages, we used the SIPP 2009 calendar year sampling weight and calculated a lower and upper bound at the 95 percent confidence level using replicate weights that took into account the complex survey design. Appendix II: Federal Initiatives and Funding Available to States to Facilitate Enrollment of Eligible Children and Implement PPACA The Children’s Health Insurance Program Reauthorization Act of 2009 (CHIPRA) and PPACA included a number of initiatives and provisions under which states may obtain federal funding to assist in enrolling eligible children, and most states have taken advantage of at least one of these. For example, CHIPRA provided incentives to states to undertake eight enrollment initiatives. Beginning in fiscal year 2009, CMS awarded performance bonuses to states that implemented at least five of the eight enrollment initiatives and also achieved specific enrollment goals. (See fig. 3.) PPACA authorized the provision of planning grants and establishment grants to assist states with the implementation of the American Health Benefit Exchanges (referred to as exchanges)— marketplaces where eligible families and individuals can purchase private health insurance. Recognizing that states will need to upgrade their Medicaid information technology systems to comply with PPACA, CMS has provided states with the opportunity to claim an enhanced Federal Medical Assistance Percentage (FMAP)—the federal share of Medicaid expenditures—through fiscal year 2015 for the costs associated with certain systems improvements, such as updates to their claims processing and enrollment systems. Specifically, instead of the 50 percent FMAP that has historically been available for most Medicaid administrative expenses, qualified states can obtain a 90 percent FMAP for the costs of implementing new information systems and a 75 percent FMAP for the costs of administering these new systems. Appendix III: GAO Contact and Staff Acknowledgments GAO Contact Staff Acknowledgments In addition to the contact named above, Susan T. Anthony, Assistant Director; Susan Barnidge; Emily Beller; Sandra George; Eagan Kemp; and Roseanne Price made key contributions to this report.

PPACA sought to increase access to affordable health insurance, and major provisions, such as a tax credit to offset the cost of private insurance premiums, will become effective in 2014. GAO estimated the extent to which (1) uninsured children would be eligible for Medicaid, CHIP, or the premium tax credit under PPACA, and (2) children would experience a change in eligibility among Medicaid, CHIP, and the premium tax credit under PPACA because of income changes. GAO also assessed CMS steps thus far to help states enroll children and related state challenges. GAO applied proposed and final 2014 PPACA eligibility rules to nationally representative 2009 data from the U.S. Census Bureau and interviewed officials from CMS and IRS, two federal agencies responsible for implementing relevant PPACA provisions, and six states that received federal funds for enrollment efforts. GAO estimates that under the 2010 Patient Protection and Affordable Care Act (PPACA), about three-quarters of approximately 7 million children who were uninsured in January 2009 would be eligible for Medicaid, the State Children’s Health Insurance Program (CHIP), or the new premium tax credit. The remaining children had family incomes too high to be eligible, were noncitizens, or would be ineligible for the premium tax credit because they would be considered to have access to affordable employer-sponsored insurance per the Internal Revenue Service’s (IRS) proposed affordability standard, in which IRS interpreted PPACA as defining affordability for an employee’s eligible family members based on the cost of an employee-only plan. Some commenters raised concerns that IRS’s interpretation was inconsistent with PPACA’s goal of increasing access to affordable health insurance as it does not consider the higher cost of family insurance and could result in some children remaining uninsured. Under PPACA, CHIP is not funded beyond 2015, and states may opt to reduce CHIP eligibility or eliminate programs in fiscal year 2020. Without CHIP, more children could become uninsured. In May 2012, IRS finalized its rule but deferred finalizing the proposed affordability standard. GAO estimates that about 14 percent of children in January 2009 who met 2014 PPACA eligibility criteria for these programs experienced a change in household income that would affect eligibility within 1 year. Changes in eligibility among children in states without policies allowing them to remain eligible for Medicaid and CHIP for a full year were estimated to be higher than in states with such policies. Frequent eligibility changes could deter enrollment if the process for changing enrollment is burdensome. The Centers for Medicare & Medicaid Services (CMS) has provided states with financial incentives and technical guidance to improve enrollment and to implement PPACA provisions. States reported challenges to enrolling eligible children, including the need for guidance to implement certain provisions—which CMS indicated was forthcoming—and state budget constraints.

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Rupert Murdoch supposedly wished his old employee Piers Morgan well, but not too much success in his new gig on CNN. The News Corp. chairman, ever mindful of Fox News' profits, needn't have worried. "Piers Morgan Tonight" is the second holdover from CNN's former management, and to say the show's debut was more polished than "Parker-Spitzer" is hardly an unqualified endorsement. Instead, Morgan was so deferential in his hour-long chat with Oprah Winfrey that the adjective she used to describe his interviewing skills -- "Surprising" -- seemed very generous indeed. "Boring" would be more accurate. Morgan -- known as an interviewer in the U.K., and an "America's Got Talent" judge in the U.S. -- didn't really press Winfrey on any front. Sure, he asked some questions about her personal life, her wealth, her role in campaigning for Barack Obama, but he let her prattle on about her "mission" and larger purpose without any mention of the messianic terms she regularly uses to discuss herself and her media empire. To her credit, Winfrey -- in saying she was in no way disappointed with Obama's presidency -- reminded Morgan that there's a "learning curve" in any job, and it would be unfair to judge him based strictly on this first program. But other than laughing too hard at her jokes and being generally pleasant, it's hard to see how this is any improvement over what Larry King was doing in the timeslot, other than being about three decades younger. The front-loaded lineup of first-week guests includes Howard Stern, Ricky Gervais and George Clooney. But any nightly (or daily) show of this kind is only as good as its host, and if this is the template, Morgan won't offer much reason to tune in on nights when he has lesser newsmakers. The premiere, notably, went up against about as partisan a night on CNN's cable rivals as you're apt to find: Sean Hannity sitting in Sarah Palin's lap (not quite literally) on Fox News' "Hannity," while Rachel Maddow hosted Michael Moore to discuss gun-control legislation on MSNBC. Hannity pitched softballs at Palin for a full half-hour, before segueing to Michael Reagan to criticize his brother's book about their father. For hard-core conservatives, that's the equivalent of having Babe Ruth and Lou Gehrig in your lineup. It would be lovely if CNN had the smarts to offer a legitimate alternative to those political poles, but based on first impressions, "Piers Morgan" isn't it. cable news news Piers Morgan Takes On Oprah, and Comes Up Short After weeks of building buzz, on Monday night it was finally time for Piers Morgan Tonight to fill television's biggest suspenders in the nine o'clock CNN slot that Larry King made famous. Morgan's interviews will clearly be more personal and pointed. He came across as polite and intelligent, and clearly does his homework. But his choice of Oprah Winfrey as his first guest was a drastic error. From the hotel room interview set — more Barbara Walters Fascinating People than anything resembling King's familiar dark-room-and-desk setup — to his obsequious bowing and scraping, the interview with the reigning queen of TV made him look like a commoner. The thing is, Morgan's no amateur. He's a journalist with decades of experience in print and television — not that he appeared that way in a sedate hotel room filled with flowers and cigar boxes. (There's a reason Barbara uses rooms like this, and Diane Sawyer, and the good people of 60 Minutes. Their interviews are soft. They're restrained. Piers is big and loud. His normal set, which it seems like he'll start using later this week for a sit-down with Howard Stern, is much more suited to him.) Not being an amateur is one thing. Trying to face off with Oprah Winfrey is quite another. She is a legendary interviewer and an alpha dog, and she put Piers through his paces. Nine minutes in, she was telling him exactly what he wasn't going to get out of her, saying, "You're wasting your time with the Stedman thing!" She talked directly to the camera. After Piers asked whether she had ever been in therapy, she turned the query right around on him and went on the attack. When he asked her what advice she'd give to Eagles quarterback Michael Vick, she calmly stated that she'd be saving that for when she got Vick on her own show. And near the end of the interview, she just told him what question he should ask her. (It was, "What do you do best?" When Piers asked it, Oprah gave a canned, hokey answer.) Piers's final plea was, "How did I do?" It was the first time he'd asked it explicitly, but it seemed like the whole interview was about seeking Oprah's approval. He constantly asked how it was going, and backed off easily when Oprah rebuffed him — never pushing for something deeper or truer from the gifted talker. "C'mon," he said, asking about the biggest check she ever wrote to the IRS. "Give the new boy a break." His giggling style might have come across as charming in the studio on his own turf, or in an interview with someone less intimidating. Here it made him seem sycophantic and almost childish. When he reached out to her and said, "Everything you touch turns to gold. Could you touch me?" I almost turned off the television. It wasn't all bad, though. There were signs of life in his interviewing style. He asked questions that real people would want to ask Oprah, even though she'd been asked many of them before. Questions like "Do you feel regal?" and "Do you like being famous?" provoked interesting answers. Likewise, "How many times have you been properly in love?" Her painful story of going to live with her father as a secretly pregnant 14-year-old felt poignant and honest. And he got her to talk a little bit about being rich, which was funny and refreshing. "I'm not sitting around counting it," Oprah said, brushing aside a question about her total worth. "I bet you know exactly how much you're worth!" Piers shot back. "Yes I do," she conceded. So how did she know? "Because I'd already counted it," she said. That was Piers's mistake in having Oprah on for his very first episode. He doesn't yet know how much he's worth in this new chair at CNN. But Oprah knows exactly how much she's worth, and it's definitely more than this untested — if bright and charming — British import. The gap in confidence, even muffled as it was by flowers and soft lighting, was glaring. The premiere of Piers Morgan Tonight revealed host Piers Morgan to be clever, tenacious, vain, a flatterer, and fitfully funny. Oh, and he had a big-name first guest: Oprah Winfrey, who talked about contemplating suicide at age 14, her quest to interview Michael Vick, and how many times she’s had her heart broken (twice). Morgan pumped up his subject shamelessly: “She’s the biggest, richest, most powerful star in the world!” “You would make a fantastic mother!” “Everything you touch is a hit — could you just touch me?” Morgan, reaching for the highest compliment a fawning British subject could pay a woman, said Winfrey was “the American Queen.” Oprah replied, regally: “I will accept it.” The America’s Got Talent judge has a bit of a doughy face and a small mouth (this is what happens when you host a talk show with at least as many close-ups on you as on your guest — people look more closely at you), and his clever-schoolboy demeanor serves him well. The hosts revealed they were both trying to book dog fancier Michael Vick as a guest, and Morgan offered to bet her “100 British pounds” on who’ll snap him up first. “Make it two,” said Oprah coolly. Oprah gassed on and on about her newly launched OWN network, which was doubtlessly a big reason she agreed to the highly publicized interview. But she was more interesting, of course, when she inadvertently revealed her ego — she said her role in life is nothing less than to “evolve the consciousness” of people. More interesting was her self-definition: “I am a Negro, formerly, born in 1954, in Mississippi when it was an apartheid state” and that therefore it was “a miracle,” what she’s attained. She spoke about losing a baby when she was 14, saying that the loss was “a relief” because “I thought I was going to have to kill myself,” given the strict rules she was raised under at home. In general, Winfrey was probably as open as she’s ever been with an interviewer, which is to say, not much, but Morgan extracted some nice moments, even if he had to beg: “Give a new boy a break.” As for whether the “new boy” is a worthy replacement for Larry King… come on, you didn’t watch Larry King Live, did you? Simply by having facts about his subject at hand and speaking coherently, Morgan aced King on his opening night. I’m looking forward to seeing him interview Howard Stern tomorrow night. Did you watch Piers Morgan Tonight? Twitter: @kentucker Piers Morgan (Credit: GETTY) If he'd watched this, Ricky Gervais might just have needed a very large handkerchief. The debut of fellow Englishman Piers Morgan's talk show on CNN, the one in which he officially succeeded Larry King, showed none of Gervais' pointed exposition of celebrity at the Golden Globes. Instead, Morgan's debut was a wonderful advertisement. A wonderful advertisement for Oprah Winfrey's new OWN TV channel. It was an advertisement in which Piers Morgan largely served as the announcer. It was an advertisement in which the Celebrity Apprentice positively begged to be allowed into the inner sanctum of Celebrity's Board of Directors. It even began with an advertisement for Oprah's new OWN channel, as if everyone should understand from the outset that this was product placement, not some in depth exploration of America's most popular woman. Morgan's nerves showed in his voice, his eyes and his desperation to have physical contact with his guest. Oprah looked benignly in his direction, as if he was a sweet, ambitious nephew whose mom had asked someone in the upper echelons of television to help him with his career. She did her best. Hers, as she said, is the Love Brand. So she tried to sprinkle a little love-dust upon his tightly-woven eyebrows. She called him good. Twice. But she also told him that he was going to get nothing newsworthy, nothing controversial, nothing titillating. In fact, by the end of the hour's show, what Oprah gave the well-prepped Morgan was nothing special. Nothing other than a schooling in what it was to know your self and your brand and to be able to control how you are seen. Morgan, in his British talk show, was able to get a mediocre singer and an equally mediocre prime minister to show genuine tears. Here, he was more successful in getting Oprah to show genuine pity at the obvious methods he was using in the hope of eliciting a headline, a snippet of her life story that had never before been revealed. Several times, she forced Morgan into the nervous laughter of a stair salesman who suddenly realizes he's in a ranch house. Oddly, his reputedly large and healthy ego nudged him to try and equate himself with her. Oprah explained that the end of her popular afternoon show wasn't the end of her career. She said: "I'm just getting started". Morgan's nervous schoolboy jaw-jerked him into "Aren't we all?" As if the two could somehow be compared. Morgan clearly felt that flattery would get him everywhere. "You are the American Queen," he told Oprah, sounding so very much like Tony Blair naming the dead Diana Spencer "The People's Princess". Perhaps he might have tried to tell her something she doesn't know. The blue cards he clutched in his no doubt ever-moisting palms seemed to hold no key, no provocation that Oprah couldn't have seen coming without so much as the opening of an eye or the sniffing of a breeze. At one point, as if telling her audience not to worry, she spoke directly to the camera, rather than to him. She wasn't going to forget who she was. And neither would he. By the end, he was asking her how well he had done. She patted him on the head. Metaphorically, of course. Kipling's The Man Who Would Be King tells the story of two British adventurers who become Kings in a remote part of Afghanistan. This Man Who Would Be King is a British man is trying to become King in a remote part of Atlanta. He will feel vindicated in merely having been anointed with Oprah's presence. His better tests will come should he manage to squeeze some snafu-laden news from an unsuspecting notable. Perhaps Paris Hilton might admit she doesn't really like sex. Oprah described her message like this: "I am the messenger to deliver the message of hope and redemption." The Love Brand tried to give Morgan hope that he would not be in need of redemption. She tried to make him believe that, one day, he will wake up and declare: "Bloody Hell, I'm Piers Morgan", just as he wondered whether she wakes up and thinks: "Bloody hell, I'm Oprah." Wednesday, Morgan gets to interview Howard Stern. That should be far more his style. Oh, and Thursday it's Ricky Gervais. Let's hope he tells Morgan what really went on in the restroom at the Golden Globes.

The obnoxious Piers Morgan familiar to reality show fans was nowhere to be seen as Piers Morgan Tonight made its debut in Larry King's old time slot on CNN, say critics. Morgan was fawning and deferential during his highly publicized interview with Oprah Winfrey. Some early reaction: "Morgan's nerves showed in his voice, his eyes and his desperation to have physical contact with his guest," writes Chris Matyszczyk at CBS, who predicts the real test for the host will come later this week, with guests including Ricky Gervais and Howard Stern. Oprah did surrender some details about her life story, but she was allowed to set the tone of the interview, which became largely a plug for her own network, Brian Lowry writes at Variety. As for his interviewing skills, which Oprah described as "surprising?" Quips Lowry, "'Boring' would be more accurate." "His giggling style might have come across as charming... in an interview with someone less intimidating," writes Chris Rovzar for New York. "Here it made him seem sycophantic and almost childish. When he reached out to her and said, 'Everything you touch turns to gold. Could you touch me?' I almost turned off the television." Ken Tucker at Entertainment Weekly writes that, "In general, Winfrey was probably as open as she's ever been with an interviewer, which is to say, not much." But despite the softball interview, Tucker believes Morgan has already shown himself to be a more than worthy successor to King. "Simply by having facts about his subject at hand and speaking coherently, Morgan aced King on his opening night," he writes.

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Summarize the decision of the discussion on the name chosen of the product. Project Manager: Can I close this? User Interface: Uh we don't have any changes, do we? Project Manager: Oh, okay. User Interface: So no. {vocalsound} Project Manager: {vocalsound} There we go. Okay, here we are again. Detailed design {disfmarker} oh, come on. Well {disfmarker} Ah {gap} s Forgot to insert the minutes, but it's about the same thing we discussed before. Uh {disfmarker} Could open that anyway, think. Other design {disfmarker} anyway, we took as {disfmarker} we took w we took rubber as as the material last time. We also {gap} that you're just busy with it. Took the advanced chip to t uh implement the advanced features. Well, we discussed the design, no sharp corners, we rounded it off, like you see on the {gap} other screen, which is fine. Um {gap} we agreed that the colour should be b uh yellow and black. Yellow in the back because it's m trendy, more trendy than black anyway. So {vocalsound} then we ca yeah. We agreed that we would implement both the L_C_D_ and speech recognition, but I'll get to that in a moment.'Cause some changes in the finances have left us implications anyway. So so, like I said, we had no insight in finances, no prices, Marketing: Hmm. Project Manager: but we have'em now, and it's bad. Anyway. We are Oh. Prototype presentation, well first you guys built the prototype. So {vocalsound} you could {gap} {disfmarker} could present that. But um let's see what be handy to do. Nee {disfmarker} no, you just go ahead and present the {disfmarker} w we'll scrap it later because {disfmarker} {gap} What? Industrial Designer: I think it's more or less the same as we had. User Interface: It's basically what we agreed upon, Marketing: Hmm? Project Manager: Oh that's User Interface: but just a little bit more specified. Industrial Designer: No much s Project Manager: hasn't changed that much, huh? Industrial Designer: No no no, not at all. Project Manager: I didn't expect anyway {gap}. You just coloured it. {vocalsound} User Interface: Uh s Final design. {vocalsound} Basically in {gap} {disfmarker} what we discussed, cover and buttons will be made of rubber, yellow colour, black components, as you can see right over here. Project Manager: Mm-hmm. I like the menu. User Interface: We chose a different type of colour for the menu. A bit darker yellow so that it com really shows in this keypad. Project Manager: Mm-hmm. User Interface: If you put them all black, it's not really that good a contrast. Project Manager: And I suppose the the the yellow is not printed on the on the rubber. It's it's part of the rubber, I suppose. User Interface: So {disfmarker} Probab Project Manager: I think that's more I think that's more durable anyway than printed on to {disfmarker} User Interface: Yeah. That's the be Industrial Designer: Hmm. User Interface: And it {disfmarker} I guess it's more easier to just paint it on the rubber Industrial Designer: Yeah, of course. User Interface: than to uh {disfmarker} Industrial Designer: That's uh the integration story again. Marketing: Mm yeah. Okay. User Interface: So we have it's a bit round shaped, Project Manager: {vocalsound} Oh yeah. User Interface: that's what we had uh {disfmarker} We chose the buttons to be uh teletext, okay button, favourite channel and the mute. Project Manager: Mm-hmm. User Interface: So that's basically what we chose there. Project Manager: Okay. User Interface: If you have anything to add, please interrupt me. Industrial Designer: No, uh this is just a description of what we see there. Project Manager: Yeah. Industrial Designer: So {disfmarker} User Interface: {vocalsound} Oh. Industrial Designer: Speaks for itself. Marketing: Yeah. User Interface: That's pretty much it. Project Manager: {vocalsound} Okay. Now it's my time to ruin everything. Well, not ruin everything, but {disfmarker} no, nah. User Interface: Oh sorry. {vocalsound} Project Manager: Finances, that's what we have here, what you drew. We have battery power, we have advanced chips and the sam the sensor. The sample sensor and uh {disfmarker} for speak recognition anyway. So which {disfmarker} you see the {disfmarker} which is de o one of the most expensive parts. So {disfmarker} well, we have sin one curve, {vocalsound} a design. Rubber design. And we had a special colour. Suppose yellow is a special colour. So just half a Euro for {gap} {disfmarker} You have pushbuttons and an L_C_D_ display. You have the total of seventeen Euros in production cost, Industrial Designer: Hmm. Project Manager: which is higher than the twelve and a half that we are permitted to use. So, Marketing: Hmm. Project Manager: easy. What do we scrap. Well think I had the best solution that I came up with is just to s take out the speech recognition. Industrial Designer: I d User Interface: I'd say that too. Industrial Designer: Yeah. Project Manager: Because the L_C_D_ has more support on customer side. Industrial Designer: Hmm. Project Manager: There are ninety one percent of uh the people, or something like that. But ninety percent who favour an L_C_D_ display, and only sixty percent that favour speech recognition. I think it's also harder to {disfmarker} User Interface: Uh we don't really have a extra function with the speech sample, Marketing: Yeah. User Interface: which you can't do with a normal remote control, Industrial Designer: Hmm. Project Manager: {gap}. So I ju User Interface: which people already do. So {disfmarker} Project Manager: I took that out. So {disfmarker} and so it's still stuck with thirteen, so I had to take out the special colour I suppose. And, yeah, I didn't see anything else I could take out. Yeah, I could take out the push-buttons, Marketing: Pushbut Project Manager: but we need those. So, generally what I came up with, in order to be cou to to have production cost of twelve and a half Euros, spe scrap speech recognition Industrial Designer: Huh. {vocalsound} {vocalsound} Marketing: Special colour, yeah. Project Manager: and the separate covers can account for the {disfmarker} if people want it, we'll just {disfmarker} then we'll do it in black. We'll just deliver it in black, have the {disfmarker} it has all the function that it's supposed to have, and if you want it {disfmarker} if you want the custom design, then you can buy the separate covers. User Interface: Well, Project Manager: You make it d orange or whatever you want. User Interface: I'd {disfmarker} I tend to disagree with you on that, because the trend issue was a big issue when we started designing this. Project Manager: It was a big issue, but {disfmarker} User Interface: So can't we just basically extend it to thirteen? Project Manager: I'll just go back. Uh let's just {disfmarker} let's see what {disfmarker} okay, let's just see what we {disfmarker} no, we we have to be under twelve and a half. Marketing: Yeah, it {disfmarker} Project Manager: It {disfmarker} it's not {disfmarker} Marketing: The p Project Manager: uh the project is a no-go if we go over twelve and a half, Industrial Designer: Okay, but there's another problem. Marketing: And the p Project Manager: so. User Interface: Okay. Industrial Designer: But there's another problem. Marketing: What {disfmarker} Industrial Designer: If we take another cover, for instance black, then we also need another button frame,'cause black and black doesn't work obviously. Project Manager: I think you {disfmarker} that's what you were ass assigned to do really, to to see how b th both those work together. Industrial Designer: Huh. Huh. Yeah. Project Manager: So I think {disfmarker} yeah, it's {disfmarker} I think it's y one of the {disfmarker} it's a good way to um to help people uh to make {disfmarker} to keep the product trendy too. Industrial Designer: Hmm. Project Manager: Just keep {disfmarker} you just make new covers for the {disfmarker} for it, Industrial Designer: Hmm. Project Manager: like we agreed before. Industrial Designer: Right. I agree. Project Manager: And everything that's left is is the basic function that uh that we want our product to have. Because the expensive parts are in either the advanced chip. But we need that for the L_C_D_ display. User Interface: Yeah. We do. Industrial Designer: Mm-hmm. Project Manager: Then again, we have the L_C_D_ display, which is also expensive. B yeah, but those go together. And yeah, we could take out the curve. Industrial Designer: Or say let's lose rubber, take plastic. User Interface: We could take out a curve indeed. Project Manager: Could {disfmarker} we could take out the curve. Is that an option? Industrial Designer: Yes. Marketing: {gap}. Project Manager: For you? Industrial Designer: Although we are demolishing a little bit the style. Marketing: But uh the {disfmarker} and {disfmarker} User Interface: I think the colour is more important than the really the curve, Project Manager: Yeah. Industrial Designer: But {disfmarker} Project Manager: {vocalsound} User Interface: because if you just end up with an entirely black remote control {disfmarker} Project Manager: I think it's {disfmarker} it it does ruin it, Marketing: Yeah. The people {disfmarker} Project Manager: but the fact that I t took that decision or t Industrial Designer: Yeah. Project Manager: Took this example actually, not really decision, but the example is because we do offer the um {vocalsound} the possibility of adding your own custom covers. So you can change {gap} any colour you want. So it's just you deliver a basic remote control with a possibility to change you into whatever you want. Industrial Designer: Can we then not also uh change the material? We take plastic for the basic cover Project Manager: You can take plastic, Industrial Designer: and {disfmarker} Project Manager: but I d it's something that's stuck into my mind is that {disfmarker} something that really came forward from the marketing research is that people like the the the the squishy feeling of {disfmarker} the spongy feeling of the {disfmarker} Marketing: Spongy, yeah. Industrial Designer: We can put those to the to the other covers. Project Manager: and it really makes it {disfmarker} also makes it different from the existing remote controls, Marketing: Yeah. Project Manager: because they're all plastic. Marketing: And {disfmarker} Project Manager: So which in in turn {disfmarker} {vocalsound} Industrial Designer: That's true. Project Manager: Rubber would increase durability Industrial Designer: But {disfmarker} Project Manager: because it doesn't break. Industrial Designer: okay. But what do you then suggest we'd lose? Because we have to lose two things and {disfmarker} I guess. Project Manager: I al like I said, I lost the speech recognition and I lost the special colour, Marketing: But {disfmarker} {vocalsound} Industrial Designer: Yes. Project Manager: which would make this black a black and grey. Industrial Designer: Okay, and that's enough? Project Manager: Yeah, that's that that that's enough, because User Interface: So black and grey is okay. Project Manager: I guess those are the basic colours. Marketing: But {disfmarker} Project Manager: So {disfmarker} Oh. User Interface: {vocalsound} Which we can fabricate, Industrial Designer: Hmm. Project Manager: I think those are basic col They want to {disfmarker} User Interface: okay. Marketing: The people want to pay for for it, so why why uh {vocalsound} do we have to keep us uh uh um on the twelve and a half? Project Manager: To ensure the profit. That {disfmarker} that's th that's the order. We're just uh {disfmarker} we're the project team and we got our our orders from the pro from the boss of our company Marketing: Yeah. Project Manager: which say we don't wanna spend more than twelve fifty for this. Marketing: But we can take a risk. Project Manager: But that's not for our {disfmarker} that's not our decision to take. We have a budget of twelve fifty per product. User Interface: No, we basically {disfmarker} Marketing: Okay, yeah. Industrial Designer: Hmm. Project Manager: So {disfmarker} User Interface: We need to stick to that. Project Manager: Stick that. I don't think it's really bad either. I mean if we we have the the backup of {disfmarker} or the backup design thing Marketing: I hope the people will like it, Project Manager: to have {disfmarker} Marketing: but {disfmarker} Project Manager: I think they would do. Th I think they do like because yo we {disfmarker} you {disfmarker} we agree upon that the that the the the cover thing was a nice idea, Marketing: {gap} {disfmarker} Project Manager: because p you could have all sort of designs while at the same time just manufacturing one product, one basic product which you could turn into any any taste you want. Marketing: Yeah. Project Manager: So I think it's the best solution to make those cu custom covers for the design aspect Industrial Designer: Perhaps we should make m Marketing: Yeah. {vocalsound} Project Manager: and keep the functionality between {disfmarker} of {disfmarker} within the th the boundaries of the your f uh your budget. Industrial Designer: Huh. Marketing: The first sheet. Project Manager: So {disfmarker} Industrial Designer: Perhaps we should make clear to our customer that we had to do this to stay under the cost. And that's {disfmarker} uh they know that this is an option and that we had to drop the option to stay under the cost, that they know that. Project Manager: {vocalsound} Well I don't think {disfmarker} Yeah. Is it worth {disfmarker} is it is it {disfmarker} Industrial Designer: Perhaps they decide tha User Interface: But they don't {disfmarker} Project Manager: does it mean anything to the customer? Like, it {disfmarker} like, we don't care {disfmarker} we don't care that you had to {disfmarker} Industrial Designer: Of course. Perhaps {gap} they uh {disfmarker} no, but perhaps they think uh okay, the cover is such a nice idea, uh let's {disfmarker} that that then they uh that allow us to make some more costs. Project Manager: True, Industrial Designer: We ca we uh we can at least tell them that {disfmarker} Project Manager: but we did we didn't get that. So I think it's {disfmarker} Industrial Designer: You don't know that. Project Manager: it should either be a pack, maybe we sh that should be sold in in the s in stores with with a standard cover or something. User Interface: Well {disfmarker} Industrial Designer: Huh. Project Manager: But {disfmarker} Industrial Designer: No, I'm not uh talking about that cost {gap} but the one that g has given us the order to design this. We could at least m uh make it like this, like you said, Project Manager: They could, but uh {disfmarker} Industrial Designer: and then tell them okay, we had to drop this and that, just that you know. It is an {disfmarker} still an option, but {vocalsound} not for this price. Project Manager: It's an option, but {disfmarker} yeah, it's true. So actually uh it's not that much of an increase, but yeah. We cannot contact them. User Interface: And if we {disfmarker} Project Manager: It's just the order that we got. Industrial Designer: Exactly, Project Manager: So that's what we gotta go with. Industrial Designer: but {disfmarker} Marketing: Yeah. Project Manager: So it's either one fi just just to get it f just to get it through final, it's either {vocalsound} turned into plastic, drop the squishy feel, make it make it more breakable, Marketing: Yeah. Industrial Designer: Hmm. Project Manager: um or turn it yellow. So {disfmarker} {vocalsound} It's uh something we have to decide on. Industrial Designer: I'd say lose the curve and the colour Project Manager: I say lose the curve. Oh that's true, Industrial Designer: and {disfmarker} Project Manager: we could lose the c I forgot that, yeah, sorry. Uh the curve. So {disfmarker} {vocalsound} User Interface: So which curve is that ba Project Manager: That's just this one just d this is the banana curve. User Interface: that's basically that curve. Industrial Designer: Hmm. Project Manager: So this would this would be straight. User Interface: So we could u still have the comfort. Marketing: Yeah, that's better. Project Manager: No, uh {disfmarker} no, that would be a curve inside the thing, I guess. No, would ju then it would just be a straight remote. Just like {vocalsound} like that. Industrial Designer: Hmm. Project Manager: Which would, yeah, turn it into something far more ordinary. {gap} we could make it yellow then, User Interface: I second that. Project Manager: but {disfmarker} You second that, you second that we lose the curve. User Interface: {vocalsound} No, that it would turn out to be a pretty straight-forward remote control. Project Manager: Okay, yeah. User Interface: So that's not really that {gap} {disfmarker} Marketing: Yeah. Yeah. Project Manager: So I think it would be a good idea to keep the curve {vocalsound} to separate it from the rest of the remote control world, so to speak. Industrial Designer: Hmm. Project Manager: So we keep the curve. So the only only solution is either to use the l y lose the yellow or lose the rubber. User Interface: I would {disfmarker} Project Manager: And I'm in favour of keeping the rubber, because it has more more advantages than the colour yellow has. Industrial Designer: {vocalsound} Oh. I agree. User Interface: Yeah. I would say {disfmarker} I would agree with you on the colour, Industrial Designer: No. Marketing: Yep. User Interface: because that's an extra option, an extra service we can deliver for a little bit of more money. Project Manager: Mm-hmm. Yeah, um User Interface: So we can always do that. Project Manager: I guess people are willing to pay for that. So I think we can take that option and just {disfmarker} with uh with the idea in the back of our head that you can customise your remote control. Industrial Designer: Hmm? Marketing: Yeah. {vocalsound} Project Manager: So I think that would still make it a nice product. Industrial Designer: Yes. Project Manager: Okay, we're final on that. So {vocalsound} it's too bad we can't make the whole super thing. But anyways we're here. Um yeah. User Interface: Which is basically what we discussed. Project Manager: This we discussed just now. That's just now {disfmarker} just {disfmarker} we could just discuss how the project went. I mean, was kind of {disfmarker} Marketing: And I want to do that. Project Manager: I sort of expected that everything would turn out this way, but because you {disfmarker} yeah, everything cannot be for free. We didn't {disfmarker} I think it was too bad we didn't have the financial info the last time. Because that was {disfmarker} I th User Interface: Yes, Industrial Designer: Huh. Project Manager: it was really essential really User Interface: we could have {disfmarker} Project Manager: to ma because we spent uh uh entire stage designing a product of which we had no idea what it would cost. Industrial Designer: Hmm. Project Manager: So we just put something {disfmarker} Marketing: Yeah. Project Manager: I think it's really nor not in stroke with reality actually. Marketing: {vocalsound} Project Manager: So {disfmarker} Industrial Designer: Me too, I felt a bit blind throughout the project, Project Manager: {vocalsound} Yeah. Industrial Designer: because in the beginning I had no list of available materials, Project Manager: Yeah, I think {gap} {disfmarker} would have been. Marketing: Yeah. Project Manager: Materials would be ok Industrial Designer: and then I d had not list of available c finances. Marketing: But {disfmarker} Project Manager: at least the last meeting I would have expected had to have that. Industrial Designer: So {disfmarker} Project Manager: So I suppose {disfmarker} Marketing: Let's um {vocalsound} see {gap} um {disfmarker} Project Manager: Yeah, let's see if it sells. I mean I suppose this sells, because it's very {disfmarker} {vocalsound} very extended. Marketing: Um {disfmarker} Project Manager: But {disfmarker} User Interface: Well I hope it sells. {vocalsound} Marketing: Let's {disfmarker} Uh {disfmarker} Project Manager: I suppose it sells, Industrial Designer: Hmm. Project Manager: because it's good. Marketing: Oh. Project Manager: I mean it's got everything for the for the reasonable price, because we didn't know what it's gonna cost anyway. Industrial Designer: Hmm. Project Manager: {vocalsound} Marketing: Hmm. Okay, let's eval evaluate uh the product of us, our design. Um I have some {disfmarker} {vocalsound} uh a method, a requirements and scale of. I uh will pre present uh some statements and we will decided together wha what {disfmarker} if it's true or false Project Manager: Okay. Marketing: and uh then we see uh if the requirements of the user are fulfilled or not. Project Manager: {vocalsound} Have been met, okay. Marketing: And I will uh make a new blank sheet Project Manager: {vocalsound} Yeah. Marketing: So so the buttons, the look and feel. I thought it was okay, but the advanced uh settings, um screen, audio and channel {disfmarker} Project Manager: {vocalsound} They're stuck under menu. User Interface: Which are basically accessible through the menu button. Marketing: We are not {disfmarker} Project Manager: For the menu. I think those are totally met, Industrial Designer: Hmm. Project Manager: because we we really took them for the {disfmarker} they have the feel they want, Marketing: Oh the menu button is it. User Interface: Yeah. Marketing: Hi Oh, okay. Project Manager: they have the simplicity they want. Marketing: Then it's all uh {disfmarker} S Project Manager: I think it's very uh very well met. Either two or one maybe. Industrial Designer: One. Marketing: it's true. Project Manager: I think we took that {disfmarker} everything they wanted into consideration. Marketing: Yeah. Project Manager: So it could either be a two or a one. Marketing: So d Oh wait. Uh pen. Project Manager: One and a half. {vocalsound} User Interface: Which is not an option. {vocalsound} Marketing: The p Oh yeah, Project Manager: Just create our own option. {vocalsound} Marketing: it's red, okay, but {disfmarker} Look and feel is everybo it's true. Project Manager: Yeah. Marketing: So {disfmarker} Anyone? And the next one {vocalsound} uh {disfmarker} yeah, when it's lost uh you can find it. Project Manager: It's perfect. Even for deaf people, yeah. Marketing: It's {disfmarker} Project Manager: It's {disfmarker} I don't think it's perfect, but we did everything possible to to get it back. Industrial Designer: Hmm. User Interface: To make it that way, yeah. Project Manager: Because if it's stuck in you couch, you can see the light. Maybe you can hear it. But I mean we tried, Marketing: Yeah. Project Manager: so I think it {disfmarker} that's {disfmarker} that deserves a one. Definitely. Marketing: And it's and it's {disfmarker} yeah. To {gap}. That's okay then. And the next one. How is that? Uh w we had {disfmarker} we don't have an uh manual, Industrial Designer: Manual. Project Manager: {vocalsound} Marketing: yeah. Industrial Designer: I think the L_C_D_ display could be a little bit more difficult then a normal remote control, Marketing: But I think that's a part of it. But {disfmarker} Project Manager: I'd use an {gap} remote control. User Interface: Mm yeah. Industrial Designer: but then again, it's for young people. So {disfmarker} Marketing: Yeah, an L_C_D_, it tells a lot about uh {disfmarker} User Interface: And it's pretty straight-forward, Industrial Designer: Yeah, I th Project Manager: It's pretty straight-forward, uh-huh. User Interface: you have a navigation {disfmarker} no keys to navigate through the L_C_D_ menus. Industrial Designer: Exactly. No, that's true. I think it won't be a big problem. Marketing: Yeah. User Interface: So {disfmarker} Marketing: So it's a one User Interface: One I d no, Marketing: or a {disfmarker} {vocalsound} I don't know. User Interface: actu Project Manager: I think {disfmarker} but we didn't even {disfmarker} Marketing: For the advanced uh settings. Project Manager: there was no issue on making a manual actually. We didn't {disfmarker} {gap} really discuss it, Marketing: No okay, that {disfmarker} uh that's true. Project Manager: but I don't think it takes {disfmarker} no, it really does doesn't take time to learn, I think. We took it s it's so easy, User Interface: No, it it is pretty straight-forward. Marketing: Oh, so it {disfmarker} Project Manager: we have so little button, everything speaks for itself really. So I think that's {disfmarker} Industrial Designer: Ah. Um {disfmarker} Project Manager: yeah, we didn't {disfmarker} it's either two or one, I guess. Marketing: Takes no ti Project Manager: Maybe it's a two, because d uh the L_C_D_ is a little is a little new and there is {disfmarker} there are some option hidden under the menu button. Industrial Designer: With the more important functions on. Marketing: Yeah. Project Manager: So I might make this a two instead of a one, I guess. User Interface: Yeah. Yeah. Marketing: And the L_C_D_, you have to see it. Project Manager: So just make that a two. Marketing: Um mm {disfmarker} Oh, it's a little bit learning. Okay. Uh yeah {vocalsound} it's uh a little bit same. Industrial Designer: Mm-hmm. Marketing: But it tells you or not? Project Manager: You can use the L_C_D_ in a good way. I think so. I think it's perfect, the w where it is, what it can do, if it useful. I think so. Marketing: But wha w Industrial Designer: Mm-hmm. Marketing: oh, yeah. What are we uh displaying on the L_C_D_ screen? Just uh only the channels and {disfmarker} or {disfmarker} Industrial Designer: {gap} the menus uh {disfmarker} Things like brightness and uh those kind of things we've put in the menu, Marketing: What uh? Industrial Designer: because we have no buttons for those. User Interface: Well, basically the menu options indeed. But {disfmarker} Marketing: Oh, in the L_C_D_ screen. Project Manager: No, y I mean in the L_C_D_ screen, the small screen. What does it display? Industrial Designer: Yes. Marketing: And for a channel selection, uh {gap} {disfmarker} or that's not {disfmarker} Project Manager: Well I thought it was I thought it was {disfmarker} I thought that people wanted previews on their {disfmarker} I'm not sure if that even possible, Marketing: Yeah, I thought I thought too Project Manager: but {disfmarker} Marketing: but {disfmarker} Project Manager:'cause it's {disfmarker} this requires a quite quite a bit of band-width. Marketing: yeah. Project Manager: I don't think it's possible really. But the {disfmarker} they didn't really define in what should be used for. User Interface: No. Marketing: Maybe a T_V_ guide or something in your L_C_D_ uh {disfmarker} Project Manager: But I think in for example like T_V_ guides, I think that's {disfmarker} that th that you can transmit through it and everything. Just for extra information on your programmes. Industrial Designer: Mm. Marketing: Yeah. Yeah, it must be clear then what {disfmarker} what what for we use it. Project Manager: But also things like like like menus or p how about preferences of your {disfmarker} uh with configuring your remote control for favourite {disfmarker} your favourite channel for example, how do you configure that. User Interface: Yeah. Marketing: Yeah. Project Manager: So that could be done by L_C_D_ display. I think it's good. No, maybe it's not a one because it's {disfmarker} we're not using it perfectly. We didn't give it {disfmarker} I don't thing over-discussing. Now we gave it enough thought though. I think we d should just lower this. Marketing: Yeah. Project Manager: Maybe maybe it's a three though. We could've used it more effectively probably. Marketing: Yeah, indeed. Project Manager: {vocalsound} Marketing: So everybody's agree with an uh three on it, Project Manager: Yeah, we are using it, User Interface: Yeah. Two or three. Industrial Designer: Yes. Marketing: it's {disfmarker} Project Manager: but it's not Marketing: W Project Manager: it's not poorly used, but it's not efficiently used, I think. User Interface: So {disfmarker} Marketing: Yeah. Project Manager: We could have ev even lost {vocalsound} the selection button and uh done everything via L_C_D_ selection. Marketing: Yeah. Project Manager: It's {disfmarker} now it's just extra to illustrate im uh extra features, Marketing: Yeah, {gap} I {disfmarker} Project Manager: but okay. Marketing: A three. Industrial Designer: Nah, it's not really {disfmarker} only an extra. User Interface: You can {disfmarker} seven. {vocalsound} {gap} {vocalsound} Industrial Designer: No menus. Marketing: Ah, nothing, that's {vocalsound} {disfmarker} A seven. Uh that's uh {disfmarker} Industrial Designer: Think about {disfmarker} Project Manager: Can you talk to remote control? User Interface: Or we could say it {disfmarker} {vocalsound} Project Manager: Well, it can't talk anymore. So we scrap that. Marketing: Yeah. {vocalsound} User Interface: Or we could say neutral, Project Manager: Oh yeah {disfmarker} {vocalsound} User Interface: we {disfmarker}'cause we scratched the {disfmarker} Project Manager: Just to be a prick, User Interface: C Project Manager: but of course you can talk to your remote control, it doesn't do anything. Marketing: {gap} Yeah yeah yeah yeah. Project Manager: But you c {vocalsound} you can talk to it. Marketing: Not with the speech recognition. Uh yeah, all the trends and no colours uh anymore. So {disfmarker} Project Manager: Well, we did take everything into consideration of course. Uh the shape i shape is i Marketing: Yeah, {gap} {disfmarker} uh um only in the curves. Project Manager: I think we {disfmarker} yeah, I think that's okay. Marketing: But the colours, we don't have special colours on it. User Interface: No, we don't have the colour. Project Manager: Yeah, {gap} special co but we took into consideration the fact that it's customisable User Interface: So I {disfmarker} Marketing: Yeah, Project Manager: to the fashi Marketing: but we {vocalsound} {disfmarker} User Interface: Yes, but the end product {disfmarker} So {disfmarker} Industrial Designer: Hmm. Marketing: yeah, we don't have it, so d Project Manager: We don't have it {disfmarker} we do have it, Marketing: In the end product. Project Manager: it's just sold as a package. Industrial Designer: But {disfmarker} M Project Manager: It does {disfmarker} it's not part of the basic product. Industrial Designer: Changing covers is also trend that we followed. Project Manager: It {disfmarker} that that's what I call trendy. I mean the shape is trendy. User Interface: {vocalsound} Project Manager: The the sh the the functions are trendy. It's just the colours that are not supporting the basic model. Because you ha Marketing: Now {disfmarker} Project Manager: it's just not affordable at the moment. User Interface: Maybe we should go with a two then, Marketing: But it's not a one. User Interface: because it's not perfect, because we can't do it initially, Marketing: Yeah. User Interface: but we {disfmarker} {vocalsound} Project Manager: It's possible, Industrial Designer: Mm-hmm. Marketing: Oh. {vocalsound} Project Manager: but you have to pay extra for the for the possibility of having it in a f a different colour. Marketing: Oh well {disfmarker} Oops. Project Manager: {vocalsound} Marketing: Oh it's a two, User Interface: Yeah. Marketing: right? Industrial Designer: Mm-hmm. Marketing: On the last one. Uh that n that's all. Project Manager: Overall score. {vocalsound} User Interface: Overall score. Marketing: Overall. {vocalsound} It's um Project Manager: One two three. {gap} sixteen. Marketing: {vocalsound} ten, sixteen three {disfmarker} uh two Project Manager: {vocalsound} Two two point some two point something. Marketing: two point seven or something like that. Industrial Designer: Hmm. Marketing: I don't know why. User Interface: Ten, sixteen, divided by {disfmarker} Project Manager: {gap}. Industrial Designer: Six. Marketing: Six. User Interface: Is two two third. Project Manager: Two and two thirds. Marketing: Um {disfmarker} So Project Manager: {vocalsound} Marketing: we can say it's it's {disfmarker} the product is {disfmarker} it's okay. Project Manager: It's okay, but {vocalsound} that's yo m Marketing: Y not {disfmarker} Industrial Designer: Mm-hmm. Project Manager: mostly it's it's influenced by the fact that we didn't have enough resources to implement speech recognition. User Interface: There's {disfmarker} Industrial Designer: Mm-hmm. Project Manager:'Cause yeah, that gives you a seven, which ruins your your average. Marketing: Yeah. Project Manager: Without that it would be like under {disfmarker} it wouldn't {disfmarker} yeah, it would be under two. So I think we have {disfmarker} even with this it's reasonable. Marketing: Woah. {vocalsound} User Interface: Yeah, if we make it into a four, as in neutral, because we didn't implement it, so we can't say that we {disfmarker} {vocalsound} that it's really not well implemented. We come out on a average of two one eighth. Marketing: Yeah. Project Manager: Well I think it's {disfmarker} two is okay. User Interface: So which is pretty w good. Industrial Designer: Mm-hmm. Project Manager: Yeah, two is pretty good. User Interface: It's at least on the positive side. Project Manager: Definitely. User Interface: So {disfmarker} We could definitely have done better if we've had more resources, Industrial Designer: Hmm, of course. Marketing: Yeah. User Interface: but {disfmarker} Project Manager: Yeah, I think it's probably {disfmarker} {gap} I Industrial Designer: Mm-hmm. Marketing: Yeah. Project Manager: I do admit that we d {vocalsound} did miss a little {disfmarker} or didn't sp didn't talk {gap} {disfmarker} talk enough about the possibility of the L_C_D_ display. We could have used it more efficiently, Marketing: Yeah. Project Manager: we just didn't think of it that way. User Interface: Yeah. Marketing: Yeah, with {gap}. . . # User Interface: True. Project Manager: So {gap} {disfmarker} like I said, {vocalsound} changing channels, everything hidden in your L_C_D_ display, so you just need the navigation buttons to do everything. Marketing: The scale. Industrial Designer: But I think for this price, this is {disfmarker} it's really a reasonable product. Project Manager: I think we div I think we did very well, Industrial Designer: It's a good product. Project Manager: uh ev even if you look at this score, we did quite well. Marketing: Yeah. Industrial Designer: Oh. Oh. Yeah. Marketing: With an L_C_D_ screen {gap}. Project Manager: It just looking for improvements what what you could have improved. So. {disfmarker} Industrial Designer: But if pep people really want speech recognition, then they must be prepared to pu to pay more, because it's cannot be done for this. Project Manager: {vocalsound} They sh they should get kids, and just stick'em in T_V_ and say change the channel. {vocalsound} Marketing: Yeah, User Interface: {vocalsound} Marketing: you can make'em another one. Industrial Designer: Hmm yeah. User Interface: {vocalsound} Marketing: {vocalsound} Industrial Designer: Ah but for this price uh you cannot ask that. Project Manager: I don'think so. Uh it's just not {disfmarker} it it's not affordable. Industrial Designer: You cannot th think of that {gap} {disfmarker} Project Manager: Or your sh you should lose the L_C_D_ screen probably, Industrial Designer: No, it's not. Project Manager: but I think that's {disfmarker} I think the L_C_D_ screen is more worth than speech recognition. Industrial Designer: Mm-hmm. Oh It's also more attractive. Project Manager: Definitely. Okay, that was that. Marketing: Yeah. Yeah. Project Manager: So that's the final product without the speakers, I guess. User Interface: So did you {disfmarker} Project Manager: Let's see, what was left in the the {disfmarker} Another one. {vocalsound} Hmm. {vocalsound} Yeah, Marketing: {vocalsound} Project Manager: we evaluate the product. {gap} {disfmarker} General project, what's i in {disfmarker} For example, I thou I thought we were pretty creative in what we created. We took the whole new approach of making exchangeable cover for example, which I thought was pretty creative, because it was never never ever listed somewhere. Industrial Designer: Hmm. Marketing: Favourite channel. Project Manager: Well {disfmarker} Anyways. Yeah, leadership is up to you. I mean perhaps I screwed up because I d {vocalsound} put a put a speech recognition into it. But that's not for me to decide. Marketing: Yeah, I know. Yeah. Project Manager: I think we did pretty well as team-work though. Because, yeah was very hard to work with one another if you cannot communicate in the meantime, Industrial Designer: Yes. Hmm. Project Manager: because when I got the when I got the input for the financial results, initially of course I wanted to contact you. Industrial Designer: Hmm. Marketing: Yeah, you're working separate. Industrial Designer: Yeah. Project Manager: Say, look, this is {disfmarker} you're doing the wrong thing, Marketing: Yeah. Industrial Designer: Huh. Project Manager: you're s you're wasting your time now, because we're implementing stuff that we cannot afford. User Interface: Yeah. Industrial Designer: Hmm. Marketing: Yeah, yeah yeah. Project Manager: So it would be better if y if there was more communication between uh {disfmarker} Industrial Designer: Hmm. Marketing: Yeah yeah yeah. Direct uh communication with {disfmarker} yeah. Project Manager: because that's that's what would w you {disfmarker} what you would normally do, either call or email someone. User Interface: And we could share information which we received. Project Manager: So that was too bad Industrial Designer: Hmm. Project Manager: con was impossible here anyways. {gap}. Industrial Designer: That's the same thing that I had in the beginning. Everybody was using materials that s I didn't have. Project Manager: It didn't have Industrial Designer: So {disfmarker} Project Manager: or didn't knew what they costs or whatever. Marketing: Yeah. Project Manager: There was just too little information about what things actually cost and if you could use them. Industrial Designer: Oh. User Interface: Yeah. Industrial Designer: Hmm. Project Manager: So that was a little unclear I suppose. I think a SMARTboard SMARTboard is pretty cool. I think uh s especially for design issues, it's very easy just to give your give your thoughts a little {disfmarker} it's easier to share them. Marketing: {vocalsound} My handwriting is little bit {disfmarker} yeah. Yeah. User Interface: Although for actual design I'd say the response time should be a little bit higher, Industrial Designer: Hmm. Project Manager: It's a little less {disfmarker} it {disfmarker} the response time is le it's very bad. User Interface: because {disfmarker} Industrial Designer: Hmm. Project Manager: It's good to visualise everything, but I think the response time should {disfmarker} could be a lot better. User Interface: The digital pen was definitely better to draw my ideas and to further elaborate on that. Marketing: But th that's {disfmarker} Project Manager: Definitely. Industrial Designer: Mm-hmm. Project Manager: Yeah, it's true. Marketing: Yeah, okay. User Interface: So {disfmarker} Industrial Designer: But there's uh also one problem with this I noticed. Uh you have to finish a page before going to a n Project Manager: No, you don't have to. No, you don't. Marketing: No. Project Manager: I jin I didn't check the finish button. Industrial Designer: Oh. Marketing: You can {disfmarker} Project Manager: I just {disfmarker} you just ditch it and you can copy it or whatever. Marketing: Done and then it's okay. Industrial Designer: Okay, I saw that uh {disfmarker} Project Manager: Uh only if you uh check the notes or {vocalsound} press done. Then it um {disfmarker} then you can {disfmarker} then it exports to Word automatically. Industrial Designer: Hmm. Project Manager: But it's not necessary to check either one of those two. Industrial Designer: Yeah, Project Manager: You can just preview your p you can just preview your page in the in the programme. Industrial Designer: but I made {disfmarker} Marketing: Oh, okay. Industrial Designer: Okay, but I made three pages Marketing: Okay, yeah. Industrial Designer: and they were not finished. And when the third one was finished, I wanted to download it and then it was not possible anymore, because you have to close all the pinnits uh the pages before going further. {gap} {disfmarker} Project Manager: Okay, before starting a ne a new page. Marketing: Okay. Project Manager: Okay, that could be b. Industrial Designer: Exactly. So we cannot work on more than one page at same time. That's not possible. Marketing: Oh. Project Manager: Okay. Industrial Designer: You have to finish it completely, Marketing: Hmm. User Interface: Oh can you? Okay. Industrial Designer: then download it, it's {disfmarker} then start a new one. Project Manager: Yeah, okay. Industrial Designer: That's not very uh handy, Project Manager: That's {disfmarker} Industrial Designer: but if you know that, then it's not a problem. Project Manager: Yeah, it's understandable, okay. {vocalsound} Any new ideas? Yeah, more communication between {vocalsound} between uh {disfmarker} that's the thing I noticed, that communication is very um very important, Industrial Designer: {vocalsound} Marketing: Important to mm {disfmarker} Project Manager: because if you get new information, it's essential f for the other team-mates to know that as soon as possible, because you would avoid making {disfmarker} doing extra work, because you were doing extra work now uh m working on the on the speech recognition, you have limitation both on the technical {gap} {disfmarker} on the d on the design side. Marketing: Yeah. Project Manager: So I think that could have been better. But that's {disfmarker} I think it's more of a a setting here that you cannot communicate than uh {gap} than somewhere else. Industrial Designer: Hmm. Project Manager: So {disfmarker} User Interface: Yeah, well it could also possibly be {disfmarker} well, is it a more real-time information base, so we can all see {vocalsound} which information is available to one another. Project Manager: Yeah, I think so. And l less p less spam probably. I'm not sure i I'm not sure you got spammed as well, Industrial Designer: Mm-hmm. Project Manager: but I get spammed like every t every two minutes there was a {disfmarker} there was another email about master classes or something. Industrial Designer: Ah. Well {disfmarker} Project Manager: So {disfmarker} Industrial Designer: Hmm. Project Manager: which were totally useless actually. I thought I should probably look into them, {vocalsound} but they were all useless. So I just {disfmarker} User Interface: Well, I personally did not have that, Marketing: Mm {disfmarker} Project Manager: Oh okay. User Interface: but {disfmarker} That's probably your l description. Project Manager: {vocalsound} User Interface: But I also didn't {disfmarker} not really. But still, you had that as well. Is that we finished up the design Industrial Designer: Huh. User Interface: and then we checked the website, Industrial Designer: Yeah. User Interface: and then there was just extra information. Marketing: Yeah, after {disfmarker} After five minutes, uh {disfmarker} Industrial Designer: {gap}. User Interface: There was a little delay in the {disfmarker} {gap} bit of a c crucial delay. Industrial Designer: Yeah, {gap} {disfmarker} Marketing: Yeah. Project Manager: I didn't have any uh more information, it's just always the same here. So that's that's kind of a {disfmarker} Industrial Designer: Mm. Marketing: Email uh {disfmarker} Project Manager: It would change, but not for me. So I'd {disfmarker} I had no extra information to go on that one than what you give me actually. Industrial Designer: Hmm. Project Manager: I couldn't do any research myself Industrial Designer: Hmm. Project Manager: or {disfmarker} I see, that's {disfmarker} Industrial Designer: Hmm. Project Manager: yeah, w I could have done a little extra work probably, then {disfmarker} Marketing: {gap} it's {disfmarker} Project Manager: {vocalsound} But I was busy enough anyway. So {disfmarker} Industrial Designer: Hmm. Marketing: {vocalsound} Yeah. Project Manager: Any new ideas found? Or is that a {gap}'cause {disfmarker} {vocalsound} uh yeah, it's {disfmarker} well, Industrial Designer: No. Project Manager: probably is. User Interface: How much time do we have for this anyway? Project Manager: I have no clue. That's like {disfmarker} oh, but it {disfmarker} {vocalsound} {vocalsound} Should i Industrial Designer: {gap}. Project Manager: if the project is evaluated and it was it was in b within budget, we should celebrate. So {disfmarker} {vocalsound} User Interface: {vocalsound} Yeah. {vocalsound} Industrial Designer: {vocalsound} Okay, Marketing: {vocalsound} {vocalsound} Industrial Designer: bring out the beer. User Interface: Champagne. Project Manager: Uh okay, think that's about it. Uh {disfmarker} Marketing: I want one for my own. {vocalsound} Project Manager: I'm not sure what we should still do though uh t let's see what {disfmarker} all your tasks were finished, right? What you ha from your assistant. So let's {disfmarker} Marketing: Yeah. Industrial Designer: I have no more email. My coach is uh being very silent now. Project Manager: Okay, Marketing: Yeah, Project Manager: I should {disfmarker} I think I sh Marketing: my personal coach i Project Manager: I still have the the total report to finish up. I think we took very little time now, because {disfmarker} {vocalsound} Yeah, we're in agreement, everything {disfmarker} the design is okay. The one thing we missed though, we don't have a product name. Marketing: What {disfmarker} Industrial Designer: No, Project Manager: How about you cook a {disfmarker} how about you cook up a product name? User Interface: Product name. Industrial Designer: we haven't think above {disfmarker} about that. Marketing: {vocalsound} Yeah, name. Industrial Designer: Huh. It's better than thi I think than a serial number. Sony uh T_R_ something uh f means nothing to me. Uh {disfmarker} Project Manager: Just {disfmarker} Marketing: Or fruit name. Project Manager: oh, think of a catchy name. I'll be working on this until the beep {disfmarker} until it beeps. So {disfmarker} Industrial Designer: Like fruit names. Marketing: Fruit name or something like that. Project Manager: What? Fruit? Marketing: The banana remote or something. Project Manager: You don't want it to resemble a banana. Marketing: {vocalsound} I don't know. Yeah, it's the form of it. Project Manager: It's not yellow anyway. User Interface: The bana'cause it's not yellow anymore. Project Manager: It's not yellow anymore. Marketing: Yeah {disfmarker} oh, yeah. {gap} {disfmarker} Project Manager: It is curved, but {disfmarker} Marketing: Uh yeah. {vocalsound} Uh {disfmarker} User Interface: Well, uh I was going for the R_C_ deluxe, but it's not really a catchy name or anything, Project Manager: No, User Interface: it's more {disfmarker} Project Manager: it's {disfmarker} Industrial Designer: {vocalsound} Uh at least it's not something with numbers. Project Manager: Hmm. Industrial Designer: Numbers are so meaningless to the people. I mean {disfmarker} Marketing: Yeah. User Interface: Something with our {gap} company name, Marketing: That's true. User Interface: can we do anything with that? Industrial Designer: {vocalsound} {gap}. User Interface: Maybe there's something on the website which will help us out. Marketing: Reaction, Real Reaction. Project Manager: {vocalsound} Industrial Designer: Real Reaction. User Interface: The reaction deluxe. Project Manager: Real Reaction future R_C_. {vocalsound} Step into the future of of remote controlling your T_V_. {vocalsound} User Interface: {vocalsound} Is that a name or a c campaign? {vocalsound} Marketing: {vocalsound} Project Manager: No that's a that's a catchy slogan. User Interface: Yeah. {vocalsound} Project Manager: Control your remote control. User Interface: Or the {disfmarker} The real reactor. Industrial Designer: Real react. Project Manager: I go for future R_C_ probably. Something like {disfmarker} It's short f Industrial Designer: The Real Reactor, I don't find that uh that bad at all. Marketing: {gap}. Project Manager: Real reactor? Industrial Designer: Yeah. Project Manager: Uh that that's User Interface: You should write it down as a {disfmarker} an option. Industrial Designer: Because our name is Real Reaction. Project Manager: That makes me think of different {vocalsound} products than a remote control really. Marketing: {vocalsound} {gap}. User Interface: {vocalsound} Project Manager: I'm not sure. Real reaction in a real {disfmarker} Marketing: Zapping. The {disfmarker} User Interface: So that's one option. Project Manager: Real reactor. Didn't notice. Industrial Designer: I'm looking for things in the name. Project Manager: Mm. Industrial Designer: So that the first three letters are s the same. R_E_A_ R_E_A_. User Interface: Should I write the banana down or {disfmarker} {vocalsound} Project Manager: I take f Marketing: Yeah, sure. Project Manager: yeah, take a banana. User Interface: Sure? Marketing: {vocalsound} The banana. Project Manager: Hmm. Marketing: Remote. Banana recei R_C_. Industrial Designer: The triple R_. Real Reaction remotes control. Triple R_. Marketing: Remote. User Interface: Well I {disfmarker} Marketing: R_ three C_. User Interface: Uh do you mean it like {disfmarker} Industrial Designer: {gap}? Marketing: R_ three C_. Industrial Designer: {gap} yeah. User Interface: You mean it like this? Industrial Designer: Yeah, that {disfmarker} Marketing: Real Reaction Remote Control. R_ three C_. Oh yeah. Industrial Designer: {gap}. {gap}. Project Manager: No, not like that. It should be it should be longer, because it's not a product name that you f print on a box. Industrial Designer: I think {disfmarker} triple R_. Project Manager: Just write out triple, like a word triple R_C_, triple stripe {disfmarker} Oh. Triple dash R_ dash s s C_. Industrial Designer: Doesn't sound {gap}? Marketing: Yeah, triple R_. Industrial Designer: Yeah. Ah. Marketing: Triple R_C_. The triple R_C_, yeah. Project Manager: Yeah. Marketing: R_ s R_ three C_. {gap}. Project Manager: {vocalsound} R_ dash C_. User Interface: Dash C_? Industrial Designer: I think I like it like this more. Project Manager: Dash. Triple R_ or triple R_C_? User Interface: Like a C_ right now or a dash in a C_? Marketing: Triple R_ dash. Project Manager: How about do both? User Interface: {vocalsound} Project Manager: Sure if it looks stupid. Uh I think that the the R_C_ together takes away the the the image of {disfmarker} it's a triple {disfmarker} Industrial Designer: Hmm. Project Manager: Uh the first the first one looks like it's a triple remote control, Industrial Designer: Mm. Marketing: That {disfmarker} Project Manager: but it's only a single remote control. And it's especially on the triple R_ that's important. The Real Reaction Remote. Industrial Designer: I would {disfmarker} huh. I would lose the C_ Marketing: {vocalsound} Yeah, this {disfmarker} yeah. Industrial Designer: and just name it triple R_. User Interface: Is it triple R_C_s? No. Project Manager: {gap} {disfmarker} Industrial Designer: It sounds like uh thinking about two different things and combining it. Marketing: Triple remote. Industrial Designer: I would just say triple R_s triple R_ Marketing: Yeah. Project Manager: Yeah, triple R_ {gap} yeah, you can {disfmarker} User Interface: Well, that's another option. Industrial Designer: That's also short, catchy. Marketing: It's okay. Project Manager: {vocalsound} Yeah, triple R_. User Interface: Okay, so which ones are we going to scratch definitely? Marketing: {vocalsound} The banana. {vocalsound} Banana. {vocalsound} Project Manager: Banana remote. {vocalsound} Industrial Designer: Banana. {vocalsound} User Interface: {vocalsound} I say this one as well. Marketing: Yeah, the deluxe. Project Manager: I think we're all in agreement about the triple R_. Industrial Designer: {gap}. Project Manager: I think triple R_ is cool. Industrial Designer: Yes. User Interface: Triple R_? Marketing: The r triple R_. Project Manager: And it looks cool when you print it in font, looks pretty cool. User Interface: Triple R_ it is. Marketing: Yeah. {vocalsound} Industrial Designer: {gap} did you do now? Project Manager: Just like this {gap} just {disfmarker} and you just print triple R_, it looks {disfmarker} doesn't look bad, Industrial Designer: Yeah. Project Manager: it's short, it's okay. Industrial Designer: Yeah. Project Manager: So have to write my report now, I guess. Um {disfmarker} Um {disfmarker} Yeah, so we have everything. We have the product, we have the costs, Industrial Designer: Yep. Project Manager: we have the possibility of everything. Marketing: It can't work. That will not {disfmarker} Project Manager: Okay. I think it's adjourned. Retire to my lair and finish the report. That was a short meeting. But efficient though. Industrial Designer: Mm-hmm. The boss is always the last one to go home. So {disfmarker} Project Manager: Probably. See. Okay, User Interface: {vocalsound} Project Manager: goodbye. Marketing: Okay. Industrial Designer: See you in a minute. Marketing: Damn. I will write that one in a Word uh document. Industrial Designer: Okay. Project Manager: Could you guys draw me a picture of the final design to put on the cover of the report? User Interface: Yeah, sure. Marketing: {vocalsound} Industrial Designer: Can't we take this one? Marketing: Oh sh Industrial Designer: Otherwise we have to do it all over again. Marketing: Um {disfmarker} User Interface: I don't really know whether we can save it as a picture or no. Industrial Designer: Is it okay if I try? Is that okay with you? User Interface: Sure. Marketing: Yeah, okay, I will ask you when uh I need the information. Industrial Designer: I'll put it back in a minute. Marketing: So it's {disfmarker} oh. Industrial Designer: Okay, it has been saving something, but where to I don't know. Marketing: Uh {disfmarker} Oh. Merge. Industrial Designer: Oh, can I say exp yes, I can. Marketing: Sucks. Industrial Designer: Export as J_ PEG. {gap}. Okay, can I not put this wherever I wants. My document is the wrong one, huh. Marketing: {gap} {disfmarker} Yeah, but {disfmarker} User Interface: Network places. Marketing: I don't know. Smart {disfmarker} no. Industrial Designer: {gap}. Marketing: Ma it's maybe it's not on the network of uh the rest. User Interface: {vocalsound} Industrial Designer: I don't think so. Marketing: That one is. {gap}. User Interface: I wouldn't pick that one, no. Industrial Designer: Document and settings. Marketing: {vocalsound} Industrial Designer: That's a pity. That means that we have to gonna draw it again. Are you gonna do that? User Interface: Sure. Industrial Designer: Okay. User Interface: Oh. Industrial Designer: That {disfmarker} Yes. Okay. Okay. Okay. Yes, that's correct. Marketing: Yeah. Industrial Designer: {vocalsound} Okay. Marketing: Okay. Industrial Designer: No. Oh, it's export. Marketing: Oh yeah, {gap}. {gap} {disfmarker} Industrial Designer: Okay. Marketing: Can I see scores? Industrial Designer: Oh, of course. Marketing: Uh, {gap} one one, two threes, two {disfmarker} Industrial Designer: Sorry. Marketing: Okay, then we'll {disfmarker} overall, two points. Yes. User Interface: I see you later. Marketing: Yeah. Project Manager: {vocalsound} Marketing: Mm.

They agreed to cross out'banana remote','the deluxe'from the options. They all agreed on the'triple R'without arguments, because the group thought it looked cool when it was printed out. In addition, it was short and catchy.

qmsum

SECTION 1. SHORT TITLE. This Act may be cited as the ``Victims of Domestic Abuse Insurance Protection Act of 1996''. SEC. 2. DEFINITIONS. As used in this Act: (1) The term ``domestic abuse'' means the occurrence of one or more of the following acts between former or current household or family members (including in-laws or extended family), spouses or former spouses, individuals engaged in or formerly engaged in a sexually intimate relationship, a caretaker and the person taken care of, a perpetrator of sexual assault and the victim of the assault, or a stalker or a sex offender in relation to the person being stalked or person against whom the offense was or is being committed: (A) Attempting to cause or intentionally, knowingly, or recklessly causing the other person bodily injury, physical harm, severe emotional distress, psychological trauma, rape, sexual assault, or involuntary sexual intercourse. (B) Engaging in a course of conduct or repeatedly committing acts toward the other person, including following the person without proper authority and under circumstances that place the person in reasonable fear of bodily injury or physical harm. (C) Subjecting the other person to false imprisonment or kidnapping. (D) Attempting to cause or intentionally, knowingly, or recklessly causing damage to property so as to intimidate or attempt to control the behavior of the other person. (2) The term ``domestic abuse-related medical condition'' means a medical condition sustained by the subject of domestic abuse which arises in whole or in part out of an action or pattern of domestic abuse in relation to the subject of domestic abuse. (3) The term ``domestic abuse status'' means the fact or perception that a person is, has been, or may be a subject of domestic abuse, irrespective of whether the person has sustained domestic abuse-related medical conditions. (4) The term ``insurance policy'' means any policy, contract, or certificate of insurance (whether for health benefits, life insurance benefits, property and casualty benefits, or otherwise) issued by an insurer and subject to the insurance laws and regulations of a State, and includes an endorsement or rider to such a policy, contract, or certificate and includes a contract of health benefits issued by a health maintenance organization. (5) The term ``insured'' means a party named on an insurance policy as the person with legal rights to the benefits provided by the policy. For group insurance, such term includes a person who is a beneficiary covered by a group policy or certificate. (6) The term ``insurer'' means any person or legal entity (including a health carrier or life, disability, and property and casualty insurer) engaged in the business of insurance and subject to the insurance laws and regulations of a State, and includes agents, brokers, adjusters, and third party administrators and includes health maintenance organizations and similar organizations subject to regulation by a State for insolvency. (7) The term ``subject of domestic abuse'' means-- (A) a person to whom an act of domestic abuse is directed, (B) a person who has had prior or current injuries, illnesses, or disorders that resulted from domestic abuse, or (C) a person who seeks, may have sought, or had reason to seek-- (i) medical or psychological treatment for domestic abuse, or (ii) protection (including court-order protection) or shelter from domestic abuse. (8) The term ``group health plan'' has the meaning given such term in section 607(1) of the Employee Retirement Income Security Act of 1974 (29 U.S.C. 1167(1)). (9) The terms ``beneficiary'' and ``participant'' have the meanings given such terms in section 3 of the Employee Retirement Income Security Act of 1974. SEC. 3. PROHIBITION OF UNFAIR DISCRIMINATION AGAINST SUBJECTS OF DOMESTIC ABUSE. (a) In General.--An insurer or group health plan may not, directly or indirectly, engage in any of the following acts or practices on the basis that the applicant or insured, or any person employed by the applicant or insured or with whom the applicant or insured is known to have a relationship or association, or a beneficiary or participant in the plan is, has been, or may be the subject of domestic abuse: (1) Denying, refusing to issue, renew or reissue, or canceling or otherwise terminating an insurance policy or coverage under the group health plan; or restricting or excluding coverage under the policy or plan; or adding a premium differential to any insurance policy or for coverage under the plan on such basis. (2) Excluding or limiting coverage for losses or denying a claim incurred by an insured or participant or beneficiary as a result of domestic abuse on the basis of the insured's, participant's, or beneficiary's abuse status, except (in the case of an insurer) as otherwise permitted or required by State laws relating to acts of abuse committed by life insurance beneficiaries. (3)(A) Subject to subparagraph (B), terminating coverage for a subject of domestic abuse because coverage was originally issued or provided in the name of the abuser and the abuser has divorced, separated from, or lost custody of the subject of domestic abuse or the abuser's coverage has terminated voluntarily or involuntarily and, with respect to health insurance coverage or coverage under a group health plan, the subject of domestic abuse does not qualify for extension of coverage under part 6 of subtitle B of title I or the Employee Retirement Income Security Act of 1974, section 4980B of the Internal Revenue Code of 1986, or title XXII of the Public Health Service Act. (B) Nothing in subparagraph (A) prohibits the insurer or group health plan from requiring the subject of domestic abuse to pay the full premium for the subject's coverage under the policy or plan or from requiring, as a condition of health insurance coverage or coverage under the plan, that the subject of domestic abuse reside or work within its service area if such requirements are applied to all insureds of the insurer with respect to such coverage or to all participants and beneficiaries. (C) The insurer may terminate group health insurance coverage after the continuation coverage required by this paragraph has been in force for 18 months if it offers conversion to an equivalent individual plan. (D) The continuation of health coverage required by this paragraph shall be satisfied by coverage under part 6 of subtitle B of title I or the Employee Retirement Income Security Act of 1974 (29 U.S.C. 1161 et seq.), section 4980B of the Internal Revenue Code of 1986, or title XXII of the Public Health Service Act provided to a subject of domestic abuse and is not intended to be in addition to any extension of coverage provided under such part, section, or title. (b) Limitation on Use or Transfer of Information.-- (1) In general.--An insurer or group health plan (or a contractor with an insurer or group health plan) may not use, disclose, or transfer information relating to an individual's abuse status, or medical condition which the insurer or plan knows or has reason to know is abuse-related, or an individual's family, household, social, or employment relationship with a subject of domestic abuse except to the extent necessary for the direct provision of health care services, compliance with abuse reporting laws, or (in the case of an insurer) compliance with an order of an entity with authority to regulate insurance or an order of a court of competent jurisdiction. Nothing in this paragraph shall be construed as limiting or precluding a subject of domestic abuse from obtaining the subject's own medical records from an insurer or group health plan. (2) Access to information by subject of domestic abuse.--A subject of domestic abuse may provide evidence of domestic abuse to an insurer or group health plan for the limited purpose of facilitating treatment of an domestic abuse-related condition or demonstrating that a condition is domestic abuse- related. Nothing in this paragraph shall be construed as authorizing an insurer or plan to disregard such provided evidence. SEC. 4. EXPLANATION OF REASONS FOR ADVERSE ACTIONS. An insurer or group health plan that takes any adverse action relating to any insurance policy or coverage under a group health plan of a subject of domestic abuse on the basis of a claim or medical condition that the insurer or plan knows or has reason to know is abuse-related, shall explain the reason for its action to the applicant or insured or individual in writing. Reference to general underwriting practices or guidelines does not constitute a specific reason. SEC. 5. SPECIAL RULE FOR LIFE INSURANCE. Nothing in this Act shall be construed to prohibit a life insurer from declining to issue a life insurance policy on the life of an individual if the applicant or prospective owner of the policy is or would be designated as a beneficiary of the policy, and if-- (1) the applicant or prospective owner of the policy lacks an insurable interest in the insured; or (2) the applicant or prospective owner of the policy is known, on the basis of police or court records, to have committed an act of domestic abuse in relation to the individual. SEC. 6. SUBROGATION WITHOUT CONSENT PROHIBITED. Except where the subject of domestic abuse has already recovered damages, subrogation of claims resulting from domestic abuse is prohibited without the informed consent of the subject of domestic abuse. SEC. 7. COMPLIANCE WITH INSURANCE PROTOCOLS FOR SUBJECTS OF DOMESTIC ABUSE. An insurer shall develop, file with the applicable regulatory authority, and adhere to, protocols specifying how employees, contractors, agents, and broker of the insurer will pursue an insurance action (including claims investigation and subrogation) that may impact the safety of a subject of domestic abuse involved with that action. SEC. 8. ESTABLISHMENT OF STANDARDS FOR INSURERS. (a) In General.--If, within the 90-day period beginning on the date of the enactment of this Act, the National Association of Insurance Commissioners adopts a Model Act and Regulations that establish standards for insurers with respect to the requirements under this Act, such standards shall apply to insurers in carrying out this Act. (b) Fallback Federal Standards.--If the NAIC does not adopt such an Act and Regulations within the period specified in subsection (a), the Secretary of Health and Human Services shall promulgate, not later than 60 days after the end of such period, standards for insurers to carry out this Act. SEC. 9. ENFORCEMENT FOR INSURERS. (a) State Enforcement.-- (1) In general.--Each State may establish under State law a regulatory program that provides for the application and enforcement of standards for insurers equal to or more stringent than the standards established under section 8 to carry out this Act. (2) Review.--The Secretary periodically shall review State regulatory programs to determine if they continue to substantially meet the requirements specified in paragraph (1). If the Secretary finds that a State regulatory program no longer substantially meets the requirements, before making a final determination, the Secretary shall provide the State an opportunity to adopt such a plan of correction as would permit the State regulatory program to continue to meet such requirements. If the Secretary makes a final determination that the State regulatory program, after such an opportunity, fails to substantially meet such requirements, the provisions of subsection (b) shall apply to insurers in that State. (b) Federal Fallback Enforcement.--In the case of an insurer, with respect to insurance policies issued in a State which does not have an approved regulatory program in effect under subsection (a)(1), that the Secretary of Health and Human Services determines fails to comply with an applicable standard established under section 8, the insurer is subject to a civil money penalty of not to exceed $25,000 for each such violation. The provisions of section 1128A of the Social Security Act (other than the first sentence of subsection (a) and other than subsection (b)) shall apply to a civil money penalty under the previous sentence in the same manner as such provisions apply to a penalty or proceeding under section 1128A(a) of such Act. SEC. 10. ENFORCEMENT WITH RESPECT TO GROUP HEALTH PLANS. The provisions of this Act insofar as they relate to group health plans shall be deemed to be provisions of title I of the Employee Retirement Income Security Act of 1974 for purposes of applying such title. With respect to group health plans, the Secretary of Labor shall enforce the requirements of this Act in the same manner as provided for under sections 502, 504, 506, and 510 of the Employee Retirement Income Security Act of 1974 (29 U.S.C. 1132, 1134, 1136, and 1140). SEC. 11. EFFECTIVE DATE. (a) In General.--Subject to subsection (b), the requirements of this Act shall take effect on July 1, 1997, or, in the case of insurers and if later, the date specified in subsection (b), and shall apply to actions occurring on or after such date. (b) Special Rule.--In the case of a State which the Secretary of Health and Human Services identifies as-- (1) requiring State legislation (other than legislation appropriating funds) in order for insurance policies to meet standards established under section 8 or for the State insurance commissioner to perform the functions described in section 9(a), but (2) having a legislature which is not scheduled to meet in 1997 in a legislative session in which such legislation may be considered, the date specified in this subsection is the first day of the first calendar quarter beginning after the close of the first legislative session of the State legislature that begins on or after the date of the enactment of this Act, and in which legislation described in paragraph (1) may be considered. For purposes of the previous sentence, in the case of a State that has a 2-year legislative session, each year of such session shall be deemed to be a separate regular session of the State legislature.

Victims of Domestic Abuse Insurance Protection Act of 1996 - Prohibits insurers and group health plans from engaging, directly or indirectly, in specified discriminatory acts or practices on the basis that the applicant or insured, or any person employed by the applicant or insured or with whom the applicant or insured has a relationship or association, or a beneficiary or plan participant, is, has been, or may be the subject of domestic abuse. Sets forth limitations upon the use or transfer of information among insurers or group health plans with respect to such subjects. Requires the insurer or group health plan to furnish a written explanation of any adverse action taken on a claim or medical condition that the insurer has reason to know is abuse-related. Prescribes parameters within which a life insurer may decline to issue a life insurance policy on the life of an individual if the applicant or prospective policy owner is or would be the designated beneficiary, and if such applicant or prospective policy owner: (1) lacks an insurable interest in the insured; or (2) is known in police or court records to have committed an act of domestic abuse in relation to the individual. Prohibits subrogation of claims resulting from domestic abuse without the informed consent of the subject of domestic abuse. Mandates that: (1) an insurer develop protocols specifying how employees, agents, and brokers will pursue an insurance action that may affect the safety of a subject of domestic abuse involved with that action; and (2) the Secretary of Health and Human Services promulgate standards for insurers to implement this Act if the National Association of Insurance Commissioners fails to adopt a Model Act that establishes standards to implement this Act. Prescribes guidelines for State and Federal enforcement of this Act, including a civil money penalty for violations.

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Background This background discusses (1) the distribution network for natural gas pipelines, (2) the key federal environmental laws that may be involved in the permitting process for these pipelines, and (3) the key stakeholders that may be involved in the permitting process. Distribution Network for Natural Gas Pipelines A compressor station is a facility that helps the transportation process of natural gas from one location to another. Natural gas, while being transported through a gas pipeline, needs to be constantly pressurized in certain distance intervals. The compressor station compresses the natural gas, thereby providing energy to move the gas through the pipeline. The gas in compressor stations is normally pressurized by special turbines, motors, and engines. Pipeline companies install compressor stations along a pipeline route, typically every 40 to 100 miles. transmission pipelines. PHMSA estimates that there are roughly 2 million miles of distribution pipelines, most of which are intrastate pipelines, in the United States. These pipelines are considered outside of FERC’s jurisdictional responsibilities. Federal Environmental Laws That May Be Involved in the Pipeline Permitting Process Several federal environmental laws and agencies may come into play in the permitting process for natural gas pipelines, depending on the proposed route for the pipeline. The principal laws involved include the National Environmental Policy Act, the Clean Water Act, the Endangered Species Act, and the National Historic Preservation Act. Pub. L. No. 91-190, 83 Stat. 852 (1970), codified as amended at 42 U.S.C. §§ 4321-4347 (2011). Intent in the Federal Register. The Notice of Intent acts as the formal announcement of the project to the public and interested federal, state, tribal, and local agencies. The lead agency is then required to determine the scope of the project. During this scoping process, the lead agency consults with resource agencies—such as the Corps or the Department of the Interior’s Fish and Wildlife Service (FWS)—to identify issues and alternatives to be analyzed in the EIS and allocate assignments for assistance in preparing the EIS. The lead agency will also identify other environmental review and consultation requirements under state, tribal, or local laws. Next, the lead agency prepares a draft EIS and solicits comments from the public; incorporates these comments into a final EIS; and issues a Record of Decision. Among other things, the Record of Decision—which is the final step for agencies in the EIS process—identifies (1) the decision made; (2) the alternatives considered during the development of the EIS, including the environmentally preferred alternative; and (3) plans to mitigate environmental impacts. Environmental assessment (EA). The lead agency prepares an EA when it is not clear whether a proposed project will have significant environmental impacts. An EA is intended to be a concise analysis that, among other things, briefly provides sufficient evidence and analysis for determining whether to prepare an EIS. If during the development of an EA, the lead agency determines that the proposed project will cause significant environmental impacts, the lead agency will stop producing the EA and, instead, produce an EIS. However, an EA typically results in a finding of no significant impact, and this finding is reported in a document that presents the reasons for the agency’s conclusion that no significant environmental impacts will occur when the proposed project is implemented. This finding is typically based on the use of mitigation measures. Categorical exclusion. The proposed pipeline project is classified as a categorical exclusion if a federal agency determines that the project falls within a category of activities that has already been determined to have no significant environmental impact. Under a categorical exclusion, the agency generally does not need to prepare an EIS or EA. NEPA regulations require federal agencies to make diligent efforts to involve the public in the preparation and implementation of NEPA documents. Under these regulations, agencies must provide a public comment period for a draft EIS; there is no corresponding requirement for an EA, but agencies may provide a public comment period. Clean Water Act.requirements of the Clean Water Act, one goal of which is to eliminate the addition of pollutants to waters of the United States. Section 404 of the Clean Water Act requires, among other things, that projects involving the discharge of dredged or fill material into waters of the United States must obtain a permit; this permit is typically issued by the Corps. Gas pipelines may involve such discharges when, for example, they are constructed within a riverbed, stream, or wetland. Additionally, pipeline construction may be subject to Section 402 of the Clean Water Act, which prohibits the discharge of pollutants into waters of the United States without a National Pollutant Discharge Elimination System (NPDES) permit. Pipeline construction is also subject to Section 401 of the Clean Water Act, which requires anyone seeking a permit for a project that may affect water quality to seek approval from the relevant state water quality agency. Pipeline projects may also be subject to many Endangered Species Act. agencies to ensure that any action they authorize, fund, or carry out is not likely to jeopardize the continued existence of a species listed as threatened or endangered under the act, or destroy or adversely modify its critical habitat. To fulfill this responsibility, the agencies must, under some circumstances, formally consult with FWS or the Department of Commerce’s National Marine Fisheries Service (NMFS) when the actions they authorize may affect listed species or designated critical habitat. Formal consultations generally result in the issuance of biological opinions by FWS or NMFS. The biological opinions contain a detailed discussion of the effects of the action on listed species or critical habitat and FWS’s and NMFS’s opinions on whether the pipeline company has ensured that its action is not likely to jeopardize the continued existence of the species or adversely modify critical habitat. In cases where a pipeline project as proposed is likely to either jeopardize the species or cause the destruction or adverse modification of its critical habitat, the opinions are to provide a “reasonable and prudent alternative” to avoid jeopardy or adverse modification that FWS or NMFS believes the pipeline company could take in implementing the action. Pub. L. 93-205, 87 Stat. 884 (1973), codified at 16 U.S.C. §§ 1531-1544 (2011). National Historic Preservation Act.Preservation Act (NHPA) requires federal agencies to take into account the project’s effect on any historic site, building, structure, or other object that is listed on the National Register of Historic Places. The Advisory Council on Historic Preservation oversees implementation of the Section 106 NHPA authority. In general, the advisory council delegates much of its authority under NHPA to state historic preservation offices. These offices identify historic properties and assess and resolve adverse effects on them under NHPA. Section 106 of the National Historic Rivers and Harbors Act of 1899. Harbors Act of 1899, projects such as pipelines that could affect navigable waters of the United States must receive authorization from the Corps. Specifically, the Corps regulates any work or structures in, over, or under navigable waters or any work that may affect the course, location, or condition of those waters. Stakeholders That May Be Involved in the Pipeline Permitting Process Pub. L. No. 69-560, 44 Stat. 1010; Pub. L. No. 71-520, 46 Stat. 918. FERC facilitate the timely development of pipeline projects.approves the construction of interstate pipelines by issuing a certificate of public convenience and necessity, which includes conditions that the pipeline company receive all required federal authorizations before beginning construction, if it has not already done so. FERC does not become involved in the permitting process for intrastate pipelines. Federal resource agencies. Federal resource agencies are responsible for managing and protecting natural and cultural resources such as wetlands, forests, wildlife, and historic properties. Virtually all applications for pipeline projects require some level of coordination with one or more of the following federal agencies, as well as others, to satisfy requirements for environmental review: The Advisory Council on Historic Preservation seeks to promote the preservation, enhancement, and sustainable use of the nation’s historic resources. For proposed natural gas pipeline projects, the Advisory Council on Historic Preservation reviews and provides comments on those pipeline projects that may affect properties listed or eligible to be listed on the National Register of Historic Places pursuant to the NHPA. The Bureau of Indian Affairs is responsible for, among other things, approving rights of way across lands held in trust for an Indian or Indian tribe. In addition, the Bureau of Indian Affairs must consult and coordinate with any affected tribe. The Bureau of Land Management (BLM) is principally responsible for issuing right-of-way permits authorizing natural gas pipelines to cross federal lands.federal agency, such as National Forest System lands, as well as BLM lands, BLM is responsible for issuing an authorization. When pipelines cross the lands of another The Corps has the authority to issue permits for the discharge of dredged or fill material into waters of the United States under Section 404 of the Clean Water Act. The Corps also has jurisdiction over structures or work in navigable waters of the United States under Section 10 of the Rivers and Harbors Act. If any activity could affect a federal project, such as a levee, dam, or navigation channel, permission from the Corps is required in accordance with Section 14 of the Rivers and Harbors Act of 1899. EPA is responsible for administering a wide variety of environmental laws. EPA’s responsibilities for the pipeline permitting process include commenting on EISs under the Clean Air Act; it also has the authority to participate in the Section 404 permit process. FWS is generally responsible for implementing the Endangered Species Act, among other laws, for freshwater and terrestrial species that may be affected by a pipeline construction project. The Forest Service is responsible for managing 193 million acres of National Forest System lands, through which many thousands of miles of natural gas pipelines cross. If a proposed pipeline crosses more than one federal agency’s lands, BLM issues a right-of-way permit. In cases where the pipeline only crosses National Forest System lands, the Forest Service issues a special- use authorization. NMFS implements, among other things, the Marine Mammal Protection Act and the Endangered Species Act for most marine species and anadromous fish (i.e., fish that spend portions of their life cycle in both fresh and salt water). State resource agencies. State-level agencies are generally responsible for managing and protecting a state’s natural and cultural resources. State resource agencies, such as state environmental or water quality agencies, as is the case with their federal counterparts, participate in and review assessments of environmental impacts in accordance with their responsibilities under federal or state laws. In some cases, federal agencies have delegated authority to state resource agencies for carrying out federal laws. Additionally, state historic preservation offices advise and consult with federal and other state agencies to identify historic properties and assess and resolve adverse effects to those properties under the NHPA. Tribal governments. As part of the planning and review process for pipeline projects, federal agencies engage in government-to- government consultation between American Indian Tribes and Alaska Native Corporations. Consultation is a deliberative process that aims to create effective collaboration and informed federal decision making. Tribal consultations can be a factor in the overall pipeline project schedule. Local governments. Local governments involved in natural gas pipeline projects may include counties or municipalities that are empowered by state law or constitution to carry out provisions to protect the environment or safety of local citizens. This may include requiring soil and erosion plans or zoning laws. Public interest groups. Public interest groups, such as Earthjustice, Delaware Riverkeeper, and the Pipeline Safety Trust, advocate for a number of issues, including the environment and public safety. They may comment on a proposed pipeline project during, for example, the NEPA process or any state processes that include public comment periods. Private citizens. Private citizens can provide comments and opinions in venues like public hearings. Like public interest groups, private citizens may comment on a proposed pipeline project during, for example, the NEPA process or any state processes that include public comment periods. The Interstate and Intrastate Pipeline Permitting Processes Can be Complex, with Multiple Stakeholders and Steps Both the interstate and intrastate pipeline permitting processes are complex in that they can involve multiple federal, state, and local agencies, as well as public interest groups and citizens, and include multiple steps. The interstate permitting process involves three key phases: a voluntary pre-filing phase, an application phase, and a post- authorization phase with multiple steps. According to stakeholders we spoke with, the interstate process is consistent because FERC acts as a lead agency in coordinating multiple stakeholders. The intrastate process can also include multiple stakeholders and steps. However, those stakeholders and steps vary from state to state, and most states do not have a lead agency coordinating the process. The Interstate Permitting Process Involves Three Phases, and Stakeholders Report It Is Consistent because It Has a Lead Agency We identified three key phases in the interstate permitting process for natural gas pipelines: pre-filing, application, and post-authorization. During these phases, federal, state, and local agencies, as well as public interest groups and citizens, may play a role in approving or commenting on the application for a permit to construct interstate pipelines. According to some industry representatives we spoke with, the interstate permitting process can be time-consuming, depending on the size and complexity of a proposed project, but it is consistent because FERC, as the lead agency, assists in coordinating with other stakeholders on the NEPA environmental analysis. In 2002, FERC established a pre-filing phase to facilitate and expedite the review of natural gas pipeline projects through early coordination with FERC and cooperating agencies (see fig. 1). The intent of this phase is to involve stakeholders sooner so that potential issues can be identified and resolved earlier, thereby taking less time overall. Use of this phase is voluntary, and FERC must approve a company’s request for pre-filing. For those projects that are less complex, such as those that do not involve federal lands, endangered species, or crossings of waters of the United States, applicants may choose not to use the pre-filing phase. According to FERC officials, in 2012, 67 percent of applicants for major interstate pipeline construction projects chose to use this phase. In the pre-filing phase, FERC and the applicant focus on gathering the necessary information for the environmental analysis, which may involve numerous federal, state, and local agencies and is typically the most complex and time-consuming step of the permitting process. Once FERC approves a company’s request to use the pre-filing phase for a project, agency staff notify other potential cooperating agencies that FERC has approved the use of the pre-filing phase and hold a planning or information meeting with the applicant and the agencies to discuss land and resource issues and concerns. FERC and the agencies also discuss the agencies’ ability to commit to an environmental review schedule. FERC will then work with the applicant and those agencies that are to have a role in the permitting process to initiate the NEPA scoping process—that is, the process of defining and refining the scope of an EIS or EA and the alternatives to be investigated—and begin the environmental analysis. Applicants are to hold “open house” meetings in the vicinity of the proposed project to share information about the project with the public. FERC staff often attends these meetings to answer any questions about the FERC permitting process and to invite the public to participate in the process at future dates. According to FERC’s website, applicants may incorporate proposed mitigation measures into the project design from comments received during these meetings. After these meetings, FERC will issue a Notice of Intent in the Federal Register for the preparation of an EA or EIS and seek additional public comments. FERC staff may also hold public scoping meetings for major projects that require an EIS or EA. Information given by the public during scoping meetings can help the company prepare environmental mitigation measures. According to industry representatives we spoke with, FERC’s pre-filing process was helpful at resolving potential problems earlier in the process, but other stakeholders said the pre-filing process is confusing and may limit public input. For example, one natural gas industry representative noted that the pre-filing phase has made the overall process less complicated. Another stated that it has resolved potential project “derailers,” such as issues with routing the pipeline through areas with endangered species, and has saved time for obtaining a permit. In addition, another industry representative said that early identification of stakeholders also increases coverage of potential resource impact issues so that appropriate surveys, mitigation practices, coordination with local and state requirements, and planning for habitat management or conservation can be coordinated with proposed project construction timelines. On the other hand, some state officials and representatives of public interest groups were more skeptical of the pre-filing phase. One representative of an environmental group said the public is unaware of the pre-filing phase and suggested that FERC and other stakeholders specifically reach out to environmental groups during the pre-filing phase if they want to resolve potential issues early in the process. However, another representative from an environmental group commended FERC for establishing an e-mail notification system that enables the public to sign up for e-mails on the progress of a specific project. Once pre-filing activities are completed or, if the applicant chooses to forgo the pre-filing phase, the applicant submits an application for a certificate of public convenience and necessity to FERC (see fig. 2 for steps in the application phase). FERC issues a Notice of Application, which includes the following: the unique number assigned to the project; the ways in which stakeholders, including the public, can become involved in the proceedings; and the methods for filing comments with FERC. There are several factors taken into account when FERC establishes a schedule for the environmental review, including the scope and complexity of the project, the requirements of any cooperating agencies, and the requested time frame of the applicant. Schedules may be adjusted if new concerns are identified, new information is introduced, or the number of comments received is greater than anticipated. However, FERC has no authority to enforce that schedule with cooperating agencies. FERC then analyzes the information in the application and begins the scoping process for those proposed projects that did not use the pre-filing phase or continues the scoping process for those proposed projects that did use the pre-filing phase. If a company did not use the pre-filing phase, FERC will begin the scoping process and consult with cooperating agencies to gather information. Next, FERC will issue a Notice of Intent to prepare either an EA or EIS. FERC, along with any cooperating agencies, will prepare either an EA or a draft EIS, depending on the potential environmental effects of the project. Cooperating agencies are responsible for assisting FERC in the preparation of the EA or EIS for those issues that fall within their jurisdiction. For example, if a project impacts waters of the United States, the Corps is likely to participate in the development of the EA or EIS because it is responsible for the regulation of activities in jurisdictional waters of the United States and would need to evaluate proposed impacts to those waters to inform a permit decision pursuant to its authorities under Section 404 of the Clean Water Act and/or Section 10 of the Rivers and Harbors Act of 1899. The environmental analysis incorporates the necessary information from all federal agencies in one document. While FERC may issue the certificate of public convenience and necessity before all federal permits, certificates, or authorizations are complete, it will not grant the authority to construct a pipeline without these federal authorizations. Pipeline companies must coordinate with the relevant agencies to ensure that these permits, certifications, and authorizations are completed. This may happen during the application phase or after FERC issues its certificate. Some states have developed written agreements with federal agencies that establish a process for carrying out their roles in consultation, review, and compliance with one or more federal laws. In some cases, state agencies have received the authority from federal agencies to implement federal laws and regulations. For example, the Clean Air Act gives EPA the authority to limit emissions of air pollutants, such as nitrogen oxides and methane, that result from constructing and operating natural gas compressor stations and pipelines. Such emission limits are established through a preconstruction permit issued by EPA, or, in some cases, by a state or local agency that has received authority from EPA to issue Clean Air Act permits in its jurisdiction. According to EPA, at least 75 percent of preconstruction permits are issued by state and local agencies, and EPA’s regional offices issue the remaining preconstruction permits. In areas where the state agency issues the clean air permits under the rules of their state implementation plan, EPA provides minimal oversight because the state is the permitting authority and therefore has primacy over decision making. In addition, state agencies may have delegated authority to process and issue federal Water Quality Certifications, required under Section 401 of the Clean Water Act, and Consistency Concurrences, under the Coastal Zone Management Act. Environmental permits issued by federal agencies can also vary by state or by region. For example, the Corps issues two types of permits to authorize activities under Section 404 of the Clean Water Act and Section 10 of the Rivers and Harbors Act of 1899: (1) individual permits, and (2) general permits. The type of permit used depends on the type and extent of proposed impacts on aquatic resources and whether a general permit is available to authorize such impacts. The Corps issues individual permits for specific projects that may have more than minimal impacts on aquatic resources, either individually or cumulatively, or are not otherwise authorized by general permits. The Corps issues general permits for activities resulting in no more than minimal adverse effects on the aquatic environment. The following three types of general permits are used for natural gas pipeline construction projects that require the discharge of dredged or fill material into waters of the United States and/or work or structures affecting the course, location, or condition of navigable waters: Nationwide permit. This type of general permit is intended to streamline and expedite the evaluation and approval process throughout the nation for certain types of activities that have only minimal impacts, both individually and cumulatively, on the aquatic environment. Activities that meet the terms and conditions of this type of permit, such as natural gas pipeline construction projects, are already authorized by the Corps. The Corps district verifies that the project meets the conditions outlined in the applicable nationwide permit. Corps headquarters, rather than one of the 38 district offices, issues these permits. However, one of the Corps’ eight division offices may add regional conditions to these permits in order to protect local aquatic ecosystems or to minimize adverse effects on ecologically critical areas or other valuable resources. Regional general permits. This type of permit authorizes activities that commonly occur in a particular region and that are expected to have a minimal impact on waters of the United States, but that do not warrant national authorization. Corps district offices issue this type of permit. Programmatic general permits. This type of general permit is established in those states or localities where there is a similar existing state, local, or other federal agency regulatory program. It is designed to avoid regulatory duplication. These types of permits may allow activities, including work in waters of the United States associated with pipeline projects, to have greater impact on waters than the nationwide general permits, provided there is still no more than minimal adverse effect on the environment. The programmatic general permit will identify those impacts that may be verified by the state or other entity with no review by the Corps, as well as any activities that may require notification to the Corps before verification is provided. Once the programmatic general permit is issued, the state or local agencies review proposed projects to verify that the proposed activities meet the terms and conditions of the permit, coordinating with the Corps’ district offices as necessary. Corps district offices receive annual reports from state and local agencies regarding the use of the programmatic general permits. Districts also retain the right to review any proposed project they determine may not meet the terms and conditions of the programmatic general permit. Most Corps districts primarily use nationwide permits to authorize work in waters of the United States in association with pipeline construction activities. Eight districts have developed regional general permits for certain activities, that may include pipeline construction, and six districts have developed state programmatic general permits. According to a Corps headquarters official, Corps districts may use different permitting mechanisms in different states to evaluate work in waters of the United States in association with pipeline projects. The regulations allow for this flexibility to account for regional differences in the aquatic environment, endangered species, historic sites, state regulations, or other factors. For example: In Pennsylvania, Corps district offices will generally rely on the Pennsylvania State Programmatic General Permit-4, under which the Pennsylvania Department of Environmental Protection verifies certain impacts that may occur in waters of the United States from the construction of some pipelines if the project meets certain criteria.According to Corps district officials, the Corps does not use a nationwide permit for these types of impacts because doing so would duplicate a similar state permit. Officials in the Corps’ Fort Worth district office said they typically use a nationwide permit to authorize work in waters of the United States in association with pipeline construction. Officials said they have not considered the use of a programmatic general permit because there are no similar permitting programs or authorizations required by the state of Texas. In Florida, Corps district officials issue both nationwide permits and regional general permits for work in waters of the United States in association with pipeline construction. Headquarters officials said the use of a programmatic general permit has not been considered because state regulatory processes are not similar enough to develop such a permit. In addition to coordinating with federal agencies on the environmental analysis, FERC may work with state resource agencies and local governments during the permitting of a natural gas pipeline. For example, an interstate natural gas pipeline project that runs through Pennsylvania would require several federal, state, and local permits, licenses, approvals, and certifications, as shown in table 1. However, some state and local actions are preempted—that is, they are superseded or overridden by federal law—because the actions conflict with federal law. For example, state certificates of necessity and convenience, which otherwise may be issued by state public utility commissions or other state agencies, are preempted because FERC’s certificate of public convenience and necessity supersedes the state’s. The process differs slightly depending on whether an EA or EIS is prepared, but in either case, FERC, acting as the lead agency, issues either a draft EIS or EA, and obtains public comments on the environmental analysis that was completed. FERC will respond to those comments, and issue its order either approving or denying the certificate of public convenience and necessity. According to representatives of one environmental group we spoke with, the public is not given sufficient time to intervene in the pipeline permitting process and often must hire attorneys to help them raise a motion with the agency because the process is complicated. According to representatives from several interest groups we spoke with, citizens are often unable to take these additional steps. However, FERC officials said that, while the agency establishes a deadline for timely motions to intervene, a motion to intervene can still be considered once the deadline has passed. Officials also said that an entity would be well-advised to file a motion to intervene as soon as possible. State officials we spoke with said that citizens are not well informed of the complicated interstate pipeline permitting process. Once FERC has issued a certificate of public convenience and necessity or denied an application, the applicant or the party to the proceeding can request that FERC rehear the case or take FERC to court over the outcome of the case. Otherwise, in order to proceed, the pipeline company must file an implementation plan with FERC including, but not limited to, how the company will implement any environmental mitigation actions identified in the environmental analysis, the number of environmental inspectors the company will assign to the project to ensure that mitigation measures are implemented, and procedures the company will follow if noncompliance occurs. FERC must give written authorization before construction can begin. Following that authorization, the pipeline company must file weekly status reports with FERC documenting inspection and compliance until all construction activities are completed. In addition, FERC is to regularly inspect the construction. Section 7 of the Natural Gas Act grants the right of eminent domain when FERC issues a certificate of public convenience and necessity; the pipeline company therefore has the right to acquire the property for that project by eminent domain if it cannot acquire the necessary land by agreement or if it cannot agree with the landowner on the compensation to be paid for the land. The Intrastate Permitting Process Varies by State, and Most States We Reviewed Do Not Use a Lead Agency If a new intrastate natural gas pipeline construction project does not cross a state border, then the responsibility for approval of pipeline routes falls to the individual states, and FERC does not play a role in siting the pipeline. The permitting process for these pipelines varies from state to state and may involve many federal, state, and local stakeholders. Unlike the interstate process, the intrastate process in most of the states we reviewed does not use a lead agency to authorize and coordinate siting and environmental reviews. As is the case with the interstate permitting process, pipeline companies must consider two issues when planning an intrastate natural gas pipeline: land acquisition and the need to identify the siting authority that oversees the location and route for that pipeline. To acquire rights to the land necessary to build the pipeline, pipeline companies will generally attempt to negotiate right-of-way agreements with individual landowners along the intended route. If negotiations fail, the companies may seek to acquire the land through eminent domain proceedings. There is no uniform standard for right-of-way agreements and eminent domain authority, and procedures vary by state. However, BLM will process permits for intrastate natural gas pipelines located on federal lands administered by the Bureau. Of the 11 states we reviewed, 5 have agencies charged with siting intrastate natural gas pipelines. These 5 states require advance approval of the location and the route of the pipeline. The remaining 6 do not have siting agencies that require advance approval of location and route. Table 2 shows these differences among the states we examined. As the table shows, the requirements of the application process differ from Florida—which generally requires state certification before constructing certain intrastate natural gas pipelines—to Texas, which does not require pipeline companies to obtain a permit to construct an intrastate pipeline and which gives natural gas utility pipeline companies statutory right of eminent domain without any prior state approval. According to public interest and industry group representatives we spoke with, the intrastate process for permitting and siting pipelines needs to be more transparent. In many states, it is difficult to determine the process for pipeline siting and whether the state has an agency with siting authority. They also told us that the intrastate process is challenging to navigate without an agency that takes the lead on siting and coordinating the environmental review, as FERC does at the interstate level. Additionally, representatives from two public interest groups we spoke with explained that it is more difficult for the public to comment on proposals for intrastate pipelines because the state processes are not transparent, and the public may not learn about pipelines until after they have been approved. The availability of eminent domain authority can also change how companies deal with land owners and, as a result, can change land owners’ perspective on the process as a whole, according to the public interest group representatives. Federal agencies become involved in the intrastate natural gas pipeline permitting process if federally protected resources have the potential to be affected by a project. For example, the Corps becomes involved when a proposed pipeline will be constructed in aquatic resources over which it has jurisdiction and FWS becomes involved if the route crosses an area with a plant or habitat on the federal list of threatened or endangered species. State environmental laws and regulations are applicable to intrastate pipelines. However, in 10 of the 11 states we reviewed, no single entity is responsible for coordinating all of the environmental reviews, including federal and state authorizations, during the intrastate permitting process. For example, in Rhode Island, the Energy Facility Siting Board is the authority for approving the siting and construction of natural gas pipelines; the pipeline company is responsible for obtaining all necessary permits, including all permitting and licensing under the jurisdiction of the state’s Department of Environmental Management. Conversely, the New York State Public Service Commission is the lead agency for the siting of intrastate natural gas pipelines. This department coordinates with other affected state agencies and local governments on the permitting process—one stop licensing. Time Frames for Interstate and Intrastate Pipeline Permitting Processes Vary Because of Multiple Factors For interstate pipelines, FERC’s public record information system contains documents that provide dates associated with the phases of the permitting process; however, FERC does not track the time it takes to complete the process. FERC officials said data on processing time frames is of limited use when planning a project because the variability among projects can make them incomparable. Using the information available on interstate natural gas pipeline projects certified from January 1, 2010, to October 24, 2012, we determined that the average processing time from pre-filing to certification for interstate natural gas pipeline projects was 558 days, and the processing times ranged from 370 to 886 days. These projects varied in size and function and included pipelines, pipeline expansions, compressor stations, and other pipeline facilities. For projects that begin in the application phase, the average processing time from formal filing to certification was 225 days for this period. The processing times for these projects, which tended to be for compressor stations and smaller pipeline expansions, ranged from 63 to 455 days. For intrastate pipelines, because the permitting process varies by state, the time frames for those processes may also vary. As is the case with interstate pipelines, time frames associated with permitting of intrastate pipelines may also vary because of differences in stakeholders, siting, and environmental factors and range in the amount of time to complete the permitting process. Some state agencies gave us estimates of time frames for specific parts of the process, but we found little comprehensive data on the intrastate permitting process in the states we reviewed. Comprehensive data are probably not available because most states do not have a lead agency that coordinates all the reviews necessary to complete the permitting process. For example, North Dakota state officials estimated that the siting part of the permitting process for intrastate pipelines takes just over 3 months; however, these 3 months do not include the time associated with any federal or state environmental reviews that may be necessary for pipeline projects. A New York state official estimated that the entire intrastate permitting process, including siting and all environmental reviews, takes 60 to 90 days for small pipelines, 3 to 6 months for medium pipelines, and 12 to 18 months for large pipelines. However, according to the official, these time frames vary depending on the complexity of the project and public opposition. The following factors can further affect the time frame for an interstate or intrastate pipeline project’s permitting process, as our stakeholders explained: Corps Section 404 Clean Water Act and Section 10 Rivers and Harbors Act permitting. The Corps does not have statutory deadlines or time frames for evaluating applications for natural gas pipelines or other types of regulated activities. However, the Corps has two performance measures specific to the timing of permit decisions. For standard individual permits, the Corps has a goal of completing its reviews and making permit decisions for 50 percent of permit applications within 120 days from receiving complete applications. In fiscal year 2011, the Corps reported that it had issued a decision on 71 percent of these applications within 120 days. The Corps has a goal of processing 75 percent of general permits within 60 days from receiving a complete request. In fiscal year 2011, the Corps reported that it had acted on 90 percent of these requests within 60 days. However, a headquarters official explained that the Corps collects information on time frames for reviewing applications and issuing decisions for all utility projects under Section 404 of the Clean Water Act and Section 10 of the Rivers and Harbors Act and does not separate data specific to natural gas pipelines from its reviews of other utility projects. According to Corps officials, application review can take longer for a number of reasons, such as the time it takes to receive all necessary information from the applicant and the time it takes for other agencies to complete decisions necessary for the Corps to finalize its review. For example, according to a Corps district official and Pennsylvania state officials, the Pennsylvania Department of Environmental Protection had, in recent years, a backlog of applications that delayed the transfer of applications to the Corps, but that backlog has been cleared. Pennsylvania officials said this backlog had probably occurred because the number of pipeline applications doubled since hydraulic fracturing of Marcellus Shale began in Pennsylvania. FWS and NMFS review under the Endangered Species Act. Federal reviews required under the Endangered Species Act can also affect time frames for the evaluation of natural gas pipeline projects. These projects can be permitted under the act in two ways. First, under section 7 of the act, federal agencies must ensure that any action they carry out (or actions of a nonfederal party that require a federal agency’s approval, permit, or funding) is unlikely to jeopardize the continued existence of a listed species or destroy or adversely modify its critical habitat. To fulfill this responsibility, federal agencies must consult with either FWS or NMFS (whichever agency has jurisdiction) when their actions may affect listed species or critical habitat. Formal consultations generally result in the issuance by FWS or NMFS of reports known as “biological opinions,” which discuss in detail the effects of proposed actions on listed species and their critical habitat, as well as that agency’s opinion on whether a proposed action is likely to jeopardize a species’ continued existence or destroy or adversely modify its critical habitat. The opinion also determines the quantity or extent of anticipated “incidental take”—that is, take that is not intentional but occurs nonetheless as a result of carrying out an agency action. FERC consults with FWS or NMFS under section 7 of the Endangered Species Act for the construction of interstate natural gas pipelines. For actions without a federal nexus (i.e., no federal funding, permit, or license), section 10 of the Endangered Species Act provides an avenue for entities to obtain permits for activities—such as the construction of a natural gas pipeline or a highway—that may result in the take of a listed species. An applicant for a permit is to submit a habitat conservation plan that shows the likely impact of the planned action; steps taken to minimize and mitigate the impact; funding for the mitigation; alternatives considered and rejected; and any other measures FWS or NMFS may require. According to representatives of an industry association we spoke with, their members report successful coordination of consultations under section 7 of the act because a federal agency, such as FERC for interstate pipelines and BLM for some intrastate pipelines, can assist the pipeline company in establishing long-term mitigation plans and other requirements for section 7 approval. The section 10 review process is less preferable, according to representatives, because the pipeline company is responsible for coordinating the relevant federal and state agency reviews and permits before the section 10 review is completed, which takes more time than a section 7 consultation. Delays in state and local government reviews. State and local permitting and review processes can take time and affect federal decision-making time frames because some federal agencies cannot issue their permits until state and local governments have completed their own permitting processes. For example, permits for federal programs delegated to states, such as section 401 of the Clean Water Act, can take time for state agencies to review and are needed for the Corps to issue an individual permit or verify a general permit. According to a Corps official and state officials, some states experience delays in completing these reviews. Overlap of federal, state, and local environmental processes. According to representatives of an industry association we spoke with, jurisdictional overlaps between federal, state, and local agencies force pipeline companies to obtain environmental permits or approvals from more than one level of government for the same activity. In some cases, the pipeline company must coordinate the pipeline route with the requirements for permits and reviews required by up to four different authorities at the federal, state, county, and municipal level. For example, these representatives stated, EPA’s regional office serving Alabama requires that ordinances be adopted to create a local construction storm water permitting program to regulate the same construction sites that the Alabama Department of Environmental Management already regulates under its statewide program. According to these representatives, natural gas pipeline projects throughout the state of Alabama are required to comply with the state issued general permit as well as overlapping permits for the same activities in any of the 67 counties and hundreds of small towns that their projects may pass through. These industry representatives reported project delays and resource allocation constraints because several layers of reviews and permits involving various federal, state, and local stakeholders often take place to address the same environmental issues for the same natural gas project. However, according to representatives of public interest groups we spoke with, efforts to combine federal, state, and local processes can undermine the opportunity for public comment. Incomplete applications. Officials in all of the Corps district offices that we spoke with reported that incomplete applications may delay their review because applicants need time to revise their information. Applications are considered incomplete for a variety of reasons. For example, the application may be missing jurisdictional information (i.e., where the waters of the United States are located relative to the project) or the applicant may miscalculate impacts. Officials from a state resource agency told us that environmental consultants, hired and given processing deadlines by pipeline companies, may submit incomplete applications in order to meet those deadlines. According to a Corps headquarters official, if applicants do not submit all of the appropriate documentation, the permit process may be delayed. Project opposition. Public opposition and litigation can lengthen the time needed to review a pipeline project or even lead to the cancellation of a project. For example, public interest groups can work with the public to request extended comment periods and public hearings for proposed natural gas pipeline projects that may adversely affect the environmental resources in the area. Natural Gas Pipeline Stakeholders Identified Management Practices to Improve the Permitting Process According to officials from federal and state agencies and representatives from industry and public interest groups we interviewed, several management practices could be implemented to help overcome some of the challenges of a complex permitting process identified by these stakeholders. These practices would help overcome the challenges involved in implementing an efficient permitting process and obtaining public comments on pipeline projects. In this regard, in March 2012, the president signed Executive Order 13604, which aims to institutionalize best practices and reduce the amount of time required to make permitting and review decisions for infrastructure projects, including pipelines. Stakeholders we spoke with and the administration, in its plan for implementing the executive order, identified the following management practices as effective, among others: Ensuring a lead agency is coordinating the efforts of federal, state, and local permitting processes for intrastate pipelines. Representatives from industry and public interest groups we interviewed noted that the interstate process is better coordinated than intrastate processes because FERC is designated as the lead agency for the environmental review of a pipeline project, but there is no similar lead agency in the intrastate permitting process. Representatives of a public interest group noted that the absence of a lead agency also makes it difficult for the public to become involved in the permitting process because citizens often do not know which agency to contact about a pipeline project. In that regard, in July 2001, the Interstate Oil and Gas Compact Commission and the National Association of Regulatory Utility Commissioners’ pipeline siting working group recommended that each state establish a coordinating effort within the governor’s office to monitor and assist in expediting the permitting process, while eliminating duplication of activities among state and local permitting entities. They further recommended that states identify all participants in the permitting process, consider naming a lead agency to monitor processing schedules within existing regulatory requirements, and determine information that needs to be communicated to the public. Ensure effective collaboration of the numerous stakeholders. Stakeholders we interviewed emphasized the importance of collaboration among the numerous stakeholders involved in the permitting process. Some federal officials noted delays occur in the permitting process when stakeholders do not collaborate effectively. For example, a federal agency’s permitting process may be delayed if it receives insufficient information from a cooperating agency. The federal plan for implementation of Executive Order 13604 identified several examples of best practices to enhance interagency coordination. Some federal agencies have memorandums of understanding or agreements with other agencies to establish collaborative relationships that relate to the permitting process. For example, as described earlier, FERC and nine other agencies signed an interagency agreement for early coordination of required environmental and historic preservation reviews to encourage the timely development of pipeline projects. FERC and FWS also have a memorandum of understanding that focuses on avoiding or minimizing adverse impacts on migratory birds and strengthening migratory bird conservation through enhanced collaboration. Providing planning tools to help companies plan routes for pipelines and avoid sensitive environmental resources. Industry representatives we spoke with noted that there is a need for technology tools that can aid in the proper routing of pipelines when companies are planning a project. Such tools should involve mapping software and best practices for specific areas of the country so that agencies do not need to reassess environmental impacts each time a company plans a project. These tools would also allow the project to be routed with the fewest environmental impacts at an early stage in the pipeline company’s design process. Without such tools, it is difficult for pipeline companies to route a project given the various federal, state, and local requirements that are not available in a single location. For example, FWS is currently developing such a tool—the Information, Planning and Conservation (IPaC) System—that is expected to let companies determine whether there are any endangered and threatened species in a potential project area and obtain information about the measures the companies can take to help protect and conserve those species when designing and constructing a project. This system is expected to help companies make better routing decisions early on, eliminating the need to modify project plans later in the permitting process. The federal plan to implement Executive Order 13604 selected IPaC as an example of a best practice to “reduce surprises and help project proponents make better informed design decisions early, when there is more flexibility to make minor modification with minimum disruption of the project goals.” Another planning tool that was mentioned as making the process more efficient by industry representatives was the Pennsylvania Natural Diversity Inventory Environmental Review Tool, which screens proposed projects to identify, avoid, or mitigate impacts on federal or state-identified threatened or endangered species. Industry representatives said this tool has been helpful to determine potential adverse impacts and plan mitigation. In addition, BLM designates pipeline corridors as part of its land use planning process. According to BLM officials, corridors reduce environmental impacts by allowing projects to share access roads and use previously disturbed areas. They also reduce the need for new data collection and land use plan amendments. Offering industry the option to fund contractors or agency staff to expedite the permitting process. Industry representatives said that many pipeline companies are willing to fund contractors or agency staff to speed up their application review process, which has slowed because of increasing numbers of energy projects and fewer agency resources. For example, stakeholders cited FERC’s practice of allowing applicants to fund a third-party contractor to review applications and assist the agency in preparing NEPA environmental documents. The third-party contractor is selected by and works under the supervision of FERC officials but is paid by the pipeline company. Other federal agencies have similar practices that allow applicants to offer funding assistance during the permitting process. A FWS official said this outside support is essential for agencies given the heavy workload and short time frames associated with pipeline projects. However, not all agencies have Congressional authority to accept funds. For instance, according to Corps officials, the agency cannot accept funds from private entities and can only accept funds from non-Federal public entities under specific circumstances. Increase the opportunities for public comments. According to representatives of some public interest groups and some state officials we interviewed, the public needs to have more opportunities to comment on a proposed pipeline project during the permitting process. A representative from one group observed that, while the typical NEPA process for public input allows the public to comment throughout the environmental review, FERC only offers a brief period for formal public comments. Representatives of other groups mentioned that, because the pipeline permitting process is complicated, it is difficult for the public to know when and how to comment and that additional information from the applicant, FERC, and states would be helpful. The implementation plan for Executive Order 13604 includes multiple best practice examples to improve outreach and education of the public. For example, the Department of the Interior is developing a web-based clearinghouse for environmental information on energy resource development. This clearinghouse is to provide environmental best practices, methods for conducting environmental assessments to aid in decision making, links to applicable federal and state laws related to energy development, and information on the various impacts of energy development projects. Agency Comments and Our Evaluation We provided a draft of this report for review and comment to the Departments of Agriculture, Defense, and the Interior; EPA; and FERC. The Department of Agriculture provided written comments in which they generally agreed with the overall findings of the report. The written comments are presented in appendix II of this report. The Department of Defense generally agreed with the overall findings of the report and provided technical or clarifying comments, which we incorporated as appropriate. The Department of the Interior and FERC provided technical or clarifying comments, which we incorporated as appropriate. EPA indicated that they had no comments on the report. We are sending copies of this report to the appropriate congressional committees; the Secretaries of Agriculture, Defense, and of the Interior; the Administrator of EPA; the Chairman of FERC; and other interested parties. In addition, the report is available at no charge on the GAO website at http://www.gao.gov. If you or your staff have any questions about this report, please contact me at (202) 512-3841 or ruscof@gao.gov. Contact points for our Offices of Congressional Relations and Public Affairs may be found on the last page of this report. GAO staff who made key contributions to this report are listed in appendix III. Appendix I: Objectives, Scope, and Methodology Our objectives for this review were to determine (1) the processes necessary for pipeline companies to acquire permits to construct interstate and intrastate natural gas pipelines; (2) information available on the time frames associated with the natural gas pipeline permitting process; and (3) stakeholder-identified management practices, if any, that may improve the permitting process. For purposes of this report, we consider the permitting process to involve steps companies need to take to obtain a permit, authorization, certificate, or approval from a federal, state, or local entity in order to construct a natural gas pipeline. To understand processes and permits required to construct natural gas pipelines at the federal level, we reviewed relevant federal laws and regulations, as well as agency documentation, such as the interagency agreement between the Federal Energy Regulatory Commission (FERC) and nine other federal agencies regarding their coordination during the review process for the National Environmental Policy Act and efforts to facilitate the development of natural gas pipeline projects. In addition, we reviewed literature on natural gas pipeline permitting issues and previous relevant GAO reports. We interviewed officials with regulatory responsibilities at FERC, the Army Corps of Engineers (Corps), the departments of Agriculture and of the Interior, and the Environmental Protection Agency. We also interviewed a range of other knowledgeable individuals—including representatives of public interest groups, such as the Pipeline Safety Trust and Delaware Riverkeeper Network; and representatives of industry groups, such as the American Gas Association and the American Petroleum Institute—whom we identified as having expertise related to the permitting of natural gas pipelines. To determine the processes for obtaining permits to construct natural gas pipelines at the state level, we selected a nonprobability sample of states for further review. We developed the following list of criteria to use as a tool for determining which states to include in our review: size of pipeline network (miles of pipe); amount of natural gas production (trillion British thermal units); amount of natural gas consumption (trillion British thermal units); natural gas inflow capacity (Million Cubic Feet per Day); natural gas outflow capacity (Million Cubic Feet per Day); recommendations from federal agency officials and other knowledgeable individuals. Because we anticipated that states differ in their pipeline permitting processes, it was important to include states that ranked both high and large on the selection criteria, as well as states that ranked are low and small. We selected states by identifying the top five and the bottom five of each selection factor. For example, in considering the size of the pipeline network, we identified the five states with the most miles of pipeline and the five states with the fewest miles of pipeline. We also identified the states that were of congressional interest, recommended by a federal agency, and/or other knowledgeable individuals we spoke with. The states selected in our review are those that were most frequently recommended and/or identified in our ranking process. We selected states that were recommended and/or identified in our ranking process at least four times to be included in our review. Twelve states were above this threshold—California, Colorado, Delaware, Florida, Louisiana, New York, North Dakota, Oklahoma, Pennsylvania, Rhode Island, Texas, and Vermont. Louisiana was later omitted from our review because of limited response from the state. For our selected states, we reviewed relevant documentation and conducted interviews with state agency officials and officials at Corps district offices in California, Florida, Pennsylvania, and Texas. Because our sample was a nonprobability sample, the information we obtained is not generalizable to all states but provides illustrative information. To identify the information available on the time frames associated with the natural gas pipeline permitting process, we conducted interviews with federal officials, industry associations, and public interest groups. In addition, we reviewed documents contained in FERC’s eLibrary, which is a record information system of electronic versions of documents issued and received by FERC on natural gas pipeline projects. FERC provided us with information on projects certified from January 1, 2009, to October 24, 2012. Owing to time and resource constraints, we limited our review to projects certified since January 1, 2010, and used eLibrary to access documents that contained information on the pre-filling date, traditional filling date, and certification date of these projects. In addition, we conducted interviews with FERC officials to determine the completeness of the documents contained in the system. We conducted this performance audit from May 2012 to February 2013, in accordance with generally accepted government auditing standards. Those standards require that we plan and perform the audit to obtain sufficient, appropriate evidence to provide a reasonable basis for our findings and conclusions based on our audit objectives. We believe that the evidence obtained provides a reasonable basis for our findings and conclusions based on our audit objectives. Appendix II: Comments from the Department of Agriculture Appendix III: GAO Contacts and Staff Acknowledgements GAO Contact Acknowledgments In addition to the individual named above, key contributors to this report included Karla Springer, Assistant Director; Pedro Almoguera; Cheryl Arvidson; Cindy Gilbert; Griffin Glatt-Dowd; Holly Sasso; Carol Herrnstadt Shulman; Barbara Timmerman; and Jeremy Williams.

Recent growth in domestic natural gas production, particularly due to increased production from shale, is resulting in an increase in the pipelines needed to transport that gas. Constructing natural gas pipelines requires clearing and maintaining rights-of-way, which may disturb habitat and historical and cultural resources. These resources are protected under a variety of federal, state, and local regulations implemented by multiple agencies. The laws, regulations and stakeholders involved in the permitting process depend on where the pipeline is constructed. FERC is the lead federal agency in approving interstate pipelines, coordinating with federal, state, and local agencies, but FERC is not involved in the approval of intrastate pipelines. In response to the Pipeline Safety, Regulatory Certainty, and Job Creation Act of 2011, GAO determined (1) the processes necessary to acquire permits to construct interstate and intrastate natural gas pipelines, (2) information available on the time frames associated with the natural gas pipeline permitting process, and (3) stakeholder-identified management practices that may improve the permitting process. GAO reviewed relevant laws and regulations and interviewed federal officials, state officials from a nonprobability sample of 11 states, and representatives from natural gas industry associations and public interest groups. GAO makes no recommendations in this report. The Departments of Agriculture and Defense generally agreed with the findings, and the other agencies had no comments Both the interstate and intrastate natural gas pipeline permitting processes are complex and can involve multiple federal, state, and local agencies, as well as public interest groups and citizens, and include multiple steps. The interstate process involves a voluntary pre-filing phase, an application phase, and a post-authorization phase with multiple steps that stakeholders reported to be consistent among projects because the process is led by the Federal Energy Regulatory Commission (FERC). FERC coordinates with federal, state, and local agencies that have statutory and regulatory authority over various environmental laws and regulations. For example, if a proposed pipeline may affect endangered species, FERC coordinates with the U.S. Fish and Wildlife Service, which reviews the impacts on such species. The intrastate process can also involve multiple stakeholders and steps, but, unlike in the interstate process, GAO found that the stakeholders and steps vary by state. For example, of the 11 states GAO reviewed, 5 have agencies charged with approving the route of natural gas pipelines and require advance approval of the location and route, and the remaining 6 do not. Pipeline companies must also comply with various federal and state environmental laws and regulations; however, in most of the 11 states, no one agency is charged with coordinating the implementation of these laws and regulations as FERC is for the interstate process. Time frames associated with the interstate and intrastate permitting processes vary because of multiple factors, according to stakeholders. For the interstate process, FERC does not track time frames, citing the limited usefulness of such data. GAO analyzed public records and found that, for those projects that were approved from January 2010 to October 2012, the average time from pre-filing to certification was 558 days; the average time for those projects that began at the application phase was 225 days. For the intrastate process, because processes vary by state, the time frames of those processes may also vary. GAO found little comprehensive data on the intrastate process. According to GAO's discussions with stakeholders, several factors can affect the time frame for the permitting process of a given project, including different types of federal permits or authorizations, delays in the reviews needed by governmental stakeholders, and incomplete applications. For example, state and local permitting and review processes can affect federal decision-making time frames because some federal agencies will not issue their permits until state and local governments have completed their own permitting processes, according to some stakeholders. Officials from federal and state agencies and representatives from industry and public interest groups told GAO that several management practices could help overcome challenges they associated with an efficient permitting process and obtaining public input: (1) ensure a lead agency is coordinating the efforts of federal, state, and local permitting processes for intrastate pipelines, (2) ensure effective collaboration of the numerous stakeholders involved in the permitting process, (3) provide planning tools to assist companies in routing pipelines and avoiding sensitive environmental resources, (4) offer industry the option to fund contractors or agency staff to expedite the permitting process, and (5) increase the opportunities for public comments.

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Everybody knows that Silicon Valley has become an economic powerhouse over the past quarter-century, but Houston’s boom is less appreciated. Joel Kotkin of Chapman University points out that over the past decade, Houston has outperformed every major metropolitan area in income growth, population growth and migration. Since 2000, the city’s employment figures have risen by 32 percent, ranking it No. 1 in percentage job growth. In August, Houston issued more single-family housing permits than all of California. The Bay Area and Houston share a strategic asset: engineers. The two regions rank first and second in the country in engineers per capita. Beyond that, they are thriving on the basis of very different growth models. Obviously, the Bay Area is driven by technology. Houston’s growth is driven by energy. More than 5,000 energy-related companies are located there. The Bay Area is a tightly regulated city. Houston has no formal zoning code, though, as the city gets more affluent, more rules are being written. The Bay Area is beautiful in the way urbanists like, while Houston is mostly ugly, in the way fast-food chains like. The Bay Area is densely populated and great for walking, while Houston is sprawling, though much of the development over the past few years has been high-density hipster infill. The Bay Area is the hands-down winner when it comes to creativity and charm. But it’s a luxury region, unaffordable and wildly unequal. Houston wins when it comes to livability, especially for people who want to have children. Kotkin, who has become an evangelist for the Houston model, points out that Houston is possibly the most ethnically diverse city in America. It’s more egalitarian than San Francisco. African-Americans and Hispanics there have high home ownership rates. Houstonians also enjoy a pretty high standard of living. If you take annual earnings per job and adjust it for the local cost of living, then Houston ranks top among major cities. Advertisement Continue reading the main story Over the past few years, liberals and conservatives have been arguing over which growth model is best. But, of course, there’s no need to choose. Both models are more or less working. What we’re seeing, it seems to me, is a profusion of economic growth models in different parts of the country — a net increase in economic pluralism and diversity. Perhaps even more than in the past, cities are specializing, turning into global hubs for a specific economic sector. This diversity is an enormous economic advantage for the country, and an enormous social and political challenge. As the country diversifies economically, it segments socially and politically. Each economic sector attracts different kinds of people and nurtures different kinds of values. The specialization of output means that every place becomes more like itself. First Draft Political news and analysis from the staff of The New York Times. Sign-up for free NYT Newsletters Morning Briefing News to start your day, weekdays Opinion Today Thought-provoking commentary, weekdays Cooking Delicious recipes and more, 5 times a week Race/Related A provocative exploration of race, biweekly Please verify you're not a robot by clicking the box. Invalid email address. Please re-enter. You must select a newsletter to subscribe to. Sign Up Receive occasional updates and special offers for The New York Times's products and services. Thank you for subscribing. An error has occurred. Please try again later. View all New York Times newsletters. In addition, as society gets more educated, it segments further. Educated people are more polarized politically than less educated people. Educated people are also more likely to move around and tend to move in with people like themselves. Over the past few decades, we’ve seen increases in residential segregation along political, income and cultural lines. As the years go by, politics more and more resembles these underlying divisions. I used to think that this was basically a centrist country and that political polarization was an elite phenomenon. But most of the recent evidence suggests that polarization is deeply rooted in the economic conditions and personal values of the country. Washington is not the cause of polarization; America is. The irony is that something good about America (economic pluralism) is contributing to something bad (segmentation and political trench warfare). Which more or less explains the midterm elections. The 2014 campaign has been the most boring and uncreative campaign I can remember. Democrats cry, “My Republican opponent is an extremist loon!” Republicans cry, “My Democratic opponent once shook hands with President Obama!” There’s not even a Contract With America, nor many policy suggestions of any sort. Most campaigns just remind preconvinced voters how bad the other party is. One result of the election is already clear. Political representation will more closely resemble the underlying social segmentation. Right now there are a lot of red states with Democratic senators. After this election, there will be fewer — probably between four and nine fewer. The election is about sorting people more tightly into their pre-existing boxes. People often compare this era to the progressive era — a time of economic transition with wide inequality and political rot. But that was an era of centralizing economic forces. This is an era of economic pluralism and political segmentation. People in San Francisco and Houston are achieving success while pursuing different economic models. It probably doesn’t make much sense to govern them intrusively from Washington as if they were engaged in the same project. It's not that there's no drama. It's that there's not much chance of big changes in national policy, no matter what happens. Pundits know two things about next month’s midterm elections. First, Republicans will likely take the Senate. Two, the elections stink. In a recent column, David Brooks declared, “The 2014 campaign has been the most boring and uncreative campaign I can remember.” Washington Post blogger Chris Cillizza responded with a post titled, “David Brooks thinks 2014 is the ‘most boring’ campaign he can remember. It’s even worse than that.” Brooks blames this dullness on the fact that Democrats and Republicans won’t change their minds. (“Polarization is deeply rooted in the economic conditions and personal values of the country.”) Cillizza blames it on the fact that both Democrats and Republicans can’t stand Washington. (“The American public are in a stage of disenchantment and anxiety that is historically unique.”) But the best answer I’ve seen comes from Ezra Klein, who argues that “elections are about stakes.” And the stakes in this one just don’t seem so high. At a micro-level, after all, the 2014 elections have plenty of drama. In Kentucky, the Republican leader in the Senate has been fighting for his political life all year. In Kansas, an entrenched incumbent could lose to a little-known independent who won’t say which party he’ll caucus with. It’s even possible we won’t know who controls the Senate until December or January, when Louisiana and Georgia hold runoffs. The dullness comes from this election’s lack of a compelling macro-theme. Yes, there are national refrains: Democrats in state after state call their Republican opponents heartless misogynists; Republicans call their Democratic opponents Obama clones. But there’s no big national issue on which voters feel that they can change the country’s course. It’s not that candidates today are more cynical or homogenized than in midterms past. It’s that the subjects they’re discussing cynically and homogenously don’t matter as much. A look at the last four midterms makes the point. In November 1998, Bill Clinton’s impeachment hung in the balance. After proving initially wary of the issue, in the campaign’s final week the National Republican Congressional Committee launched an advertising campaign in 30 swing districts aimed at rallying the GOP base. One spot showed two mothers asking each other, “What did you tell your kids?” But the ads backfired. The people most energized by the specter of impeachment were Democrats, especially African Americans. (It was the 1998 impeachment struggle that launched Moveon.org—as in “move on to something more important than impeachment.”) After expecting to win numerous House seats, the GOP ended up losing five. Speaker Newt Gingrich was forced to resign. Democratic Senator Robert Torricelli of New Jersey declared that as a result of the election, “Any serious effort to remove President Clinton from office is effectively over.” In 2002, the stakes were just as high. Roughly a month before Election Day, the House and Senate had voted to authorize President George W. Bush to invade Iraq. But because Bush had agreed to a last-ditch United Nations inspection effort, and because he was still seeking UN approval for military action, many still believed an invasion could be averted. The midterms were thus cast as a referendum on war. Like 2014, 2002 had plenty of micro-drama. After voting against the war, progressive hero Paul Wellstone died in a plane crash a week before Election Day. In Georgia, Republicans ran ads featuring Osama bin Laden and Saddam Hussein in an effort to depict Vietnam Veteran and triple amputee Max Cleland as soft on homeland security. But what made these local storylines nationally compelling was their connection to the still unresolved question of whether America would go to war. By 2006, America was in the middle of another fierce debate about Iraq: Not whether to get in, but whether to get out. In a Washington Post poll two weeks before Election Day, a plurality of Americans called Iraq their top concern. And in exit polls, 55 percent of voters said they wanted some or all U.S. troops withdrawn. As in 2002, the dramatic local stories—Senator Joe Lieberman’s loss to an anti-war challenger in the Connecticut Democratic primary, ex-Marine Jim Webb’s defeat of potential presidential contender George Allen in Virginia—connected to this national debate in a way the key races this year do not. As it turned out, the victorious congressional Democrats did not force George W. Bush to rapidly withdraw U.S. troops from Iraq. But the prospect that they might gave the election its passion. There's little reason a Republican Senate would substantially change American policy toward ISIS or Ebola. Finally, the 2010 midterms occurred in the wake of the greatest economic collapse since the Great Depression and the largest expansion of the American welfare state since the Great Society. And back then, far more than today, both these subjects seemed up for grabs. Many Republicans believed that by winning Congress in 2010 and then the presidency in 2012, they could repeal, or at least cripple, Obamacare. With the deficit skyrocketing, the nascent Tea Party warned that only by radically changing course could America avoid becoming Greece. The size and scope of federal intervention in the economy seemed to hang in the balance in a way it had not since the 1930s. All the dramatic races that year—Sharron Angle’s effort to unseat Majority Leader Harry Reid in Nevada, Marco Rubio’s driving Charlie Crist out of the Republican Party in Florida, Rand Paul’s shocking victory in Kentucky—connected to that theme. I remember watching MSNBC on election night as liberal pundits speculated about whether the barbarians now at the gates would force a debt default that sent the global economy into meltdown. For conservatives, it was enthralling. For liberals, it was terrifying. It certainly wasn’t boring. Today, these domestic-policy debates are more muted. Republicans still denounce Obamacare, but few still believe they can repeal it. Big partisan differences about the size of government remain, but with the deficit going down and Republicans no longer willing to go to the brink over the debt limit, the crisis atmosphere of 2010 has faded. Overseas, Americans worry about Ebola and ISIS, but those threats don’t divide them along partisan lines like Iraq. There’s little reason to believe that electing a Republican Senate would substantially change American policy toward either. Don’t get me wrong. I’m not saying next month’s elections don’t matter. They do. As Annie Lowrey has predicted, a Republican Senate—if elected—will work mightily to prevent President Obama from using his executive authority to implement a broad range of government regulation. But these potential fights are mostly too narrow and too technical to grab public attention. Americans just don’t believe that as much hinges on their vote as did in 1998, 2002, 2006, or 2010. For many pundits, that makes this election boring. For many ordinary Americans, I suspect, it’s something of a relief. Even Fed Chairman Ben Bernanke is bored with this election. (J. Scott Applewhite/AP) David Brooks thinks this election stinks. Here's how he described it in a column Tuesday: The 2014 campaign has been the most boring and uncreative campaign I can remember. Democrats cry, “My Republican opponent is an extremist loon!” Republicans cry, “My Democratic opponent once shook hands with President Obama!” There’s not even a Contract With America, nor many policy suggestions of any sort. Most campaigns just remind preconvinced voters how bad the other party is. Brooks is right. Having watched lots and lots of TV ads and talked to lots and lots of candidates, pollsters, media consultants and other political professionals over the last 22 (or so) months, it's become clear that at the center of this election is a whole lot of nothing. On the one side you have a Democratic party whose enthusiasm for the "hope" and "change" promised by Barack Obama six years ago has withered, and which hopes to hang on to its Senate majority by running away from him. On the other are Republicans who are entirely content to simply be the not-Obama party, trumpeting their plans to roll back some of his most controversial accomplishments but without any real plans of their own about what to do. That lack of ideas — new or otherwise — has created a remarkable sameness (and vanilla-ness) in the rhetoric being used by the two parties' candidates all over the country. Republicans like to remind voters how their Democratic opponent voted with Obama "99" or "97" or "93" percent of the time. Democrat after Democrat argues that Republicans want to rob women of their rights, roll back the clock to the "Mad Men" era and reward the wealthy on the backs of the poor. In virtually every high-profile race — no matter what state it is being held in or whether it's an open seat or a challenger taking on an incumbent — you hear variations on those attacks. And almost nothing else. It's a race to the bottom with the emphasis on tearing the other guy (or gal) down. Of course, there's some level of "same as it ever was" at work here. Campaigns — and especially midterms — don't tend to be the ones in which the two parties battle it out in the arena of "big" ideas. Back in 2006 — when Democrats won a sweeping victory in the House — they pushed a policy platform called "Six for '06" but, in truth, that was largely a set of platitudes, not a specific blueprint for what they would do if given control. Even the much-ballyhooed "Contract With America," which is widely credited with keying the Republican House takeover in 1994, was more of a broad policy statement than a set of specific ideas. (The entire "contract" runs a total of two pages.) But the thing that's different this time around is that the American public are in a stage of disenchantment and anxiety that is historically unique. Faith in institutions — from Congress to the presidency to the Supreme Court to big business — is at or near record lows. A majority of people believe the country is headed in the wrong direction — and have felt that way for years. Add it all up and you get an anxiety-filled electorate, anxious not only about the various challenges (threats?) we face in this country and around the world but also about the ability of our leaders, in politics and outside of it, to do anything about it. In a world of the Islamic State, an aggressive Russia, turmoil in the Middle East, Ebola fears, an economy not recovering as fast as anyone wants and a growing belief that the American Dream is dying (or dead), there's no one out there offering a vision of what the future could — and should — look like. The election is boring, sure. But it's more than that. It's vapid and inconsequential at a time when the world's challenges suggest a need for something more. We now live in an era of political smallness mismatched to the big-ness of the societal issues we face. It's no wonder everyone is so anxious about the future. It's as fuzzy as it's been in a very long time. Midterm elections rarely excite the general public, but 2014 is shaping up to be an especially underwhelming cycle for many Americans. With about a month remaining in the congressional races, 15% are very closely following news about the midterms — down from similar periods before the elections in 2010 (25%) and 2006 (21%). Our new survey, which was fielded Oct. 2-5, found that an additional 22% of the public said they are following the midterm election news fairly closely, 25% said not too closely, and 39% said not at all. As Pew Research has tracked midterm news interest throughout the year, attention to the elections consistently has lagged behind what it was four years ago. In eight surveys this year, news interest in the midterms has never topped 16% in a given week. So what’s behind the public’s collective shrug? Perhaps Americans have gotten used to the idea of partisan control of at least one chamber of Congress being on a knife’s edge. This time, it’s the Senate, where the Democrats’ majority is very much in jeopardy. In 2010, it was the House, which moved from Democratic to Republican control. Four years earlier, the Democrats wrested control of both the House and Senate from the GOP. Another possible explanation is that so many news stories outside electoral politics are capturing the headlines in recent days. Last week, other topics in the news captured more of the public’s attention, including the deadly spread of the Ebola virus in Africa and around the world (36%), U.S. airstrikes against ISIS (31%) and problems with the Secret Service (21%). Back in March, when we first asked this year, the public was more interested in the missing Malaysian jetliner, Russia’s annexation of Crimea and the continued rollout of the 2010 health care law. In general, interest in midterm elections — with no national races and only one-third of the states voting for the Senate — vastly trails that of presidential years. One month out from the 2012 presidential election, 45% of adults were paying close attention to the campaign; in 2008, even more (57%) were following news about the race for the White House. Though news interest isn’t as high as the last midterm cycle, other metrics show that some other attitudes of the public are similar to past midterm years. For instance, about half of registered voters (51%) last month said they had given “quite a lot” of thought to the coming elections, compared with 50% in 2010 and 45% in 2006. In that same survey, 40% of registered voters said they were more enthusiastic than usual about voting in this congressional election, which is between the 37% who said that in September 2006 and the 47% in October 2010. And with four weeks to go until most ballots are cast, there is still time for Americans to tune in. For one thing, local TV will have broadcast an estimated $2.6 billion of ads by the time the midterms are over. Topics: News Interest, Election News, 2014 Election Pew calls it the "Meh Midterm": with weeks left in the 2014 election, only about 15 percent of Americans are closely following news about the midterms. That's a lot lower than the (still pretty low) 25 percent who were closely following the news at this point in the 2010 midterm, or the 26 percent who were closely following the news at this point in the 2006 midterm. The finding isn't a fluke: "In eight surveys this year, news interest in the midterms has never topped 16% in a given week," reports Pew. Ouch. It's not the election's fault. The campaign itself is as fascinating and close as any in memory. Republicans are slightly favored to take the Senate — but Democrats could easily hold it. No one actually knows what will happen if Republicans cut Democrats down to 48 seats, but independents Greg Orman (Kansas) and Larry Pressler (South Dakota) win their elections —which they might! This buzzing uncertainty about the basic meaning of the election night results is rare. The 2014 election is doing its best to be interesting. Washington will still suck either way The problem is it can't change the basic post-election reality: Washington will still suck either way. Pew's results back my impression of Washington right now. The midterm isn't dominating the city in the way elections usually do. Ebola is a bigger issue, as is ISIS. And that, really, is the problem for this election: it doesn't seem likely to really change anything anyone cares about. Elections are about stakes. The 2006 midterm election was about the Democrats' promise — quickly broken, but still — to end the Iraq War. The 2010 election was about the GOP's promise to block President Obama's agenda and cut the deficit. Senate Democrats want to win, but they're not too worried about losing These elections felt consequential because they were consequential. Democrats really could have defunded the Iraq War if they won in 2006. Republicans really could — and did — freeze Obama's agenda after winning in 2010. But neither Republicans nor Democrats have been able to make the case that the plausible 2014 outcomes will change much. Democrats won't take back the House, and without the House, they can't kickstart Obama's agenda. Republicans might win the Senate, and that'll give them the leverage to do...what they're already doing. More to the point, perhaps, the 2014 election feels ephemeral. Senate Democrats want to win, but they're not too worried about losing. They note that they're defending only 10 seats to the GOP's 24 in 2016 — and doing so amidst the more favorable demographics of a presidential election. If Democrats lose the Senate by a seat or two this year, they're likely to take it back in 2016. And it's not as if much is going to get done in the meantime. A Supreme Court Justice could retire or die The easiest argument for consequence that either party has is a morbid one: a Supreme Court Justice could retire or die. In that event, the 2014 election may prove to be one of the most consequential in recent American history. But for some reason, no candidate has adopted "Four Supreme Court Justices are over 70!" as their campaign slogan. This is a cramped and shortsighted view of why elections matter. For one thing, nominations are still flowing through the Senate, and the federal bench will look rather different depending on whether Harry Reid or Mitch McConnell is Senate Majority Leader. For another, the crucial majorities that pass legislation are built over many elections. A seat Democrats lose in Colorado in 2014 might be the vote that President Hillary Clinton needed to pass universal pre-k in 2017. A seat Republicans lose in Kansas might be the seat President Mike Pence needed to secure a majority. The 2014 election, in other words, may not lead to much action in 2015, but it could prove decisive in 2017. You never know. But the people who aren't paying attention to the election probably aren't reading this column, either.

The consensus heading into crunch time of the midterm elections is that they're... boring. (See here and here, or check out a recent Pew poll showing the anemic level of interest among Americans-only about 15% are closely following things.) Midterms always lack the punch of a presidential election, of course, but this year seems worse than any recent one, writes Peter Beinart at the Atlantic. The problem, he argues, is that while 2014 has a number of dramatic races on the local level, nothing is grabbing people in more of a big-picture sense. "There's no big national issue on which voters feel that they can change the country's course." Go back to 1998, for instance, when the possibility of Bill Clinton's impeachment hung over the election. In 2002, the nation fretted about invading Iraq, and four years later, it fretted about leaving. In 2010, the economic collapse overshadowed all. But this year, "Americans just don't believe that as much hinges on their vote." Control of the Senate? Sure, "Democrats want to win, but they're not too worried about losing," writes Ezra Klein at Vox. Even if they do, "they're likely to take it back in 2016. And it's not as if much is going to get done in the meantime." People right now are worried about Ebola and ISIS, he adds, and that gets to heart of the problem of the 2014 election: "It doesn't seem likely to really change anything anyone cares about." Click for Klein's full column, or Beinart's.

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Background Contracts are generally considered to be physically complete once all option provisions have expired, the contractor has completed performance, and the government has accepted the final delivery of supplies or services. Physically completed contracts should then be closed within time frames set by the FAR—6 months for firm-fixed-priced contracts and 36 months for flexibly-priced contracts. The FAR prohibits the closing of contract files if the contract is in litigation, under appeal, or where the contract is being terminated and termination actions have not been completed. Flexibly-priced contracts take longer to close because additional steps must be taken during the closeout process; for example, audits on costs incurred and settlement of the contractor’s final indirect cost rates. Contracting officers and DCAA need to ensure that costs incurred by the contractor and charged to the government are allowable, allocable, and reasonable. On flexibly-priced contracts, contracting officers need to establish final indirect cost rates based on the contractor’s incurred costs. Indirect cost rates are a mechanism for establishing the proportion of indirect costs—such as a contractor’s general and administrative expenses—that can be charged to a contract. See figure 1 for the contract closeout process. Federal acquisition regulations require contractors to submit proposals that include information on all of their flexibly-priced contracts in a fiscal year. DCAA uses a checklist to determine whether a proposal is adequate, which, among other items, includes various cost schedules, subcontract information, and information on contracts that would be ready for closeout. DCAA may determine that a contractor’s incurred cost proposal is inadequate for a variety of reasons, such as incomplete or inaccurate information, and request that the contractor revise and resubmit the incurred cost proposal. This process may take several iterations before the proposal is deemed adequate. DCAA categorizes the proposals based on the total value of the proposal, called the auditable dollar value (ADV), which is the sum of all of the costs on flexibly-priced contracts for that contractor during the fiscal year. Figure 2 depicts key steps in the incurred cost audit process. There is not a one-to-one relationship between an incurred cost audit and an individual contract. In a single fiscal year, a contractor may incur costs on multiple flexibly-priced contracts, and all of these contracts would be included in the contractor’s proposal. Further, since the period of performance on an individual contract may span several years, an audit of each of the contractor’s incurred cost proposals for those years needs to be conducted to provide the information necessary to close one flexibly-priced contract. Prior Work by GAO and the Inspectors General Our prior work has highlighted some challenges at DOD in closing out contracts, as well as challenges at DCAA regarding incurred cost audits. For example, In September 2009, we reported on problems with DCAA’s audit quality and recommended DCAA improve audit quality guidance and develop a risk-based audit approach. DOD and DCAA generally agreed with the recommendations. As a result, DCAA required more testing and stricter compliance with government auditing standards that added staff time to complete audits. Additionally, as DCAA’s workload increased and resources remained relatively constant, auditors prioritized time-sensitive activities, such as audits to support new awards, and incurred cost audits were not completed, creating a backlog. In September 2011, we found that DOD’s ability to close the contracts it awarded to support efforts in Iraq and Afghanistan was hindered by several factors, including limited visibility into over-age contracts and DCAA workforce shortfalls. DOD concurred with our recommendations and revised its guidance on contract closeout in a contingency environment to require regular monitoring and assessment of the progress of closeout activities. In November 2011, we found that DCMA faced workforce challenges that caused delays in conducting timely quality assurance, audits of contractor processes, and contract closeout activities. Additionally, we found that DCAA had workforce challenges that affected its ability to conduct business system audits. We recommended that DCMA and DCAA identify and execute options to assist with audits and improve transparency of the current status of contractor business systems. DOD generally concurred with the recommendations and has initiated some actions to address them. In December 2012, we found that DOD components—including the Army, Navy, and Air Force—did not prioritize contract closeout and had limited data on the extent of their contract closeout backlog. We also reported that DOD’s efforts to close its large, flexibly-priced contracts were hindered by the backlog of DCAA’s incurred cost proposals. We recommended that DOD components establish baseline data and performance measures related to contract closeout. DOD concurred with our recommendations and the components have since established performance measures on contract closeout. The challenges faced by DOD in closing out contracts are not recent. In 2001, the DOD Inspector General issued a report that found weaknesses in the closeout process, including inadequate monitoring of contracts that could be closed, inattention to closeout requirements, and erroneous data about contracts available for closeout. Further, challenges in closing contracts are not exclusive to DOD. In recent years, the Inspectors General at several federal agencies, including the National Aeronautics and Space Administration (NASA), State, and the Department of Transportation, among others—have reported on the issues and challenges related to contract closeout. For example, In February 2014, the NASA Inspector General found that delays in closing contracts were due to the workload at DCAA, that some funds were not being deobligated in a timely manner, and that the closeout process was not uniform across the agency. For example, the Inspector General reported that contract personnel at the various NASA offices used different guidance when closing out contracts, which impaired their ability to share information and work across the agency; In November 2014, the Inspector General at State found that the agency did not have systems in place for tracking contingency contracts in Afghanistan nearing completion or which had funds that were expired or were available for deobligation. The Inspector General further found that State had not established comprehensive procedural guidance for contract closeout or ensured existing guidance was accurate for these contingency contracts in Afghanistan. According to State acquisition officials, State revised its policies and guidance regarding contract closeout. For example, the Foreign Affairs Handbook was updated to include procedures on how to address common difficulties in closing contracts; and In July 2015, the Inspector General at the Department of Transportation found that the agency had not implemented oversight procedures or performance measures on contract closeout to assess whether the components were complying with closeout requirements. Effective Management of Contract Closeout Varied Across the Selected Agencies Selected Agencies and Components Had Varying Levels of Contract Closeout Data and Some Established Related Goals and Performance Measures The five agencies and selected components we reviewed varied widely in ensuring that contracts were closed within the time frames prescribed by federal acquisition regulations. None of the five agencies we reviewed had all of the following: (1) centralized data on the number of contracts needed to be closed out; (2) information on where the contracts were in the closeout process; (3) established agency-wide contract closeout- related goals; and (4) established performance measures to assess progress toward achieving these goals. Most agencies delegated responsibility for contract administration, including closing out contracts, to their components. We found that some components within these agencies had at least three of these elements. For example, DCMA, which manages contract closeout for contracts that have been delegated to it, had each of these elements in place, while the Air Force, Navy, and Army each had contract closeout data and had established goals and performance measures, but lacked data on where contracts were in the closeout process. DHS also had information on the number of contracts eligible or overdue for closeout and had initiatives underway to reduce the number of low-risk, firm-fixed-priced contracts but did not have initiatives for higher risk contracts, including those involving flexibly-priced contracts. According to the federal standards for internal control, management should use quality information to make informed decisions and evaluate the entity’s performance in achieving key objectives and addressing risks. To help meet the FAR expected time frames for closing out contracts, using data on the number of firm-fixed-priced and flexibly-priced contracts that are eligible or overdue for closeout and where contracts are in the closeout process can help agencies identify where additional management attention is needed in order to close the contracts. Further, establishing goals and performance measures to assess progress toward achieving these goals can be an important tool in demonstrating leadership commitment. Having information on the scope of the issue and identifying the challenges to closing contracts could help agency-level management tailor approaches to specifically address causes as to why contracts remain open, notably if they are similar across the various components within the agency. In addition, establishing goals and performance measures ensure that sufficient management attention is paid to contract closeout. As shown in table 1, agencies varied in having each of these elements. A recurring issue highlighted in our prior work, as well as in this review, is that contract closeout was not a priority for either agency management or contracting officers. Agency officials and contracting officers noted the focus for contracting officers is to award contracts for the goods and services needed to support agency operations and missions, and that closing out contracts is largely viewed as an administrative task that staff get to when time is available. Further, agency acquisitions officials we spoke with on this review noted that their ability to focus attention on contract closeout was affected by resource constraints, including workforce challenges and sequestration. At the agency level, DOD has focused management attention on contract closeout, but does not have agency-wide data in place and does not have insight into the components’ goals and performance measures. In September 2014, Defense Procurement and Acquisition Policy established the Contract Closeout Working Group to improve and streamline the contract closeout process, including policy revisions and technology updates to its systems. DOD officials noted that while the department has limited insight as to the total number and value of contracts needing to be closed, it is the responsibility of the components and contracting offices to manage contract administration, including closeout. According to the federal standards for internal control, management should use quality information to make informed decisions and evaluate the entity’s performance in achieving key objectives and addressing risks—such as identifying improper payments and utilizing unspent funds elsewhere. DOD does not have the ability to track contract closeout centrally, and the components use a number of different contract management systems. The lack of insight into contracts that need to be closed, including where they are in the closeout process, hinders DOD’s oversight and ability to develop targeted approaches to address the causes as to why contracts remain open—especially if there are similar issues across the agency and could make it difficult to identify areas that may need improvement. Further, without DOD oversight and monitoring of the performance measures set at the component level, DOD will not be able to assess agency-wide progress in managing contract closeout. At the component level, we found that each of the DOD components we reviewed had data on the number of contracts to be closed and had goals and performance measures in place. However, with the exception of DCMA, the components were unable to track—and therefore address— challenges as to where contracts were in the closeout process, such as how many contracts were awaiting DCAA audits or needed action to be taken by their contracting staff. For example, Starting in 2013, the Army established a contract closeout task force in an effort to reduce over-age contracts. In fiscal year 2015, the Army set an overall goal of getting its over-age contracts down to 70 percent or below of the total contracts due for closeout. Further, the Army has established specific percentage goals for its contracting activities. According to the Army’s Contracting Enterprise Review for the third quarter of fiscal year 2017, only one out of the Army’s five contracting activities was on track to meet its fiscal year 2017 goal. Overall, the Army reported that it had a total of 231,627 firm-fixed- priced and flexibly-priced over-age contracts, which constituted about 86 percent of the total contracts due for closeout. The Army does not have the data broken out by contract-type. In January 2016, the Navy established a goal for each of its contracting activities of reducing the number of over-age contracts by 5 percent from the end of 2016 and 10 percent cumulatively for 2017. In May 2017, the Navy conducted its first annual review on over-age contracts with its senior leadership and reported that 6 of the 10 contracting activities met the 2016 goal. Overall, the Navy reported it had a total of 74,453 firm-fixed-priced and 10,637 flexibly-priced over- age contracts as of December 2016 across its 10 contracting activities, including the Marine Corps. In 2015, the Air Force established a goal to eliminate its over-age contracts by fiscal year 2020. To do so, the Air Force established a goal for fiscal year 2016 of reducing the number of contracts needing to be closed out by 10 percent; in fiscal year 2017, the goal rose to 20 percent. The Air Force reported that for fiscal year 2016, the 10 percent reduction goal was met for firm-fixed-price contracts. For flexibly-priced contracts, however, the Air Force reports two categories of contracts—“cost” and “other”—and reported that it had a percentage increase of over-age “cost” contracts from 69 percent to 72 percent and an increase for “other” contracts from 82 percent to 85 percent. As of June 2017, the Air Force reported 33,844 firm-fixed- priced and a combined total of 21,036 flexibly-priced contracts due for closeout across its 15 contracting activities. For firm-fixed-priced contracts, DCMA established a goal of reducing its over-age contracts by 50 percent in fiscal year 2016, which it met. DCMA further established a goal that it would have no more than 869 over-age firm-fixed-priced contracts after fiscal year 2017 and no more than 350 after fiscal year 2018. For flexibly-priced contracts, DCMA established a goal from fiscal year 2016 through fiscal year 2020 to reduce its over-age flexibly-priced contracts by an additional 20 percent each year. As of March 2017, DCMA reported that it had 70,322 firm-fixed-priced and flexibly-priced over-age contracts and is generally on track to meeting its goals for both firm-fixed-priced and flexibly-priced contracts as of May 2017. DLA runs monthly reports of contracts that are recommended for closeout, and, in January 2017, DLA acquisition officials reported that 784 firm-fixed-priced contracts were recommended for closeout. Most of DLA’s contracts are firm-fixed-priced contracts, with few flexibly- priced contracts. DLA has instituted a shorter goal for closing out a firm-fixed-priced contract—within 120 days—as opposed to the FAR time frames of 180 days of contract completion. DLA officials stated that the agency consistently meets this goal, having approximately 99 percent of its contracts closed within the shorter time frame. According to DLA acquisition officials, management attention over the last 2 years resulted in a reduction of the number of contracts in the backlog, with the intention of preventing another backlog from reoccurring. DOD officials noted that DOD has several department-wide initiatives to help components address their contract closeout backlogs. For example, in December 2013, DOD implemented a policy change, increasing the obligation threshold from $150,000 to $500,000 for contracts that could qualify for automatic closeout. To qualify, contracts must be under $500,000, firm-fixed-priced, and not have certain contract clauses that require contracting officers to take action, such as patents. For contracts meeting these criteria, DOD systems automatically closed these contracts. In August 2015, DOD added a contract closeout module in one of its data systems to identify and automatically close contracts that were not covered by other automated closeout processes. This initiative leverages implementation of the Procurement Data Standard across the various DOD contract writing systems to improve visibility and accuracy of contract-related data needed to determine whether automated closeout can occur. DOD reported that over 12,000 contracts were closed in fiscal year 2016 across the department using the new module. DOD is also working to ensure that contracts closed in its contract writing systems, for example the Standard Procurement System, are reflected as closed in other data systems, such as Electronic Document Access. Further, DOD awarded a contract with the AbilityOne program in 2010 for contract closeout support services. The AbilityOne program provides career opportunities for people who are blind or have severe disabilities, including service-disabled veterans. The program also trains and employs wounded veterans to support contract closeout activities. In September 2015, DOD awarded a follow-on 5-year contract to AbilityOne to provide continued contract closeout support services. The contract has a not-to- exceed value of $75 million. DOD officials stated that since it started, more than 317,000 contracts—across the various DOD components— would be closed through the AbilityOne contract as of May 2017. Department of Health and Human Services HHS management does not have information on the extent the agency has contracts due for closeout or where contracts are in the closeout process. Having such information could help the agency in its oversight of contract closeout by identifying and addressing the causes as to why contracts remain open—such as determining if the issues affecting contract closeout are similar across the components. According to HHS acquisition officials, this information is managed at the component-level. While there is value in components tracking and managing their own progress, HHS will not be able to assess agency-wide progress in managing contract closeout without oversight and monitoring of the performance measures set at the component level. According to the federal standards for internal control, management should use quality information to make informed decisions and evaluate the entity’s performance in achieving key objectives and addressing risks—such as recovering improper payments or identifying unspent funds for use elsewhere. While various HHS components reported that they are taking various actions to address contract closeout, such as establishing goals and performance measures to track their progress in closing contracts, we focused on CMS—which accounted for about 30 percent of contract dollars in fiscal year 2015 for HHS. CMS tracks the number and type of contracts that are overdue for closeout. According to senior CMS acquisition officials, in October 2014 they established a closeout goal of closing 2,250 contracts per year. In fiscal year 2016, CMS surpassed this goal and closed 2,831. As of June 2017, CMS had already met this goal for fiscal year 2017, closing 2,653 contracts and reported that it had 2,244 firm-fixed-priced and 2,867 flexibly-priced over-age contracts that still needed to be closed. In addition, CMS also issues monthly reports on contracts due for closeout that are shared among the various CMS offices. CMS acquisition officials stated that having management-level attention had a positive effect in identifying and addressing issues affecting contract closeout. Our review found that DHS management made a commitment to address contract closeout challenges, gained insight into the extent of its contract closeout backlog, and has initiatives underway to address at least a portion of its contract closeout backlog. It has not, however, established goals and performance measures to assess its progress in reducing its contract closeout backlog. To respond to a material weakness identified in the agency’s 2014 annual Financial Report, senior DHS management initiated an effort in March 2016 to identify the extent of the department’s contract closeout backlog. This effort, jointly led by the Chief Procurement Officer and the Chief Financial Officer, used DHS’s contract reporting system to pull data from FPDS-NG and identify contracts that had a period of performance end date that had elapsed beyond 6 months for firm-fixed-priced contracts and beyond 36 months for flexibly-priced contracts. According to a March 2016 memorandum, DHS estimated that it had approximately 382,000 over-age contracts—those that were beyond the FAR set time frames for closeout. DHS determined that 352,000 (about 92 percent) of the over- age contracts were considered “low-risk” contracts—contracts awarded using simplified acquisition procedures or firm-fixed price contracts with the remaining 30,000 being flexibly-priced contracts. DHS has ongoing efforts to address over a quarter of its low-risk, firm- fixed-priced contracts. From the list of 352,000 low-risk contracts, DHS financial management officials identified 5,695 over-age firm-fixed-priced contracts with unliquidated obligations of $50,000 or less. DHS officials then verified the list of these contracts with their components’ acquisition and financial management staff to verify the unliquidated obligation amounts and confirm that the contracts were ready for closeout. DHS published the list of verified contracts in a October 2016 Federal Register notice, requested contractors to submit any outstanding invoices associated with these contracts within 60 days of publication, and, if it did not receive any outstanding invoices, indicated that it planned to close the contracts. DHS acquisition officials estimated that by August 2017 about 100,000 (about 28 percent) low-risk, over-age contracts would be closed through this effort. According to the DHS officials, the initiative was focused on closing older contracts where the funds have already expired and did not collect information on the amount of funds that were deobligated off of these over-age contracts. Further, DHS acquisition officials stated that they received feedback from DHS components that the effort was helpful in reducing some of the administrative paperwork that allowed them to close these low-risk contracts. DHS officials also stated that in fiscal year 2015, they implemented a separate initiative to address unliquidated obligations that targeted contracts in each DHS component with the highest amount of unliquidated obligations, which resulted in a deobligation of $164 million from those contracts. According to the DHS officials, they also have several efforts related to flexibly-priced contracts, including efforts to coordinate with DCAA on the status of audits, developing a tool to track the audits and providing additional guidance and training to close the contracts. DHS has not, however, established goals and performance measures to assess the department’s overall progress in reducing the total number of firm-fixed-priced or flexibly-priced contracts that need to be closed. Further, DHS does not have insight—either at the agency-level or the component-level—as to where these contracts are in the closeout process that would help identify where there are challenges in the process. This hinders the agency’s ability to target its approaches to address the causes as to why contracts remain open and could make it difficult to identify areas that may need improvement. Additionally, without goals and performance measures, DHS officials will not be able to track progress agency-wide on closing contracts over time. According to the federal standards for internal control, management should use quality information to make informed decisions and evaluate the entity’s performance in achieving key objectives and addressing risks—such as recovering improper payments or identifying unspent funds for use elsewhere. DOJ management does not have agency-wide information on its contracts that are eligible or overdue for closeout. Having such information could help the agency in its oversight of contract closeout by identifying and addressing the causes as to why contracts remain open. The Deputy Assistant Attorney General for Policy, Management, and Planning, who also serves as DOJ’s Senior Procurement Executive, is responsible for implementing agency-wide procurement policy and other management initiatives. DOJ acquisition officials told us, however, that DOJ is decentralized, and it is up to each bureau to manage contract closeout—including the implementation of policies, monitoring closeout efforts, as well as establishing any goals and performance measures. While there is value in components tracking and managing their own progress, DOJ will not be able to track the department’s overall progress across the agency on closing contracts or determine if the issues affecting contract closeout are similar across the components and address them at an agency-wide level without information on the number and type of contracts that need to be closed, as well as goals and performance measures. According to federal standards for internal control, management should use quality information to make informed decisions and evaluate the entity’s performance in achieving key objectives and addressing risks—such as recovering improper payments or identifying unspent funds for use elsewhere. The three DOJ components that we reviewed had varying levels of information on their contract closeout backlog. For example, senior FBI acquisition officials told us that while the FBI has procedures in place for contract closeout and for removing unliquidated obligations, the FBI does not have management-level oversight on contracts that need to be closed. According to the FBI acquisition officials, the FBI does not centrally track contract closeout information and would have to go to individual contracting offices for information. The FBI accounts for about 22 percent of DOJ’s contract dollars. BOP, which accounted for about 36 percent of DOJ’s contract dollars in fiscal year 2015, generally lacked centralized information on the status of contracts needing to be closed out. ATF, which accounted for about 3 percent of DOJ’s contract dollars in fiscal year 2015, identified 58 firm-fixed-priced contracts that had a total of approximately $4.3 million dollars in unliquidated obligations that needed to be closed out. ATF was one of the first bureaus in DOJ to implement DOJ’s Unified Financial Management System (UFMS). According to ATF acquisition officials, they use UFMS to identify contracts that need to be closed based on the elapsed period of performance date and if the contracts have unliquidated obligations. In addition, ATF has two staff dedicated to closing contracts and senior ATF acquisition officials meet quarterly to discuss the progress of contracts due for closeout. ATF does not have the ability, however, to use UFMS to identify contracts that do not have unliquidated obligations. Further, FBI, BOP, and ATF do not have specific goals and performance measures in place. Having agency-wide information on contracts due for closeout could help DOJ in its oversight of contract closeout by identifying and addressing challenges that could be similar across its components. State management does not have information on the extent of contracts that are eligible or overdue for closeout across the agency or where the contracts are in the closeout process. Having such information could help the agency in its oversight of contract closeout and address challenges at an agency-wide level. Further, State has not established goals and performance measures to assess its progress in reducing its over-age contracts. While State does not have information on the total number of contracts due for closeout, in 2009 it established a contract closeout team that tracks contracts for which it provides closeout support at the request of contracting officers. As of November 2016, the contract closeout team was working on 128 contracts due for closeout. State acquisition officials stated that they are working to improve their ability to track when newly awarded contracts become eligible for closeout. In October 2016, State implemented a pilot to identify contracts based on the period of performance end date to identify the number of contracts ready for closeout on a quarterly basis. The pilot added new data fields into its system for contracts awarded or modified since October 2016. This is intended to help contracting officers monitor their contracts and move forward in the closeout process. The pilot ended in April 2017, and State acquisition officials expect full implementation within 18 months. While this initiative can have positive outcomes if implemented as planned, it does not pertain to contracts awarded prior to October 2016. For those older contracts, the new fields will not be applicable, limiting State’s insight into those contracts. The lack of information on the full scope of contracts that need to be closed and where they are in the contract closeout process, coupled with the absence of goals and performance measures, means that State will not be able to track its progress across the agency on closing contracts over time. Further, without this information, it could hinder the agency’s ability to target its approaches to address the causes as to why contracts remain open and make it difficult to identify areas that may need improvement. According to the federal standards for internal control, management should use quality information to make informed decisions and evaluate the entity’s performance in achieving key objectives and addressing risks—such as recovering improper payments or identifying unspent funds for use elsewhere. Selected Civilian Agencies’ Responses to Prohibition on DCAA Conducting Incurred Cost Audits for Non-Defense Agencies While DOD is generally required to use DCAA for contract audit support services, DCAA also provided these services to civilian agencies— including DHS, State, and HHS—on a reimbursable basis. As noted previously, the NDAA for Fiscal Year 2016 included a provision prohibiting DCAA from performing audit support services on behalf of other federal agencies until DOD certified that DCAA had reduced its incurred cost audit inventory to below 18 months. Starting in January 2016 DCAA notified the civilian agencies for which they had planned to do reimbursable work in fiscal year 2016 that it would no longer be able to perform audits for them until it met the statutory requirements. This affected approximately 500 audits that DCAA had planned to perform. DCAA had to coordinate with the agencies that it does work for to determine if DOD had audit responsibility over certain contractors. For contractors that did not fall under DOD cognizance, the other agencies had to identify alternate means to meet their contract audit needs. Several of the agencies we reviewed took actions to obtain audit services. For example, DHS established blanket purchase agreements with private auditing firms to conduct incurred cost audits for its contractors, while State issued orders off of an existing one. DHS acquisition officials stated that since the vast majority of DHS’ contractors are also DOD contractors, the agency intends to continue to rely on DCAA for audits where it is already performing efforts for DOD and can provide a timely response. In instances where DCAA services are not available or if DCAA cannot provide a timely response, DHS plans to use the private firms for contractors for which they have cognizance. As of July 2017, DHS had not awarded any orders off of its blanket purchase agreements. State awarded four orders off of an existing blanket purchase agreement for incurred cost audit support from a private accounting firm. State acquisition officials stated that they received two incurred cost audit reports and will begin the process of negotiating rates with the contractor. They also stated that that they will continue to use DCAA because DOD has cognizance over many of its contractors. HHS officials stated that some of their components use DCAA for incurred cost audits, but others are using alternate options such as conducting the audit work internally or contracting out to private firms for audit support services. Further, in April 2016, the Office of Management and Budget sent out a survey to federal agencies to gauge the effect of the DCAA prohibition and determine the agencies’ audit needs. The survey determined that there was enough of a need for contract audit support services that, in August 2016, the Federal Aviation Administration—in coordination with the Office of Management and Budget—led a civilian agency working group to address this gap. Since then, the working group conducted market research to identify the extent to which civilian agencies relied on DCAA or private sector providers to perform financial audits on their behalf, the extent to which the private sector can address the need for contract audit support services, and potential contract approaches to meet the needs of civilian agencies. For example, the working group determined that federal agencies spent about $100 million annually on contract audit support services, either through reimbursable work performed by DCAA or through contracts awarded to private accounting firms. The working group is working with the Office of Management and Budget and the General Services Administration on a contract solution that maximizes existing contracts already available across multiple agencies using the General Services Administration’s Federal Supply Schedules program. Further, the working group is preparing an ordering guide to assist agencies with placing contracts for contract audit related services. The guide, expected to be completed by August 2017, will also identify best practices to address concerns regarding the quality of audits. DCAA Made Progress in Reducing Its Incurred Cost Audit Backlog, but Opportunities Exist for Improvement DCAA made progress in reducing its inventory of incurred cost proposals awaiting audit by about half since fiscal year 2011 and has closed more than three-quarters of its oldest proposals—those submitted for years prior to fiscal year 2014. This reduction was due to several initiatives that DCAA implemented in recent years, such as risk-based sampling, conducting multi-year audits, and dedicating more staff resources to conduct incurred cost audits. DCAA did not, however, meet its goal of having 2 years of incurred cost proposals in its inventory by fiscal year 2016 and may not be able to meet its revised goal to do so by the end of fiscal year 2018. Further, our work identified two areas in which DCAA may be missing opportunities or currently lacks information to help identify additional ways to reduce its inventory. These areas include: (1) assessing actions for reducing the amount of time it takes for DCAA to begin an incurred cost audit and establishing related performance measures to assess its progress and (2) evaluating the use of multi-year auditing and establishing related performance measures. DCAA Reduced Total Inventory, Including Its Backlog of Incurred Cost Proposals Awaiting Audit DCAA has reduced its overall inventory of incurred cost proposals awaiting audit from about 31,000 in fiscal year 2011 to about 14,000 as of the end of fiscal year 2016. Over that same time period, DCAA reduced what it characterizes as its backlog of old incurred cost proposals—those proposals submitted for fiscal year 2013 and prior—from 21,000 to below 5,000. DCAA did not, however, meet its original goal of having a 2-year inventory of audit proposals—eliminating its backlog of proposals older than 2 years—by fiscal year 2016 and acknowledged that meeting its revised goal to do so by the end fiscal year 2018 will be challenging. DCAA policy officials stated that they were unable to meet the goal of eliminating the backlog due to resource constraints, including workforce challenges, such as hiring freezes. Overall, as of the end of fiscal year 2016, DCAA’s total inventory included 14,208 incurred cost proposals, representing approximately $825 billion in auditable dollar value (ADV) (see figure 3). DCAA attributes its progress in reducing its total inventory as well as its backlog of incurred cost proposals awaiting audits to various efforts, such as management attention in prioritizing incurred cost audits, as well as two specific initiatives—the implementation of a risk-based approach to identify proposals for audit and multi-year audits, in which multiple proposals are done under a single audit. DCAA has reduced its inventory primarily through the use of a risk-based approach to conducting audits. Under this approach, DCAA focused its resources on conducting audits of proposals that it deemed high-risk or exceeded $250 million in ADV. According to DCAA policy officials, DCAA auditors are supposed to make the risk assessment concurrently when determining that a proposal is adequate. Factors that DCAA considers when conducting a risk assessment include whether a specific risk was identified by an external source—such as a contracting officer—or the audit team has identified a specific risk that has a material impact to the proposal being assessed, business system deficiencies, and prior audit experience with the contractor, among others. DCAA officials stated that DCAA audits all proposals that are deemed high risk regardless of ADV. As of the end of fiscal year 2016, DCAA’s data indicates contractors had submitted 9,309 incurred cost proposals that were either deemed adequate by DCAA or were awaiting an adequacy review. DCAA reported it had made a risk assessment on 8,426, or about 91 percent, of those proposals. DCAA policy officials stated that several factors contributed to the gaps on the status of proposals, such as instances where audits on proposals for earlier years for a contractor are ongoing and DCAA would need to consider the results of those audits when assessing risk for proposals for later years. For incurred cost proposals that were deemed low-risk or were $250 million or below in ADV, DCAA would audit a certain percentage of those proposals, with the percentages varying by different strata. As a result, DCAA conducts far fewer audits on low-risk, lower-dollar value proposals than it did prior to initiating the risk-based approach in 2012. For low-risk and lower-dollar value proposals that were not sampled, DCAA issues a low-risk memorandum that recommends the contracting officers use his or her authority to determine the contractor’s final indirect cost rates and proceed with closing the contract. Unless they are assessed to be high-risk, DCAA will close the majority of these proposals with a low-risk memorandum. Since the risk-based initiative was implemented in 2012, DCAA issued a total of 18,292 low-risk memorandums to close out proposals, compared to a total of 9,641 incurred cost audit reports. In developing the risk-based approach, DCAA assessed the costs associated with performing audits at different ADV against the savings associated with identifying unallowable or questioned costs. DCAA determined that it had a higher return on investment for higher value ADV proposals and that the return on investment was negative for audits conducted on lower-dollar proposals. For example, DCAA reported that even under its risk-based approach, it conducted 767 audits on incurred cost proposals with ADVs of $1 million or less from fiscal years 2014 through 2016, but expended approximately $18 million more in staff resources than the government received by identifying unallowable or questioned costs. DCAA policy officials stated they regularly assess results and, if appropriate, revise the sampling percentages. DCAA policy officials also noted that the use of multi-year auditing— through which it combines audits of two or more incurred cost proposals into a single audit—has helped reduce the inventory. According to DCAA’s data, multi-year auditing reduced the average number of hours to conduct an audit by 40 percent over conducting separate single-year audits. DCAA, however, does not actively track at the agency level how many proposals have been closed or are planned to be closed using this process. DCAA policy officials stated that DCAA’s management information system does not have a specific field to collect information on open proposals that are planned to be closed using multi-year audits. Instead, DCAA policy officials stated that they can determine the number of proposals closed through multi-year auditing once the audit reports have been issued. DCAA reported that it used multi-year audits to close 1,232 and 1,536 incurred cost proposals, in fiscal years 2015 and 2016, respectively, which constituted about 13 percent and 19 percent, respectively, of the total number of incurred cost proposals closed in those years. DCAA Has Not Developed Certain Performance Measures for Its Incurred Cost Proposals While DCAA has made progress in reducing its inventory of incurred cost proposals awaiting audit, our work identified two areas in which DCAA may be missing opportunities or lacking information to help identify additional ways to reduce its inventory. These areas include: (1) assessing actions for reducing the amount of time it takes for DCAA to begin audit work and establishing related performance measures to monitor its progress and (2) evaluating the use of multi-year auditing and establishing related performance measures. Federal standards for internal control call for the establishment of clear, consistent objectives and the identification and analysis of what measures will be used to determine if an agency is achieving those objectives. DCAA’s data for fiscal year 2016 indicate that once a contractor submits an adequate incurred cost proposal, it took DCAA on average 885 days— or nearly 2 and a half years—before DCAA completed the incurred cost proposal audit. Further, our analysis found that DCAA’s backlog of contractor proposals submitted for 2013 and prior years includes 51 adequate proposals that have $1 billion or more in ADV submitted by at least 15 of DOD’s largest contractors for which audits have not been completed. The number of days from the date these 51 proposals were determined adequate ranged from 78 to 2,206 days at the end of fiscal year 2016, meaning that a contractor submitted an adequate cost proposal more than 6 years ago but DCAA has not yet completed the audit. According to DCAA policy officials, staff availability is the primary factor for the delay before starting audit work. For example, proposals closed in fiscal year 2016 waited in DCAA’s queue an average of 747 days before the start of audit work. From the time that DCAA initiated the audit—which it defines as the date DCAA holds an entrance conference with the contractors—it took DCAA about 138 days on average to complete the audit in fiscal year 2016. For the average days that DCAA took to complete incurred cost audits from fiscal years 2011 through 2016, see figure 4. DCAA policy officials attributed the delay in initiating an audit once adequacy is determined to several factors, including the lack of staff, and when adequate proposals are submitted—the majority of which are received in June each year, which leaves little time to take action before the end of the fiscal year. Further, DCAA officials noted that, historically, DCAA used a “6-24-6” month framework for conducting incurred cost audits. DCAA officials noted that the FAR provides contractors 6 months to submit an incurred cost proposal, and, if DCAA is able to complete its audit of that proposal within 24 months, contracting officers will have 6 months to close out flexibly-priced contracts. Delays in receiving an adequate proposal will affect contracting officers’ ability to close out the contracts in a timely manner. Even though the 6-24-6 framework is not being met in practice, DCAA has not established specific goals for initiating audits, nor assessed whether the 6-24-6 framework under which it currently operates should be revised to take into account the realities of the time frames for contractors to submit adequate proposals or DCAA’s own staffing shortages. Assessing and implementing options to reduce the amount of time DCAA takes to begin its incurred cost audit work and establishing performance measures could help DCAA further reduce its inventory. Complicating DCAA’s ability to plan and initiate audit work are proposals submitted by contractors that are determined to be inadequate. While DCAA has started, or could start audit work on almost 90 percent of the backlog of 4,328 incurred cost proposals which were considered adequate, more than 10 percent—or 452—proposals were still considered inadequate. For example, we identified 10 proposals of $1 billion or more in ADV submitted for fiscal years 2011 through 2013 by major defense contractors that were still considered inadequate as of September 2016. These 10 proposals collectively amounted to about $36 billion in ADV, or about 9 percent of DCAA’s total amount of ADV in its backlog. For fiscal years 2014 through 2016, about 45 percent of the incurred cost proposals submitted by contractors were considered inadequate. Figure 5 depicts the extent to which proposals associated with DCAA’s incurred cost inventory were considered adequate, which includes proposals pending an adequacy review, and proposals that were considered inadequate. DCAA officials acknowledged that they do not currently have insight into the reasons why DCAA determined that a contractor’s proposal was inadequate, the number of times that a contractor submits revised proposals until it is deemed adequate, or the length of time it takes to receive an adequate proposal, but they noted they recently began an initiative to do so. Additionally, DCAA recently began to study the feasibility of developing a web-based submission portal for incurred cost proposals that could allow contractors the option to submit their proposals with real time visibility and guidance on common issues. This could lessen the number of times proposals are returned by DCAA as inadequate since contractors could identify potential issues prior to initially submitting proposals. Additionally, DCAA does not actively track how many proposals are planned to be closed using multi-year audits. These audits accounted for 19 percent of the total number of incurred cost proposals closed in fiscal year 2016, and, according to DCAA policy officials, DCAA would like to continue the use of multi-year audits to gain work efficiencies by combining proposals under one audit. DCAA has not, however, fully evaluated how the process could be improved nor established related performance measures, such as the number of proposals closed, ADV examined, the timeliness of the audits, or its impact on contractors. Federal standards for internal control call for the establishment of clear, consistent objectives and the identification and analysis of what measures will be used to determine if an agency is achieving those objectives. Industry representatives noted that multi-year audits take more effort on their part to support—especially for older proposals—and do not enable them to correct deficiencies in a timely fashion. DCAA is aware of these concerns and has sought contractor input about the efficacy and usefulness of multi-year audits, but has not done a comprehensive assessment, including how, if at all, the use of multi-year audits affect industry or determined how the process could be improved. As a result, it would be difficult for DCAA to assess if there are areas to multi-year auditing where additional efficiencies could be gained. Conclusions We found that closing out contracts is not the highest priority for contracting officers that are charged with awarding and administering contracts for products and services to meet mission needs. Yet this is a critical step to ensure the government receives the goods and services it purchases at the agreed upon price and, if done in a timely manner, provides opportunities to utilize unspent funds for other needs. Most of the agencies we reviewed delegated responsibility for closing out contracts to their components; however, none of the five agencies and only one of the components we reviewed had the critical elements that would assist them in overseeing their efforts to more effectively manage their respective contract closeout backlog. Having centralized information on the number and type of contracts that need to be closed out and where the contracts are in the closeout process could help management address the causes as to why contracts remain open in order to reduce the contract closeout backlog. While agencies may tailor their approaches to their specific needs and organizational structures, federal internal control standards require that agency management use quality information to make informed decisions and evaluate the entity’s performance in achieving key objectives and addressing risks—especially if the risks and challenges are similar across the agency. DCAA’s investment of significant management attention and resources, as well as its use of risk-based approaches to conducting audits, has enabled DCAA to significantly reduce both its overall inventory and its backlog of older incurred cost proposals. Doing so should help contracting officers close more of their outstanding flexibly-priced contracts, enable DCAA to focus more of its resources on other audit responsibilities, and reduce some of the burden on industry. Despite this progress, DCAA has 14,208 incurred cost proposals in its inventory, representing approximately $825 billion in ADV as of fiscal year 2016; hence, DCAA cannot afford to miss opportunities to further improve its incurred cost audit processes and timeliness. In this regard, DCAA has not assessed options and has not established performance measures for reducing the length of time to begin audit work on incurred cost proposals; the primary reason for the delay is due to the availability of DCAA staff to begin the audit work. Further, DCAA has not fully assessed or established performance measures on the use of multi-year audits, which it hopes to expand to help further reduce its inventory. Without such information, DCAA will be missing opportunities to assess options for further reducing its inventory of incurred cost proposals. Recommendations for Executive Action To enhance management attention to closing out contracts, we are making the following seven recommendations, one to each of the five agencies in our review and two to DCAA to manage its incurred cost inventory. The Secretary of Defense should develop a means for department- wide oversight into components’ progress in meeting their goals on closing contracts and the status of contracts eligible for closeout. (Recommendation 1) The Secretary of Health and Human Services should develop a means for department-wide oversight into components’ progress in meeting their goals on closing contracts and the status of contracts eligible for closeout. (Recommendation 2) The Secretary of Homeland Security should develop a means, either at the agency or the component level, to track where the contracts are in the closeout process, and establish goals and performance measures for closing contracts. (Recommendation 3) The Attorney General should direct the Senior Procurement Executive to ensure the development of a means to track data on the number and type of contracts eligible for closeout and where the contracts are in the closeout process, as well as a means to assess—at the agency or component level—progress by establishing goals and performance measures for closing contracts. (Recommendation 4) The Secretary of State should develop a means at the agency level to track data on the entirety of the number and type of contracts eligible for closeout, where the contracts are in the closeout process, and establish goals and performance measures for closing contracts. (Recommendation 5) The Director, DCAA should assess and implement options for reducing the length of time to begin incurred cost audit work and establish related performance measures. (Recommendation 6) The Director, DCAA should comprehensively assess the use and effect of multi-year audits on both DCAA and contractors and establish related performance measures. (Recommendation 7) Agency Comments and Our Evaluation We provided a draft of this report to DOD, DHS, State, HHS, and DOJ for review and comment. With the exception of DHS, the agencies concurred with our recommendations. DOD, DHS, and State provided written comments, which are reprinted in appendixes I-III, respectively, and summarized below. In comments provided in emails from the respective audit liaisons, DOJ and HHS concurred with the recommendations. DOD, DHS, and DOJ also provided technical comments, which we incorporated as appropriate. DOD concurred with our recommendation to develop a means for department-wide oversight into components progress in meeting their goals on closing contracts and the status of contracts eligible for closeout. Additionally, DOD concurred with our recommendations that the Director, DCAA, assess and implement options for reducing the length of time to begin incurred cost audit work and to comprehensively assess the use and effect of multi-year audits. DOD also agreed that the length of time to start an incurred cost audit should be minimized to the maximum extent practicable. DOD noted that in addition to the initiatives identified in our report, DCAA will continue to assess options to improve timeliness and implement actions to do so. Further, DOD agreed to conduct a more comprehensive analysis regarding the use and effect of multi-year audits on the contractors being audited as well as customers relying on the audit reports. DOD plans to complete these actions by March 31, 2018. Our draft report also included a recommendation that DCAA develop a means to centrally track risk assessments determinations. In responding to our draft report, DCAA provided additional information on how risk determinations were tracked and provided supporting data. We verified that information and, as a result, removed the recommendation and incorporated the data into the report as appropriate. DHS did not concur with our recommendation to develop a means to track where contracts are in the closeout process and establish related performance goals and measures. DHS agreed that contract closeout was a critical step in the procurement process, but noted that contract closeout activities are limited to available resources. DHS stated that the agency does not have a tool to track where contracts are in the closeout process and that obtaining such a tool would be resource-intensive. Further, DHS noted that having such a tool would not provide DHS with an effective way to remove bottlenecks from the closeout process. DHS noted, however, that it is committed to improving the closeout process and intends to establish a working group to assess current close out metrics and related performance measures. Additionally, the working group will assess the list of contracts eligible for closeout, monitor the progress of reducing the backlog and determine whether existing tools are available for obtaining information on the closeout process. The working group will also recommend improvements based on the availability of resources for closeout actions by November 30, 2018. While we did not call on DHS to obtain a new tracking tool, we believe that the planned efforts of the working group could meet the intent of our recommendation. State agreed with our recommendation to develop a means to track where contracts are in the closeout process and establish related performance goals and measures. State noted that it anticipates developing goals and performance measures by December 2019. We are sending copies of this report to the Secretary of Defense; the Secretaries of the Army, Navy, and Air Force; the Director, Defense Contract Audit Agency; the Director, Defense Contract Management Agency; the Director, Defense Logistics Agency; the Secretaries of Health and Human Services, Homeland Security, and State; the Attorney General; appropriate congressional committees; and other interested parties. This report will also be available at no charge on GAO’s website at http://www.gao.gov. If you or your staff have any questions concerning this report, please contact me at (202) 512-4841 or by e-mail at dinapolit@gao.gov. Contact points for our Offices of Congressional Relations and Public Affairs may be found on the last page of this report. Key contributors to this report are listed in appendix V. Appendix I: Comments from the Department of Defense Appendix II: Comments from the Department of Homeland Security Appendix III: Comments from the Department of State Appendix IV: Defense Contract Audit Agency’s (DCAA) Average Inventory Calculation Congress enacted a provision in the National Defense Authorization Act (NDAA) for Fiscal Year 2016 which prohibited the Defense Contract Audit Agency (DCAA) from conducting audits for non-defense agencies unless the Secretary of Defense certified that DCAA’s backlog of incurred cost audits was less than 18 months of incurred cost inventory. The law defined DCAA’s incurred cost inventory as the level of contractor incurred cost proposals from prior fiscal years that were currently being audited by DCAA. In September 2016, the Under Secretary of Defense (Comptroller) certified to Congress that DCAA had reduced its incurred cost proposal inventory below the required 18 month threshold to an average of 17.6 months. To support this determination, DCAA used its total inventory of incurred cost proposals that were in its inventory as of August 2016. DCAA made several adjustments to this total inventory to remove those incurred cost proposals for which it could not conduct an audit (i.e. were determined to be inadequate) and removed the number of reimbursable proposals (since these proposals are primarily for non-defense agencies which DCAA was prohibited from doing by the NDAA for Fiscal Year 2016) and direct-cost only work. Using the revised number, DCAA then took the number of elapsed days since the contractor submitted an adequate incurred cost proposal and divided by 30 days to approximate the number of elapsed months. The number of elapsed months was then averaged across the inventory to arrive at their total inventory calculation. Appendix V: GAO Contact and Staff Acknowledgments GAO Contact Staff Acknowledgments In addition to the contact named above, Bruce Thomas (Assistant Director), Anh Nguyen (Analyst-in-Charge), Peter Anderson, Giny Cheong, William Cordrey, Lorraine Ettaro, Kurt Gurka, Julia Kennon, Elisha Matvay, and Roxanna Sun made major contributions to this report.

Closing contracts is a key step in the contracting process. GAO and others have previously reported that large numbers of contracts were not closed within time frames set by federal regulations, which can expose the government to financial risk. DCAA's backlog of audits of contractors' incurred cost proposals contribute to the delays in closing out flexibly-priced contracts. GAO was asked to review the extent of the contract closeout backlog at federal agencies. In addition, a House Armed Services Committee report included a provision for GAO to assess DCAA's incurred cost audit backlog. This report addresses the extent to which (1) selected federal agencies effectively manage contract closeout, and (2) DCAA effectively manages its incurred cost audit backlog. GAO selected five agencies based on the number of contracts awarded and dollars obligated in fiscal year 2015. GAO analyzed documents and interviewed acquisition officials to assess how contract closeout is managed. GAO also analyzed data on DCAA's incurred cost audit backlog. The effectiveness of management efforts to reduce the number of contracts overdue for closeout varied across five agencies GAO reviewed-the Departments of Defense, Health and Human Services, Homeland Security (DHS), Justice, and State. None of the agencies had critical elements agency-wide that would help track and oversee contract closeout processes-the number and type of contracts to be closed, where the contracts were in the process, and goals and performance measures. Having such information could help management address the causes as to why contracts remain open and reduce the contract closeout backlog. Since 2011 the Defense Contract Audit Agency (DCAA) has reduced its inventory of contractors' incurred cost proposals awaiting audit by about half to 14,208, and DCAA has significantly reduced its backlog of older proposals-those for 2013 and prior-as of September 2016. To do so, DCAA used a risk-based approach to reduce the number of audits and began conducting multi-year audits, in which two or more incurred cost proposals are closed under a single audit. Nevertheless, DCAA did not meet its initial goal of eliminating its backlog by fiscal year 2016, and DCAA officials stated that they are unlikely to meet its revised goal by the end of fiscal year 2018. Further, GAO found that in fiscal year 2016, DCAA averaged 885 days from when a contractor submitted an adequate incurred cost proposal to when the audit was completed. The lag was due to limited availability of DCAA staff to begin audit work, as it took DCAA an average of 138 days to complete the actual audit work (see figure). DCAA may be missing opportunities to help identify additional ways to reduce its inventory. For example, DCAA has not assessed options to reduce time to initiate audit work or comprehensively assessed how the use of multi-year audits could be improved and has not established related performance measures for both.

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This is a set of web collections curated by Mark Graham using the Archive-IT service of the Internet Archive. They include web captures of the ISKME.org website as well as captures from sites hosted by IGC.org.These web captures are available to the general public.For more information about this collection please feel free to contact Mark via Send Mail Dear Max, Your mother and I don't yet have the words to describe the hope you give us for the future. Your new life is full of promise, and we hope you will be happy and healthy so you can explore it fully. You've already given us a reason to reflect on the world we hope you live in. Like all parents, we want you to grow up in a world better than ours today. While headlines often focus on what's wrong, in many ways the world is getting better. Health is improving. Poverty is shrinking. Knowledge is growing. People are connecting. Technological progress in every field means your life should be dramatically better than ours today. We will do our part to make this happen, not only because we love you, but also because we have a moral responsibility to all children in the next generation. We believe all lives have equal value, and that includes the many more people who will live in future generations than live today. Our society has an obligation to invest now to improve the lives of all those coming into this world, not just those already here. But right now, we don't always collectively direct our resources at the biggest opportunities and problems your generation will face. Consider disease. Today we spend about 50 times more as a society treating people who are sick than we invest in research so you won't get sick in the first place. Medicine has only been a real science for less than 100 years, and we've already seen complete cures for some diseases and good progress for others. As technology accelerates, we have a real shot at preventing, curing or managing all or most of the rest in the next 100 years. Today, most people die from five things -- heart disease, cancer, stroke, neurodegenerative and infectious diseases -- and we can make faster progress on these and other problems. Once we recognize that your generation and your children's generation may not have to suffer from disease, we collectively have a responsibility to tilt our investments a bit more towards the future to make this reality. Your mother and I want to do our part. Curing disease will take time. Over short periods of five or ten years, it may not seem like we're making much of a difference. But over the long term, seeds planted now will grow, and one day, you or your children will see what we can only imagine: a world without suffering from disease. There are so many opportunities just like this. If society focuses more of its energy on these great challenges, we will leave your generation a much better world. • • • Our hopes for your generation focus on two ideas: advancing human potential and promoting equality. Advancing human potential is about pushing the boundaries on how great a human life can be. Can you learn and experience 100 times more than we do today? Can our generation cure disease so you live much longer and healthier lives? Can we connect the world so you have access to every idea, person and opportunity? Can we harness more clean energy so you can invent things we can't conceive of today while protecting the environment? Can we cultivate entrepreneurship so you can build any business and solve any challenge to grow peace and prosperity? Promoting equality is about making sure everyone has access to these opportunities -- regardless of the nation, families or circumstances they are born into. Our society must do this not only for justice or charity, but for the greatness of human progress. Today we are robbed of the potential so many have to offer. The only way to achieve our full potential is to channel the talents, ideas and contributions of every person in the world. Can our generation eliminate poverty and hunger? Can we provide everyone with basic healthcare? Can we build inclusive and welcoming communities? Can we nurture peaceful and understanding relationships between people of all nations? Can we truly empower everyone -- women, children, underrepresented minorities, immigrants and the unconnected? If our generation makes the right investments, the answer to each of these questions can be yes -- and hopefully within your lifetime. • • • This mission -- advancing human potential and promoting equality -- will require a new approach for all working towards these goals. We must make long term investments over 25, 50 or even 100 years. The greatest challenges require very long time horizons and cannot be solved by short term thinking. We must engage directly with the people we serve. We can't empower people if we don't understand the needs and desires of their communities. We must build technology to make change. Many institutions invest money in these challenges, but most progress comes from productivity gains through innovation. We must participate in policy and advocacy to shape debates. Many institutions are unwilling to do this, but progress must be supported by movements to be sustainable. We must back the strongest and most independent leaders in each field. Partnering with experts is more effective for the mission than trying to lead efforts ourselves. We must take risks today to learn lessons for tomorrow. We're early in our learning and many things we try won't work, but we'll listen and learn and keep improving. • • • Our experience with personalized learning, internet access, and community education and health has shaped our philosophy. Our generation grew up in classrooms where we all learned the same things at the same pace regardless of our interests or needs. Your generation will set goals for what you want to become -- like an engineer, health worker, writer or community leader. You'll have technology that understands how you learn best and where you need to focus. You'll advance quickly in subjects that interest you most, and get as much help as you need in your most challenging areas. You'll explore topics that aren't even offered in schools today. Your teachers will also have better tools and data to help you achieve your goals. Even better, students around the world will be able to use personalized learning tools over the internet, even if they don't live near good schools. Of course it will take more than technology to give everyone a fair start in life, but personalized learning can be one scalable way to give all children a better education and more equal opportunity. We're starting to build this technology now, and the results are already promising. Not only do students perform better on tests, but they gain the skills and confidence to learn anything they want. And this journey is just beginning. The technology and teaching will rapidly improve every year you're in school. Your mother and I have both taught students and we've seen what it takes to make this work. It will take working with the strongest leaders in education to help schools around the world adopt personalized learning. It will take engaging with communities, which is why we're starting in our San Francisco Bay Area community. It will take building new technology and trying new ideas. And it will take making mistakes and learning many lessons before achieving these goals. But once we understand the world we can create for your generation, we have a responsibility as a society to focus our investments on the future to make this reality. Together, we can do this. And when we do, personalized learning will not only help students in good schools, it will help provide more equal opportunity to anyone with an internet connection. • • • Many of the greatest opportunities for your generation will come from giving everyone access to the internet. People often think of the internet as just for entertainment or communication. But for the majority of people in the world, the internet can be a lifeline. It provides education if you don't live near a good school. It provides health information on how to avoid diseases or raise healthy children if you don't live near a doctor. It provides financial services if you don't live near a bank. It provides access to jobs and opportunities if you don't live in a good economy. The internet is so important that for every 10 people who gain internet access, about one person is lifted out of poverty and about one new job is created. Yet still more than half of the world's population -- more than 4 billion people -- don't have access to the internet. If our generation connects them, we can lift hundreds of millions of people out of poverty. We can also help hundreds of millions of children get an education and save millions of lives by helping people avoid disease. This is another long term effort that can be advanced by technology and partnership. It will take inventing new technology to make the internet more affordable and bring access to unconnected areas. It will take partnering with governments, non-profits and companies. It will take engaging with communities to understand what they need. Good people will have different views on the best path forward, and we will try many efforts before we succeed. But together we can succeed and create a more equal world. • • • Technology can't solve problems by itself. Building a better world starts with building strong and healthy communities. Children have the best opportunities when they can learn. And they learn best when they're healthy. Health starts early -- with loving family, good nutrition and a safe, stable environment. Children who face traumatic experiences early in life often develop less healthy minds and bodies. Studies show physical changes in brain development leading to lower cognitive ability. Your mother is a doctor and educator, and she has seen this firsthand. If you have an unhealthy childhood, it's difficult to reach your full potential. If you have to wonder whether you'll have food or rent, or worry about abuse or crime, then it's difficult to reach your full potential. If you fear you'll go to prison rather than college because of the color of your skin, or that your family will be deported because of your legal status, or that you may be a victim of violence because of your religion, sexual orientation or gender identity, then it's difficult to reach your full potential. We need institutions that understand these issues are all connected. That's the philosophy of the new type of school your mother is building. By partnering with schools, health centers, parent groups and local governments, and by ensuring all children are well fed and cared for starting young, we can start to treat these inequities as connected. Only then can we collectively start to give everyone an equal opportunity. It will take many years to fully develop this model. But it's another example of how advancing human potential and promoting equality are tightly linked. If we want either, we must first build inclusive and healthy communities. • • • For your generation to live in a better world, there is so much more our generation can do. Today your mother and I are committing to spend our lives doing our small part to help solve these challenges. I will continue to serve as Facebook's CEO for many, many years to come, but these issues are too important to wait until you or we are older to begin this work. By starting at a young age, we hope to see compounding benefits throughout our lives. As you begin the next generation of the Chan Zuckerberg family, we also begin the Chan Zuckerberg Initiative to join people across the world to advance human potential and promote equality for all children in the next generation. Our initial areas of focus will be personalized learning, curing disease, connecting people and building strong communities. We will give 99% of our Facebook shares -- currently about $45 billion -- during our lives to advance this mission. We know this is a small contribution compared to all the resources and talents of those already working on these issues. But we want to do what we can, working alongside many others. We'll share more details in the coming months once we settle into our new family rhythm and return from our maternity and paternity leaves. We understand you'll have many questions about why and how we're doing this. As we become parents and enter this next chapter of our lives, we want to share our deep appreciation for everyone who makes this possible. We can do this work only because we have a strong global community behind us. Building Facebook has created resources to improve the world for the next generation. Every member of the Facebook community is playing a part in this work. We can make progress towards these opportunities only by standing on the shoulders of experts -- our mentors, partners and many incredible people whose contributions built these fields. And we can only focus on serving this community and this mission because we are surrounded by loving family, supportive friends and amazing colleagues. We hope you will have such deep and inspiring relationships in your life too. Max, we love you and feel a great responsibility to leave the world a better place for you and all children. We wish you a life filled with the same love, hope and joy you give us. We can't wait to see what you bring to this world. Love, Mom and Dad This week, the richest business leaders and investors from around the world will gather in Davos, Switzerland, for the annual meeting of the World Economic Forum. In keeping with tradition, a small portion of the agenda will be devoted to global development and the plight of people living at the other end of the global income distribution. Philanthropy is one way of linking the fortunes of these disparate communities. What if some of the mega-rich could be persuaded to redistribute their wealth to the extreme poor? This question may feel hackneyed, but it deserves a fresh hearing in light of a dramatic reduction in the global poverty gap over the past several years (Figure 1). The theoretical cost of transfers required to lift all poor people’s income up to the global poverty line of $1.90 a day stood at approximately $80 billion [1] in 2015, down from over $300 billion in 1980. (Values expressed here are in 2015 market dollars.) Figure 1. Official foreign aid now exceeds the annual cost of closing the poverty gap Source: Authors’ calculations based on OECD, World Bank This reduction can be unpacked into two parts. The first is a steep decline in the number of people living below the global poverty line. This is increasingly recognized as one of the defining features of the era. A U.N. goal to halve the poverty rate in the developing world between 1990 and 2015 was nearly achieved twice over. The second and lesser-known factor is the shrinking average distance of the world’s poor from the poverty line. In 1980, the mean daily income of those living below $1.90 was $1.09. In 2012 it was 25 cents higher at $1.34. (Values expressed here in 2011 purchasing power parity dollars.) Despite this good news, global poverty still demands attention. Hundreds of millions of people continue to suffer this most acute form of deprivation. In several countries, the prospects for ending poverty over the next generation, in line with a recently endorsed successor U.N. goal, appear challenging at best. Figure 1 illustrates that in 2006, global aid flows exceeded the cost of the global poverty gap for the first time. This suggests that the elimination of extreme poverty should be possible simply through a more efficient allocation of aid. However, this confuses foreign aid’s goals and functions. The bulk of official foreign aid is used in the provision of public goods, such as physical infrastructure and strengthening institutions. Only 2 percent is directed to social payments and their administration. If the elimination of extreme poverty is to be achieved through targeted transfers, it depends on sources other than foreign aid. The main source of transfers to the poor is welfare programs run and financed by developing countries themselves. These social safety nets have emerged as an increasingly prominent instrument in the toolkit of developing economy governments. Eighty-three percent of developing economies employ unconditional cash transfer programs, although many are small in scale. Several countries are in the process of building the apparatus for more accurate targeting and authentication through the assembly of beneficiary registries and the rolling out of identity programs. In at least 10 developing countries, social safety nets have succeeded in establishing a social floor by lifting all those people under the poverty line up above the threshold. In the vast majority, however, safety nets are insufficiently targeted or generous for that purpose, reflecting not only resource constraints, but also political choices that can be resistant to change. A complementary approach is to consider the role of private mechanisms and wealth. NGOs were among the original pioneers of cash transfers in the developing world. More recently, the NGO GiveDirectly has designed a compelling new method of charitable giving that sends money directly to the poor using digital monitoring and payment technology. Its approach has received strong endorsements from independent charity assessors and has been validated by impact evaluations. Yet the scale of its existing donations remains tiny relative to the global poverty gap. This is where Davos’s global elite could come into play: What difference could a philanthropic donation from the world’s richest people make? Comparing billionaire wealth with the global poverty gap To explore this question, we begin by identifying those developing countries that are home to a least one billionaire. (Our analysis is restricted to billionaires by data, not by the potential largesse of the world’s multi-millionaires. We focus our attention on billionaires in the developing world given the traditional focus of philanthropy on domestic causes.) Let’s assume that the richest billionaire in each country agrees to give away half of his or her current wealth among his or her fellow citizens, disbursed evenly over the next 15 years, roughly in accordance with the Giving Pledge promoted by Bill Gates. That money would be used exclusively to finance transfers to poor people based on their current distance from the poverty line. Transfers would be sustained at the same level for the full 15-year period with the aim of providing a modicum of income security that might allow beneficiaries to sustainably escape from poverty by 2030. Table 1 summarizes the key results. In each of three countries—Colombia, Georgia, and Swaziland—a single individual's act of philanthropy could be sufficient to end extreme poverty with immediate effect. Swaziland is an especially striking case as it is among the world’s poorest countries with 41 percent of its population living under the poverty line. In Brazil, Peru, and the Philippines, poverty could be more than halved, or eliminated altogether if the billionaires could be convinced to match Mark Zuckerberg’s example and increase their donation to 99 percent of their wealth. Table 1. The potential impact on poverty of individual billionaire giving pledges Country Cost per year to close the poverty gap Wealthiest billionaire Net worth Poverty rate pre-transfer Poverty rate post-transfer Nigeria $12,070 m A. Dangote $14,700 m 45% 43% Swaziland $85 m N. Kirsh $3,900 m 41% 0% Tanzania $1,645 m M. Dewji $1,250 m 40% 39% Uganda $1,035 m S. Ruparelia $1,100 m 33% 32% Angola $1,277 m I. dos Santos $3,300 m 28% 25% S. Africa $1,068 m J. Rupert $7,400 m 18% 14% Philippines $648 m H. Sy $14,200 m 12% 3% Nepal $144 m B. Chaudhary $1,300 m 12% 8% India $5,839 m M. Ambani $21,000 m 12% 10% Guatemala $215 m M. Lopez Estrada $1,000 m 12% 10% Venezuela $870 m G. Cisneros $3,600 m 11% 9% Georgia $40 m B. Ivanishvili $5,200 m 10% 0% Indonesia $845 m R. Budi Hartono $9,000 m 9% 6% Colombia $444 m L. C. Sarmiento $13,400 m 7% 0% Brazil $1,223 m J. P. Lemann $25,000 m 4% 1% Peru $95 m C. Rodriguez-Pastor $2,100 m 3% 1% China $3,072 m W. Jianlin $24,200 m 3% 2% Source: Authors’ calculations based on Forbes, International Monetary Fund, PovcalNet, and the World Bank. Poverty rates post-transfer calculated based on average distance of the poor from the poverty line. In other countries—Nigeria, Tanzania, Uganda, and Angola—the potential impact on poverty is only modest. A number of factors account for differences between countries, but two factors that penalize African countries are especially noteworthy. First, the depth of poverty in Africa remains high, with 15 percent of the population living on less than $1.00 a day; and second, Africa has relatively high prices compared to other poor regions, which means more dollars are required to deliver the same amount of welfare. For those nations that have more than one billionaire, an alternative scenario is that the country’s club of billionaires makes the pledge together and combines resources to tackle domestic poverty. This would end poverty in China, India, and Indonesia—countries that rank first, second, and fifth globally in terms of the absolute size of their poor populations. The last two columns of Table 2 describe the results. Table 2. The potential impact on poverty of collective billionaire giving pledges Country Cost per year of closing the poverty gap No. of Billionnaires Net Worth Poverty rate pre-transfer Poverty rate post-transfer Nigeria $12,070 m 5 $22,900 m 45% 42% Swaziland $85 m 1 $3,900 m 41% 0% Tanzania $1,645 m 2 $2,250 m 40% 38% Uganda $1,035 m 1 $1,100 m 33% 32% Angola $1,277 m 1 $3,300 m 28% 25% S. Africa $1,068 m 7 $28,550 m 18% 2% Philippines $648 m 11 $51,300 m 12% 0% Nepal $144 m 1 $1,300 m 12% 8% India $5,839 m 90 $294,250 m 12% 0% Guatemala $215 m 1 $1,000 m 12% 10% Venezuela $870 m 3 $9,600 m 11% 7% Georgia $40 m 1 $5,200 m 10% 0% Indonesia $845 m 23 $56,150 m 9% 0% Colombia $444 m 3 $18,500 m 7% 0% Brazil $1,223 m 54 $181,050 m 4% 0% Peru $95 m 6 $8,750 m 3% 0% China $3,072 m 213 $564,700 m 3% 0% Source: Authors’ calculations based on Forbes, IMF, PovcalNet, and the World Bank. Poverty rates post-transfer calculated based on average distance of the poor from the poverty line. This exercise is of course laden with simplifying assumptions. [2] It is intended to provoke discussion, not to provide definitive figures. Moreover, it is open to debate whether transfers represent the most cost-effective way of sustainably ending poverty, the extent to which transfers ought to be targeted, the efficacy of building private transfer programs alongside public safety nets, and whether cash transfers represent the most appropriate use of billionaires’ philanthropy. What is less contestable is that a falling global poverty gap presents an opportunity for more systematic efforts for poverty reduction. This raises the question: How low does the poverty gap have to fall before we explicitly design programs to bring the remaining poor above the poverty line? We would argue that we are already beyond this point, not least in countries that remain a long way from ending poverty. Were a billionaire at Davos to commit to using his or her wealth in this fashion, it could trigger a powerful demonstration effect of innovative solutions—not just for other billionaires, but for countries that are currently at risk of being left behind. [1] The cost of the global poverty gap in 2015 is an overestimate compared with the World Bank’s tentative poverty estimate for the same year. This is due to a different treatment of Nigeria. For this exercise, we rely on data from the 2009/10 Harmonized Nigeria Living Standards Survey reported in PovcalNet, despite its well-documented problems, whereas the Bank draws on the 2010/11 General Household Survey. [2] Simplifying assumptions include: zero administrative costs in identifying the poor, assessing their income, and administering payments with no leakages, or no portion of those costs being borne by billionaires; the efficacy of administering miniscule transfers to those who stand on the margin of the poverty line; and no change in the cost of closing the poverty gap in a country over time, whether due to population growth, an increase or decrease in poverty, or a change in prices relative to the dollar.

As the world's richest and most powerful people leave the World Economic Forum in Davos, Switzerland, there was growing discussion about the impact the world's wealthiest could have, single-handedly, on the poverty rates of entire nations. One analyst at the Brookings Institution crunched some numbers and found that, if the richest resident of three countries-Swaziland, Colombia, and Georgia-distributed half their wealth over the next 15 years to those who live below the poverty line according to how far below it they reside, no one would live below the poverty line in that time. Ditto if multiple billionaires (as opposed to just one) in each of six other countries pool their resources. "This might all seem far-fetched," notes Quartz, "but some of the world's richest people are already giving their money away." There's the example of Bill and Melinda Gates-who have given away $30 billion and have 100 others signed on to donate the majority of their wealth through the Giving Pledge. Similarly, Mark Zuckerberg and wife Priscilla Chan greeted the birth of their daughter with an open letter vowing to give away 99% of their Facebook shares, currently valued at $45 billion. The Brookings analyst hopes activity like this "could trigger a powerful demonstration effect of innovative solutions-not just for other billionaires, but for countries that are currently at risk of being left behind." The news comes as Oxfam International finds that the richest 1% of the world is just 62 people, and that their combined wealth equals that of the entire bottom half of the rest of the world (3.7 billion people and growing).

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SECTION 1. SHORT TITLE. This Act may be cited as the ``Tribal General Welfare Exclusion Act of 2013''. SEC. 2. INDIAN GENERAL WELFARE BENEFITS. (a) In General.--Part III of subchapter B of chapter 1 of the Internal Revenue Code of 1986 is amended by inserting before section 140 the following new section: ``SEC. 139E. INDIAN GENERAL WELFARE BENEFITS. ``(a) In General.--Gross income does not include the value of any Indian general welfare benefit. ``(b) Indian General Welfare Benefit.--For purposes of this section, the term `Indian general welfare benefit' includes any payment made or services provided to or on behalf of a member of an Indian tribe (or any spouse or dependent of such a member) pursuant to an Indian tribal government program, but only if-- ``(1) the program is administered under specified guidelines and does not discriminate in favor of members of the governing body of the tribe, and ``(2) the benefits provided under such program-- ``(A) are available to any tribal member who meets such guidelines, ``(B) are for the promotion of general welfare, ``(C) are not lavish or extravagant, and ``(D) are not compensation for services. ``(c) Definitions and Special Rules.--For purposes of this section-- ``(1) Indian tribal government.--For purposes of this section, the term `Indian tribal government' includes any agencies or instrumentalities of an Indian tribal government and any Alaska Native regional or village corporation, as defined in, or established pursuant to, the Alaska Native Claims Settlement Act (43 U.S.C. 1601 et seq.). ``(2) Dependent.--The term `dependent' has the meaning given such term by section 152, determined without regard to subsections (b)(1), (b)(2), and (d)(1)(B). ``(3) Lavish or extravagant.--The Secretary shall, in consultation with the Tribal Advisory Committee (as established under section 3(a) of the Tribal General Welfare Exclusion Act of 2013), establish guidelines for what constitutes lavish or extravagant benefits with respect to Indian tribal government programs. ``(4) Establishment of tribal government program.--A program shall not fail to be treated as an Indian tribal government program solely by reason of the program being established by tribal custom or government practice. ``(5) Ceremonial activities.--Any items of cultural significance, reimbursement of costs, or cash honorarium for participation in cultural or ceremonial activities for the transmission of tribal culture shall not be treated as compensation for services.''. (b) Conforming Amendment.--The table of sections for part III of subchapter B of chapter 1 of such Code is amended by inserting before the item relating to section 140 the following new item: ``Sec. 139E. Indian general welfare benefits.''. (c) Statutory Construction.--Ambiguities in section 139E of such Code, as added by this Act, shall be resolved in favor of Indian tribal governments and deference shall be given to Indian tribal governments for the programs administered and authorized by the tribe to benefit the general welfare of the tribal community. (d) Effective Date.-- (1) In general.--The amendments made by this section shall apply to taxable years for which the period of limitation on refund or credit under section 6511 of the Internal Revenue Code of 1986 has not expired. (2) One-year waiver of statute of limitations.--If the period of limitation on a credit or refund resulting from the amendments made by subsection (a) expires before the end of the 1-year period beginning on the date of the enactment of this Act, refund or credit of such overpayment (to the extent attributable to such amendments) may, nevertheless, be made or allowed if claim therefor is filed before the close of such 1- year period. SEC. 3. TRIBAL ADVISORY COMMITTEE. (a) Establishment.--The Secretary of the Treasury shall establish a Tribal Advisory Committee (hereinafter in this subsection referred to as the ``Committee''). (b) Duties.-- (1) Implementation.--The Committee shall advise the Secretary on matters relating to the taxation of Indians. (2) Education and training.--The Secretary shall, in consultation with the Committee, establish and require-- (A) training and education for internal revenue field agents who administer and enforce internal revenue laws with respect to Indian tribes on Federal Indian law and the Federal Government's unique legal treaty and trust relationship with Indian tribal governments, and (B) training of such internal revenue field agents, and provision of training and technical assistance to tribal financial officers, about implementation of this Act and the amendments made thereby. (c) Membership.-- (1) In general.--The Committee shall be composed of 7 members appointed as follows: (A) Three members appointed by the Secretary of the Treasury. (B) One member appointed by the Chairman, and one member appointed by the Ranking Member, of the Committee on Ways and Means of the House of Representatives. (C) One member appointed by the Chairman, and one member appointed by the Ranking Member, of the Committee on Finance of the Senate. (2) Term.-- (A) In general.--Except as provided in subparagraph (B), each member's term shall be 4 years. (B) Initial staggering.--The first appointments made by the Secretary under paragraph (1)(A) shall be for a term of 2 years. SEC. 4. OTHER RELIEF FOR INDIAN TRIBES. (a) Temporary Suspension of Examinations.--The Secretary of the Treasury shall suspend all audits and examinations of Indian tribal governments and members of Indian tribes (or any spouse or dependent of such a member), to the extent such an audit or examination relates to the exclusion of a payment or benefit from an Indian tribal government under the general welfare exclusion, until the education and training prescribed by section 3(b)(2) of this Act is completed. The running of any period of limitations under section 6501 of the Internal Revenue Code of 1986 with respect to Indian tribal governments and members of Indian tribes shall be suspended during the period during which audits and examinations are suspended under the preceding sentence. (b) Waiver of Penalties and Interest.--The Secretary of the Treasury may waive any interest and penalties imposed under such Code on any Indian tribal government or member of an Indian tribe (or any spouse or dependent of such a member) to the extent such interest and penalties relate to excluding a payment or benefit from gross income under the general welfare exclusion. (c) Definitions.--For purposes of this subsection-- (1) Indian tribal government.--The term ``Indian tribal government'' shall have the meaning given such term by section 139E of such Code, as added by this Act. (2) Indian tribe.--The term ``Indian tribe'' shall have the meaning given such term by section 45A(c)(6) of such Code.

Tribal General Welfare Exclusion Act of 2013 - Amends the Internal Revenue Code to exclude from gross income, for income tax purposes, the value of an Indian general welfare benefit. Defines "Indian general welfare benefit" as any payment made or services provided to or on behalf of a member of an Indian tribe under an Indian tribal government program if: (1) such program is administered under specified guidelines and does not discriminate in favor of members of the governing body of the Indian tribe; and (2) the program benefits are available to any tribal member, are for the promotion of general welfare, are not lavish or extravagant, and are not compensation for services. Directs the Secretary of the Treasury to: (1) establish a Tribal Advisory Committee to advise the Secretary on the taxation of Indians, (2) establish and require training and education for Internal Revenue Service (IRS) field agents on federal Indian law and the implementation of this Act, and (3) suspend audits and examinations of Indian tribal governments and members of Indian tribes and waive any interest or tax penalties related to the exclusion from gross income of Indian general welfare benefits.

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Background Industrial security integrates information, personnel, and physical security to protect classified information entrusted to contractors. The goal is to ensure that contractors’ security programs detect and deter espionage and counter the threat posed by adversaries seeking classified information. According to DSS, attempts by foreign agents to obtain information from contractors have increased over the last several years and are expected to increase further. The NISP is the governmentwide program to assure federal agencies that contractors adequately protect classified information. The NISP was established by executive order in 1993 to replace industrial security programs operated by various federal agencies. Under the national program, contractor facilities must be cleared prior to accessing classified information and must implement certain safeguards to maintain their clearance. DOD is responsible for clearing facilities and monitoring contractors’ protection of classified information. DOD, with concurrence from the Department of Energy, Nuclear Regulatory Commission, and Central Intelligence Agency, issued the National Industrial Security Program Operating Manual (NISPOM) in 1995. The NISPOM prescribes the requirements, restrictions, and safeguards that contractors are to follow to prevent the unauthorized disclosure—or compromise—of classified information. DSS administers the NISP on behalf of DOD and 24 other agencies through its Industrial Security Program. DSS’s Industrial Security Program, which is one of DSS’s three core mission areas, oversees more than 11,000 contractor facilities to assure U.S. government customers that their classified information is protected. By clearing a facility, DSS has determined that the contractor facility is eligible to access classified information at the same or lower classification level as the clearance granted—Confidential, Secret, or Top Secret. Under the NISP, a facility is a grouping of buildings related by function and location that form an operating entity. Facilities include manufacturing plants, laboratories, offices, and universities. They range in size from small offices that are owned and operated by one person to huge manufacturing complexes that are one of many owned by a large corporation. According to DSS, about half of the cleared facilities have been approved by DSS to store classified information on site, while the other facilities access classified information at a government site or at another facility approved for storage. DSS’s industrial security representatives serve as the primary points of contact with cleared facilities and are responsible for ensuring that contractors have security programs that comply with the NISPOM. The 240 industrial security representatives are assigned to 23 field offices spread throughout the country, where field office chiefs supervise their work. Representatives’ oversight involves educating facility personnel on security requirements, accrediting information systems that process classified information, approving classified storage containers, and assisting contractors with security violation investigations. DSS representatives also conduct periodic security reviews to assess whether contractor facilities are adhering to NISPOM requirements and to identify actual and potential security vulnerabilities. Security reviews are scheduled annually for facilities that store classified information and every 18 months for facilities that do not have classified information on site. In overseeing and assisting contractors, the representatives are to follow the procedures contained in the Industrial Security Operating Manual, which DSS issued to guide its personnel in administering the NISP. For example, the manual specifies how representatives should conduct security reviews to evaluate the quality of a facility’s security program and how contractor facilities’ reports of security violations should be handled. DSS Does Not Evaluate the Effectiveness of Its Oversight DSS relies on performance goals and measures that do not provide it a basis for assuring government customers that its oversight of contractor facilities mitigates the risk of information compromise. Instead of focusing on the overall results of its oversight and the protection of classified information, DSS evaluates its performance in terms of indicators, such as the number of security reviews completed on time. Further, while industrial security representatives maintain paper files on the quality of contractor security programs and the types of security violations that result in compromises of classified information, DSS does not analyze this information, and the manner in which it is maintained does not lend itself to such analysis. Without this analysis, DSS is limited in its ability to detect trends in the protection of classified information across facilities, to determine sources of security vulnerabilities, and to identify those facilities with the greatest risk of compromise. DSS’s Performance Goals and Measures Do Not Indicate If Mission Is Being Achieved Although DSS has reported that it has met or exceeded many of its performance goals, DSS has no basis for determining whether it is fulfilling its overall industrial security mission. DSS’s industrial security mission, as stated in its current Fiscal Year 2000-2005 strategic plan, is to (1) ensure that all contractor facilities overseen by DSS properly protect classified information in their possession and (2) assure government customers that facilities are eligible to receive classified information and have systems in place to protect the classified information. However, DSS currently does not have performance goals and measures that would indicate whether DSS is fulfilling this mission. DSS assesses its industrial security program based on the: percentage of security reviews completed, percentage of security reviews that covered all pertinent areas of contractors’ security programs, length of time needed to clear contractor facilities for access to classified information, and length of time needed to clear contractor personnel for access to classified information. Such indicators are important. For example, according to DSS officials, the indicator pertaining to the completion of security reviews provides government customers assurances that industrial security representatives are monitoring their contractors. The timeliness of clearances also matters because the facility and its personnel cannot access classified information in support of a government contract until DSS has cleared them. For each of the indicators, DSS established specific performance goals. While DSS did not meet all of its goals related to the timeliness of contractor facility and personnel clearances, it met or exceeded the goals related to security reviews. For example, DSS’s goal is to conduct annual security reviews of 98 percent of the facilities that store classified information on site. In fiscal year 2002, the most recent year for which data are available, DSS reported meeting this goal. DSS also reported that it exceeded the goal of having 75 percent of its security reviews cover all pertinent areas within contractor facilities’ security programs. Based on a review of selected security review reports, DSS determined that 86 percent of its security reviews conducted in fiscal year 2002 covered all pertinent areas and accurately reflected the contractor facilities’ overall security posture. However, DSS measured its achievement of this goal based on field office chiefs’ selection and review of about 550 of the approximately 9,000 reports completed by industrial security representatives. This review does not focus on the quality of the facilities’ security programs or the representatives’ review of those programs. Instead, it is used to determine the completeness of the reports. These current goals and measures alone do not enable DSS to determine whether its oversight is effectively ensuring that contractors protect classified information. There are no goals related to how well facilities are protecting classified information, which would provide an indication as to whether DSS is achieving its mission. For example, while DSS evaluates the completeness of security review reports submitted by industrial security representatives, it does not evaluate its performance in terms of the ratings and number of findings that result from security reviews. Nor does DSS evaluate its performance in terms of the frequency of security violations and information compromises occurring at contractor facilities. By not assessing its performance based on factors such as facility compliance with NISPOM requirements, DSS cannot determine whether its oversight efforts are contributing to an increase or decrease in facilities’ compliance and the protection of classified information. DSS’s Lack of Analysis Limits Its Ability to Determine If Its Oversight Reduces the Risk of Information Compromise DSS maintains records on how well contractor facilities protect classified information but does not analyze these records. There are no programwide analyses of violations reported by facilities or results of DSS’s reviews of facilities. Further, the manner in which DSS maintains records on facilities’ security programs—geographically dispersed paper-based files— does not lend itself to analysis. Industrial security representatives maintain a file folder on each facility they oversee. According to DSS officials, the information contained in these file folders represents the official record on each contractor facility. The folders are the primary means for documenting information on facilities’ security programs and representatives’ interactions with those facilities. The folders contain, in paper copy form, information such as the facility’s clearance level, identity of the facility owner, results of the last two security reviews, and facility’s reports on security violations. Folders are kept with their respective industrial security representatives throughout the country. An analysis of the types of security violations reported by facilities, their causes, or corrective actions taken would require a manual review of each file folder. According to DSS officials, DSS has not conducted such an analysis in recent years nor has it made any other attempt to identify the most common violations of the NISPOM or their causes. As a result, DSS does not know whether certain types of violations are increasing or decreasing or why such changes may be occurring. For example, DSS officials told us that anecdotal evidence indicates that there are an increasing number of security violations involving unsecured e-mail transmission of classified information. However, DSS has no basis for knowing what percentage of facilities have had such violations or how significant any increase has been. By not analyzing the information contained in the file folders, DSS is unable to identify patterns of security violations across all facilities based on factors such as the type of work conducted at the facility, the facility’s government customer, or the facility’s corporate affiliation. Officials at several contractor facilities informed us that their security procedures are developed and managed at the corporate level and, therefore, all facilities owned by the corporation follow the same procedures. As a result, security problems at one facility may indicate a more general, corporatewide vulnerability. For example, an industrial security representative attributed a series of violations at a facility owned by a large corporation to that facility’s inadequate security education program. However, facility security officials told us that their education program was developed at the corporate level, rather than by that facility. Because DSS does not track violations and their causes across facilities, there was no way to readily determine whether use of the corporate security education program resulted in violations at other facilities. DSS recently created a new database to track the number of security violations reported by facilities. Industrial security representatives are required to enter into the database which facility reported the violation, which field office is responsible for the facility, and the industrial security representative’s determination regarding whether information was compromised. According to DSS officials, DSS will use the new database to calculate the number of security violations nationwide and by region and to track the amount of time representatives take to make a determination after receiving facilities’ violation reports. However, because of the limited data it will contain, the database cannot be used to identify common types and causes of security violations reported by facilities. DSS also does not analyze information on the quality of facility security programs, such as ratings and the number and types of findings from DSS’s security reviews. While DSS officials expressed interest in eventually analyzing security review ratings and findings, they told us the new database currently lacks this capability. DSS has not manually reviewed the file folders and analyzed security review ratings to determine, for example, whether the number of facilities meeting NISPOM requirements is increasing or if security programs for facilities owned by one corporation have consistently lower ratings than those owned by another corporation. DSS also has not analyzed the security review findings to identify the number and most common types of findings. As a result, DSS cannot identify patterns of security review findings across all cleared facilities on the basis of the type of work they perform, their size, or corporate ownership. DSS Does Not Always Comply with NISP Requirements after a Possible Compromise of Information Industrial security representatives often failed to determine whether security violations by facilities resulted in the loss, compromise, or suspected compromise of classified information or made determinations that were not in accordance with approved criteria. Such determinations are important because if classified information is lost, compromised, or suspected of being compromised, the affected government customer must be notified so it can evaluate the extent of damage to national security and take steps to mitigate that damage. Even when representatives made an appropriate determination, they often took several weeks and even months to notify the government customer because of difficulties in identifying the customer. As a result, the customer’s opportunity to take necessary corrective action was delayed. Industrial Security Representatives Failed to Make Appropriate Determinations for Many Reported Security Violations The NISPOM requires a facility to investigate all security violations. If classified information is suspected of being compromised or lost, the facility must provide its DSS industrial security representative with information on the circumstances of the incident and corrective actions taken to prevent future occurrences. The industrial security representative is to then review this information and, using the criteria specified in DSS’s Industrial Security Operating Manual, make one of four final determinations: no compromise, suspected compromise, compromise, or loss. Table 1 outlines the criteria for each determination. If a determination other than no compromise is made, the Industrial Security Operating Manual directs the representative to inform the government customer about the violation so a damage assessment can be conducted. However, as shown in figure 1, for 39 of the 93 security violations that we reviewed, industrial security representatives made no determinations regarding the compromise or loss of classified information. For example, in two cases where the same facility reported the improper transmission of classified information via e-mail, DSS made no determinations even though the facility reported the possibility of compromise in both cases. In eight cases at another facility, employees repeatedly failed to secure a safe room to ensure the protection of classified information. DSS made no determinations in any of the eight cases. In the absence of a determination, the industrial security representatives did not notify the government customers of these violations. The government customers, unaware of the violations, could not take steps to assess and mitigate any damage that may have resulted. For 54 of the 93 violations we reviewed, representatives made determinations regarding the compromise or loss of information, but the majority were not consistent with the criteria contained in DSS’s Industrial Security Operating Manual. As figure 1 further illustrates, representatives made 24 determinations regarding compromise or loss that were consistent with the criteria contained in the manual. However, representatives made 30 inappropriate determinations, such as “compromise cannot be precluded” or “compromise cannot be determined.” Neither of these is consistent with the determinations in the manual—no compromise, suspected compromise, compromise, or loss. For example, in nine cases, the same facility reported that classified material was left unsecured, and the facility did not rule out compromise. In each of these cases, the industrial security representative did not rule out compromise but used an alternative determination. Senior DSS officials informed us that industrial security representatives should not make determinations other than the four established in the Industrial Security Operating Manual because the four have specific meanings based on accepted criteria. By not following the manual, representatives have introduced variability in their determinations and, therefore, their decisions of whether to notify the government customer of a violation. Among the 30 reported violations for which inappropriate determinations were made, industrial security representatives notified the affected government customers in 5 cases so the customers could assess and mitigate any resulting damage. These cases included three violations involving classified material that was left unsecured at the same facility. For the remaining 25 reported violations, the customers were not made aware of the violations even when the violations were similar to those reported to other customers. The failure of representatives to always make determinations consistent with the Industrial Security Operating Manual is at least partially attributable to inadequate oversight. The Standards and Quality Branch is the unit within DSS responsible for ensuring that industrial security representatives properly administer the NISP. Branch officials regularly test and review field office chiefs and representatives on NISP requirements, particularly those related to granting clearances and conducting security reviews. According to DSS officials, the results of these tests and reviews are used to design training courses that address weaknesses in job skills. However, the Standards and Quality Branch does not test or review how representatives respond to reported violations and make determinations regarding compromise. As a result, DSS does not know the extent to which representatives understand and are consistently applying Industrial Security Operating Manual requirements related to violations and, therefore, cannot make necessary revisions to training and guidance. In addition, field office chiefs are responsible for supervising and ensuring the quality of industrial security representatives’ day-to-day oversight of contractors. However, there is no specific requirement in the Industrial Security Operating Manual for field office chiefs to review their industrial security representatives’ determinations regarding reported security violations. We found no evidence that chiefs reviewed the cases in which the representatives either did not make determinations or made determinations that were inconsistent with the manual. Further, chiefs may not fully understand the manual’s criteria for determinations. For example, one field office chief we met with tracked the industrial security representatives’ processing of reported security violations by using a categorization sheet containing the inappropriate determination “compromise not precluded.” DSS Is Not Always Able to Quickly Notify Government Customers about Violations While the Industrial Security Operating Manual does not specify a time requirement for notifying government customers when classified information has been lost or compromised, DSS is frequently unable to notify customers quickly because of difficulties in identifying the affected customers. DSS notified government customers regarding 16 of the 54 reported violations for which representatives made determinations. Figure 2 shows that for 11 of these 16 violations, DSS did not notify the customer for more than 30 days after the contractor reported that information was lost, compromised, or suspected of being compromised. In one case, 5 months passed before an industrial security representative was able to notify a government customer that its information was suspected of being compromised. This delay was a result of the facility’s inability to readily determine which government customer was affected by the compromise. When a loss, compromise, or suspected compromise has been determined, the industrial security representative generally relies on the facility to identify the affected government customer. However, when the facility is operating as a subcontractor, it may not be aware of the government customer’s identity. In such instances, the subcontractor may have to work with the prime contractor to identify the government customer to provide the industrial security representative with this information. In one case we reviewed, a subcontractor made repeated attempts over a 5- month period to obtain the affected government customer’s identity from the prime contractor. In another case, an official with a subcontractor facility informed us that it was extremely difficult and time-consuming for him to identify the affected government customer, which took approximately 2 months. Such delays limit the government customer’s opportunity to assess the extent of potential damage to national security. Representatives Often Do Not Notify Facilities of Their Determinations Even Though It May Be Useful to Do So While the Industrial Security Operating Manual requires industrial security representatives to notify government customers of loss or compromise determinations, there is no requirement for representatives to inform facilities of their final determinations. However, senior DSS officials told us that they expect representatives to provide facilities with their final determinations. They explained that this helps facility officials understand what constitutes loss, compromise, or suspected compromise. Contractor security officials at one facility confirmed this by telling us that receiving determinations enables them to better understand which violations must be reported to DSS. Yet, industrial security representatives provided facilities with determinations for only 34 of the 93 reported violations we reviewed, and 18 of the 34 were inappropriate determinations. As a result of both inappropriate determinations and determinations not being provided by DSS, facility officials may misunderstand what constitutes a violation that must be reported to DSS and whether they have taken appropriate actions to contain any possible compromise and prevent future incidents. Conclusions By granting contractors access to classified information, the government has entrusted them with protecting national security. Ensuring that contractors safeguard classified information is DSS’s mission, yet DSS cannot provide adequate assurances that it is fulfilling this mission. Through its oversight, DSS cannot prevent every incident of information compromise, but unless DSS knows whether its oversight minimizes the risk of information compromise, it does not have an informed basis for managing its oversight. By not evaluating the information it maintains on how well contractors protect classified information, DSS may not realize where the risks and systemic vulnerabilities exist. Further, DSS has no basis for adjusting its resources to address emerging security weaknesses, such as the electronic transmission of classified information. Although DSS’s inability to assess its performance as well as evaluate and make changes to its oversight does not necessarily mean that contractors are not fulfilling their responsibilities under the NISP, the effectiveness of DSS’s oversight is diminished and the assurances it provides to government customers regarding the protection of their information cannot be relied on. Likewise, by not making appropriate determinations regarding compromise or loss, DSS does not always notify government customers that their information has been lost or compromised, thereby, limiting corrective actions and possibly increasing the damage to national security. Inappropriate determinations may also confuse contractors’ understanding of the reporting requirements and result in contractors not reporting incidents that should be reported. Recommendations for Executive Action To enable DSS to evaluate whether its oversight reduces the risk of information compromise, we recommend that the Secretary of Defense direct the Director, Defense Security Service, to take the following three actions: establish results-oriented performance goals and measures that would enable DSS to assess the extent to which it is achieving its industrial security mission, identify the information that needs to be analyzed to detect systemic vulnerabilities and identify trends regarding how contractor facilities protect classified information, and regularly analyze that information to make informed management decisions about the use of resources for its oversight activities and make any needed changes to those activities or procedures to reduce the risk of information compromise. In carrying out these actions, DSS will need to evaluate alternatives for creating a new system or further developing an existing system to record and analyze standard information on how well contractors protect classified information. We also recommend that the Secretary of Defense direct the Director of DSS to take the following four actions to ensure that appropriate determinations are made regarding possible information compromises and that government customers are notified of such situations in a timely manner: evaluate industrial security representatives and field office chiefs’ understanding of the criteria for making determinations regarding the compromise of classified information and revise training and guidance for representatives and chiefs based on the results of that evaluation, revise Industrial Security Operating Manual requirements to emphasize the need to apply the established determinations regarding the compromise or loss of classified information, explore the effects of establishing specific time-based criteria in the Industrial Security Operating Manual for representatives to make determinations and notify government customers, and establish mechanisms that create accountability for knowing the identity of government customers so that industrial security representatives can readily notify those customers of any loss or compromise. This could be accomplished by requiring representatives to maintain such information in their file folders or ensuring that contractors, particularly when they are subcontractors, know the identity of their government customers before an incident resulting in compromise or loss occurs. Additionally, to improve contractors’ understanding of which security violations must be reported to DSS, we recommend that the Secretary of Defense direct the Director of DSS to revise the Industrial Security Operating Manual to require industrial security representatives to inform facilities of the official determinations regarding the loss or compromise of classified information. Agency Comments and Our Evaluation In written comments on a draft of this report, DOD concurred with our recommendations. However, DOD stated that the report’s conclusion— that DSS cannot provide adequate assurances that its oversight ensures the protection of classified information by contractors—is not supported because we did not evaluate how well contractors protect classified information. While agreeing that its performance measures are not results- oriented, DOD stated that DSS is able to provide assurances regarding the protection of classified information through its security reviews. For 99 percent of security reviews, according to DOD, contractors were found to be satisfactorily protecting classified information. Additionally, DOD indicated that the problems we identified with security violations and possible information compromises were purely administrative. DOD stated it assumes that DSS’s current processes for handling security violations and possible information compromises did not leave classified information at risk. While contractors are ultimately responsible for protecting the classified information entrusted to them, DSS is charged with ensuring that contractors fulfill this obligation. Our review focused on how effectively DSS’s oversight ensures that contractors protect classified information. As explained in our report, DSS does not assess the effectiveness of its oversight based on how well contractors are protecting information from compromise nor does it analyze data to identify systemic vulnerabilities in contractors’ protection of classified information. Therefore, DSS cannot provide adequate assurances that its oversight ensures the protection of classified information. DSS is also hindered in its ability to identify and implement corrective changes to reduce the risk of information compromises resulting from security violations. In its comments, DOD stated that DSS does not have the ability to identify and analyze trends regarding how contractors protect classified information because it lacks the information technology infrastructure to conduct such analyses. We are uncertain of the basis for DOD’s statement that 99 percent of the facilities received satisfactory security review ratings because DSS officials told us during the course of our review that they do not track the facilities’ ratings. Also, by focusing only on security review ratings, DOD is overlooking other indicators—such as security review findings and incidents of possible compromise—that could enable DSS to improve its oversight. Further, the rating may not be an adequate measure of effectiveness. First, an industrial security representative can rate a facility’s security program as satisfactory even if the facility does not fully comply with the NISPOM and its failure to do so could logically lead to information compromise. Second, because DSS does not track information on security review ratings and violations, it cannot establish whether there is a correlation between a facility’s rating and the frequency and seriousness of that facility’s violations and information compromises. Finally, as we noted in our report, DSS’s security review quality metric is based not on the quality of reviews, but rather on the completeness of industrial security representatives’ reports. Also, the manner in which field office chiefs select reports for the quality review is not statistically valid and, therefore, DSS cannot draw conclusions about the quality of security review reports nationwide based on that quality review. The problems we identified with DSS’s response to security violations and possible information compromises go beyond administrative processing. Our findings focus on whether DSS has fulfilled its oversight responsibilities. As DOD noted in its comments, DSS is responsible for determining whether a violation has resulted in compromise, ensuring that the contractor took corrective action, and notifying the government customer. Yet, as discussed in our report, industrial security representatives failed, in 39 of the 93 security violations we reviewed, to determine whether the violations resulted in the loss, compromise, or suspected compromise of classified information. For an additional 30 violations, representatives made inappropriate determinations, which created variability in their decisions on whether to notify the government customer of a violation. Absent a determination consistent with the Industrial Security Operating Manual, one cannot draw conclusions on whether the contractor conducted an adequate inquiry into the violation and took corrective action to prevent its recurrence. Therefore, we cannot agree with DOD’s assumption that weaknesses in DSS’s handling of security violations did not leave classified material at risk. DOD’s comments are reprinted in appendix II, along with our evaluation of them. We are also sending copies of this report to interested congressional committees; the Secretary of Defense; the Director, Defense Security Service; the Assistant to the President for National Security Affairs; and the Director, Office of Management and Budget. We will make copies available to others upon request. In addition, this report will be available at no charge on the GAO Web site at http://www.gao.gov. If you or your staff have any questions regarding this report, please contact me at (202) 512-4841. Key contributors to this report are listed in appendix III. Appendix I: Scope and Methodology To assess the Defense Security Service’s (DSS) oversight of contractors’ implementation of the National Industrial Security Program (NISP), we reviewed Department of Defense (DOD) regulations and guidance on industrial security, including the National Industrial Security Program Operating Manual, as well as DSS policies, procedures, and guidance for overseeing contractor facilities. We also assessed DSS’s performance goals and measures contained in its strategic plan and annual report against our reports related to the Government Performance and Results Act and internal controls. We discussed the development of DSS goals, objectives, and performance metrics with DSS officials. To become more familiar with the roles and responsibilities of DSS staff, particularly as they relate to maintaining information on facility security programs, we reviewed DSS’s training materials, the Industrial Security Operating Manual, and selected facility file folders. We also discussed with DSS officials at headquarters and field locations how they use the information in the facility file folders to manage the industrial security program and oversee contractor facilities. To assess adherence to required procedures by DSS after a security violation and possible compromise of classified information, we used a case study approach. Using DSS’s Facilities Database, we selected cases from all facilities participating in the NISP as of March 2003. We reviewed the data and identified facilities that reported to DSS security violations since January 1, 2001, and selected 13 cleared facilities that varied according to size, clearance level, and geographic location. For those 13 facilities, we reviewed DSS’s official facility file folders and identified 93 reported violations. For those violations, we examined DSS’s actions to determine whether industrial security representatives and field office chiefs handled these reports in accordance with the Industrial Security Operating Manual. We also spoke with representatives and chiefs regarding the actions they take after receiving violation reports. We analyzed the information in DSS’s files on the 13 facilities and their violations to identify the determinations made by industrial security representatives, how frequently government customers were contacted, and the timeliness of government customer notification. In addition, we visited the facilities selected for our case study and interviewed those facilities’ security officials to obtain clarification and additional information about the reported security violations and actions taken by DSS. Because we did not take a statistical sample of facilities, the results from our analyses cannot be generalized. We also did not assess the reliability of the Facilities Database as a whole. However, we confirmed that the data used to select the 13 cases, specifically the facility size and clearance level, were consistent with the information in the facility files we reviewed. We performed our review from March 2003 through January 2004 in accordance with generally accepted government auditing standards. Appendix II: Comments from the Department of Defense The following are GAO’s comments on the Department of Defense’s letter dated February 12, 2004. GAO’s Comments 1. Our report recognizes that contractors are responsible for protecting classified information entrusted to them. However, the focus of the report is how well DSS is fulfilling its mission to ensure that contractors are protecting classified information. We clearly state that DSS’s inability to assess whether it is fulfilling its mission does not necessarily mean that contractors are not protecting the classified information entrusted to them. 2. We are uncertain of how DOD determined that 99 percent of cleared contractors were awarded satisfactory ratings nor do we know what time period this percentage covers and whether it has varied over time. However, for DSS to effectively manage its oversight, it needs to regularly analyze data and examine trends regarding the protection of classified information over time instead of producing the data to fulfill a one-time information request. 3. The results of our case studies can and do indicate serious weaknesses in how DSS oversees contractor facilities even though they cannot be generalized because, as discussed in appendix I, we did not take a statistical sample. 4. Our report identifies shortcomings in DSS’s ability to evaluate whether it is fulfilling its mission, make informed management decisions, and ensure that industrial security representatives properly resolve security violations and possible information compromises. Our report offers specific recommendations for improvement, all of which DOD agreed to implement. 5. It is unclear from DOD’s comments what other measures DSS relies on to determine success in accomplishing its mission. Our review assessed the goals and measures established by DSS and found that they do not provide a basis for determining whether DSS is fulfilling its mission. 6. Maintaining the prime contract numbers for all tiers of contracts in a new information management system may not be sufficient to ensure that government customers are readily notified of a loss or compromise. In at least two cases we reviewed, industrial security representatives informed subcontractor facility officials that, in addition to the prime contract number, the name and complete address of the government customer and a point of contact needed to be provided before DSS could process the violation. In one case, an official at a subcontractor facility informed the representative that such information was not readily available on the DD Form 254, which is designed to provide a contractor with the security requirements and classification guidance needed for the performance of a classified contract. Appendix III: GAO Contact and Staff Acknowledgments GAO Contact Acknowledgments In addition to the individual named above, Johana R. Ayers; Ronald T. Bell, Jr.; Lily J. Chin; Brendan S. Culley; Ian A. Ferguson; Kenneth E. Patton; and Eric E. Petersen made key contributions to this report.

Department of Defense (DOD) contractors perform numerous services that require access to classified information. With access comes the possibility of compromise, particularly as foreign entities increasingly seek U.S. military technologies. To ensure the protection of classified information, the National Industrial Security Program (NISP) establishes requirements that contractors must meet. In administering the NISP for DOD and 24 other government agencies, DOD's Defense Security Service (DSS) monitors whether 11,000- plus contractor facilities' security programs meet NISP requirements. In response to a Senate report accompanying the National Defense Authorization Act for Fiscal Year 2004, GAO assessed DSS's oversight and examined DSS's actions after possible compromises of classified information. DSS cannot provide adequate assurances to government agencies that its oversight of contractor facilities reduces the risk of information compromise. DSS is unable to provide this assurance because its performance goals and measures do not relate directly to the protection of classified information. While DSS maintains files on contractor facilities' security programs and their security violations, it does not analyze this information. Further, the manner in which this information is maintained--geographically dispersed paper-based files--does not lend itself to analysis. By not analyzing information on security violations and how well classified information is being protected across all facilities, DSS cannot identify systemic vulnerabilities and make corrective changes to reduce the risk of information compromise. When a contractor facility reports a violation and the possible compromise of classified information, DSS does not always follow established procedures. After receiving a report of a possible information compromise, DSS is required to determine whether compromise occurred and to notify the affected government agency so it can assess any damage and take actions to mitigate the effects of the suspected compromise, compromise, or loss. However, DSS failed to make determinations in many of the 93 violations GAO reviewed and made inappropriate determinations in others. In 39 of the 93 violations, DSS made no determinations regarding compromise. For 30 of the remaining 54 violations, DSS's determinations were not consistent with established criteria. As a result, government agencies are not being kept informed of possible compromises of their information. In addition, weeks or months can pass before government agencies are notified by DSS of possible information compromises because of difficulties in identifying the affected agencies. In 11 out of 16 instances GAO reviewed, it took DSS more than 30 days to notify the affected agency that its information had been lost or compromised. DSS relies on contractor facilities to identify the affected government agencies, but some facilities cannot readily provide DSS with this information because they are subcontractors that have to obtain the identity of the government agency from the prime contractors. In one case, 5 months passed before a subcontractor facility could provide DSS with the identity of the government agency whose information was suspected of being compromised. Such delays limit the government agencies' opportunity to assess and mitigate any damage from loss or compromise.

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Endophytic insects provide the textbook examples of herbivores that manipulate their host plant’s physiology, putatively altering source/sink relationships by transferring cytokinins (CK) to create ‘green islands’ that increase the nutritional value of infested tissues. However, unambiguous demonstrations of CK transfer are lacking. Here we show that feeding by the free-living herbivore Tupiocoris notatus on Nicotiana attenuata is characterized by stable nutrient levels, increased CK levels and alterations in CK-related transcript levels in attacked leaves, in striking similarity to endophytic insects. Using 15N-isotope labeling, we demonstrate that the CK N6-isopentenyladenine (IP) is transferred from insects to plants via their oral secretions. In the field, T. notatus preferentially attacks leaves with transgenically increased CK levels; plants with abrogated CK-perception are less tolerant of T. notatus feeding damage. We infer that this free-living insect uses CKs to manipulate source/sink relationships to increase food quality and minimize the fitness consequences of its feeding. Insect herbivores are under constant pressure from their host plants: they must adapt to toxic or anti-digestive defense compounds whose levels often dramatically increase in response to insect feeding; and their food source has low nitrogen to carbon ratios and a dietary value which decreases as leaves mature and senesce. Some herbivorous insects have developed strategies to overcome the low nutritional contents of their host plants and have evolved specialized mechanisms to tolerate, or even co-opt toxic plant defense metabolites for their own uses, in an apparent evolutionary arms race (Strong et al., 1984; Després et al., 2007; Heckel, 2014). Phytophagous insects can be categorized as either endophytic or free-living depending on the relationships that they establish with their host plant. This distinction is not binary and many transitional forms exist even within the same taxa. Consequently, the large differences in herbivorous lifestyles has selected for plant defense responses that counter different herbivory strategies (Kessler and Baldwin, 2002; Schuman and Baldwin, 2016). Free-living insects are mobile on their host plants, moving among plants, and frequently among different plant species. As a consequence of this mobility, they can freely choose tissues that are most nutritious or least defended, but the most nutritious tissues are often highly defended, resulting in a potential trade-off for herbivores (Ohnmeiss and Baldwin, 2000; Brütting et al., 2017). To avoid herbivore-induced defenses, free-living insects often move to other plant parts or even other host plants in response to defense activation, and the advantages of such movement are readily seen when induced defenses are abrogated (Paschold et al., 2007) or experimentally manipulated (van Dam et al., 2000). In contrast, endophytic insects develop more intimate relationships with their host plants as they are sedentary and spend a large portion of their life cycle within plant tissues. They have evolved strategies to overcome many of the plant defenses by hijacking plant metabolism and reprogramming plant physiology in their favor (Giron et al., 2016). Often the only viable plant defense is the ‘scorched earth’ response, whereby infested tissues are abscised from the plant (Fernandes et al., 2008). To date, the best-studied examples of endophytic plant-manipulating species, featured in most textbooks of plant physiology, are the gall-forming insects and leaf-miners. Gall-forming organisms, which include not only several orders of insects but also mites, nematodes and microbes, promote abnormal plant growth by reprogramming the expression of plant genes, to create novel organs that provide favorable environments for the exploiter (Stone and Schönrogge, 2003; Shorthouse et al., 2005). Advantages for the gall-formers range from an improved nutritional value, with reduced defense levels, to protection from diseases, competitors, predators, parasitoids and unfavorable abiotic conditions (Hartley, 1998; Stone and Schönrogge, 2003; Allison and Schultz, 2005; Harris et al., 2006; Saltzmann et al., 2008; Nabity et al., 2013). Manipulations of leaf-mining larvae do not result in the formation of new macroscopic structures like galls but they are often revealed during senescence of host tissues, where ‘green islands’ appear around the active feeding sites (Engelbrecht, 1968; Engelbrecht et al., 1969; Giron et al., 2007; Kaiser et al., 2010). Such green islands maintain a high level of photosynthetic activity typical of non-senescent leaves, thus providing nutrition for the larvae which feed on them (Behr et al., 2010; Body et al., 2013; Zhang et al., 2016). In this way, green islands reflect a battle between plant and infesting insect during the nutrient recovery phase that precedes abscission. The host plant tries to recover nutrients from the senescent leaf, whereas the insect tries to maintain a nutritious environment so as to complete its development. The most likely effectors used by insects to manipulate a plant’s normal physiological response to wounding are phytohormones, since significant levels of some well-known wound-responsive phytohormones, including cytokinins (CKs), abscisic acid (ABA) and auxins, have been found in the body and salivary secretions of a number of gall-forming insects (Mapes and Davies, 2001; Straka et al., 2010; Tooker and De Moraes, 2011; Yamaguchi et al., 2012; Tanaka et al., 2013; Takei et al., 2015), as well as in the bodies and labial glands of leaf-mining larvae (Engelbrecht et al., 1969; Body et al., 2013). Amongst these phytohormones, CKs deserve additional discussion due to their role in the formation of green islands (Engelbrecht, 1968; Engelbrecht, 1971; Engelbrecht et al., 1969; Giron et al., 2007; Kaiser et al., 2010; Body et al., 2013; Zhang et al., 2017). CKs are adenine derivatives which play a key role in the regulation of plant growth and development (Sakakibara, 2006). They are known for their capacity to increase photosynthetic activity (Jordi et al., 2000), determine sink strength (Mok and Mok, 2001) and inhibit senescence (Richmond and Lang, 1957; Gan and Amasino, 1995; Ori et al., 1999). More recently, CKs have been shown to regulate herbivory-induced defense signaling (Schäfer et al., 2015b; Schäfer et al., 2015c; Brütting et al., 2017). The long history of investigating CKs in the formation of green islands dates back to the late 1960’s, to reports of increased levels of CKs in affected tissues (Engelbrecht, 1968; Engelbrecht et al., 1969). In the last decade, studies on the leaf-mining larvae of Phyllonorycter blancardella identified CKs as the causative factors for the ‘green island’ phenomenon (Giron et al., 2007; Kaiser et al., 2010; Body et al., 2013; Zhang et al., 2017). These studies suggested that insects could be the source of phytohormones used to manipulate plant physiological responses. However, a clear demonstration of the ability of insects to transfer CKs to a host plant remains elusive. To assess whether an insect actively transfers CKs to manipulate plant physiology, we studied the interactions between the well-established ecological model-plant Nicotiana attenuata and one of its most abundant specialist herbivores, Tupiocoris notatus. N. attenuata is a wild diploid tobacco species native to southwestern North America. T. notatus is a free-living, 3–4 mm mirid bug (Miridae, Heteroptera) specialized to tobacco species and a few other solanaceous plants including Datura wrightii. It is a piercing-sucking cell-content feeder that damages the surface of the leaves without removing foliar material. Its feeding behavior is in sharp contrast with the feeding behavior of a well-studied specialist herbivore of N. attenuata, the lepidopteran Manduca sexta, whose chewing larvae cause extensive tissue damage and a well characterized defense response (Baldwin, 1998; Kessler and Baldwin, 2001; Kessler et al., 2004; Steppuhn et al., 2004; Zavala et al., 2004; Schuman et al., 2012). When plants are attacked by M. sexta, specific insect-derived fatty acid-amino acid conjugates elicit a defense response regulated by a burst of jasmonic-acid (JA) (Baldwin, 1998; Halitschke et al., 2001; Kessler et al., 2004). This jasmonate burst triggers the accumulation of defense metabolites like nicotine, caffeoylputrescine, diterpene-glycosides and tripsyin-proteinase inhibitors. It has also strong effects on the regulation of primary metabolism (Voelckel and Baldwin, 2004): sugars, starch and total soluble proteins readily decrease in the attacked leaves (Ullmann-Zeunert et al., 2013; Machado et al., 2015), as does photosynthesis (Meza-Canales et al., 2017). In contrast, infestation with T. notatus in nature, surprisingly, does not decrease plant fitness (Kessler and Baldwin, 2004), despite resulting in damage to large portions of photosynthetically active leaf area. Tissues around T. notatus feeding sites have increased rates of photosynthesis per chlorophyll content that may compensate for the damage caused by herbivore feeding, resulting from an active ingredient of the oral secretion of T. notatus which remains to be identified (Halitschke et al., 2011). We previously observed increased damage by T. notatus in tissues that were enriched in CKs through the transgenic manipulation of N. attenuata CK metabolism, using plants expressing a dexamethasone (DEX) -inducible construct driving transcription of the CK biosynthesis gene, isopentenyltransferase (IPT, i-ovipt). Individual DEX-treated leaves of field-grown plants suffered more damage from T. notatus than did mock-treated leaves. This led to the hypothesis that increased CK levels promote better nutritional quality, which in turn increases T. notatus feeding damage (Schäfer et al., 2013). Here, we report that T. notatus adults and nymphs contain high concentrations of two CKs. When confined to feeding on single N. attenuata leaves, concentrations of CKs increase in attacked leaves throughout the feeding period, with consequences for nutrient concentrations. Using 15N-labeled tracers, we demonstrate that T. notatus transfer CKs to the leaves on which they feed. Finally, we analyzed how changes to CK metabolism in plants affected T. notatus feeding preferences. We conclude that CK-dependent manipulation of plant metabolism is not only a strategy used by gall-forming insects or leaf-miners, but also employed by this free-living insect, which directly transfers CKs at feeding sites to manipulate its host plant. To characterize the defensive response of N. attenuata to mirid attack, we analyzed jasmonate hormones and defense metabolites that are known to be induced by M. sexta, as well as T. notatus feeding (Kessler and Baldwin, 2004). Continuous feeding by T. notatus (Figure 1a) causes visible damage to N. attenuata leaves (Figure 1b) and triggers defense responses in attacked leaves (Figure 1c–j). Three days of T. notatus feeding induced levels of the defense metabolites nicotine and caffeoylputrescine (CP), as well as trypsin proteinase inhibitor activity (TPI) (Figure 1c–e). T. notatus feeding also elevated the levels of jasmonic acid (JA), its precursor cis- (+) −12-oxophytodienoic acid (OPDA) and its bioactive isoleucine conjugate (JA-Ile) (Figure 1f–h). Interestingly, there was also a significant increase in salicylic acid (SA), but no influence on abscisic acid (ABA) (Figure 1i, j). JA and JA-Ile levels triggered by T. notatus feeding remained elevated for up to six days when mirids were confined to feed on a single leaf (Figure 1—figure supplement 1a–c). Their concentrations remained higher than controls even when T. notatus were free to move to other parts of the plant, although they steadily decreased over the six days (Figure 1—figure supplement 1d–f) These results demonstrate that N. attenuata’s response to T. notatus involves activation of JA signaling and downstream defense responses. Feeding by M. sexta is detrimental to N. attenuata fitness. It causes reduction of photosynthesis in attacked leaves (Halitschke et al., 2011; Meza-Canales et al., 2017) and a decrease in sugar and total soluble protein (TSP) contents (Ullmann-Zeunert et al., 2013; Machado et al., 2015). In contrast, T. notatus feeding seems to increase photosynthetic activity in attacked leaves, when accounting for tissue damaged by the feeding (Halitschke et al., 2011). We measured the impact of continuous T. notatus feeding over several days on the nutritional quality of the attacked leaves. We analyzed TSPs, sugar and starch levels, as well as measuring photosynthetic rates and chlorophyll contents of leaves over a period of 144 hr. Visibly heavily damaged leaves did not show significant decreases in nutrient levels when mirids were confined to feed on a single leaf with a small plastic cage (Figure 2a). TSP levels decreased with time in a clipcage but mirid feeding did not have a significant influence (Figure 2b). Furthermore, we did not observe any significant changes in starch, sucrose, glucose or fructose (Figure 2c–f). Although we did not observe changes in carbohydrate levels, photosynthesis was significantly reduced in attacked leaves (Figure 2—figure supplement 1b). In contrast, mirid feeding had no effect on chlorophyll contents (Figure 2—figure supplement 1c). When entire plants were heavily infested (Figure 2—figure supplement 2a), changes in nutrient levels in the plant became apparent only for TSP levels, which decreased after mirid feeding (Figure 2—figure supplement 2b). Conversely, levels of starch, sucrose, glucose and fructose were not affected by mirid feeding (Figure 2—figure supplement 2c–f). Both chlorophyll contents and photosynthetic rates significantly decreased after T. notatus whole-plant attack (Figure 2—figure supplement 3a–c). In summary, when only twenty mirids were allowed to feed on a single leaf the overall nutritional quality was not altered, although the feeding damage was visibly severe. In contrast, during a more extreme mirid infestation in which entire plants were severely attacked, TSP levels of attacked leaves decreased, but sugar and starch contents remained unchanged. However, no overall apparent increased photosynthetic activity was observed. An allocation of nutrients from unattacked to attacked tissue may explain the observation that even heavy T. notatus feeding only marginally influenced nutrient levels in attacked leaves. If this inference is correct, then mirid feeding likely influences the source/sink relationships of the host plant. Cytokinins (CKs) are known to regulate source/sink relationships and stabilize nutrient levels in tissues fed on by endophytic insects. Recently, we showed that these phytohormones also play a role in plant defense, since M. sexta herbivory, wounding, and JAs can increase the levels of cZ-type CKs in N. attenuata (Schäfer et al., 2015c; Brütting et al., 2017). As we did not see a strong decrease in nutrients after mirid feeding, it was especially interesting to investigate CK metabolism during T. notatus attack. When entire plants were attacked, mirid feeding significantly increased the accumulation of NaCKX5 transcripts, which code for a CK oxidase/dehydrogenase responsible for CK degradation (Figure 3a). Transcript levels of NaZOG2, which codes for a CK glucosyltransferase responsible for CK inactivation, as well as transcripts of NaLOG4 (Figure 3—figure supplement 1b), which is involved in CK biosynthesis, also increased after mirid feeding. In contrast, transcript levels of the isopentenyltransferase NaIPT5, which catalyzes the rate-limiting step of CK biosynthesis, were reduced after mirid feeding (Figure 3—figure supplement 1c). T. notatus feeding did not change levels of the CK response regulator NaRRA5 (Figure 3—figure supplement 1d). The levels of the different types of CKs varied depending on time and whether mirids attacked single leaves or entire plants. When entire plants were infested with T. notatus, overall leaf CK contents gradually increased over time (Figure 3b). Levels of cis-zeatin (cZ) (Figure 3—figure supplement 2a), cis-zeatin riboside (cZR) (Figure 3—figure supplement 2d), trans-zeatin (tZ) (Figure 3—figure supplement 2b) and trans-zeatin riboside (tZR) (Figure 3—figure supplement 2e) were significantly higher after T. notatus attack. In contrast, levels of N6-isopentenyladenine (IP) remained unaffected by mirid feeding (Figure 3—figure supplement 2c) and levels of N6-isopentenyladenosine (IPR) decreased in attacked leaves (Figure 3—figure supplement 2f). This decrease was significant in the first 24 hr after the initiation of mirid attack and disappeared at later harvest times (p<0. 05 in TukeyHSD post hoc test). When mirids were only allowed to feed on a single leaf, we did not observe changes in levels of summed CKs over the whole time series (Figure 3—figure supplement 3b). However, Bonferroni-corrected t-tests of single time point revealed increased levels at the last time point harvested, after 144 hr of feeding (tt: p=0. 026). The changes in individual CKs only partially overlapped with those observed during whole-plant feeding. There was a significant increase in cZ (Figure 3—figure supplement 3c), but levels of cZR decreased (Figure 3—figure supplement 3f). IP levels, which did not change during whole-plant feeding, were significantly higher overall after single-leaf feeding, although pairwise comparisons for each time point did not reveal significant changes at any given time point. tZ, tZR and IPR remained unaffected by mirid feeding when only single leaves were attacked (Figure 3—figure supplement 3d, g, h). Interestingly, in both experiments overall CK levels remained unchanged or were increased, despite the concomitant increases in transcripts of genes related to CK degradation. From these results, we infer that CKs are involved in the observed nutritional stability during mirid feeding; this hypothesis prompted a more detailed analysis of the origin of these CKs. Mirid attack enhanced the levels of cZ and cZR as was previously found for M. sexta herbivory, wounding, and JA application (Schäfer et al., 2015c; Brütting et al., 2017); but in contrast to these other types of elicitations, long-term mirid feeding and the associated JA accumulation did not decrease IP levels. This was particularly surprising given that CK degradation and inactivation processes appeared to have been activated by mirid feeding. We analyzed CK levels in T. notatus to determine whether these insects could themselves provide a source of CKs. We found very high levels of IP and IPR in extracts from the insect bodies (Figure 3c). While concentrations of IPR were comparable to those in leaves (around 1 pmol per g fresh mass (FM) ), concentrations of IP exceeded those of leaves by up to three orders of magnitude: while levels in leaves ranged from 0. 01 to 0. 1 pmol g FM−1, levels in insects were usually between 1 and 5 pmol g FM−1 and attained values as high as 16 pmol g FM−1. Insects collected from N. attenuata plants in their natural habitat at a field site in Utah, USA, also contained high amounts of IP: in a pooled sample of ten insects, we measured 18. 26 pmol IP per g FM-1. Mirids contained high IP and IPR levels in their bodies independently of their sex, developmental stage, or food source (Figure 3—figure supplement 4). The sole significant difference was that IP concentration in nymphs was about half as high as in adult males and females, but nymphs still had concentration several times those found in leaves (Figure 3—figure supplement 4a). To evaluate if CKs levels remained stable when T. notatus was no longer feeding on its host plant, we reared insects for five days either on artificial diet (containing no CKs) or on plants. Insects raised on artificial diet had IP levels in their body that were not different from levels in insects raised on plants; IPR levels were also unchanged (Figure 3—figure supplement 4b). Although the source of CKs in T. notatus remains unknown, we hypothesize that IP and IPR found in T. notatus body could be used by the mirid to counter the decrease in IP levels in attacked leaves that is commonly observed in response to long-term JA elicitation or M. sexta feeding. To evaluate whether T. notatus could transfer CKs to the plant, we conducted 15N- labeling experiments. We grew plants in hydroponic culture with 15N-labeled KNO3 as the only source of nitrogen. We furthermore created a stock of T. notatus insects that were 15N-labeled by raising them for an entire generation on 15N-grown plants. We then performed two different types of experiments to trace the origin of CKs in T. notatus attacked leaves: we either used 15N-grown plants that we exposed to 14N-labeled insects (Figure 4 and Figure 4—figure supplement 1) or we used 14N-grown plants and exposed them to 15N-labeled insects (Figure 4—figure supplements 2 and 3). CKs are adenine derivatives that contain five nitrogen atoms. Therefore, CKs produced by 15N-labeled plants or insects harbored five 15N and are readily distinguished from 14N-labeled CKs by mass spectrometry (Figure 4—figure supplements 4 and 5). In the first approach, we used a low-infestation setup by placing 20 15N-labeled T. notatus adults in a small cage on the leaf of a 14N-grown plant for five days. After four days of continuous feeding, we found detectable amounts of 15N-labeled IP (and IPR) in the leaves (Figure 4—figure supplements 2 and 3): around 2. 35 fmol [15N5]-IP per g FM, which represent the 3. 3% of the [15N5]/[14N5]-IP ratio (Figure 4—figure supplement 2d). [15N5]-labelled IP and IPR could only have originated from the insects, as the natural abundance of 15N is below 0. 4%, and IP (or IPR) with five 15N would occur about once in a trillion molecules. From these values, one mirid feeding on a leaf for five days could account for a transfer of at least 0. 12 fmol IP per g FM−1 (Supplementary file 1), assuming that CK transport, degradation or conversion to other CK forms can be excluded. In the reverse experiment, we used 15N-grown plants and insects raised on 14N-grown plants (Figure 4 and Figure 4—figure supplement 1). We placed 15N-grown plants in cages where T. notatus were reared on 14N-grown plants. These 15N-grown plants were switched to a new cage with infested 14N-grown plants once per day to ensure that they were always attacked by 14N-labeled insects and to prevent the accumulation of 15N in the 14N-labeled insects. After 5 days, an average of 48% of the [15N5]/[14N5]-IP ratio was 14N labeled and therefore originating from the insects (Figure 4). In this stronger induction setup, IPR transfer from the insect to the plant was also already detected after 24 hr and accounted to 19% of the [15N5]/[14N5]-IP ratio after 5 d (Figure 4—figure supplement 1). To evaluate how IP and IPR were transferred to the leaf during feeding, we analyzed the CK contents of the oral secretions and frass of T. notatus, which we considered the most likely means of transfer. Mirids were fed on sugar solutions covered with parafilm, which allowed the insects to penetrate the film with their stylets while preventing evaporation and preventing either insects or their frass from being immersed in the liquid. We then measured CKs in the sugar solution, which contained substances transferred by the oral secretions, as well as in the surface wash, which contained insect excretions (frass). We found large amounts (high signal intensity) of IP mainly from the oral secretions (from the sugar solution that mirids had fed on) and much lower amounts in the frass of the mirids (from the surface wash) (Figure 5). IPR was found in oral secretions and in frass in similar amounts (Figure 5—figure supplement 1). These results clearly demonstrate that T. notatus is able to transfer CKs (mainly IP) to its host plant. The most likely means of transfer would be via the salivary secretion produced during feeding, although we cannot rule out a smaller contribution of feces, which are sticky and tend to cover infested leaves. In nature, T. notatus feeds on young N. attenuata tissues, such as younger stem leaves and young growing leaves. This feeding pattern was inferred from the damage distributions observed on plants in both nature and the glasshouse (Figure 6—figure supplement 1a), as well as in two-choice assays (Figure 6—figure supplement 1b). The young leaves preferred by T. notatus are typically rich in CKs (Brütting et al., 2017). To evaluate how CK metabolism affects the interaction of N. attenuata with T. notatus, we used transgenic N. attenuata plants that were either enhanced in CK production (i-ovipt) or silenced in CK perception (irchk2/3). Transgenic i-ovipt plants that contain a dexamethasone (DEX) -inducible promotor system coupled to an IPT gene were produced as previously described (Schäfer et al., 2013; Schäfer et al., 2015b), and allowed a DEX-mediated induction of CK overproduction. irchk2/3 plants, fully characterized in Schäfer et al., (2015b) are silenced for two of three CK receptors. T. notatus prefers leaves of i-ovipt plants which have been treated with DEX and therefore have higher levels of CKs (Figure 6a). If T. notatus is given the choice between empty vector (EV) and irchk2/3 plants, mirids show a strong preference for EV plants, as shown in the lower damage levels on irchk2/3 plants (Figure 6b). Furthermore, we found pronounced differences in the reaction of the plants to the damage caused by T. notatus feeding. Mirid attack caused necrotic lesions in irchk2/3 plants, comparable to a pathogen-induced hypersensitive response, whereas this did not occur in WT, EV or i-ovipt plants (Figure 6c). To better understand the feeding preferences of T. notatus, we measured nutrient levels in irchk2/3, DEX-induced i-ovipt plants and EV plants (Figure 7). Starch and sucrose did not differ among the lines (Figure 7c, f). However, i-ovipt plants had higher concentrations of protein, free amino acids, glucose and fructose than did irchk2/3 plants (Figure 7a, b, d, e). The i-ovipt plants tended to have higher nutrient levels than did EV plants but the results were only statistically significant for glucose concentrations (Figure 7d). In contrast, irchk2/3 plants tended to have lower nutrient levels compared to EV, but these were only significantly lower for fructose concentrations (Figure 7e). From these results, we conclude that CKs play a dual role in the T. notatus-N. attenuata interaction: as important determinants of tissue palatability for T. notatus by enhancing nutrient contents, but also as important tolerance factors that allow plants to suffer negligible or lower fitness consequences of mirid attack than they would otherwise. We unambiguously demonstrated that T. notatus transfers two types of CKs, IP and its riboside IPR, to N. attenuata providing the first clear demonstration of CK transfer from an insect to a plant. IP has been generally considered one of the most active natural CKs based on classical activity assays (Gyulai and Heszky, 1994; Sakakibara, 2006) and high concentrations of IP have been previously reported in plant-manipulating endophytic insects, like leaf miners and gall-formers (Engelbrecht, 1968; Engelbrecht et al., 1969; Mapes and Davies, 2001; Straka et al., 2010; Yamaguchi et al., 2012; Body et al., 2013; Tanaka et al., 2013). We also showed that oral secretions, and in much lower amounts, frass of T. notatus, contained IP and IPR, thus providing a possible means of transfer. Concentrations of total CK content of N. attenuata leaves steadily increased during long-term T. notatus feeding, consistent with the observation that mirids transferred CKs to plants. The overall reconfiguration of the transcriptional activity of genes involved in CK degradation, inactivation, and biosynthesis upon T. notatus feeding did not correlate with the apparent changes in CK concentrations. This suggests that N. attenuata might activate a type of CK detoxification in response to mirid feeding and CK introduction. Additional support for the existence of a mechanism that counter-balances mirid-injected CKs comes from the observation that the leaf concentration of IP and IPR, the two CKs transferred by T. notatus, were unaffected or only slightly changed in plants by mirid feeding. This was surprising, considering that we estimated that after five days of whole-plant infestation, roughly half of the total IP in attacked leaves originated from mirids. Understanding to what extent the observed changes in cytokinin signaling result from mirid-mediated CK transfer is further complicated by the fact that CK levels respond as part of the herbivory-inducible defense signaling (Schäfer et al., 2015a; Schäfer et al., 2015c; Brütting et al., 2017). The dual role of CKs in plant growth and defense highlights the complexity of the fine regulation of CKs needed to regulate plant physiological responses. Not only CK quantities, but also CK structures, and the hormonal balance with other phytohormones may influence changes in metabolism upon insect feeding (Giron et al., 2013). Demonstrable effects on host plants induced by endophytic insects include alterations of plant morphology, changes in the nutritional quality of the affected tissues and the inactivation of plant defenses surrounding the attack sites (Giron et al., 2016). Whereas alterations of plant morphology are associated with only some endophytic insects, for example gall-formers, control of the nutritional quality of the infested tissues seems to be a common feature of all endophytic insect-plant interactions. N. attenuata leaves maintain their nutritional quality despite being heavily damaged by T. notatus feeding: only total soluble proteins (TSPs) decreased with heavy infestation, as in the whole-plant experiments, whereas concentrations of glucose, fructose, sucrose and starch remained unchanged. Previous studies in N. attenuata showed that wounding and application of oral secretions (OS) of M. sexta as well as M. sexta feeding reduced glucose and fructose concentrations by inhibiting soluble invertases. Such reductions are JA-dependent, and abrogated in transgenic lines impaired in JA production (Machado et al., 2015). A negative influence of jasmonates on plant primary metabolism has also been suggested by studies in a number of other plants (Babst et al., 2005; Skrzypek et al., 2005; van Dam and Oomen, 2008; Hanik et al., 2010; Tytgat et al., 2013). Hence, the fact that T. notatus feeding activated JA-signaling, but did not negatively influence soluble monosaccharide concentrations, suggested that an additional counterbalancing alteration in the primary metabolism of N. attenuata occurs during T. notatus feeding. Similar to what has been observed for carbohydrates, wounding and M. sexta OS application results in a 91% reduction of total soluble proteins (TSPs) in young rosette leaves (Ullmann-Zeunert et al., 2013), the same leaf stage used in this study. After 144 hr of continuous T. notatus infestation, during which proteins should be heavily depleted by mechanical cell-content damage – in contrast to the minor damage associated with OS elicitation (Halitschke et al., 2001) – TSP reductions were only ca. 75%. More surprisingly, a smaller T. notatus infestation (twenty mirids confined on a single leaf) did not change TSP contents at all during 144 hr of continuous feeding. These results are consistent with microarray analysis that compared expression patterns induced by T. notatus and M. sexta, which revealed that mirid-specific transcriptional responses occurred largely in primary metabolism (Voelckel and Baldwin, 2004). Thus, during T. notatus feeding, plant’s primary metabolism seems to be influenced by mechanisms different from the classical JA-mediated herbivory and wound responses. In contrast to the apparent sugar and starch homeostasis observed during T. notatus feeding and to the observations of Halitschke et al. (2011), we observed a decrease in photosynthetic rates during continuos mirid feeding. Reduced photosynthetic rate is a general response observed in a number of plant-insect interactions (Zhou et al., 2015) as well as in N. attenuata. Wounding and elicitation with M. sexta OS rapidly decrease photosynthetic CO2 assimilation and this reduction is mediated by the JA-precursor OPDA (Meza-Canales et al., 2017). We think that the discrepancy between our work and results from Halitschke et al. (2011) likely results from differences in the experimental protocols used: (1) we used a very heavy infestation, (2) the leaf area used to measure the photosynthetic rates of mirid-attacked leaves included both damaged and undamaged areas, (3) we did not normalize photosynthetic rates to intact undamaged leaf tissue. In any case, the reduction in the overall photosynthetic rates observed during T. notatus feeding was not consistent with unchanged starch and sugar levels and, together with the observation that T. notatus transfers CK during feeding, suggests the inhibition of senescence and/or transport of nutrients to the attacked leaves. Manipulation of plant defenses is another phenomenon often observed in endophytic insect-host plant interactions (Giron et al., 2016). We showed that T. notatus feeding activated N. attenuata JA-dependent defense pathways in a way consistent with previous studies; the increases in defense metabolites induced by T. notatus feeding were comparable to those elicited by M. sexta attack (Kessler and Baldwin, 2004). T. notatus is well-adapted to the specialized metabolism of N. attenuata; it prefers wild-type plants which are less susceptible to invasion by other herbivores, rather than those impaired in JA biosynthesis with reduced defense metabolites (Fragoso et al., 2014). Insects counter the presence of toxic metabolites in their host plants by detoxification or sequestration of toxic substances (Heckel, 2014), and the detoxification ability of T. notatus is suggested by the observation that it accumulates transcripts encoding detoxification enzymes in response to JA-dependent defenses (Crava et al., 2016). This fact, togheter with the finding that defense pathways were not down-regulated during the T. notatus feeding, point out that down-regulation of plant defense may not benefit T. notatus as much as its manipulation of its host’s nutritional status. Choice assays demonstrated that T. notatus is attracted to plant tissues with enhanced CK levels, both when CK levels were naturally higher, as in young plant tissues, and when CKs are experimentally increased using DEX-inducible transgenic plants (Schäfer et al., 2013). When CK perception was impaired as in the irchk2/3 line, mirids preferred WT or EV plants over the transgenic plants as shown by their different damage levels. This preference for higher CK levels and against irchk2/3 plants could either be a direct effect of CKs or – more likely – an indirect effect of CK-related processes. A direct attraction to CKs in insects has been discussed (Robischon, 2015) but to our knowledge there is no direct evidence that insects perceive CKs. Consistent with the second hypothesis, CK levels were not reduced in irchk2/3 plants compared to those of the EV line (Schäfer et al., 2015c). Thus, we infer that T. notatus prefers metabolites positively associated with the CK pathway. These might be molecules produced either by N. attenuata primary or specialized metabolism. For example, T. notatus is attracted to quercetin (Roda et al., 2003), and some related phenolic compounds are influenced by CK levels (Schäfer et al., 2015b; Brütting et al., 2017). Consistent with the primary metabolism hypothesis, we showed that nutrient levels of transgenic lines correlated with T. notatus preference. This inferred preference for nutrients is also consistent with T. notatus damage distribution on whole plants, which is concentrated on young, CK-rich and nutrient-rich tissues. These tissues are also better defended (Brütting et al., 2017) suggesting a possible trade-off between palatability and anti-digestive effects of the diet. However, specialized detoxification mechanisms likely allow T. notatus to feed with impunity on otherwise well-defended tissues (Crava et al., 2016), thus allowing T. notatus’s feeding choice to reflect the nutritional quality, rather than the defensive status, of its host. Our results provide evidence that a free-living insect transfers CKs which manipulates its host plant’s metabolism, likely for its own benefit. CK-mediated plant manipulation strategies have only been known so far from endophytic insects (Giron et al., 2016). The low mobility and intimate associations of endophytic insects with their host plants provides the selective environment for the evolution of mechanisms that allow them to manipulate host plant physiology and/or morphology. Species known for CK-dependent manipulation of host plants are not closely related to each other, and span several orders: Lepidoptera, Hymenoptera, Hemiptera and Diptera. The most studied examples are Lepidopteran leaf-miners which cause the green island phenomenon, like Phyllonorycter blancardella (Giron et al., 2007; Kaiser et al., 2010; Body et al., 2013; Zhang et al., 2017) or Stigmella argentipedella (Engelbrecht, 1968; Engelbrecht, 1971; Engelbrecht et al., 1969). Among gall-forming organisms, two species of the genus Bruggmannia are also capable of producing green islands (Fernandes et al., 2008). Other gallers that manipulate plant morphology can be found among hymenopterans, such as gall-wasps, dipterans such as gall-midges and gall-flies, and hemipterans such as psyllids and gall-aphids. Among these, a role for CKs in gall formation has been shown for the dipterans Eurosta solidaginis (Mapes and Davies, 2001) and Rhopalomyia yomogicola (Tanaka et al., 2013), the hymenopteran Dryocosmus kuriphilus (Matsui et al., 1975) and sawflies of the genus Pontania (Yamaguchi et al., 2012) and the hemipterans Pachypsylla celtidis (Straka et al., 2010) and the galling-aphid Tetraneura nigriabdominalis (Takei et al., 2015). The fact that species from different orders have developed similar mechanism of plant manipulation suggests either an ancient evolutionary origin or a convergent evolutionary trait. We propose that such CK-dependent manipulation is more widespread than previously thought, and is also shared with free-living insects like T. notatus. CKs transferred by T. notatus could originate from its host plant and be sequestered by the insect but also they could be synthesized by the insect itself or its associate endosymbionts. In fact, CKs are also produced by organisms other than plants, like fungi (Chanclud et al., 2016), bacteria (Costacurta and Vanderleyden, 1995) and nematodes (Siddique et al., 2015). It is thought that IP and IPR can be derived from tRNA, and this suggests that the substrate for CK biosynthesis is shared by all organisms (Persson et al., 1994), virtually enabling insects to produce CKs. The most recent studies on CK-mediated manipulation of plant physiology by insects suggested a role of endosymbiotic bacteria in CK production (Kaiser et al., 2010; Giron et al., 2013; Zhang et al., 2017). Antibiotic feeding experiments have revealed that endosymbionts like Wolbachia are the most likely producers of CKs in the leaf-miner P. blancardella (Kaiser et al., 2010; Body et al., 2013). Yet, this bacterium is unlikely responsible for CK production in T. notatus, as Wolbachia was not be detected in mirids from the same glasshouse colony used in our experiments, and was identified only very rarely from insects collected from the field (Adam et al., 2017). Free-living phytophagous insects are thought not to manipulate their host plant’s physiology to enhance the nutritional quality of their diet, as they are free to move to the best feeding locations on a plant. This work provides evidence of the ability of a free-living insect to introduce CKs into their host during feeding to maintain a better nutritional environment. We suggest that this mechanism may be commonly found in other free-living species and that it combines the benefits of the two different lifestyles: the ability to move, hide and choose the best feeding locations, and to manipulate the host plant via CK-transfer. Clarifying the details of the origins of the T. notatus-transferred CKs and studying their role in nature will provide new insights into the complex interactions that occur during plant-herbivore interactions. All chemicals used were obtained from Sigma‐Aldrich (St. Louis, MO, US) (http: //www. sigmaaldrich. com/), Merck (Darmstadt, Germany) (http: //www. merck. com/), Roth (Karsruhe, Germany) (http: //www. carlroth. com/) or VWR (Radnor, PE, US) (http: //www. vwr. com), if not mentioned otherwise in the text. CK standards were obtained from Olchemim (Olomuc, Czech Republic) (http: //www. olchemim. cz), dexamethasone (DEX) from Enzo Life Sciences (Farmingdale, NY, US) (http: //www. enzolifesciences. com/), HCOOH for ultra-performance LC from Fisher Scientific (Hampton, NH, US) (http: //www. fisher. co. uk/) or from Honeywell Riedel-de HaënTM (Morris Plains, NJ, US) (http: //www. riedeldehaen. com/), and GB5 from Duchefa (Haarlem, The Netherlands) (http: //www. duchefa-biochemie. nl/). We used the 31st inbred generation of Nicotiana attenuata (Torr. ex S. Wats.) originating from the ‘Desert Inn’ population from the Great Basin Desert (Washington County, UT, US) as wild-type (WT) plants (Baldwin et al., 1994). Transgenic plants were generated from WT N. attenuata, as described by Krügel et al., 2002) by Agrobacterium-mediated transformation. Empty vector transformed plants (EV) (line A-04-266-3) were used as controls in experiments that included other transgenic lines. The transgenic line irchk2/3 was transformed with a construct harboring inverted-repeat gene fragments to silence the expression of two of the three known CK receptor homologs NaCHK2 and NaCHK3 (Chase Domain Containing Histidine Kinase 2 and 3) and was previously characterized (Schäfer et al., 2015b). We used line A-12–356, which had a silencing efficiency of about 50%. The i-ovipt line (A-11–92 x A-11–61) contains a gene encoding the rate-limiting step of CK biosynthesis, the isopentenyltransferase (IPT) from Agrobacterium tumefaciens (Tumor morphology root; Tmr). The IPT gene is controlled by the pOp6/LhGR expression system, which allows transcriptional up-regulation in a specific tissue by the application of DEX (Schäfer et al., 2013). Application of DEX to the leaves of the plant induces the transcription of IPT which locally increases CK levels. DEX was dissolved in lanolin paste with 1% DMSO at a final concentration of 5 µM. For control treatments, we used 1% DMSO in lanolin. The lanolin paste was applied to the petioles of the leaves 24 hr prior to other treatments as previously described (Schäfer et al., 2013). Seeds were sterilized and germinated on Gamborg B5 media as described by Krügel et al. (2002) with modifications as previously described (Brütting et al., 2017). For soil growth conditions, ten days after germination, plants were first transplanted to TEKU pots and 10 days later into 1 L pots. For hydroponic growth conditions, the plants were transferred after 12 days into 50 mL hydroponic culture single pots and 10 days later into 1 L hydroponic containers. Conditions for hydroponic culture were previously described (Ullmann-Zeunert et al., 2012) as were conditions for soil growth (Brütting et al., 2017). For the damage determination experiment in the glasshouse, single plants were grown in 4 L pots. Plants were maintained under glasshouse conditions (22–27°C; ca. 60% RH, 16: 8 light: dark regime) as previously described (Adam et al., 2017). To prevent T. notatus infestation of the main glasshouse facility of the MPI-COE, we maintain the colony in a separate glasshouse located in Isserstedt, Germany, approximately 7 km from the main glasshouse facility. Plants were germinated in the main glasshouse facility and transferred to the Issersted glasshouse just before plants began to flower, when plants had main stems about 25 cm tall. In both glasshouses, plants were maintained at comparable growth conditions. After transferring plants to the Isserstedt glasshouse, we allowed for at least two days of acclimation before initiating experiments. The colony of T. notatus (Distant, 1893) (Figure 1,1A) originated from insects caught in the vicinity of the Brigham Young University/Max Planck field station at Lytle Ranch Preserve in the Great Basin Desert (Washington County, UT, US) and was annually refreshed with insects caught from the same field site. The colony was maintained in cages made of acrylic glass (2 × 1 × 1 m) with a fine mesh for air circulation. Cages were maintained under the same glasshouse growth conditions that were used for the cultivation of N. attenuata (27°C; ca. 60% RH, 18: 8 light: dark regime). We fed insects with hydroponically grown WT N. attenuata plants. Fresh plants were provided weekly and remained in the cages for several weeks to allow nymphs to hatch from eggs laid in the older plants. Insects were collected from the cage for experiments using an insect exhauster. Prior to being clip-caged onto N. attenuata leaves, insects were anaesthetized with CO2. For the artificial diet we dissolved amino acids (L-alanine, 50 mg; L-arginine, 30 mg; L-cysteine, 20 mg; glycine, 20 mg; L-histidine, 30 mg; L-leucine, 30 mg; L-lysine, 20 mg; L-phenylalanine, 30 mg; L-proline, 80 mg; L-serine, 100 mg; L-tryptophan, 500 mg; L-tyrosine, 10 mg; L-valine, 40 mg; L-asparagine, 200 mg; L-aspartic acid, 200 mg; L-glutamine, 500 mg; L-glutamic acid, 300 mg; L-isoleucine, 20 mg; L-methionine, 10 mg; L-threonine, 100 mg), sugars (glucose, 400 mg; fructose, 150 mg; sucrose, 800 mg) and vitamins (Vanderzant Vitamin Mix, 650 mg) in 40 mL water and sterile filtered the solution. Additionally, we prepared an agar solution (1 g Agar-Agar in 60 mL water) which was sterilized by autoclaving. After cooling the liquid agar solution to approximately 60°C in a water bath, we added the nutrient solution and aliquoted the diet under sterile conditions in single 0. 5 mL micro-centrifuge tubes, where it solidified. These tubes were stored at 4°C until use. For experiments, T. notatus were placed in plastic boxes (10 × 6 × 6 cm) covered with paper tissue and sealed with a perforated lid. In each box, 15 to 20 mirids were placed with a tube containing the artificial diet as the sole food source in addition to a source of water. The diet was exchanged with fresh diet every day. The shaded boxes were kept in the glasshouse. After 5 days the surviving mirids were collected, flash-frozen in liquid nitrogen and stored at −80°C until CK extraction. T. notatus oral secretions were collected as previously described (Halitschke et al., 2011) with minor modifications. In brief, we placed 15 to 20 mirids in a single plastic box (10 × 6 × 6 cm) covered with paper tissue and sealed with a perforated lid. In each box, we placed an inverted scintillation vial lid filled to the brim with sugar solution (~3 mL, 40 mM glucose) as the sole food and water source. Lids were covered by a stretched thin layer of Parafilm (Neenah, WI, US) which allowed for stylet penetration. After 24 hr, we collected the lids, removed the sugar solution with a syringe and carefully dissolved (with MeOH) the frass spots deposited on the parafilm. As a control, similarly packaged sugar solutions in boxes lacking mirids maintained under the same conditions were used. Frass and sugar solution samples originating from the exposure to approximately 100 mirids were pooled. Pooled sugar solutions were freeze-dried overnight. Prior to CK extraction, extraction buffer was used to dissolve the evaporated sugar solution. To measure the defense responses of N. attenuata to T. notatus feeding, whole plants were exposed to mirid attack in the T. notatus rearing cages and damaged lamina from the first (lowest) stem leaf were collected after three days of feeding. Control plants were placed in a similar but mirid-free cage under same conditions. Collected lamina pieces were flash-frozen in liquid nitrogen and stored at −80°C until analysis. For kinetic analysis of CKs, JA, JA-Ile, primary metabolites and photosynthetic rates during T. notatus attack, we used two different experimental setups which differed in the area damaged by T. notatus, and the number of T. notatus used to inflict the damage to the N. attenuata plants. In the first setup, only one leaf per plant was exposed to T. notatus. We enclosed twenty adults on the first (lowest) stem leaf in a round plastic clip-cage (7 cm dimeter, 5 cm height). Clip-cages had holes covered with a fine mesh for air ventilation. Control plants received empty clip-cages to control for the effects of caging leaves. We also sampled leaves from plants without clip-cages. Before sampling, mirid mortality was scored, and samples with more than 50% mortality were discarded. Control and damaged leaf lamina were harvested at nine time-points from separate plants (0,1, 3,24,48,72,96,120 and 144 hr), flash-frozen in liquid nitrogen and kept at −80°C until analysis. In the second setup, the entire aboveground plant was exposed to mirids, by placing the plants directly into the T. notatus rearing cage; control plants were placed in a similar, empty cage. Damaged lamina from the first (lowest) stem leaf were sampled at seven time-points from separate plants (0,24,48,72,96,120 and 144 hr). Both experiments were started in the morning (09: 00 – 12: 00) and each harvest time represented at least three replicate plants. For each experiment, phytohormone concentrations (CKs, JA and JA-Ile), sugars (sucrose, fructose and glucose), starch, total soluble proteins, photosynthetic rates and chlorophyll contents were measured. Caffeoylputrescine and nicotine were extracted and determined by UHPLC-ToF-MS by analyzing extracted ion chromatograms as previously described (Schäfer et al., 2015c; Brütting et al., 2017). For extraction, 80% MeOH (v/v) was used for approximately 100 mg of frozen and ground leaf material from each sample (exact tissue masses were recorded). Values are presented as peak area * g FM−1. TPI activity was determined using a radial diffusion assay (Jongsma et al., 1994; van Dam et al., 2001) with approximately 50 mg of frozen and ground leaf lamina (exact tissue masses were recorded). TPI activity was normalized to leaf protein content. The protein content of the extracts used for the TPI assay was determined using a Bradford-assay (Bradford, 1976) on a 96-well microtiter plate. Total soluble proteins were extracted from 50 mg of frozen ground leaf lamina (exact tissue masses were recorded) in 0. 5 ml 0. 1 M M Tris-HCl (pH 7. 6) following the protocol described by Ullmann-Zeunert et al. (2012). Protein concentrations were measured using a Bradford assay (Bradford, 1976) on a 96-well microtiter plate using bovine serum albumin (BSA) as standard. Free amino acids were extracted from leaf lamina by acidified MeOH extraction [MeOH: H2O: HCOOH 15: 4: 1 (v/v/v) ] and analyzed by liquid chromatography coupled to a triple quadrupole MS (Bruker EVOQ Elite, Bruker Daltonics, Bremen, Germany; www. bruker. com), as previously described (Schäfer et al., 2016). Glucose, fructose, sucrose and starch were determined following the protocol described by Machado and colleagues (Machado et al., 2015). Briefly, 100 mg plant tissues (exact tissue masses were recorded) were extracted first with 80% (v/v) ethanol and later twice with 50% (v/v) ethanol, each by incubation for 20 min at 80°C. Supernatants from all extractions were pooled, and sucrose, glucose and fructose were quantified enzymatically as previously described (Velterop and Vos, 2001). The remaining pellets were used for an enzymatic determination of starch content (Smith and Zeeman, 2006). Net CO2 assimilation rate was measured with a LI-COR LI-6400/XT portable photosynthesis system (LI-COR Inc., Lincoln, NE, US). All measurements were conducted using a 2 cm2 chamber, at constant CO2 (400 µmol CO2 mol air−1), light (300 μmol m−2 s−1 PAR), temperature (25–26°C) and relative humidity (20–40%). Measurements of photosynthetic rates of leaves with clip-cage were specifically done on the area included in the clip-cage. We measured photosynthetic rates of control leaves and leaves damaged by T. notatus. Leaves with clip-cages were analyzed in the covered area shortly after removal of the clip cage. Chlorophyll was quantified using a Minolta SPAD Chlorophyll meter 502. Chlorophyll content is displayed in arbitrary SPAD units. Each sample value is the mean of chlorophyll content measured at three different random spots from each analyzed leaf. Leaves with clip cages were analyzed in the covered area shortly after the removal of the clip cage. RNA was extracted with TRIzol (Thermo Fisher Scientific, Waltham, MA, US), according to the manufacturer’s instructions. cDNA was synthesized by reverse transcription using oligo (dT) primer and RevertAid reverse transcriptase (Thermo Fisher Scientific). Real-time qPCR was performed using actin as a standard on a Mx3005P qPCR machine (Stratagene, San Diego, CA, US) using a qPCR Core kit for SYBR Green I No ROX mix (Eurogentec, Seraing, Belgium). The primer sequences are provided in Supplementary file 2. Levels of defense signaling compounds after three days of T. notatus herbivory were quantified as described by Kallenbach et al., 2010). JA, JA-Ile, OPDA and SA were analyzed as described by Kallenbach and colleagues (Kallenbach et al., 2010) and ABA as described by Dinh and colleagues (Ðinh et al., 2013). Kinetics of CKs, JA and JA-Ile during 144 hr of T. notatus attack was measured as previously described (Schäfer et al., 2016). In brief, phytohormones were extracted from ca. 100 mg of fresh ground leaf material (exact sample masses were recorded) using acidified methanol and purified on reversed phase and cation exchange solid-phase extraction columns. The measurements were done via liquid chromatography coupled to a triple quadrupole MS (Bruker EVO-Q Elite) equipped with a heated electrospray ionization source. The method was extended for the detection of 15N-labeled CKs. The parent → product ion transitions for 15N labeled CKs are listed in Supplementary file 3. Chromatograms of IP, [D6]-IP, [15N5]-IP as well as IPR, [D6]-IPR and [15N5]-IPR are shown in Figure 4—figure supplement 4 and 5. The same extraction method was used for CK extraction from T. notatus, using approximately 10 mg of ground material (five pooled samples of ca. 20 adults; exact sample masses were recorded). N. attenuata plants with more than 98% of their total nitrogen content as 15N were obtained following the protocol described by Ullmann-Zeunert and colleagues (Ullmann-Zeunert et al., 2012). Briefly, twelve days after germination, plants were transferred into individual 50 mL hydroponic culture containers with Ca (15NO3) 2 as the sole nitrogen source. Ten days later, the plants were transferred to 1 L hydroponic culture chambers with the same 15NO3- concentration as of K15NO3. Once per week, the plants were fertilized with 1 mM K15NO3 and the containers were maintained at 1 L with distilled water. To generate 15N labeled T. notatus, we reared insects for an entire generation on fully 15N-labelled N. attenuata plants. Two-hundred adult females were transferred to a 47. 5 × 47. 5 × 93 cm insect cage with four 15N-labelled N. attenuata plants in the early elongation stage of growth. Females were allowed to lay eggs for four days and subsequently removed. 15N-labelled plants were fertilized once a week (as described above), and after three weeks two fresh 15N-labelled plants were added to the insect cage. One week after the first adults emerged, the 15N labeled mirids were collected and used for the cytokinin transfer experiment. Two types of experiments were conducted to quantify the transfer of cytokinins from mirids to N. attenuata plants. In the first, twenty 15N-labelled T. notatus adults were quickly anesthetized with CO2 prior to clip-caging on a lower stem leaf of a 14N-grown plant, with one clip-cage per plant. We collected and froze in liquid nitrogen the leaf lamina corresponding to the area included in the clip-cage and thus damaged by mirids at different time-points: 0,3, 6,24,48,72,96 and 120 hr. Each harvest time included at least three replicate plants. Samples were stored at −80°C until analysis. In the second type of experiment, five 15N-labelled N. attenuata plants were placed in the mirid rearing cage. One lower stem or rosette leaf per plant was harvested at 0,3, 6,24,48,72,96 and 120 hr, thus each harvest time represented five replicate plants. Plants were transferred once per day to a different cage to ensure that mirids did not accumulate 15N-labeled metabolites. We separated the leaf lamina from the mid-rib and froze the lamina in liquid nitrogen, and stored the samples at −80°C until analysis. In the field, the damaged area on different leaf types was estimated as % of the total leaf area. The proportion of damaged leaf area for each leaf was visually estimated and the leaves were grouped into three different types (Figure 6—figure supplement 1a). Finally, we calculated average leaf damage for all leaf types: rosette leaves, the first (oldest) three stem leaves and all younger stem leaves and side branches. Similarly, T. notatus damage distribution within the plant was evaluated under controlled conditions in the glasshouse. In this experiment, a total of seven WT plants were used to form four replicates. The replicates consisted of three pairs of WT plants and one single plant, where each replicate was placed in one cage, meaning that one cage represented one replicate with no matter if there were one or two plants inside the cage. The plants in each cage were exposed to adults of T. notatus (n = 10 insects/plant) for one week. T. notatus infestations continued for an additional two weeks, where in the last week, five insects/plant were added to each cage. T. notatus damage was estimated from high resolution pictures of 15 leaves per plant at standardized rosette, mid stem, and young stem positions. Using Photoshop (Adobe), the damage was evaluated and expressed as a percentage of total damage per plant. For choice assays conducted in the field between young and fully mature leaves, we collected insects from native populations at our field station in Utah, USA. Ten to fifteen T. notatus adults were placed in a plastic cup. The cup was connected to two other plastic cups (Figure 6—figure supplement 1b), one enclosing a fully mature stem leaf and the other enclosing young, growing leaves (apical meristem and young leaves which had not yet completed the sink-source transition). To prevent desiccation, leaf petioles were submerged in water in a 2 mL plastic microcentrifuge tube. As the insects are night-active, after one night (12 hr) during which the insects could choose between the two containers, we counted the number of mirids in each. For choice assays between WT and irchk2/3 plants, we placed plants in a large mesh-enclosed cage in the glasshouse (3 × 4 × 1. 6 m) into which 500 T. notatus were released. The damage on each plant (as described above) was estimated 10 days later. Data from choice assays on i-ovipt plants were taken from a previously published dataset (Schäfer et al., 2013). Plants were either treated with pure lanolin (LAN) as a control or with DEX-containing lanolin as described above. We treated the first (oldest) ten stem leaves of a flowering plant and placed one DEX- and one LAN-treated plant in one 47. 5 × 47. 5×93 cm insect cage. About 100 T. notatus adults were added to the cage, and the damaged leaf area was estimated after 10 days. The average damage level from all 10 treated leaves from each plant was counted as one replicate. Data were analyzed using R 3. 3. 1 (2016-06-21; http: //www. r-project. org). Statistical tests and number of replicates, as well as transformations to data in order to meet assumptions of a test (homoscedasticity, normality), are provided in the figure legends. Normality of data sets was assessed by Shapiro–Wilk tests and homoscedasticity by Levene' s test. If not mentioned otherwise, time course data were analyzed with ANCOVA with mirid feeding as factor and time as continuous explanatory variable. If the response variable was not linearly dependent on time we used two-way ANOVAs (TWA) with mirid feeding and time as factors. In all the analyses of experiments with the data from clip-cages, we only used data from control clip-cages and clip-cages with mirids. Differences were considered significant when p<0. 05.

Many insects use plants for food and for shelter. To protect themselves, plants often develop defense mechanisms that deter or debilitate their attackers, such as producing toxins or storing nutrients away from the attacked tissues. But some insects manage to counter the plants' defense responses. Such species are often less mobile and spend a large part of their life in a restricted area of the plant, for example, inside plant tissues. Also known as 'endophytic' animals, these insects can even manipulate the signaling system in a plant, such as a class of plant hormones called cytokinins, which help plants to grow and to develop seeds and nutrient-storing fruits or young leaves. Researchers have previously assumed that endophytic animals target cytokinins because they are restricted to living in certain areas within the plant, and - unlike 'free-living' insects - lack access to other, potentially more nutritious feeding sites. By modifying cytokinins, the location-bound insects could create their own 'nutrient pool'. Until now, it was unclear how insects transfer cytokinins to a plant and if this ability was restricted to endophytic insects. To investigate this further, Brütting, Crava et al. studied the response of coyote tobacco plants infested with a free-living insect, the sap-sucking bug Tupiocoris notatus. The experiments revealed that the insects' bodies contained large quantities of a type of cytokinin, even when insects were raised on artificial diets. Brütting, Crava et al. then developed a method to clearly distinguish cytokinins present in the insects from those produced by the plants to test whether T. notatus can transfer these plant hormones during feeding. The results showed that similar to endophytic insects, T. notatus injects cytokinins into the attacked leaves, presumably to create a stable nutritious environment. Insects represent the largest and most diverse group of organisms on Earth, including many crop pests. Despite their detrimental impact on the environment and the health of the farmers, pesticides are used predominantly for pest control. A better understanding of how insects use cytokinins to increase the nutritional value of the leaves may help us to find ways to increase the crop's tolerance to insect attacks.

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Gay-rights activist and award-winning author Larry Kramer is 79 and in failing health, but that won’t defuse the impact of his latest bombshell project: the first 800-page instalment of a two-part history of America that tells of the secret gay life of figures from Alexander Hamilton, George Washington and Abraham Lincoln to Mark Twain, Herman Melville and Richard Nixon. The American People: Volume 1, subtitled Search for My Heart, has taken nearly 40 years to complete and may prove to be one of the most provocative historical, or pseudo-historical, accounts of American history. Kramer, who is co-founder of Aids services group Gay Men’s Health Crisis and the Aids Coalition to Unleash Power (Act Up), as well as a chronicler of queer life with plays including The Normal Heart and The Destiny of Me, said the book is a labour of love designed to counter what he feels to be the exclusion of gays – or gay life – from history books. “It may look like fiction, but to me, it’s not,” Kramer told the New York Times last week. “Most histories have been written by straight people. There has never been any history book written where the gay people have been in the history from the beginning.It’s ridiculous to think we haven’t been here for ever.” The American People, Volume I: Search for My Heart is causing consternation among historians, who say there is little evidence to back Kramer’s claims. Ron Chernow, author of an epic 2004 biography of Alexander Hamilton – the statesman Kramer claims was at least bisexual if not entirely gay – cautions against “ransacking history in service of a political agenda”. Kramer also claims that John Wilkes Booth assassinated Abraham Lincoln not because he was unhappy that the South was losing the civil war, but because Lincoln had spurned him. “You only have to look at photographs of Wilkes and [co-conspirator] Lewis Powell to see that they’re full of their own beauty. We call it gaydar – the thing straight historians don’t have. Or take Mark Twain. He had a huge gay life.” Kramer has a history of initiating high-profile disputes. He had a war of words with Tony Kushner over acknowledging Lincoln’s orientation in his screenplay for Steven Spielberg’s 2012 biopic of the civil war president. He scrapped with Barbra Streisand over her planned film adaptation of The Normal Heart. The author insisted it should include gay sex; Streisand retorted that her intention was “to promote the idea of everyone’s right to love. Gay or straight!” The American People is likely to rankle with historians as there is no evidence many of historical figures were gay. He claims Lincoln biographer Doris Kearns Goodwin became “hysterical” at the suggestion of homosexual tendencies in the president but this was, he added in an interview with NPR, “only because she didn’t write it first”. The book, which has been labelled as a novel to avoid legal complications, has divided US reviewers. LGBT magazine The Advocate said that at points in Kramer’s book “the reader will feel like the audience at Springtime for Hitler”. The New York Times calls it “a far-reaching historical exposé”. The first volume spans pre-Columbian Florida, Puritans and early settlements (brimming with same-sex desire), the American revolution, the civil war and the years leading to the second world war. It includes a history of syphilis, hepatitis, hatred, ostracism, concentration camps, the CIA, and a shadowy disease. The disease is named as Aids in the second volume, due in 2017, which will bring the history to the present. Facebook Twitter Pinterest Larry Kramer with film star Julia Roberts backstage at the Emmy awards last year. Photograph: Todd Williamson/Invision/AP Aaron Hicklin, editor of Out magazine, said young members of the LGBT community are becoming interested in their history, a history that will not automatically be passed on because so many of the elder generation died young of HIV/Aids. Whether it is absolutely accurate or not, The American People speaks to a need across gay and straight communities to revise historical accounts from which sexual orientation was absent. This can be seen in the mainstream – Benedict Cumberbatch as Alan Turing in The Imitation Game, for instance, or the story of Harvey Milk in Milk (2008) – but rarely goes further back in time. “The gay historical timeline tends to go back as far as Oscar Wilde and no further,” said Hicklin. “There were pioneers before us but no one took the time to write about them, and there’s an appetite to claim a history that has been hidden from us.” From some quarters there have been calls for a exclusively gay branch of archaeology directed solely at establishing sexual orientation (Kramer claims traces of semen found in the stools of English settlers prove his point). But where gay rights warriors sought to define themselves as gay in defiance of the stigma around Aids, Hicklin considers that sexual orientation is no longer a singular marker of identity. “Young people feel much more a part of mainstream society, so a lot has been forgotten about the price that was paid. “For Larry Kramer, it’s important to carry on being the archetypal activist who wants to queerify history and bring it out into the public domain.” Perhaps the most remarkable aspect of The American People is that it was written at all. Kramer began writing it in 1975, soon after publication of his groundbreaking novel Faggots. He shelved it until 2000, when his liver was failing and he was given weeks to live. By 2010, Kramer having had a liver transplant, the manuscript had swelled to 4,000 pages. Kramer’s editor at Macmillan says he is in no doubt that writing is what keeps him alive. Kramer believes the injustice is continuing. Thirty five years since HIV/Aids was identified there is still no cure, and violence against gay people is increasing in several parts of the world. “We should have our own army as gays,” he told The Advocate. “It’s lovely that we can get married, but that’s really small potatoes compared to what we don’t have, which is equality.” “Magnum opus” doesn’t seem like a robust enough phrase to describe the scope of “The American People,” which stretches back to the prehistoric swamps of the Everglades and concludes, in the second volume, in contemporary New York City. When Farrar acquired the two volumes in 2010, the narrative had swelled to around 4,000 pages. Advertisement Continue reading the main story Blending farce and tragedy, autobiography and fiction, it opens as Fred Lemish, a stand-in for Mr. Kramer, is struggling to finish writing a book titled “The American People,” a far-reaching historical exposé that describes Alexander Hamilton, George Washington, Abraham Lincoln, Mark Twain and other major historical figures as gay. “He’s been struggling with this history for many years,” Mr. Kramer writes of his fictional counterpart. Mr. Kramer said he was driven to write the book because he had long felt that gays had been excluded from history books, written out or ignored. “Most history is written by straight people, and they don’t have gaydar,” Mr. Kramer said during an interview at his apartment in Greenwich Village. “People say, ‘Can you prove to me that George Washington was gay?’ and I say, ‘Can you prove to me that he wasn’t?’ ” (As evidence, Mr. Kramer notes that Washington “was surrounded by men, and he designed all their uniforms himself.”) In the novel, and in conversation, Mr. Kramer criticizes historians and scholars like Stacy Schiff, Ron Chernow and Doris Kearns Goodwin for glossing over homosexuality in American history. If he had had his way, he would simply have called the book a work of history rather than fiction, he said. “Farrar Straus said call it a novel, that way the lawyers will leave you alone,” he said. “But I believe everything in the book is true. It may look like fiction, but to me, it’s not.” Mr. Chernow, the author of a well-regarded 2004 biography of Alexander Hamilton, said he was surprised to be called out in Mr. Kramer’s novel, particularly as he brings up in his book the possibility that Hamilton might have been bisexual. “I’m glad that Larry Kramer is raising the issue, I’m just mystified at why he’s attacking me, when I thought he would have applauded the fact that I take this seriously,” Mr. Chernow said. “It’s a legitimate issue for historians or novelists, but we also have to be careful not to ransack history in service of a political agenda.” As for Mr. Kramer’s theory about Washington, Mr. Chernow countered that it was common in that era for generals to design soldiers’ uniforms. If conflicting early reviews are any indication, “The American People” is likely to be as polarizing and controversial as Mr. Kramer’s other works. While Kirkus Reviews praised the novel as “breathtakingly well-written,” others have taken a dimmer view. “To call it a rough read at times would be an understatement,” a reviewer wrote in Publishers Weekly. Photo “It’s like being hit by a truck, the book is so all over the place,” the novelist Andrew Holleran, who has known Mr. Kramer since the 1970s, said of the book. “What I admired as a writer was the imaginative energy and the amount he takes on.” Jonathan Galassi, the president and publisher of Farrar, Straus and Giroux, said the book struck him as ambitious and provocative, both as a work of literature and as an act of protest. Advertisement Continue reading the main story “This book is, in a way, a culmination of all his artistic activity,” he said. “Larry is a great polemicist and a fighter, and this book is part of his polemic about gay history and the roles of gays in our society. He’s still fighting and he’s using art to do it.” Please verify you're not a robot by clicking the box. Invalid email address. Please re-enter. You must select a newsletter to subscribe to. Sign Up Receive occasional updates and special offers for The New York Times's products and services. Thank you for subscribing. An error has occurred. Please try again later. View all New York Times newsletters. Mr. Kramer never set out to be a political activist and said that in some ways he was ill-suited to the role. “They say I’m obnoxiously noisy and angry, but I’m actually a bit of a shy person,” he said. Early in his career, Mr. Kramer wanted to write comedy. After graduating from Yale, he worked in the film industry, and was nominated for an Oscar for his screenplay adaptation of the D. H. Lawrence novel “Women in Love” (1969). In 1978 he published a controversial novel, “Faggots,” a satire of rampant drug use and promiscuity among gays in New York. “It’s meant to be a comic novel — it made me laugh,” he said. His mission changed in the early 1980s, when H.I.V. and AIDS began spreading rapidly among gays and claimed the lives of two friends. He was a founder of the Gay Men’s Health Crisis, a nonprofit support organization for those affected by the disease, and later started Act Up, a protest group that brought attention to the AIDS crisis. His activism spilled into his writing, with plays like “The Normal Heart” and “The Destiny of Me,” which center on gay characters confronting the AIDS epidemic and take aim at indifferent politicians and institutions. Mr. Kramer’s activism was closely and messily intertwined with his personal life. In 1989, he learned he was H.I.V. positive and suffering from liver damage, the result of hepatitis B. He credits Dr. Anthony Fauci, director of the National Institute of Allergy and Infectious Diseases, for being among a handful of doctors who saved him by giving him experimental drugs. Mr. Kramer once vilified Dr. Fauci in print, calling him “an incompetent idiot” and a murderer, for his early approach to fighting the spread of AIDS. “The man who I criticize for allowing the virus to spread is the man who saved my life,” Mr. Kramer said. Mr. Kramer’s acerbic and belligerent style has often alienated friends and supporters. He and the playwright Tony Kushner had a blowout over Mr. Kushner’s screenplay for the Steven Spielberg movie “Lincoln” (2012), because Mr. Kramer felt that Mr. Kushner should have depicted Lincoln as a gay man. “He and I had a major falling out over this,” Mr. Kramer said. “I so wanted him to put that in the movie.” (In “The American People,” Mr. Kramer strikes back with his own fictional Lincoln, who has a male lover and often lustily utters “mighty fine” after their encounters.) These days, Mr. Kramer seems much less bellicose, though no less passionate. He speaks in a tremulous near-whisper and relies on a hearing aid and a cane. He spent much of last year in hospitals being treated for abdominal infections and almost died twice, he said. He was bedridden in 2013 when he and his longtime partner, the architect David Webster, were finally married in the intensive care unit of NYU Langone Medical Center. Mr. Kramer’s writing has taken on a greater urgency lately. He has been writing for five or six hours every day, seven days a week. His progress is visible in the towering stacks of manuscript pages that cover a large table in his living room, which doubles as a work space. He is also working on a screenplay for a sequel to “The Normal Heart” and is the subject of an HBO documentary, “Larry Kramer: In Love and Anger,” which will be shown in June, just after his 80th birthday. As he works toward finishing what he views as the defining work of his career, Mr. Kramer said he hoped to be remembered for his art as much as his activism. “It goes against the grain in this country to do both, and that’s why I’m not taken seriously,” he said. “I want this book to be taken seriously as a work of art and a work of thought.” Kramer on Kramer Photography By Benedict Evans Larry Kramer’s long awaited new novel, The American People: Volume I, subtitled Search for My Heart, is finally seeing the light of day this April. It’s just the first volume, though “just” may not be the right word, since it’s 800 pages. There are times when the reader will feel like the audience at “Springtime for Hitler.” One will also find oneself laughing out loud, thinking hard, and being thrilled that someone has taken on American history from the viewpoint of gay people. The book is the history of syphilis, hepatitis, hatred, ostracism, the settling of America, concentration camps — American and German — Jews, the CIA, and something called The Underlying Condition (which we suspect will become, in the second volume, AIDS). It begins in pre-Columbian Florida, with monkeys in the Everglades, and goes on to the Puritans, the American Revolution, the Civil War, and World War II, ascribing same-sex desire to George Washington, Alexander Hamilton, Mark Twain, Abraham Lincoln, and so many other figures central to American history that there is no point in listing them here. It also contains the moving saga of a young boy growing up in Washington, D.C., during World War II, who learns he’s a “sissy.” In Washington, Kramer writes, “Moderation is everything,” but in this novel moderation has no place; with hundreds of characters, from nuns to Nazis, this book is in essence a fantasia on American history. How historians will receive it is hard to predict — but Kirkus Reviews called it “breathtakingly well written,” and the Publishers Weekly reviewer said it left him wondering “what the hell will happen next.” To find out, I sat down with Larry in his apartment on Washington Square Park in Manhattan. I just finished reading your new novel, The American People: Volume I. Is it true that you started writing it in 1975? I started when I finished Faggots, long before AIDS came along, and I kind of put it to the side. I wrote a couple of plays in between, so it was done in bits and pieces over the years. And it wasn’t until I got really sick, when I had the liver transplant [in 2000], that I got serious about making it as a whole. Because I didn’t think I was going to live. Now I’ve got to finish Volume II. If you started it before AIDS had even emerged, what was the impetus then to write the book? Was it the idea that gays have been written out of history? Oh, I don’t know. Why does every gay writer start out? To write his Proust? And so I wrote my Proust. The title comes from that speech by Reagan which really did hit me, where I knew he was talking about “the American people,” and I knew that I was not part of that crowd that he was talking about. It was so obvious. There has never been any history book written where the gay people have been in history since the beginning. It’s ridiculous to think that we haven’t been here forever. First, I started doing research into people who I felt were important, like Washington and Lincoln, and — So far we’ve got George Washington, Alexander Hamilton, the Marquis de Lafayette, Baron von Steuben, Lincoln, Andrew Jackson, Franklin Pierce, James Buchanan, Ralph Waldo Emerson — which really surprised me — Hawthorne, Melville, John Wilkes Booth, Mark Twain, Eleanor Roosevelt, of course, James Forrestal, Richard Nixon, Herbert Hoover. You’re writing three kinds of history in this book. There were times when you would quote from a book you had read, and you would acknowledge the author and the source. To me, that’s just history. Then there were times when I was in a kind of phantasmagoria nightmare scene, and I thought, This is not history anymore; we’re now in Larry’s imagination. And there’s the most deceptive category, the middle, in which it seems to me that you were mixing real source material with imagination. You didn’t want to write a history in which you simply extracted little-known facts about gay people in history and put it together as straight history? No, originally I didn’t want to call it a novel, I just wanted to call it The American People and let people figure out what it was. There isn’t anything in the book that I don’t agree with, or have some belief in, and if I couldn’t find some source that would give me the right to say it, I said my version of it, as with John Wilkes Booth. Did you ever look at the pictures of all the guys who were charged with murdering Lincoln? You go on about how good-looking Lewis Powell [a co-conspirator of Wilkes Booth] was. He was just a real hot number. A hot number, and he knew it. They were such an unlikely lot of people. And the women had nothing to do with it, and how did they even get together? I had never read anything where they’ve been able to convince me why they were all in the same group somehow. Just to say they were Southerners and all that shit. I didn’t buy that. So what you’re saying in a sense is that you used gaydar? I used gaydar. What else have I got? This is a book that starts in the pre-Columbian era in the United States, with monkeys in the Everglades in a kind of James Michener way. You’re taken back to the foundation of the continent, and there’s a great deal about colonial times, the American Revolution, the Civil War, up to the ’50s and the McCarthy era — and that’s just Volume I. So it’s this huge, sprawling, wide-ranging thing, which halfway through breaks off into a pretty conventional story about a neighborhood in Washington, D.C., in which one of the characters, Daniel Jerusalem, grows up dealing with the fact that his father beats him up and has called him a sissy. What then became clear to me was this was really about where did AIDS come from. And by the end of Volume II I will tell you where it came from, and what I think caused it. And what should have been done, that wasn’t done. Because, we are gay, they wouldn’t do it. I think most people know or expect this book to be about AIDS. But this is also about German scientists that came to the United States, it’s about the science of eugenics, it’s about Henry Ford, it’s about anti-Semitism, and it’s about the four horsemen of the apocalypse, which are amoebas — what is the line? I love this line. Piss, shit, amoebas and — Piss, shit, and it’s the history of blood, it’s the history of diseases, it’s the history of concentration camps. You found a way to write about anything that interested you. The history of syphilis is in this book. How did you know when to leave stuff out? It was once much longer and it was much wilder. I can write in a crazy way, making things up, and I made a lot of drugs’ names up and symptoms up, and then one of the editors, along the way said: “You really don’t have to make that up. Why don’t you just use the real one?” What are you expecting reactions to be? You got very good reviews in Kirkus and [Publisher’s Weekly]. Oh unbelievable. I always expect to get trashed for everything. I never got a good review before in my life. Even Normal Heart. How much of Volume II is written? I’m writing. It has to come out a year after this one. And also, the phenomenal success of the movie of The Normal Heart has touched me a lot. It was one of HBO’S biggest shows ever. And you realize, more people saw my play in those two hours that one night, than you could fill a theater with for years. Larry Kramer and his husband, David Webster I’m writing it now.It’s all I live for. If I didn’t have my writing right now, I would go crazy. Truly, I don’t feel well.I don’t think that way. I faced death a couple times, and that had me look at this book differently. Imagine what it’s like facing death. David [Webster, Kramer’s husband] said I almost died three times.My relationship with Tony has been very complicated because I wanted him to deal with Lincoln [in the script he wrote for Spielberg’s movie] and he didn’t want to indicate in the movie that he was gay.Oh yeah, we speak now. We were both upset by it because we were both very fond of each other. But I do get self-righteous about some things.Well, because it wasn’t happening to us; it was happening to them. There’s no question that AIDS has not been attended to because it was conceived of as a gay disease. That’s a long time, 35 years, for there still to be so little known about what’s happening to us. They still have no idea of how to cure it. Ebola comes along and they all rush over there immediately, and the same people who should have found the stuff for HIV were doing Ebola, and it’s going away, it’s disappearing, in a very short amount of time, because they are really attending to it. This government is still not attending to HIV. I don’t care what anybody says. Not nearly as many people will ever get sick from Ebola as we lost to AIDS.Yeah. I came to realize that Hitler got the idea for the Final Solution from the eugenics movement. We gave him that, because that was an American thing. We have killed a lot of people over the years.Oh, I don’t think we’re ever behind anything. Certainly America is getting nastier. Look at Guantanamo. Look at all the people we’re murdering everywhere.We should have our own army as gays. I’m quite disappointed in where we are. I mean, it’s lovely that we can get married, but that’s really small potatoes compared with what we don’t have, which is equality.Oh, heavens no, not with them. We don’t have any organizations that I look to. ACT UP, I think, was the one great thing that came out of it, and then they destroyed each other. But that was an actual major accomplishment what they achieved, in getting all the drugs out. And we did it. No one else did it for us. HRC is a crock of shit. Because they got a lot of money and what do they do with it? There is no other model except fighting back. Fight. It’s not anything you negotiate; it’s a thing you threaten. “If you don’t do X and Y, we won’t do X and Y.” That’s how you get power. We don’t have power, and I don’t see any organization that has any power. And that’s why ACT UP worked. Once we got it together, we learned how to threaten, and play the good cop/bad cop business, which I had learned in the movie business! Every organization should have both, a good cop and a bad cop. And the good cop makes the initial negotiation, and if it doesn’t work, you send out the bad cop to fight. We are not good fighters, gay people.Powerful is the Koch brothers, who manage to get, through the Supreme Court, the ability to give as much money as you want to any kind of political group, which is destroying everything. Power is all about money. Power is about not sitting back.[You’re] making that a basis of gay life, and it shouldn’t be. That was our big problem, that we fought for the wrong things.Yeah. There’s more to life than whether you can go to a leather bar. That’s part of why we got in all the trouble. I have mixed feelings about Truvada. I’m afraid that people will use it for the wrong reason. But that’s no reason not to be glad that it’s there. To take it as a prophylactic, just so you can go out and fuck at the Mineshaft. So you can take a pill and not worry. And that’s, again, what caused all this trouble we’re in, in the first place. A lot of people died in 35 years. And I guess I came to realize that I’m angry that I’ve been allowed to die. I was much more hopeful earlier on. I’m not saying I’m not hopeful, but I’m not hopeful. And part of what depresses me is how passive most of the gay population is about this issue. So now we have Truvada and you can get laid on Saturday night, and surely, we deserve more than that from 35 years of waiting. We could have so much if we just used the power that was there to be taken, if we could just learn how to take it. Why are there still so few people saying that? Why hasn’t there ever been another Larry Kramer? And I don’t mean that as self-serving. But Larry, what do we want power for? We want power to have happiness in our own lives. I live in a little town in Florida now, and over the years my street has changed. I now have two women living together two houses down. And a man across the street, a new neighbor, came over right away, and, in a nice way, basically said, “I have no trouble with gay people.” It has trickled down to this little street in this little town in Florida where I feel less endangered. You must not accept that as enough, or as all that you’re fighting for. Things have improved in some ways. Oh that’s what people say: Why are you complaining? You have so much now. Well, I don’t think we do. Who’s the guy who just came out? Joel Grey? Been trying to get him to do that for 20 years. He replaced Brad Davis in Normal Heart at the Public. So I’ve known him a long time. I’m one of the first people he sent the article to, that he’d come out. I don’t know why, now, at age 82 [laughs]. We’re so much better than most people, and we’re not getting our due for it from them, or even from each other — how much we contribute to the culture of this country. And none of that has been bought with power. It’s been bought with talent. And what would we get if we combined the talent with the amount of money that’s available in this population? How second rate the gay people who made it in the government are. When you look at the list of gay presidents, for instance, with the exception of Washington and Lincoln, all the other ones are really just jokes. Who do you think is the most powerful gay person right now, behind the scenes or in front of the scenes, in governmental life or public life? I have no idea. There are a couple of exceedingly rich gay men who have foundations, such as Tim Gill, Jon Stryker. What is that money buying? We have to be able to get to the people with power, and we still can’t do that. You can’t call the president and see him like you should be able to. And [Bill] Clinton did us more harm than good. What do you think about Hillary Clinton? I hope she gets elected. I think she’s been around long enough and knows how to play the game that needs to be played. I think she would be more available to us than anyone else. How do you feel about the state of gay literature these days? I don’t follow it very closely. I belong to Lambda Literary, and they put out quite a good newsletter. I still don’t see us tackling the big themes. Why is everything sexual? Why is everything about love affairs that do or do not work? We seem very limited in our ambitions. That’s why this book is so extraordinary to me. It’s such an act of chutzpah, of taking on absolutely everything. What else is there to take on? What else is there to do with your time? You’ve experienced as much of life as I have. Did you like the book? Well, it’s wonderful that I’m alive to do it. I came close to dying, in this last year. You didn’t hear the wonderful story of our marriage? I had the judge, and we were going to be married on my apartment terrace with just a few close friends, and that was on a Monday, and I was taken to the hospital on Tuesday, and Wednesday we were married in intensive care at NYU, after we got permission from the head of NYU, to allow me to be married there. I couldn’t sign my name. I wrote X’s. I was so out of it. [Laughs] And that was my wedding. I mean I was out of it. I’m terrified that it’ll come back somehow. And for all intents and purposes, they don’t know what causes it. It’s not unrelated to HIV, but it’s not AIDS. So, it’s been hard, keeping a grasp on all of these things. I’m grateful that you’re doing this at this moment cause it’ll help the book...and I have been working on it a long time. It’ll be nice to have a place to rest and stay and be remembered. Why do you write?I was a late bloomer in terms of writing. I was not writing like Ed White was out of the cradle.Over such a long period of time, yes. I can’t wait to get back to it.It starts in the ’50s, and goes on from there. What we haven’t talked about is J. Edgar Hoover, who’s really very prominent in Volume I. Who was monstrous, but gay, and responsible for an amazing number of antigay things in this country. He lived across the street from my best friend, in Washington. And, of course, all the files were burned by the faithful secretary when he died. He ran a whorehouse for men in Washington during the war. There’s a lot in the book about people in power who were gay, who used it against gays, and Roosevelt was surrounded by people who didn’t like us. Nobody’s hands were clean.Reagan, hands down, no contest. What with his being responsible for not attending to gays and AIDS deaths, he was responsible for killing more people than Hitler or Stalin.Successful activism is about being angry enough and loud enough to be heard. Choose your issues and your targets, and go after them in any way you think you can. ACT UP chose drugs into bodies. We knew little about the many things we had to learn enough about to be successful, so we taught ourselves. Identify your enemies and go after them with threats. Numbers are nice but one person stationed holding a poster up in a strategic location can be effective. You must not be afraid to be obnoxious or to concern yourself with what others might think of you, particularly other gays. You have a mission. You must care passionately about this mission and make it clear and concise. Do not water it down by including too many items on your agenda. This is not all that complicated. Anger, passion, and volume are your weapons. We all have these within us. The courage to let it come out is the necessary frosting for this cake. Be bold. You’d be surprised how strong you are capable of being.

George Washington and Abraham Lincoln were gay. So were Alexander Hamilton, Herman Melville, and Richard Nixon-at least according to a new book that the author considers true but his publisher is selling as fiction to avoid legal problems, the Guardian reports. Needless to say, Larry Kramer's 800-page The American People, Volume 1: Search for My Heart is ruffling feathers. Kramer claims, for example, that Washington was gay in part because he designed soldiers' uniforms, the New York Times reports, and John Wilkes Booth killed Abraham Lincoln because the president spurned him. "You only have to look at photographs of Wilkes and [co-conspirator] Lewis Powell to see that they're full of their own beauty," says Kramer. "We call it gaydar-the thing straight historians don't have." Now at least one historian is calling him out on some details (generals used to make soldiers' uniforms, apparently). But Kramer, 79, who has long endured health problems and speaks in a whisper, describes a bigger picture: "Most histories have been written by straight people," he says. "There has never been any history book written where the gay people have been in the history from the beginning." Kramer is well-known for having started groups around the AIDS crisis in the 1980s, writing plays about gay life, and challenging Lincoln screenwriter Tony Kushner to reveal the Civil War president's orientation. Now Kramer is calling for "our own army as gays" to fight anti-gay repression worldwide, the Advocate reports. "I mean, it's lovely that we can get married," he says, "but that's really small potatoes compared with what we don't have, which is equality."

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Mr. Partilla, then a 42-year-old triathlete and a president of media sales at Time Warner, recognized a kindred dynamo. “She’s such a force,” he said. “She rocks back and forth on her feet as if she can’t contain her energy as she’s talking to you.” Advertisement Continue reading the main story The connection was immediate, but platonic. In fact, as they became friends so did their spouses. There were dinners, Christmas parties and even family vacations together. So Ms. Riddell was surprised to find herself eagerly looking for Mr. Partilla at school events — and missing him when he wasn’t there. “I didn’t admit to anyone how I felt,” she said. “To even think about it was disruptive and disloyal.” What she didn’t know was that he was experiencing similar emotions. “First I tried to deny it,” Mr. Partilla said. “Then I tried to ignore it.” But it was hard to ignore their easy rapport. They got each other’s jokes and finished each other’s sentences. They shared a similar rhythm in the way they talked and moved. The very things one hopes to find in another person, but not when you’re married to someone else. Ms. Riddell said she remembered crying in the shower, asking: “Why am I being punished? Why did someone throw him in my path when I can’t have him?” In May 2008, Mr. Partilla invited her for a drink at O’Connell’s, a neighborhood bar. She said she knew something was up, because they had never met on their own before. “I’ve fallen in love with you,” he recalled saying to her. She jumped up, knocking a glass of beer into his lap, and rushed out of the bar. Five minutes later, he said, she returned and told him, “I feel exactly the same way.” Then she left again. As Mr. Partilla saw it, their options were either to act on their feelings and break up their marriages or to deny their feelings and live dishonestly. “Pain or more pain,” was how he summarized it. Photo “The part that’s hard for people to believe is we didn’t have an affair,” Ms. Riddell said. “I didn’t want to sneak around and sleep with him on the side. I wanted to get up in the morning and read the paper with him.” Advertisement Continue reading the main story With that goal in mind, they told their spouses. “I did a terrible thing as honorably as I could,” said Mr. Partilla, who moved out of his home, reluctantly leaving his three children. But he returned only days later. Then he boomeranged back and forth for six months. The pain he had predicted pervaded both of their lives as they faced distraught children and devastated spouses, while the grapevine buzzed and neighbors ostracized them. Please verify you're not a robot by clicking the box. Invalid email address. Please re-enter. You must select a newsletter to subscribe to. Sign Up Receive occasional updates and special offers for The New York Times's products and services. Thank you for subscribing. An error has occurred. Please try again later. View all New York Times newsletters. “He said, ‘Remind me every day that the kids will be O.K.,’ ” Ms. Riddell recalled. “I would say the kids are going to be great, and we’ll spend the rest of our lives making it so.” The problem was she could not guarantee that. All they had were their feelings, which Ms. Riddell described as “unconditional and all-encompassing.” “I came to realize it wasn’t a punishment, it was a gift,” she said. “But I had to earn it. Were we brave enough to hold hands and jump?” They did jump. Both officially separated from their spouses by late 2008, though they waited until July 2009 before moving in together. “I didn’t believe in the word soul mate before, but now I do,” said Mr. Partilla, who is 46 and in January is to become a chief operating officer of Dentsu, a Japanese advertising agency. They finalized their divorces this year. “I will always feel terribly about the pain I caused my ex-husband,” said Ms. Riddell, 44 and working freelance. “It was not what I ever would have wished on him.” Or on her children. Advertisement Continue reading the main story “My kids are going to look at me and know that I am flawed and not perfect, but also deeply in love,” she said. “We’re going to have a big, noisy, rich life, with more love and more people in it.” On Nov. 15, the couple were legally wed at the Marriage Bureau in New York by Blanca Martinez of the City Clerk’s office. Then on Dec. 11, Ms. Riddell donned a Nicole Miller strapless gown for a small ceremony in the presidential suite of the Mandarin Oriental New York hotel. As if on cue, the hotel room phone rang as she began to recite her vows. Mr. Partilla’s 10-year-old daughter answered. “We’re in the middle of a wedding,” she informed the caller, while her younger two siblings and two soon-to-be step-siblings spun off like small planets freed from the pull of gravity. “This is life,” said the bride, embracing the messiness of the moment along with her bridegroom. “This is how it goes.” vows Times No Longer Even Euphemizing Spouse-Dumping ShareThis Counter Email Only one year ago, we noted the Times' gross habit of including people in the "Vows" column who very clearly cheated on and abandoned a former spouse or lover before getting hitched to the current one, then went on to revel in it in the Times, so their exes can relive that period in all its painful glory. But that practice seems practically innocent compared to today's "Vows:" See, time was, the Times would say a couple "faced many obstacles to romance," or "the road to love was bumpy," when what they meant was that there were other human lives involved. In 2009, affairs and spouse-dumping would still pop out at you in the column - abruptly, perhaps, but rarely with unmitigated pride. But now, with print media facing its struggles and competing with saucier online writing, the spouse-dumping goes right in the lede: What happens when love comes at the wrong time? Carol Anne Riddell and John Partilla met in 2006 in a pre-kindergarten classroom. They both had children attending the same Upper West Side school. They also both had spouses. Part “Brady Bunch” and part “The Scarlet Letter,” their story has played out as fodder for neighborhood gossip. But from their perspective, the drama was as unlikely as it was unstoppable. Once a subject of shame, this affair is made to sound like a veritable harlequin novel. Except, in reality, it was clearly still awkward and sort of sad: The connection was immediate, but platonic. In fact, as they became friends so did their spouses. There were dinners, Christmas parties and even family vacations together. The couple claims that they didn't have a sexual affair, but they did have an encounter that their spouses couldn't have been too happy about: "I’ve fallen in love with you,” he recalled saying to her. She jumped up, knocking a glass of beer into his lap, and rushed out of the bar. Five minutes later, he said, she returned and told him, “I feel exactly the same way.” Then she left again. You sort of expect this "Vows" to hook right at some point; for the former spouses to be quoted saying that they've accepted things, they're all friends now, the kids are okay. Because, you know, Carol Ann Riddell and John Partilla opted to have this tale put in print. We don't doubt that these things just happen, but we do question the decision to celebrate it in the face of your exes and children, right in the New York Times. Especially when the "Brady Bunch" part turned out to be pretty shaky. Because it sounds like they're by no means all friends: “He said, ‘Remind me every day that the kids will be O.K.,’ ” Ms. Riddell recalled. “I would say the kids are going to be great, and we’ll spend the rest of our lives making it so.”The problem was she could not guarantee that. All they had were their feelings, which Ms. Riddell described as “unconditional and all-encompassing.” “My kids are going to look at me and know that I am flawed and not perfect, but also deeply in love,” she said. “We’re going to have a big, noisy, rich life, with more love and more people in it.” Wedding announcements in The New York Times often elicit a certain amount of resentment and schadenfreude, but the reaction to yesterday’s “Vows” column was of a whole different order of magnitude. That column told the story of TV reporter Carol Anne Riddell and advertising executive John Partilla, who divorced their longtime spouses and split up their families in order to be with each other. In addition to strong condemnation from numerous bloggers and many of the paper’s own commenters, the article, as a first of sorts for the Times, invited a number of questions. Why were the ex-spouses of the newlyweds not mentioned by name in the story? Did the reporter call them for comment, as basic journalistic practice would dictate? Why did the Times open up the comment board when most Vows stories are off-limits? And above all, what were the couple thinking in telling their story in a space normally reserved for feel-good, soft-focus meet-cute tales? “We did this because we just wanted one honest account of how this happened for our sakes and for our kids’ sakes,” Riddell told me. “We are really proud of our family and proud of the way we’ve handled this situation over the past year. There was nothing in the story we were ashamed of.” Riddell says the backlash is “sort of surprising to me. I think people are focusing a lot on the negative, but there was a lot of positive.” But, she notes, “we’ve had a lot of people say to us how brave we are to do this, how commendable it was that we were as honest as we were.” Indeed, while most Times commenters expressed disapproval, there were also some like the one who wrote “Thank you Carol and John for the honest reflection…I wish them a long and happy marriage.” Riddell declines to say whether she or her husband asked for their exes’ consent to tell their story in the Times, or at least notified them that it was in the works. “I really don’t want to wade into this any further than we already have,” she says. “It’s not helpful to anybody.” She says, however, that she and her groom did not ask the paper not to interview the exes or negotiate any other preconditions: “They made their own decisions on that front.” So did the story’s author, Devan Sipher, seek comment from the exes? “I would assume not, but I don’t really know,” says Riddell. Asked that same question, a Times spokeswoman says, “We do not comment on the process of editing and reporting including who was and was not contacted for interviews related to a specific story. The Vows/Wedding column adheres to the standards of the Times.” The paper’s Weddings/Celebrations editor, Robert Woletz, did not return a message; nor did the exes, who, like their former spouses, both have high-level jobs in the media industry. (In both cases, the first marriage was also written up in the Times.) That leaves the question of why this story, unlike most “Vows” columns, was opened for reader comments. “We open up comments on articles that are engaging to readers,” says the Times spokeswoman. “This is a very interesting story.” That is undoubtedly true — though it would be even more true had the exes been interviewed as well. iStockphoto/Crisma Turns out godless left-wing urban sodomites can get morally uppity too. With its genteel, Jane Austen-like formula, the New York Times' "Vows" section is the nation's most reliable weekly comedy of manners, a tale in which sexual chemistry and inevitable misunderstandings lead to showers of rice instead of restraining orders and inflammatory Facebook status updates. The usual narrative goes as follows: Unlikely yet well-heeled duo (vegetarian/hunter, conservative/liberal, city slicker/country mouse) meets cute. Duo encounters obstacles -- distance, job demands, a history of meth abuse -- amusingly related in wry, illuminating anecdotes from friends and family. Couple overcomes said obstacles and winds up at the altar -- usually with their kids or a dog in tow, and pledges to be each other's "force field of awesome." New York Times readers feel mixture of romantic hopefulness and oddly satisfying, "Jesus Christ, who are these pretentious tools?" disdain while polishing off their morning Lavazzas. But while previous "Vows" columns have skirted with drama, controversy and even a hint of the verboten, Sunday's column really took the proverbial fondant-enrobed, lemon curd-filled, multi-tiered cake. From the first sentence -- "What happens when love comes at the wrong time?" -- readers knew this was to be a story full of intrigue, ripe for finger wagging. The celebratory couple, former local news reporter Carol Anne Riddell and media executive John Partilla, met in 2006 in their children's classroom -- while they were both married to other people. A friendship blossomed between the two families, and all that closeness soon kindled a feeling more than platonic between Riddell and Partilla. They fell in love, and though Riddell says, "We didn’t have an affair," they did begin a long, emotionally grueling courtship that eventually led to much social ostracism, two divorces, a small, private ceremony last month, and then, you know, a splashy story in the New York Times on Sunday. It didn't take long for the gasps of outrage to rise up. As New York magazine noted, "We do question the decision to celebrate it in the face of your exes and children, right in the New York Times" while Gawker facetiously noted the "Homewrecking Couple's Scandalous New York Times Wedding Announcement." And Slate's film critic Dana Stevens minced no words, calling it "staggeringly monstrous." But though Gothamist says that "Vows" isn't "usually a place where you reel in disgust," that's almost always an appropriate response to its "We're so wacky and unconventional, in our Fortune 500 way!" weekly litany of dynasty-making. The novelty this time is the openness of the narrative -- one that does not follow a more conventionally appropriate form. And there is undeniably something deeply unsettling about a father saying of his brand-new second wife, "I didn’t believe in the word soul mate before, but now I do." Finding a "soul mate" is wonderful, but divorce is crappy, especially when offspring are involved. The reality that Mom and Dad do not love each other -- and that they've likely been pretending otherwise for a while -- is a cruel blow for anyone, whether the child is 2 or 42. To have that sting instigated at least in part by the arrival of another person on the scene, and to have that relationship then become the subject of croissant-nibbling conversation at breakfast tables across the land -- yow, that's got to hurt. Nearly 200 comments later, many Times readers agree. The National Review unsurprisingly called the tale "heartbreaking," tut-tutting, "Make sure you have the kind of lovingly rigorous support system -- faith, friends, family -- that will remind you that there might be another choice other than succumbing to feelings vs. dishonesty." There might be. Then again, there might not. Because love, as the "Vows" column proves, is not always neat, wise or even good. And what this particular story leaves out, surely for the sake of the spurned exes, is the state of Riddell and Partilla's respective prior unions. The drama of a pair of home-wreckers mangling up each other's families is certainly juicy, but who's to judge whatever loneliness or pain already existed in both their lives before they found each other? Who's to say that sticking with something that may no longer be happy or fulfilling or even healthy is for the ultimate best? And who's to say that everybody isn't exponentially better off now? The real head-scratcher isn't that the couple found each other; marriages bust up and people pair off anew every day. It's the fact that they chose to go so public with their joyous new union, and that the Times leapt so eagerly on a story so guaranteed to turn off plenty of readers, that's perplexing. Is it all just narcissism combined with whopping insensitivity? Can't rule it out, but maybe it's something else. As many of us have learned through hard-won experience, the great beauty of the modern era is that we now have the world's most efficient mechanism for the ripping off of Band-Aids at our fingertips. We can tweet about cancer. We can announce that we just had a miscarriage in a Facebook update. And we can tell friends and neighbors, "My mate and I fell in love while we were married to other people." People don't have to like it, and don't have to approve, but at least they can know, so they don't have to whisper when we run run into them at Fairway. It must be easier to give a public version of events than to repeat it forever to every curious friend. Would being ashamed make Riddell and Partilla any less married right now -- or would being quiet about it just make other people a little more comfortable? While the "Vows" column will no doubt continue to inspire shudders of revulsion on a reliable, seven-day cycle, I can't take particular umbrage with a story of two people who did not heroically triumph over illness or age or prior heartbreak, who were instead flawed and mistake-prone and know what it feels like to hurt their families and lose friends. Because as romantic and frothy as the "Vows" section often is, a wedding is not always simply a happy ending. Sometimes it's just a messed-up true story.

The New York Times' normally genteel "Vows" column hit a nerve on Sunday, with its tale of John Partilla and Carol Riddell-who each left spouses to get hitched. It's "part Brady Bunch, part The Scarlet Letter," the paper cooed. Bloggers and commenters tore the paper a new one. "These things happen," says New York magazine, "but we do question the decision to celebrate it in the face of your exes and children." Now Partilla tells the New York Post he regrets the whole thing. "We can't control other people's judgments," he says, but if they'd known the reaction they'd get, "we obviously would not have shared our life in any way publicly." So why did they do it at all? "We just wanted one honest account of how this happened," Riddell tells Forbes. Heck, Mary Elizabeth Williams of Salon doesn't see why this is worse than typical vomit-inducing Vows column. "A wedding is not always simply a happy ending," she writes. "Sometimes it's just a messed-up true story."

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Recently, we reported on a new class of naphthoquinone derivatives showing a promising anti-trypanosomatid profile in cell-based experiments. The lead of this series (B6,2-phenoxy-1,4-naphthoquinone) showed an ED50 of 80 nM against Trypanosoma brucei rhodesiense, and a selectivity index of 74 with respect to mammalian cells. A multitarget profile for this compound is easily conceivable, because quinones, as natural products, serve plants as potent defense chemicals with an intrinsic multifunctional mechanism of action. To disclose such a multitarget profile of B6, we exploited a chemical proteomics approach. A functionalized congener of B6 was immobilized on a solid matrix and used to isolate target proteins from Trypanosoma brucei lysates. Mass analysis delivered two enzymes, i. e. glycosomal glycerol kinase and glycosomal glyceraldehyde-3-phosphate dehydrogenase, as potential molecular targets for B6. Both enzymes were recombinantly expressed and purified, and used for chemical validation. Indeed, B6 was able to inhibit both enzymes with IC50 values in the micromolar range. The multifunctional profile was further characterized in experiments using permeabilized Trypanosoma brucei cells and mitochondrial cell fractions. It turned out that B6 was also able to generate oxygen radicals, a mechanism that may additionally contribute to its observed potent trypanocidal activity. Overall, B6 showed a multitarget mechanism of action, which provides a molecular explanation of its promising anti-trypanosomatid activity. Furthermore, the forward chemical genetics approach here applied may be viable in the molecular characterization of novel multitarget ligands. Among the tropical diseases, there are maladies whose etiological agents belong to the Trypanosomatidae family of the Protista, order Kinetoplastea, that are responsible for infections concentrated in the poorest, mainly rural areas of the planet, and that are grouped under the name of “most neglected diseases” [1]. In particular, parasites of the genus Trypanosoma are responsible for Chagas' disease in Latin America and sleeping sickness in sub-Saharan Africa [2]–[5]. Because of their occurrence in low-income and middle-income countries, these diseases do not have high visibility in Western societies, although sleeping sickness is among the neglected tropical diseases with the highest rates of death [6]. Vaccine development has been hampered by either the high degree of antigenic variation as exhibited by the bloodstream dwelling African trypanosome, Trypanosoma brucei, and the localization of the American trypanosome, Trypanosoma cruzi, within cells of the human host, despite a successful experimental oral vaccine based on attenuated T. cruzi has been reported [7]. In this context, chemotherapy still represents a viable option for treatment of these infections [8]. However, the majority of the currently available drugs are decades old (some back to 1920) and have, unfortunately, many limitations, including high toxicity and the emergence of drug resistance. The latter issue has called for designing innovative approaches to drug discovery for infections by trypanosomes [9], [10]. A major role in this respect is played by combination therapy, which has been shown to be a possible strategy for both preventing and overcoming chemotherapy-induced resistance [11]. A logical alternative to combination therapy is the development of drugs able to hit multiple targets [12], [13]. Such multitarget compounds are single chemical entities that can provide the same pharmacological profile as drug combinations, but potentially with fewer side effects. In fact, when two or more drugs are administered as a combination, there is a possibility that the drugs may interact with each other (drug-drug interaction). This interaction could increase or decrease the effective concentration of one of the drugs or, more frequently, could even enhance the adverse effects. Indeed, single multitarget compounds have a much simpler pharmacokinetic profile than combination therapy, also prevent possible side effects due to drug-drug interactions, greatly simplify the therapeutic regimen, with positive consequences for patient adherence and caregiver compliance, and finally an overall improved selectivity. Furthermore, the easier and cheaper manufacturing and formulation of a single active pharmaceutical ingredient would make multitarget drugs inherently more cost-effective and widely accessible than combinations [14]. It should be mentioned that if there is any synergism or additive effect among the targets, then the effective dosage of a multitarget drug is most likely lower than that of a single-target drug. When lowering the therapeutic dose, however, it will be crucial to find a balance between decreasing the dose to avoid side effects and keeping it sufficiently high to prevent the development of resistance. On these premises, it has been proposed that against trypanosomatid-borne diseases such compounds may prove more efficacious, tolerable, and affordable than the available arsenal of drugs [12], [15]. Naphthoquinone and other quinone derivatives have been reported as one of the major natural product classes with significant activity against Trypanosoma [16]–[18]. For instance, lapachol exhibits a marked anti-trypanosomal profile, while displaying no serious toxic effects in humans [19]. In view of the well-known biological properties of this class of compounds, it is highly possible that naphthoquinones exert their anti-trypanosomatid profile by means of a multitarget mechanism. Indeed, a multitarget profile for this class of compounds is easily conceivable, because quinones, like many other natural products, provide plants with potent defense chemicals with an intrinsic multifunctional mechanism of action [20]. Furthermore, it can be hypothesized that in addition to a possible target-related mechanism, the general free-radical-generation mechanism of quinones – probably also at the basis of their general cytotoxicity – may contribute to multitarget profile of these molecules [21] [22]. Indeed, it has been reported in the literature that parasitic protists are particularly sensitive to oxidative stress [23]. In this field, we have recently reported on the preparation of a focused library of 16 compounds based on the 1,4-naphthoquinone and 1,4-anthraquinone natural occurring scaffolds [24]. From this small compound collection, several molecules were active against Trypanosoma and Leishmania at low concentrations. Some of the derivatives exhibited potency in the nanomolar range, with one (2-phenoxy-1,4-naphthoquinone, B6 in Figure 1) displaying an ED50 value of 80 nM against Trypanosoma brucei rhodesiense, as assessed in experiments using in vitro cultured parasites. It also showed a selectivity index (ratio of the compound' s ED50 values on mammalian cell lines and trypanosomes) of 74 [24], which is very close to the specifications required by WHO/TDR to be considered an anti-trypanosomatid hit [25]. However, the molecular mechanism and the target (s) responsible for the biological profile of this class of compounds have remained undisclosed. Here, by means of a chemical proteomics approach, we aimed at identifying the putative molecular target (s) of B6. In particular, using its immobilized derivative 1 (Fig. 1), we isolated its targets from parasite extracts. Then, by means of biochemical experiments, we verified the ability of the molecule to bind to recombinant forms of the identified targets. In light of the general property of naphthoquinones to generate free radicals, we finally analyzed oxygen consumption in permeabilized trypanosomes and production of reactive oxygen species (ROS) in trypanosome mitochondrial cell fractions. This allowed us to elucidate additional B6 potential mechanisms of action, as chemical proteomics is not suited to identify non-protein targets. Before covalently attaching B6 to the solid support through a linker, we analyzed which site (s) of the molecule were more appropriate for linking purposes. To this end we synthesized derivatives 1–3 (Fig. 1), which carry in different positions amino or hydroxyl groups that can be easily exploited as anchor points. The synthesis was carried out according to the procedure reported for B6 [24], and which relies on the substitution of a 2-bromonaphthoquinone with the corresponding phenol. Synthesis of 2- (4-amino-phenoxy) -[1], [4]naphthoquinone (1). To a stirred solution of 4-amino-phenol (0. 46 g, 4. 2 mmol) in 90 ml dimethylformamide (DMF) potassium carbonate (1. 70 g, 12. 3 mmol) was added. After stirring for 1 h at room temperature, 2-bromo-[1], [4]-naphthoquinone (1. 0 g, 4. 2 mmol) was added. After stirring for further 3 h, the reaction was diluted with water and ice (500 ml) and the resulted brownish solid was collected by filtration to give 0. 49 g of crude 1, which was crystallized by EtOH/water (40% yield). IR (Nujol) 3421,3382,1680,1635,1609,1508,1204,980,718 cm−1; 1H NMR (300 MHz, CDCl3) δ 8. 25-8. 22 (m, 1H), 8. 12-8. 09 (m, 1H), 7. 80-7. 78 (m, 2H), 6. 95 (d, 2H, J = 8. 5 Hz), 6. 76 (d, 2H, J = 8. 5 Hz), 6. 04 (s, 1H), 3. 77 (br s, 2H, exchangeable with D2O); HRMS (ES) m/z calculated for C16H11NO3Na 288. 0637, found 288. 0639 [M++Na+]. Synthesis of 5-hydroxy-2-phenoxy-[1], [4]naphthoquinone (2). It was synthesized in 33% yield from phenol and 2-bromo-5-hydroxy-[1], [4]-naphthoquinone, following the procedure reported for 1 and crystallization from methylene chloride/petroleum ether. 1H-NMR (CDCl3,400 MHz): δ 5. 88 (s, 1H), δ 7. 13 (d, J = 8. 4,2H), δ 7. 26-7. 35 (m, 2H), δ 7. 47 (t, J = 7. 6,2H), δ 7. 61 (t, J = 8. 0,1H), δ 7. 74 (d, J = 7. 2,1H), δ 12. 10 (s, exch, 1H); 13C-NMR (CDCl3,400 MHz): δ 113. 17,114. 40,119. 91,121. 22,125. 56,127. 03,130. 70,131. 30,135. 90,152. 77,161. 30,161. 34,179. 43,191. 07; MS (ESI+) m/z: 289 [M++Na+]. Synthesis of 8-hydroxy-2-phenoxy-[1], [4]naphthoquinone (3). It was synthesized in 35% yield from phenol and 2-bromo-8-hydroxy-[1], [4]-naphthoquinone, following the procedure reported for 1 and crystallization from methylene chloride/petroleum ether. 1H-NMR (CDCl3,400 MHz): δ 5. 94 (s, 1H), δ 7. 13 (d, J = 8. 8,2H), δ 7. 26-7. 34 (m, 2H), δ 7. 47 (t, J = 7. 6,2H), δ 7. 59-7. 67 (m, 2H), δ 11. 79 (s, exch, 1H); 13C-NMR (CDCl3,400 MHz): δ 114. 18,119. 22,121. 22,124. 30,126. 98,130. 70,132. 20,137. 48,152. 77,160. 38,162. 25,184. 25; MS (ESI+) m/z: 289 [M++Na+]. The synthesized compounds were then tested against T. b. rhodesiense parasites as previously reported [24], and their activities are shown in Figure 1 along with that of B6. Because of its superior trypanocidal activity, 1 was selected for further immobilization studies. For identification of the B6 targets, T. b. rhodesiense was used. The parasites were isolated from blood of an infected mouse (received from the Swiss Tropical and Health Institute, Basel, Switzerland). Parasite lysates were prepared as described previously [26]. Briefly, 5×108 cells were lysed in 200 µl lysis buffer consisting of 160 µl PBS (38 mM Na2HPO4,2 mM NaH2PO4,29 mM NaCl, pH 8. 0) containing 44 mM glucose and 40 µl of lysis buffer concentrate (100 mM HEPES, pH 7. 5,750 mM NaCl, 5% Triton-X-100,10 mM tris (2-carboxyethyl) phosphine (TCEP), 50% glycerol, and 1 µl/ml protease cocktail (Sigma). After short periods of sonication the mixture was centrifuged for 10 min at 14,000 g, the supernatant was recovered and stored in aliquots of 100 µl at −80°C, until needed. The protein concentration, determined using the Bradford dye assay with BSA as reference protein, was found to be 8. 4 mg/ml. 1 was immobilized to epoxy-activated Sepharose 6B using a modified protocol described earlier [26]. To this end, swollen and thoroughly washed matrix was resuspended in two volumes of 35 mM ligand dissolved in a solution containing 50% dioxane/50% H2O (solution 1). Coupling was performed for 48 h at 40°C using 40 µl of swollen resin mixed with 80 µl of ligand solution. After coupling, the matrices were centrifuged for 1 min at 3000 g, and then washed 4–5 times with 10 volumes (400 µl) of solution 1. Afterwards, remaining free epoxides were reacted by adding 1 ml of a solution containing 50% dioxane and 50% acetic acid (pH 3. 2) and incubation for 20 h at 40°C. After incubation, the samples were centrifuged at 3000 g for 2 min and the supernatants collected. The resin was finally washed in five rounds, each with 10 volumes of solution 1. Again all supernatants were collected. Supernatants of all steps were collected in a 25. 0 ml volumetric flask and used to determine the amount of ligand washed off. Direct absorbance of the scans of the immobilized ligand on the matrix resuspended in 50% glycerol solution (v/v) clearly confirmed successful coupling (data not shown). The amount of inhibitor bound to the matrix was determined by back calculation of the amount of compound applied and amount recovered. Routinely 2–3 µmol/ml of compound were bound. A control matrix was prepared without ligand and treated as described above. Before incubation with the lysate the matrices were washed twice with water and then equilibrated in the lysate buffer by washing each resin three times with the lysate buffer. The lysate (100 µl; diluted to 2. 1 mg/ml protein using PBS) was incubated with the ligand-bound matrix for 2 h at 4°C while mixing at 700 rpm. Then, the matrix was washed 5 times with the lysate buffer while mixing for 90 sec at 1400 rpm. The control matrix was incubated with the same amount of lysate and treated equally. Finally, both matrices were washed with a solution containing 5 mM HEPES (pH 7. 0) and sent to the Functional Genomics Center of Zurich for protein identification directly from the matrices. To this end, resins were re-suspended in 50 µl trypsin solution (10 ng/µl trypsin in 10 mM Tris-HCl, 2 mM CaCl2, pH 8. 2) and incubated at 37°C overnight. Supernatants were separated and beads extracted twice with 5% formic acid in 10% acetonitrile. All three supernatants of the corresponding matrices were combined, dried, and then dissolved in 25 µl 0. 1% formic acid. 2 µl were injected via an autosampler and run with two different gradients (A and B) for LC/ESI/MS/MS-QTOF analysis. Database searches were performed by using the ProteinLynx Global Server (Swiss Prot, all species) and Mascot (Swiss Prot, excluding the major eukaryotic species from the search) search programs. Only hits with enough independent peptide spectra to give a probability of over 95% were considered for the study. The expression construct has been obtained by subcloning the corresponding GK gene (isolated from T. b. brucei strain Lister 427) from the formerly designed pET15-TbGK plasmid [27] into vector pET28 to create the final expression plasmid pET28-TbGK (unpublished data). TbGK was then expressed in E. coli BL21 (DE3) -CodonPlus-RIL grown in 1 liter LB medium supplemented with kanamycin (50 µg/ml) and chloramphenicol (34 µg/ml) for 16 h at 37°C. Expression was induced by adding 1 mM isopropyl β-d-thiogalactanopyranoside (IPTG), and growth was continued for another 2 h at 37°C. Bacteria were harvested (20 min, 4°C, 5000 rpm), resuspended in a mixture of 96% buffer A (20 mM Tris-HCl, 200 mM NaCl, pH 7. 6) and 4% buffer B (20 mM Tris-HCl, 200 mM NaCl, 500 mM imidazole, pH 7. 6), and supplemented with a small amount of DNase. The suspension was passed twice through a French Press, centrifuged (20 min, 4°C, 9800 rpm), and the clarified crude extract was applied onto a 5 ml Ni-chelating column. TbGK was eluted using a 10 column volumes linear imidazole-gradient using buffer A and buffer B. Eluted fractions were analyzed by SDS-PAGE. Fractions containing TbGK were combined, supplemented with 2 mM CaCl2 and 10 U/ml thrombin to cleave off the histidine tag. After overnight incubation at 16°C, the digested protein was concentrated and desalted using a Superdex 75 column equilibrated with buffer A. Protein concentration was determined at 280 nm (extinction coefficient = 81650 M−1 cm−1). The protein was diluted to a concentration of 2 mg/ml and stored at 4°C. The TbGK activity was measured using a modified version of a continuous enzyme-coupled spectrophotometric assay developed previously [28]. Briefly, the ATP consumption associated with glycerol phosphorylation was coupled to the oxidation of NADH via the coupled pyruvate kinase/lactate dehydrogenase enzyme pair. The assays were performed in 1. 0 ml triethanolamine/HCl buffer (0. 1 M triethanolamine, 2. 5 mM MgSO4,10 mM KCl, 5 mM glycerol, 0. 1 mM ATP, 2. 2 mM phosphoenolpyruvate, 0. 1% DMSO, pH 8. 0), at 25°C in the presence of 5 U pyruvate kinase, 3 U lactate dehydrogenase, and 150 ng TbGK. The concentration of B6 was varied in the range of 1 nM to 10 µM. The reaction was started by adding 0. 42 mM NADH. The IC50 value of B6 was calculated as the mean of three independent experiments. T. b. brucei glyceraldehyde-3-phosphate dehydrogenase (TbGAPDH) was expressed using expression vector pET28a carrying the GAPDH gene which had been subcloned from a former expression clone (T. b. brucei strain Lister 427) based on pET3a [29] using NdeI and BamHI restriction enzymes (unpublished results). A culture of E. coli strain BL21 (DE3) harboring an expression plasmid with TbGAPDH was grown at 37°C in 100 ml of LB medium. When the OD at 600 nm was between 0. 5 and 0. 8, expression was induced by addition of 1 mM IPTG. Growth was continued overnight. TbGAPDH was purified essentially as above reported for TbGK with the only difference that the clarified crude extract was applied onto a Talon resin column (Talon, Clontech), and that the protein of interest was eluted with 200 mM imidazole. TbGAPDH was identified by SDS-PAGE and Coomassie blue staining. The kinetics of the TbGAPDH reaction was monitored using a continuous enzyme-coupled spectrophotometric assay, as previously described [29]. In brief, TbGAPDH activity was measured following the NADH oxidation at 340 nm, in a coupled assay with 3-phosphoglycerate kinase and using a Jasco V-550 spectrophotometer. All measurements were performed at 25°C in 0. 01 M triethanolamine, pH 7. 6,1. 7 mM ATP, 1 mM EDTA, 100 mM KCl, 5 mM MgSO4,1. 7 mM NaHCO3,25 µg 3-phosphoglycerate kinase. The IC50 values were determined in a final volume of 1 ml in the presence of 120 ng TbGAPDH and 5. 6 mM 3-phosphoglycerate (3-PGA), while varying the B6 concentration after a pre-incubation of enzyme plus inhibitor for 10 min. The reaction was started by the addition of 0. 2 mM NADH. Each point of the curve was measured in triplicates and the value for IC50 was estimated from graphically plotted dose-response curves. The type of inhibition was determined with respect to NADH and 3-PGA in consideration of Michaelis-Menten steady-state conditions. To investigate the inhibition mechanism with respect to NADH, TbGAPDH was incubated for 10 min at room temperature with different inhibitor concentrations (0–15 µM) in a total volume of 1 ml. The reaction was initiated by the addition of 5. 6 mM 3-PGA and NADH (ranging from 5 µM to 200 µM). The inhibition mechanism with respect to 3-PGA was determined in a similar way. To this end, TbGAPDH was incubated with varying inhibitor concentrations (10 nM–100 µM). The reaction was initiated by the addition of 0. 2 mM NADH and 3-PGA (ranging from 50 µM to 5. 6 mM). The α and Ki values were obtained from Dixon and secondary plots. The reported values represent the mean of two independent experiments. For a comprehensive analysis of the effect of simultaneous inhibition of GAPDH and GK on the glycolytic flux by B6, we used a mathematical model. Modeling was done with the T. brucei glycolysis model [30] in the open-source software package PySCeS [31] with Python 2. 6. In the model version used here, the equations for the cytosolic and glycosomal adenylate kinases were replaced with mass action kinetics consistent with the equilibrium constant of 0. 442 mM [32], the glycerol-3-phosphate oxidase (GPO) has access to the glycosomal pools of glycerol-3-phosphate (G3P) and dihydroxyacetone phosphate and phosphoglycerate mutase has access to the glycosomal pool of 3-PGA. These adjustments hardly affected control distribution and the glycolytic flux in this version of the model is equally sensitive to glycerol inhibition at anaerobic conditions as the model described in [30]. To simulate titration of a non-competitive inhibition, the Vmax of GAPDH and/or GK was multiplied by, using a Ki for GAPDH of 4 µM (based on measurements in this paper: 3. 6 or 5. 0 µM depending on the substrate that was varied) and for GK 0. 90 µM (as reported in this paper). To simulate anaerobic conditions, the Vmax of the short mitochondrial respiratory chain (in the model referred to as GPO, consisting of a mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase, ubiquinone and the alternative oxidase (TAO) ) was set to zero. Bloodstream- and procyclic-form T. b. brucei cells (strain Lister 427, cell line 449) were cultured up to the exponential growth phase and homogenates were obtained by grinding the washed parasite pellets with silicon-carbide abrasive grain (mesh 300) in disruption buffer containing 250 mM sucrose, 25 mM Tris-HCl and l mM EDTA, pH 7. 8 (STE buffer). Differential centrifugation [33] was performed as follows: the suspension was taken up in another 3 ml STE buffer and centrifuged at 30 g for 3 min. The supernatant, representing the cell homogenate, was centrifuged at 1,500 g for 10 min giving the nuclear fraction. The post-nuclear supernatant was then centrifuged at 5,000 g for 10 min giving the large-granular (mitochondria-enriched) fraction as pellet. This fraction was resuspended in 300 µl STE buffer. Oxygen consumption was monitored in a thermostated vessel at 37°C with a Clark electrode (oxygraph). Measurements in permeabilized bloodstream-form T. brucei were done in buffer 1 (96. 9 mM NaCl, 3. 1 mM KCl, 5 mM MgCl2,2 mM Na2HPO4,90 mM Tris at pH 7. 5). Batches of 2×107 cells were pelleted, washed in buffer 1 and incubated for 5′ on ice in 1 ml of buffer 1 containing 1 ng digitonin to permeabilize the cells. Subsequently the permeabilized cells were pelleted and washed twice with buffer 1 without digitonin after which the pellet was taken up in 1 ml buffer 1 and transferred to the oxygraph. Substrates and inhibitors were added as described in the text. 2 mM salicylhydroxamic acid (SHAM) was used to completely inhibit the T. b. brucei alternative oxidase (TAO) that is part of the GPO. B6 and SHAM were dissolved in DMSO. The DMSO concentration used in these oxygen consumption measurements was less than 0. 7%. The method used to measure H2O2 production in T. b. brucei mitochondria is based on the fluorogenic probe 2′, 7′-dichlorodihydrofluorescein diacetate (H2DCFDA) which emits an intense green fluorescence only after deacetylation and subsequent oxidation. ROS production by the mitochondrial fraction of bloodstream form T. b. brucei was measured in a 96-well microtiter plate using a fluorescence plate reader (Victor Wallace multiplate reader). In each well, 0. 25 mg mitochondrial protein/ml and 5 µM H2DCFDA to a final volume of 0. 2 ml with 10 mM Tris-HCl, 50 mM KCl, 1 mM EDTA, pH 7. 5 were present. The reaction, performed at 25°C, was started by the addition of 10 mM G3P, in the presence and absence of 10 µM B6. We used bloodstream and procyclic forms of T. b. brucei strain Lister 427, cell lines 449 [34], constitutively expressing the E. coli tetracycline (Tet) repressor gene via the chromosomally integrated plasmid pHD449 that also confers phleomycin resistance. Bloodstream forms were cultured in HMI-9 medium containing 10% heat-inactivated foetal calf serum (Invitrogen) and 0. 18 µg/ml phleomycin (Cayla) at 37°C under water-saturated air with 5% CO2. Procyclic trypanosomes were grown in SDM79 medium [35] supplemented with 15% foetal calf serum and 0. 5 µg/ml phleomycin at 28°C under water-saturated air with 5% CO2. The glucose-depleted SDM-80 medium, first employed by Lamour et al. [36], was supplemented with 5 µg/ml hemin, 9% (v/v) dialyzed “glucose free” heat-inactivated foetal calf serum (Sigma) and 1% (v/v) of normal heat-inactivated foetal calf serum (Invitrogen). T. b. rhodesiense in vitro growth inhibition activity of 1–3 (Fig. 1) was assessed using bloodstream form of STIB 900 strain, following the procedure reported in Orhan et al. [37]. The anti-trypanosomal activity tests were also performed on T. b. brucei according to the “Long Incubation Low Inoculation Test” (LILIT) [38], [39] using cultured bloodstream and procyclic forms of T. b. brucei strain Lister 427, cell line 449 [34]. It should be noted that T. b. rhodesiense, used in the initial inhibition activity assays, and T. b. brucei, used in all molecular and biochemical studies and in some growth inhibition assays performed at a later stage, are different subspecies that at the molecular level are almost identical. The essentially only difference is that the former subspecies is human pathogenic, due to resistance to a lytic factor present in human serum, whereas the latter subspecies is susceptible to the lytic factor. In addition, variable expression levels of proteins may give rise to differences in drug susceptibilities between subspecies. Docking of B6 was carried out using the crystal structure of TbGAPDH (PDBid: 2X0N) [40]. The binding pocket was defined as 10 Å from Cys166. Tautomeric states of histidines and the positions of asparagine and glutamine side chain amidic groups were optimized to improve the hydrogen bonding pattern. Polar hydrogen atoms were also optimized. The adopted force field was a modified version of the ECEPP/3 force field [41]. B6 was assigned the MMFF force field atom types and charges [42]. Docking simulations were carried out by means of the Biased Probability Monte Carlo stochastic optimizer as implemented in ICM [43], [44]. The molecular conformation of the system was described by means of internal coordinate variables. The other binding site residues were represented by pre-calculated 0. 5 Å spacing potential grid maps, representing van der Waals potentials for hydrogens and heavy atoms, electrostatics, hydrophobicity, and hydrogen bonding, respectively. The van der Waals interactions were described by a smoother form of the 6–12 Lennard-Jones potential with the repulsive contribution capped at a cutoff value of 4 kcal/mol. Poses from Monte Carlo sampling were rescored by means of the standard ICM empirical scoring function [45]. A typical target isolation project begins with structure-activity relationship (SAR) studies, in which various portions of the small molecule of interest are modified to determine which one (s) can be used as points of attachment to a solid matrix [46]. It is important to note that several small molecules that have no sites appropriate for modification are not suited for affinity-based target isolation [47]. In our case, we successfully modified B6 to accommodate functional groups through which it could be covalently linked to the resin. We investigated three positions for the introduction of reactive amino or hydroxyl groups: one on the phenoxy moiety (compound 1) and two on the napthoquinone portion (compounds 2 and 3) (Fig. 1). Subsequent biological studies showed that the chemical modifications performed on B6 resulting in 1 and 3 did not have a dramatic impact on the observed anti-Trypanosoma profile. Conversely, 2 showed a significant decrease of the trypanocidal activity (Fig. 1). In particular, as 1 was only 7 times less active than B6, we assumed that 1 might retain B6-like binding properties. Thus, we next performed affinity chromatography and target identification studies using 1. A T. b. rhodesiense lysate was prepared as described in the Methods section, and 1 was immobilized on an affinity chromatography column. After LC separation using two gradients and subsequent mass spectrometry and database searches, four trypanosome proteins were identified with a probability of more than 95%, and which were found only on the coupled matrix but were absent in the control experiment (Table S1): i. e., (i) glycosomal glycerol kinase (TbGK; SwissProt accession code Q9NJP9), (ii) tubulin beta chain (P04107), (iii) tubulin alpha chain (P04106), and (iv) glycosomal glyceraldehyde-3-phosphate dehydrogenase (TbGAPDH; P22512) (Table S1). Two proteins, TbGK and TbGAPDH, were selected for further studies as putative targets of B6, while tubulins were excluded because of the fact that they are well known to interfere with affinity chromatography studies due to their high abundance [48]. Indeed, unspecific association of T. b. brucei tubulin with affinity matrix has been observed in a similar affinity work by Mercer et al. [49]. For the chemical validation of both TbGK and TbGAPDH as putative targets of B6, the proteins were recombinantly expressed and purified to near-homogeneity. After purification of TbGK the SDS-PAGE analysis revealed a band of apparent high purity and with a subunit molecular weight corresponding to that of TbGK (56 kDa) (Fig. 2A). The purified protein indeed possessed TbGK activity, which was inhibited by B6 with an IC50 value of 0. 90±0. 30 µM (Fig. 3A). Expression and purification of TbGAPDH afforded pure and active enzyme exhibiting the expected subunit weight of 42 kDa (Fig. 2B). The inhibition assay showed that B6 was also a good inhibitor of this enzyme, with an IC50 value of 7. 25±1. 62 µM (Fig. 3B), strongly indicating that TbGAPDH interaction with immobilized compound 1 indeed was specific, and that TbGAPDH was not retained due to its well-known tendency to be isolated by non-specific association with chromatography resins [26]. These experiments confirmed the chemical proteomics results. In fact, both TbGK and TbGAPDH could be inhibited by B6, and therefore both represent possible molecular targets of this naphthoquinone derivative. To investigate the metabolic effect of combined and separate non-competitive inhibition of GAPDH and/or GK, we performed simulations with the bloodstream-form T. brucei glycolysis model, based on the Lister 427 strain [30]. Inhibition of GAPDH and GK together has a strong effect on the ATP production flux (Fig. 4A). Earlier results showed that 20–40% inhibition of glycolytic ATP production flux will result in 50% inhibition of the growth rate [50]. In the simulations, 40% inhibition of the ATP production flux is reached at an inhibitor concentration of 7 µM. This is only 3. 5-fold lower than the experimentally determined ED50 of 24. 8 µM for B6 on cultured bloodstream-form Lister 427 trypanosomes (see Methods and below for details). Modeling the inhibition of GAPDH or GK separately for aerobic glycolysis showed that GAPDH inhibition alone could be sufficient for the full effect (Fig. 4A). This can be expected from the fact that under aerobic conditions, there is only a small flux to glycerol. However, under anaerobic conditions, glucose is broken down to equimolar amounts of pyruvate and glycerol [50]. Therefore, we also did simulations under anaerobic conditions. In this context, B6 had an even a stronger effect (Fig. 4B), but now GK inhibition alone would give an equally strong effect on the ATP production flux as the combined inhibition of GAPDH and GK. We conclude, based on the modeling, that inhibition of GK is therefore only important when the parasite experiences periods of low oxygen tension. Under aerobic conditions prevailing in most part of the blood circulation system, the non-competitive inhibition of GAPDH should be sufficient for maximal effect of B6. Therefore, we further focused on the effect of B6 on GAPDH. Kinetic studies were performed to investigate the mechanism by which B6 inhibits TbGAPDH. Figure 5 shows TbGAPDH activity with respect to NADH (Fig. 5A) or 3-PGA (Fig. 5B) as substrate and at increasing concentrations of inhibitor B6. Double reciprocal plots showed B6 to act as a non-competitive inhibitor: the fitted Ki was similar when NADH or 3-PGA was varied (Ki of 3. 60±0. 57 µM for variation of NADH and Ki of 4. 99±1. 70 µM for variation of 3-PGA, exhibiting an α value of 0. 6 and 0. 4, respectively). The factor α describes the effect of the inhibitor on the affinity of the substrate toward the enzyme and the effect of the substrate on the inhibitor affinity for the enzyme [51]. We measured the IC50 value of B6 after a pre-incubation of TbGAPDH with the inhibitor for 10 min and then diluted them 10-fold to the final concentration for the assay. After 1∶10 dilution, the compound was able to inhibit TbGAPDH with an IC50 value close to that obtained under standard assay conditions (IC50 = 9. 98±3. 53 µM with dilution and IC50 = 7. 25±1. 62 µM without dilution; see Fig. 3B). The long pre-incubation time necessary for any B6 effect and the observation that IC50 value is not significantly affected by dilution suggested a possible tight or covalent binding inhibition mechanism for the compound [52]. A possible explanation for this behavior could be related to the cysteine trap mechanism of naphthoquinones previously reported for similar compounds [53]. In particular, we might hypothesize that B6 could trap Cys166, which has been shown to play a crucial role in GAPDH' s catalytic activity [54]. However, we cannot rule out that based on the observed non-competitive inhibition kinetics, B6 binds GAPDH outside the catalytic pocket. To investigate the possible covalent bond interaction, we tried to detect the B6/GAPDH complex by ESI-TOF and MALDI-TOF mass spectroscopies. However, this peptide fragment was not ionized enough to be detected by mass spectrometry under our test conditions (data not shown). Since mass-spectroscopic analysis was not helpful for clarification of the covalent bonding interaction between Cys166 and B6, we next exploited molecular docking to predict the plausible binding mode of B6 in the GAPDH active site. A docking simulation was performed using B6 and the available three-dimensional structure of TbGAPDH. The docking results (Fig. 6A) clearly showed that B6 could be suitably placed in the TbGAPDH active site to undergo a nucleophilic attack from the Cys166 side chain, and consequently form a covalent adduct, which can be responsible for the overall inhibition mechanism of B6. In Figure 6B, two possible mechanisms by which the covalent adduct could be formed are shown. The Cys166 thiol undergoes 1,4-Michael addition to B6 to form the corresponding thioether-substituted hydroquinone. Alternatively, a reaction at C3 with the phenate displacement as leaving group and formation of the substitution product may take place. Both of these mechanisms have already been reported for phenoxybenzoquinone derivatives as VEGFR inhibitors [53]. Tests of growth inhibition by B6 were performed on procyclic trypanosomes cultured in medium containing glucose (SDM79 medium), resulting in glycolytic activity for the cells' main energy supply, or without glucose (SDM80 medium), where the proline is the main substrate for free energy production through mitochondrial metabolism. Under these latter conditions, the glycosomal GAPDH would still be needed for gluconeogenesis, to synthesize glucose 6-phosphate for production of glucoconjugates, but the gluconeogenic flux is assumed to be much lower than the glycolytic flux when glucose is present. It was observed that cells grown under the condition of active glucose metabolism were somewhat more susceptible for inhibition by B6 (ED50 = 0. 25±0. 09 µM) than the cells relying on proline metabolism as their main source of free energy (ED50 = 0. 67±0. 16 µM). This is in support of our observation that TbGAPDH, a crucial enzyme of the trypanosomal glycolytic pathway, may be an intracellular target of B6. Although this difference in ED50 values is relatively small, it may still be significant. In addition, the difference between ED50 and IC50 values (see previous paragraph), may suggest that there should be another target, which plays an important role in causing B6' s trypanocidal activity. Naphthoquinones have been shown to be able to generate free radicals (mainly ROS) at the mitochondrial level [55]. To determine whether B6 could exert its trypanocidal action also through this mechanism, respiration was monitored in permeabilized T. b. brucei bloodstream-form cells in the presence of B6 and/or SHAM (Fig. 7). SHAM is an inhibitor of TAO, which acts as the final electron acceptor in bloodstream form T. b. brucei. The differences in absolute oxygen consumption rate with 10 mM G3P as electron donor between individual experiments are probably due to different extents of permeabilization, and hence we also included the results in percentages (Fig. 7B–D, lower panels). As expected, 2 mM SHAM inhibited respiration completely (Fig. 7A–B). When B6 was added to permeabilized T. b. brucei respiring on G3P, the oxygen consumption rate increased. This happened both in the absence and presence of SHAM (Fig. 7A–C), showing that B6 is not just relieving SHAM inhibition. The oxygen consumption in the presence of SHAM plus B6 was the same irrespective of whether B6 was added after or together with SHAM (compare Fig. 7B and D). Clearly, B6 resulted in non-TAO mediated oxygen consumption. This finding might be explained by taking into account the capability of B6 to react with molecular oxygen, likely as a consequence of ROS production. Addition of an extra 2–4 mM SHAM did not relieve this B6-mediated oxygen consumption (Fig. 7B–D). To prove the capability of B6 to generate ROS, we measured the production of radicals during respiration in a mitochondrial fraction of T. b. brucei (bloodstream form) in the presence and absence of B6 (Fig. 8). Indeed, in this experiment, production of ROS, due to the reactivity of B6 when it was reduced by the mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase, was detected. After 40 min, B6, in the presence of G3P, was able to generate radicals in a significant way compared to the control experiments. B6 caused a considerable production of ROS during the respiration of T. b. brucei bloodstream-form mitochondria, but not in bovine heart submitochondrial particles (Table S2 and Fig. S1). To provide support for the notion that the trypanocidal effect of B6 could in part be attributed to the generation of toxic ROS, we made use of an available bloodstream form T. b. brucei cell line in which glucose-6-phosphate dehydrogenase (G6PDH) expression can be knocked down by RNA interference (RNAi) [56]. Previously, it has been shown that this cell line grows equally well as wild-type cells under normal (reducing) growth conditions, but it is highly susceptible to ROS when the G6PDH expression is partially knocked down, an effect attributed to decreased NADPH production [57]. Indeed, administration of B6 to cells induced for decreased expression of G6PDH by RNAi showed that these cells are much more susceptible. The ED50 for the induced RNAi cell line is 0. 25 µM, whereas for the non-induced RNAi cell line, it was 10. 8 µM. This clearly showed that B6 could increase mitochondrial ROS production as a further molecular mechanism at the basis of its trypanocidal profile. Neglected tropical diseases are a huge health emergency, which requires remarkable efforts in the search for novel drug candidates to combat them. In fact, the drug discovery pipeline in the field of neglected tropical infectious diseases is almost dry, and fast technologies should be exploited to identify novel classes of potential drug candidates. A possible integrated strategy could be the use of parallel synthesis to generate libraries of small organic molecules combined with fast phenotypic assays that allow testing hundreds of compounds in a reasonable amount of time. This strategy could rapidly provide new hit candidates that can be further progressed to the hit-to-lead and lead optimization steps of the drug discovery process. Thus, it is currently considered equally productive as the target-based approach [58] and it also show higher strengths when searching for multitarget ligands [13]. However, to rationally carry out the two latter steps, information on the molecular target (s), and possibly on the hit-target binding mode can be of paramount importance, as any modification on the hit scaffold can be rationally guided by computational and biophysical/biochemical methods. Therefore, once a hit compound has been identified by means of cell-based experiments, it is fundamental to try identifying the potential target (s) by means of bioanalytical approaches. In this respect, chemical proteomics has emerged as a promising method to fish out targets from cell lysates using affinity chromatography [26]. Indeed, chemical proteomics has been shown to be highly suitable in complementing cell-based experiments, and to provide fundamental information for accelerating, in a rational manner, the progress of new classes of compounds through the drug discovery process [59]–[61]. It should be noted that such an approach can be particularly suited for compounds that form covalent adducts or bind tightly to protein targets. In addition, non-protein targets (e. g. radical oxygen production, DNA, etc.) can be missed by using this approach. Here, we have reported on the application of chemical proteomics techniques aiming at the identification of the potential molecular targets of a new class of naphthoquinone derivatives. They have recently been characterized, by means of cell-based experiments, to act as trypanocidals, but the molecular target (s) of this class of compounds remained undisclosed. We have here identified two potential targets (TbGAPDH and TbGK) of B6, a representative member of this class of compounds. Subsequent biochemical assays have clearly demonstrated that B6 is able to inhibit both targets with IC50 values in the micromolar range. However, mathematical modeling has shown that GK inhibition contributed to B6 trypanocidal activity only under low oxygen conditions. In fact, this enzyme under most physiological conditions does not play an essential role in the trypanosome' s metabolism, and is thus considered as a sub-optimal drug target, which may become important when inhibited in conjunction with other enzymes (i. e. TAO) [62]. Conversely, GAPDH is a vital parasitic enzyme and a well-validated molecular target for antiparasitic drug discovery [62]. To fully account for the nanomolar profile of B6 as assessed by phenotypic cell-based assay, other mechanisms had to be evoked. In light of this, and based on a vast literature reporting that quinones and naphthoquinones can interact with the mitochondrial respiratory chain [55], [63]–[65], we have performed further experiments using trypanosomal isolated mitochondria and permeabilized cells. In this way, we could demonstrate that B6 was also able to interfere with the respiratory chain by generating ROS, and supporting the likelihood that B6 interacts with additional targets located in T. brucei' s mitochondrion. We also observed an ED50 of B6 for bloodstream-form T. b. brucei strain Lister 427 of 24. 8 µM, whereas the ED50 of procyclics of the same strain grown with glucose as free-energy source is 0. 25 µM. The fact that glucose-grown procyclic trypanosomes are 100-fold more susceptible than glucose-grown bloodstream-form cells strongly suggests that in procyclic trypanosomes inhibition of the glycosomal GAPDH is not the dominant cause of death. The nature of the dominant target of B6 in the procyclics, other than GAPDH, remains to be determined. An alternative possibility is that the bloodstream-form, whose natural habitat is the human body, expresses a more elaborate system to deal with ROS generated by the host than the procyclics that live in the insect' s midgut. Consequently, if B6 generates important ROS in cells of both life-cycle stages, the cultured bloodstream-forms may also be better equipped to deal with it than the cultured procyclic cells. In addition, ROS production might be higher in procyclics, as respiratory chain complexes I and III, which are well-known sites of ROS production, are not expressed in the bloodstream forms of T. brucei. As for GK, its inhibition likely only marginally contributed to the final trypanocidal activity of our compound. However, glycerol may be an additional substrate in the blood and, although of much less use than glucose (lower concentration, and lower ATP yield), glycerol consumption may relieve the effect of glycolysis inhibition. In this respect, inhibition of GK by B6 may have relevance in vivo as it might prevent temporal rescue of the parasite by glycerol utilization or under transient anaerobic condition. In conclusion, naphtoquinones like B6 can be considered a promising class of natural-like multitarget compounds against T. brucei, which warrants further studies to definitely elucidate its multitarget profile against parasitic protists.

The multitarget approach can represent a promising strategy for the discovery of innovative drug candidates against neglected tropical diseases. However, multitarget drug discovery can be very demanding, because of the highly time-consuming step related to the fine balancing of the biological activities against selected targets. An innovative workflow for discovering multitarget drugs can be envisioned: i) design and synthesis of natural-like compounds; ii) test them using phenotypic cell-based assays; iii) fishing potential targets by means of chemical proteomics. This workflow might rapidly provide new hit candidates that can be further progressed to the hit-to-lead and lead optimization steps of the drug discovery process. The two latter steps can benefit from information on the molecular target (s), which may be identified by chemical proteomics. Herein, we report on the elucidation of the mode of action of a new series of anti-trypanosomal naphthoquinone compounds, previously tested using cell-based assays, by means of chemical proteomics, classical biochemistry, molecular and system biology.

lay_plos

2 children among more than 100 people held hostage in Houston stash house HOUSTON—More than 100 suspected illegal immigrants were found living in deplorable conditions in a south Houston stash house Wednesday, police said. The men, women and children were held against their will by smugglers or coyotes. One officer described the inside of the home in the 14700 block of Almeda School Road as "awful." "There is no hot water in the house. There is a toilet that partially works—one bathroom for in excess of 100 people," said HPD spokesman John Cannon. Police said there was human waste all over the house. The victims included a 24-year-old pregnant woman and her two children, ages 5 and 7. "The children were scared," said Noland Luke, a neighbor. "They brought them out, they crying and carrying on. Feel sorry for them." The woman’s mother called authorities after a coyote, who was paid to bring them across the border from Mexico, refused to release the immigrants until he got more money. Police investigated her complaint and discovered one of the largest stash houses they’ve ever seen. Deadbolt locks on the inside doors kept the victims from being able to leave the one-story home. The hostages -- 94 adult males, 14 adult females and the two children—will be turned over to the custody of ICE. Five smugglers were arrested by Houston police. There were about 250 chickens living outside the home. Still, neighbors said they never suspected anything. "I only see them going in and out. That’s it," said Ellea Johnson. "I never had an idea it was like this." Johnson was especially disturbed about the children. "These kids aren’t going to forget this, they’re not," She said. "They need counseling for this. They’re young, elementary school children. It’s going to stick with them." HELD RESPONSIBLE. OKAY, THANK YOU MUCH. AN EXCLUSIVE LOOK INSIDE THE BUSTED STASH HOUSE IN SOUTH HARRIS COUNTY WHERE POLICE SAY MORE THAN 100 CAPTIVES WERE STUFFED INSIDE THIS TINY HOME WITH NO RUNNING WATER AND A BROKEN TOILET. LOCAL 2'S PHIL ARCHER TAKES US ON AN EXCLUSIVE TOUR OF THIS HOUSE OF HORROR. Reporter: THE HOUSE IS JUST 1284 SQUARE FEET. BUT MORE THAN 100 PEOPLE WERE HELD HERE AGAINST THEIR WILL. SOME LOCKED INSIDE FOR WEEKS, WHEN HOUSTON POLICE AND FEDERAL AGENTS FOUND THEM WEDNESDAY. THE MEN WERE SHOELESS AND STRIPPED TO THEIR UNDERWEAR TO PREVENT THEM FROM ESCAPING. THEY WERE FORCED TO LIVE LIKE ANIMALS. WE TOOK A CAMERA IN TODAY TO FIND THE GEAR OVER, DARK AS A CAVE -- THE INTERIOR, DARK AS A CAVE, THE WINDOWS COVERED THE SHEETS, THE FLOOR A HEAP OF TRASH. THERE WERE A FEW DIRTY BOX SPRING MATTRESSES SCATTERED IN A COUPLE OF BEDROOMS. PEOPLE WERE SQUEEZED INTO EVERY INCH OF SPACE AVAILABLE IN THIS TINY TWO-BEDROOM HOUSE. POLICE FOUND MORE THAN A DOZEN WOMEN AND 19 CHILDREN AMONG THEM IN A HOUSE WITHOUT RUNNING WATER AND ONLY ONE BROKEN TOILET. IMMIGRATION AGENTS TOLD THE HOUSE HOMELAND SECURITY COMMITTEE MEETING IN HOUSTON TODAY THAT FIVE SMUGGLERS NOW FACE AN ARRAY OF CHARGES. FIVE SUBJECTS THAT ARE GOING TO BE CHARGED IN FEDERAL COURT FOR A VARIETY OF FEDERAL OFFENSES INCLUDING HOSTAGE TAKING, UNLAWFUL POSSESSION OF A FIREARM, CONSPIRACY TO HARBOR ILLEGAL ALIENS, AND THAT IS HAPPENING AS WE SPEAK. Reporter: WHEN FEDERAL AGENTS GOT IN YESTERDAY, THEY FOUND PEOPLE LITERALLY ON TOP OF EACH OTHER. IT'S EASY TO SEE THE SQUALOR. BUT WHAT THE CAMERA CAN'T CONVEY IS THE SMELL. THE SMELL OF DOZENS OF UNWASHED BODIES. IT'S THE SMELL OF DESPERATION AND POVERTY. AGENTS SAY ONE WOMAN WAS HELD HERE FOR AT LEAST 15 DAYS. SHE AND THE OTHER SAFE NOW IN FEDERAL CUSTODY. THEY ARE ILLEGAL IMMIGRANTS, BUT NOW ALSO CRIME VICTIMS. THAT STATUS COULD ALLOW THEM TO AVOID DEPORTATION AND REMAIN IN THE U.S. PHIL ARCHER, KPRC LOCAL 2. HOUSTON - Federal agents said a total of 115 immigrants were being held at a suspected stash house in south Harris County by five smuggling suspects who are now in custody. Special agent Brian Moskowitz with U.S. Immigration and Customs Enforcement told members of the U.S. House Committee on Homeland Security, meeting today in Houston, that the suspects will be charged with “a variety of federal offenses, including hostage taking, unlawful possession of a firearm and conspiracy to harbor illegal aliens.” Houston police made the discovery Wednesday morning in the 14700 block of Almeda School Road. Police said they had been conducting surveillance at the home after they received a report of a kidnapping Tuesday night. Police said they were contacted by the family of a 24-year-old woman who reported that a human smuggler, or "coyote", had failed to show up at a meeting place where they were supposed to turn over the woman and her two children, ages five and seven. According to Houston police, the investigation led to the home on Almeda School Road, where they pulled over a vehicle that was seen leaving Wednesday morning. Police said inside the suspects' vehicle they found two guns and evidence of a human smuggling operation. Speaking to the same committee Thursday, Houston Police Chief McClelland said, “We believed someone's life was in jeopardy." Once inside, the officers found a "sea of people," ranging in age from teens to adults. "Bodies upon bodies, people stacked on top of each other. Dirty, filthy, conditions," said HPD spokesman John Cannon. Police said a total of 94 men and 14 women were discovered, including one pregnant woman who was taken to a hospital for treatment. "I can't believe it, can't believe because you never see that," said one neighbor. The men were wearing only underwear and no shoes, so they couldn't escape, police said. The doors were locked from the outside. An ICE official told Local 2 it was the largest operation it's encountered in five years. Authorities said 500 chickens were also found on the property. Local 2 reporter Phil Archer toured the interior of the house Thursday, and found it dark as a cave, with the exception of a few, bare light bulbs. The windows were covered with sheets. The floor was a heap of blankets, garbage bags and trash. A few, dirty box spring mattresses were scattered in the two bedrooms and a built-on extension. The immigrants were also detained and were taken to an ICE detention facility. Once there, they'll be fingerprinted and undergo medical screenings. Each person will also have a one-on-one interview. Some may be returned to their home countries, but decisions will be made on a case-by-case basis. All of the them will have the opportunity to go before an immigration judge. And all will have an opportunity to apply for a “U-Visa,” which allows illegals who are also victims of crime to live and work legally in the U.S. for four years. Page 1 of 1 More than 100 people were found Wednesday morning inside a house at 14711 Almeda School Road in Southeast Houston, Wednesday, March 19, 2014. Police found the group, who are all presumed to be in the country illegally -- at a squalid "stash house" in southeast Houston. Police say it appears to be human smuggling operation. More than 100 people were found Wednesday morning inside a house at 14711 Almeda School Road in Southeast Houston, Wednesday, March 19, 2014. Police found the group, who are all presumed to be in the country illegally -- at a squalid "stash house" in southeast Houston. Police say it appears to be human smuggling operation. More than 100 people were found Wednesday morning inside a house at 14711 Almeda School Road in Southeast Houston, Wednesday, March 19, 2014. Police found the group, who are all presumed to be in the country illegally -- at a squalid "stash house" in southeast Houston. Police say it appears to be human smuggling operation. More than 100 people were found Wednesday morning inside a house at 14711 Almeda School Road in Southeast Houston, Wednesday, March 19, 2014. Police found the group, who are all presumed to be in the country illegally -- at a squalid "stash house" in southeast Houston. Police say it appears to be human smuggling operation. More than 100 people were found Wednesday morning inside a house at 14711 Almeda School Road in Southeast Houston, Wednesday, March 19, 2014. Police found the group, who are all presumed to be in the country illegally -- at a squalid "stash house" in southeast Houston. Police say it appears to be human smuggling operation. More than 100 people were found Wednesday morning inside a house at 14711 Almeda School Road in Southeast Houston, Wednesday, March 19, 2014. Police found the group, who are all presumed to be in the country illegally -- at a squalid "stash house" in southeast Houston. Police say it appears to be human smuggling operation. More than 100 people were found Wednesday morning inside a house at 14711 Almeda School Road in Southeast Houston, Wednesday, March 19, 2014. Police found the group, who are all presumed to be in the country illegally -- at a squalid "stash house" in southeast Houston. Police say it appears to be human smuggling operation. More than 100 people were found Wednesday morning inside a house at 14711 Almeda School Road in Southeast Houston, Wednesday, March 19, 2014. Police found the group, who are all presumed to be in the country illegally -- at a squalid "stash house" in southeast Houston. Police say it appears to be human smuggling operation. More than 100 people were found Wednesday morning inside a house at 14711 Almeda School Road in Southeast Houston, Wednesday, March 19, 2014. Police found the group, who are all presumed to be in the country illegally -- at a squalid "stash house" in southeast Houston. Police say it appears to be human smuggling operation. More than 100 people were found Wednesday morning inside a house at 14711 Almeda School Road in Southeast Houston, Wednesday, March 19, 2014. Police found the group, who are all presumed to be in the country illegally -- at a squalid "stash house" in southeast Houston. Police say it appears to be human smuggling operation. More than 100 people were found Wednesday morning inside a house at 14711 Almeda School Road in Southeast Houston, Wednesday, March 19, 2014. Police found the group, who are all presumed to be in the country illegally -- at a squalid "stash house" in southeast Houston. Police say it appears to be human smuggling operation. More than 100 people were found Wednesday morning inside a house at 14711 Almeda School Road in Southeast Houston, Wednesday, March 19, 2014. Police found the group, who are all presumed to be in the country illegally -- at a squalid "stash house" in southeast Houston. Police say it appears to be human smuggling operation. More than 100 people were found Wednesday morning inside a house at 14711 Almeda School Road in Southeast Houston, Wednesday, March 19, 2014. Police found the group, who are all presumed to be in the country illegally -- at a squalid "stash house" in southeast Houston. Police say it appears to be human smuggling operation. More than 100 people were found Wednesday morning inside a house at 14711 Almeda School Road in Southeast Houston, Wednesday, March 19, 2014. Police found the group, who are all presumed to be in the country illegally -- at a squalid "stash house" in southeast Houston. Police say it appears to be human smuggling operation. More than 100 people were found Wednesday morning inside a house at 14711 Almeda School Road in Southeast Houston, Wednesday, March 19, 2014. Police found the group, who are all presumed to be in the country illegally -- at a squalid "stash house" in southeast Houston. Police say it appears to be human smuggling operation. More than 100 people were found Wednesday morning inside a house at 14711 Almeda School Road in Southeast Houston, Wednesday, March 19, 2014. Police found the group, who are all presumed to be in the country illegally -- at a squalid "stash house" in southeast Houston. Police say it appears to be human smuggling operation. More than 100 people were found Wednesday morning inside a house at 14711 Almeda School Road in Southeast Houston, Wednesday, March 19, 2014. Police found the group, who are all presumed to be in the country illegally -- at a squalid "stash house" in southeast Houston. Police say it appears to be human smuggling operation. More than 100 people were found Wednesday morning inside a house at 14711 Almeda School Road in Southeast Houston, Wednesday, March 19, 2014. Police found the group, who are all presumed to be in the country illegally -- at a squalid "stash house" in southeast Houston. Police say it appears to be human smuggling operation. More than 100 people were found Wednesday morning inside a house at 14711 Almeda School Road in Southeast Houston, Wednesday, March 19, 2014. Police found the group, who are all presumed to be in the country illegally -- at a squalid "stash house" in southeast Houston. Police say it appears to be human smuggling operation. More than 100 people were found Wednesday morning inside a house at 14711 Almeda School Road in Southeast Houston, Wednesday, March 19, 2014. Police found the group, who are all presumed to be in the country illegally -- at a squalid "stash house" in southeast Houston. Police say it appears to be human smuggling operation. More than 100 people were found Wednesday morning inside a house at 14711 Almeda School Road in Southeast Houston, Wednesday, March 19, 2014. Police found the group, who are all presumed to be in the country illegally -- at a squalid "stash house" in southeast Houston. Police say it appears to be human smuggling operation. More than 100 people were found Wednesday morning inside a house at 14711 Almeda School Road in Southeast Houston, Wednesday, March 19, 2014. Police found the group, who are all presumed to be in the country illegally -- at a squalid "stash house" in southeast Houston. Police say it appears to be human smuggling operation. More than 100 people were found Wednesday morning inside a house at 14711 Almeda School Road in Southeast Houston, Wednesday, March 19, 2014. Police found the group, who are all presumed to be in the country illegally -- at a squalid "stash house" in southeast Houston. Police say it appears to be human smuggling operation. More than 100 people were found Wednesday morning inside a house at 14711 Almeda School Road in Southeast Houston, Wednesday, March 19, 2014. Police found the group, who are all presumed to be in the country illegally -- at a squalid "stash house" in southeast Houston. Police say it appears to be human smuggling operation. More than 100 people were found Wednesday morning inside a house in the 14000 block of Almeda School Road in Southeast Houston, Wednesday, March 19, 2014. Police found the group, who are all presumed to be in the country illegally -- at a squalid "stash house" in southeast Houston. Police say it appears to be human smuggling operation. More than 100 people were found Wednesday morning inside a house at 14711 Almeda School Road in Southeast Houston, Wednesday, March 19, 2014. Police found the group, who are all presumed to be in the country illegally -- at a squalid "stash house" in southeast Houston. Police say it appears to be human smuggling operation. (Cody Duty / Houston Chronicle) More than 100 people were found Wednesday morning inside a house at 14711 Almeda School Road in Southeast Houston, Wednesday, March 19, 2014. Police found the group, who are all presumed to be in the country illegally -- at a squalid "stash house" in southeast Houston. Police say it appears to be human smuggling operation. (Cody Duty / Houston Chronicle) Five accused smugglers suspected of concealing the 115 people who were discovered last week in a south Harris County "stash house" have been charged by federal authorities. Court papers describe an extortion operation enforced by threats of violence, including sexual abuse, where undocumented immigrants paid for transportation to locations across the United States but were sidetracked inside a small Pearland-area house while even more money was demanded from their relatives for their release. Authorities who discovered the single-story residence described a house of horrors where armed guards held dozens of nearly naked people hostage while their families scraped together unexpected ransoms. Federal officials said they suspect Jose Aviles-Villa, Jonathan Solorzano-Tavila, Antonio Barruquet-Hildiberta, Jose Cesmas-Borja and Eugenio Cesmas-Borja were running the operation. Each man is charged with one count of hostage-taking and an additional count of violence with a firearm. They will have their initial court appearances before a Houston federal magistrate on Tuesday afternoon, followed by a material witness hearing where 12 people are slated to testify, records show. Monday's court filings reveal details about how authorities discovered the operation on Wednesday. A woman contacted the Houston Police Department about the possible kidnapping of her daughter and two small grandchildren. The tipster said she had paid smugglers $15,000 to transport her three relatives to Chicago. The grandmother told investigators that a smuggler in Houston told her that if she didn't pay an additional $13,000 that they would "make her family disappear," a criminal complaint against the five accused men said. The information led authorities to a 1,284-square-foot house on Almeda School Road where they stopped a red Ford Mustang leaving the property. While obtaining identification from two men in the front seat and two women in the back, officers observed "a black semi-automatic handgun protruding from beneath the front passenger seat," the complaint said. The group was detained, and officers recovered two handguns from the car. The two men later were identified as Aviles-Villa and Jose Cesmas-Borja. When other HPD officers conducting surveillance on the house heard about the stop, they decided to make a health and welfare check on the residence. As they approached, three men "ran across the yard, jumped over a fence and fled into a wooded field." Those men were identified as Solorzano-Tavila, Barruquet-Hildiberta and Eugenio Cesmas-Borja. People held inside said the men were armed stash house guards who "were holding them against their will until their families paid their transportation fees." A third group of officers saw several men in their underwear inside, then contacted local U.S. Department of Homeland Security officials upon believing that they had located the woman reported missing and her two children. The three were positively identified a short time later. In all, authorities found 99 males and 16 females, including one who was pregnant. More than one dozen of the people held hostage were juveniles. Some of the people, who had been held since at least Feb. 1, said they were destined for places including New York and Georgia, court papers said. Last week, a Houston Police Department spokesman described rooms littered with plastic trash bags and clothes, a single toilet and an unbelievable stench. Occupants ranged in age from 5 to 47 and were from El Salvador, Mexico, Guatemala and Honduras. Most of the inner and outer doors of the home had dead bolt locks, court papers said, and the majority of the windows were covered with plywood from inside. A wooden paddle, a stun gun, two shotguns and ammunition were found inside the house, along with a shotgun and rifle outside nearby. The paddle was used to "intimidate" the confined people and prevent them from escaping, court papers said. Clothing and shoes were locked inside a closet to prevent the immigrants from running away. Upon being read their Miranda rights in Spanish, the defendants explained their roles in the operation and said they were paid $600 to $1,750 each week for their services, according to the criminal complaint. The raid marked the largest discovery of undocumented people in the Houston region in seven years and underscored the area's role as a hub for smuggling people into Texas and the rest of the country. The last remarkable local residential bust happened in 2012, when more than 80 people were found in an Alief-area home in far southwest Harris County.

Houston police last Wednesday uncovered a grim building covered in human waste and packed with 115 people held hostage inside. Court papers filed in the case against five alleged human smugglers provide more insight into how the "stash house" came to be, and how police uncovered it. The Houston Chronicle reports that the tip came from a worried grandmother, who reached out to local police after being contacted by a smuggler who demanded $13,000 in order to transport her daughter and two young grandchildren to Chicago-a trip the woman said her daughter had paid the smugglers $15,000 to facilitate. Pay the ransom, the grandmother was told, or the smugglers would "make her family disappear." KPRC previously reported the 24-year-old and her children didn't show up at a prescribed meeting place last Tuesday night. The grandmother's tip brought police to the Pearland-area home; neighbors told KHOU they had no inkling of what was going on inside, but it didn't take police long to figure it out: One set of officers stopped a Ford Mustang that was exiting the property and noticed "a black semi-automatic handgun protruding from beneath the front passenger seat"; other officers saw three men jump a fence and flee; still a third group observed men wearing only underwear inside, and called the Department of Homeland Security. Why the lack of clothing? Per the filing, the alleged smugglers locked up their hostages' clothes and shoes to make fleeing difficult; deadbolt locks, boarded-up windows, a wooden paddle, stun gun, and firearms also assisted to that end. Those held there, some for at least six weeks, hailed from El Salvador, Mexico, Guatemala, and Honduras. An initial court appearance is today scheduled for the five accused, who are charged with hostage-taking and violence with a firearm.

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Monocytes are circulating short-lived macrophage precursors that are recruited on demand from the blood to sites of inflammation and challenge. In steady state, classical monocytes give rise to vasculature-resident cells that patrol the luminal side of the endothelium. In addition, classical monocytes feed macrophage compartments of selected organs, including barrier tissues, such as the skin and intestine, as well as the heart. Monocyte differentiation under conditions of inflammation has been studied in considerable detail. In contrast, monocyte differentiation under non-inflammatory conditions remains less well understood. Here we took advantage of a combination of cell ablation and precursor engraftment to investigate the generation of gut macrophages from monocytes. Collectively, we identify factors associated with the gradual adaptation of monocytes to tissue residency. Moreover, comparison of monocyte differentiation into the colon and ileum-resident macrophages revealed the graduated acquisition of gut segment-specific gene expression signatures. The recent past has seen major advance in our understanding of the diverse origins of tissue macrophages, as well as their discrete maintenance strategies. Macrophages were shown to arise from three distinct developmental pathways that differentially contribute to the tissue compartments in the embryo and adult (Ginhoux and Guilliams, 2016). In the mouse, embryonic tissue macrophages first develop from primitive macrophage progenitors that originate in the yolk sac (YS). In the brain, YS-macrophage-derived macrophages persist throughout adulthood, while in most other tissues these cells are replaced by fetal monocytes that derive from multi-potent erythro-myeloid progenitors (EMP). Definitive hematopoiesis commences from E10. 5 with the generation of hematopoietic stem cells (HSC) that first also locate to the fetal liver, but eventually seed the bone marrow (BM) to maintain adult hematopoiesis. Most EMP-derived tissue macrophage compartments persevere throughout adulthood without major input from HSC-derived cells; however, in certain barrier tissues, as well as selected other organs, like the heart, embryonic macrophages are progressively replaced by HSC-derived cells involving a blood monocyte intermediate (Varol et al., 2015). Monocytes are continuously generated in the BM involving a sequence of developmental intermediates, before extravasation into the circulation (Ginhoux and Jung, 2014; Mildner et al., 2016). Once in the blood, murine classical Ly6C+ monocytes have a limited half-life (Yona et al., 2013). On demand, Ly6C+ monocytes can be rapidly recruited to sites of injury and challenge, where they complement tissue-resident macrophages and dendritic cells. In absence of challenge, some Ly6C+ monocytes give rise to vasculature-resident Ly6C- cells, which patrol the vessel walls (Auffray et al., 2007). These cells display macrophage characteristics including extended life spans (Yona et al., 2013; Mildner et al., 2017). In addition, Ly6C+ monocytes replenish the above-mentioned selected steady state tissue macrophage compartments, including the gut and skin (Ginhoux and Jung, 2014). Given their mobility, plasticity and key role in pathologies, the manipulation of monocytes and their differentiation could bear considerable therapeutic potential. However, monocyte differentiation into tissue resident cells remains incompletely understood. Gut macrophages, which reside in the connective tissue underlying the gut epithelium, the lamina propria, are considered key players for the maintenance of intestinal homeostasis. As such, they constantly sense their environment and respond to the unique microbiota and food challenge (Bain and Mowat, 2011; Zigmond and Jung, 2013). Recent studies revealed that monocyte-derived lamina propria macrophages comprise in mice two populations, that is short-lived cells and long-lived cells with self-renewing capacity (Shaw et al., 2018); the latter population might also include remnants of embryonic populations, as could additional intestinal long-lived macrophage populations that reside near blood vessels, nerves and in the Peyer' s Patches (De Schepper et al., 2018). Evidence for macrophages with different half-lives is also emerging for the human small intestine (Bujko et al., 2018). Collectively, these findings highlight the existence of considerable macrophage heterogeneity, not only between different organs, but also within given tissues. Monocyte differentiation into intestinal macrophages involves phenotypic changes with respect to Ly6C, CD64 and MHCII expression, a sequence described as ‘monocyte waterfall’ (Tamoutounour et al., 2012). Mature steady-state gut macrophages tolerate the commensal microbiota and food antigens (Bain and Mowat, 2011; Zigmond and Jung, 2013). Their relative unresponsiveness is thought to rely on regulatory circuits that balance the expression of pro- and anti-inflammatory gene products, such as cytokines and molecules participating in pattern recognition receptor signaling cascades. Macrophages located in different tissues display characteristic enhancer landscapes and gene expression profiles, which have been attributed to the exposure of instructing factors of the microenvironment they reside in Amit et al. (2016). Gut segments display distinct anatomy, function and microbiota load (Mowat and Agace, 2014) and macrophages of small and large intestine hence also likely differ. Despite the known distinct susceptibility of the colon and ileum to pathology, such as to the IL10R deficiency (Glocker et al., 2009; Zigmond et al., 2014) and in ulcerative colitis, so far no comparative analysis of their macrophages has been reported. Likewise, our general understanding of monocyte-derived tissue resident macrophages remains scarce and is largely restricted to settings of inflammation. Here we investigated monocyte differentiation into intestinal macrophages in the small and large intestine. Using adoptive monocyte transfers into macrophage-depleted recipients (Varol et al., 2007; Varol et al., 2009), we synchronized the macrophages in terms of development, isolated colonic and ileal macrophages and subjected them to transcriptome profiling. Our data establish the distinct identities of gut segment resident macrophages and shed light on the kinetics and gradual gene expression of specific factors for their establishment of their identities. Tissue macrophages display distinct gene expression profiles and enhancer landscapes (Amit et al., 2016). This holds also for intestinal macrophages residing in small and large intestine. Transcriptomes of Ly6C+ BM monocytes, that is the macrophage precursors, and transcriptomes of their progeny in colon and ileum displayed 6200 genes differentially expressed at least 2-fold across all analyzed data sets out of a total of 12345 detected genes (Figure 1—figure supplement 1A–C). 2255 genes were expressed in monocytes and down-regulated in macrophages (cluster I). Conversely, cluster II comprised genes whose expression was absent from monocytes, but shared by both small and large intestinal macrophages. Finally, 1087 and 987 genes were either preferentially or exclusively expressed in ileal or colonic macrophages, respectively. To further characterize adult monocyte-derived gut macrophages, we took advantage of an experimental system involving monocyte engraftment of macrophage-depleted animals (Varol et al., 2007; Varol et al., 2009). Analysis of transferred cells at different intervals from engraftment allows the study of intestinal macrophage development over time, since monocyte differentiation is synchronized. For the cell ablation we used [CD11c-DTR > WT] BM chimeras, in which diphtheria toxin (DTx) receptor (DTR) transgenic intestinal macrophages can be conditionally ablated by DTx injection (Varol et al., 2007; Varol et al., 2009) (Figure 1A). Two days prior to monocyte transfer, DTx was applied to the recipients to clear their intestinal macrophage niche and mice were subsequently treated with DTx every second day. The monocyte graft was isolated from BM of Cx3cr1GFP/+ mice (Jung et al., 2000) and defined as CD117- CD11b+ CD115+ Ly6C+ GFPint cells; donor animals also harbored an allotypic marker (CD45. 1) (Figure 1A, Figure 1—figure supplement 1A). Grafted cells could be visualized in recipient gut tissue and underwent expansion, as reported earlier (Varol et al., 2009) (Figure 1—figure supplement 2). Intestinal tissues of recipient mice were harvested on day 4,8 and 12 post engraftment (Figure 1A) to isolate graft-derived macrophages according to CD45. 1 and CX3CR1/GFP expression; these cells were CD11b+ CD64+ Ly6C- and mainly CD11chi (Figure 1B). Of note, specifics of the system preclude harvest of graft-derived cells at later time points (Varol et al., 2009). The monocyte graft and colonic and ileal macrophage populations were subjected to bulk RNA-seq using MARS-Seq technology (Jaitin et al., 2014). Gene expression profiling revealed robust changes already at day four post engraftment; changes appeared to be tissue- rather than time-specific, with the exception of two day 4 samples of ileal macrophages, which clustered with the colonic samples (Figure 1C, Figure 1—figure supplement 3A). Transcriptomes of the retrieved engrafted macrophages lacked expression of pro-inflammatory genes, such as Saa3, Lcn2, Il1b, Il6 and Tnf, as opposed to cells retrieved from colitic animals treated with Dextran Sulfate Sodium (DSS) (Okayasu et al., 1990) (Figure 1—figure supplement 4). This supports the earlier notion that the cell ablation results in a transient tissue response, but the monocyte transfer system mimics cell differentiation close to steady-state conditions (Varol et al., 2009). Comparative transcriptome analysis of grafted monocytes, their progeny retrieved from the recipient intestines, and resident ileal and colonic macrophages that were independently retrieved from Cx3cr1GFP/+ mice revealed 4213 differentially expressed genes (DEG) (>4 fold differences in any pairwise comparison among a total of 12878 genes) (Figure 1D). 748 genes were exclusively expressed in the monocyte graft, including Ly6c2, Ccl6 and Cebpe (Cluster I) (Figure 1D, E). 793 genes shared expression in graft-derived cells and gut macrophages in colon and ileum (Cluster II). These included Irf6, Gata6, Ly6A and Smad7. Cluster III comprised 634 genes that were either exclusively or preferentially expressed in colonic macrophages, such as Pparg, Foxa1 and Tlr1 (Figure 1E). Cluster IV comprised 539 genes exclusively or preferentially expressed in ileal macrophages, such as Nos2, Sox4 and Mmp9 (Figure 1E). Metascape analysis (Zhou et al., 2019) highlighted that genes associated with cluster II, II and IV were associated with distinct pathways, particularly with respect to epithelial cell communication and cell metabolism (Figure 1—figure supplement 3B). We also identified genes that differed in expression between engrafted and resident macrophages. Specifically, 796 and 467 genes displayed high or low expression levels in monocytes, respectively, were highly expressed in resident colonic macrophages, but either low or absent in the graft-derived cells (Cluster V and VI). Finally, cluster VII comprised 236 genes that were expressed in monocytes, and down-regulated in resident gut macrophages, but not in the grafted cells. Volcano plot analysis revealed that early monocyte differentiation (graft vs day 4) was characterized by abundant changes in gene expression in both colon and ileum. In the colon 3407 genes were up- and 3608 genes were down-regulated; in the ileum 2975 genes were up- and 3206 genes were down-regulated, with up to 10 fold change (Figure 1F). Later time points (day 4 to day 8 and day 8 to day 12) were characterized by less pronounced alterations, both with respect to the number of DEG and their fold change (Figure 1G, H). In the colon 289 genes (66% of all significantly-changed genes) were induced between day 4 and 8 and only 111 genes (19%) were up-regulated between day 8 and 12. In the ileum this trend was reversed, with 95 genes (21%) up-regulated between day 4 and 8, but 321 genes (85%) induced between day 8 and 12. Collectively, this establishes that monocytes that enter distinct gut segments rapidly acquire characteristic transcriptomic signatures. Our experimental set up allows us to focus on cells that entered the gut in a defined time window. Interestingly, even by day 12, monocyte graft-derived cells differed in expression profiles when compared to resident ileal and colonic macrophages (Figure 2A). Differences included genes absent from, and exclusively expressed by engrafted cells (Figure 1D clusters V - VII, Figure 2B–D). These differences might be attributable to incomplete macrophage maturation or to the recently reported heterogeneity of the intestinal macrophage compartment (Shaw et al., 2018; De Schepper et al., 2018). Notably, the majority of the cells we retrieve likely comprise lamina propria/mucosa resident macrophages, rather than the less abundant macrophages of the submucosa or muscularis layer (Shaw et al., 2018; De Schepper et al., 2018). Generation of a CD4+ Timd4+ subpopulation of lamina propria macrophages was reported to require prolonged residence in the tissue (Shaw et al., 2018). Although grafted cells in both colon and ileum acquired with time some expression of a hallmark of the long-lived cells, the phosphatidyl serine receptor Timd4 (Tim4) (Figure 2B, Figure 1D cluster VI), other signature genes, such as Cd209f, C2 and Rusc2 (Shaw et al., 2018), were not expressed in the time frame analyzed here (Figure 2B). The hypothesis that 12 days were insufficient for engrafted macrophages to acquire the full gene expression profile of resident cells was furthermore corroborated by the delayed onset of MHCII (encoded by H2-ab1) expression, one of the markers for intestinal macrophage maturation (Tamoutounour et al., 2012; Schridde et al., 2017) (Figure 2C). Engrafted macrophages were also characterized by lack of expression of Irf1 and the member of the TAM receptor kinase family Axl (Figure 2C). Genes, whose expression was low to absent in resident macrophages from both colon and ileum, but prominent in the engrafted cells (Figure 1D cluster VII), included the ones encoding ribosome-associated proteins and histones (Figure 2D). As previously shown (Varol et al., 2009), the reconstitution of emptied intestinal tissue with monocyte-derived cells involves clonal expansion, that is less likely to occur in physiological setting. In line with this notion, out of the 6383 genes significantly differing between engrafted and resident macrophages from either colon or ileum, 897 were annotated with a Gene Ontology (GO) term associated with ‘proliferation’ and ‘cell cycle’; 99 of these DEG were low to absent in resident macrophages from both tissues retrieved from non-engrafted Cx3cr1GFP/+ mice, such as Hmga1 (Figure 1D). We recently reported the requirement of IL10Ra on colonic macrophages for gut homeostasis. Mice lacking this cytokine receptor on intestinal macrophages develop severe gut inflammation in the colon, but not the ileum (Zigmond et al., 2014; Bernshtein et al., 2019), as do children that harbor an IL10RA deficiency (Glocker et al., 2009). Interestingly, Il10ra was not induced in engrafted colonic macrophages, even 12 days after tissue entry, while engrafted ileal macrophages displayed Il10ra transcripts as early as day 4 (Figure 2E). Expression of the cytokine IL10 itself was almost absent from monocytes and engrafted macrophages in both tissues, but significantly present in resident macrophages retrieved from non-engrafted Cx3cr1GFP/+ mice, to a larger extent in the colon than the ileum (Figure 2E). Of note, Il1b expression displayed a similar pattern in line with an earlier suggestion that IL1 might induce macrophage IL-10 expression (Foey et al., 1998). Likewise, genes induced following macrophage exposure to Il10, such as Ccr5 (Houle et al., 1999) and Socs3 (Cassatella et al., 1999) displayed similar expression patterns. This suggests that in our model the IL10/IL10R axis is inactive in newly differentiated macrophages and established only after further maturation in the tissue. Flow cytometric analysis of colon tissue of WT C57BL/6 mice identifies a small population of CD11b+ Ly6C+ MHCII- cells that likely represent recent monocyte immigrants into the tissue (Figure 2—figure supplement 1A). These rare cells probably entered the lamina propria to maintain the steady state macrophage pool of the intestine, before differentiating and have been referred to as P1 population of a ‘monocyte waterfall’ (Tamoutounour et al., 2012). Global RNAseq analysis of this population, alongside the Ly6C+ MHCII- macrophages revealed that, like the day four graft, these endogenous gut immigrants down-regulated monocyte markers and gained a gut macrophage signature, including expression of the Forkhead transcription factor (TF) FoxD2 and the nuclear receptor Nr3c2 (Figure 2—figure supplement 1B, C). Endogenous gut immigrants did not display a signature indicating proliferation, such as Rpl10 or Hmga1. However, like the grafted cells, these early colonic immigrants lacked signature genes associated with long-lived gut macrophages, including Timd4 and CD209f, as well as expression of Il10ra and Il10. To further corroborate our data, we used a distinct cell ablation model and performed adoptive monocyte transfers into DTx-treated [CX3CR1-DTR > WT] BM chimeras (Diehl et al., 2013; Aychek et al., 2015). The gene lists of upregulated and down regulated genes in macrophages retrieved from engrafted [CD11c-DTR > WT] and [CX3CR1-DTR > WT] chimeras showed with 71% and 59%, respectively, considerable overlap (Figure 2—figure supplement 2). While our adoptive transfer inherently aims at the reconstitution of ablated cells which differ in the two models, the observed coherence suggests robustness of the approach. Collectively, these data show that despite some differences, monocyte graft-derived cells recapitulate the ‘monocyte waterfall’ (Tamoutounour et al., 2012). We next focused on factors that might be involved in the generation of segment-specific macrophages, that is genes whose expression differed between colonic and ileal macrophages. 458 genes were up-regulated during monocyte differentiation in a segment-specific manner – 351 in colonic macrophages and 107 in ileal macrophages (Figure 3A). Monocyte differentiation into ileal macrophages was accompanied by induction of Gata and Hbox TF family members, such as Gata5 and Hbox3 (Figure 3A), as well as genes encoding the chemokine Ccl5 and the chemokine receptor CCR9. Monocytes that entered colon tissue preferentially up-regulated Foxd2, the nuclear receptor Nr3c2, and the dominant negative helix-loop-helix protein Id2 (Figure 3A). Of the genes, which were specifically down-regulated in only one intestinal segment, 78 genes followed this trend in colonic and 99 in ileal macrophages. The latter down-regulated genes included 7 TFs, such as Foxp1 and Trim16, while Arid5a and Elk3 were specifically down-regulated in the colon (Figure 3B). Many genes related to immune reaction and response to challenge displayed higher expression in ileal macrophages than their colonic counterparts. Examples are: Arid5a, whose gene product regulates IL6 (Masuda et al., 2016); Elk3, which encodes a member of the ETS TF family and was reported to modulate the phagocytosis of bacteria by macrophages (Tsoyi et al., 2015) and Ano6, that is down-regulated in colonic macrophages (Figure 3B), and reportedly supports microbiocidal activity of macrophages involving P2X7 receptor signaling (Ousingsawat et al., 2015). In contrast, the enzyme Sod1, which was reported to impair macrophage-related parasite killing in cutaneous Leishmaniasis (Khouri et al., 2009), showed lower expression in ileal macrophages (Figure 3B). Another interesting group of genes are those, which are expressed in monocytes, but further up-regulated in one tissue upon differentiation and down-regulated in the other. These genes might encode factors whose expression is incompatible with segment-specific macrophage fates. 54 such genes were expressed in colonic engrafted macrophages and silenced in their ileal counterparts; 15 genes followed an opposite trend (Figure 3C). Only one TF was found in both groups, KLF4 (Figure 3C). Aldh2 encoded by Aldh2, mostly known for its role in alcohol detoxification, was recently reported to play a role in repression of ATP6V0E2, which is critical for proper lysosomal function, autophagy, and degradation of oxidized LDL (Zhong et al., 2019) (Figure 3C). Hdac7 encoded by Hdac7 and up-regulated in ileal macrophages and down-regulated in colonic macrophages, was reported to interfere with the myeloid gene expression pattern and to inhibited macrophage-specific functions (Barneda-Zahonero et al., 2013) (Figure 3C). Finally, Fmnl3 participates in filopodia generation (Harris et al., 2010) (Figure 3C). Collectively, these data establish that monocytes establish gut segment specific gene expression patterns, likely under the influence of local cues. Transcriptomes of colonic and ileal macrophages 4 days after monocyte tissue entry were alike, with many genes sharing expression in both tissues when compared to their monocyte progenitors. Overall, by day 4, expression of 2007 genes was down-regulated more than 2-fold in the monocyte graft following differentiation into macrophages in both tissues (out of 12485 genes expressed). 2404 genes were induced in both the colon and ileum, arguably as part of a generic transcriptome signature of intestinal macrophages (Figure 4A). Notably, 919 of the genes induced during monocyte differentiation into generic intestinal macrophages displayed very low prior expression in monocytes – below 50 reads (Figure 4B). In contrast, expression of fewer transcripts (183) seemed to be actively silenced during the differentiation process, as seen in the violin blot in Figure 4B. This implies that monocytes actively acquire macrophage identities by de novo mRNA synthesis, while much of the monocytic gene expression is compatible with the differentiation process. The top 5 GO-terms associated with genes up-regulated in intestinal macrophages related to cell adhesion and migration processes (Figure 4C), including the chemokine Cxcl1, Ptprk which regulates cell contact and adhesion, and the integrin Itga6 (Figure 4D). The top 5 GO-terms associated with down-regulated genes in macrophages included cell cycle and division, as well as mRNA processing and DNA repair (Figure 4C, D), for example DNA polymerase beta (Polb), a cell cycle checkpoint regulator (Rad17) and an RNA helicase (Setx). Collectively, these data are in line with the transformation of circulating monocytes into non-migratory tissue resident cells and suggest a role for DNA damage-associated molecules during the differentiation process. Gene expression changes are driven by TFs. In the case of intestinal macrophages, three major TF families seem to participate in monocyte-macrophage differentiation: CCAAT-enhancer-binding proteins (C/EBPs), E2 transcription factors (E2F) and early growth response TFs (Egr). Four of the Cebp family members (Cebpa, b, d, g) and 5 E2F family members (E2f2,4, 6,7, 8) were significantly down-regulated upon monocyte differentiation into macrophages (Figure 5A). In contrast, Egr1,2 and 3 were up-regulated. Other down-regulated TFs included the regulators of immune response Bach1, Bcl6, Irf9, Nfatc3, Nfkb1 and Rela, as well as the master macrophage TF Spi1 (PU. 1) (Figure 5B). The list of induced genes was enriched with homeobox TFs such as Sox13, Pbx1, Foxa1 and others (Figure 5C). In addition, this group comprised TFs that had previously been reported to be critical for the development of other tissue macrophages, such as the nuclear receptor LXRα encoded by Nr1h3 for splenic macrophages and Gata6 for peritoneal macrophages (Varol et al., 2015; Rosas et al., 2014). Monocytes are generated in the BM to be subsequently disseminated throughout the body via blood vessels. Under inflammation, the cells are rapidly recruited to the site of injury. In absence of challenges, Ly6C+ monocytes can have distinct fates. A fraction of them gives rise to vasculature-resident Ly6C- ‘patrolling’ cells (Auffray et al., 2007). Other cells contribute to the homeostatic replenishment of selective tissue macrophage compartments (Figure 6A). To gauge the impact of the blood environment, as compared to a solid tissue such as the intestine, on the differentiation process, we next compared transcriptomes of Ly6C- blood cells and gut macrophages to their Ly6C+ monocyte precursors (Figure 6—figure supplement 1). A heat map of all 1303 genes, whose expression significantly differed between Ly6C+ monocytes compared to blood- and gut tissue-resident cells (day 4), revealed five clusters (Figure 6B). Monocyte progeny, whether in vasculature or tissue shared signatures, showed considerable similarities as reflected in the expression pattern of two thirds of the genes (66. 7%). Specifically, clusters I and II comprised 780 genes that were down-regulated upon Ly6C+ monocyte differentiation, including hallmark monocyte markers, such as Ly6c, Ccr2 and Mmp8 and Myd88 (Figure 6B, C). 160 genes were induced in both blood- and tissue resident monocyte-derived cells, albeit to different extend, including as Pparg, Ets2 and Tgfbr2 (Cluster III) (Figure 6B, D). Cluster IV and V spanned one third of the genes differentially expressed by Ly6C+ monocytes and their progenies, but distinct in tissue and vascular resident cells. Specifically, cluster IV comprised 253 genes up-regulated in blood-resident cells and down-regulated in gut macrophages. This included Csf2ra, Nfkb1, Il10ra and Spi1 (PU. 1). Cluster V comprised 110 genes induced in gut macrophages but not vasculature-resident cells, such as the mitochondrial master regulator Ppargc1b and the metalloprotease Adam19 (Figure 6B). Concerning TFs, Cebpb was induced in Ly6C- blood monocytes, as was previously reported (Mildner et al., 2017), while Cebpa and Cebpd were down-regulated in both blood- and gut-resident cells (Figure 6E). Irf family members 7,8 and 9 were down-regulated during Ly6C+ monocyte differentiation in blood and gut. Collectively, these data establish that vasculature-resident Ly6C- monocytes and gut macrophages that derive both from Ly6C+ monocytes display considerable overlap in transcriptomic signatures, but also display gene expression patterns that are likely associated with their specific environments. Adult tissue macrophages can derive from distinct origins (Ginhoux and Guilliams, 2016; Varol et al., 2015). Most tissue macrophages are currently believed to be generated in the embryo from EMP via a fetal liver monocyte intermediate and subsequently maintain themselves through self-renewal. Selected macrophages residing in barrier tissues, such as gut and skin, however rely on constant replenishment from blood monocytes. Here we report the study of this macrophage generation from monocyte precursors. Following tissue damage and infection, classical monocytes, defined as CD14+ cells in humans, and Ly6C+ cells in mice critically contribute to inflammatory reactions by promoting and resolving acute challenges (Ginhoux and Jung, 2014; Mildner et al., 2016). At the sites of injury, monocytes can give rise to cells with both macrophage and DC features. Monocyte differentiation during inflammation has been studied in various pathophysiological settings, including experimental auto-immune encephalitis (Yamasaki et al., 2014; Masuda et al., 2016), colitis (Rivollier et al., 2012; Zigmond et al., 2012) and others (Avraham-Davidi et al., 2013). To study less well understood physiological monocyte differentiation in absence of overt inflammation, we took advantage of an experimental system that allows synchronized reconstitution of macrophage compartments by monocyte engraftment (Varol et al., 2007; Varol et al., 2009). The majority of changes in gene expression, both in fold change and numbers, occurred in the transition from monocytes to either colonic or ileal macrophages at day four post transfer, that is immediately after extravasation. Engrafted colonic and ileal macrophages segregated in gene expression according to tissue. Tissue-specific macrophage imprinting occurs hence early during development, shortly after tissue infiltration. Interestingly, two samples of engrafted ileal macrophages clustered with the colonic samples rather than the ileal ones, suggesting that the colonic signature is default. Colonic macrophages at day 12 post engraftment were also more distinct from endogenous colonic macrophages, compared to their ileal counterparts, which implies that the mature colonic gene signature might take longer time to develop or could be more heterogeneous. Recent studies have noted heterogeneity within murine blood monocytes, in particular with respect to an intermediate between the Ly6C+ and Ly6- populations (Mildner et al., 2017). While we currently cannot formally rule out that, colonic and ileal macrophages could hence derive from distinct precursors, we consider this however unlikely. Mowat and colleagues have reported a transcriptome analysis of monocytes and colonic macrophages, including intermediates of the ‘waterfall’ (Schridde et al., 2017). The authors highlighted the critical role of TGFb in the differentiation process. While a comparison of these data to ours confirmed the late onset of genes that characterize long-lived gut macrophages, the use of the distinct platforms and distinct experimental set up precluded further direct alignment. Of note, monocyte-derived cells are in our system synchronized with respect to development and therefore allow additional temporal resolution, especially with respect to final population of the scheme (P4), which comprises in the cited study (Schridde et al., 2017) a heterogeneous conglomerate. With their extended half-life, Ly6C- monocytes that patrol the endothelium, have been proposed to represent vasculature-resident macrophages (Ginhoux and Jung, 2014). Indeed, these cells shared gene signatures with the gut resident macrophages, such as the reduction in IRF TFs following monocyte differentiation and induction of characteristic macrophage genes, such as PPARg and TGFbR. However, the comparison of these blood-resident cells to gut tissue-resident macrophages revealed also considerable differences likely associated with the residence in vasculature and the solid tissue, respectively. Though anatomically close, the small and large intestine represent very distinct tissues, including structural dissimilarities, such as the extended ileal villi and Peyer’s Patches, characteristic distinct abundance of immune cells, as well as different luminal microbiome content (Mowat and Agace, 2014). Highlighting these differences, ileum and colon display also unique susceptibility to perturbations, as for instance to a IL10R deficiency (Zigmond et al., 2014; Bernshtein et al., 2019). Our comparative analysis of colonic and ileal macrophages, including their generation from monocytes, might provide critical insights into the mechanism underlying segment-specific pathology resistance or - susceptibility in the gut. Together with earlier reports (Mildner et al., 2017; Schridde et al., 2017), our data sets can provide a starting point for hypothesis-driven experiments. To conclude, we characterized here monocyte-derived intestinal macrophages generated under conditions avoiding overt inflammation. We highlight specific genes and TFs which are regulated following monocyte differentiation to generic or segment-specific intestinal macrophages. By comparing transcriptomes of early intestinal macrophages and blood-resident Ly6C- cells, we show that the populations which share a common ancestor – the Ly6C+ blood monocytes – show considerable overlap in gene expression, while they also display adaptation to their specific environments. Our data provide a gateway and reference point to further studies on monocyte differentiation to macrophages. Mice were kept in a specific-pathogen-free (SPF), temperature-controlled (22 ± 1°C) facility on a reverse 12 hr light/dark cycle at the Weizmann Institute of Science. Food and water were given ad libitum. Mice were fed regular chow diet (Harlan Biotech Israel Ltd, Rehovot, Israel). The following mice strains all on C57BL6 background were used: Cx3cr1gfp/+ mice (Jung et al., 2000), CD11c-DTR transgenic mice (B6. FVB-Tg [Itgax-DTR/GFP] 57Lan/J) (Jung et al., 2002) and CX3CR1-DTR transgenic mice (Diehl et al., 2013). BM chimeras were generated by engraftment of 7–10 weeks old recipient mice that were irradiated the day before with a single dose of 950 cGy using a XRAD 320 machine (Precision X-Ray (PXI). Femurs and tibiae of donor mice were removed and BM was flushed with cold PBS. BM was washed with cold PBS twice and filtered by 100 μm filter. BM cells were suspended in PBS and 5 × 106 cells were injected IV into irradiated recipient. Mice were handled and experiments were performed under protocols approved by the Weizmann Institute Animal Care Committee (IACUC) in accordance with international guidelines. Femurs and tibias of donor mice were removed and BM was flushed with cold PBS. BM was washed with cold PBS twice and filtered by 100 μm filter. Cells were suspended with PBS and loaded on equal amount of Ficoll (GE healthcare). Tubes were centrifuged 920 g in room temperature for 20 min without breaks and Buffy coats were collected and washed with cold PBS. CD11c-DTR > wt]. Cells were stained and sorted according to the following markers: CD117- CD11b+ CD115+ Ly6C+ GFPint. BM chimeras were treated with 18 ng / gram bodyweight Diphtheria toxin (DTx) (Sigma-Aldrich, Cat # D0564) for two consecutive days before transfer. At the day of transfer mice were injected with 106 BM monocytes IV. At days 1,3, 5,7 and 9 after transfer mice were injected with 9 ng / gr bodyweight DTx. Intestines were removed and fecal content flushed out with PBS; tubes were opened longitudinally and cut into 0. 5 cm sections. Pieces were placed in 5ml/sample (up to 300gr of tissue) of Hanks' Balanced Salt Solution (HBSS) with 10% heat-inactivated FCS/FBS, 2. 5 mM EDTA and 1 mM DL-Dithiothreitol ( (DTT), Sigma-Aldrich Cat# D9779) and incubated on a 37°C shaker at 300 rpm for 30 min to remove mucus and epithelial cells. Following incubation, samples were vortexed for 10 s and filtered through a crude cell strainer. Pieces that did not pass the strainer were collected and transferred to 5 ml/sample of PBS +/+ with 5% heat-inactivated FCS/FBS, 1 mg/ml Collagenase VIII (Sigma-Aldrich Cat# C2139) and 0. 1 mg/ml DNase I (Roche Cat# 10104159001). Tissue was incubated in a 37°C shaker at 300 rpm for 40 min (colon) or 20 min (ileum) in the solution. After incubation, samples were vortexed for 30 s until tissue was dissolved, then filtered through a crude cell strainer. The strainer was washed with PBS - /- and centrifuged in 4ºC, 375G for 6 min. Cells were stained and subjected to FACS analysis or sorting. Blood was retrieved from the vena cava, immediately placed in 150 U/ml Heparin and loaded on Ficoll (GE healthcare). Tubes were centrifuged 920 g in room temperature for 20 min without breaks and Buffy coats were collected and washed with cold PBS. Cells were sorted according to the following parameters: CD45+ CD11b+ CD115+ Ly6C+/-. Samples were suspended and incubated in staining medium (PBS without calcium and magnesium with 2% heat-inactivated Fetal Calf/Bovine Serum (FCS/FBS) and 1 mM EDTA) containing fluorescent antibodies. Following incubation, cells were washed with staining buffer only or staining buffer with DAPI, centrifuged, filtered through 80 μm filter and read. For FACS analysis, LSRFortessa (BD Biosciences) was used. For cell sorting, FACSAria III or FACSAria Fusion (BD Biosciences) were used. Results were analyzed in FlowJo software (Tree Star). Staining antibodies (clones indicated within brackets): anti-CD45 (30-F11), CD11b (M1/70), CD115/CSF-1R (AF598), Ly-6C (HK1. 4), CD64/FcγRI (X54-5/7. 1), CD11c (N418), anti-I-Ab (MHCII) (AF6-120. 1), DAPI. RNA-seq of populations was performed as described previously (Diehl et al., 2013; Jaitin et al., 2014). Cells were sorted into 100 µl of lysis/binding buffer (Life Technologies) and stored at 80°C. mRNA was captured using Dynabeads oligo (dT) (Life Technologies) according to manufacturer’s guidelines. A derivation of MARS-seq (Jaitin et al., 2014) was used to prepare libraries for RNA-seq, as detailed in Shemer et al. (2018). RNA-seq libraries were sequenced using the Illumina NextSeq 500. Raw reads were mapped to the genome (NCBI37/mm9) using hisat (version 0. 1. 6). Only reads with unique mapping were considered for further analysis. Gene expression levels were calculated and normalized using the HOMER software package (analyzeRepeats. pl rna mm9 -d < tagDir > count exons -condenseGenes -strand + -raw). Gene expression matrix was clustered using k-means algorithm (MATLAB function kmeans) with correlation as the distance metric. PCA was performed by MATLAB function pca. Gene ontology was performed by DAVID (https: //david. ncifcrf. gov). Data on molecules and pathways was partly obtained by Ingenuity Pathway Analysis (IPA), Ingenuity Target Explorer, Qiagen and Metascape Pathway analysis (Zhou et al., 2019). Results are presented as mean ± SEM. Statistical analysis was performed using Student’s t test. * p-value<0. 05 ** p-value<0. 01 *** p-value<0. 001.

Macrophage cells play a crucial role in keeping the body free of disease-causing microbes and debris. They surveille the tissues, detect and clear infections, and tidy up dead cells. Most internal organs contain a population of macrophages that move into the organ during development and then persist throughout an organism's life. However, tissues in contact with the outside world, such as the gut, need a constant supply of fresh macrophages. This supply depends on immune cells called monocytes moving into these tissues from the blood and maturing into macrophages when they arrive. The macrophages in the gut have a challenging job to do. They need to be able to detect infections amongst healthy gut bacteria and foreign food particles. Macrophages from other tissues would overreact if they encountered this complicated environment, but gut macrophages learn to tolerate their surroundings by switching genes on and off as they mature. The exact combination of genes macrophages in the gut use depends on whether they are in the small or large intestine, which have different anatomies and resident microbes. To understand how monocytes mature into macrophages in the gut, previous studies have focused on what happens during an infection. However, it remains unclear how monocytes develop into mature gut macrophages in the healthy gut. To address this question, Gross-Vered et al. have looked at mice in which gut macrophages can be killed when a drug is applied. This made it possible to replace the mice's own macrophages with fluorescently labelled cells derived from monocytes. Fluorescent monocytes were introduced into the bloodstream and arrived in the small and large intestine after the drug had been administered. Gross-Vered et al. then collected cells derived from these labelled monocytes and examined the genes that they were using. This revealed that once the monocytes entered the gut they began sensing their new environment and switching thousands of genes on and off. These changes happened rapidly at first and continued more gradually as the macrophages matured. Comparing the fluorescent macrophages from the small and large intestines revealed many similarities, but there were also hundreds of genes that differed. In the small intestine, macrophages switched on genes involved in catching and consuming bacteria, whereas macrophages in the large intestine, which has more resident healthy bacteria, turned on fewer of these bacteria-eating genes. Inflammatory bowel disorders like ulcerative colitis and Crohn' s disease both involve gut macrophages. Comparing the genes that macrophages use in the healthy and diseased gut may reveal information about these disorders. For example, ulcerative colitis only affects the large intestine, so understanding how and why the monocytes mature differently there could shed light on new ways to treat the disease.

lay_elife

FIELD OF THE INVENTION [0001] The invention relates to a device for irradiating absorbing liquids, for example wastewater that is to be disinfected, with UV radiation. PRIOR ART [0002] Devices for treating sewage treatment plant effluent for the purpose of reducing the germ count have long been known. The underlying research was published in 1984 by Scheible et al. in the publications of the Environmental Protection Agency with the title “Ultraviolet Disinfection of Waste Waters from Secondary Effluent and Combined Sewer Overflows”. Various concepts of disinfection of sewage treatment plant effluent using UV radiation were investigated in detail both theoretically and practically in the course of this research, and conclusions were drawn from it. Furthermore, requirements were formulated for the design and operation of a UV installation for wastewater disinfection. The following special characteristics of the medium (wastewater) were taken into account: high microbiological loading of the medium poor UV transmittance of the medium tendency of the medium to form sediments high content of suspended matter in the medium high throughputs of up to several 1000 m 3 per hour. [0008] A UV irradiation installation for wastewater disinfection is in principle an irradiation zone installed in the discharge point of the sewage treatment plant, through which the water flows freely or is forced to flow continuously, and the water flowing through is irradiated with UV radiation of a particular wavelength by means of suitable electrically operated sources of UV radiation. Therefore it is necessary that each volume element of the water receives a sufficient radiation dose. The radiation dose is defined as the radiation energy with microbiocidal action that is applied per unit area. The characteristics of the wastewater stated above can be converted to the following requirements for the design of UV irradiation installations: uniform illumination of the irradiation zone avoidance of large layer thicknesses of the irradiated water uniform flow through the irradiation zone as few hydraulic dead zones as possible in the irradiation zone adjustment of the height of the water level with fluctuating throughputs as few internals as possible in the water, which favour deposits of suspended matter easy access for cleaning and lamp changing. [0016] Since the throughputs are as a rule high, irradiation units in the free surface flow, i.e. in the open channel, are considered almost exclusively for the UV irradiation of biologically purified wastewater. An open discharge channel, as a rule provided by the builder, is equipped with an irradiation installation that forms an arrangement of as a rule cylindrically shaped UV light sources uniformly covering the flow cross-section of this discharge channel. Depending on the arrangement of the cylindrical UV radiation sources, a distinction is made between: installations with a vertical arrangement of UV radiation sources. Here the cylindrical UV sources stand in quartz tubes closed on one side, in the manner of a bar screen with vertical teeth in the channel. This design has the advantage that lamp changing is possible from above, i.e. without undoing water-tight seals. A disadvantage is the unavoidable formation of rings and deposits in the zone where the water level fluctuates, which are difficult to remove. installations with UV radiation sources arranged horizontally in the direction of flow. Here the quartz tubes with the UV radiation sources inside are completely immersed and water flows round them in parallel. This thereby avoids the disadvantages of the lateral arrangement, however the water-tight seals have to be undone for lamp changing. [0019] Installations with UV radiation sources arranged horizontally in the direction of flow are largely employed in current practice. [0020] Following the aforementioned study by the Environmental Protection Agency, this technology was first launched on the market in the USA and Canada. Numerous such installations have since been installed throughout the world. [0021] In installations according to the prior art, an open rectangular discharge channel at the discharge point of the sewage treatment plant, provided by the builder, is equipped with a set of low-pressure mercury radiators (LP-Hg radiators). Therefore each radiator is provided with a cylindrical quartz jacket tube, around which the water to be treated flows. In each case a row of jacket tubes with LP-Hg radiators arranged above one another are assembled to form a module, comprising a stainless steel frame for holding the jacket tubes arranged above one another, a top cover to prevent emission of harmful UV radiation, a plug-and-socket connector for power supply and handles for removal in the vertical direction for maintenance purposes. [0022] For complete illumination of the channel cross-section, several such modules are secured parallel to one another in a frame that is mounted rigidly on the channel, and functions as support, fixing device and cable conduit. [0023] LP-Hg radiators are mercury gas discharge lamps with UV-transparent lamp tubes, which emit the greater part of their radiation in the microbiocidal triplet resonance of mercury at 253.7 nm. This type of lamp typically has an electric power of less than 100 W, even in special designs (amalgam lamp). For larger installations with a throughput of 10,000 m 3 per hour or more, an electrical energy consumption of 20 to 30 Wh per cubic metre of water can be taken as a guide value, depending on the properties of the water. At 100 W power of an LP-Hg radiator, such an installation requires 2000 to 3000 radiators. This is already possible according to the prior art by parallel or series connection of radiator assemblies, but there are the following disadvantages: no economies of scale for plant costs large space required high cost of operation and maintenance high lamp costs. [0028] For these reasons, UV treatment of larger effluent streams is very expensive according to the prior art. The logical step for lowering the cost is to increase the power per radiator, which immediately leads to reduction of the number of radiators and therefore the capital costs. [0029] With LP-Hg radiators with amalgam, power ratings up to a maximum of about 300 W can be achieved. For a further increase in power, medium-pressure mercury radiators (MP-Hg radiators) are known, and depending on their design they can have an electric power rating of 10 kW or more per radiator. They emit a quasi-continuum of UV radiation at high power density, and with reference to microbiocidal UV radiation, the generating efficiency is from approx. 12% to 15%. Previous attempts to use these high-power MP-Hg radiators for wastewater disinfection were unsuccessful, for the following reasons: non-uniform distribution of irradiation intensity owing to the high radiation power of the individual radiators increased deposits from the wastewater and burning of these deposits onto the jacket tubes of the radiators. The reason for this is the far higher wastewater-jacket tube temperature gradient for conducting away the higher thermal power of the MP-Hg radiator. risks of accidents and short-circuits through manipulation of the high-voltage connectors of the MP-Hg radiators in the water-soaked zone. [0033] There is therefore a need for cost-effective installations for UV treatment of larger wastewater streams. SUMMARY OF THE INVENTION [0034] The invention is based on the problem of proposing an installation for flow-through UV irradiation of absorbing liquids, which permits uniform irradiation of the liquid even at high throughputs. [0035] Another problem of the invention is to propose such a device that can be manufactured cost-effectively. [0036] Yet another problem of the invention is to propose a device for flow-through UV irradiation of absorbing liquids, for which the maintenance costs are reduced. [0037] The problem is solved with a device for flow-through UV irradiation of absorbing liquids, for example wastewater that is to be disinfected, by at least two assemblies of UV radiators, characterized in that one radiator assembly has a cylindrical UV light source and three concentric, cylindrical jacket tubes, with a coolant for carrying away heat from the UV radiation source flowing in a hollow space between an inner and a central jacket tube. [0038] The coolant is able to lower the temperature on the outer jacket tube, so that burning of deposits onto the jacket tube can be prevented. It is possible to increase the diameter of the outermost jacket tube, so that it becomes possible to achieve a more uniform distribution of the irradiation intensity. The diameter of the outer jacket tube is then preferably between 80 and 250 mm, and especially between 120 and 200 mm. [0039] The hollow space between the outer and the central jacket tube can be filled with UV-transparent gas. [0040] The power density of the UV radiation sources, for example MP-Hg radiators, is preferably at least 10 W UV-C radiation per cm in the axial direction. [0041] The UV radiation sources can therefore be arranged horizontally, transversely to the direction of flow, for example on a U-shaped steel shrouding, with the ends of the jacket tubes preferably sealed and mounted and provided with connections for power and coolant. To simplify maintenance, lateral service pits can be provided, giving access to the UV radiation sources and allowing them to be replaced. [0042] To prevent emission of harmful UV radiation upwards, covers are preferably provided above the radiator assemblies. [0043] The radiator assemblies can be arranged in longitudinal section in the pattern of two-dimensional close packing of spheres, in order to ensure the most uniform possible distribution of irradiation intensity. [0044] For the coolant, a coolant circulating pump and a heat exchanger for releasing heat to the absorbing liquid can be provided. [0045] Distilled water, which has very low UV absorption, can preferably be used as coolant. It is possible to add alcohol, such as ethanol or methanol, in order to prevent biological contamination of the coolant. [0046] The jacket tubes preferably comprise UV-permeable material, for example quartz glass. [0047] It is possible to provide throughput-dependent power control of the sources of UV radiation, so that despite variable throughput, the radiation intensity is always correct. BRIEF DESCRIPTION OF THE DRAWINGS [0048] Further features and advantages of the invention will become clear from the following description of a preferred example of application, referring to the appended drawings. [0049] FIG. 1 shows a top view of an example of application of a device according to the invention for the flow-through UV radiation of absorbing liquids. [0050] FIG. 2 shows a view in longitudinal section of the example of application in FIG. 1. [0051] FIG. 3 shows a cross-sectional view along line A-A in FIG. 2. [0052] FIG. 4 shows an example of application of a radiator assembly according to the invention. [0053] FIG. 5 shows a detail view of the radiator assembly in FIG. 4. [0054] FIG. 6 shows an example of the arrangement of several radiator assemblies for achieving uniform irradiation intensity. DETAILED DESCRIPTION OF THE INVENTION [0055] The invention will be described in detail in the following, on the basis of a preferred embodiment. [0056] The device according to the invention is shown in top view in FIG. 1, in longitudinal section view in FIG. 2 and in cross-sectional view against the direction of flow in FIG. 3. The direction of flow of the absorbing liquid is indicated with an arrow, but is of no importance for the functionality of the device according to the invention. A U-shaped stainless steel wall 12, which is kept dimensionally stable by external stiffeners 13 and diagonal members 14 arranged above the surface of the liquid, is installed in channel 15. On each side there is a covered pit 16, 17, through which all electrical connections, control lines and pipes or hoses of the coolant circuit are led, and are accessible for maintenance purposes. In the two lateral vertical zones of the stainless steel wall 12 there are circular openings 18 in pairs opposite one another, into which the radiator assemblies, namely outer jacket tube 1, holders and covers 8, 9 and 10 are inserted and made water-tight. The electrical connecting cables and coolant hoses are not shown. Most preferably, they are led through a cable conduit, also not shown, on the vertical stiffener 13. The irradiation zone is covered at the top via one or more stainless steel caissons 19, 20, which ensure that even at high water throughput above the uppermost radiator assembly the maximum distance from the radiator assembly predetermined by the irradiation geometry (i.e. the clearance between caisson and upper radiator assembly) is not exceeded. Conversely, a weir in the flow direction after the irradiation zone ensures instead that the bottom edge of the caisson always remains wetted and in particular the uppermost radiator assembly does not in any circumstances dry out completely or partially. The caisson can either be welded into the stainless steel wall solidly as a stabilizing cover, or it can be of detachable design to allow better accessibility to the irradiation zone for cleaning purposes. Above it, there is a walk-on cover 21, which can also serve as base plate for a switch cabinet with the power supply and the necessary control and monitoring equipment, if this is to be positioned directly on the installation. The solidly welded-in caisson 19 serves for mounting a coolant circulating pump 22 and as cable shaft, if the switch cabinet is mounted on the cover. [0057] In the flow direction, before the irradiation zone there is a heat exchanger 23, via which the heat energy of the MP-Hg radiators absorbed by the coolant is released to the liquid stream. It consists of a distribution chamber with connector for the feed line 24, plenum chamber with connector for the outlet line 25 and, between them, U-shaped copper tubes 26, which dip into the water from above and transfer the waste heat to the wastewater stream that is to be treated. [0058] The heat exchanger serves at the same time as a screen against UV radiation emitted forwards from the irradiation zone and as an obstacle to flow for evening out the flow through the irradiation zone. [0059] In the direction of flow, after the irradiation zone there is finally a baffle plate 26 pointing obliquely downwards, which on the one hand optimizes flow guidance at the top edge of the irradiation zone and on the other hand serves as a screen against UV radiation emitted backwards from the radiation zone. [0060] The radiator assembly will be explained in detail below, referring to FIGS. 4 and 5. It consists of a centrally arranged, cylindrical UV radiator, for example an MP-Hg radiator with a power rating of at least 10 W UV-C radiation per centimetre in the axial direction, which radiation source 4 is provided with a holder 5 and electrical connectors 6. An outer jacket tube 1, middle jacket tube 2 and inner jacket tube 3, preferably made of UV-permeable quartz glass, are arranged concentrically to the UV radiation source 4 and are sealed with lateral holders 8, 9 having O-ring seals 7, plus an end cover 10. In addition, connectors 11 are provided for circulation of the coolant in the hollow space between the inner jacket tube 3 and the middle jacket tube 2. [0061] The more uniform distribution of the irradiation intensity on the absorbing liquid that is possible according to the invention will be illustrated in the following as an example with a water throughput of 10,000 m 3 /h, equivalent to 2777 l/s. [0062] Let us assume the use of 50 MP-Hg radiators per 10 kW of electric power, a generating efficiency of the UV radiation of 15%, and length of the light source of 100 cm, an outside diameter of the (outer) jacket tube of 4 cm according to the prior art and a UV permeability of the wastewater of 80%/cm. [0063] Then an irradiation intensity of approx. 1.2 W/cm 2 is calculated for the outside surface of the jacket tube, an irradiation intensity of approx. 0.65 W/cm 2 at a distance of 1 cm and an irradiation intensity of approx. 0.38 W/cm 2, corresponding to less than a third, at a distance of 2 cm. [0064] If we consider the space in which the irradiation intensity drops from 100% to approx. 33%, this is approx. 3.8 l per jacket tube. With 50 radiators we get a total space of 190 l. The 2777 l/s water throughput according to this example cannot be led through such a small irradiation zone. The very high surface load with thermal energy that is to be led away leads moreover to strengthened formation and burning-on of deposits on the outer jacket tube, which in its turn leads to increasing absorption of radiation in the layer of contaminants. [0065] With an outside diameter of the outer jacket tube 1 of 10 cm (20 cm) the irradiation density in the above example on the jacket tube is 0.477 (0.238) W/cm 2 and at a radial distance of 3 cm (3.5 cm) it drops back to about a third, namely 0.153 W/cm 2 (0.08 W/cm 2 ). The associated volume per UV source is approx. 12.2 l (26 l), corresponding to 1300 l total volume for 50 radiators. Enlargement of the outer jacket tube thus permits far higher throughputs of the liquid that is to be irradiated. However, this requires effective removal, by the coolant, of the heat produced by the high-power UV radiation source. Conversely, the coolant must absorb as little UV radiation as possible itself, so that the coolant flows through a relatively “thin” hollow space between the inner jacket tube 3 and the middle jacket tube 2, preferably with distilled water as coolant on account of the low UV absorption, if necessary with addition of ethanol to prevent the development of bacteria and algae. Owing to the low UV absorption, the jacket tubes 1, 2, 3 are preferably made of quartz glass. [0066] Through the interaction of several radiator assemblies, the irradiation intensity can be evened out considerably, as shown schematically in FIG. 6. This shows a number of MP-Hg radiators with a diameter of the outer jacket tube 1 of 20 cm, which have a radiation power of 15 W/cm and a minimum distance of 7 cm between two adjacent radiators following the pattern of two-dimensional close packing of spheres. Based on an irradiation intensity of 0.238 W/cm 2 (see above) at the surface of the outer jacket tube 1, with a UV-permeability of the water of 80% per centimetre, calculation gives an irradiation intensity of max. 0.16 W/cm 2 by superimposing two radiators at point 1 and min. 0.117 W/cm 2 by superimposing three radiators at point 2. [0067] The desirable enlargement of the outer jacket tube 1 requires additional measures for removing the heat energy associated with operation of the high-power UV source. As the removal of heat by convection with downstream transfer of heat to the medium that is to be irradiated is no longer adequate at the increased distances of several centimetres with a larger jacket tube, according to the invention a total of three quartz tubes 1, 2, 3 are provided for each radiator assembly, namely the inner jacket tube 3, the middle jacket tube 2, and the outer jacket tube 1 round which the liquid that is to be irradiated flows. This offers the possibility of circulating fully desalinated water or some other sufficiently UV-permeable liquid or gaseous coolant through the hollow space between inner jacket tube 3 and middle jacket tube 2. Owing to the absorption and the high heat capacity of the coolant, the energy of the thermal radiation will largely be carried away, in contrast to cooling by a blower, and on the whole a very uniform temperature of UV source 4 will be achieved. As a result, warming of the liquid-wetted outer jacket tube 1, which promotes deposit formation, can be avoided almost completely. [0068] An important advantage of the device according to the invention is effective avoidance of deposits on the outer jacket tube round which the liquid that is to be irradiated flows. In conventional systems, all the heat energy produced in the UV source is transferred via the jacket tube to the liquid that is to be irradiated. [0069] This necessarily leads to a jacket tube-water temperature gradient. This can lead to deposition of particles on the outside surface of the jacket tube, especially at low flow velocities, so that absorption of the UV, VIS and IR radiation further increases this temperature gradient. The result is that formation of deposits is further accelerated. Finally, really dark, solid crusts form, which are difficult to remove. [0070] The heat transfer at a solid-fluid phase boundary is given by: [0000] Q=α·A ( − T ), where [0000] Q is the heat flux in watts α is the heat transfer coefficient in watts per m 2 and ° K., A is the area of heat transmission in m 2, is the temperature in the fluid in ° K., and T is the wall temperature in ° K. [0071] For a tube around which water flows at a velocity v (in m/s), the following approximation for α is given in the literature: [0000] α=(500 to 1800)×√ν, depending on surface finish. [0072] The problem of effectively keeping the outer jacket tube clean can be formulated as follows: for a given power rating, the temperature difference ( −T) given in the above equation must be kept small enough, under all operating conditions, so that the self-intensifying fouling process described above can be avoided. Investigations have shown that with the arrangement according to the invention, avoidance of fouling can largely be achieved if the thermal power that is to be carried away at an average flow velocity of 2 m/s can be kept below approx. 2000 W/m 2, corresponding to a temperature difference ( −T) of 1 to 3 kelvin for the range of α given above. [0073] Factors contributing to keeping the surface largely clear of deposits are, on the one hand, the restriction of the temperature of the outer jacket tube resulting from the interaction of the reduced thermal power per unit area and heat abstraction through the increased tangential velocity, and on the other hand the fact that the surface is kept clean by the abrasive forces of the water flowing past. [0074] At low flow velocities, if the lamp power is also reduced, the deposits only form a thin film on the surface of the jacket tube, and this is washed away again at full flow. [0075] The requirements on thermal load per unit area and the tangential velocity can be translated into concrete geometric requirements for an example of MP-Hg radiators for wastewater disinfection on the basis of the following typical data: [0000] Discharging distance: 100 cm UV-C radiation power: &gt;1200 W Depth of penetration in the water: 3 cm (return to 1/e) Required average dose: 40 mJ/cm 2 according to formula (1) given below Indication of rows of lamps one after another: at least 2 for adequate mixing. [0076] Then, from the restriction of the power per unit area to less than 2000 W/m 2 taking into account heat abstraction via the coolant of approx. 50% of the total power, we find a minimum diameter of the outer jacket tube 1 of 8 cm. Limitation of the clearance between adjacent quartz tubes can be deduced from the required tangential velocity. The following approximate formula is used for the UV dose: [0000] Dose   ( mJ  /  cm 2 ) = UV - power   ( W ) · penetration   depth   ( cm ) flow   ( 1  /  s ) ( 1 ) [0077] On solving with respect to the flow, using a minimum velocity of the water of 2 m/s we get a maximum free cross-section of 900 cm 2 per radiator, corresponding to a maximum clearance between two adjacent jacket tubes of 9 cm.

The invention relates to a device for irradiating an absorbing liquid, for example waste water to be disinfected, in a throughflow. Said device comprises at least two radiator units having one cylindrical UV radiation source and three concentric sheaths. A cooling medium for carrying off heat from the UV radiation source flows in a hollow space between the inner and the center sheath. The inventive device allows to use high-performance UV radiation sources and to evenly irradiate the zones of irradiation and prevents floating particles from settling down on the radiator units.

big_patent

Nucleocytoplasmic trafficking is emerging as an important aspect of plant immunity. The three related pathways affecting plant immunity include Nuclear Localization Signal (NLS) –mediated nuclear protein import, Nuclear Export Signal (NES) –dependent nuclear protein export, and mRNA export relying on MOS3, a nucleoporin belonging to the Nup107–160 complex. Here we report the characterization, identification, and detailed analysis of Arabidopsis modifier of snc1,11 (mos11). Mutations in MOS11 can partially suppress the dwarfism and enhanced disease resistance phenotypes of snc1, which carries a gain-of-function mutation in a TIR-NB-LRR type Resistance gene. MOS11 encodes a conserved eukaryotic protein with homology to the human RNA binding protein CIP29. Further functional analysis shows that MOS11 localizes to the nucleus and that the mos11 mutants accumulate more poly (A) mRNAs in the nucleus, likely resulting from reduced mRNA export activity. Epistasis analysis between mos3-1 and mos11-1 revealed that MOS11 probably functions in the same mRNA export pathway as MOS3, in a partially overlapping fashion, before the mRNA molecules pass through the nuclear pores. Taken together, MOS11 is identified as a new protein contributing to the transfer of mature mRNA from the nucleus to the cytosol. Plants utilize a two-layered immune system to recognize and combat pathogens. Conserved pathogen-associated molecular patterns (PAMPs), which are detected by plasma membrane localized plant pattern recognition receptors (PRRs), trigger a low amplitude defense response termed PAMP-triggered immunity (PTI) [1]. To suppress PTI, pathogens have evolved a suite of effectors (also called Avirulence or Avr proteins) [2]–[5]. The second layer of the plant immune response is initiated by the recognition of effectors or their effects, by cognate Resistance (R) proteins, leading to a strong defense response termed effector-triggered immunity (ETI) that culminates with hypersensitive response (HR) [1]: a programmed cell death event believed to restrict pathogen growth. Most cloned R genes encode Nucleotide Binding Leucine Rich-Repeat (NB-LRR) proteins with either a Toll/Interleukin1 receptor (TIR) or a Coiled-Coil (CC) -domain at their N terminus [6]. Mutant snc1 (suppressor of npr1-1, constitutive 1) plants carry a gain-of-function mutation in a TIR-NB-LRR R gene. This mutation renders the snc1 protein auto-active without the presence of pathogens. As a consequence, snc1 plants constitutively express Pathogenesis Related (PR) defense marker genes, accumulate high levels of defense phytohormone salicylic acid (SA), and are more resistant to the virulent bacterial pathogen Pseudomonas syringae maculicola (P. s. m.) ES4326 and the oomycete pathogen Hyaloperonospora arabidopsidis (H. a.) Noco2. Like most TIR-type R proteins, snc1 is fully dependent on EDS1 (Enhanced Disease Susceptibility 1) and PAD4 (PhytoAlexin Deficient 4) for its function [7], [8]. The snc1 mutant results from a point mutation in the linker region between the NB and LRR domains. This gain-of-function mutation causes activation of the ETI pathways mediated by TIR-NB-LRR R proteins. The extreme dwarf morphology of snc1 also makes it an ideal candidate to carry out genetic screens to investigate ETI components downstream of SNC1. Utilizing the unique autoimmune phenotypes of snc1, we conducted genetic screens to search for components contributing to R protein mediated immune responses. From the MOS (modifier of snc1) genetic screens, three nucleocytoplasmic pathways have been shown to affect plant immunity: Nuclear Localization Signal (NLS) -mediated nuclear protein import, Nuclear Export Signal (NES) -dependent nuclear protein export and mRNA export. MOS6, an importin α homolog involved in NLS-dependent protein import was shown to contribute to plant immunity [9]. More recently we identified MOS7, an integral nuclear pore component homologous to Nucleoporin88 (Nup88). Partial loss-of-function mos7-1 mutant plants showed increased NES-dependent protein export and exhibited reduced nuclear accumulation of the important defense regulators EDS1, NPR1 (Non-expressor of Pathogenesis Related 1) and snc1 [10]. The contribution of mRNA export to plant immunity was demonstrated in the mos3 mutant which exhibits defects in basal and R protein mediated immunity [10], [11]. MOS3/SAR3/AtNup96 is an integral nuclear pore component in the conserved Nup107–160 complex and was shown to be required for mRNA export [12]. Here we report the identification of modifier of snc1,11 (mos11), a T-DNA insertional mutant. Mutation in MOS11 partially suppresses all the autoimmune phenotypes of snc1. MOS11 encodes a protein with unknown function. However, it is evolutionarily conserved and shares homology with a human protein CIP29. Our data suggest that MOS11 localizes to the nucleus and functions in the same mRNA export pathway as MOS3. The mos11-1 mutant was identified by T-DNA tagging in the snc1 background [13]. Normally snc1 plants are dwarf, have twisted dark green leaves, accumulate high levels of the defense phytohormone SA and are resistant to the virulent oomycete H. a. Noco2 and P. s. m. ES4326 bacteria. mos11-1 partially suppressed the snc1 morphology (Figure 1A). The mos11-1 snc1 plants never grew to wild type size and their leaves remained curly (Figure 1A). In addition, semi-quantitative RT-PCR confirmed that endogenous PR1 and PR2, defense markers acting downstream of R gene activation that are constitutively expressed in snc1, are greatly reduced in mos11-1 snc1 (Figure 1B). In order to investigate how the mos11 mutation suppresses snc1 morphology and PR gene expression, we assessed whether the snc1 protein levels were affected in mos11-1 snc1 plants. We observed a slight but consistent reduction in snc1 levels in mos11-1 snc1 compared with snc1 plants (Figure 1C). Total SA levels in mos11-1 snc1 were drastically reduced compared with snc1 plants, but were still significantly higher than in wild type plants (Figure 1D). To assess whether the mos11-1 mutation affects snc1-mediated resistance to virulent pathogens, we performed infection assays with the obligate biotrophic oomycete H. a. Noco2 and P. s. m. ES4326 bacteria. While snc1 plants were resistant to H. a. Noco2 and showed very low sporulation, mos11-1 snc1 displayed intermediate susceptibility to H. a. Noco2 (Figure 1E). mos11-1 snc1 supported 13-fold more sporulation than snc1, however it was still significantly more resistant than the wild type (Figure 1E). This trend was also observed when we investigated susceptibility to P. s. m. ES4326 (Figure 1F). Taken together, these results show that mos11-1 partially suppresses all the phenotypes of snc1. The T-DNA insertion causing the mos11-1 snc1 phenotype was identified in the 5′ UTR of At5g02770 using inverse PCR (Figure 2A). To confirm that the mutant phenotypes in mos11-1 snc1 were caused by the T-DNA insertion, we cloned the wild type DNA of At5g02770 with 1992 base pairs (bp) upstream of the start codon and transformed it into mos11-1 snc1 plants. At5g02770 fully complemented the mos11-1 snc1 morphology (Figure 2B). When the T2 transgenic plants were challenged with H. a. Noco2, they were as resistant as snc1 plants (Figure 2C). These data confirmed that MOS11 is At5g02770. Another T-DNA insertion allele (SAIL_266_E03) in the first exon of At5g02770 was available from the Arabidopsis Stock Center (ABRC), which we named mos11-2 (Figure 2A). The mos11-1 single mutant was obtained from a backcross between mos11-1 snc1 and the wild type Columbia ecotype (Col-0). The mos11-1 and mos11-2 single mutants had identical morphology but were slightly different than Col-0. When mos11-1 was crossed with mos11-2, the two alleles failed to complement each other in all 29 F1 plants (data not shown), confirming that mos11-2 and mos11-1 carry lesions in the same gene. When mos11-2 was crossed with snc1, the mos11-2 snc1 double mutant exhibited a similar level of snc1 suppression as mos11-1 (data not shown), further supporting that At5g02770 is MOS11. MOS11 is a single copy gene that encodes a 214 amino-acid protein with unknown function. Using BlastP, the human protein CIP29 (cytokine induced protein 29 kDa) was found to share 38% identity and 50% similarity over the entire length of the MOS11 protein. It is striking that islands of positively charged residues, arginine and lysine, are highly conserved (see alignment in Figure S1). Although MOS11 does not have predicted NLS (Nuclear Localization Signal) motifs, it is predicted by WoLF PSORT (http: //wolfpsort. org/) to be a nuclear protein. To determine the biochemical function of MOS11, we first investigated its subcellular localization. When MOS11-GFP with its native promoter was transformed into mos11-1 snc1, among the 9 T1 progeny, all showed snc1-like morphology (Figure 3A). The T2 transgenic plants also displayed snc1-like enhanced resistance to H. a. Noco2 (Figure 3B). These data indicate that MOS11-GFP largely reflects endogenous MOS11 in the transgenic line, and therefore, the subcellular localization of MOS11-GFP should be identical to MOS11. As shown in Figure 3C, in contrast to the MOS3-GFP control, which localizes to the nuclear envelope, strong MOS11-GFP signal was observed in the nuclei of leaf epidermal and root cells, however it appears to be excluded from the nucleolus. Nuclear localization of the MOS11-GFP signal was observed in guard cells as well. Thus we conclude that MOS11 is a nuclear protein. In humans, CIP29 was shown to bind RNA (23). The two known interacting protein partners of CIP29 are the RNA helicase DDX39 and FUS/TLS. DDX39 shows 91% identity with UAP56, a Drosophila RNA helicase involved in mRNA export [14]. DDX39 also interacts with ALY and FUS/TLS, which were both shown to contribute to mRNA export [15]–[17]. These associations prompted us to investigate whether MOS11 also contributes to mRNA export. We first used dot blot hybridization to check whether the total mRNA level was affected in mos11-1 and mos11-2 plants. mos11-1 and mos11-2 are morphologically indistinguishable (Figure 4A). Decreasing RNA concentrations were used to avoid saturation. No visible differences in total RNA levels were observed (Figure 4B). To assess whether mRNA export from the nucleus to the cytoplasm was affected in mos11-1 and mos11-2 plants, we carried out poly (A) in situ hybridization with a 48-mer oligo d (T) 5′-end labeled with Alexa 488. Poly (A) signals were observed in the nucleus of Col-0 and the cytosol of all labeled cells (Figure 4C). When mos11-1 and mos11-2 were analyzed, most poly (A) signal was found in the nucleus, indicating that mRNA export is diminished in mos11 plants, resulting in mRNA accumulation in the nucleus (Figure 4C and Figure S2). Thus MOS11 is likely a contributing factor in the mRNA export pathway. Note that nucleolar oligo d (T) signal is weak in all in situ experiments (Figure 4C and Figure 5E), a region where MOS11-GFP was also absent (Figure 3C). Since mos3 was previously shown to affect mRNA export [12], we investigated the relationship between mos3-1 and mos11-1 using epistasis analysis. The morphological phenotypes associated with both single mutants are quite different; mos3-1 shows long and narrow leaves and flowers early, whereas mos11-1 has mostly wild type-like morphology. The mos3-1 mos11-1 double mutant displayed a mos3-like morphology with leaves that are slightly narrower and longer than those of the mos3 single mutant, but flowered slightly earlier than mos3-1 or mos11-1 (Figure 5A and 5B). These data suggest that in regard to flowering time regulation, MOS3 and MOS11 seem to play partially overlapping roles. In order to assess the relationship between mos3 and mos11 in immunity, we evaluated the response of mos3-1, mos11-1 and mos11-1 mos3-1 to the virulent bacteria P. s. m. ES4326. Virulent pathogens, ones not carrying Avirulence genes recognized by the plant R proteins, were used to evaluate the contribution of basal resistance to immunity. Both mos3-1 and mos11-1 mos3-1 plants exhibited enhanced susceptibility when infiltrated with virulent P. s. m. ES4326 and supported higher bacterial growth, whereas the response of the single mos11-1 mutant was similar to that of the wild type (Figure 5C), suggesting that MOS11 is not involved in basal resistance. No further enhanced susceptibility was observed for mos11-1 mos3-1, indicating that these two genes function in the same pathway to regulate immunity. It should be noted that due to the quantitative limit of infection assays, possible minor additive effects like those observed for the flowering time phenotype might have been missed. To test whether mos11 affects R protein mediated resistance, we inoculated Col-0, mos11-1 and mos11-2 with Pseudomonas syringae pv tomato (P. s. t.) DC3000 strains expressing Avirulence genes avrRps4, avrRpm1, avrRpt2 or avrPphB. Avirulent pathogens carry Avirulence genes whose products or the effects of the products in the plant cells are recognized by plant R proteins. These pathogens are used to evaluate the integrity of ETI in plants. Bacterial growth was not affected in the single mutants for any of the R protein mediated pathways (Figure S3). Thus we conclude that MOS11 is not involved in the ETI response mediated by these R proteins. We then analyzed the mRNA export status in the mos11-1 mos3-1 double mutant compared with the single mutants. Similar to the mos3-1 and mos11-1 plants, the poly (A) signal in the double mutant was mostly observed in the nucleus. However, further poly (A) accumulation was not detected in the nuclei of the double mutant (Figure 5E and Figure S2). It should be noted that in all the mRNA export deficient mutants tested, some signal, albeit much weaker than the wild type, was always observed in the cytosol, which explains why these mutations are not lethal and the plants can successfully complete their life cycle. Together, these data suggest that MOS3 and MOS11 function in the same mRNA export pathway, possibly in a partially overlapping manner. Since MOS3 is part of the nuclear pore while MOS11 localizes inside the nucleus, MOS3 probably functions downstream of MOS11 in mRNA export (Figure 6). To search for components in R protein mediated immunity, we performed genetic screens for suppressors of the autoimmune mutant snc1. mos11-1 was identified from a T-DNA mutagenized snc1 population. Mutation in MOS11 partially abolishes the dwarf stature, elevated SA levels, and enhanced resistance to H. a. Noco2 and P. s. m. ES4326 of snc1. MOS11 encodes a protein with unknown function in the plant kingdom, however, it shares 50% similarity with human CIP29. Similarities and identities are more striking in islands of conserved positively charged residues; namely lysine and arginine (Figure S1). These basic islands are known to be present in nucleic acid binding proteins directly involved in DNA/RNA binding [18]–[20]. There are a limited number of reports on human CIP29, which was initially named HCC-1 after being isolated from the hemofiltrate of patients with chronic renal failure [21] and later renamed CCL14a [22]. Overexpression of CIP29 in human embryonic kidney cell line results in slower growth. Biochemical characterization demonstrated that CIP29 could bind RNA on its own [23]. In yeast two-hybrid assays, CIP29 interacted with two DEAD-box RNA helicases, BAT1 and DDX39 [24]. Hints on the potential biological functions of CIP29 came from the study by Sugiura et al. (2007). They showed that the ATP-dependent DEAD-box RNA helicases DDX39 can co-immunoprecipitate CIP29, confirming the earlier yeast two-hybrid interaction reported by Leaw et al. (2004). Additionally they demonstrated that CIP29 can bind RNA on its own [23], and more importantly, it enhances the RNA unwinding activity of DDX39. The same group also identified FUS/TLS, a nucleic acid binding protein participating in both transcription and splicing as a physical interactor of CIP29 [23]. In addition, they detected ALY, a well-characterized mRNA export factor, in the DDX39 immunoprecipitate. CIP29 localized mainly to the nucleus although small amounts were also detected in the cytosol [25]. Very recently, Dufu et al. (2010) reported that CIP29 physically associates with ALY via UAP56 in an ATP-dependent manner to form a trimeric complex. In addition they demonstrated that efficient recruitment of CIP29 to the mRNA is splicing- and cap-dependent [26]. The functions of the CIP29 interactors, its affinity for RNA and the conserved island of positively charged residues suggest a potential role of CIP29 in RNA processing and/or RNA export, although mRNA export defects have not been shown for a cip29 mutant. Through knockout analysis with mos11 mutants, we demonstrate that MOS11 is indeed required for mRNA export. We speculate that its human counterpart CIP29 probably has a similar function. In eukaryotic cells, transcription and translation take place in two separate compartments, which allows fine-tuned regulation of biological processes. For proteins to be translated in the cytosol, the mRNA molecules must first be properly processed, assembled into an export competent mRNA ribonucleoprotein (mRNP) particle and transferred through the nuclear pore. This assembly starts at the onset of transcription. Several proteins bind to the nascent pre-mRNA. These proteins can be involved in transcription [27], capping, splicing [28], polyadenylation [29] or binding to nuclear pore proteins [30]. Most of the knowledge on mRNA export has been gained from studies of human and yeast cells. Little is known about the mechanisms of plant mRNA export (for reviews see [31], [32]), although it is believed to share high similarity with yeast and human pathways. In plants, several nuclear pore complex mutants have been shown to exhibit defects in mRNA export [12], [31]–[37]. In Arabidopsis, previously reported components shown to participate in mRNA export all localize to the nuclear envelope; these include MOS3/AtNup96, AtNup160, LOS4, AtNup1 and AtTHP1. LOS4 encodes a DEAD-box RNA helicase enriched at the nuclear rim and it is also present in the cytosol. Two point mutation alleles of los4/cryophyte were shown to affect mRNA export [38]. More recently orthologs of the TREX-2 complex, which is necessary for mRNA export in yeast and humans, have been identified in Arabidopsis. The TREX-2 complex, which is tethered to the nuclear pore complex via Nup1 in yeast, is composed of Thp1, Sac3, Cdc31 and Sus1. AtNUP1 and AtTHP1 were shown to be necessary for mRNA export, but the other three proteins do not appear to be involved in mRNA export despite evidence of a physical interaction with other TREX-2 subunits [36]. Another protein, TEX1, which is not part of the THO/TREX core complex but part of a complex associated with the TREX complex is also found in Arabidopsis [37]. In Drosophila and human, TEX associates with the helicase UAP56 and the mRNA export factor ALY [39], [40]. However, co-IP experiments using HA-tagged AtTEX did not pull-down ALY or UAP56 in Arabidopsis, but they did retrieve THO1, THO2, THO5, THO6 and THO7 [37]. Subcellular localization of MOS11-GFP revealed that MOS11 is a nuclear protein. In mos11-1, mos11-2 and mos3-1, mRNA export was similarly affected. Epistasis analysis between mos11-1 and mos3-1 suggested that these two genes function in the same mRNA export pathway. Thus we drew a simplified model of the mRNA export pathway in plants (Figure 6) where MOS11 likely functions in the early steps of the mRNA export before mRNA exits the nucleus through the nuclear pore. This is consistent with their respective subcellular localization where MOS11 is nuclear and MOS3 is limited to the nuclear rim. This indicates that MOS11 has access to the mRNA prior to MOS3. Since MOS11 is conserved in eukaryotes, its human homolog CIP29 probably also functions in a similar step in mRNA export as suggested by Dufu et al. [26]. The reason why mos11 suppresses snc1 phenotypes is not clear. mos11 single mutant plants, although slightly different from the wild type, grow to full stature, produce seeds and complete their life cycle successfully. This shows that the observed impairments in mRNA export in mos11 are not sufficient to significantly affect ontology. The snc1 mutation involves an extensive transcriptional reprogramming that results in the dramatic phenotypes displayed by snc1 plants. Among the changes observed following the auto-activation of snc1 are the induction of defence gene markers and a strong stimulation of the SA biosynthetic pathway. These changes are associated with dwarf stature and increased resistance to pathogens. It is conceivable that MOS11 could promote mRNA export of genes that are up-regulated following defence activation, possibly including R genes. In support of this hypothesis, dot blot analysis demonstrated that mos11-1 does not affect the overall level of transcription since all lines analyzed had similar levels of total mRNA. However mos11-1 does have an effect on snc1 protein level in mos11-1 snc1, possibly resulting from impaired SNC1 mRNA export leading to less mRNA for translation of the protein (Figure 1C). The detailed mechanism of MOS11 in mRNA export is not clear. Since CIP29 was recently shown to interact with ALY and UAP56 in an ATP-dependent manner [26], and it was also shown to enhance the RNA unwinding activity of DDX39 [23], we speculate that the function of MOS11 is likely to act as a co-chaperone to plant homologs of UAP56 and ALY to co-catalyze mRNA unwinding prior to its nuclear export. The specific targets of MOS11 await further investigation. All plants were grown under a 16 h light/8 h dark regime. SA extractions and measurements were carried out as previously described [41]. RNA was extracted from 2-week-old plate-grown seedlings on half Murashige and Skoog (½ MS) media using the Totally RNA kit (Ambion). 200 ng of RNA was used for reverse transcription using Superscript II (Invitrogen). RT-PCR analysis of PR1 and PR2 expression was carried out as previously described [8]. Tubulin was used as an uninduced loading control with primers 5′ACGTATCGATGTCTATTTCAACG3′ and 5′ATATCGTAGAGAGCCTCATTGTCC3′. Pathogen assays with H. a. Noco2 and P. s. m. ES4326 were carried out as previously described [7]. T-DNA screen in snc1 was described previously [13]. Total protein was extracted from 4-week-old soil-grown plants using the extraction buffer containing 100 mM Tris-HCl (pH 8), 0. 2% SDS and 2% β-mercaptoethanol. 30 µl of total protein was separated on 8% SDS-PAGE and transferred onto nitrocellulose membrane. For SNC1 total protein level, the membrane was probed with purified anti-SNC1 antibody, which was generated against a SNC1-specific peptide in rabbit [42]. The full-length genomic MOS11 gene plus 1992 bp upstream of its start codon was cloned in frame to the N-terminus of GFP in a modified pCambia1305 vector in which the GUS gene had been replaced by GFP. Plant transformation was carried out by Agrobacterium in planta dipping [43] and transformants were selected on ½ MS supplemented with 30 µg/ml hygromycin. T1 and T2 transgenic plants were then observed with a confocal microscope with propidium iodine as a cell wall/dying nuclei marker. The total RNA was extracted by RNAiso Plus (Takara, Cat No. D9108A). The RNA samples were quantified by spectrophotometry and confirmed by agarose gel electrophoresis. The RNA samples were prepared and applied to the Hybond-N+ membrane (Amersham Cat. No. RPN303B). The membrane was cross-linked for 2 minutes at 70000 micro-joules/cm2 and baked for 2 hours at 80°C. The membrane was pre-hybridized for 1 hour at 37°C in Hyb-50 (Mylab Corporation, Beijing, P. R. C.). The 18-mer oligo-dT was labeled with 32P by the T4 poly-nucleotide kinase (NEB, Cat. No. M0201) and added to the pre-hybridization buffer. After 16 hours of hybridization, the membrane was washed following Amersham Hybond-N+ manual. The signals were detected by phospho-imager using a Typhoon scanner. We adopted a protocol similar to that described in Parry et al. (2006). Briefly, 4–7 day-old plate-grown seedlings were immersed in 1 ml fixation cocktail (50% fixation buffer consisting of 120 mM NaCl, 7 mM Na2HPO4,3 mM NaH2PO4,2. 7 mM KCl, 0. 1% Tween 20,80 mM EGTA, 5% formaldehyde, 10% DMSO and 50% heptane) in a glass container and gently agitated for 30 minutes at room temperature. The sample was dehydrated twice for 5 minutes each in 100% methanol, three times for 5 minutes each in 100% ethanol and incubated for 30 minutes in ethanol: xylene (50∶50) with gentle agitation. The samples were washed twice in ethanol for 5 minutes each and twice in methanol for 5 minutes each before the samples were incubated 5 minutes in methanol: fixation buffer without formaldehyde (50∶50) and fixed in fixation buffer with 5% formaldehyde for 30 minutes. Samples were rinsed twice for 5 minutes each with fixation buffer without formaldehyde and incubated for 5 minutes with PerfectHyb Plus (Sigma). 1 ml of new hybridization buffer was added and pre-hybridized at 50°C for 1 hour. Finally, 5 pmol of 5′ end-labeled Alexa-488 48-mer oligo d (T) (Invitrogen) was added and incubated overnight in the dark. The final samples were mounted in water and fluorescence was observed with a spinning disc microscope at a magnification of 63X using the following settings: Volocity 5. 1 software, Hamamatsu C9100-50 camera, the exposure time was set to 100 ms and the sensitivity was adjusted to 176. Settings were the same for all samples.

In eukaryotic cells, mRNAs are synthesized in the nucleus where they are processed (capped, spliced, poly-adenylated, and carried toward the nuclear pore) prior to being exported to the cytoplasm for translation. Insights into mRNA export mechanisms have been gained mostly from studies on yeast and humans, while little is known about plant mRNA export. In a genetic screen for suppressors of the Arabidopsis autoimmune mutant snc1, we isolated and identified mos11 (modifier of snc1,11). MOS11 encodes a protein with unknown function in the plant kingdom, and its closest relative in animals is the human RNA binding protein CIP29, whose function is unclear. We demonstrate that MOS11 is a nuclear protein and that mos11 mutants accumulate more poly (A) mRNA in the nucleus, likely resulting from reduced mRNA export. Our data suggest that MOS11 functions before nuclear pore entry in the mRNA export pathway.

lay_plos

CROSS-REFERENCES TO RELATED APPLICATIONS This application claims the benefit of the provisional patent application Ser. No. 60/763,998 filed 2006 Feb. 1 by the present inventor. FEDERALLY SPONSORED RESEARCH None SEQUENCE LISTING None BACKGROUND This invention relates to carrying bags, specifically to carrying bags that have straps with a multimode capability. There exists many strap apparatuses that convert single strap bags to backpacks, or have multimode capabilities. U.S. Pat. No. 6,311,884, B1, Dual Strap System for Conversion of Bags to Backpacks, presents an invention that has a backpack configuration that appears to be similar to the backpack configuration of my invention. A comparison between the two inventions however yields the other invention&#39;s deficiencies. It does not have a single-strap configuration; the user detaches the swivel snap hooks on the single strap that comes with the bag and then attaches the invention to the bag with its own swivel snap hooks. This is much less convenient than my invention. U.S. Pat. No. 6,220,492, B1, Multi-Way Bag, has at least six different configurations, including a single-strap configuration and a backpack configuration. However to convert from one configuration to another, the strap needs to be detached from the bag and then reattached in a different way. Furthermore, the many guides, connectors and slits make the appearance of the bag less than desirable. U.S. Pat. No. 5,881,932, Convertible Bag, has both a single-strap configuration and a backpack configuration. However the mechanisms for conversion are rather complex; furthermore if the bag is in a horizontal state when in the backpack configuration, then it must be rotated to a vertical state in the conversion to single-strap configuration. This is an undesirable feature for many types of bags. Finally, the bag itself has hidden compartments that hold the strap, so it is expensive to produce and does not apply to existing bag designs. U.S. Pat. No. 5,415,332, Multimode Traveling Bag, has a single-strap configuration, a backpack configuration, and an over-the-head configuration. However it has an entirely different implementation than my invention. It uses a single length of strap, not doubled over to form a loop, rather than my invention, which uses a strap in the shape of a closed loop. Furthermore it does not apply to bags that open at the top, as many traveling bags do. My invention does. U.S. Pat. No. 5,577,652, Convertible Backpack, is a bag with a single strap attached by swivel snap hooks. To convert from one configuration to another, either the hooks must be detached and then reattached in a different way; or the bag, if it hangs horizontally in the backpack configuration, will then hang vertically in the single-strap configuration. U.S. Pat. No. 6,138,881, Convertible Backpack/Shoulder Bag, has a single strap with a zipper along the length of the strap. When the bag is worn as in the single-strap configuration, the zipper is closed presenting a single strap. To convert to the backpack configuration, the zipper is opened, revealing two straps. Although this is a fine approach to the multimode problem it appears only to apply to bags that are narrower at the top than at the bottom, limiting its use. Eagle Creek used to sell Convertabrief, a briefcase-like bag with backpack straps hidden in a pocket. To convert from a single-strap configuration to the backpack configuration, the hidden straps would be removed from the hidden compartment and reattached using swivel snap hooks. They now sell a product, Convertabrief ES, which also has additional features like wheels and extendable handles. It is an attractive item for the traveler, but like most of the other inventions in the prior art it is difficult to reconfigure. My invention has advantages that these other inventions do not have. Its design makes it easiest of all the inventions for the wearer to switch from one configuration to another. It may be incorporated into to most bags&#39; existing design and does not detract from the appearance of the bag. It is also the only invention that has the dual-strap configuration. It is well suited for large bags like golf club bags, duffel bags, and musical instrument cases where the strap segments may be attached to the position on the bag between the top of the bag and the bottom of the bag. SUMMARY This invention provides a carrying bag with up to three configurations: a single-strap configuration, a backpack configuration and a dual-strap configuration. The richness of the embodiments and the simplicity for the user to switch from one configuration to the other makes it an attractive choice for any bag designer. It is the only invention that offers a dual-strap configuration where the bag is worn in like a single-strap bag; however the two straps emanate from the bag, one going over each shoulder, distributing the weight of the bag. Finally it is a novel design that will facilitate its marketing. DRAWINGS There are 9 sheets with 23 figures. FIG. 1 shows my invention in the single-strap configuration. FIG. 2 shows the invention in the backpack configuration. FIG. 3 shows the invention in the dual-strap configuration. FIG. 4 a shows the rotator member in the single-strap configuration with the straps passing through it. FIG. 4 b shows the cross section of the rotator member. FIG. 5 a L and 5 a R shows the constraining member on the left and right side respectively with the strap passing through it. FIG. 5 b L and 5 b R show the constraining member components for the left and right sides. FIG. 5 c L and 5 c R show the slide member components for the left and right sides. FIG. 5 d L and 5 d R show the swivel snap hook components for the left and right side. FIG. 5 e L and 5 e R show the attachment members for the left and right sides. FIG. 6 a shows the rivet of the rotator member (second embodiment). FIG. 6 b shows a top view of the rotator member (second embodiment). FIG. 6 c shows a bottom view of the rotator member (second embodiment). FIG. 7 a shows a slide member (second embodiment). FIG. 7 b show the slide member with strap going through (second embodiment). FIG. 8 a shows the rotator member (third embodiment). FIG. 8 b shows a cross section of the rotator (third embodiment). FIG. 8 c shows the top component of the rotator member (third embodiment). REFERENCE NUMERALS 3 Carrying Bag 3 L Left side of bag 3 R Right side of bag 3 T Top of bag 3 BT Bottom of bag 3 F Front of bag 3 B Back of bag 4 Strap 5 R Constraining member on right side 5 L Constraining member on left side 7 R Right attachment member 7 L Left attachment member 9 O First strap segment 9 I Second strap segment 10 Single loop 12 Double loop 14 L Left Slide member 14 R Right Slide member 18 Rotator member 19 Strap adjustment mechanism 27 L Snap hook component of left swivel snap hook 27 R Snap hook component of right swivel snap hook 28 L Loop component of left swivel snap hook 28 R Loop component of right swivel snap hook 29 Swivel snap hook 29 L Left swivel snap hook 29 R Right swivel snap hook 32 Rotator member top component first loop 34 Rotator member top component second loop 35 Rotator member top component 42 Rotator member bottom component first loop 44 Rotator member bottom component second loop 45 Rotator member bottom component 46 Single loop opening 48 Strap loop exit point 50 Strap second end 52 Strap second end attachment place 56 Rotator member top component hole 57 Rotator member top component protuberance 58 Rivet 59 Rotator member bottom component protuberance 60 Rotator member bottom component hole 62 Rivet bottom end 64 Rivet top end 65 Top strap section 66 L Ring on left side 66 R Ring on right side 67 Bottom strap section 68 L Attachment component on left side 68 R Attachment component on right side 80 R Slide member slide component on right side 80 L Slide member slide component on LEFT side 82 L Slide member bracket component on left side 82 R Slide member bracket component on right side 84 Slide member clearance 86 Strap section 94 Rivet top end extension 96 Rotator member top component ridge 98 Rotator member top component ridge first end 100 Rotator member top component ridge second end 102 Slide swivel snap hook member 104 Slide swivel snap hook member slide component 106 Slide swivel snap hook member side bars 108 Slide swivel snap hook member loop 110 Slide swivel snap hook snap hook component 112 Strap entry point 114 Strap exit point 118 Rotator member (third embodiment) 119 Rotator member top component (third embodiment) 120 Rotator member bottom component (third embodiment) 122 Rivet (third embodiment) 124 Top strap (third embodiment) 126 Top strap entry point (third embodiment) 128 Top strap exit point (third embodiment) 130 Bottom strap (third embodiment) 132 Bottom strap entry point (third embodiment) 134 Bottom strap exit point (third embodiment) 136 Bottom component hole (third embodiment) 138 Top component hole (third embodiment) DESCRIPTION OF THE INVENTION First Embodiment The following description applies to the first embodiment of the invention. FIG. 1 shows a perspective view of the first embodiment configured in the single-strap configuration. The bag 3 has a right side 3 R, a left side 3 L, a top 3 T, and a bottom 3 B. The first embodiment consists of the bag 3, a single length of strap 4, swivel snap hooks 29 L and 29 R, a strap adjustment mechanism 19, right side constraining member 5 R, left side constraining member 5 L, and a rotator member 18. The strap adjustment mechanism 19 is constructed from the single loop 10 and a double loop 12 in the conventional manner for straps. The strap is attached to the bag 3 on the left side 3 L and the right side 3 R. The single strap 4 is divided into a first strap segment 90, which is on the outside and a second strap segment 91, which is on the inside in the single strap configuration. The two strap segments each extent form the first constraining member to the second constraining member. When the bag is in the single-strap configuration and worn over one shoulder, the two straps appear as a single strap to a casual observer. FIG. 2 shows the first embodiment in the backpack configuration. The rotator member 18 is shown in FIG. 4 a and a cross section of the rotator member 18 is shown in FIG. 4 b. FIG. 3 shows the strap apparatus in the dual-strap configuration. In this configuration the rotator member 18 is disposed close to the right swivel snap hook 28 R. The strap components are separated so that a strap segment can be put on each shoulder, with the bag hanging on a side of the wearer towards the back. FIG. 4 a shows the rotator member 18. It is composed of a top component 35, a bottom component 45 and a rivet 58. The top component 35 has two loops 32 and 34 to allow the strap to pass through. It has a hole 56 to allow the rivet to pass through. The bottom component 45 has two loops 42 and 44 to allow the strap to pass through. It has a hole 60 (hidden in FIG. 4 a ) to allow the rivet 58 to pass through. The rivet 58 passes through the hole 60 of the bottom component 45 and the hole 56 of the top component 35. It is capped on the rivet&#39;s bottom end 62 exiting from the bottom component 45 and on the top end 64 of the rivet exiting the top component 35. The three components of the swivel loop member 18 are manufactured so the top and bottom components rotate freely about the rivet after the straps are in place but will not separate. It is also manufactured so that the rotator member will not slide freely along the straps on its own by the force of gravity, but can be moved with minimal resistance by the wearer along the straps either to adjust the strap length, or to reconfigure the apparatus in the single-strap configuration, the backpack configuration or the dual-strap configuration. A protuberance 57 occurs on the top component 35 of the rotator member 18 and a second protuberance 59 occurs on the bottom component 45 of the rotator member 18. They are positioned so that when the strap apparatus is configured in the single-strap configuration, one protuberance will lie directly above the other protuberance. FIG. 4 b shows a cross section of the rotator member with the straps in place. The rivet 58 passes through the top component 35 of the rotator member and the bottom component 45 of the rotator member through the holes 58 and 60. The top half strap 65 of the strap apparatus weaves through the top component 35 of the rotator member and the bottom half 67 of the strap apparatus weaves through the bottom component 45 of the rotator member. FIG. 5 a R shows the constraining member 5 R for the right side with the strap constrained on the right side. FIG. 5 b R shows the components of the constraining member 5 R. Referring to FIG. 5 b R, the right side constraining member 5 R consists of a right slide member 14 R, a right swivel snap hook 29 R, and a right attachment component 7 R. FIG. 5 c R shows how the slide member 14 R is designed. The slide member 14 R consists of a slide component 80 R and a bracket component 82 R. In its manufacture, the slide component will allow the strap to slide through it with only slight resistance. It will also be snug enough to prevent the strap from twisting in the slide component 80 R as it slides through the slide member 14 R. The bracket component 82 R is designed to keep the slide member positioned so the slide member 14 R does not ride up the strap. FIG. 5 d R shows the structure of the right snap swivel hook 29 R. The right swivel snap hook 29 R is composed of a swivel loop 28 R that accommodate the strap and can rotate freely, and the snap hook 27 R which can be attached and removed from the attachment ring 66 R. FIG. 5 e R shows the structure of the attachment member 7 R. It is constructed with an attachment ring 66 R and an attachment component 68 R. The attachment component 68 R is attached to the right side near the top, and the attachment ring 66 R is attached to the attachment component 68 R. Referring to FIG. 5 a R, the strap 4 enters through the slide component 80 R of the slide member 14 R, then through the loop 28 R of the swivel snap hook 29 R and then back through the slide component 80 R of slide member 14 R emerging from the slide component 80 R. The bracket component 82 R prevents the slide member 14 R from riding up the strap away from the swivel snap hook. It has sufficient clearance between the strap 4 and the bracket component 82 R so it will not hinder the strap as it slides through the slide component 80 R when switching between the single-strap configuration and the backpack configuration and when adjusting the strap length. FIGS. 5 a L through 5 e L show the details of the constraining member 5 L for the left side with the strap constrained on the left side. The descriptions of these figures are entirely analogous to the right side FIGS. 5 a R through 5 e R and will not be repeated. The strap apparatus can be attached and unattached to the bag using the two swivel snap hooks 29 R and 29 R and the attachment ring 66 T and 66 L. The left swivel snap hook 29 L is attached to the bag by snapping the left swivel snap hook snap component 27 L of the left swivel snap hook 29 L onto the bag&#39;s left attachment ring 66 L. The right swivel snap hook 29 R is attached to the bag by snapping the right swivel snap hook snap component 27 R of the right swivel snap hook 29 R onto the bag&#39;s right attachment ring 66 R. Operation To change the strap apparatus configuration from single-strap configuration of FIG. 1 to the backpack configuration of FIG. 2, do the following: Referring to FIG. 1, first hold the strap apparatus from the rotator member 18 positioned at the highest point of the two strap segments when the bag held with the bottom parallel to the floor, and let the bag hang so the strap is tight. The wearer arranges the strap if necessary so the strap sides aren&#39;t twisted. The wearer&#39;s left hand grabs the first strap segment 9 O to the left of the top point with palm down. The wearer&#39;s right hand grabs the second strap segment 9 I strap with the right hand palm down. Then the wearer lifts the bag holding the strap in the two places indicated. The wearer shakes the strap slightly if necessary. The weight of the bag will cause the strap to slide thorough the slide members 14 L and 14 R and the rotator member 18 until the backpack configuration of FIG. 2 is obtained. The wearer then puts the strap apparatus with the bag attached on in the standard way backpacks are put on the shoulders with the bag at the back of the wearer. FIG. 2 shows the strap in the backpack configuration. To convert the strap apparatus from the backpack configuration to single-strap configuration, do the following. Referring to FIG. 2, take the backpack off the shoulders and hold in front of the wearer. Grab the rotator member 18 and rotate the top component so the protuberances are aligned. Then lift the rotator member 18 and shake gently until the single-strap configuration of FIG. 1 is obtained. It may be necessary to adjust the strap so the strap is not twisted. To convert the strap apparatus from the single-strap configuration to the dual-strap configuration, the wearer does the following. Referring to FIG. 1, the wearer slides the rotator member 18 along the strap until it is adjacent to the right swivel snap hook 28 R. The direction of the sliding should be away from the strap adjustment mechanism 19. Then the wearer puts the apparatus on so the bag is on one side towards the wearer&#39;s back; one strap is over the shoulder on the side of the bag, the other strap is over the other shoulder. This completes the description of the first embodiment of this invention. Second Embodiment In the second embodiment of this invention the following two modifications are made. For the first modification of this second embodiment, refer to FIG. 6 a. The rivet 58 has an extension 94 on its top end. Referring to FIG. 6 b, the top component 35 of the rotator member 18 has an added ridge 96. The bottom component 45 does not have a ridge. The ridge is attached permanently to the top component 35 so the rivet can rotate relative to the top component 35 between the ridge ends 98 and 100. It is constructed so that when the top member is situated in relation to the bottom member so the strap is in backpack configuration, the extension will may not rotate past the ridge end either at 98 or 100, depending which way the top component 35 is rotated relative to the bottom component 45. When the strap apparatus is in the single-strap configuration so the bottom component 45 and top component 35 are aligned as shown if the FIG. 6 b, the extension 94 lies approximately half way between the ridge ends 98 and 100. FIG. 6 c shows a bottom view perspective of FIG. 6 b. The rivet head 62 is attached permanently to the bottom component 45 of the rotator member 18. Note that the protuberances 57 and 59 of FIG. 4 a are missing, since in this modification, the top component 35 and bottom component 45 can only be aligned in one way. With this modification, converting from backpack configuration to single-strap configuration is facilitated. FIG. 7 a shows the second modification to this second embodiment. The left slide member 14 L and left swivel snap hook 28 L shown in FIG. 1 are combined into a single swivel snap hook slide member 102 of FIG. 7 a. The swivel snap hook slide member 102 consists of a slide component 104, two support components 106, a loop component 108 and a swivel snap hook 110. Similarly, the right slide member 14 R and right swivel snap hook 28 L are combined into a swivel snap hook slide member with the same design. FIG. 7 b shows how the strap loops through the slide swivel snap hook member 102. Compare to FIG. 5 b. In FIG. 7 b, the strap enters through the slide component 104 at entry point 112, then goes through the loop component 108 of the slide swivel snap hook member 102, and then back through the slide component 104 emerging 114. The swivel snap hook component swivels and is used to hook onto the bag loop 55 R. This completes the second embodiment of the invention. Operation The operation of the second embodiment follows that of the first embodiment. Third Embodiment FIG. 8 a shows the third embodiment of this invention. The only manner in which this embodiment differs from the first embodiment is that the rotator member 118 has a different design from the rotator member 18 of the first embodiment. Referring to FIG. 8 a, the rotator member 118 has a top component 119, a bottom component 120 and a rivet 122. The top component 119 has a flattened tube shape that will allow the top strap 124 to enter one end of the top component 126 and exit the other end 128. The bottom component has a flattened tube shape that will allow the bottom strap 130 to enter one end of the bottom component 132 and exit the other end 134. The top component 119 and the bottom component 120 are held together by a rivet 122 that allows the to components to rotate freely relative to each other. FIG. 8 b shows a cross section of the rotator member 118 of this embodiment. Referring to FIG. 8 b, the top strap 124 enters 126 the top component 119 and exits 128 the top component 119 at the other end. The bottom strap 130 enters 132 the bottom component 120 and exits 134 the bottom component 130 at the other end. The rivet 122 attaches the two components together but allows them to rotate freely. The rivet passes through the bottom component through a hole 136 its top and through the top component through a hole 138 at its bottom. FIG. 8 c shows the top component 119 of the rotator member 118 in greater detail. The bottom component 120 is has the same design. This completes the third embodiment of the invention. Operation The operation of the third embodiment follows that of the first embodiment. CONCLUSIONS, RAMIFICATIONS AND SCOPE These are not the only embodiments of my invention. While my above description contains many specificities, these should not be construed as limitations on the scope of the invention, but rather as an exemplification of the presently preferred embodiments thereof. Many other ramifications and variations are possible within the teachings of the various embodiments. For example, Subsets of the three configurations may be implemented such as implementing only the single-strap and dual-strap configurations. Shoulder pads may be added. A rope-shaped strap, a chain or any other strap used for carrying bags, may be used as a strap, which is shown in these embodiments as a flat strap. The components that attach the strap to the bag may be permanently attached to the bag; swivel snap hooks may be replaced by any other detachable connector. Mechanisms may be added to the strap segments above the bag so the two strap segments stay together for a portion of the straps when in the single-strap configuration, but will release easily when transitioning it to the backpack or dual-strap configuration. The fasteners are shown in the embodiments as rivets. Any attachment mechanism may be used such as but not limited to rivets, sewing, gluing, or fusion. In any of the embodiments the strap adjustment mechanisms may be eliminated or included, and if included may have one or two. Removable or permanent usage guides may be installed on the strap apparatus, temporarily or permanently, to assist the wearer in learning how place the hands when the wearer transitions the strap apparatus from the single-strap configuration to the backpack configuration. Thus the scope of the invention should be determined by the appended claims and their legal equivalents, and not by the examples given.

This invention presents strap apparatus that is attached to a carrying bag such as a briefcase, computer bag, or golf club bag. In the first embodiment, the strap apparatus is constructed from a single strap that is configured as a closed loop. The closed loop is constrained to the sides of the bag near the top by two constraining means; the closed loop being divided into two strap segments of approximately equal lengths. Depending on how the straps are shouldered, the bag may be worn in a single-strap configuration, a backpack configuration, or a dual-strap configuration. Transitioning from one configuration to the other is easily done by the wearer without having to remove the apparatus from the bag. Furthermore when in the single-strap configuration, the straps stay together and appear to the casual observer as a single-strap as found on an ordinary bag. The appearance of the bag when in the single-strap configuration will not reveal its multimode capability.

big_patent

Transition row metal ions are both essential and toxic to microorganisms. Zinc in excess has significant toxicity to bacteria, and host release of Zn (II) at mucosal surfaces is an important innate defence mechanism. However, the molecular mechanisms by which Zn (II) affords protection have not been defined. We show that in Streptococcus pneumoniae extracellular Zn (II) inhibits the acquisition of the essential metal Mn (II) by competing for binding to the solute binding protein PsaA. We show that, although Mn (II) is the high-affinity substrate for PsaA, Zn (II) can still bind, albeit with a difference in affinity of nearly two orders of magnitude. Despite the difference in metal ion affinities, high-resolution structures of PsaA in complex with Mn (II) or Zn (II) showed almost no difference. However, Zn (II) -PsaA is significantly more thermally stable than Mn (II) -PsaA, suggesting that Zn (II) binding may be irreversible. In vitro growth analyses show that extracellular Zn (II) is able to inhibit Mn (II) intracellular accumulation with little effect on intracellular Zn (II). The phenotype of S. pneumoniae grown at high Zn (II): Mn (II) ratios, i. e. induced Mn (II) starvation, closely mimicked a ΔpsaA mutant, which is unable to accumulate Mn (II). S. pneumoniae infection in vivo elicits massive elevation of the Zn (II): Mn (II) ratio and, in vitro, these Zn (II): Mn (II) ratios inhibited growth due to Mn (II) starvation, resulting in heightened sensitivity to oxidative stress and polymorphonuclear leucocyte killing. These results demonstrate that microbial susceptibility to Zn (II) toxicity is mediated by extracellular cation competition and that this can be harnessed by the innate immune response. S. pneumoniae is the world' s foremost bacterial pathogen and a leading cause of death in young children in developing countries [1], [2], [3]. One of the major factors associated with the incidence and severity of S. pneumoniae infections in these children is dietary zinc deficiency (a significant ongoing problem in developing countries [4], [5]). Zinc, which occurs as the divalent cation Zn (II), is the second most abundant transition metal in humans and has crucial roles in many facets of the immune system [6], [7]. The physiological concentration ranges of Zn (II) range from a few µM to over 100 µM and it has been suggested that Zn (II) interacts with up to 10% of all human proteins [8], [9], [10]. Zn (II) concentrations are elevated in response to inflammation and bacterial infection as a consequence of Zn (II) release from damaged or apoptotic cells, and from sequestering proteins such as metallothionein. Despite the requirement of Zn (II) for optimal immune function, diseases in developing countries associated with Zn (II) -poor status are predominantly acute respiratory infections, otitis media and diarrhea [7]. In recent years clinical trials of Zn supplementation have been undertaken in developing countries and meta-analyses of multiple trials [11], [12] have shown a clear association between Zn (II) supplementation and a reduction in the incidence and severity of pneumonia and diarrhea. However, to date no clear mechanism for the protective effect of Zn (II) has been identified. Zn (II) is also an essential micronutrient for bacteria, although it has significant toxicity at high concentrations [13], [14], [15], [16], [17]. However, the molecular basis of zinc toxicity remains poorly defined. Studies from our laboratory have previously demonstrated that the pneumococcal surface antigen A (PsaA) from S. pneumoniae has a clear interaction with Zn (II) [18] despite not being involved in its uptake, which occurs via a distinct Zn (II) ATP-binding cassette (ABC) permease [19], [20]. PsaA is the solute-binding protein (SBP) of a manganese-specific ABC permease encoded by the psaBCA locus. Mn (II) is an essential trace element for both prokaryotes and eukaryotes where it has roles in many cellular processes [21]. In S. pneumoniae Mn (II) regulates a diverse array of genes and has been shown to have roles in competence and in managing oxidative stress [22], [23]. Notably, the importance of Mn (II) for oxidative stress management, beyond its role in manganese superoxide dismutase, is an area of growing research. Recently, Mn (II) was shown to be able to substitute for ferrous iron in a nonredox metabolic enzyme to protect carbon metabolism under conditions of oxidative stress [24]. Intriguingly, both Mn (II) and Zn (II) are acquired by SBPs belonging to the Cluster A-1 family [25] of prokaryotic transporters [19]. The tertiary structure of the respective SBPs are very similar, although key differences are present at the metal binding sites [26]. Mn (II) acquisition, mediated by PsaA, is important for S. pneumoniae growth, proliferation and virulence [27]. In S. pneumoniae, loss of psaA results in a massive reduction of virulence in systemic, respiratory tract and otitis media murine models of infection [27], [28], [29]. The importance of Mn (II) for the virulence of bacteria has also been observed in a number of other pathogens including Bacillus anthracis, S. pyogenes, and Staphylococcus aureus [30], [31], [32]. Therefore, as the S. pneumoniae Mn (II) ABC permease PsaBCA is essential for growth in vivo, we hypothesized that Zn (II) could compete for Mn (II) binding, and thereby mediate toxicity by impairing Mn (II) acquisition. In this paper, we present the biophysical characterization of PsaA and show how Zn (II) competition for Mn (II) transport in vitro impairs growth and renders S. pneumoniae hypersensitive to oxidative and polymorphonuclear leukocyte killing. We then illustrate the significance of these findings by showing how in vivo host Zn (II) concentrations change in response to S. pneumoniae infection. Taken together, this study presents a new paradigm for the molecular basis of bacterial susceptibility to Zn (II) toxicity, due to its extracellular competition for an essential metal ion micronutrient transporter, and its exploitation in vivo by the host immune response. We first determined the binding constants (KA) of Mn (II) and Zn (II) with PsaA using isothermal titration calorimetry (ITC). Representative binding isotherms of apo-PsaA are shown in Fig. 1A and B (for further details see Text S1). The derived affinity constants (1/KA = KD) calculated for 1∶1 complexes of PsaA with Mn (II) or Zn (II) were 3. 3±1. 0 nM and 231±1. 9 nM, respectively. The ITC data indicated that Zn (II) had an affinity nearly two orders of magnitude lower than Mn (II), consistent with the role of PsaBCA in Mn (II) acquisition under physiological conditions. Nevertheless, the data suggested Zn (II) could compete for PsaA binding at high concentrations. The crystal structure of PsaA had previously been determined with Zn (II) tetrahedrally coordinated by the four metal-binding residues (His67, His139, Glu205 and Asp280) [18] but no structure for the Mn (II) -loaded protein has been reported. We crystallized Mn (II) -loaded PsaA and determined its structure at 2. 7 Å resolution (PDB ID: 3ZTT). Well-defined and continuous electron density was observed for the active site residues that tetrahedrally coordinated the metal ion (Fig. 1C and D), which was identified as Mn (II) (see Text S2 for further details). The tetrahedral coordination of Mn (II) is analogous to Zn (II) coordination in PsaA (Fig. S1) and the structures of other Cluster A-1 SBPs (Treponema pallidum TroA [33], Synechocystis 6803 MntC [34], Synechocystis 6803 ZnuA [35] and E. coli ZnuA [26]). Overall, our structural analysis indicates that PsaA is capable of binding Mn (II) and Zn (II) with only very minor accompanying structural changes in the final loaded state (Fig. S1C and D). Metal ion discrimination within the Cluster A-1 SBPs is proposed to occur through subtle differences in the binding sites, Zn (II) -binding being favoured by the presence of three N ligands (from His residues), and Mn (II) -binding being favoured by two N (His) and two O (Asp or Glu) ligands [26]. We determined, by ITC, that Zn (II) binding had a high enthalpy (ΔH = −18. 2±0. 4 kcal. mol−1) relative to Mn (II) (ΔH = −7. 5±1. 1 kcal. mol−1), as would be predicted by the Irving-Williams series for the stability of metal ion complexes [36]. However, the preference of PsaA for Mn (II) is the result of the highly unfavourable entropic contribution of Zn (II) binding (−TΔS = 9. 5±0. 4 kcal. mol−1) relative to Mn (II) (−TΔS = −4. 0±0. 9 kcal. mol−1). Thus, the binding free energies (ΔG) of the PsaA-Mn (II) and PsaA-Zn (II) complexes (−11. 5±0. 2 kcal. mol−1 and −8. 9±0. 2 kcal. mol−1, respectively) are consistent with both the preference for Mn (II) under physiological conditions and the observed difference in affinity of the protein (1 order of magnitude = 1. 41 kcal. mol−1). Mn (II) preference for PsaA is, therefore, due to differences in the entropic contributions. We employed a thermal stability assay (TSA) to measure the influence of Mn (II) - and Zn (II) -specific binding on the melting temperatures (Tm) of PsaA. This assay observes the overall stability of the folded state of the protein as a function of the Tm. Metal-ion ligands that bind and stabilise the protein' s quaternary structure induce a more thermo-stable structure, i. e. a higher Tm. ‘As prepared’ PsaA (Fig. 1E) was found to have a major transition at 62. 1°C, corresponding to the apo-protein, and a minor peak at 72. 9°C, corresponding to the co-purifying Zn (II) form (present in ‘as prepared’ PsaA) (Table S1). This interpretation was confirmed by analysis of cation-free PsaA binding site point mutants that were purified as apo-proteins (Fig. S2, Table S1). Co-incubation of PsaA with saturating Zn (II) increased the overall Tm to 72. 9°C (Fig. 1E, Table S2). In contrast, saturating Mn (II) only increased the Tm of the apo-PsaA to 65. 1°C and did not affect the Zn (II) -PsaA complex. Titration of Mn (II) with PsaA showed that apo-PsaA demonstrated a stepwise increase in Tm (Fig. 1F, Table S2) from 63. 2°C to 70. 2°C, while the PsaA-Zn (II) subpopulation (72. 9°C peak) was not affected by the Mn (II) additions. In conclusion, the TSA unexpectedly showed that Zn (II) induced greater thermal stability than Mn (II), despite Mn (II) having a higher affinity for PsaA. We then sought to determine the phenotypic effect of Zn (II) on PsaA-mediated Mn (II) uptake. This was assessed by in vitro metal ion competition assays with wild-type S. pneumoniae and the ΔpsaA mutant grown in the presence of differing ratios of Zn (II) in a semi-synthetic medium. Increasing the Zn (II): Mn (II) ratio slowed bacterial growth and at very high ratios, i. e. at 1000∶1 [1000 µM Zn (II): 1 µM Mn (II) ], S. pneumoniae growth was completely inhibited (Fig. 2A). Growth was restored, albeit heavily delayed, at Zn (II): Mn (II) ratios of 100∶1 and 250∶1, while at ratios of 50∶1 and 10∶1 Zn (II): Mn (II), S. pneumoniae growth was essentially the same as in the basal medium (Fig. 2A). Growth at inhibitory Zn (II) concentrations could be reversed by Mn (II) supplementation to a 1∶1 ratio relative to the Zn (II) concentration (Fig. 2B), consistent with a competitive effect of Zn (II) for PsaA. The inhibitory effect of Zn (II) was also observed on exponential phase growing cells (Fig. 2C). The addition of Zn (II) at ratios of 300∶1 and 1000∶1 inhibited growth of S. pneumoniae within 120 minutes (2–3 generations). Intriguingly, high ratios of Zn (II): Mn (II) slowed the growth rate of S. pneumoniae similar to that of the isogenic ΔpsaA mutant. Previously, our group has reported that the phenotypic effects of a ΔpsaA mutant include compromised Mn (II) uptake and a slower growth rate, relative to wild-type S. pneumoniae [28]. As the ΔpsaA strain lacks PsaA, and consequently has no functional high affinity Mn (II) importer, we predicted that the mutant would be insensitive to Zn (II) competition. Consistent with this hypothesis, we observed that competitive ratios of Zn (II): Mn (II), e. g. 250∶1 (Fig. 2D), had no effect on the already slower growth rate of the ΔpsaA mutant. Extracellular Zn (II) exerted a clear effect on the growth rate of S. pneumoniae. As this could occur through a number of possible routes in a complex biological system, we sought to determine if extracellular Zn (II) was influencing intracellular metal concentrations by conducting inductively coupled plasma mass spectroscopy (ICPMS) on S. pneumoniae grown in different Zn (II): Mn (II) ratios. We observed that at the competitive ratio of 100∶1, i. e. growth perturbing, the concentration of Zn (II) was essentially the same as that at the non-competitive 10∶1 Zn (II): Mn (II) ratio (Fig. 2E, Table S3). This indicated that despite the high concentration of extracellular Zn (II), intracellular Zn (II) homeostasis was not being disregulated. The intracellular concentrations of Zn (II) increased nearly two-fold in the wild-type when grown in the presence of high extracellular Zn (II) (10∶1 and 100∶1 Zn (II): Mn (II) ratios). Although this had no apparent physiological effect, as demonstrated by the 10∶1 Zn (II): Mn (II) ratio, the higher intracellular concentrations may render S. pneumoniae more susceptible to Zn (II) toxicity if Zn (II) efflux pathways were blocked or impaired. Collectively, these data support a model whereby high concentrations of extracellular Zn (II) were competitively inhibiting Mn (II) uptake and, as the intracellular Zn (II) concentration observed for the 100∶1 and 10∶1 Zn (II): Mn (II) ratios were essentially the same, it follows that the intracellular Zn (II) and hence Zn (II) toxicity was not due to toxic intracellular accumulation. The lack of significant variation in the intracellular Zn (II) concentrations between the 100∶1 and 10∶1 Zn (II): Mn (II) -grown cells suggested that Zn (II) was not transported by the Mn (II) permease, despite the affinity of PsaA for Zn (II). This inference is supported by comparisons with the ΔpsaA mutant, which lacks a Mn (II) transporter and had similar intracellular accumulations of Zn (II) to that of wild-type S. pneumoniae (Fig. 2E, Table S3). Taken together these data suggest a model where PsaA-Zn (II) binding results in an irreversible “dead end” complex, the physiological basis of which could be to prevent Zn (II) leakage into the bacterial cell through the Mn (II) transporter in the presence of high extracellular Zn (II) concentrations. In contrast, the Mn (II) -PsaA complex likely represents a reversible complex that, upon interaction with the ABC permease, delivers the metal ion into the transporter and allows apo-PsaA to be released. This model is consistent with our biochemical observations, which found that Zn (II) conferred very high thermal stabilisation on PsaA in the TSA, and that Zn (II) removal required unfolding of the protein, as it was resistant to dialysis, chelation and Mn (II) competition. ICPMS analysis of S. pneumoniae grown in the 100∶1 Zn (II): Mn (II) ratio showed a five-fold decrease in intracellular accumulation of Mn (II) (P = 0. 002) compared to the 10∶1 and 1∶1 ratios (Fig. 2F Table S3). Thus, extracellular Zn (II) was inhibiting Mn (II) uptake by S. pneumoniae, thereby inducing a phenotype similar to a ΔpsaA mutant. However, unlike the mutant strain, the 100∶1 Zn (II): Mn (II) -grown S. pneumoniae had an intact high affinity Mn (II) transporter. The residual Mn (II) uptake observed in the ΔpsaA mutant most likely occurs via non-specific transport. We then sought to determine what effect competitive ratios of Zn (II) had on PsaBCA expression. The expression of PsaBCA is regulated by PsaR, a DtxR family regulator, which represses psaBCA transcription in response to Mn (II) while de-repression occurs at low Mn (II) or high Zn (II) concentrations [37]. Under our experimental conditions, 100∶1 Zn (II): Mn (II) -grown S. pneumoniae demonstrated slower growth and decreased intracellular Mn (II) compared to 10∶1 Zn (II): Mn (II) -grown S. pneumoniae, which had unaffected growth. An analysis of psaA transcription and psaA expression found that, in 100∶1 Zn (II): Mn (II) -grown cells, both were highly up-regulated relative to the 10∶1 grown cells (Fig. S3A and S3B). As the intracellular Zn (II) concentrations in both the 100∶1 and 10∶1 Zn (II): Mn (II) -grown S. pneumoniae were essentially the same, the observed PsaR-dependent up-regulation of expression of the psaBCA operon would be most likely due to the Mn (II) starvation phenotype and similar to that previously observed in the ΔpsaA strain [37]. Mn (II) restriction in S. pneumoniae has two well established consequences, namely a reduction in oxidative stress response capability and a concomitant loss of in vivo virulence [23], [28], [29]. Therefore, to assess whether the Zn (II) -dependent inhibition of Mn (II) uptake was inducing a ΔpsaA-like phenotype, we investigated the capacity of 100∶1 Zn (II): Mn (II) -grown S. pneumoniae to tolerate oxidative stress mediated chemically, via paraquat or by human polymorphonuclear leucocytes (PMNs). Paraquat is a redox compound that generates superoxide in the cytoplasm, and survival after exposure requires effective oxidative stress management [23]. After 30 min. of paraquat exposure both the 100∶1 Zn (II): Mn (II) -grown S. pneumoniae and the ΔpsaA strain had significantly lower survival rates (31% [P = 0. 046] and 18% [P = 0. 040], respectively) when compared to the Mn (II) replete wild-type S. pneumoniae (100%) (Fig. 3A). Following the hypothesis that reduced Mn (II) accumulation results in hypersensitivity to oxidative stress, we next examined whether this heightened susceptibility to killing extended to human PMNs (Fig. 3B). Both the 100∶1 Zn (II): Mn (II) -grown S. pneumoniae and the ΔpsaA strain had significantly lower survival rates after incubation with PMN (13. 4% [P = 0. 0292] and 5. 0% [P = 0. 0285], respectively) when compared to the Mn (II) replete wild-type S. pneumoniae (22. 3%). The increased susceptibility of the ΔpsaA mutant and the 100∶1 Zn (II): Mn (II) grown wild-type S. pneumoniae demonstrates that Mn (II) has a direct role in resisting PMN killing. Taken together these data show that Zn (II) competitively inhibits intracellular Mn (II) acquisition in S. pneumoniae leading to significantly increased susceptibility to oxidative stress, similar to the ΔpsaA strain. The physiological consequences of Mn (II) deprivation, as a result of Zn (II) competition in vitro, suggested that S. pneumoniae survival in vivo could be compromised, similar to the ΔpsaA strain. Therefore, we investigated the levels of Mn (II) and Zn (II), and other cations, in tissue samples collected from naïve and S. pneumoniae-infected mice. For naïve mice, the ratios of Zn (II) to Mn (II) in niches that would be subject to colonization by S. pneumoniae did not exceed ∼60∶1 (Table 1). Thus, the observed ratios of Mn (II) to Zn (II) within these physiological niches should be permissive for Mn (II) acquisition by PsaBCA. For mice infected with S. pneumoniae, the samples harvested 48 hours post-infection showed significantly shifted metal ratios, with Zn (II) concentrations increasing in all tissues (Table 1, Fig. 3C). This effect was largely specific for Zn (II), although minor increases were observed with Fe (II) in the nasopharynx and the brain and Cu (II) in blood serum (Table S4). The increase of Zn (II) was most dramatic in blood serum and the nasopharynx, where the Zn (II): Mn (II) ratio increased to approximately 900∶1 (P = 0. 0292) and 330∶1 (P = 0. 0163), respectively. The lack of variation among the majority of other metal ions examined (Table S4) indicated that elevation of Zn (II) has a major role in host response to S. pneumoniae infection. These findings were consistent with the earlier study of Strand and co-workers [38], which found that Zn (II) -deficient mice had heavier S. pneumoniae nasopharyngeal colonization, compared to the Zn (II) replete mice, and only Zn (II) deficient mice had S. pneumoniae bacteremia. The dramatic elevation of Zn (II) observed in these two tissues, upon infection with S. pneumoniae in our study, suggested that Zn (II) could compete with Mn (II) (which remained largely unchanged) for PsaA interaction (Table 1, Fig. 3D) and, therefore, demonstrate that metal ions have a role in ameliorating bacterial propagation and dissemination in vivo. Although nearly 30% of all proteins contain metal ions [21], an excess of certain transition metal ions can exert significant toxicity. The potential roles of such metals in resisting bacterial invasion are increasingly being recognized. Notably the importance of copper in macrophages for bactericidal activity has recently been demonstrated [39]. Our present study provides the first direct evidence that extracellular Zn (II) can exert its toxicity towards S. pneumoniae by competing with Mn (II) for binding to the PsaA solute binding protein, thereby preventing the acquisition of Mn (II) via the Psa permease. While our study was being completed, Jacobsen and co-workers similarly proposed that Zn (II) toxicity results in cytoplasmic Mn (II) deficiency, but no evidence for the mechanism was provided [40]. We have shown that Zn (II) competition results in reduced Mn (II) accumulation and elicits a phenotype nearly identical that of an isogenic ΔpsaA mutant. Reduced intracellular Mn (II) perturbs bacterial growth as the capacity to manage oxidative stress, which occurs during aerobic growth, is compromised [23], [28]. This is particularly noticeable during the outgrowth of stationary phase cells, which showed impaired growth at Zn (II): Mn (II) ratios of 100∶1 and greater, while exponential phase growing cells were inhibited at higher ratios, presumably as Mn (II) was titred out by cell division. This may also indicate that Mn (II) has distinct intracellular roles at different stages of S. pneumoniae growth. Very high (>1 mM) concentrations of zinc completely bacterial inhibited growth and it is likely that under these conditions Zn (II) toxicity was mediated by mechanisms in addition to competition for PsaA. Our findings also provide a physiological rationale for the Zn (II) -dependent regulation of PsaR [37] as a mechanism for regulating expression of the Mn (II) transporter during exposure to high levels of the competing cation. Cluster A-1 SBPs are present in a broad range of pathogenic bacteria, such as S. pneumoniae, Yesinia pestis, Staphyloccocus aureus, and S. pyogenes [19], [31], [32], [41], and so the competitive mechanism of Zn (II) toxicity proposed for S. pneumoniae could also extend to these bacteria. This investigation also resolves an area of significant confusion as to the presence of Zn (II) in the original crystal structure of PsaA [18], which has remained contradictory to all of the physiological data for the role of the Psa permease in S. pneumoniae. Here we show PsaA in complex with its physiological ligand and the difference in the affinities for Mn (II) and Zn (II) confirm its physiological role in Mn (II) acquisition. Our biophysical characterization also provides an explanation for the presence of Zn (II) in the original crystal structure. The thermal stabilization data show that Zn (II) binding induces a highly stabilized from of PsaA and it is highly likely that this would preferentially crystallize in comparison to the less thermally stable apo- form of the protein. This is also consistent with the behaviour of other Cluster A-1 Mn (II) -SBPs, all of which show a preference for Mn (II) but still retain an interaction with Zn (II), albeit at a lower affinity. Furthermore, consistent with the lack of solvent access to the binding site in the metal loaded-PsaA structures, we observed that Zn (II) could not be extracted from PsaA by chelating agents, despite the protein' s lower affinity for the metal, or by competition with Mn (II). This supports our observations that, at high ratios, Zn (II) binding to PsaA could effectively inactivate the Psa permease leading to impairment of Mn (II) uptake as shown in our in vitro studies. Our study also highlights the importance of Zn (II) during in vivo infection and implicates a role for Zn (II) in the host immune response. Dietary zinc deficiency is a global health issue that affects 2 billion people [42] and, in developing countries, zinc deficiency is associated with a higher burden of respiratory disease [4], [43]. In recent years Zn (II) supplementation therapies have proven to be efficacious in reducing the incidence of respiratory infections [4], [5], [12], [43]. These studies have also shown that inadequate dietary Zn (II) can reduce mean blood serum Zn (II) concentrations by up to 76%. Thus, dietary Zn (II) -deficiency can significantly reduce the Zn (II): Mn (II) ratio in the blood and, presumably, other tissues [4], [5], [43]. The association between Zn (II) and optimal immune function is well known and our findings offer a plausible molecular explanation for the protective role of zinc. We have shown that Zn (II) concentrations were significantly elevated within 48 hours of bacterial infection in all niches colonized by S. pneumoniae, consistent with the importance of Zn (II) in the innate immune response. Furthermore, a comparison between the naïve and infected states clearly shows that the alterations in metal ion concentrations in host niches were essentially Zn (II) -specific with only isolated changes observed for other trace elements. We propose that it is likely that the observed increase in Zn (II) concentration in vivo is due to extracellular release of Zn (II) to recruit immune effector cells [44], [45] and to compete for Mn (II) acquisition. This inference is consistent with the massive elevation of the Zn (II): Mn (II) ratio in the nasopharynx and in blood serum, two niches that were hyper-susceptible to S. pneumoniae colonization in Zn (II) -deficient mice [38]. Furthermore, our observations are also consistent with the recent findings of Corbin and co-workers [13], who found that the Gram-positive pathogen Staphylococcus aureus had a similar dependence on Mn (II) in abscess tissue, where the host protein calprotectin was identified as mediating in vivo Mn (II) restriction. Assessing in vivo metal ion bioavailability remains a technical challenge, since even in blood serum, metal ions may be sequestered in proteins and other carrier molecules. Despite this caveat, which applies to this study and others such as Corbin and co-workers [13], our data directly implicate the specific elevation of Zn (II) as a major factor in host response to infection. Based on the available data we propose that, during in vivo infection, Mn (II) bioavailability could be restricted due to elevation of Zn (II): Mn (II) ratio by the release of Zn (II) from damaged or apoptotic cells and from sequestering proteins such as metallothionein. This would initially slow S. pneumoniae growth while also increasing its susceptibility to oxidative killing, as it is the ratio, not the absolute concentration of the two metal ions, that is important. This would be consistent with the requirement of Mn (II) uptake for S. pneumoniae infection in vivo, as the ΔpsaA mutant is totally avirulent [27], [28], [29]. Zn (II) was released during infection at high ratios relative to Mn (II) in the nasopharynx and in blood serum. In our in vitro studies, these ratios were inhibitory and it is not unreasonable to speculate that these ratios in vivo could obfuscate Mn (II) -PsaA interactions and thereby induce a Mn (II) -starvation phenotype in the invading pathogen. The host release of Zn (II) would have the additional benefit of also driving recruitment of leukocytes and other components of the innate immune response [44], [45]. The cumulative effect of this would be that elevation of the Zn (II): Mn (II) ratio in vivo would also increase the susceptibility of S. pneumoniae to oxidative killing by immune effector cells, such as PMNs, as we have shown in vitro, thereby facilitating more efficient defence against the invading pathogen. PMNs are a major component of the innate response and in addition to their oxidative killing mechanisms they contain large stores, up to 50% of their total protein, of the metal binding protein calprotectin [46]. Corbin and co-workers [13] first postulated that calprotectin could be released and directed at bacterial pathogens as a mechanism for restricting metal ion availability in vivo. Supporting this inference, they observed that calprotectin-deficient mice had increased abscess formations and bacterial burdens relative to infected wild-type animals. Release of calprotectin by PMNs during the immune response could also have a significant effect on the Zn (II): Mn (II) ratio, by sequestering Mn (II). This would increase the susceptibility of invading bacteria to oxidative killing mechanisms, consistent with both the earlier suggestions of Corbin and co-workers [13] and in our model for in vivo Mn (II) starvation of the invading bacterium. Collectively, this study demonstrates the biophysical basis of metal specificity for an essential bacterial cell-surface protein and its importance for bacterial propagation. We have provided direct evidence of how the usually non-toxic metal ion Zn (II) mediates toxicity to S. pneumoniae, namely by competition for Mn (II) acquisition leading to intracellular Mn (II) starvation. Furthermore, we have provided a molecular explanation for how this could be harnessed and exploited by the innate immune system to heighten the bacterium' s sensitivity to immune effector cells. This has great significance in the context of the nutritional importance of Zn (II) and its association with optimal immune function. All procedures performed in this study were conducted with a view to minimising the discomfort of the animals, and used the minimum numbers to generate reproducible and statistically significant data. All experiments were approved by the University of Adelaide Animal Ethics Committee (Animal Welfare Assurance number A5491–01; project approval number S-2010–001) and were performed in strict adherence to guidelines dictated by the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. High-level expression and purification was conducted as described previously [47]. PsaA was enriched for Mn (II) by harvesting cells and purification in the presence of 5 mM MnSO4. SDS-PAGE and western blotting was conducted as described previously [28]. Demetallated (apo) PsaA was prepared from Mn (II) -enriched PsaA (50 µM) that was heated to 62. 5°C in the presence of 0. 5 mM EDTA for 5 minutes. The sample was then cooled for 10 min, centrifuged and 18,000 × g for 10 min and the supernatant desalted on a PD10 column (GE Healthcare). The sample was analyzed for Zn (II) and Mn (II) content by boiling 10 µM protein at 95°C for 20 min in 3. 5%, 7% or 14% HNO3. Samples were analyzed on an Agilent 7500cx ICP-MS (Adelaide Microscopy, University of Adelaide). Routine treatment used 3. 5% HNO3 as no differences were found in the quantity of metal released. Apo-PsaA was defined as having less than 10% total metal content on a mol metal/mol PsaA basis. Point mutants were constructed by overlap extension PCR, essentially as described previously [48]. The mutants were confirmed to be in-frame by PCR and sequence analysis using primers flanking each gene in question. The primers used for construction and validation of the mutants are listed in Table S5. Overlap extension products were then cloned, in frame, into the respective restriction sites in pQE30 (Qiagen, Germany). ITC was performed using a VP-ITC unit (Microcal, USA) with apo-PsaA, 4. 5,10,15 or 20 µM, in 20 mM sodium phosphate buffer, pH 6. 5 at 25°C. Samples were centrifuged and degassed prior to analysis. MnSO4 was injected at 3 min intervals with 56 injections of 4 µl with a 10 second injection time. ZnSO4 was injected at 3 min intervals with 28 injections of 10 µl with a 20 second injection time. A stir rate of 307 per min was used in both experiments. Data were analyzed using the Origin 7. 0 software (Microcal) and the parameters (± SEM) determined. The thermal shift assay was based on the Thermfluor assay [49] conducted using 10 µM PsaA in 100 mM MOPS, pH 7. 2,150 mM NaCl, 5× SYPRO Orange (Invitrogen) in the presence of 10 µM to 1000 µM metal, using a Roche LC480 Real-Time Cycler. For further details see Text S3. Frozen stock S. pneumoniae D39 were prepared as described previously [28]. For in vitro growth measurements, frozen stock culture was added to C+Y with 1 µM MnSO4 and supplemented with ratios of metal ions as specified. The starting A600 was 0. 05 for all cultures. For the Zn (II) challenge experiment a frozen stock culture was added to C+Y with 1 µM MnSO4 to a starting A600 of 0. 05 and grown to an A600 of 0. 3. Cells were washed with C+Y, pre-warmed to 37°C, and then reinoculated into C+Y with 1 µM Mn SO4 and supplemented with 100 µM, 300 µM or 1 mM ZnSO4, to an A600 of 0. 2. Cell growth was then monitored at A600. All analyses were carried out in at least biological triplicate. Bacteria prepared in this manner were washed twice, each in 10 volumes of PBS, before western blotting. For ICPMS analyses cells were washed 3 times, 14,000 x g for 10 min, in PBS +5 mM EDTA and then washed 3 times with PBS. The dry cell mass was determined and the material boiled at 95°C for 20 min in 3. 5%, 7% or 14% HNO3. The metal-ion containing supernatant was collected by centrifugation at 14,000 x g for 30 min and metal content determined on an Agilent 7500cx ICPMS (Adelaide Microscopy, University of Adelaide). Routine treatment used 3. 5% HNO3 as no differences were found in the quantity of metal released. Outbred 5–6 week old female CD1 (Swiss) mice were used in these experiments, under the approval of the Animal Ethics Committee of The University of Adelaide. For metal content determination in naïve mice (n = 5), samples of nasopharynx, lungs, blood and brain were harvested as described previously [50], using PBS only (without trypsin or EDTA). For metal content determination in mice challenged with pneumococci (n = 10) were infected intranasally with approx. 107 c. f. u. of S. pneumoniae D39, following the procedures described previously [50]. Mice were sacrificed at 48 hr-post-infection and samples of nasopharynx, lungs, blood and brain of each mouse were harvested and processed as described previously [50], again using PBS only. Tissue samples were analyzed for mass, except blood serum, and boiled in 3. 5% HNO3 for 20 min at 95°C. Blood serum was diluted in 7% HNO3 and boiled for 20 min at 95°C. The metal-ion containing supernatant was collected by centrifugation at 14,000 x g for 30 min and metal content determined on an Agilent 7500cx ICPMS (Adelaide Microscopy, University of Adelaide). The tissue concentrations of metal ions were calculated using the mass or volume of tissue dissolved in HNO3. One-step relative quantitative real time RT-PCR using a Roche LC480 Real-Time Cycler, was performed as described previously [48]. The psaA primers are listed in Table S4 and were used at a final concentration of 200 nM per reaction. 16S rRNA was employed as a control. Amplification data were analyzed using the comparative critical threshold (2−ΔΔCT) method [48]. Manganese-enriched IMAC-purified protein was further purified on a HiLoad 26/60 column. The protein was eluted in 0. 2 M MnSO4,0. 2 M NaCl and 20 mM HEPES/NaOH, pH 7. 5 at 20°C. The peak-containing fractions were analyzed for purity by SDS-PAGE and selected fractions were concentrated to 18 mg. ml−1 using Amicon Ultra-4 centrifugal devices (Millipore). The crystals of the Mn (II) -bound form were optimized by several rounds of microseeding utilizing the nuclei of Mn (II) -bound PsaA crystals obtained by vapour diffusion in hanging drops at 18°C essentially as conducted previously [18]. The seed was introduced into a drop equilibrated overnight and containing 1 µl of protein and 1 µl of reservoir solution containing 33% PEG 1500,0. 15 M SPG buffer pH 4. 0,0. 2 M MnSO4 suspended over 0. 5 ml of reservoir solution. SPG buffer was produced by mixing succinic acid, sodium dihydrogen phosphate, and glycine in the molar ratios 2∶7∶7 and pH adjusted by adding 10 N NaOH. Crystals grew to the maximum size of 200 µm x 200 µm x 100 µm within 7–10 days. For data collection, the crystals were cryoprotected with 20% glycerol before being flash-cooled by rapid immersion in liquid nitrogen. The diffraction data were collected on a single crystal on the MX2 microfocus beamline of the Australian Synchrotron [51] (Melbourne, Australia) operating at 6545 eV (for details and refinement statistics see Table S6). For further details of the data collection and analysis see Text S3. Bacteria were grown to an A600 = 0. 3 in minimal media with or without Zn (II) supplementation, washed 3 times with PBS +2. 5 mM EDTA to remove excess cations and then 3 times with PBS. Cells were incubated for 30 minutes with 60 mM paraquat (Sigma-Aldrich) and then serially diluted and plated on blood-agar. Plates were incubated overnight at 37°C +5% CO2. Survival was calculated as a percentage of colonies at 30 minutes compared to 0 minutes. The PMN killing assay was adapted from [52], and further details are provided in Text S3.

Dietary zinc deficiency is a global health problem affecting almost two billion people. Infectious diseases associated with zinc deficiency include respiratory infections caused by bacteria, and notably, Streptococcus pneumoniae, which is responsible for more than 1 million deaths annually. The association between zinc and immunity is well known, but the mechanism by which zinc provides protection against infectious diseases has remained a mystery. Previously, we found that manganese was essential for S. pneumoniae growth and its ability to cause disease. Intriguingly, we also observed that zinc could bind to the manganese transport protein. Therefore, we sought to determine if zinc could inhibit manganese transport, and to observe what the effects would be on S. pneumoniae. We found that zinc prevented manganese uptake. This slowed bacterial growth and rendered it hypersensitive to immune cell killing. We also observed that, during S. pneumoniae infection in mice, zinc released by the host increased to concentrations that could compete for manganese uptake. Our study provides direct evidence for how zinc is toxic to bacteria by preventing manganese uptake. Furthermore, we show how this could be harnessed by the immune system, thereby providing a scientific basis for the protective effect of zinc against infectious diseases.

lay_plos

Bracoviruses are symbiotic viruses associated with tens of thousands of species of parasitic wasps that develop within the body of lepidopteran hosts and that collectively parasitize caterpillars of virtually every lepidopteran species. Viral particles are produced in the wasp ovaries and injected into host larvae with the wasp eggs. Once in the host body, the viral DNA circles enclosed in the particles integrate into lepidopteran host cell DNA. Here we show that bracovirus DNA sequences have been inserted repeatedly into lepidopteran genomes, indicating this viral DNA can also enter germline cells. The original mode of Horizontal Gene Transfer (HGT) unveiled here is based on the integrative properties of an endogenous virus that has evolved as a gene transfer agent within parasitic wasp genomes for ≈100 million years. Among the bracovirus genes thus transferred, a phylogenetic analysis indicated that those encoding C-type-lectins most likely originated from the wasp gene set, showing that a bracovirus-mediated gene flux exists between the 2 insect orders Hymenoptera and Lepidoptera. Furthermore, the acquisition of bracovirus sequences that can be expressed by Lepidoptera has resulted in the domestication of several genes that could result in adaptive advantages for the host. Indeed, functional analyses suggest that two of the acquired genes could have a protective role against a common pathogen in the field, baculovirus. From these results, we hypothesize that bracovirus-mediated HGT has played an important role in the evolutionary arms race between Lepidoptera and their pathogens. Unlike bacteria, which have obtained a notable proportion of their genes through the acquisition of sequences from distantly related organisms, eukaryotes are generally thought to evolve mainly through the modification of existing genetic information [1]. However evidence of horizontal gene transfer (HGT) in eukaryotes is accumulating and is recognized as an important factor in their evolution and acquisition of novel traits [2–5]. The majority of events reported concerns transposable elements, DNA sequences capable of excising or copying themselves from one genomic locus to integrate into another locus [6]. Genome sequencing has revealed that eukaryotes have also acquired DNA from symbionts and parasites, probably because the intimacy of these relationships favours DNA exchange. For example, numerous insect and nematode genomes contain sequences originating from Wolbachia [7,8] an endocellular bacteria widespread in insect populations infecting, in particular, host germ line cells [9]. Recently, a systematic investigation of HGT events in three available lepidopteran genomes (Bombyx mori, Danaus plexippus and Heliconius melpomene) revealed multiple ancient HGT events from bacteria and fungi to these lepidopteran genomes [10]. Here we present an original mode of HGT between two insect orders based on the integrative properties of a virus (bracovirus) that has evolved within parasitic wasp genomes for ≈100 million years and that is used to facilitate the development of their progeny in caterpillars by inhibiting host immune defenses. In one case, we could demonstrate the direction of the transfer based on the presence of a sequence important for the virus life cycle. This is a rare example where the likely mechanism of HGT can be established in an animal system. Moreover we present functional analyses suggesting that some of the transferred genes have been recycled by Lepidoptera to protect them against a common viral pathogen. Bracoviruses play a central role in parasite-host interactions involving parasitic wasps and their caterpillar hosts. Bracoviruses are injected by parasitic wasps into their hosts along with wasp eggs. These wasps develop during their larval stage within the body of their lepidopteran hosts. Tens of thousands of species of wasps belonging to the braconid family and parasitizing a large diversity of lepidopteran species are each associated with a specific bracovirus [11]. All these associations originated from a single integration event of a nudivirus genome in a common ancestor of the wasps [12]. Since this integration ≈100 MYA, the genes involved in virus particle production have been dispersed in the wasp genome, they are no longer packaged in the particles that contain genes encoding virulence factors. Moreover the endogenous chromosomally transmitted virus has evolved depending on its contribution to parasitism success, resulting in a specific set of virulence genes packaged in the particles in the different wasp lineages [13]. These viruses are now essential for successful development of the wasp larvae within lepidopteran hosts [13–15]. Viral replication and particle production occur exclusively in the wasp ovaries from endogenous viral elements present in the wasp genome. The particles, that contain dsDNA circles harbouring the virulence genes, constitute the major component of the fluid injected with the eggs into the parasitized caterpillar host during wasp oviposition. Once in the host body the particles enter lepidopteran host cells and the host cellular machinery expresses these virulence genes. Viral products ensure wasp larvae survival in the lepidopteran body by interfering with caterpillar host immune responses and development [16,17]. The dsDNA circles packaged in the particles are produced from chromosomally transmitted proviral segments stably integrated in the wasp genome [18–21]. The typical eukaryotic organization of the genes transferred by the particles [22] and their lack of similarity with viral genes suggest they originate from the wasp genome, which could be demonstrated for a few of them by phylogenetic analyses [23]. However many genes have diverged in their sequence from insect genes, to the extent that they are currently no more closely related to wasp genes than to mammalian genes [24,25]. Many other bracovirus genes have unknown origins and display no similarities to genes in data banks except with other bracovirus sequences. For example, Cotesia congregata bracovirus (CcBV) encodes 26 bracovirus specific gene families (named BV1 to BV26) [18]. We previously reported that some viral circles were found to be reintegrated in the genome of different geographic strains of the wasp Cotesia sesamiae [13,26]. The occurrence of circle integrations back into wasp genomes probably reflects a broad integration ability of circles since it was recently shown that integration into the DNA of parasitized lepidopteran host cells is a part of the bracovirus life cycle. Indeed it was shown that Microplitis demolitor bracovirus circle integration into lepidopteran Pseudoplusia includens DNA occurs by a specific mechanism involving a conserved viral site named Host Integration Motif [27]. During integration the circles are opened specifically at this site, resulting in integrated forms readily distinguishable from that of the proviral form [27]. The analysis of Cotesia sesamiae bracovirus (CsBV) reintegrated circles suggests that the same mechanism was involved in their integration back into the wasp genome [13,26]. Parasitized caterpillars represent most of the time an evolutionary dead-end since parasitoid wasps inhibit metamorphosis [28] and the host usually does not survive parasitism [29]. However it is conceivable that some hosts might successfully defend themselves against the parasite by interrupting wasp oviposition, eliminating the eggs or killing the larvae, resulting in the reproduction of Lepidoptera that have been infected by bracoviruses. Parasitoid wasps could also target non-host species and fail to interfere with their development [30]. We can speculate that caterpillar escape from the fatal issue after virus injection could allow bracovirus particle entry into germ cells and in rare cases stable integration of circles into lepidopteran genomes. To determine if bracovirus sequences could indeed be integrated into lepidopteran genomes, we compared the DNA sequences packaged in the particles of Cotesia congregata bracovirus (CcBV), the genome of which is almost completely characterized [18], to a series of genomes from non-host Lepidoptera of the parasitoid wasp C. congregata and from Manduca sexta, a regular host. Here, in contrast to a previous bioinformatic analysis listing a series of bracovirus insertions, most of them relatively short [31], we searched for large nucleotide stretches (more than 500 bp long) that could encode potentially domesticated genes by lepidopteran species and evaluated the evolutionary meaning of these integrations by functional analysis of two of the transferred genes. Similarity searches allowed the identification of bracovirus DNA insertions in the genomes of the monarch (Danaus plexippus), the silkworm (Bombyx mori), the beet armyworm (Spodoptera exigua) and the fall armyworm (Spodoptera frugiperda) but not in the genome of tobacco hornworm (M. sexta), the regular host of Cotesia congregata. All these insertions were characterized by the presence of large stretches of nucleotide sequences strikingly similar to those of bracoviruses (close to 90% identities at the nucleotide level) flanked by lepidopteran-specific sequences. Insertions include genes but also in some cases parts of bracovirus circles, the organization of which has been conserved, indicating the direction of HGT was from bracovirus to Lepidoptera. Moreover, in one insertion a regulatory signal involved in dsDNA circle production in the wasp has been retained, constituting an unambiguous signature of the bracoviral origin of the sequence since bracovirus replication is non autonomous and occurs exclusively in the wasp ovaries. Altogether our data indicate that bracoviruses have been a recurrent source of genes for Lepidoptera. Moreover functional analyses provide convergent results suggesting that some of these genes might contribute to protect larvae against baculoviruses, deadly pathogens that threaten them in the field, which would explain why they have been maintained in lepidopteran genomes. Unexpected levels of similarities were observed between sequences of several lepidopteran genomes and bracoviruses. The level of similarity is in the range of that found for homologous genes coding for highly conserved proteins such as histone H4, almost invariant from plants to animals. However this similarity is unlikely to be due to conservative selection since the encoded genes are conserved only in a limited number of phylogenetically closely related lepidopteran species. In this study we report the presence of these bracovirus-related sequences in several lepidopteran genomes and discuss the possible mechanisms involved in their acquisition. Compared to a previous report describing bracovirus DNA insertions in the monarch and silkworm genomes [31] we provide here an in depth analysis of the structure of the bracoviral and lepidopteran flanking sequences. We show that monarch insertions are fixed in the species, that their presence in the lineage is ancient and that they have undergone rearrangements since their integration. By measuring selection pressures using genomes from individuals of 80 monarch and 8 related species we show that the selection acting on these genes is mainly conservative, which suggests the domesticated Ben genes could play a role in monarch physiology. In addition we report for the first time HGT and domestication of bracovirus sequences in Lepidoptera of the Spodoptera lineage. Moreover we present functional analysis on 2 unrelated genes suggesting the transferred genes could protect the Lepidoptera against a viral pathogen. High similarities observed could be due in theory either to DNA sequence transfer from bracovirus to lepidopteran genomes or vice versa. Wasp larvae containing a bracovirus as an endogenous virus have an intimate relationship with Lepidoptera since they develop within the body of their hosts, for this reason it is possible that acquisition of lepidopteran genes by bracoviruses can occur. Accordingly horizontal transfer of a Mariner like transposable element (MLE) shared by a parasitoid wasp and its host was previously reported. In this case, the direction of the transfer was supposed to be from Lepidoptera to Hymenoptera based on the presence of this transposon in closely related species of the lepidopteran host and its absence in a closely related parasitoid species [46]. Another horizontal transfer of a transposable element (Helitron) was reported between Copidosoma floridanum an endoparasitoid wasp (not associated with a bracovirus) and the Lepidoptera Trichoplusia ni suggesting that parasitism might favor horizontal transfer of TEs [47] but the direction of the transfer was not determined in that study. Similarly, Thomas et al., (2010) also found evidence of horizontal transfer of Helitrons in bracoviruses and Bombyx mori [48]. One of the insertions described here is particularly informative regarding the direction of the transfer because it contains a regulatory sequence typical of bracoviruses (see Fig 5). The sequences named Direct Repeat Junction (DRJ) that terminate all bracovirus proviral segments are conserved among BVs [18]. These direct repeats are involved in dsDNA circle production [49]. During viral replication, large molecules are amplified that serve as precursors for the production of individual circles, produced by a recombination between the DRJs [19]. As a result, a single DRJ (resulting from the recombination) is present on a circle. This recombination process was confirmed recently by inactivation of two Tyrosine recombinase genes (vlf1 and int-1) using RNA interference, which resulted in impairment of circle formation [50]. The presence of a DRJ in the BV2-5 insertion in the S. exigua genome constitutes an unambiguous signature of its bracoviral origin since these regulatory elements are specific of the bracovirus life cycle. This clearly demonstrates that the BV2-5 sequence originated from the bracovirus and was acquired by the lepidopteran genome. The direction of the other horizontal transfers, although not as clearly proven, also appears to be more likely from bracovirus to Lepidoptera genomes, because only a limited number of closely related lepidopteran species harbour these sequences. Moreover bracovirus life cycle features suggest they are involved in horizontal transfer. Indeed bracovirus circles have been shown to enter cells of all tissues tested [51,52] and to integrate into the DNA of lepidopteran host cells as a part of the wasp life cycle [27]. Several components of the virus particles belong to the integrase family (VLF1, INT1, INT2) [53] and thus potentially mediate integration. During parasitism, bracoviruses do not replicate in host tissues and therefore integration into host cell DNA may allow persistence of bracovirus DNA in lepidopteran larvae that continue to develop [27]. It was previously shown that a side effect of this integration mechanism was to allow circle integration events back into germline cells of the wasp [26]. This was indicated by the analysis of bracovirus sequences in Cotesia sesamiae genome. Strikingly, segments homologous to CcBV circle 10 were found in two different genomic locations in C. sesamiae strains of Kenya [26]. Sequence comparison of circular and reintegrated viral forms [13,26] indicated that circle integration likely involved the same mechanism as the one described for the integration of bracovirus circles into lepidopteran host genomic DNA during parasitism, using specific sites on the circle (the Host Integration Motifs) [27]. The occurrence of circle integration into lepidopteran host germline DNA resulting in sequence transfer between bracovirus and Lepidoptera is likely another consequence of this viral integration mechanism. Although we did not find integration of complete circles such as those described in the wasp genome, the largest Ben9 encoding region in Danaus plexippus corresponds to more than half of C25 sequence and has retained in this Lepidoptera the bracovirus organisation with two genes (RnaseT2 and Ben9) separated by non-coding sequences [18]. We hypothesize that bracovirus insertions correspond to remnants of circles integrated in Lepidoptera genomes that have been subject to many rearrangements since their integration. Indeed it is likely that after circle integration bracovirus sequences are lost, unless they provide a selective advantage to the insect. Therefore, identification of complete circles in genomes, corresponding to recently integrated sequences, not fixed in the species, might require more diverse template sources than the very limited number of individuals used for lepidopteran genome sequencing. The insertions described in this paper are most probably all ancient. For example, Ben9 was already present in the common ancestor of the Danaina subtribe 5 MYA [35]. Moreover evidence that rearrangements have occurred is provided by the comparison of the two Ben9 gene containing regions, one having conserved a larger part of the bracovirus non-coding sequence than the other. The insertion in the B. mori genome has also been obviously rearranged since a stretch of lepidopteran specific DNA separates bracovirus sequences in two parts. BV2-5, Se-BLL2 and SF2. 5 insertions in Spodoptera spp correspond mostly to single genes, which could represent an ultimate stage of domestication, most of the sequence of the circle having been lost. It is also possible that a broader mechanism than virus-mediated integration, such as DNA repair, which is involved for example in transgenic mice production [54], might have resulted in the insertion of fragments of bracovirus circles in Lepidoptera. However it should be noted that in all cases described in this study the insertions correspond exclusively to sequences from bracovirus circles: we did not find any stretch of wasp sequence flanking or separating bracovirus DNA sequences in the lepidopteran genomes. Thus although integration of wasp DNA could be possible in theory, given that the wasp larvae develop within lepidopteran hosts, we did not search for, nor find evidence of wasp DNA (non-viral) integration in this study. The presence of bracovirus sequences in lepidopteran genomes is apparently a paradox given that infected larvae are considered as an evolutionary dead-end (see Fig 10). For example, CcBV has been shown to induce alteration of host developmental programming resulting in inhibition of metamorphosis, even when experimentally injected in a lower amount than during wasp oviposition [28]. Accordingly, we found no evidence for HGT of CcBV genes in M. sexta, a common host of C. congregata but instead genes having similarities with other polyDNAviruses [33]. Some host species might be less susceptible to the effect of bracoviruses on development or could have developed resistance mechanisms, and therefore “live to tell the tale” after parasitism and injection of particles (Fig 10). However we propose that the main route of bracovirus gene acquisitions by Lepidoptera could be through parasitoid wasp stinging of non-host species (Fig 10). In the field, the host range of the wasp C. congregata corresponds to several species of sphingidae, but in laboratory conditions it was shown to oviposit in non-host species such as the noctuidae Trichoplusia ni [30]. Such behaviour might offer the opportunity for bracovirus DNA to integrate into genomes of lepidopteran lineages that do not belong to the host range of bracovirus-associated wasps (such as species of the monarch lineage for example) and to “escape” bracovirus induced host development arrest. In this context the cellular machinery of Lepidoptera appear to be sufficiently conserved to express a bracovirus gene normally adapted for expression in a different lepidopteran family. Indeed, the conservation of Ben9 and BV2-5 intron splicing in two different lepidopteran families (sphingid/nymphalids and sphingid/noctuids respectively, Fig 1, Fig 2), illustrates that these genes can be “ready to be expressed” even in non-target species. Although not recorded in the field to our knowledge, oviposition in non-hosts may happen since parasitoids, such as Cotesia species, attacking aggressive caterpillars do not have the time to intensively examine the potential host before oviposition. In rearing conditions, they sometimes lay their eggs into other adult wasps, which shows the lack of specificity in their choice [55]. Conservation of bracovirus genes in lepidopteran genomes is likely associated with an increase in insect fitness due to the expression of the viral genes. This hypothesis is sustained by functional studies with the SeBLL2 and BV2-5 proteins from S. exigua showing they have an impact on baculovirus infection. These results suggest that host domestication of these bracoviral genes might increase insect protection to this natural pathogen playing a role in regulating population dynamics in the field [56,57]. We have found that the interference of recombinant BV2-5 with the cellular cytoskeleton dynamics has a strong impact on the baculovirus producing this protein suggesting BV2-5 could confer larval protection against baculovirus infection. This hypothesis is corroborated by the fact that an S. exigua BV2-5 bearing strain is less susceptible to baculovirus infection than the European population bearing the BV2-5 truncated form (S6 Fig). However the genetic background between the two strains is probably different and other approaches such as the use of CRISPR/Cas9 technology to produce S. exigua lines by knocking out of BV2-5 or restoring the functional BV2-5 will be required to unambiguously demonstrate the protective function of this protein, after baculovirus infection. The alteration of a fundamental cellular component such as cytoskeleton dynamics probably also induces a cost. S. exigua is a Palearctic species, which was introduced in America in 1876 probably from Europe [58]. The fact that Lepidoptera now collected in Europe encode a truncated form of BV2-5 suggests that a recent mutation has spread in this population. It is tempting to speculate that BV2-5-mediated baculovirus protection might induce a cost leading for example to increased susceptibility to other pathogens such as bacteria or parasitoids. The frequency of one or the other form of BV2-5 might depend on the abundance and local selective pressure exerted by pathogens and/or parasites and the cost might also explain why BV2-5 has been lost in S. frugiperda while it was detected in S. litura a more recent species in the Spodoptera lineage. In any case BV2-5 coding sequence is more conserved than the other part of the bracovirus insertion suggesting the gene as Ben genes in the monarch is generally under conservative selection and not neutrally transmitted. In addition to BV2-5, we have observed that another gene of bracovirus origin Se-BLL2 can also confer certain level of protection in experimental conditions against both viral forms of baculovirus, occluded derived virions (responsible of the primary infection) and budded viruses (responsible of the systemic infection of larvae). C-type lectins are carbohydrate-binding proteins playing a range of functions in multiple organisms [59]. In general, PDV lectins are able to specifically recognize carbohydrates on the surface of the endoparasitoid eggs and, thus, inhibit the recognition of the eggs by the lepidopteran host recognition proteins [60]. Although little is known about the response of C-type lectins to viral invaders, crustacean lectins have been reported to be related to the antiviral defense [61,62]. In lepidopterans, the only example of antiviral response involving C-type lectins was reported by Chai et al. [63]. Our experiments have shown that BLL2 action is interfering with the initial viral entrance into the Sf21 cells (Fig 8B and 8C). According to these results, it is likely that antiviral action of Se-BLL2 is due to its interaction with viral or host cell membrane glycoproteins involved in viral binding and entrance. Nevertheless, additional studies will be needed to define the exact mode of action of BLL proteins as well as their possible role in the host interaction with viral and non-viral pathogens and parasitoids. In any case it should be noted that the acquired genes do not confer a complete protection against baculovirus infection and our study confirm that S. exigua larvae are indeed susceptible to baculovirus infection (Fig 9B). According to the literature the susceptibility of Spodoptera spp depend on many factors such as the larval stage [64], the type of plant hosting the insects [65], the geographical origin of the insects, and even on the midgut microbiota composition [66]. Many individuals ingesting a sublethal dose of OBs can survive with a covert infection (larvae harbouring baculovirus but not displaying the disease symptoms) the incidence of which can be over 50% in the field for Spodoptera exigua [56]. Little is known on the molecular aspects of this phenomenon but BV2-5 effect on cytoskeleton dynamics could possibly contribute to this latency. Taken together a large number of factors can modulate insect susceptibility and given the high incidence of baculovirus infection in the field being even only less susceptible can have a great impact at the population fitness. In the context of a host-pathogen arms race any new trait that confers an advantage to any of the competitors is susceptible to be incorporated into the gene pool. Altogether our results strongly suggest that two acquired genes can confer an advantage against viral infection although the comprehensive analysis of the molecular function of the identified proteins is awaited and we cannot completely exclude at this stage that they could have other functions. Ben genes also probably have a role for the Lepidoptera since they have been maintained in different lineages and we have shown that in the monarch they are mostly under conservative selection. We have described in this report several insertions of bracovirus DNA sequences in a series of lepidopteran genomes. In mammals a few examples have been described of integrated retrovirus receptor genes conferring a specific protection against new infections by related viruses using the same cell entry mechanism [67,68]. Recently, this concept of genes acquired and domesticated by hosts to protect against related virus infections has been shown to operate also for a Bornavirus (negative strand RNA virus) [69]. Virus resistance conferred by expression of viral genes in plants has also been described. Indeed, transgenic plants expressing viral gene constructs can exhibit resistance to infection by the virus [70,71]. Here, we extend this concept of an organism using pathogen genetic resources as a protection against other pathogens, to insects. Indeed, we show that domestication of different bracovirus genes most likely confers protection to Lepidoptera against baculoviruses, a common pathogen in the field. What is very original compared to previous reported cases is the use of viral sequences as a protection against a distantly related virus. Indeed, most of the viral sequences inserted into host genomes that were hypothesized to confer a protection are effective against closely related viruses. The protection mechanisms are based on the expression of defective proteins of viral origin that are able to interact with those of the pathogenic virus and thus interfere with cell entry [72], replication [73] or interfere by producing small RNAs inducing destruction of virus transcripts having highly similar sequences [74,75]. Since baculovirus infection of the host could be lethal for the parasitoid [76], it might be speculated that the function of some of the bracoviral genes domesticated by Lepidoptera was already to protect the parasitized larvae against baculovirus infection. This might provide an explanation for both the unusual ability to interfere with distantly related virus infections and the fact that the bracovirus genes have conserved the same structure after their integration into Lepidoptera genomes. A specific bracovirus circle integration mechanism into lepidopteran host DNA operating during parasitism and resulting occasionally in circle reintegration into wasp genome has been previously characterized [27]. This mechanism is likely involved in HGTs between Hymenoptera and Lepidoptera, although it is also possible that some of the sequences might have been integrated through DNA repair. Once integrated into lepidopteran genomes, bracovirus genes are readily domesticated by Lepidoptera since they are already adapted for expression in lepidopteran tissues during parasitism. Indeed the majority of the CcBV genes expressed during parasitism were shown to possess an insect structure with an arthropod transcription start site, at least one intron and polyadenylation signals [22] and we showed here that the splicing machinery of different Lepidoptera families can produce the same mRNAs from a bracovirus gene containing introns. Altogether the ability of bracoviruses to mediate integration, the fact that bracovirus gene structure is adapted to expression in Lepidoptera and that bracovirus circles have acquired different gene sets depending on the wasp lineage suggest we are only seeing the tip of the iceberg and that numerous cases of integration and domestication of bracovirus sequences will be identified with the exponential rise of genomic data provided by new generation sequencing. Thus this phenomenon is not merely a curiosity but has most likely played an important role in the arms race between Lepidoptera and their pathogens. Sequences sharing high similarity with Bracovirus sequences in lepidopteran genomes were identified using the 35 CcBV circles [18] as queries in megablast analysis (NCBI) against whole genome shotgun contig data banks (wgs at NCBI) restricted to lepidopteran genome sequences. Unlike the bioinformatic study which recently reported numerous short insertions of bracovirus sequences in lepidopteran genomes [31] we focused here on bracovirus-like sequences more than 1 kb long and encoding at least one gene. A blast analysis between proviral integrated circle sequences and the different contigs identified was used to determine the precise location of high scoring pairs (HSP) reported in Fig 1. HSP and annotated sequences were visualised using DNAPlotter [77]. Homologous transcribed sequences were then searched for in the Transcriptome Shotgun Assembly (TSA) database at NCBI, for Spodoptera and Bombyx sequences (no data was found in TSA for Danaus plexippus). It should be noted that although all the identified insertions are very closely related to CcBV since the donor circle can be identified in many cases, this does not necessarily indicate that C. congregata was the donor species since only a handful of bracovirus packaged genomes have been sequenced to date among those associated with the estimated 1200 species of the Cotesia genus [78]. Specimen of Danaina subtribe were kindly provided by David Smith. They were collected randomly in the field in the late 1990s, killed in ethyl ethanoate vapour immediately before storage in 95% ethanol [34]. They were stored for ≈6 years at -20°C then for 9 years at room temperature (D. Smith, personal communication). Adult D. plexippus were collected in Australia (voucher number, vn: 396), D. genutia in Thailand (vn: 430), D. chrysippus chrysippus in Oman (vn: 262), T. septentrionis septentrionis in Malay Peninsula (vn: 216). More recently, D. plexippus plexippus caterpillars were sampled in August 2012 in Valcartier, Canada (46°56’52”N, 71°29’50”W). Four colonies of S. exigua, derived from different geographic locations, were continuously reared on artificial diet at 25± 3°C with 70±5% relative humidity and a photoperiod of 16h light: 8h dark. The FRA strain was supplied by M. Lopez-Ferber, (INRA St. Christol les Alés, France) [79]. The ALM strain was established from successive collections from southern Spain [80]. XEN-R strain was obtained from cotton fields in Pattville, AL. (USA) and was later selected for resistance to Bacillus thuringiensis [81,82]. SUI population was provided by Andermatt Biocontrol AG (Grossdietwil, Switzerland). The DNA from the Mexican population was provided by P. Caballero (Universidad Publica de Navarra). Finally, DNA representing S. exigua from Japan was obtained from the cell line Se301 originally derived from insects collected in Japan (Hara, et al. 1995). After grinding frozen samples in liquid nitrogen, DNAs from D. plexippus larvae were extracted by C. Béliveau using QiaAMP DNA mini kit (Qiagen) and were sent by M. Cusson (Québec, Canada). For specimens of Danaina subtribe, 20 mg of tissues were dried at 37°C to eliminate ethanol, frozen in liquid nitrogen and ground with a pellet and a mortar previously refrigerated at -80°C. DNA was then extracted using the QiAmp DNA Mini Kit (Qiagen) following the supplier’s instructions. To compensate for partial degradation of DNA from old samples, primers were designed for amplification of short fragments. A 35-cycle PCR (94°C for 60 s; 50°C for 60 s; 72°C for 60 s) was performed with 10 pmol of each primer, 0. 2 mM dNTP (MP Biochemicals), 1. 5 mM MgCl2,0. 5 unit Goldstar (Eurogentec) and 20 ng of genomic DNA. PCR products (8 μl) were run on 1. 5% agarose gels. The EF1α gene was used as a control of DNA sample quality. Successful amplifications of Ben9 insertions (Fig 2) were obtained: for D. plexippus using primers Ben9 12F and Ben9 12R (176 bp fragment, S1 Table); for D. chrysippus and D. genutia using primers Ben9 13F and Ben9 13R (173 bp fragment); for and T. septentrionis using Ben9 14F and Ben9 14R (123 bp fragment); amplifications of Ben4 insertion (S1 Fig) was obtained using Ben4 2F and Ben4 2R (223 bp fragment). It should be noted that the PCR analyses did not allow us to discriminate between the two Ben9 insertions (Fig 1 (A) and 1 (B) ). Total RNA was isolated from 5th instar S. exigua larvae using RNAzol reagent (Molecular research centre, INC) as described in the manufacturer’s protocol. One μg of each RNA was DNase treated (Invitrogen) and reverse transcribed into cDNA with oligo (dT) and hexamer primers using Super Script II Reverse Transcriptase from Invitrogen. D. plexippus RNA was prepared using TRIzol reagent (Invitrogen) and sent by M. Cusson (Québec, Canada), it was further treated by rDNase (NucleoSpin RNA, Macherey-Nagel) to eliminate residual DNA until no PCR amplification of a control gene could be detected from the sample. For D. plexippus, a total of 1μg of RNA was reverse transcribed into cDNA with oligo (dT) primers using Super Script II reverse transcriptase or Omniscript RT kit (Qiagen). PCR amplifications from cDNA of the different genes were performed using standard protocols and specific primers (S1 Table). For the BV2-5 alleles, initial sequences were obtained from the transcriptome of S. exigua larvae exposed to different types of pathogens [40]. Two primers flanking the coding sequence were designed and used to amplify this sequence from cDNAs originating from different populations and cells. RT-PCR-amplified alleles of BV2-5 were directly sequenced or cloned into pGEM-T Easy vector (Promega) and sequenced using standard primers. At least two independent clones were sequenced for each insect population. Selection pressures operating on D. plexippus Ben4 and Ben9 genes were measured using Illumina data available at NCBI (SRA data bank) and corresponding to 80 individuals from different wild populations and 8 individuals from other Danaus species (D. erippus, D. chrysippus, D. eresimus, D. gilippus) [30] in order to identify molecular signatures that might be associated with particular selection pressures. The reads from D. plexippus samples were mapped onto the reference genome of D. plexippus [30] with Bowtie2 (2. 2. 4) [83] using default parameters. The Ben regions were then extracted with Samtools [84]. A consensus sequence was obtained for each sample (minimum coverage of 5 reads and minimum frequency of a variant for the individual to be considered as heterozygote = 0. 25). Sequences with a missing base ratio above 50% due to heterogeneity in the sequencing were discarded. Reads corresponding to the two Ben9 copies could not be separated due to high similarity, therefore Ben9 was analyzed as a single gene. We performed de novo assembly of the reads from the other species using Velvet 1. 2. 07 [85] and a k-mer length of 31 and the Ben genes were identified by BLAST. All the sequences were aligned using MAFFT [86], the sequences with a STOP codon were corrected in order to use them for the selection analyses. The global dN/dS (ω) ratios was measured using two different methods, AnalyseCodonData implemented in HyPhy [87] and Codeml from PAML [88] with a means ω ratio for all branches (model = 0) and one ω value for all sites (NSsites = 0). Then, the ω ratio was measured for each site along the alignment, in order to identify regions with a particular selection signature, using the SLAC method implemented in HyPhy (significance thresold: p-value ≤ 0. 1) and Codeml from PAML [88] with a means ω ratio for all branches (model = 0) and selection model (NSsites = 2), which classify the sites into three classes (ω = 0 neutral, 0<ω< 1 under negative selection and ω>1 under positive selection). These analyses were performed using the phylogeny based on genome wide SNP data [30]. The phylogenetic tree shown in Fig 3 was built based on the common region of Ben4 and Ben9 using PHYML and substitution model HKY85 and 1000 bootstraps. A universal genome walking kit (Clontech) was used for the sequencing of the whole integrated BV2-5 and Se-BLL2 contigs. For this purpose, three S. exigua genomic DNA libraries [89] were subjected to primary and secondary PCRs using the general primers provided by the kit and specific primers designed to amplify 5’ and 3’ flanking regions of the BV2-5 and Se-BLL2 open reading frames (ORFs) (S1 Table). The amplified fragments were purified, cloned into the pGEM-T Easy vector and sequenced. The presence and abundance of mRNA of Se-BV2-5 and Se-BLL2 in different larval tissues were analyzed by quantitative reverse transcription PCR (qPCR). Briefly, total RNAs from fat body, midgut and hemocytes were isolated from untreated 5th instar larvae using the RNAzol reagent (Molecular research center, INC) as described in the manufacturer’s protocol. A total of 1 μg RNA was reverse transcribed into cDNA with oligo- (dT) primer using SuperScript II reverse transcriptase (Invitrogen). cDNAs were used to determine the level of transcripts for each gene by qPCR. Reactions were carried out using an ABI Prism 7700 thermocycler from Applied Biosystems. SYBR green Ex Taq master mix (Clontech) was employed in a total volume of 20 μl. Specific primers for each gene were designed by Primer Express Software (Applied Biosystems) (S1 Table). For each gene, at least three biological replicates were employed. Data are presented as fold change using the method of 2-ΔΔCt and normalized to the internal control gene, ATP synthase. The effect of baculovirus infection on the expression pattern of the lectins in the larval midgut was determined. Third instar larvae were orally infected with S. exigua nucleopolyhedrovirus (SeMNPV). Each larva was fed with 104 occlusion bodies (OBs). Midguts of treated and untreated larvae were collected 72 h after treatment [66]. Total RNA from the treated and control larvae were collected as described above. cDNA was reverse-transcribed and the presence and abundance of the mRNA was determined using qPCR as described above. The full ORF of the two main allelic forms of BV2-5 (complete and truncated) were amplified by PCR from cDNA obtained from S. exigua FRA and Xen-R larvae, respectively. They were cloned into pFBD-pH vector, downstream of the p10 promoter to generate pFBD-pH-BV2-5 (for the complete form) and pFBD-pH-BV2-5t (for the truncated form) vectors. pFBD-pH refers to the dual vector pFBD (Clontech) containing the AcMNPV polyhedrin gene downstream of the PH promoter. In order to generate recombinant baculoviruses, Escherichia coli strain DH10Bac that contains the AcMNPV ΔCC bacmid [90] and the pMON7124 helper plasmid [91] was transformed with pFBD-pH-BV2-5, pFBD-pH-BV2-5t, or pFBD-pH plasmids according to a standard procedure described for the Bac-to-Bac system (Invitrogen). Recombinant bacmids were selected based on white-blue screening of DH10Bac colonies and the positive clones were confirmed by PCR. Bacmid DNAs were isolated from bacterial cells according to standard procedure and used to transfect S. frugiperda ovary-derived cell line Sf21 using Insect Gene Juice Transfection Reagent (Novagen). Four to six days post transfection, the recombinant ΔCC-pH, ΔCC-pH-BV2-5 and ΔCC-pH-BV2-5t bacmid-derived viruses were collected and multiplied to produce high-titer stocks for further experiments. Another type of construct was generated to study the cellular localization of BV2-5 by expressing this protein fused to GFP. Primers containing BglII and EcoRI were designed to amplify the BV2-5 gene from the pFBD-pH_BV2-5. The obtained fragment was sub-cloned into pGEM-T Easy, double digested with BglII and EcoRI, and cloned in p166AcV5-Se8-GFP [92] in order to obtain the fusion gene BV2-5_GFP in the plasmid p166AcV5-Se8-BV2-5GFP. Subsequently, the GFP gene and the recombinant BV2-5GFP gene were amplified using specific primers that contained the NotI and PstI restriction sites (Forward BV2-5GFP: 5’ TTGCGGCCGCATGTTGCCTATTACC3’; Forward GFP: 5’ CTGCGGCCGCATGGGCAAAGGAGAAGAACTTT3’; Reverse: 5’AGCTGCAGTTACGACCAGCCGCCGCTGGCATCT3’). Both genes were cloned under the ph promoter of pFBD to generate pFBD-GFP and pFBD-BV2-5GFP, which were used to transpose into the AcMNPV bacmid as previously described (S4 Fig). Cellular localization of the BV2-5 protein was determined using the recombinant baculovirus expressing BV2-5 fused to GFP (AcMNPV-BV2-5GFP). Previously to the confocal analysis, Sf21 cells were maintained in Grace’s medium (Invitrogen) supplemented with 10% fetal bovine serum and 0. 5% penicillin/streptomycin at 27°C. A first set of cells was infected with AcMNPV-BV2-5 GFP and a second set with AcMNPV-GFP at a multiplicity of infection (MOI) of 10. A third set of cells was infected with AcMNPV-GFP and treated with 5 μM latrunculin A (Sigma Aldrich) 12 hours post infection (hpi). A fourth group of cells was maintained without any treatment as a negative control. Seventy-two hpi, cells were pelleted by centrifugation for 2 min at 3000xg and fixed with 4% paraformaldehyde (PFA) for 20 min. Then, the cells were washed twice with PBS and permeabilized for 10 min with 0. 2% Triton X-100 in PBS-BSA 10%. After another step of PBS washing, cellular actin was stained overnight at 4°C with phalloidin-tetramethylrhodamine B isocyanate TRITC (Sigma Aldrich). Finally, the cells were washed, stained with DAPI (4’, 6’-diamidino-2-phenylindole) to visualize the nucleus of cells and fixed by dakocytomation fluorescent mounting medium (Dakocytomation). Mounted cells were observed under confocal microscope (FV1000, OLYMPUS). Effect of BV2-5 on baculovirus multiplication in cell culture was determined by a one-step growth curve assay. Sf21 cells were infected with the different recombinant baculoviruses at an MOI of 2. After infection, cells were washed and incubated in fresh medium. At different time points, an aliquot of medium was harvested and the viral titer (amount of budded viruses) in each sample was determined by qPCR. For that purpose, viral DNAs were extracted using Prepman reagent (Applied Biosystems) following the manufacturer protocol and were quantified by comparing the obtained Ct values against a standard curve of known viral concentration. Three independent replicates were performed for each sample. Recombinant Se-BLL2 was expressed and produced in an Escherichia coli expression system and purified with affinity chromatography using the HiTrap Chelating HP column (GE Healthcare). In order to test the effect of BLL2 on baculovirus infection, the purified protein was added in different concentrations (50 μg/mL, 10 μg/mL, and 1μg/mL) to AcMNPV-GFP virions in presence of 10mM CaCl2 and the mixture was incubated for 2 hours. After that, the mixture of virus-lectin was then used to infect Sf21 cells (MOI of 0. 5). Similarly, other sets of cells were infected with AcMNPV-GFP or AcMNPV-GFP incubated with 10 mM CaCl2 as controls. Thirty-six hours post infection, percentage of cells showing GFP was determined for the different treatments in order to compare the virus entry to cells. In addition, an aliquot of the medium was harvested at different time points, and the virus titer was determined for each sample and time point by qPCR as described above. S. exigua third instar larvae were infected with Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) by the drop-feeding method. Occlusion bodies (OBs) (5x105) from SeMNPV were added to a solution containing sucrose and phenol red colorant (10% and 0. 05%, respectively) in presence or absence of purified Se-BLL2 (0. 15mg/mL). The larvae were allowed to drink from the virus and control solution in Petri dishes and then transferred individually to the assay plates. Mortality was then recorded every 12 h until death or pupation of all the larvae. Sixteen larvae were used for each treatment and three independent replicates were performed. Mortality was expressed as the percentage of dead larvae. The time to death was assessed by comparing the mortality curves using the Kaplan Meier method (GraphPad Prism 5). The statistical significance was determined using the log-rank analysis (Mantel-cox test). Insect bioassays comparing susceptibility to SeMNPV in two different populations (SUI and MEX) of S. exigua were performed as described above at a final dose of 103 OBs/larva. The putative ORFs were determined with the EditSeq program from DNASTAR and the homologs in other insect species were obtained using BLAST comparison at NCBI (http: //www. ncbi. nlm. nhi. gov), Silkbase (http: //silkworm. genomics. org. cn), Manduca sexta genome project (http: //agripestbase. org/manduca) and LepidoDB (http: //www6. inra. fr/lepidodb). The predicted amino acid sequences were aligned using the ClustalX software [93] and visualized in GenDoc program [94]. Evolutionary distance was calculated for aligned sequences by Maximum-likelihood method and the phylogenetic trees were conducted with the MEGA5 program [95]. Reliability of an inferred tree was determined using bootstrap test (1000 replicates). For a clearer view of the branches, bootstrap values are reported over 100. For the Lectins comparison, the names and accession numbers of proteins compared were as follows. Sf lectin 3–1 (Sf1H08856-3-1) and Sf lectin 5–1 (Sf2H07501-5-1): Spodoptera frugiperda proteins obtained from Spodobase (http: //bioweb. ensam. inra. fr/spodobase); littoralis_C2971: S. littoralis lectin-like protein; Sl_lectin: lectin-like protein from S. litura; Se-BLL1-6: S. exigua bracovirus-like lectins (KP406769-74). CsMBV CTL CrPDV HP, CvBV L, CpPDV lectin, CrBV lectin, CcV3, CvBV 2L: C-type-lectins from bracoviruses of Cotesia species, (AGO14401. 1), (BAC55179. 1), (AEE09593. 1), (AAS10157. 1), (AAO74641. 1), (CCQ71085. 1), (AEE09562. 1), Gi-CTLD, Gi-LRP, Gf CTLD2, Gf CTLD3, Gf CTLD4: C-type-lectins from bracoviruses of Glyptapanteles species (ABK56997. 1), (ABK56993. 1), (ACE75074. 1), (ACE75072. 1), (ACE75071. 1). Nv HLPB, Mr LBP, Mr HLBP1, Mr HLPB, Mr_HLBP C-type-lectins from Hymenoptera, Nasonia vitripennis, Megachile rotundata Megachile rotundata, Microplitis demolitor (XP_001599898. 2), (XP_003708137. 1), (XP_003701025. 1), (XP_003706756. 1), (XP_003704952. 1) (XP_008555202. 1), Se-LL1,2 and 3: Spodoptera exigua lepidopteran-like lectins 1,2 and 3 (KP406775-77), Bm CTL19: B. mori C-type lectin 19 (NP_001165396. 1), Bm CTL21: B. mori C-type lectin 21 (NP_001037056. 1), Ms IML4: Manduca sexta immunolectin 4 (AAV41237. 2), Ms_IML 4 2: M. sexta immunolectin 3 (AAV41236. 1), Ms IML2: M. sexta immunolectin 2 (AAF91316. 3), Ha CTL8: Helicoverpa armigera C-type lectin 8 (AFI47453. 1), Ha LCT6: H. armigera C-type lectin 6 (AFI47451. 1), Ha CL2: H. armigera C-type lectin 2 (ACI32834. 1), Of_IML: Ostrinia furnacalis immunolectin (ABZ81710. 1), Lo IML1: Lonomia oblique immunolectin 1 (AAV91436. 1), Lo L3: Lonomia oblique lectin 3 (AAV91450. 1), Ap_CTL: Antheraea pernyi C-type lectin (AGN70857. 1), Mc_CTL: Mamestra configurata C-type lectin (AEA76325. 1), Pr_CTL: Pieris rapae C-type lectin (AEO52696. 1), As_CTL: Anopheles stephensi C-type lectin galactose binding (ACP43727. 1), Ae_CTL: Aedis aegypti C-type lectin (ABF18196. 1), Md_UP: Musca domestica uncharacterized protein LOC101901048 (XP_005189940. 1), Dv_GJ: Drosophila virilis GJ17272 (XP_002051932. 1), Dm BCTL: Drosophila melanogaster C-type lectin 27kD, isoform B (NP_001260046. 1), De_GG: Drosophila erecta GG24353 (XP_001968708. 1), Dm CTL: Drosophila melanogaster C-type lectin 27kD, isoform A (NP_608858. 3), Dy_GE: Drosophila yakuba GE14680 (XP_002087961. 1), Dw_GK: Drosophila willistoni GK23915 (XP_002064562,1), Dmoj GI: Drosophila mojavensis GI15343 (XP_002001743,1). For the BV2-5 comparision, the names and accession numbers are: S. exigua_BV2-5: S. exigua BV2-5 (KP406767); S. littoralis BV2-5: S. littoralis BV2-5; S. litura BV2: S. litura BV2-5 (GBBY01010418. 1); CcBV_BV2-5: C. congregata bracovirus hypothetical protein 3 segment 25 (CCQ71080. 1); ; GI_HP1 and GI_HP2: Glyptapanteles indiensis hypothetical proteins L1_00460 and L1_00290 (ABK57032. 1 and ABK57015. 1); GF_CHP1 and GF_CHP2: Glyptapanteles flavicoxis hypothetical proteins (ACE75094. 1 and ACE75115. 1). S. frugiperda genomic bacs at NCBI, Genbank acc: FP340419. 1 and FP340412. 1 for BV2-5 and Se-BLL2, respectively.

Eukaryotes are generally thought to evolve mainly through the modification of existing genetic information. However, evidence of horizontal gene transfer (HGT) in eukaryotes-the accidental acquisition of a novel gene from another species, allowing acquisition of novel traits-is now recognized as an important factor in their evolution. We show here that in several lineages, lepidopteran genomes have acquired genes from a bracovirus that is symbiotically used by parasitic wasps to inhibit caterpillar host immune defences. Integration of parts of the viral genome into host caterpillar DNA strongly suggests that integration can sporadically occur in the germline, leading to the production of lepidopteran lineages that harbor bracovirus sequences. Moreover, some of the transferred bracovirus genes reported here originate from the wasp genome, demonstrating that a gene flux exists between the two insect orders Hymenoptera and Lepidoptera that diverged ≈300 MYA. As bracovirus gene organisation has evolved to allow expression in Lepidoptera, these transferred genes can be readily domesticated. Additionally, we present functional analyses suggesting that some of the acquired genes confer to caterpillars a protection toward baculovirus, a very common pathogen in the field. This phenomenon may have implications for understanding how caterpillars acquire resistance against baculoviruses used in biological control.

lay_plos

People who reported eating fast food in the last 24 hours had elevated levels of some industrial chemicals in their bodies, according to a new analysis of data from federal nutrition surveys. The study is the first broad look at how fast food may expose the public to certain chemicals, called phthalates, that are used to make plastics more flexible and durable. The chemicals, which don’t occur in nature, are common in cosmetics, soap, food packaging, flooring, window blinds, and other consumer products. The Centers for Disease Control says "phthalate exposure is widespread in the U.S. population." Though the health consequences of encountering these substances aren’t fully known, scientists have increasingly focused on their effects on health and development, particularly for pregnant women and children. Research in rats has shown that they can disrupt the male reproductive system, and there’s evidence for similar effects in humans. The latest research suggests that fast food is a significant source of the chemicals, which may leach into food from machinery used in processing or packaging, or from gloves worn by workers. "Right now there are few choices for individuals who are interested in reducing their exposure, and there’s also not very much regulation” of phthalates, said Ami Zota, assistant professor of environmental and occupational health at the George Washington University Milken Institute School of Public Health. In the U.S., "research happens once they’ve been introduced in commerce, rather than before,” she said. Zota and colleagues from GW analyzed data from almost 9,000 people who participated in federal nutrition surveys between 2003 and 2010. Participants answered detailed questions about what they ate in the last 24 hours and gave urine samples that were analyzed for byproducts that indicated the presence of three chemicals. The study was published in the journal Environmental Health Perspectives, which is supported by the National Institutes of Health. For two of the three substances Zota examined—phthalates designated as DEHP and DiNP—there was a significant relationship between fast-food intake and exposure. People who ate more fast food had more evidence of phthalates in their urine. The third chemical they measured was Bisphenol A, or BPA, which is commonly used to line aluminum cans. That wasn't significantly correlated with fast-food intake. It’s difficult to determine what the health risks of phthalates are. The American Chemistry Council says that they’ve been thoroughly studied and “phthalates used in commercial products do not pose a risk to human health at typical exposure levels.” The Environmental Protection Agency, in a 2012 Phthalates Action Plan, notes that it is "concerned about phthalates because of their toxicity and the evidence of pervasive human and environmental exposure to them." Japan banned vinyl gloves in food preparation over concerns about DEHP, and the European Union has limited the use of the chemicals in food products and toys. Some phthalates, including DEHP, were restricted in children’s toys in the U.S. by a 2008 law. The latest study is based on snapshot surveys, rather than following people over time. So it can’t establish that eating fast food caused people to have elevated levels of phthalates, but the association is strong. It also doesn’t tell us anything about the health effects of potential exposure to the chemicals from eating fast food. The most important business stories of the day. Get Bloomberg's daily newsletter. The cost of endocrine-disrupting chemicals, a group that includes phthalates, in the EU is estimated to be €163 billion annually, or 1.28 percent of the EU’s gross domestic product, according to a recent paper by Leo Trasande, associate professor of pediatrics, environmental medicine, and population health at NYU School of Medicine. He said the study out today "adds further data to support the notion that people should avoid eating highly processed or highly packaged foods.” That doesn't mean just junk food, he notes. Canned vegetables or organic milk that’s been piped through plastic tubing could carry the same chemical risks. “It’s not fair to say, ‘Oh, these exposures only happen if you eat unhealthy foods.' " Zota said that for people interested in reducing their exposure, “common-sense approaches will take you a long way. Eat organic when you can. If you can’t still, try to eat fresh vegetables,” she said. "Try to eat low on the food chain." People who reported consuming more fast food in a national survey were exposed to higher levels of potentially harmful chemicals known as phthalates, according to a study published by researchers at Milken Institute School of Public Health (Milken Institute SPH) at the George Washington University. The study, one of the first to look at fast-food consumption and exposure to these chemicals, appears in the journal Environmental Health Perspectives. "People who ate the most fast food had phthalate levels that were as much as 40 percent higher," says lead author Ami Zota, ScD, MS, an assistant professor of environmental and occupational health at Milken Institute SPH. "Our findings raise concerns because phthalates have been linked to a number of serious health problems in children and adults." Phthalates belong to a class of industrial chemicals used to make food packaging materials, tubing for dairy products, and other items used in the production of fast food. Other research suggests these chemicals can leach out of plastic food packaging and can contaminate highly processed food. Zota and her colleagues looked at data on 8,877 participants who had answered detailed questions about their diet in the past 24 hours, including consumption of fast food. These participants also had provided researchers with a urinary sample that could be tested for the breakdown products of two specific phthalates--DEHP and DiNP. Zota and her colleagues found that the more fast food participants in the study ate the higher the exposure to phthalates. People in the study with the highest consumption of fast food had 23.8 percent higher levels of the breakdown product for DEHP in their urine sample. And those same fast food lovers had nearly 40 percent higher levels of DiNP metabolites in their urine compared to people who reported no fast food in the 24 hours prior to the testing. The researchers also discovered that grain and meat items were the most significant contributors to phthalate exposure. Zota says the grain category contained a wide variety of items including bread, cake, pizza, burritos, rice dishes and noodles. She also notes that other studies have also identified grains as an important source of exposure to these potentially harmful chemicals. In addition, the researchers also looked for exposure to another chemical found in plastic food packaging--Bisphenol A or BPA. Researchers also believe exposure to BPA can lead to health and behavior problems, especially for young children. This study found no association between total fast food intake and BPA. However, Zota and her colleagues found that people who ate fast food meat products had higher levels of BPA than people who reported no fast food consumption. This study fits into a bigger field of ongoing research showing that phthalates are in a wide variety of personal products, toys, perfume and even food. In 2008 Congress banned the use of phthalates in the production of children's toys because of concerns about the health impact of these chemicals. But Zota notes that DEHP and DiNP are two phthalates still in use despite concerns that they leach out of products and get into the human body. Studies of the health impact of exposure to these chemicals have suggested they can damage the reproductive system and they may lead to infertility. Large studies that might conclusively link phthalates in fast food and health problems could take years to conduct. In the meantime, Zota offers some common sense advice. She notes that frequent consumption of fast food is not recommended because such foods contain higher amounts of fat, salt and calories. "People concerned about this issue can't go wrong by eating more fruits and vegetables and less fast food," Zota suggests. "A diet filled with whole foods offers a variety of health benefits that go far beyond the question of phthalates." Environ Health Perspect; DOI:10.1289/ehp.1510803 Recent Fast Food Consumption and Bisphenol A and Phthalates Exposures among the U.S. Population in NHANES, 2003–2010 Ami R. Zota, Cassandra A. Phillips, and Susanna D. Mitro Author Affiliations open Department of Environmental and Occupational Health, Milken Institute School of Public Health, George Washington University, Washington, DC, USA PDF Version (233 KB) Abstract About This Article Supplemental Material Background: Phthalates and bisphenol A (BPA) are widely used industrial chemicals that may adversely impact human health. Human exposure is ubiquitous and can occur through diet, including consumption of processed or packaged food. Objective: To examine associations between recent fast food intake and BPA and urinary metabolites of di(2-ethylhexyl) phthalate (ΣDEHPm) and diisononyl phthalate (DiNPm) among the U.S. population. Methods: We combined data on 8,877 participants from the National Health and Nutrition Examination Survey (NHANES 2003–2010). Using 24-hr dietary recall data, we quantified: a) fast food intake [percent of total energy intake (TEI) from fast food]; b) fast food-derived fat intake (percent of TEI from fat in fast food); and c) fast food intake by food group (dairy, eggs, grains, meat, and other). We examined associations between dietary exposures and urinary chemical concentrations using multivariate linear regression. Results: We observed evidence of a positive, dose–response relationship between fast food intake and exposure to phthalates (p-trend < 0.0001) but not BPA; participants with high consumption (≥ 34.9% TEI from fast food) had 23.8% (95% CI: 11.9%, 36.9%) and 39.0% (95% CI: 21.9%, 58.5%) higher levels of ΣDEHPm and DiNPm, respectively, than nonconsumers. Fast food-derived fat intake was also positively associated with ΣDEHPm and DiNPm (p-trend < 0.0001). After adjusting for other food groups, ΣDEHPm was associated with grain and other intake, and DiNPm was associated with meat and grain intake. Conclusion: Fast food may be a source of exposure to DEHP and DiNP. These results, if confirmed, could inform individual and regulatory exposure reduction strategies. Citation: Zota AR, Phillips CA, Mitro SD. 2016. Recent fast food consumption and bisphenol A and phthalates exposures among the U.S. population in NHANES, 2003–2010. Environ Health Perspect 124:1521–1528; http://dx.doi.org/10.1289/ehp.1510803 Address correspondence to A.R. Zota, Department of Environmental and Occupational Health, Milken Institute School of Public Health, George Washington University, 950 New Hampshire Ave. NW, Suite 414, Washington, DC 20052 USA. Telephone: (202) 994-9289. E-mail: azota@gwu.edu We thank K. Robien for her input on the manuscript. This study was funded by the National Institute of Environmental Health Sciences (R00ES019881). The authors declare that they have no actual or potential competing financial interests and that their freedom to design, conduct, interpret, and publish research is not compromised by any controlling sponsor. Received: 24 September 2015 Revised: 2 December 2015 Accepted: 10 March 2016 Published: 13 April 2016 Note to readers with disabilities: EHP strives to ensure that all journal content is accessible to all readers. However, some figures and Supplemental Material published in EHP articles may not conform to 508 standards due to the complexity of the information being presented. If you need assistance accessing journal content, please contact ehponline@niehs.nih.gov. Our staff will work with you to assess and meet your accessibility needs within 3 working days. Supplemental Material PDF (178 KB) Note to readers with disabilities: EHP has provided a 508-conformant table of contents summarizing the Supplemental Material for this article (see below) so readers with disabilities may determine whether they wish to access the full, nonconformant Supplemental Material. If you need assistance accessing journal content, please contact ehponline@niehs.nih.gov. Our staff will work with you to assess and meet your accessibility needs within 3 working days. Supplemental Table of Contents PDF (104 KB) Introduction Related EHP Article Phthalates in Fast Food: A Potential Dietary Source of Exposure Wendee Nicole Phthalates are a class of high production volume industrial chemicals that are ubiquitously used in commerce. High-molecular-weight phthalates, such as di(2-ethylhexyl) phthalate (DEHP), are used as plasticizers to impart flexibility in polyvinyl chloride (PVC) materials such as food packaging, flooring, and medical devices (U.S. EPA 2012). In recent years, other phthalates, including diisononyl phthalate (DiNP), have been replacing DEHP in these applications due, in part, to legislation limiting the use of DEHP in certain applications (European Chemicals Agency 2012). Bisphenol A (BPA) is a high production volume chemical used to make polycarbonate plastics and epoxy resins, found in food and beverage cans as well as thermal receipt paper (Calafat et al. 2008). Phthalates and BPA can leach, migrate, or off-gas from products over time and enter the human body via ingestion, inhalation, and dermal absorption. Once in the body, phthalates and BPA are quickly metabolized and excreted in urine, with elimination half-lives less than 24 hr (Johns et al. 2015; Vandenberg et al. 2007). Human exposure to these chemicals is widespread (CDC 2013). Urinary metabolites of DEHP and DiNP are detected in 98% of the US general population (Zota et al. 2014) with higher exposures observed in children (Koch et al. 2004; Wittassek et al. 2011). Urinary metabolites of BPA are detected in 90% of the U.S. population with higher exposures observed in non-Hispanic Blacks, children, females, and those of lower socioeconomic status (Calafat et al. 2008; Nelson et al. 2012). Experimental animal studies demonstrate that DEHP and DiNP have endocrine-disrupting properties because of their anti-androgenic effects on the male reproductive system (National Research Council 2008). Human exposure to DEHP has been associated with adverse reproductive, neurobehavioral, and respiratory outcomes in children (Braun et al. 2013; Ejaredar et al. 2015) and metabolic disease risk factors such as insulin resistance in adolescents and adults (James-Todd et al. 2012; Attina and Trasande 2015). Though epidemiologic evidence of DiNP is less complete, recent studies report associations between exposure and similar health outcomes including adverse respiratory and metabolic outcomes in children (Bertelsen et al. 2013; Attina and Trasande 2015). BPA is also a suspected endocrine disrupter, and experimental and human evidence suggest that BPA is a reproductive toxicant (Peretz et al. 2014). In addition, prenatal BPA exposure has also been associated with adverse neurobehavioral outcomes in children (Mustieles et al. 2015). Given the concern over chemical toxicity, it is important to identify modifiable sources of exposure that may be targeted for exposure reduction strategies. Simulated exposure modeling, observational epidemiologic studies, and intervention studies all suggest that diet is an important exposure pathway for both high-molecular-weight phthalates and BPA (Carwile et al. 2011; Geens et al. 2012; Rudel et al. 2011; Serrano et al. 2014; Trasande et al. 2013; Wormuth et al. 2006). For example, a recent review of dietary and non-dietary exposures to BPA concluded that food sources contributed to more than 90% of overall BPA exposure among non-occupationally exposed individuals (Geens et al. 2012), and a second study that examined five individuals in Bochum, Germany fasting for 48 hr found that diet was the most significant source for DEHP and DiNP exposures as well (Koch et al. 2013). Food is likely contaminated with phthalates and BPA during processing (Cao 2010; Geens et al. 2012). Phthalates have been shown to leach into food from PVC in materials like tubing used in the milking process, lid gaskets, food preparation gloves, conveyor belts and food packaging materials (Cao 2010; Serrano et al. 2014). In fact, an intervention study reported that urinary BPA and DEHP were reduced by 66% and 53–56%, respectively, when participants’ diets were restricted to food with limited packaging (Rudel et al. 2011). Foods high in fat, such as dairy and meat, may be more contaminated by high-molecular-weight phthalates that are more lipophilic such as DEHP (Serrano et al. 2014). Fast food may be an important source of exposure to phthalates and BPA because it is highly processed, packaged, and handled. A recent study of children 1–5 years old found that those who ate one or more fast food meals a week had greater DiNP and butylbenzyl phthalate exposures than did those who ate less than one meal a week (Watkins et al. 2014); however, that study was limited by the small age range of participants and the imprecise measure of fast food consumption. In this study, we investigated the association between recent fast food consumption (derived from 24-hr dietary recall data) and exposure to high-molecular-weight phthalates (DEHP and DiNP) and BPA in the U.S. population using data from the National Health and Nutrition Examination Survey (NHANES). We focus on DEHP and DiNP because previous work shows that estimated dietary exposures to these phthalates are higher than other commonly studied phthalates (Sakhi et al. 2014). We hypothesized that increased consumption of fast food will be associated with higher urinary levels of BPA and the metabolites of these two phthalates. Methods Study Population We used data from the 2003–2004, 2005–2006, 2007–2008, and 2009–2010 cycles of NHANES, a nationally representative survey and physical examination of the civilian, non-institutionalized U.S. population conducted by the Centers for Disease Control and Prevention (CDC). The study sample included all participants ≥ 6 years old who completed a 24-hr dietary recall survey and provided a urine sample for phthalate or BPA analysis. The National Center for Health Statistics Research Ethics Review Board approved the study protocol. All participants gave informed consent; parents or guardians provided consent for participants < 18 years of age. There were 10,506 participants with urinary measurements of phthalate metabolites and creatinine. We sequentially excluded participants who did not self-identify as non-Hispanic White, non-Hispanic Black, or Mexican American/Hispanic (n = 495); who were missing information on household income (n = 702); who were missing information on body mass index (BMI; n = 89); or who were missing kilocalorie data (n = 343) resulting in a final sample size of 8,877 study participants for the DEHP analyses. The final sample size for the DiNP analyses was 6,629 because the DiNP oxidative metabolite, monocarboxyoctyl phthalate (MCOP), was not measured in 2003–2004. Most chemical analytes are measured in approximately one-third of the overall NHANES population, and phthalate metabolites and BPA were measured in different subpopulations. There were 10,418 participants with urinary BPA measurements. Using our sequential exclusion criteria, we removed 1,626 participants and an additional 3 participants who were missing creatinine measures. The final sample size for BPA analyses was 8,789 study participants. Urinary Chemical Analysis Phthalate metabolites and total BPA (free plus conjugated species) were measured in spot urine samples collected during the in-person exam at the Mobile Examination Center (MEC) and stored at –20°C until they were shipped to the CDC’s National Center for Environmental Health (Atlanta, GA) for analysis. Analytical methods have been described in detail elsewhere (CDC 2013). Briefly, chemical analytes were quantified in urine using solid-phase extraction coupled online with high-performance liquid chromatography and tandem mass spectrometry and expressed as wet weights (ng/mL). The limits of detection (LOD) ranged from 0.2 to 1.2 ng/mL for the phthalate metabolites and varied across study cycles. Therefore, we assumed the maximal LOD for each phthalate metabolite in our analysis to facilitate aggregation of data across study cycles (Zota et al. 2014). The LOD for BPA was 0.4 ng/mL for all study cycles. For both phthalates and BPA, concentrations below the LOD were substituted with the LOD divided by the square root of two. All analytes were detected in more than 90% of the study population except for mono(2-ethylhexyl) phthalate (MEHP; 64% > LOD). To approximate DEHP exposure, we calculated a summary metric (ΣDEHPm) equal to the molar sum of four DEHP metabolites: MEHP, mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), and mono(2-ethyl-5-carboxypentyl) phthalate (MECPP) using an approach previously described in Zota et al. (2014). DiNP exposure was characterized by its oxidative metabolite MCOP (referred to as DiNPm throughout the manuscript). Dietary Assessment The primary 24-hr dietary recall interview was administered in person at the MEC by dietary interviewers. Detailed protocols are described elsewhere and are briefly summarized here (CDC 2002). Survey participants ≥ 12 years old completed the dietary interview on their own, and proxy-assisted interviews were conducted with children 6–11 years old. During the interview, participants were prompted to report an uninterrupted listing of all foods and beverages consumed in a 24-hr period the day before the interview (midnight to midnight). This information was used to estimate the types and amounts of foods and beverages consumed as well as intakes of energy, nutrients, and other food components from those foods and beverages. Analysts at the National Center for Health Statistics (NCHS) in partnership with the U.S. Department of Agriculture’s (USDA) Food Surveys Research Group processed the intake data using the USDA’s Food and Nutrient Database for Dietary Studies (CDC 2002, 2007). Their output included publicly available datasets that summarize total nutrients for each participant as well as individual food profiles. The dietary recall interview also asked about the source of each food item. Fast food was defined as food obtained from restaurants without waiter/waitress service, or from pizza restaurants regardless of waiter/waitress service. All carryout and delivery food was also considered fast food. Foods from all other sources, including restaurants with waiters/waitresses, bars, taverns, lounges, vending machines, and mail or packaged foods, were not considered fast food (CDC 2007). For this analysis, we extracted 24-hr intake of kilocalories and grams of fat, in total and from fast food specifically, by participant. We also extracted kilocalories of fast food by the following food groups as designated by the USDA: dairy, eggs, grains, meat, other (examples of fast food items in each food group are provided in Table 1). Statistical Analysis Analyses were conducted in SAS (version 9.3; SAS Institute Inc., Cary, NC). Because we combined four survey cycles, we calculated new sample weights for each participant according to the NCHS analytical guidelines (CDC 2002). The degrees of freedom for our study sample were calculated by subtracting the number of clusters in the first level of sampling (strata) from the number of clusters in the second level of sampling (PSUs) (National Center for Health Statistics 2006). Based on our degrees of freedom, we used the following critical values from the t distribution for the calculation of all confidence intervals (CIs): DEHPm (1.99); DiNPm (2.01); and BPA (1.99). All analyses were adjusted for the non-random sampling design and the sample population weights. A (two-sided) p-value < 0.05 was considered statistically significant. Consumption of fast food was modeled several ways. First, we compared those who ate any fast food to those who did not. Second, we calculated fast food intake as the percent of total energy intake (TEI) from fast food in the 24-hr period. Third, we calculated fast food-derived fat intake as the percent of TEI from kilocalories of fat in fast food in the 24-hr period. Intake of fast food and fast food-derived fat were modeled as the following three categories: none, low, and high; where the low and high categories are divided at the weighted median of the exposed population. As a sensitivity analysis, we separately adjusted our final fast food intake models for intake from vending machines and from restaurants (percent of TEI; modeled as none, low, high) to account for other potential sources of packaged and processed food. We additionally examined fast food intake by the five USDA food groups. First, we examined each food group separately, dividing the percent of TEI from fast food for each food group into three categories (none, low, high). Second, to account for potential confounding by non-fast food sources, we adjusted each food group by its non-fast food counterpart (e.g., percent of TEI from fast food meat was adjusted for percent of TEI from non-fast food meat). Percent of TEI from non-fast food sources were also modeled as three categories (none, low, high). Third, to account for potential confounding by other food groups, we included all five of the fast food groups in the same model. We used multivariate linear regression models to examine the association between fast food consumption and urinary chemical concentrations. We performed natural log-transformation of chemical concentration data prior to regression analysis to improve normality and stabilize the variance. All regression models included natural log–transformed urinary creatinine [a marker of urine dilution (Barr et al. 2005)] and the following demographic variables: age (continuous); sex, race/ethnicity [non-Hispanic white, non-Hispanic black, or Hispanic (including Mexican American)], BMI [underweight (< 18.5), normal (18.5–24.99), overweight (25–29.99) and obese (> 30)], poverty:income ratio (PIR; the ratio of household income to poverty threshold adjusted to family size and inflation; [< 1 (i.e., beneath the poverty threshold), 1–3, and > 3], and NHANES survey cycle (2003–2004, 2005–2006, 2007–2008, 2009–2010). From these regression models, we estimated: a) percent difference in urinary chemical concentrations by fast food consumption as (e(β) – 1) × 100% with 95% CIs estimated as (e(β ± critical value × SE) – 1), where β and SE are the estimated regression coefficient and standard error, respectively; and b) least squares geometric means of urinary chemical concentrations by fast food consumption as e(least squares means) with 95% CIs as e(least squares mean ± critical value × SE), where the least square means is the mean of urinary chemical concentrations by fast food intake after adjustment for covariates. When exposure was modeled as three categories, percent differences were estimated by comparing each of the upper two groups to the lowest group. Additionally, a test for trend was performed by modeling the integer value of each exposure category (i.e., 0, 1, 2) as an ordinal term, and using its p-value as a test of departure from the null hypothesis of no linear trend. We tested for potential effect modification by age, sex, race/ethnicity, and household income by including multiplicative interaction terms in the statistical models. To further assess whether our results were specific to fast food, we also examined associations of TEI (tertile categories: low, medium, high) and total fat intake (percent of TEI; tertile categories: low, medium, high) with urinary chemical concentrations. Results Approximately one-third of the 8,877 participants reported consuming fast food on the day prior to their urine sample collection. Participants who ate fast food were more likely to be < 40 years old, male, and non-Hispanic black and to have higher TEI and total fat intake. Fast food consumers had higher levels of ΣDEHPm, DiNPm, and BPA than did nonconsumers. This difference was statistically significant for phthalates but not for BPA (Table 2). We observed evidence of a positive, dose–response association between fast food intake and ΣDEHPm (p for trend < 0.0001) (Table 3). Compared to nonconsumers, low consumers had 15.5% (95% CI: 6.3%, 25.6%) and high consumers had 23.8% (95% CI: 11.9%, 36.9%) higher levels of ΣDEHPm, respectively. Fast food-derived fat intake was also significantly associated with ΣDEHPm (p for trend < 0.0001) but smaller in magnitude than the association with fast food intake. For example, high consumers of fast food-derived fat had 18.9% (95% CI: 8.9%, 29.9%) higher levels of ΣDEHPm compared to nonconsumers. Furthermore, total fat intake (of all foods) but not TEI was associated with ΣDEHPm. Consumers in the highest tertile had 17.3% (95% CI: 9.2%, 26.0%) higher levels of ΣDEHPm, respectively, than consumers in the lowest tertile (Table 3). Similar to ΣDEHPm, there was evidence of a positive dose–response association between fast food intake and DiNPm (p for trend < 0.0001) (Table 3). Compared to nonconsumers, low consumers had 24.8% (95% CI: 12.9%, 37.9%) and high consumers had 39.0% (95% CI: 21.9%, 58.5%) higher levels of DiNP, respectively. The association with fast food-derived fat intake and DiNPm was smaller in magnitude than the association with fast food intake. Furthermore, total fat intake but not TEI was associated with DiNPm; however, this association was not monotonic and only the middle tertile estimate was significant (Table 3). In sensitivity analyses, adjustment for restaurant intake increased the associations between fast food intake and ΣDEHPm and DiNP by 10% to 20% (see Table S1) while there was virtually no change in the main effects after adjustment for vending machine intake. Next we calculated the association between fast food intake specific to each USDA food group with ΣDEHPm. All food groups were frequently consumed except for eggs, which were consumed in fast food meals by only 2% of the population. When each food group was modeled individually, we observed significant associations for all food groups except for eggs, although none of the associations were monotonic (Table 4; Model 1). These associations were virtually unchanged when each food group model was further adjusted for intake of non-fast food sources from the same food group (Table 4; Model 2). However, when all the food groups were examined together in the same model, only the individual categories of high grain and low other intake remained significant. Furthermore, the association between grain intake and ΣDEHPm became monotonic with a significant test for trend (p for trend = 0.01) (Table 4, Model 3). Similar to ΣDEHPm, we observed significant associations between DiNPm and all food groups except for eggs when each group was modeled individually (Table 5, Model 1). When each food group was further adjusted for intake of non-fast food sources from the same food group, associations were virtually unchanged (Table 5, Model 2). When all the food groups were examined together in the same model, we observed significant, monotonic associations for grain and meat intake (p for trend < 0.05). Those with high fast food grain and meat consumption had 21.9% (95% CI: 9.4%, 35.8%) and 20.1% (95% CI: 3.0%, 40.1%) higher levels of DiNPm, respectively, than did nonconsumers of those fast food groups (Table 5, Model 3). Table 5 – Association between recent fast food consumption by food group and urinary concentrations of DiNPm in the U.S. general population, NHANES 2005–2010 (n = 6,629). View Table (HTML Version) View larger image (TIF File) There was a monotonic, dose–response association between fast food meat and BPA in all three models (p for trend ≤ 0.01) (see Table S2). When all the food groups were examined together in the same model (see Table S2, Model 3), those with high fast food meat intake had 11.9% (95% CI: 2.0%, 22.7%) higher levels of BPA, respectively, than did nonconsumers of fast food meat. Low egg intake was significantly associated with BPA in all three models although the estimates for high egg intake were near the null and the linear test for trend was not significant. Lastly, the highest tertile for grain intake was negatively associated with BPA across all three models. Tests for interaction by sex and household income were not significant. Tests for interaction by age were not significant for ΣDEHPm or BPA, but were significant for DiNPm (p interaction < 0.05) (Figure 1; see also Table S3). Exposure to DiNPm was not associated with fast food intake in children, but was positively associated with fast food intake in adolescents and adults. Tests for interaction by race/ethnicity were not significant for DiNPm or BPA, but were significant for ΣDEHPm (p interaction < 0.05) (Figure 1; see also Table S4). Exposure to ΣDEHPm was positively associated with fast food intake in all three racial/ethnic groups; however, the association did not reach statistical significance among Hispanics. Moreover, the highest tertile estimate for fast food intake was greater in magnitude for non-Hispanic blacks compared to non-Hispanic whites or Hispanics. Figure 1 – Association [LSGM (95% CI)] between fast food intake (percent of TEI) and urinary phthalate metabolite concentrations in the U.S. general population by (A) age for DiNPm (n = 6,629; p interaction = 0.02) and (B) race/ethnicity for ΣDEHPm (n = 8,877; p interaction = 0.04). Estimates in Figure A are from linear regression models of interactions between fast food intake and age group adjusted for urinary creatinine, sex, race/ethnicity, BMI, PIR, and NHANES survey cycle. Estimates in Figure B are from linear regression models of interactions between fast food intake and race/ethnicity adjusted for urinary creatinine, age, sex, BMI, PIR, and NHANES survey cycle. Corresponding percent change estimates are provided in Tables S3 and S4. View larger image (TIF File) Discussion In this cross-sectional study of the U.S. population, we found a consistent, positive association between recent fast food consumption and phthalates exposure. Furthermore, there was evidence of a monotonic, positive dose–response; participants with high fast food intake had 20–40% higher urinary concentrations of phthalate metabolites than did nonconsumers. To our knowledge, this is the largest study to date on fast food consumption and biomarkers of environmental chemical exposure and the first to use a population-based sample. We did not find an association between total fast food consumption and BPA. Our null finding may, in part, be explained by the fact that the intake of BPA from non-canned foods is likely minimal compared to BPA from canned foods and nondietary sources (e.g., contact with thermal receipts) (Geens et al. 2012). We did find a significant, monotonic association between fast food meat intake and BPA, which corresponds to the small but growing evidence suggesting that hamburgers may be a source of BPA exposure. In a 2008 total diet study in Quebec, Canada, levels of BPA in fast food composite samples were generally low (1–3 ng/g) except for in the hamburger (10.9 ng/g) (Cao et al. 2011). In a study of 491 Mexican-American pregnant women, those who consumed hamburgers three or more times a week had 20% higher urinary BPA levels than did nonconsumers of hamburgers (Quirós-Alcalá et al. 2013). While fast food consumption was associated with exposure to both phthalates, our results highlight some important differences between DEHP and DiNP. The magnitude of the associations was 40–50% higher for DiNP than for DEHP. Moreover, associations for fast food-derived fat and DiNP were more pronounced than associations for TEI or total fat intake and DiNP suggesting DiNP contamination sources may be specific to fast food. In contrast, the magnitude of the association for highest intake of fast food-derived fat and total fat were similar for DEHP suggesting that some DEHP contamination sources may be common to all high fat foods. While many of the studies on dietary intake of phthalates do not include DiNP (Cirillo et al. 2011; Colacino et al. 2010; Schecter et al. 2013; Trasande et al. 2013), our results are consistent with a recent study of 296 children from Ohio ages 1–5 years that reported positive associations between fast food consumption and urinary metabolites of DiNP but not DEHP (Watkins et al. 2014). Additionally, a recent study of 10 phthalates in Norwegian food items found that DiNP was the most detected phthalate, and that estimated dietary exposures were also highest for DiNP (Sakhi et al. 2014). Our results show an interesting parallel with the changing trends in U.S. phthalate exposure as measured in biomonitoring data; between 2001 and 2010, ΣDEHP metabolites decreased by 37% and MCOP (DiNP metabolite) increased by 149% (Zota et al. 2014). Collectively, this work suggests that DiNP may be replacing DEHP in food contact materials. Future studies should further examine the human health effects associated with DiNP exposure and its potential synergy with other phthalates such as DEHP. Our analysis of fast food consumption by food groups found that intake of grain items was significantly associated with DiNP and DEHP with evidence of a monotonic, positive dose response. Meat intake was also associated with DiNP and DEHP, but the association with DEHP did not remain significant after adjusting for other food groups. Many food monitoring studies report higher phthalate residues in high fat foods, including oils, meat, and dairy (Cao 2010; Serrano et al. 2014). In line with these data, some epidemiological studies show positive associations between consumption of meats, fats, and dairy products and DEHP (Serrano et al. 2014). The robust association between fast food grain items and both phthalates in our study may, in part, be a result of the way the food items were classified. The grains category was heterogeneous and included a wide variety of items (Table 1) such as bread, cake, pizza, burritos, rice dishes, and noodles. Similar to our results, however, the recent Norwegian food monitoring study also identified grains to be the largest contributor to estimated daily intake of DEHP and DiNP, suggesting that grains consumption may be a true source of exposures (Sakhi et al. 2014). For example, since grain products are found on the exterior of foods such as pizza or burritos, they may be in greater contact with packaging materials. Future research should further investigate phthalate content in specific fast food menu items or by fast food chains to further characterize dietary exposures to phthalates. Our findings also suggest that associations between fast food intake and phthalate exposure are not uniform across the population. The association between DiNP and fast food was observed in adolescents and adults, but not in children 6–11 years old, potentially reflecting differences in exposure sources or behavior between groups. Furthermore, the association between fast food intake and DEHP exposure was more pronounced in blacks than in Hispanics, which may be linked to the higher consumption of fast food calories among blacks (see Table S4), or to differences in the types of fast food meals consumed between racial/ethnic groups. Future research could expand on these findings by examining how the contribution of dietary and non-dietary sources to phthalates exposures varies by race/ethnicity or socioeconomic status. The complexity and variability of fast food production makes it difficult to identify the sources of high-molecular-weight phthalates, though some likely sources have been suggested, including PVC gloves, PVC tubing, and plastic packaging. Food monitoring and duplicate diet studies conducted in Japan found that use of disposable PVC gloves during the preparation and packaging of meals was a major source of dietary intake of DEHP and that sterilizing the gloves with alcohol increased DEHP migration (Tsumura et al. 2001a, 2001b). The same study team also demonstrated a decline in DEHP levels in prepared meals after the ban of DEHP in PVC gloves in Japan (Tsumura et al. 2003). PVC tubing and plastic packaging may play a role since many of the food items available at fast food restaurants are prepared in bulk quantities at central supply facilities and then shipped to individual restaurants where they are cooked, reheated, or assembled (Schlosser 2012). For example, an Italian study that compared levels of DEHP and di-n-butyl phthalate in school meals before and after the food was packaged found that packaging increased phthalate concentrations by more than 100% (Cirillo et al. 2011). Future research should further characterize the role of food production, processing, and handling in dietary phthalate exposure. One main study limitation is the cross-sectional design of NHANES; thus, we cannot infer a causal relationship between fast food consumption and urinary phthalate metabolites. Future research should confirm our findings using a longitudinal study design that includes assessment of phthalates exposure in individuals before and after the consumption of fast foods. Other study limitations included the reliance on a single spot urine sample. While multiple urine samples or a 24-hr total urine sample is ideal, the collection of a single spot urine within 24 hr of the reported dietary consumption corresponds well with the short elimination half-lives of phthalates and BPA. We also relied on self-reported dietary data, which may have been prone to errors in reporting. However, the measurement error arising from both the urine sample collection and dietary recall is likely nondifferential. Lastly, the fast food category encompassed a wide variety of food items and establishments, which makes it difficult to isolate specific types of food production or menu items that may be underlying the observed associations. However, there are also advantages to use of NHANES dietary data. The dietary interview used a specific definition of fast food that eliminates subjectivity by the participant, and the broad array of food choices included in the fast food category increases the generalizability of our results. There are several notable strengths to our study. To our knowledge, this is the first study to use NHANES dietary recall data to examine the association between fast food consumption and urinary measures of environmental chemicals in the U.S. population. This study design allowed us to evaluate the associations among a large, representative sample of the U.S. population that included children. Moreover, we were able to quantify population-level consumption of fast food using information on energy and nutrients. Finally, our findings were robust to sensitivity analyses including adjustment for other potential sources of processed or packaged food as well as several different categorizations of fast food intake. Conclusions In conclusion, our findings suggest that fast food consumption may be a source of exposure to DEHP and DiNP, but not BPA, among the general population. These results, if confirmed in future longitudinal studies, may have great public health significance given the recommendations by various scientific and governmental bodies to limit exposure to phthalates due to concern over potential adverse health effects (Chronic Hazard Advisory Panel on Phthalates and Phthalate Alternatives 2014; U.S. EPA 2012). 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Phthalates Action Plan (Revised). Available: https://www.epa.gov/sites/production/files/2015-09/documents/phthalates_actionplan_revised_2012-03-14.pdf [accessed 15 September 2015]. Vandenberg LN, Hauser R, Marcus M, Olea N, Welshons WV. 2007. Human exposure to bisphenol A (BPA). Reprod Toxicol 24:139–177. Watkins DJ, Eliot M, Sathyanarayana S, Calafat AM, Yolton K, Lanphear BP, et al. 2014. Variability and predictors of urinary concentrations of phthalate metabolites during early childhood. Environ Sci Technol 48:8881–8890. Wittassek M, Koch HM, Angerer J, Brüning T. 2011. Assessing exposure to phthalates – the human biomonitoring approach. Mol Nutr Food Res 55:7–31. Wormuth M, Scheringer M, Vollenweider M, Hungerbühler K. 2006. What are the sources of exposure to eight frequently used phthalic acid esters in Europeans? Risk Anal 26:803–824. Zota AR, Calafat AM, Woodruff TJ. 2014. Temporal trends in phthalate exposures: findings from the National Health and Nutrition Examination Survey, 2001–2010. Environ Health Perspect 122:235–241, doi: 10.1289/ehp.1306681.

People who eat fast food may have higher levels of potentially infertility-causing industrial chemicals in their body, according to a new study. Bloomberg reports researchers were looking specifically at DEHP and DiNP, two types of phthalates found in everything from cosmetics to window blinds. They studied nutrition data from nearly 9,000 people and found those who got more than 35% of their total energy intake from fast food in the previous 24 hours had DEHP and DiNP levels 24% and 39% higher respectively. Phthalates may be entering fast food products through packaging, gloves worn by workers, or machinery used to process the food. Meat and grain products from fast-food restaurants were shown to be the most associated with higher phthalate levels in diners, according to a press release. "Our findings raise concerns because phthalates have been linked to a number of serious health problems in children and adults," researcher Ami Zota says in the press release. Previous studies show those problems include possible damage to the male reproductive system. While the American Chemistry Council says phthalates aren't harmful, the EPA is concerned about them, Japan has banned them in food-prep gloves, the EU has limited their use in food, and a 2008 US law restricted them in children's toys. Further studies are needed to see if phthalates in fast food are responsible for any health problems. But Zota has some solid advice either way: "People concerned about this issue can't go wrong by eating more fruits and vegetables and less fast food."

multi_news

CROSS-REFERENCE TO RELATED APPLICATION [0001] Applicant claims priority from Provisional patent applications 60/311,440, 60/311,441, and 60/311,610, all filed Aug. 10, 2001. BACKGROUND OF THE INVENTION [0002] Earplugs are commonly produced by punching a plug out of a plate of material or molding individual earplugs in individual molds. It is also possible to form earplugs by extruding material that is cut into earplugs. U.S. Pat. No. 5,753,015 describes feeding a small diameter core of round cross-section through an extrusion head while resilient foam material is extruded around the core, to provide a continuous extrusion. As the extrusion cools, it is cut into discrete pieces of about 25 mm length to thereby form individual earplugs. Patent publication WO 02/26465 describes extruding foamable material that will form a slow recovery foam, through an extrusion head, and using a knife blade to cut the extrusion whenever it projects by about 25 mm from the extrusion head, to thereby form individual earplugs. In both cases, the individual earplugs resulting from cutting the extrusion as it emerges from the extrusion head, must be packaged. Earplugs which were very easily packaged and withdrawn from the packaging would be of value. SUMMARY OF THE INVENTION [0003] In accordance with one embodiment of the present invention, an earplug arrangement and method for forming it are provided, which enables low cost production, storage, shipping, and dispensing. The earplug arrangement includes multiple earplugs connected in series to form a chain of earplugs. The chain of earplugs is formed by an extrusion of foamable material which is stored in a plurality of loops, with individual earplugs obtained by severing the last earplug in the chain from the rest of the chain. During extrusion, locations of minimum diameter are formed at uniform spacings of about 1 cm to 5 cm to define the opposite ends of earplugs. This facilitates bending of the chain to store it in loops or turns, and facilitates severing of the last earplug from the chain. [0004] In one arrangement, the chain include a core of elastomeric material which is at least twice as stiff as the material of the foam covering that was extruded around the core. The core resist column-like collapse when the earplug is pressed into the ear canal. The core also holds the chain together and allows the ends of the earplugs to be of a small diameter less than one-fourth the maximum diameter along the extrusion, to more clearly define the individual earplugs and facilitate cutting of earplugs from the chain. [0005] The novel features of the invention are set forth with particularity in the appended claims. The invention will be best understood from the following description when read in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS [0006] [0006]FIG. 1 is a partial sectional side elevation view showing a process and equipment for producing a chain of earplugs and also showing a portion of such chain of earplugs, where the chain comprises a single material. [0007] [0007]FIG. 2 is a side elevation view of one earplug of the chain of FIG. 1, after it has been cut from the chain. [0008] [0008]FIG. 3 is a front elevation view of compressing apparatus of FIG. 1, taken on line 3 - 3 thereof. [0009] [0009]FIG. 4 is a sectional side view of a method and equipment for generating a chain of earplugs of another embodiment of the invention, with a core of material that is stiffer than the extruded covering lying around the core, and showing a portion of the chain of earplugs. [0010] [0010]FIG. 5 is a side elevation view of one of the earplugs of the chain of FIG. 4, after it has been cut from the chain. [0011] [0011]FIG. 6 is a sectional view taken on line 6 - 6 of FIG. 5. [0012] [0012]FIG. 7 is a sectional side view showing a method and equipment for producing a chain of earplugs of another embodiment of the invention, and showing a portion of the chain of earplugs. [0013] [0013]FIG. 8 is a side elevation view showing one of the earplugs of the chain of FIG. 7 after it has been severed from the chain. [0014] [0014]FIG. 9 is a sectional view taken on line 9 - 9 of FIG. 8. [0015] [0015]FIG. 10 is a partially sectional isometric view showing a holder for holding a chain of earplugs and dispensing them. [0016] [0016]FIG. 11 is a sectional view of a portion of the holder of FIG. 10. [0017] [0017]FIG. 12 is a partially sectional isometric view of a holder of another embodiment of the invention, with a chain of earplugs therein. [0018] [0018]FIG. 13 is an isometric view of a chain of earplugs stored in zig-zag loops. [0019] [0019]FIG. 14 is a partially sectional side elevation view showing a method and equipment for constructing a chain of earplugs of another embodiment of the invention, and showing a portion of the chain, wherein only the front ends of the earplugs are rounded. DESCRIPTION OF THE PREFERRED EMBODIMENTS [0020] [0020]FIG. 1 illustrates an extrusion head 10 with an opening 12 through which flowable polymer material 14 is extruded. The polymer material 14 is a foamable material, and after passing out through the opening 12, the material expands in diameter as it foams, until it reaches a maximum diameter D and solidifies to become a resilient solid foam 16. The diameter D is about 12 mm so it can fit snugly into a person&#39;s ear canal and block sound. Accordingly, the extrusion 18 is constructed so it can be cut into individual earplugs. [0021] In accordance with the present invention, applicant does not cut the earplugs as they emerge or soon after they emerge from the extrusion head 10, but instead leaves a long extrusion 18 which forms a chain of earplugs 19 for easier storage and dispensing. A compressor 20 is located close to the extrusion head 10 to pinch the extrusion emerging from the opening 12. This leaves locations 22 of minimum diameter E at spacings S of between about 1 cm and 5 cm so individual earplugs 24 between adjacent locations 22 are long enough to be easily inserted into the ear canal and pulled out without excessive waste of material. The compression occurs close to the extrusion head before the extruded material has fully solidified (preferably within 5 cm of the extrusion head), and the compressor does not heat the extrusion. [0022] [0022]FIG. 3 shows one form of compressor 20, which includes four compressor elements or dies 31 - 34 and actuators 36 for moving the compressor elements simultaneously close together toward the extrusion axis 37 to compress the extrusion and then away from the extrusion. This results in the locations 22 of small diameter. The particular compressor elements have sides 38 that abut one another. It is also possible to have compressor elements that overlap one another. The compressor 20 is preferably located close to the extrusion head, where the foamable material has not foamed to its full diameter D. As a result, foamable material on either side of the compression location continues to foam and grow so as result in each earplug 24 having rounded ends 40. The particular foam material 16 is preferably a slow recovery foam material, which can be rolled to a small diameter and inserted into the ear canal, and which resiliently expands over a period such as thirty seconds to fill the ear canal and block it. If a resilient foam of the rapid recovery type, which recovers to its full diameter within one second, is used, then earplugs of such material are difficult to insert into the ear canal unless means are provided to prevent column-like collapse. [0023] [0023]FIG. 2 illustrates one earplug 24 which results from a pair of cuts at two locations 22 of the chain of FIG. 1. The earplug has rounded ends 40 and has a small protuberance or nipple 44 at each of its rounded ends where it was cut. The earplugs are not cut from the chain until at least one hour, and usually at least one day, after the extrusion is formed when the chain is of room temperature. [0024] [0024]FIG. 4 shows a system 50 for producing an extrusion 52 that forms a chain of earplugs 54 of another embodiment of the invention. As a foamable and flowable material 60 is extruded through an opening 62 of the extrusion head 64, a core 70 of elastomeric material at least twice as stiff as the foam is fed through the extrusion opening 62. The result is the extrusion 52 that includes the core 70 and a covering 72 of resilient polymer foam, the foam material preferably being a fast recovery foam rather than a slow recovery foam. A compressor 74 similar to the compressor 20 of FIG. 3 is provided to create locations of small diameter that are spaced apart by about 1 to 5 cm, and where the extrusion later can be cut to form individual earplugs. The compressor 74 preferably compresses the foamable material to substantially eliminate it at the location 76, so the location 76 consist of only the core 70. In practice, a small amount of the covering material lies around the core at the locations 76. The narrow locations facilitate cutting of earplugs from the chain, and make the chain easy to bend into loops, to turns for storage. [0025] [0025]FIG. 5 illustrates one of the earplugs 80 which has been cut from the chain 54 of earplugs of FIG. 4. The earplug has rounded ends 82, except for nipples 84 where the chain was cut at the narrow location 76. FIG. 6 shows that the diameter K of the core 70 is less than half and preferably less than one-quarter the diameter J of the covering. The core 70 is highly useful to prevent column-like collapse as one end, referred to as the front end of the earplug, begins to enter the ear canal as the opposite rear end is pressed forwardly. Without the core 70, it is very difficult to install a resilient foam earplug into the ear canal. In FIG. 6 the core diameter K is about 2 mm, which is no more than one-fourth the earplug maximum diameter D of about 12 mm. [0026] [0026]FIG. 7 illustrates a system 100 of another embodiment of the invention, which is similar to that of FIG. 4, except that a core 102 is provided which is in the form of a sleeve. As shown in FIG. 9, the sleeve-shaped core 102 has a gap 104, which is preferably no more than 90°, which allows covering material 110 of resilient fast-recovery foam to flow into the inside of the sleeve 102 to fill it. A compressor 72 similar to compressor 20 of FIG. 3, compresses the covering 110 of resilient foam that surrounds the sleeve 102, at intervals spaced about 1 cm to 5 cm apart. This leaves locations 112 where the chain of earplugs 114 can be bent to form into a loop, where an earplug can be easily severed form the chain, and where the ends 116 of the earplugs are rounded. FIG. 8 shows one of the earplugs 120 of the diameter D, with protrusions or nipples 122 at its opposite ends. FIG. 9 shows that the diameter P of the sleeve is about 3 mm which is less than half and no more than about one-fourth the maximum diameter Q of the earplug. [0027] [0027]FIG. 14 illustrates a modified apparatus 130 with a compressor 72 adjacent to the extrusion head 132, and another compressor 134 spaced from the extrusion head. The compressor 72 forms earplug rounded front ends 140 that enter the ear canal, and locations 142 of reduced diameter where the earplugs can be cut. The other compressor 134 is spaced from the extrusion head 132 and forms the rear ends of the earplugs so they are less rounded, but have narrow locations 152 where the earplugs can be severed. The chain of earplugs 152 has alternate minimum diameter locations 142, 152 that are different. [0028] [0028]FIG. 10 illustrates a chain of earplugs 160 in a holder 162 which includes a cylindrical drum 164 and a box 166 that surrounds the drum. The drum is rotatably mounted on the box so it can be turned to move the end of the chain at the last earplug 170 of the chain, out through a dispenser opening 172. A cutter 174 which has a blade operated by depressing a handle, cuts earplugs from the end of the chain. The chain of earplugs extends in a plurality of loops, or turns of 360 degrees about the drum axis 176, with considerable bending occurring at the narrowed locations of the chain. The chain of earplugs has a length of at least 50 cm and preferably at least 100 cm, to contain at least ten earplugs and preferably at least 20 earplugs (and more preferably at least 100 earplugs). Each of the turns extending around the drum contains at least five earplugs. [0029] [0029]FIG. 11 shows one example of a cutter 180. The cutter includes a pair of blades 182, 184. A handle 186 can be depressed to move the blade 182 beyond the axis 190 of the chain of earplug 192. A mechanism (not shown) moves the other blade 184 simultaneously in the opposite direction across the first blade 182 to shear an earplug between them. A pair of advancing wheels 192, 194 advance the chain of earplugs. [0030] [0030]FIG. 12 shows another holder 200 which includes a stationary centerpiece 204 that is preferably in the form of a cylinder or partial cone, and with a chain of earplugs 206 wrapped in a plurality of turns about the centerpiece. A box 210 surrounds the chain of earplugs, and has an opening 212 through which an end portion 214 of the chain can extend. A retainer 216 retains the ends of the chain, and the last earplug 220 of the chain can be cut off at the retainer 216. [0031] [0031]FIG. 13 shows still another arrangement, where a chain of earplugs 230 is stored in back-and-forth loops such as 232, 234, each of which may be considered to be a complete turn. [0032] Thus, the invention provides earplugs that can be manufactured and stored in a low cost and efficient manner, and a method for constructing the earplugs. The earplugs are formed as an extrusion of about 12 mm diameter to fit snugly in a human ear canal, and can be stored as by wrapping it in a plurality of loops or turns, and with individual earplugs being dispensed by cutting a length of about 1 cm to 5 cm from the extrusion. The extrusion preferably is formed with locations of reduced diameter spaced by between about 1 cm and 5 cm apart, where the extrusion can be readily cut and which provides increased flexibility to the chain to bend it into a turn. The extrusion is preferably formed by compressing it to form the narrow locations, with the compression occurring close to the extrusion head where the foamable material has not yet completely foamed so ends of the earplugs continue to foam and form rounded ends. It is possible to provide a chain of earplugs without narrowed locations, but at least 50 cm long so it can be cut into at least 10 earplugs. The extrusion can include a core which is a solid core or sleeve (a sleeve preferably has a gap) to stiffen the earplug, and with the thickness of resilient foam covering material that surrounds the core having a reduced thickness less than one-quarter maximum thickness at the locations of minimum diameter. It is possible to have slight compressions between opposite ends of the earplug to stiffen it. [0033] Although particular embodiments of the invention have been described and illustrated herein, it is recognized that modifications and variations may readily occur to those skilled in the art, and consequently, it is intended that the claims be interpreted to cover such modifications and equivalents.

An earplug arrangement enables low cost manufacture and storage of earplugs and convenient dispensing of individual earplugs. The earplugs are formed from an extrusion of a maximum diameter of about 12 mm and with narrow locations spaced apart by about 1 to 5 cm to separate the extrusion into a chain of at least ten earplugs, where the last earplug of the chain of earplugs formed by the extrusion can be cut from the rest of the chain for insertion into a person&#39;s ear canal. The chain of earplugs is stored in a plurality of loops in a container, as in a plurality of loops wrapped about a cylinder. The extrusion can include a stiffening core of stiff first material and an extruded covering of a soft resilient foam second material surrounding the core, the thickness of the second material being less than half as great at the narrowed locations as at maximum diameter locations while the core is of uniform cross-section along the entire length of the extrusion.

big_patent

That afternoon with Rima in the forest under the mora tree had proved so delightful that I was eager for more rambles and talks with her, but the variable little witch had a great surprise in store for me. All her wild natural gaiety had unaccountably gone out of her: when I walked in the shade she was there, but no longer as the blithe, fantastic being, bright as an angel, innocent and affectionate as a child, tricksy as a monkey, that had played at hide-and-seek with me. She was now my shy, silent attendant, only occasionally visible, and appearing then like the mysterious maid I had found reclining among the ferns who had melted away mist-like from sight as I gazed. When I called she would not now answer as formerly, but in response would appear in sight as if to assure me that I had not been forsaken; and after a few moments her grey shadowy form would once more vanish among the trees. The hope that as her confidence increased and she grew accustomed to talk with me she would be brought to reveal the story of her life had to be abandoned, at all events for the present. I must, after all, get my information from Nuflo, or rest in ignorance. The old man was out for the greater part of each day with his dogs, and from these expeditions he brought back nothing that I could see but a few nuts and fruits, some thin bark for his cigarettes, and an occasional handful of haima gum to perfume the hut of an evening. After I had wasted three days in vainly trying to overcome the girl's now inexplicable shyness, I resolved to give for a while my undivided attention to her grandfather to discover, if possible, where he went and how he spent his time. My new game of hide-and-seek with Nuflo instead of with Rima began on the following morning. He was cunning; so was I. Going out and concealing myself among the bushes, I began to watch the hut. That I could elude Rima's keener eyes I doubted; but that did not trouble me. She was not in harmony with the old man, and would do nothing to defeat my plan. I had not been long in my hiding-place before he came out, followed by his two dogs, and going to some distance from the door, he sat down on a log. For some minutes he smoked, then rose, and after looking cautiously round slipped away among the trees. I saw that he was going off in the direction of the low range of rocky hills south of the forest. I knew that the forest did not extend far in that direction, and thinking that I should be able to catch a sight of him on its borders, I left the bushes and ran through the trees as fast as I could to get ahead of him. Coming to where the wood was very open, I found that a barren plain beyond it, a quarter of a mile wide, separated it from the range of hills; thinking that the old man might cross this open space, I climbed into a tree to watch. After some time he appeared, walking rapidly among the trees, the dogs at his heels, but not going towards the open plain; he had, it seemed, after arriving at the edge of the wood, changed his direction and was going west, still keeping in the shelter of the trees. When he had been gone about five minutes, I dropped to the ground and started in pursuit; once more I caught sight of him through the trees, and I kept him in sight for about twenty minutes longer; then he came to a broad strip of dense wood which extended into and through the range of hills, and here I quickly lost him. Hoping still to overtake him, I pushed on, but after struggling through the underwood for some distance, and finding the forest growing more difficult as I progressed, I at last gave him up. Turning eastward, I got out of the wood to find myself at the foot of a steep rough hill, one of the range which the wooded valley cut through at right angles. It struck me that it would be a good plan to climb the hill to get a view of the forest belt in which I had lost the old man; and after walking a short distance I found a spot which allowed of an ascent. The summit of the hill was about three hundred feet above the surrounding level and did not take me long to reach; it commanded a fair view, and I now saw that the belt of wood beneath me extended right through the range, and on the south side opened out into an extensive forest. "If that is your destination," thought I, "old fox, your secrets are safe from me." It was still early in the day, and a slight breeze tempered the air and made it cool and pleasant on the hilltop after my exertions. My scramble through the wood had fatigued me somewhat, and resolving to spend some hours on that spot, I looked round for a comfortable resting-place. I soon found a shady spot on the west side of an upright block of stone where I could recline at ease on a bed of lichen. Here, with shoulders resting against the rock, I sat thinking of Rima, alone in her wood today, with just a tinge of bitterness in my thoughts which made me hope that she would miss me as much as I missed her; and in the end I fell asleep. When I woke, it was past noon, and the sun was shining directly on me. Standing up to gaze once more on the prospect, I noticed a small wreath of white smoke issuing from a spot about the middle of the forest belt beneath me, and I instantly divined that Nuflo had made a fire at that place, and I resolved to surprise him in his retreat. When I got down to the base of the hill the smoke could no longer be seen, but I had studied the spot well from above, and had singled out a large clump of trees on the edge of the belt as a starting-point; and after a search of half an hour I succeeded in finding the old man's hiding-place. First I saw smoke again through an opening in the trees, then a small rude hut of sticks and palm leaves. Approaching cautiously, I peered through a crack and discovered old Nuflo engaged in smoking some meat over a fire, and at the same time grilling some bones on the coals. He had captured a coatimundi, an animal somewhat larger than a tame tom-cat, with a long snout and long ringed tail; one of the dogs was gnawing at the animal's head, and the tail and the feet were also lying on the floor, among the old bones and rubbish that littered it. Stealing round, I suddenly presented myself at the opening to his den, when the dogs rose up with a growl and Nuflo instantly leaped to his feet, knife in hand. "Aha, old man," I cried, with a laugh, "I have found you at one of your vegetarian repasts; and your grass-eating dogs as well!" He was disconcerted and suspicious, but when I explained that I had seen a smoke while on the hills, where I had gone to search for a curious blue flower which grew in such places, and had made my way to it to discover the cause, he recovered confidence and invited me to join him at his dinner of roast meat. I was hungry by this time and not sorry to get animal food once more; nevertheless, I ate this meat with some disgust, as it had a rank taste and smell, and it was also unpleasant to have those evil-looking dogs savagely gnawing at the animal's head and feet at the same time. "You see," said the old hypocrite, wiping the grease from his moustache, "this is what I am compelled to do in order to avoid giving offence. My granddaughter is a strange being, sir, as you have perhaps observed--" "That reminds me," I interrupted, "that I wish you to relate her history to me. She is, as you say, strange, and has speech and faculties unlike ours, which shows that she comes of a different race." "No, no, her faculties are not different from ours. They are sharper, that is all. It pleases the All-Powerful to give more to some than to others. Not all the fingers on the hand are alike. You will find a man who will take up a guitar and make it speak, while I--" "All that I understand," I broke in again. "But her origin, her history--that is what I wish to hear." "And that, sir, is precisely what I am about to relate. Poor child, she was left on my hands by her sainted mother--my daughter, sir--who perished young. Now, her birthplace, where she was taught letters and the Catechism by the priest, was in an unhealthy situation. It was hot and wet--always wet--a place suited to frogs rather than to human beings. At length, thinking that it would suit the child better--for she was pale and weakly--to live in a drier atmosphere among mountains, I brought her to this district. For this, senor, and for all I have done for her, I look for no reward here, but to that place where my daughter has got her foot; not, sir, on the threshold, as you might think, but well inside. For, after all, it is to the authorities above, in spite of some blots which we see in their administration, that we must look for justice. Frankly, sir, this is the whole story of my granddaughter's origin." "Ah, yes," I returned, "your story explains why she can call a wild bird to her hand, and touch a venomous serpent with her bare foot and receive no harm." "Doubtless you are right," said the old dissembler. "Living alone in the wood, she had only God's creatures to play and make friends with; and wild animals, I have heard it said, know those who are friendly towards them." "You treat her friends badly," said I, kicking the long tail of the coatimundi away with my foot, and regretting that I had joined in his repast. "Senor, you must consider that we are only what Heaven made us. When all this was formed," he continued, opening his arms wide to indicate the entire creation, "the Person who concerned Himself with this matter gave seeds and fruitless and nectar of flowers for the sustentation of His small birds. But we have not their delicate appetites. The more robust stomach which he gave to man cries out for meat. Do you understand? But of all this, friend, not one word to Rima!" I laughed scornfully. "Do you think me such a child, old man, as to believe that Rima, that little sprite, does not know that you are an eater of flesh? Rima, who is everywhere in the wood, seeing all things, even if I lift my hand against a serpent, she herself unseen." "But, sir, if you will pardon my presumption, you are saying too much. She does not come here, and therefore cannot see that I eat meat. In all that wood where she flourishes and sings, where she is in her house and garden, and mistress of the creatures, even of the small butterfly with painted wings, there, sir, I hunt no animal. Nor will my dogs chase any animal there. That is what I meant when I said that if an animal should stumble against their legs, they would lift up their noses and pass on without seeing it. For in that wood there is one law, the law that Rima imposes, and outside of it a different law." "I am glad that you have told me this," I replied. "The thought that Rima might be near, and, unseen herself, look in upon us feeding with the dogs and, like dogs, on flesh, was one which greatly troubled my mind." He glanced at me in his usual quick, cunning way. "Ah, senor, you have that feeling too--after so short a time with us! Consider, then, what it must be for me, unable to nourish myself on gums and fruitlets, and that little sweetness made by wasps out of flowers, when I am compelled to go far away and eat secretly to avoid giving offence." It was hard, no doubt, but I did not pity him; secretly I could only feel anger against him for refusing to enlighten me, while making such a presence of openness; and I also felt disgusted with myself for having joined him in his rank repast. But dissimulation was necessary, and so, after conversing a little more on indifferent topics, and thanking him for his hospitality, I left him alone to go on with his smoky task. On my way back to the lodge, fearing that some taint of Nuflo's evil-smelling den and dinner might still cling to me, I turned aside to where a streamlet in the wood widened and formed a deep pool, to take a plunge in the water. After drying myself in the air, and thoroughly ventilating my garments by shaking and beating them, I found an open, shady spot in the wood and threw myself on the grass to wait for evening before returning to the house. By that time the sweet, warm air would have purified me. Besides, I did not consider that I had sufficiently punished Rima for her treatment of me. She would be anxious for my safety, perhaps even looking for me everywhere in the wood. It was not much to make her suffer one day after she had made me miserable for three; and perhaps when she discovered that I could exist without her society she would begin to treat me less capriciously. So ran my thoughts as I rested on the warm ground, gazing up into the foliage, green as young grass in the lower, shady parts, and above luminous with the bright sunlight, and full of the murmuring sounds of insect life. My every action, word, thought, had my feeling for Rima as a motive. Why, I began to ask myself, was Rima so much to me? It was easy to answer that question: Because nothing so exquisite had ever been created. All the separate and fragmentary beauty and melody and graceful motion found scattered throughout nature were concentrated and harmoniously combined in her. How various, how luminous, how divine she was! A being for the mind to marvel at, to admire continually, finding some new grace and charm every hour, every moment, to add to the old. And there was, besides, the fascinating mystery surrounding her origin to arouse and keep my interest in her continually active. That was the easy answer I returned to the question I had asked myself. But I knew that there was another answer--a reason more powerful than the first. And I could no longer thrust it back, or hide its shining face with the dull, leaden mask of mere intellectual curiosity. BECAUSE I LOVED HER; loved her as I had never loved before, never could love any other being, with a passion which had caught something of her own brilliance and intensity, making a former passion look dim and commonplace in comparison--a feeling known to everyone, something old and worn out, a weariness even to think of. From these reflections I was roused by the plaintive three-syllable call of an evening bird--a nightjar common in these woods; and was surprised to find that the sun had set, and the woods already shadowed with the twilight. I started up and began hurriedly walking homewards, thinking of Rima, and was consumed with impatience to see her; and as I drew near to the house, walking along a narrow path which I knew, I suddenly met her face to face. Doubtless she had heard my approach, and instead of shrinking out of the path and allowing me to pass on without seeing her, as she would have done on the previous day, she had sprung forward to meet me. I was struck with wonder at the change in her as she came with a swift, easy motion, like a flying bird, her hands outstretched as if to clasp mine, her lips parted in a radiant, welcoming smile, her eyes sparkling with joy. I started forward to meet her, but had no sooner touched her hands than her countenance changed, and she shrunk back trembling, as if the touch had chilled her warm blood; and moving some feet away, she stood with downcast eyes, pale and sorrowful as she had seemed yesterday. In vain I implored her to tell me the cause of this change and of the trouble she evidently felt; her lips trembled as if with speech, but she made no reply, and only shrunk further away when I attempted to approach her; and at length, moving aside from the path, she was lost to sight in the dusky leafage. I went on alone, and sat outside for some time, until old Nuflo returned from his hunting; and only after he had gone in and had made the fire burn up did Rima make her appearance, silent and constrained as ever. On the following day Rima continued in the same inexplicable humour; and feeling my defeat keenly, I determined once more to try the effect of absence on her, and to remain away on this occasion for a longer period. Like old Nuflo, I was secret in going forth next morning, waiting until the girl was out of the way, then slipping off among the bushes into the deeper wood; and finally quitting its shelter, I set out across the savannah towards my old quarters. Great was my surprise on arriving at the village to find no person there. At first I imagined that my disappearance in the forest of evil fame had caused them to abandon their home in a panic; but on looking round I concluded that my friends had only gone on one of their periodical visits to some neighbouring village. For when these Indians visit their neighbours they do it in a very thorough manner; they all go, taking with them their entire stock of provisions, their cooking utensils, weapons, hammocks, and even their pet animals. Fortunately in this case they had not taken quite everything; my hammock was there, also one small pot, some cassava bread, purple potatoes, and a few ears of maize. I concluded that these had been left for me in the event of my return; also that they had not been gone very many hours, since a log of wood buried under the ashes of the hearth was still alight. Now, as their absences from home usually last many days, it was plain that I would have the big naked barn-like house to myself for as long as I thought proper to remain, with little food to eat; but the prospect did not disturb me, and I resolved to amuse myself with music. In vain I hunted for my guitar; the Indians had taken it to delight their friends by twanging its strings. At odd moments during the last day or two I had been composing a simple melody in my brain, fitting it to ancient words; and now, without an instrument to assist me, I began softly singing to myself: Muy mas clara que la luna Sola una en el mundo vos nacistes. After music I made up the fire and parched an ear of maize for my dinner, and while laboriously crunching the dry hard grain I thanked Heaven for having bestowed on me such good molars. Finally I slung my hammock in its old corner, and placing myself in it in my favourite oblique position, my hands clasped behind my head, one knee cocked up, the other leg dangling down, I resigned myself to idle thought. I felt very happy. How strange, thought I, with a little self-flattery, that I, accustomed to the agreeable society of intelligent men and charming women, and of books, should find such perfect contentment here! But I congratulated myself too soon. The profound silence began at length to oppress me. It was not like the forest, where one has wild birds for company, where their cries, albeit inarticulate, have a meaning and give a charm to solitude. Even the sight and whispered sounds of green leaves and rushes trembling in the wind have for us something of intelligence and sympathy; but I could not commune with mud walls and an earthen pot. Feeling my loneliness too acutely, I began to regret that I had left Rima, then to feel remorse at the secrecy I had practiced. Even now while I inclined idly in my hammock, she would be roaming the forest in search of me, listening for my footsteps, fearing perhaps that I had met with some accident where there was no person to succour me. It was painful to think of her in this way, of the pain I had doubtless given her by stealing off without a word of warning. Springing to the floor, I flung out of the house and went down to the stream. It was better there, for now the greatest heat of the day was over, and the weltering sun began to look large and red and rayless through the afternoon haze. I seated myself on a stone within a yard or two of the limpid water; and now the sight of nature and the warm, vital air and sunshine infected my spirit and made it possible for me to face the position calmly, even hopefully. The position was this: for some days the idea had been present in my mind, and was now fixed there, that this desert was to be my permanent home. The thought of going back to Caracas, that little Paris in America, with its Old World vices, its idle political passions, its empty round of gaieties, was unendurable. I was changed, and this change--so great, so complete--was proof that the old artificial life had not been and could not be the real one, in harmony with my deeper and truer nature. I deceived myself, you will say, as I have often myself said. I had and I had not. It is too long a question to discuss here; but just then I felt that I had quitted the hot, tainted atmosphere of the ballroom, that the morning air of heaven refreshed and elevated me and was sweet to breathe. Friends and relations I had who were dear to me; but I could forget them, even as I could forget the splendid dreams which had been mine. And the woman I had loved, and who perhaps loved me in return--I could forget her too. A daughter of civilization and of that artificial life, she could never experience such feelings as these and return to nature as I was doing. For women, though within narrow limits more plastic than men, are yet without that larger adaptiveness which can take us back to the sources of life, which they have left eternally behind. Better, far better for both of us that she should wait through the long, slow months, growing sick at heart with hope deferred; that, seeing me no more, she should weep my loss, and be healed at last by time, and find love and happiness again in the old way, in the old place. And while I thus sat thinking, sadly enough, but not despondingly, of past and present and future, all at once on the warm, still air came the resonant, far-reaching KLING-KLANG of the campanero from some leafy summit half a league away. KLING-KLANG fell the sound again, and often again, at intervals, affecting me strangely at that moment, so bell-like, so like the great wide-travelling sounds associated in our minds with Christian worship. And yet so unlike. A bell, yet not made of gross metal dug out of earth, but of an ethereal, sublimer material that floats impalpable and invisible in space--a vital bell suspended on nothing, giving out sounds in harmony with the vastness of blue heaven, the unsullied purity of nature, the glory of the sun, and conveying a mystic, a higher message to the soul than the sounds that surge from tower and belfry. O mystic bell-bird of the heavenly race of the swallow and dove, the quetzal and the nightingale! When the brutish savage and the brutish white man that slay thee, one for food, the other for the benefit of science, shall have passed away, live still, live to tell thy message to the blameless spiritualized race that shall come after us to possess the earth, not for a thousand years, but for ever; for how much shall thy voice be our clarified successors when even to my dull, unpurged soul thou canst speak such high things and bring it a sense of an impersonal, all-compromising One who is in me and I in Him, flesh of His flesh and soul of His soul. The sounds ceased, but I was still in that exalted mood and, like a person in a trance, staring fixedly before me into the open wood of scattered dwarf trees on the other side of the stream, when suddenly on the field of vision appeared a grotesque human figure moving towards me. I started violently, astonished and a little alarmed, but in a very few moments I recognized the ancient Cla-cla, coming home with a large bundle of dry sticks on her shoulders, bent almost double under the burden, and still ignorant of my presence. Slowly she came down to the stream, then cautiously made her way over the line of stepping-stones by which it was crossed; and only when within ten yards did the old creature catch sight of me sitting silent and motionless in her path. With a sharp cry of amazement and terror she straightened herself up, the bundle of sticks dropping to the ground, and turned to run from me. That, at all events, seemed her intention, for her body was thrown forward, and her head and arms working like those of a person going at full speed, but her legs seemed paralysed and her feet remained planted on the same spot. I burst out laughing; whereat she twisted her neck until her wrinkled, brown old face appeared over her shoulder staring at me. This made me laugh again, whereupon she straightened herself up once more and turned round to have a good look at me. "Come, Cla-cla," I cried; "can you not see that I am a living man and no spirit? I thought no one had remained behind to keep me company and give me food. Why are you not with the others?" "Ah, why!" she returned tragically. And then deliberately turning from me and assuming a most unladylike attitude, she slapped herself vigorously on the small of the back, exclaiming: "Because of my pain here!" As she continued in that position with her back towards me for some time, I laughed once more and begged her to explain. Slowly she turned round and advanced cautiously towards me, staring at me all the time. Finally, still eyeing me suspiciously, she related that the others had all gone on a visit to a distant village, she starting with them; that after going some distance a pain had attacked her in her hind quarters, so sudden and acute that it had instantly brought her to a full stop; and to illustrate how full the stop was she allowed herself to go down, very unnecessarily, with a flop to the ground. But she no sooner touched the ground than up she started to her feet again, with an alarmed look on her owlish face, as if she had sat down on a stinging-nettle. "We thought you were dead," she remarked, still thinking that I might be a ghost after all. "No, still alive," I said. "And so because you came to the ground with your pain, they left you behind! Well, never mind, Cla-cla, we are two now and must try to be happy together." By this time she had recovered from her fear and began to feel highly pleased at my return, only lamenting that she had no meat to give me. She was anxious to hear my adventures, and the reason of my long absence. I had no wish to gratify her curiosity, with the truth at all events, knowing very well that with regard to the daughter of the Didi her feelings were as purely savage and malignant as those of Kua-ko. But it was necessary to say something, and, fortifying myself with the good old Spanish notion that lies told to the heathen are not recorded, I related that a venomous serpent had bitten me; after which a terrible thunderstorm had surprised me in the forest, and night coming on prevented my escape from it; then, next day, remembering that he who is bitten by a serpent dies, and not wishing to distress my friends with the sight of my dissolution, I elected to remain, sitting there in the wood, amusing myself by singing songs and smoking cigarettes; and after several days and nights had gone by, finding that I was not going to die after all, and beginning to feel hungry, I got up and came back. Old Cla-cla looked very serious, shaking and nodding her head a great deal, muttering to herself; finally she gave it as her opinion that nothing ever would or could kill me; but whether my story had been believed or not she only knew. I spent an amusing evening with my old savage hostess. She had thrown off her ailments and, pleased at having a companion in her dreary solitude, she was good-tempered and talkative, and much more inclined to laugh than when the others were present, when she was on her dignity. We sat by the fire, cooking such food as we had, and talked and smoked; then I sang her songs in Spanish with that melody of my own-- Muy mas clara que la luna; and she rewarded me by emitting a barbarous chant in a shrill, screechy voice; and finally, starting up, I danced for her benefit polka, mazurka, and valse, whistling and singing to my motions. More than once during the evening she tried to introduce serious subjects, telling me that I must always live with them, learn to shoot the birds and catch the fishes, and have a wife; and then she would speak of her granddaughter Oalava, whose virtues it was proper to mention, but whose physical charms needed no description since they had never been concealed. Each time she got on this topic I cut her short, vowing that if I ever married she only should be my wife. She informed me that she was old and past her fruitful period; that not much longer would she make cassava bread, and blow the fire to a flame with her wheezy old bellows, and talk the men to sleep at night. But I stuck to it that she was young and beautiful, that our descendants would be more numerous than the birds in the forest. I went out to some bushes close by, where I had noticed a passion plant in bloom, and gathering a few splendid scarlet blossoms with their stems and leaves, I brought them in and wove them into a garland for the old dame's head; then I pulled her up, in spite of screams and struggles, and waltzed her wildly to the other end of the room and back again to her seat beside the fire. And as she sat there, panting and grinning with laughter, I knelt before her and, with suitable passionate gestures, declaimed again the old delicate lines sung by Mena before Columbus sailed the seas: Muy mas clara que la luna Sola una en el mundo vos nacistes tan gentil, que no vecistes ni tavistes competedora ninguna Desdi ninez en la cuna cobrastes fama, beldad, con tanta graciosidad, que vos doto la fortuna. Thinking of another all the time! O poor old Cla-cla, knowing not what the jingle meant nor the secret of my wild happiness, now when I recall you sitting there, your old grey owlish head crowned with scarlet passion flowers, flushed with firelight, against the background of smoke-blackened walls and rafters, how the old undying sorrow comes back to me! Thus our evening was spent, merrily enough; then we made up the fire with hard wood that would last all night, and went to our hammocks, but wakeful still. The old dame, glad and proud to be on duty once more, religiously went to work to talk me to sleep; but although I called out at intervals to encourage her to go on, I did not attempt to follow the ancient tales she told, which she had imbibed in childhood from other white-headed grandmothers long, long turned to dust. My own brain was busy thinking, thinking, thinking now of the woman I had once loved, far away in Venezuela, waiting and weeping and sick with hope deferred; now of Rima, wakeful and listening to the mysterious nightsounds of the forest--listening, listening for my returning footsteps. Next morning I began to waver in my resolution to remain absent from Rima for some days; and before evening my passion, which I had now ceased to struggle against, coupled with the thought that I had acted unkindly in leaving her, that she would be a prey to anxiety, overcame me, and I was ready to return. The old woman, who had been suspiciously watching my movements, rushed out after me as I left the house, crying out that a storm was brewing, that it was too late to go far, and night would be full of danger. I waved my hand in good-bye, laughingly reminding her that I was proof against all perils. Little she cared what evil might befall me, I thought; but she loved not to be alone; even for her, low down as she was intellectually, the solitary earthen pot had no "mind stuff" in it, and could not be sent to sleep at night with the legends of long ago. By the time I reached the ridge, I had discovered that she had prophesied truly, for now an ominous change had come over nature. A dull grey vapour had overspread the entire western half of the heavens; down, beyond the forest, the sky looked black as ink, and behind this blackness the sun had vanished. It was too late to go back now; I had been too long absent from Rima, and could only hope to reach Nuflo's lodge, wet or dry, before night closed round me in the forest. For some moments I stood still on the ridge, struck by the somewhat weird aspect of the shadowed scene before me--the long strip of dull uniform green, with here and there a slender palm lifting its feathery crown above the other trees, standing motionless, in strange relief against the advancing blackness. Then I set out once more at a run, taking advantage of the downward slope to get well on my way before the tempest should burst. As I approached the wood, there came a flash of lightning, pale, but covering the whole visible sky, followed after a long interval by a distant roll of thunder, which lasted several seconds and ended with a succession of deep throbs. It was as if Nature herself, in supreme anguish and abandonment, had cast herself prone on the earth, and her great heart had throbbed audibly, shaking the world with its beats. No more thunder followed, but the rain was coming down heavily now in huge drops that fell straight through the gloomy, windless air. In half a minute I was drenched to the skin; but for a short time the rain seemed an advantage, as the brightness of the falling water lessened the gloom, turning the air from dark to lighter grey. This subdued rain-light did not last long: I had not been twenty minutes in the wood before a second and greater darkness fell on the earth, accompanied by an even more copious downpour of water. The sun had evidently gone down, and the whole sky was now covered with one thick cloud. Becoming more nervous as the gloom increased, I bent my steps more to the south, so as to keep near the border and more open part of the wood. Probably I had already grown confused before deviating and turned the wrong way, for instead of finding the forest easier, it grew closer and more difficult as I advanced. Before many minutes the darkness so increased that I could no longer distinguish objects more than five feet from my eyes. Groping blindly along, I became entangled in a dense undergrowth, and after struggling and stumbling along for some distance in vain endeavours to get through it, I came to a stand at last in sheer despair. All sense of direction was now lost: I was entombed in thick blackness--blackness of night and cloud and rain and of dripping foliage and network of branches bound with bush ropes and creepers in a wild tangle. I had struggled into a hollow, or hole, as it were, in the midst of that mass of vegetation, where I could stand upright and turn round and round without touching anything; but when I put out my hands they came into contact with vines and bushes. To move from that spot seemed folly; yet how dreadful to remain there standing on the sodden earth, chilled with rain, in that awful blackness in which the only luminous thing one could look to see would be the eyes, shining with their own internal light, of some savage beast of prey! Yet the danger, the intense physical discomfort, and the anguish of looking forward to a whole night spent in that situation stung my heart less than the thought of Rima's anxiety and of the pain I had carelessly given by secretly leaving her. It was then, with that pang in my heart, that I was startled by hearing, close by, one of her own low, warbled expressions. There could be no mistake; if the forest had been full of the sounds of animal life and songs of melodious birds, her voice would have been instantly distinguished from all others. How mysterious, how infinitely tender it sounded in that awful blackness!--so musical and exquisitely modulated, so sorrowful, yet piercing my heart with a sudden, unutterable joy. "Rima! Rima!" I cried. "Speak again. Is it you? Come to me here." Again that low, warbling sound, or series of sounds, seemingly from a distance of a few yards. I was not disturbed at her not replying in Spanish: she had always spoken it somewhat reluctantly, and only when at my side; but when calling to me from some distance she would return instinctively to her own mysterious language, and call to me as bird calls to bird. I knew that she was inviting me to follow her, but I refused to move. "Rima," I cried again, "come to me here, for I know not where to step, and cannot move until you are at my side and I can feel your hand." There came no response, and after some moments, becoming alarmed, I called to her again. Then close by me, in a low, trembling voice, she returned: "I am here." I put out my hand and touched something soft and wet; it was her breast, and moving my hand higher up, I felt her hair, hanging now and streaming with water. She was trembling, and I thought the rain had chilled her. "Rima--poor child! How wet you are! How strange to meet you in such a place! Tell me, dear Rima, how did you find me?" "I was waiting--watching--all day. I saw you coming across the savannah, and followed at a distance through the wood." "And I had treated you so unkindly! Ah, my guardian angel, my light in the darkness, how I hate myself for giving you pain! Tell me, sweet, did you wish me to come back and live with you again?" She made no reply. Then, running my fingers down her arm, I took her hand in mine. It was hot, like the hand of one in a fever. I raised it to my lips and then attempted to draw her to me, but she slipped down and out of my arms to my feet. I felt her there, on her knees, with head bowed low. Stooping and putting my arm round her body, I drew her up and held her against my breast, and felt her heart throbbing wildly. With many endearing words I begged her to speak to me; but her only reply was: "Come--come," as she slipped again out of my arms and, holding my hand in hers, guided me through the bushes. Before long we came to an open path or glade, where the darkness was not profound; and releasing my hand, she began walking rapidly before me, always keeping at such a distance as just enabled me to distinguish her grey, shadowy figure, and with frequent doublings to follow the natural paths and openings which she knew so well. In this way we kept on nearly to the end, without exchanging a word, and hearing no sound except the continuous rush of rain, which to our accustomed ears had ceased to have the effect of sound, and the various gurgling noises of innumerable runners. All at once, as we came to a more open place, a strip of bright firelight appeared before us, shining from the half-open door of Nuflo's lodge. She turned round as much as to say: "Now you know where you are," then hurried on, leaving me to follow as best I could.

Abel, certain that the old man has been less than honest with him, determines to learn about Rima's history from Nuflo because he knows now that the girl will not willingly reveal the whole truth. Rima, moreover, has become very aloof after their meeting at the mora tree. Nuflo disappears with his dogs for long hours during the day, and Abel is suspicious because Rima's guardian seldom returns with any sizable quantity of nuts and fruits from his expeditions. After scrambling through the woods and then falling asleep for a time, Abel finally spies Nuflo cooking an animal. One mystery, then, is easily solved: Rima, refusing to eat meat and not allowing Nuflo to kill any of the forest animals, has thereby deprived the old man of his pleasure in enjoying the taste of meat. But Nuflo, with his dogs, hunts animals and eats the meat without her knowledge. Abel suspects that Rima's sensitivity to odors and her domination of the woodland have betrayed Nuflo in his secret, but the old man is convinced that he is safe. Realizing that he can "blackmail" Nuflo into telling him about Rima's past, Abel prods the old man with questions about the girl, but the wily peasant lies his way out of the trap. He insists that Rima is not a surviving member of a lost race but that her senses have been acutely developed because of living outdoors almost all the time; he denies that the bird language is really so different. Abel does not believe Nuflo and is less sympathetic toward hima as a result of the latter's evasive behavior; he is, in fact, increasingly angry as he starts back. On the way to the hut, Abel hears Rima, but she again avoids him when he starts forward to meet her. He cannot understand her changed attitude and her continual avoidance of him. Abel is depressed when Rima finally makes her appearance inside the hut, "silent and constrained as ever." Hurt by Rima's neglect, Abel once more decides to play the same game: He will leave for a while to see if she misses him. Abel returns to the village of the Parahuari Indians but is surprised to find the site abandoned. He soon surmises from the evidence of orderly decampment that the Indians are visiting some neighbors, a usual procedure among the tribes. Abel is happy to be alone and reflects contentedly about his adventures so far, but he shortly starts to miss Rima and to regret his abrupt departure. He is disturbed in his tranquility by the appearance of Cla-cla, the old woman, who has been compelled to leave the other Indians on their trip because of her ill health. Despite her suspicions of Abel, which in turn arouse his fears about the Indians' hostility, Cla-cla accepts Abel as a companion for her miserable solitude. They sing and chat gaily, and the evening turns into a festive occasion for both to forget their sorrows. The next morning, however, Abel is so lonesome for the sight of Rima that he determines to go back to the forest without delay. Cla-cla's pleadings and the threat of an oncoming storm do not deter Abel from his decision. Escaping from the old woman, Abel hastens into the woodland, but he loses his sense of direction when the storm lessens visibility. Drenched by the driving rain, Abel is rescued by Rima, who had been waiting faithfully for him. But Rima reverts to her withdrawn attitude as soon as they come safely in sight of Nuflo's place.

booksum

Group 2 Innate lymphoid cells (ILC2s) are innate cells that produce the TH2 cytokines IL-5 and IL-13. The importance of these cells has recently been demonstrated in experimental models of parasitic diseases but there is a paucity of data on ILC2s in the context of human parasitic infections and in particular of the blood dwelling parasite Schistosoma haematobium. In this case-control study human peripheral blood ILC2s were analysed in relation to infection with the helminth parasite Schistosoma haematobium. Peripheral blood mononuclear cells of 36 S. haematobium infected and 36 age and sex matched uninfected children were analysed for frequencies of ILC2s identified as Lin-CD45+CD127+CD294+CD161+. ILC2s were significantly lower particularly in infected children aged 6–9 years compared to healthy participants. Curative anti-helminthic treatment resulted in an increase in levels of the activating factor TSLP and restoration of ILC2 levels. This study demonstrates that ILC2s are diminished in young helminth infected children and restored by removal of the parasites by treatment, indicating a previously undescribed association between a human parasitic infection and ILC2s and suggesting a role of ILC2s before the establishment of protective acquired immunity in human schistosomiasis. Innate lymphoid cells are a recently described cell type that has transformed our understanding of the role of innate immune responses in the generation of adaptive immune responses. Group 2 Innate lymphoid cells (ILC2s) produce the classical TH2 cytokines IL-5 and IL-13 [1–3] and have been shown to be crucial for protective immune responses in experimental helminth infection [4–6]. ILC2s play an important role as an early source of IL-13 and are crucial for timely worm expulsion in mice infected with the murine helminth parasite Nippostrongylus brasiliensis. However, to date there is only one study in humans which characterised ILCs in a small population of patients with diverse filarial infections [7]. The interaction of helminth parasites and the host immune system has been extensively studied in experimental models and naturally infected humans. In experimental models, the immune responses to helminths, including schistosome infections, is polarized towards a type 2 immune response (reviewed in [8–10]). However, research over the last decade has shown that the establishment of helminth infections requires modulation of host type 2 responses, which is mediated by several different mechanisms including regulatory T cells, regulatory B cells and the immuno-modulatory cytokines IL-10 and TGF-β [11–15]. To a large extent these observations have been supported by evidence from human studies [16–18]. Type 2 immune responses have been shown to involve innate immune cells including dendritic cells, basophils and alternatively activated macrophages [19–21]. However in human, TH2 responses to helminth infections are less pronounced [22,23]. In addition, humans infected with helminths show alterations of cellular responses such as regulatory T cells, dendritic cells and CD4+ T Cell responses [16,24,25] and these responses vary with age (and thereby with history of infection with helminths). Thus, the specific characteristics of the human type 2 response to helminths are more complex than in experimental models meaning that observations from the experimental setting must be validated in natural human infections. Human ILC2s are negative for lineage markers (Lin-), but positive for the hematopoietic cell marker CD45 [26]. IL-7 is essential for the development of ILC2s [5,27] and therefore CD127 (IL-7Rα) is a main marker for characterising ILC2s. Furthermore human ILC2s express the ‘chemokine receptor homologous molecule expressed on TH2 cells’ (CRTH2 = CD294) a marker of TH2 cells [28] and expressed the NK cell receptor CD161 (NKR-P1A) [26], whereas CD117 (c-kit) is mainly expressed on ILC3 and only on a subset of ILC2s [2]. ILC2s were initially described in fetal and adult gut and lung, but have also been identified in peripheral blood [26], but the function of blood ILC2s has not yet been investigated in detail. Changes in proportions of blood ILC2s could reflect either a global modulation of ILC2s or changes in the potential to migrate to tissues. ILC2s are dependent on the TH2-associated transcription factors GATA-3 [29,30] and RORα [27,31] and the ‘thymic stromal lymphopoietin’ TSLP enhances GATA-3 expression and subsequent production of IL-4, IL-5 and IL-13, especially if supplied in combination with IL-33, but less efficiently with IL-25 [32]. Changes of systemic IL-33 during helminth infection have been recently reported [33]. The presence of ILC2s in human peripheral blood has been demonstrated in healthy individuals and in patients with mild or severe asthma [34,35]. Furthermore, in mouse models ILC2s as well as IL-25 and IL-33, have been shown to be involved in allergic immune responses [36–41]. The aim of this study was to determine if levels of blood ILC2s change in the context of a parasitic infection in a human population, thereby providing evidence that ILC2s are important in human parasitic diseases. The study focused on the helminth parasite of the genus Schistosoma (blood-flukes) which causes schistosomiasis, a neglected tropical disease affecting about 240 million people mainly in Sub-Saharan Africa [42]. The most prevalent form is urogenital schistosomiasis caused by Schistosoma haematobium. The heaviest burden of this helminthic disease occurs in children, who typically acquire infection in the first year of life [43,44]. Infection accumulates with age, and in most populations peaks around the age of 9–14 years, followed by a decrease in infection [45,46] which has been attributed to the development of protective acquired immunity [47,48]. Therefore immune epidemiological analyses covering these different stages of infection have been shown to be important in the analysis of the immune response towards helminth infections [16,24,25]. In this study we describe for the first time, alterations of ILC2 proportions in human blood during natural infection with schistosomes in a heterogeneous population (different ages to capture the different infection dynamics of rising, peaking and declining infection levels) and the effect of removing the helminth parasites through curative anti-helminthic drug treatment on ILC2 proportions. To elucidate pathways involved in changes of ILC2s, levels of the ILC2 activating factors TSLP and IL-33 were analysed. The study received institutional and ethical approval from the Ethical Review Board of the University of Zimbabwe and the Medical Research Council of Zimbabwe respectively. Permission to conduct the study in the selected area was obtained from the Provincial Medical Director, the District Educational Officer and School Heads. The aims and procedures of the study were explained to participants and their parents/guardians (in the case of children) in the local language, Shona, with written informed consent and assent obtained from the participants or parents/guardian before enrolment into the study and before administration of the anti-helminthic drug. Participants were free to drop out at any time of the study and parents/guardian could withdraw their children from the study. After sample collection, anti-helminthic treatment with the standard dose of praziquantel was offered to all participants and administered by a local physician. This study was conducted in Magaya village in the Murehwa district of the Mashonaland East Province of Zimbabwe (31°91’E; 17°63’S) as part of a larger study investigating the immuno-epidemiology of human urogenital schistosomiasis of which several aspects have been published [24,25,49,50]. Samples used within this study were collected between September and November 2008. Previous studies in this area and national surveys indicated low prevalence’s of S. mansoni and soil-transmitted helminths (STH), whereas the S. haematobium prevalence was high (>50%) [51,52]. The area is mesoendemic for Plasmodium infection [53]. Magaya is a rural village where subsistence farming is the predominant occupation. Due to lack of safe water and sanitation facilities, participants are in frequent contact with fresh water, which harbours the parasite life stage (cercariae) infective to the human host. Frequency of water contacts of participants, their history of anti-helminthic treatment and residential history in the schistosome endemic area were documented by questionnaires. To be included in the cross-sectional part of the study, participants had to meet the following criteria: a) been lifelong residents of the study area, b) not have previously received anti-helminthic treatment, c) be negative for S. mansoni, STH, Plasmodium falciparum (ensuring that the confounding effects of these parasites were excluded from the study) and HIV, d) have provided at least two urine and two stool samples on consecutive days for parasitological analysis and a blood sample sufficient for PBMC and plasma isolation. From the participants who fulfilled these criteria a group of 72 people were selected covering an age range of 6–18 years with 24 people in each of following three age groups: 6–9,10–13,14–18 years. Within each age group equal numbers of uninfected and infected people were selected providing a S. haematobium prevalence of 50%. Furthermore participants were age and sex matched between the uninfected and infected groups. Egg positive samples were chosen to have comparable infection intensities between the three age groups. The resulting study group is shown in Table 1. The effect of removing the parasites on ILC2s and levels of the activating factors TSLP and IL-33 were determined in a cohort study where participants were treated with the anti-helminthic drug praziquantel at the recommended dose of 40 mg/mL and followed up 6 weeks later (before re-infections reach patency) as recommended [54]. To be included in this study participants had to meet the following criteria a) been included in the cross-sectional study described above, b) been positive for infection with S. haematobium parasites at baseline, c) been treated with the anti-helminthic drug praziquantel, d) have provided at least two urine and two stool samples for a parasitology check and have been confirmed negative for infection with S. haematobium parasites 6 weeks after treatment, e) provided a blood sample for isolation of PBMC and plasma at the 6 week post-treatment survey. Twelve participants, 9 males and 3 females, with a mean age of 9. 58 years (range 6–13 years) and a mean infection intensity of 43. 0 eggs/10 mL (range 3. 0–158. 3) before treatment fulfilled these criteria. At least two urine and two stool samples were collected over three consecutive days (between 9am and 1pm). Infection with S. haematobium was determined by filtration of 10 mL urine and microscopic analysis following the standard urine filtration procedure [55]. Infection with S. mansoni and STH was determined in stool samples using the Kato-Katz method [56], with the results confirmed in a random subset of stool samples by the formol-ether concentration technique [57]. Up to 20 mL of venous blood was collected into heparinised blood collection tubes and a further five mL into EDTA coated tubes. Blood was analysed for HIV using the rapid test ‘DoubleCheckGoldTM HIV 1&2’ (Orgenics) and positive samples were re-tested using ‘Determine HIV ½ Ag/Ab Combo’ (InvernessMedical). Blood smears were stained with Giemsa, microscopically examined for Plasmodium falciparum and checked using a serological test (Paracheck-PF®, Orchid Biomedical Systems). Heparinised blood was used for the isolation of PBMCs through density gradient centrifugation LymphoprepTM (Axis-Shield). PBMC were cryopreserved in 10% DMSO (Sigma) and 90% fetal calf serum (Lonza). 1x106 PBMCs were surface stained using the following anti-human antibodies: FITC-conjugated lineage cocktail (BD Bioscience) containing CD3, CD14, CD16, CD19, CD20, CD56, FITC-conjugated anti-CD11c (clone 3. 9), FITC-conjugated anti-CD123 (clone 6H6), e450-conjugated anti-CD161 (HP-3G10), APC-e780-conjugated anti-CD127 (eBioRDR5; all eBioscience), VioGreen-conjugated anti-CD45 (clone 5B1), APC-conjugated anti-CD117 (A3C6E2), PE-conjugated anti-CD294 (BM16; all Miltenyi Biotec). At least 4x105 stained PBMCs were acquired on a FACSCantoIITM (BD Bioscience) and analysed using FlowJo7 software (TreeStar). Previous experiments confirmed that cryopreserved PBMCs did not include any basophils and mast cells using this phenotyping approach. Plasma levels of TSLP and IL-33 by ELISA were analysed using commercially available kits (eBioscience and RnD systems respectively). IL-4, IL-5 and IL-13 were measured using established protocols [50,58]. Since the data did not meet the assumptions of parametric tests, all statistical analyses were carried out using non-parametric tests. For the comparison of two groups (egg negative versus egg positive, male versus female), Mann-Whitney U test was applied, for multiple groups the Kruskal-Wallis test followed by a post-hoc comparison (Mann-Whitney U test with Bonferroni correction) was used. For comparison of paired data (pre- versus post-treatment) the Wilcoxon signed-rank test was applied. Correlations were tested using a Spearman' s rho analysis. All statistical analysis was carried out using IBM SPSS v19 and p-values were taken as significant if ≤ 0. 05. To identify human peripheral blood ILC2s, Mjösberg and co-workers [26] depleted PBMCs from T cells, B cells and monocytes, followed by a flow cytometric analysis of the remaining cells. This approach was not practical here, due to a much larger sample size used in the present study, and as cell numbers were limited. We therefore directly stained whole PBMCs with a lineage cocktail, which also included the NK cell marker CD56. PBMCs were gated on live cells and duplicates excluded (Fig. 1A). Gated single cells were analysed by a Lin cocktail and CD45, which showed a Lin- negative population expressing CD45. Within this population a CD45hi and a CD45int population, could be clearly distinguished. To identify human ILC2s, Lin-CD45+ PBMCs were gated on CD127+ (Fig. 1B). Within Lin-CD45+CD127+ cells a defined population of CD294+CD161+ cells could be identified (Fig. 1C). CD294+CD161+ cells contained both a CD117+ and a CD117- subset (Fig. 1D). In reverse CD127+CD117+ ILCs did not exclusively define CD294+ and CD161+ cells (Median of CD294+CD161+ in CD127+CD117+: 45. 6, range 13. 1–74. 7) and it is known that CD117+ compromise both ILC2s and ILC3s. Of note, CD127+CD294+CD161+ subsets are all CD45hi. In summary, although not completely homogenous Lin-CD45+CD127+CD294+CD161+ cells define a population of human blood ILC2s, hence CD127+CD294+CD161+ ILC2s were used in subsequent analyses. Next, the effect of current schistosome status on the proportions of blood ILC2s was analysed. The analyses showed that CD127+CD294+CD161+ ILC2s were all significantly lower in schistosome-infected individuals (p = 0. 027) when levels of these cells were expressed as percentages of lin-CD45+CD127+ (Fig. 2A). This was also true if ILC2s were expressed as percentages of lin-CD45+ (Median: 4. 8 egg negative (ve-), 3. 1 egg positive (ve+) p = 0. 044). In addition proportions of ILC2s were negatively correlated to the intensity of infection determined by egg count (r = −0. 300, p = 0. 005 using non-parametric Spearman correlation). Of note, CD127+CD117+ ILCs show a comparable pattern (Median: 13. 7 egg ve-, 7. 5 egg ve+, p = 0. 001). To determine if these differences were consistent across age groups undergoing different schistosome infection dynamics, the population was divided into three age groups, 6–9,10–13 and 14–18 year olds reflecting rising, peaking and declining infection levels. This analysis showed that ILC2s in infected participants were significantly lower than those in uninfected people of the youngest age group (Fig. 2B). This difference was not significant in children aged 10–13 years and proportions of ILC2s were comparable in the oldest age group aged 14–18 years. Comparable results were obtained if ILC2s levels were correlated to infection intensity. The youngest age group showed a strong negative correlation between infection intensity and ILC2 proportions (r = −0. 483, p = 0. 008), whereas a weaker negative correlation was observed in 10–13 old participants (r = −0. 403, p = 0. 025) and with no significant correlation in the 14–18 years old (r = −0. 045, p = 0. 418). CD127 (IL-7Rα) was used for identification of ILC2s, allowing the actual expression levels (MFI) of CD127 on ILC2s to be measured. Expression levels of CD127 did not show significant variation between infected and uninfected people for all age groups (6–9 years: p = 0. 514; 10–13 years: p = 0. 114; 14–18 years: p = 0. 551). Of note, levels of the systemic effector TH2 cytokines IL-4, IL-5 and IL-13 did not show any association with peripheral ILC2s (i. e. ILC2s to IL-4: r = −0. 113 (p = 0. 346); to IL-5 r = 0. 138 (p = 0. 248 and to IL-13 r = 0. 016 (p = 0. 894) using non-parametric Spearman correlation). Systemic TH2 cytokines were positively associated to infection intensity only in the oldest age group (IL-4: r = 0. 488, p = 0. 008; IL-5: r = 0. 400, p = 0. 027; IL-13: r = 0. 497, p = 0. 007). Detailed data for the TH2 cytokines are presented in S1 Fig with IL-5 and IL-13 only significant in children 14–18 years of age. To test if the differences in the ILC2 proportions were related to the presence of schistosome parasites, schistosome adult worms were removed by curative treatment and analysed in 12 children aged 6–13 years who were S. haematobium positive prior to treatment and had cleared infection 6 weeks later were followed up and proportions of ILC2s were determined. The overall proportions of their ILC2 cells increased significantly post-treatment as shown in Fig. 3A. Older egg negative participants have putatively developed a protective immune response and have been referred to in other studies as endemic normal and a high worm specific IgE/IgG4 ratio is a marker for schistosome-specific resistance [25]. Curative treatment by chemotherapy has been shown to accelerate the development of a protective immune response [59], hence post-treatment levels of ILC2 were compared to egg ve- individuals aged 14–18 years. Post-treatment, levels of ILC2 rose up to levels observed in untreated 14–18 year old participants who were egg negative prior to treatment (Fig. 3B). After dividing these 12 children into two groups of 6–9 and 10–13 years of age, ILC2s increased in 5 out of 7 and in 4 out of 5 children, respectively. The increase of ILC2 levels were inversely related to the pre-treatment levels of these cells, with the lowest pre-treatment proportions exhibiting the highest magnitude of change post-treatment. We have previously reported this phenomenon in changes in antibody levels in children exposed to S. mansoni infection [59]. Maintenance and activation of ILC2s has been reported to depend on the cytokines IL-25, IL-33 and TSLP. Pre-treatment plasma levels of IL-33 and TSLP were measured, but no significant difference was observed when egg negative and egg positive children were compared regardless of the age group investigated (Fig. 4A, B). However TSLP levels increased significantly 6 weeks after curative treatment (Fig. 4C). In contrast, the level of IL-33 remained unchanged following treatment (Fig. 4D). In parasitic infections, ILC2s have been described in experimental models of helminth infections demonstrating their role as an early source of the cytokine IL-13 and in worm expulsion [4,6, 60–62]. Given these results, this study aimed to determine if ILC2s populations are altered during human parasite infections using an immuno-epidemiological approach reflecting the complex dynamics during human parasite infections and specifically focusing on young children with accumulating infection. Hence, included in the study are young children who have yet to acquire schistosome infection (negative for schistosome eggs and parasite-specific antibodies), infected young and older children (schistosome egg positive) and older children putatively resistant to schistosome infection (schistosome egg negative but positive for schistosome-specific IgG and IgE [63]). First, this study confirms that ILC2s can be identified in human peripheral blood using Lin-CD45+CD127+ cells in combination with CD161+ and CD294+ in a population of young African children. This study, shows for the first time that proportions of human blood ILC2s are diminished during an infection with trematodes. The strongest effect was observed in the youngest age group. Reductions in proportions of ILC2s could theoretically arise in two ways; due to a reduction in generation/maintenance of the cells in people infected with S. haematobium parasites. Alternatively, ILC2s could migrate and accumulate at the site of infection or to the tissues where eggs get trapped, initiating a localised immune response thereby leading to a reduction of the cells in peripheral blood. Furthermore reduced levels of CD127 could indicate a reduced responsiveness to IL-7 and thereby ILC2 survival. However, expression levels did not show any variation which supports reduced responsiveness to IL-7. In this present study ILC2s are quantified as proportions. Changes in ILC2 proportions due to an increase of other cell types within Lin-CD45+ cells cannot be excluded. However, our results are consistent with those from a previous study on blood ILC2s, indicating that in a TH2 mediated disease, ILC2s are lower in patients compared to controls [35]. In contrast a recent study showed in 21 people with diverse filarial infections (Loa loa, Wuchereria bancrofti and with Onchocerca volvulus) an increase of ILCs characterised by CD127+CD117+ [7]. However infected people were aged 25–66 years and therefore significantly older than even in the oldest age group investigated in this study, in which ILC2s were also slightly higher in egg positive compared to egg negative people, which may become significant if older people analysed. Interestingly, reduced ILC2s were mainly observed in the youngest children, whereas in older children (14–18 years) there were no differences between egg negative and positive individuals. These differences are likely to be related to the dynamics of infection and the immune response due to ILC2s may have different dynamics compared to the development of protective acquired immunity. Age-related changes of cellular, antibody and cytokine pattern in the context of schistosome infection have been reported previously [24,64,65] and such pattern are supported by theoretical models of helminth infections [66]. These changes have been taken to represent population changes from susceptibility to infection the development of protective acquired immunity [66]. Parasitological and immuno-epidemiological studies show that the egg negative children in the youngest age group in this population have yet to acquire infection (no parasite specific—IgM responses which are indicative of exposure to parasite life stages). The remaining study population are either currently infected (indicated by the presence of parasite eggs) or have had previous infection and are now putatively immune (indicated by the absence of parasite eggs in combination with the presence of parasite-specific immune responses associated with resistance to infection such as anti-worm antibodies (IgE, IgG1, IgE/IgG4 ratio) and parasite specific cellular responses (IL-5) as previously reported [25,58,67]). Thus, in this population, the youngest egg negative children are the only ones who have not yet experienced a schistosome infection, and the observed changes in ILC2s in this age group upon infection, suggests that these cells are important in the early phase of the immune response, when the TH2 pathway is initially triggered, rather than in the later phases of infection, when TH2 responses become established. This is consistent with our results from studies of the myeloid dendritic cells in this population [24], which show a similar pattern. Indeed it has been shown that ILC2s, in particular, provide an early source of IL-13 during experimental helminth infection [4]. Dynamic changes in the immune response to schistosomes are well documented and age-related changes of cellular immune responses have been previously reported in this and other study populations [16,24,25]. Clearance of S. haematobium infection following curative treatment with the anti-helminthic drug praziquantel caused an increase in ILC2 proportions, recovering to levels comparable to egg negative 14–18 year old participants, who have already developed natural protective immunity against parasites [25]. This is in keeping with earlier studies showing that chemotherapy accelerates the development of protective acquired immunity against schistosomes [59] as well as studies from this population showing that chemotherapy induced cellular responses associated with resistance against re-infection [50]. However due to the small sample size post-treatment, this result requires further verification. Both IL-33 and TSLP are important in ILC2 activation and propagation. There was no significant association with systemic levels of IL-33 or TSLP with schistosome infection prior to treatment. Hence systemic levels of these cytokines do not explain the alterations in blood ILC2s and do not indicate a different activation status. However, if changes in ILC2 proportion are due to migration, IL-33 and TSLP might still play a role if regulated locally (in which case local levels of the cytokines would not necessarily be reflected systemically). We have obtained supportive evidence for the importance IL-33 by analysing the effect of a specific IL-33 single nucleotide polymorphisms (SNP) showing that allele variation influences schistosome infection intensity without influencing systemic IL-33 levels (manuscript in preparation). In addition, systemic TH2 cytokines IL-4, IL-5 and IL-13 did not show significant associations with peripheral ILC2s. However, if changes in ILC2s are due to migration, ILC2 activation could be still initiated locally and play an important role in the immune response. This is supported by the finding that systemic TH2 cytokines are only associated to infection in the oldest age group, indicating that a classical TH2 response is more important and reflected on systemic levels in older chronically infected individuals. Further studies are required to analyse the activation status of ILC2s in infected people relative to their expression levels of GATA-3, IL-5 and IL-13 as well as receptors for IL-33 and TSLP. Recently is has been reported that in mouse models IL-9 and amphiregulin are important in the function of ILC2s [68,69]. However, data in humans are sparce and both IL-9 and amphiregulin were not incorporated when this study was carried out. Increased levels of TSLP were observed 6 weeks after treatment, which suggests that TSLP may be involved in the increase of ILC2s following treatment, but no change in IL-33 was observed after treatment. This latter result contradicts previous reports of an increase in IL-33 several weeks after treatment [33]. Further studies are required to clarify the role of IL-33 in human infections with S. haematobium and should also include IL-25, which was not analysed during this study due to limitations in blood levels that could be collected safely from the younger children. In summary, this is the first study to report peripheral blood ILC2s being diminished in young children suggesting that ILC2s may play a role in the immune responses to human helminth infection before the establishment of schistosome protective immunity. Clearly further mechanistic studies are required to determine how ILC2 levels are diminished in helminth infection and how they are restored following removal of the helminth infection by curative treatment. Nonetheless, this study validates the paradigms demonstrated in experimental studies of an association between parasitic infection and ILC2 cells.

Understanding how immune responses are generated is critical for vaccine development. There are comparatively few studies on the interface between the innate and adaptive immune system in generating protective immune responses. Infections with helminth parasites, a cause of neglected tropical diseases, have a huge collective impact on public health in affected developing countries. Helminths are associated with a complex type 2 immune response mediated by cytokines characteristically produced by adaptive T helper 2 cells (TH2). However in recent years a newly described type of innate immune cells has been shown to produce TH2 cytokines. This cell type was subsequently called 'Group 2 innate lymphoid cells' (ILC2s). The importance of these cells has been demonstrated in experimental models of helminth infection as well as in allergic diseases. The present study describes changes in human ILC2s during the human helminth infection schistosomiasisas (bilharzia). Our study shows that the proportions of ILC2s, were lower in young, but not older infected children when compared to uninfected participants, suggesting a role for ILC2s before the establishment of helminth protective acquired immunity. Furthermore ILC2s were restored after curative treatment of the helminth infection. Human mechanistic studies will determine if the association between ILC2s and schistosome infection is causal or a marker of resistance to infection.

lay_plos

Mr Verloc, going out in the morning, left his shop nominally in charge of his brother-in-law. It could be done, because there was very little business at any time, and practically none at all before the evening. Mr Verloc cared but little about his ostensible business. And, moreover, his wife was in charge of his brother-in-law. The shop was small, and so was the house. It was one of those grimy brick houses which existed in large quantities before the era of reconstruction dawned upon London. The shop was a square box of a place, with the front glazed in small panes. In the daytime the door remained closed; in the evening it stood discreetly but suspiciously ajar. The window contained photographs of more or less undressed dancing girls; nondescript packages in wrappers like patent medicines; closed yellow paper envelopes, very flimsy, and marked two-and-six in heavy black figures; a few numbers of ancient French comic publications hung across a string as if to dry; a dingy blue china bowl, a casket of black wood, bottles of marking ink, and rubber stamps; a few books, with titles hinting at impropriety; a few apparently old copies of obscure newspapers, badly printed, with titles like _The Torch_, _The Gong_--rousing titles. And the two gas jets inside the panes were always turned low, either for economy's sake or for the sake of the customers. These customers were either very young men, who hung about the window for a time before slipping in suddenly; or men of a more mature age, but looking generally as if they were not in funds. Some of that last kind had the collars of their overcoats turned right up to their moustaches, and traces of mud on the bottom of their nether garments, which had the appearance of being much worn and not very valuable. And the legs inside them did not, as a general rule, seem of much account either. With their hands plunged deep in the side pockets of their coats, they dodged in sideways, one shoulder first, as if afraid to start the bell going. The bell, hung on the door by means of a curved ribbon of steel, was difficult to circumvent. It was hopelessly cracked; but of an evening, at the slightest provocation, it clattered behind the customer with impudent virulence. It clattered; and at that signal, through the dusty glass door behind the painted deal counter, Mr Verloc would issue hastily from the parlour at the back. His eyes were naturally heavy; he had an air of having wallowed, fully dressed, all day on an unmade bed. Another man would have felt such an appearance a distinct disadvantage. In a commercial transaction of the retail order much depends on the seller's engaging and amiable aspect. But Mr Verloc knew his business, and remained undisturbed by any sort of aesthetic doubt about his appearance. With a firm, steady-eyed impudence, which seemed to hold back the threat of some abominable menace, he would proceed to sell over the counter some object looking obviously and scandalously not worth the money which passed in the transaction: a small cardboard box with apparently nothing inside, for instance, or one of those carefully closed yellow flimsy envelopes, or a soiled volume in paper covers with a promising title. Now and then it happened that one of the faded, yellow dancing girls would get sold to an amateur, as though she had been alive and young. Sometimes it was Mrs Verloc who would appear at the call of the cracked bell. Winnie Verloc was a young woman with a full bust, in a tight bodice, and with broad hips. Her hair was very tidy. Steady-eyed like her husband, she preserved an air of unfathomable indifference behind the rampart of the counter. Then the customer of comparatively tender years would get suddenly disconcerted at having to deal with a woman, and with rage in his heart would proffer a request for a bottle of marking ink, retail value sixpence (price in Verloc's shop one-and-sixpence), which, once outside, he would drop stealthily into the gutter. The evening visitors--the men with collars turned up and soft hats rammed down--nodded familiarly to Mrs Verloc, and with a muttered greeting, lifted up the flap at the end of the counter in order to pass into the back parlour, which gave access to a passage and to a steep flight of stairs. The door of the shop was the only means of entrance to the house in which Mr Verloc carried on his business of a seller of shady wares, exercised his vocation of a protector of society, and cultivated his domestic virtues. These last were pronounced. He was thoroughly domesticated. Neither his spiritual, nor his mental, nor his physical needs were of the kind to take him much abroad. He found at home the ease of his body and the peace of his conscience, together with Mrs Verloc's wifely attentions and Mrs Verloc's mother's deferential regard. Winnie's mother was a stout, wheezy woman, with a large brown face. She wore a black wig under a white cap. Her swollen legs rendered her inactive. She considered herself to be of French descent, which might have been true; and after a good many years of married life with a licensed victualler of the more common sort, she provided for the years of widowhood by letting furnished apartments for gentlemen near Vauxhall Bridge Road in a square once of some splendour and still included in the district of Belgravia. This topographical fact was of some advantage in advertising her rooms; but the patrons of the worthy widow were not exactly of the fashionable kind. Such as they were, her daughter Winnie helped to look after them. Traces of the French descent which the widow boasted of were apparent in Winnie too. They were apparent in the extremely neat and artistic arrangement of her glossy dark hair. Winnie had also other charms: her youth; her full, rounded form; her clear complexion; the provocation of her unfathomable reserve, which never went so far as to prevent conversation, carried on on the lodgers' part with animation, and on hers with an equable amiability. It must be that Mr Verloc was susceptible to these fascinations. Mr Verloc was an intermittent patron. He came and went without any very apparent reason. He generally arrived in London (like the influenza) from the Continent, only he arrived unheralded by the Press; and his visitations set in with great severity. He breakfasted in bed, and remained wallowing there with an air of quiet enjoyment till noon every day--and sometimes even to a later hour. But when he went out he seemed to experience a great difficulty in finding his way back to his temporary home in the Belgravian square. He left it late, and returned to it early--as early as three or four in the morning; and on waking up at ten addressed Winnie, bringing in the breakfast tray, with jocular, exhausted civility, in the hoarse, failing tones of a man who had been talking vehemently for many hours together. His prominent, heavy-lidded eyes rolled sideways amorously and languidly, the bedclothes were pulled up to his chin, and his dark smooth moustache covered his thick lips capable of much honeyed banter. In Winnie's mother's opinion Mr Verloc was a very nice gentleman. From her life's experience gathered in various "business houses" the good woman had taken into her retirement an ideal of gentlemanliness as exhibited by the patrons of private-saloon bars. Mr Verloc approached that ideal; he attained it, in fact. "Of course, we'll take over your furniture, mother," Winnie had remarked. The lodging-house was to be given up. It seems it would not answer to carry it on. It would have been too much trouble for Mr Verloc. It would not have been convenient for his other business. What his business was he did not say; but after his engagement to Winnie he took the trouble to get up before noon, and descending the basement stairs, make himself pleasant to Winnie's mother in the breakfast-room downstairs where she had her motionless being. He stroked the cat, poked the fire, had his lunch served to him there. He left its slightly stuffy cosiness with evident reluctance, but, all the same, remained out till the night was far advanced. He never offered to take Winnie to theatres, as such a nice gentleman ought to have done. His evenings were occupied. His work was in a way political, he told Winnie once. She would have, he warned her, to be very nice to his political friends. And with her straight, unfathomable glance she answered that she would be so, of course. How much more he told her as to his occupation it was impossible for Winnie's mother to discover. The married couple took her over with the furniture. The mean aspect of the shop surprised her. The change from the Belgravian square to the narrow street in Soho affected her legs adversely. They became of an enormous size. On the other hand, she experienced a complete relief from material cares. Her son-in-law's heavy good nature inspired her with a sense of absolute safety. Her daughter's future was obviously assured, and even as to her son Stevie she need have no anxiety. She had not been able to conceal from herself that he was a terrible encumbrance, that poor Stevie. But in view of Winnie's fondness for her delicate brother, and of Mr Verloc's kind and generous disposition, she felt that the poor boy was pretty safe in this rough world. And in her heart of hearts she was not perhaps displeased that the Verlocs had no children. As that circumstance seemed perfectly indifferent to Mr Verloc, and as Winnie found an object of quasi-maternal affection in her brother, perhaps this was just as well for poor Stevie. For he was difficult to dispose of, that boy. He was delicate and, in a frail way, good-looking too, except for the vacant droop of his lower lip. Under our excellent system of compulsory education he had learned to read and write, notwithstanding the unfavourable aspect of the lower lip. But as errand-boy he did not turn out a great success. He forgot his messages; he was easily diverted from the straight path of duty by the attractions of stray cats and dogs, which he followed down narrow alleys into unsavoury courts; by the comedies of the streets, which he contemplated open-mouthed, to the detriment of his employer's interests; or by the dramas of fallen horses, whose pathos and violence induced him sometimes to shriek pierceingly in a crowd, which disliked to be disturbed by sounds of distress in its quiet enjoyment of the national spectacle. When led away by a grave and protecting policeman, it would often become apparent that poor Stevie had forgotten his address--at least for a time. A brusque question caused him to stutter to the point of suffocation. When startled by anything perplexing he used to squint horribly. However, he never had any fits (which was encouraging); and before the natural outbursts of impatience on the part of his father he could always, in his childhood's days, run for protection behind the short skirts of his sister Winnie. On the other hand, he might have been suspected of hiding a fund of reckless naughtiness. When he had reached the age of fourteen a friend of his late father, an agent for a foreign preserved milk firm, having given him an opening as office-boy, he was discovered one foggy afternoon, in his chief's absence, busy letting off fireworks on the staircase. He touched off in quick succession a set of fierce rockets, angry catherine wheels, loudly exploding squibs--and the matter might have turned out very serious. An awful panic spread through the whole building. Wild-eyed, choking clerks stampeded through the passages full of smoke, silk hats and elderly business men could be seen rolling independently down the stairs. Stevie did not seem to derive any personal gratification from what he had done. His motives for this stroke of originality were difficult to discover. It was only later on that Winnie obtained from him a misty and confused confession. It seems that two other office-boys in the building had worked upon his feelings by tales of injustice and oppression till they had wrought his compassion to the pitch of that frenzy. But his father's friend, of course, dismissed him summarily as likely to ruin his business. After that altruistic exploit Stevie was put to help wash the dishes in the basement kitchen, and to black the boots of the gentlemen patronising the Belgravian mansion. There was obviously no future in such work. The gentlemen tipped him a shilling now and then. Mr Verloc showed himself the most generous of lodgers. But altogether all that did not amount to much either in the way of gain or prospects; so that when Winnie announced her engagement to Mr Verloc her mother could not help wondering, with a sigh and a glance towards the scullery, what would become of poor Stephen now. It appeared that Mr Verloc was ready to take him over together with his wife's mother and with the furniture, which was the whole visible fortune of the family. Mr Verloc gathered everything as it came to his broad, good-natured breast. The furniture was disposed to the best advantage all over the house, but Mrs Verloc's mother was confined to two back rooms on the first floor. The luckless Stevie slept in one of them. By this time a growth of thin fluffy hair had come to blur, like a golden mist, the sharp line of his small lower jaw. He helped his sister with blind love and docility in her household duties. Mr Verloc thought that some occupation would be good for him. His spare time he occupied by drawing circles with compass and pencil on a piece of paper. He applied himself to that pastime with great industry, with his elbows spread out and bowed low over the kitchen table. Through the open door of the parlour at the back of the shop Winnie, his sister, glanced at him from time to time with maternal vigilance.

Meet Mr. Verloc, who's leaving his brother-in-law in charge of his London shop as he steps out for a little walk. The brother-in-law doesn't sound all that capable of running the place, but truth is that Verloc doesn't really care all that much about his "ostensible" business. The shop is attached to his house, which is a grimy brick building in London. The windows of the shop contain pictures of naked dancing girls, as well as "packages in wrappers like patent medicines; closed yellow paper envelopes". In other words, these are packages that are really discrete, and you might already be realizing that Mr. Verloc runs some sort of pornography shop. Nice first impression on the reader, right? Imagine what people would've thought in 1907. The book describes the characters that frequent Verloc's shop, and doesn't paint a flattering picture of them. They tend to be either anxious young men or older gents with muddy clothes and their collars turned up to hide their faces. A bell at the door usually brings Mr. Verloc from out of the house and into the shop. He isn't the warmest of clerks; but hey, it's the pornography business, and customers aren't likely to care one way or the other. Sometimes, it is actually Mrs. Verloc who answers the bell. She is described as an attractive woman "with a full bust, in a tight bodice, and with broad hips". Sometimes she makes younger customers uncomfortable, and they leave after buying only a ridiculously overpriced bottle of ink. You also learn that there are "evening visitors," who come into the shop and don't buy anything, but instead "lif up the flap at the end of the counter in order to pass into the back parlour". The reader isn't quite sure who these dudes are, or why they've going into the Verloc's home, but Winnie doesn't seem to mind. Winnie's mother is an old woman who can barely move because of her swollen legs. She's had a long life, and at one point ran a boarding house in another part of London. She's been a widow for a while now, and she's happy that Winnie married Mr. Verloc, who seems like a nice man who can take care of them all. Winnie's mother remembers how Mr. Verloc used to come to her boarding house every now and then, always arriving from somewhere outside England. When he used to come, he would eat his breakfast in bed and "remain wallowing there" until noon every day. He would leave in the early afternoon and come back in the early morning, maybe three or four a.m., sounding like he'd been talking all night. The old woman doesn't really like the new neighborhood, but she is happy that her daughter is secure, along with Stevie. So why, we might already wonder, is she so worried about Stevie? Well it turns out that Winnie's brother has what people today would call a mental disability. Stevie has tried to hold jobs in the past, but he seems unable to keep them because he gets too easily distracted. On one occasion, he actually set off fireworks in the stairwell of an office building and caused a huge commotion. Winnie only found out afterwards that "two other office-boys in the building had worked upon his feelings by tales of injustice and oppression till they had wrought his compassion to the pitch of that frenzy". In other words, two smarter young men tricked Stevie into lighting off the fireworks out of protest for the terrible treatment they'd received. At this point, Conrad shows you that Stevie can be easily manipulated. Stevie doesn't seem to cause Mr. Verloc much hassle. For the most part, he stays in his room at the rear of the Verloc house and spends his days drawing circles with a pencil and mathematical compass. While he does this, Winnie "glance at him from time to time with maternal vigilance", which suggests that she's very protective of him.

booksum

CROSS-REFERENCES TO RELATED APPLICATIONS This application is a continuation of U.S. application Ser. No. 12/659,963 filed Mar. 26, 2010, which is a continuation of U.S. patent application Ser. No. 10/533,940 (issued as U.S. Pat. No. 8,006,691) filed May 4, 2005, which is a national stage application of PCT/AU04/00810, filed Jun. 21, 2004 in English, which claims the benefit of Australian Application No. 2003903139, filed Jun. 20, 2003, Australian Application No. 2003905136, filed Sep. 22, 2003, and Australian Application No. 2004901008, filed Feb. 27, 2004, each incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION This invention relates to breathable gas supply apparatus, and particularly but not exclusively to such apparatus for use in Continuous Positive Airway Pressure (CPAP) treatment of conditions such as Obstructive Sleep Apnea. It will be described herein in its application to CPAP treatment apparatus, but it is to be understood that the features of the invention will have application to other fields of application, such as mechanical ventilation and assisted respiration. BRIEF SUMMARY OF THE INVENTION The advantages of incorporating humidification of the air supply to a patient are known, and CPAP machines are known which incorporate humidifying devices. One of the objects of the invention is to provide a simple and compact breathable gas supply apparatus incorporating a humidifier which is simple and economic in its construction, compact, and easy to use. Other objects and advantages of the invention will be described throughout the specification. It is to be understood that apparatus described herein contains a number of advances on the prior art, many of which independent inventions, although they contribute together to the realisation of the general object expressed above. The apparatus described herein incorporates novel aspects of architecture which contribute to a reduction in size compared with known units having similar performance. Techniques for noise reduction and damping are described which enable such a smaller machine to have noise performance which is at least as good as known lager machines. The apparatus described achieves full integration of the humidifier with the flow generator, in the sense that air flow, electrical and, if required, data connection between the flow generator and the humidifier are provided automatically upon the physical engagement of the two devices, without the need for any other process of interconnection. In such an integrated device, provisions to guard against flowback of water from the humidifier tank to the flow generator are important, and novel sealing arrangements, and novel arrangements for minimising the occurrence of flowback while at the same time improving the uptake of water vapour in the humidifier are also described. The humidifier is readily detached and replaced on the machine, and has very few parts to be disassembled during cleaning. Also described herein are improved, modular, devices for enabling data connection with the apparatus, including the connection of data storage devices such as memory cards, smart cards, communication ports and the like to be selectively attached by the user or by medical personnel. The various aspects of the invention will now be described with reference to the accompanying illustrations, which show a presently proposed embodiment. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a general view of breathable gas apparatus embodying the various features of the invention; FIG. 2 shows the flow generator of the apparatus; FIG. 3 shows the humidifier unit; FIG. 4 is a cutaway view of the flow generator; FIG. 5 is a rear view of the humidifier; FIG. 6 is an exploded view of components of the flow generator; FIG. 7 is an underneath view of a chassis forming part of the flow generator; FIG. 8 is a rear view of the chassis; FIG. 9 is a general view of a fan forming part of the flow generator; FIG. 10 is an underneath view of the fan; FIG. 11 is a cross-sectional view of the fan; FIG. 12 shows the humidifier in partly disassembled state; FIG. 13 is an underneath view of the tank of the humidifier; FIG. 14 is an underneath view of the tank showing an alternative valve; FIG. 15 is a view of the tank cover; FIG. 16 is an underneath view of the tank cover; FIG. 17 is an underneath view of a modified tank cover; and FIG. 18 shows an exemplary modular connector arrangement; FIG. 19 shows an exemplary cover for an exemplary modular connector arrangement; FIG. 19A shows an exemplary modular connector arrangement with the exemplary cover of FIG. 19 ; FIG. 20 shows an exemplary modular connector arrangement; and FIG. 21 shows an exemplary modular connector arrangement. DETAILED DESCRIPTION OF THE INVENTION The illustrated apparatus comprises a flow generator 50 and a humidifier 150, shown in their assembled condition in FIG. 1. As shown in FIG. 2, the flow generator engages with the separable humidifier at an engagement face 52, from which protrudes an air connector 53 for the delivery of air from the fan to the humidifier container, and electrical connectors 54 for the delivery of power to the humidifier heater described below. The face 52 also carries a pair of slots 55 which are engaged by corresponding tongues 156 provided on the humidifier engagement face 157 ( FIG. 5 ) by which the flow generator 50 and humidifier 150 are connected together, as will be described in more detail below. Externally, the flow generator 50 is also provided with an LCD screen 58 and associated keys 59 by which the user can set the operating parameters of the unit. The flow generator 50 has an external case of rigid plastics material moulded in two parts, a top case 60 and a bottom case 61. The lower edge of the top case 60 is stepped and flanged at 62 to mate with the periphery of the bottom case 61. Overmoulded with the rigid plastics body of the bottom case 61 is a rubber sealing flange 63, which locates between and seals against the cases 60 and 61 on the one hand, and the outer surface of a chassis 64 described further below. Formed in the bottom case 61 by walls which join the outer wall of the case are the lower portions 65 and 67 of, respectively, a power supply cavity and a resonator cavity. The upper portions 66 and 68 of these cavities are formed in the chassis 64, described below. The chassis 64 is formed with a peripheral wall 69 flanged around its lower edge to engage with the inner periphery of the overmoulded sealing flange 63. The chassis 64 includes a downwardly extending fan cavity 70 in which is mounted the fan 90 described below. This cavity 70 is formed by moulded side walls 71 and base 72, which are formed by moulding thermoplastic around an inserted stainless steel liner 73. The fan cavity 70 opens to the upper surface of the chassis 64 to enable insertion of the fan 90, this opening being closed by a lid 74. Like the cavity 70, the lid 74 has an imbedded stainless steel plate insert moulded within a thermoplastics material, and at its edges the lid is provided with co-molded elastomer sealing edges. The formation of the cavity 70 by insert moulding from differing materials provides very effective acoustic damping, as does the combination by co-moulding of the hard and soft plastics described already and further described below. In this aspect of the present invention, the use of co-moulding or overmoulding in the combination of materials of different, preferably widely different, stiffness and different, preferably widely different, density has been found to be particularly advantageous in providing acoustic damping. The upper portion 66 of the power supply cavity is formed by a side wall 75 extending downwardly from the roof of the chassis 64, which sealingly engages the opposed wall of the lower portion 65 of this cavity. Preferably, the lower wall is provided for this purpose with a co-moulded or overmoulded rubber sealing flange similar to the sealing flange 63. The power supply compartment is thus sealed against the ingress of moisture from the interior of the unit in the case of backflow from the humidifier. Similarly, the air path is sealed from the power supply compartment. The interior is at the same time acoustically sealed from the power supply cavity, which may not be completely sealed from the exterior, due to the necessity of providing mains power input and low voltage power output to the humidifier, via connectors 54 mounted in apertures 78 and 80 respectively in the rear and front walls of the cavity, and if necessary the venting of the compartment to outside air for cooling. Supported on the top of the chassis 64, in the space formed between the chassis and the top of the top case 60 is a printed circuit board 81 carrying the electronic control components of the unit. At the rear of the board 81 an edge connector 82 and a sliding connector are accessible from a connector aperture 83 in the rear of the case 60, providing for modular connector arrangements to be described in more detail below. Also provided in the rear wall of the top case is an air inlet 84, and this communicates with an air inlet passage 85 formed in the roof of the upper portion 66 of the power supply cavity, this passage in turn opening through the inner side wall of that cavity at 87 to the air space surrounding the fan cavity 70 in the interior of the unit. Air drawn into the unit by the fan will thus pass over the roof of the power supply and thereby assist in the dissipation of heat generated by the power supply. A removable air filter body 85 A containing a replaceable filter element attaches to the inlet 84, as shown in FIGS. 2 and 6. From the air space surrounding the fan cavity 70, inlet air passes to the fan cavity via an inlet tube 88 depending from a horizontal extension of the side wall 71 of the fan cavity. The fan cavity and the space surrounding it and enclosed by the upper and lower cases form a pair of serially connected volume mufflers, and the dimensions of the inlet tube 88 and the air passage 85 are chosen to optimise the noise attenuation produced by these mufflers, within the constraint of avoiding unacceptable air flow restriction. It will now be convenient to describe the features of the fan, which are shown in FIGS. 9 to 11. The fan 90 comprises a motor 91, preferably brushless DC motor, provided with a coaxial impeller 92, mounted vertically within a fan housing consisting of a cover 93 and a base 94. An air inlet 95 is provided in the floor of the base 94 on the impeller axis, and cavities in the cover and base form a volute 96 which leads from the impeller to an air outlet 97. The cover and base 93 and 94 are joined by means of slotted tabs 98 which extend upwardly from the base to snap over stepped ribs 99, the tabs 98 being further located by fitting between parallel ribs on the cover 93. The joint between the cover 93 and the base 94 is sealed by an elastomeric sealing ring 101. The bottom surface of the fan housing base 94 is provided with radial stiffening ribs 102, and overmoulded to the base 94 is an elastomer damping member 103 which covers that bottom surface between the ribs 102, and extends around the edge of the base by a flange portion 104 and peripherally spaced tabs 105. By overmoulding to the rigid plastic base 94 an elastomer of much lower stiffness and much lower density substantial acoustical damping is provided to the fan housing. Moulded integrally with the rigid plastics portion of the fan housing base are feet 106 which extend through the overmoulded elastomer damping member 103 to receive helical mounting springs (not shown) by which the fan is mounted on the base 72 of the fan cavity. The degree of size reduction which is an objective of the present invention requires great care to be taken to minimise the transmission of noise and vibration, particularly from the motor and the impeller of the fan 90. The mounting springs are therefore chosen to ensure minimal transmission of the vibration frequencies encountered during operation. This is achieved by choosing the springs with reference to the mass of the fan 90, such that the natural frequency of the system comprising the springs and the fan is at least approximately one tenth of the vibration frequency encountered when the motor is running at its lowest operating speed. The air outlet 97, upon the introduction of the fan into the fan cavity, is connected by means of a thermoplastic elastomer coupling member with an air outlet passage 109 which extends from the side wall of the fan cavity to a connecting nozzle 110 extending through an aperture 111 provided for this purpose in the front face of the flow generator. The fan 90 therefore floats within its cavity 70 in the chassis 64 with minimum acoustic coupling to the remainder of the flow generator. The characteristics of the mounting springs and the coupling member are chosen to minimize the transmission of characteristic vibration frequencies of the fan. The air outlet passage 109 is formed in the roof of the upper portion 68 of the resonator cavity. Holes 112 communicating with the resonator cavity are provided in the floor of the passage 109 where it crosses this cavity, which acts in the manner of a Helmholtz resonator. By adjusting the dimensions and number of the holes 112, the frequency response of the resonator can be adjusted for optimum noise cancellation. If desired, a second Helmholtz resonator cavity can be provided opposite the upper portion 68 of the resonator cavity, if the dimensions of the top case 60 allow this. The novel use of Helmholtz resonators for noise attenuation contributes to the success in achieving significant size reduction in the flow generator of the present invention. As shown in FIG. 12, the humidifier 150 comprises a base unit 151 designed for simple attachment to and detachment from the flow generator 50, and a tank 152 which is similarly attachable to and detachable from the base unit. The rear face of the base unit 151 has a peripheral flange 153 which seats in a corresponding peripheral recess 113 surrounding the front face of the flow generator 50 when the two units are brought together by linear movement towards each other. The tongues 156 are moveable vertically and resiliently urged downwardly, so that these tongues engage in the slots 55 and snap home to engage the two units by means of the downwardly extending fingers 158 at the ends of the tongues. An air flow passage 160 passes through the humidifier engagement face 157 and opens to the front wall of the base unit. This passage is surrounded at the rear wall with a cylindrical connecting portion 161 which receives the nozzle 110 of the flow generator upon engagement of the two units. The inner surface of the portion 161 is provided with a sealing device such as a layer of elastomer or other soft resilient material. The rear face of the base unit also carries a connector 162, in this embodiment a pair of flat male blade connectors, for engagement with a mating connector on the front face of the flow generator, to provide power to the humidifier heater from the power supply in the power supply cavity. Although not shown in the illustrated embodiment, the respective faces may also carry further interconnecting devices, where other electrical or data connections are required to be established between the flow generator and the humidifier or downstream devices including the air conduit or the mask. Such devices may take the form of optically coupled devices, or connectors of other suitable kinds. The use of such an opto-coupling connector enables the implementation of a simple protocol for communications between the flow generator and the humidifier. For example, the current flow levels of the flow generator can be sent to the humidifier controller which then adjusts the operation of the humidifier according to a predetermined algorithm. Within the humidifier base unit 151 but not shown here is provided a variable power supply for a heating element which heats a circular metal pad 163. A control knob 164 is provided on the upper surface of the unit for adjustment of the heat supplied to the pad 163. A semicircular wall 165 surrounds the rear part of the pad 163, and carries at its upper edge an inwardly directed flange 166. The pad 163 stands proud of the surrounding base surface 168. It will be observed that the air flow passage 160 opens to the front face of the base unit at the foot of a circular recess 167 of larger diameter, corresponding to the diameter of the tank air inlet 175 described below. The effect of this is to provide a vertical offset between the air flow passage 160 and the inlet 175, with the former lower than the latter in the normal orientation of the unit. This configuration assists in the prevention of backflow as will be described below. It is to be observed that the axial offset in question could be achieved in other ways. The recess 167 is provided with a sealing layer of elastomer or other sealing material. The tank 152 comprises a cover 170 which is preferably of a transparent plastics material, a metal base 171 preferably of stainless steel, a base flange 172 which functions to couple the cover and the base, and a sealing gasket 173 which locates between the base of the cover and the metal base 171. The periphery of the base flange 172 is dimensioned to slide into engagement with the wall 165 of the base unit and beneath the flange 166 to engage the tank with the base unit, and the tank cover 170 is provided with a cylindrical air inlet 175 extending from its side wall. The inlet 175 is dimensioned to fit sealingly within the recess 167 when the tank is engaged with the base unit as described above and as shown in FIG. 3. An air outlet 176 extends upwardly from the roof of the cover 170 for connection with an air hose for the delivery of humidified air to the patient. The metal base 171 seats within the base flange 172 which is provided with a central aperture, so that the bottom of the metal base 171 is exposed to contact the pad 163 when the tank is engaged with the base unit. The metal base 171 is thus heated by the heating element of the base unit. To assist in achieving good heat transfer between the pad 163 and the base 171, the former is resiliently biased upwardly, for example by means of a spring or springs (not shown). This has the further advantage of providing for positive retention of the tank in the base unit, by providing around the central aperture in the base flange, a downwardly directed rim (not shown) which will initially depress the heating plate as the tank is moved into position on the base unit, and which forms a central space into which the heating plate moves under its spring pressure, upon full engagement of the tank with the base unit. In alternative embodiments not illustrated here, the tank may be provided with locking detents for retention on the base unit. The lower edge of the cover 170 and the inner edge of the base flange 172 are provided with bayonet type engagement formations 177 and 178 respectively, so the tank components can be assembled and disassembled simply by relative rotation of the cover and the base flange. To assist in this operation, a peripheral groove 179 is provided in the base of the base flange 172, and this groove is interrupted at intervals by finger-engaging bridges 180. The inner wall of the groove 179 protects the user&#39;s fingers against accidental contact with the metal base 171, in case removal of the cover is carried out while the base is still hot. The tank is intended to be filled via the air outlet 176, and the apparatus may be provided with a filling bottle with a spout dimensioned for a convenient fit with that outlet. Such a bottle may be provided with a spout of the kind incorporating an air bleed passage which will allow the tank to fill to the correct predetermined height. In alternative embodiments, other filling arrangements may be employed. The correct filling height is also indicated by filling level graduations 184 scribed or otherwise marked on the wall of the cover 170. As will be seen in FIG. 16, the air inlet 175 of the cover 170 extends within the cover in the form of an arcuate passage 181, to open to the interior of the cover at a point beyond, in the direction of air flow, the outlet 176. The open end 183 of the passage 181 is directed obliquely towards the inner wall of the cover. The outlet 176 is, furthermore, between the convexly curved side of the passage 181. This configuration has several important consequences. Firstly the curvature of the passage 181 and the oblique orientation of its outlet 183 will induce a swirling action on the air mass within the tank, as the air moves around the tank to escape from the outlet 176. This swirling action will enhance the uptake of water vapour from the water contained in the tank. Secondly the configuration minimises the risk of water from the tank flowing back into the air inlet passage should the tank be tilted while containing water. Whenever the orientation of the tank is such that the air outlet 176 is below the outlet 183, water will flow into the air outlet before it will flow into the inlet passage, and whenever the air outlet 176 is above the outlet 183, then except in the case of inversion of the tank, water will not escape via the arcuate passage 181 unless the tank has been filled with a volume of water which is greater than that which is contained within the sector of the tank below a tangent to the convex surface of the passage 181. This can be avoided by appropriate setting of the heights of the filling level graduations 184. Should water escape into the passage 181 due to inversion of the humidifier while it is engaged with the flow generator, its path to the air flow passage 160 will be blocked by the dam formed by the face of the recess 167, which will then be below the air flow passage 160. FIG. 17 shows a modified form of tank cover in which a downwardly extending wall 187 is provided across the end of the arcuate passage 181, this wall extending in a curved wall 188 beyond the outlet 183. The curved wall 188 assists in the formation of a swirling air flow within the tank, while both walls 187 and 188 tend to protect the outlet 183 against wave action within the tank during transport. If desired, further security against backflow can be provided by locating a non-return valve at an appropriate point. An example of this is shown in FIG. 14, where a valve comprising a flexible membrane 185 supported on a spider 186 is placed in the mouth of the tank air inlet 175. In the illustrated embodiment the arcuate passage 181 is shown as a low profile passage of substantially rectangular cross-section. An alternative approach is to continue this passage as a cylindrical passage having a diameter similar to that of the air passage leading from the flow generator to the humidifier. The advantage of this will be to avoid the introduction of impedance to the flow of air through the humidifier. Generally speaking it is desirable to minimise pressure drop through the humidifier, to avoid interfering with diagnostic or monitoring functions in the flow generator, for example the detection of snoring, which require the detection of sound transmitted back through the system from the patient. The enhanced uptake of water vapour achieved by inducing the swirling of air as it passes through the tank enables, in an alternative embodiment of the invention, the elimination of the heating of the water in the tank 152. In such an embodiment the heating element and its controls, and the heat transfer components including the pad 163 and the metal tank base 171 are eliminated, and the humidifier becomes a simpler, passive, device. FIGS. 18 to 21 show various forms of modular data connections foreshadowed earlier, utilizing the connector aperture 83 in the rear of the flow generator housing. The connector aperture 83 is provided in the wall of a rectangular recess 115. An arcuate depression 123 is provided in the upper surface of the unit above the recess 115 to facilitate removal of closure elements from the depression, as described below. Where the flow generator in question is not intended to be employed with any data connection, the connector aperture 83 is closed off by a blank closure element 117, shaped to fit into the recess 115. This element snaps into the recess by means of lower tabs 118 and an upper tab 119 which fit corresponding depressions such as 122 in the walls of the recess 115, to close the connector aperture 83 and conform to the contours of the surrounding surface of the unit. Complementarily shaped closure elements can be provided for the reception of different kinds of data devices. Shown in FIG. 20 is an element 116 a provided with a slot for the reception of a smart card 120. The element 116 a or the printed circuit board itself may carry the necessary smart card socket. Shown in FIG. 21 is an element 116 b provided with a DB type data socket. In this case the element 116 b is contoured to provide a lower front recess 121 to facilitate gripping of the associated plug. Other forms of element 116 can be provided to enable the connection of devices such as memory cards and pre-programmed devices as required. This facility furthermore enables a wide range of devices to be integrated with the apparatus in modular fashion, for example a clock display which may utilise the system clock contained in the flow generator controller, a voice activation unit, oximetry, ECG and other diagnostic aids, a sound recorder, a light. It is emphasised that the forgoing disclosure has sought to describe many innovations in flow generator and humidifier design, and it is foreshadowed that these will be the subject of separate claims to protection in applications claiming the priority of this document. While the invention has been described in connection with what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not to be limited to the disclosed embodiment, but on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

A humidifier has a base unit with an engagement face that is configured to interface with a flow generator. The humidifier also has a tank configured to be removably received by the base unit and hold a volume of liquid. The tank has a side wall with an air inlet. The humidifier further has an air flow passage configured to receive an air connector of the flow generator at the engagement face of the base unit. The air flow passage is axially offset from a tank inlet. In addition, a cross-section of the airflow passage inlet and a cross-section of the tank air inlet are substantially perpendicular to a horizontal plane.

big_patent

SECTION 1. SHORT TITLE. This Act may be cited as the ``Nuclear Regulatory Commission Reorganization Plan Codification and Complements Act''. TITLE I--REPLACEMENT OF REORGANIZATION PLAN SEC. 101. GENERAL FUNCTIONS. (a) Functions.--Those functions of the Nuclear Regulatory Commission (in this title referred to as the ``Commission'') concerned with-- (1) policy formulation; (2) rulemaking, as defined in section 553 of title 5 of the United States Code, except that those matters set forth in 553 (a)(2) and (b) which do not pertain to policy formulation orders or adjudications shall be reserved to the Chairman of the Commission; (3) orders and adjudications, as defined in section 551 (6) and (7) of title 5 of the United States Code; and (4) approving the distribution of appropriated funds according to programs and purposes proposed by the Executive Director for Operations, shall remain vested in the Commission. A majority of the Commission may determine, in an area of doubt, whether any matter, action, question, or area of inquiry pertains to one of these functions. Any member of the Commission may request such a vote. Any member of the Commission may propose a policy matter for consideration by the Commission. All members of the Commission shall have full, unfettered, timely, and equal access to information pertaining to its functions. The performance of any portion of these functions may be delegated by the Commission to a member of the Commission, including the Chairman of the Commission (in this title referred to as the ``Chairman'') and to the staff. (b) Officers and Employees.-- (1) Officers.--With respect to the following officers or successor officers duly established by statute or by the Commission, the Chairman shall initiate the appointment, subject to the approval of the Commission, and the Chairman or a member of the Commission may initiate an action for removal, subject to the approval of the Commission by majority vote: (A) Executive Director for Operations. (B) Chief and Deputy Chief Financial Officer. (C) General Counsel. (D) Director of the Office of Commission Appellate Adjudication. (E) Secretary of the Commission. (F) Director of the Office of Public Affairs. (G) Director of the Office of Congressional Affairs. (H) Director of the Office of International Programs. (I) Chief Administrative Judge and members of the Atomic Safety and Licensing Board Panel. Any performance evaluation or rating of the officers listed in subparagraphs (A) through (I) shall be determined by a majority vote of the members of the Commission. (2) Replacement of officers.--(A) In the event of a vacancy in a position described in paragraph (1), the Chairman may designate an acting officer for a maximum of 60 days, after which any further extension must be approved by the Commission. If, at the end of 60 days, the Commission has not approved the appointment of an officer proposed by the Chairman, or the Chairman has not proposed one, any Commissioner may initiate the appointment subject to approval of the Commission. (B) With respect to the following officers or successor officers duly established by statute or by the Commission, the Chairman, after consultation with the Executive Director for Operations, shall initiate the appointment, subject to the approval of the Commission, and the Chairman, or a member of the Commission may initiate an action for removal, subject to the approval of the Commission by majority vote: (i) Director of the Office of Nuclear Reactor Regulation. (ii) Director of the Office of Nuclear Material Safety and Safeguards. (iii) Director of the Office of Nuclear Regulatory Research. (iv) Director of the Office of Nuclear Security and Incident Response. (v) Director of the Office of New Reactors. (vi) Director of the Office of Federal and State Materials and Environmental Management Programs. (vii) Director of the Office of Investigations. (viii) Director of the Office of Enforcement. (3) Appointment of advisory committee on reactor safeguards.--The Chairman or a member of the Commission shall initiate the appointment of the Members of the Advisory Committee on Reactor Safeguards, subject to the approval of the Commission. The provisions for appointment of the Chairman of the Advisory Committee on Reactor Safeguards and the term of the members shall not be affected by the provisions of this title. (4) Delegation of staff supervision functions.--The Commission shall delegate the function of appointing, removing, and supervising the staff of the following offices or successor offices to the respective heads of such offices: Executive Director for Operations, General Counsel, Secretary of the Commission, Chief Financial Officer, Office of Commission Appellate Adjudication, Office of Congressional Affairs, Office of Public Affairs, and Office of International Programs. The Commission shall delegate the functions of appointing, removing, and supervising the staff of the following panels and committee to the respective Chairmen thereof: Atomic Safety and Licensing Board Panel and Advisory Committee on Reactor Safeguards. (c) Commission Member Offices.--Each member of the Commission shall appoint, remove, and supervise the personnel employed in his or her immediate office. (d) Performance of Functions.--The Commission shall act as provided by section 201(a)(1) of the Energy Reorganization Act of 1974 (42 U.S.C. 5841(a)(1)) in the performance of its functions as described in subsections (a) and (b) of this section. SEC. 102. CHAIRMAN. (a) Functions.--Except as otherwise provided in section 101, all functions of the Commission shall rest with the Chairman. The Chairman shall be the official spokesman for the Commission and, as such, shall represent the policies determined by a majority of the Commission. (b) Additional Functions.--The Chairman shall also be the principal executive officer of the Commission, and shall be responsible to the Commission for assuring that the Executive Director for Operations and the staff of the Commission (other than the officers and staff referred to in section 101 (b)(4) and (c)) are responsive to the requirements of the Commission in the performance of its functions; shall determine the use and expenditure of funds of the Commission, in accordance with the distribution of appropriated funds according to programs and purposes approved by the Commission; shall present to the Commission for its consideration the proposals set forth in paragraph (3); and shall be responsible for the following functions, which the Chairman shall delegate, subject to the Chairman's direction and supervision, to the Executive Director for Operations unless otherwise provided by this Act: (1) Administrative functions of the Commission. (2) Distribution of business among such personnel and among administrative units and offices of the Commission. (3) Preparation of proposals for the reorganization of the major offices of the Commission. (4) Appointing and removing, without any further action by the Commission, all officers and employees under the Commission other than those whose appointment and removal are specifically provided for by section 101 (b) and (c). (c) Governing Principles.-- (1) In general.--The Chairman as principal executive officer and the Executive Director for Operations shall be governed by the general policies of the Commission and by such regulatory decisions, findings, and determinations, including those for reorganization proposals, budget revisions, and distribution of appropriated funds, as the Commission may by law, including this title, be authorized to make. (2) Full and current information.--The Chairman and the Executive Director for Operations shall have joint responsibility insuring that the Commission is fully and currently informed about matters within its functions. (3) Failure to act in accordance.--If a majority of Commissioners determine that the Chairman has not acted in accordance with paragraph (1) or (2), such Commissioners shall provide written notice of the determination to the President and provide copies thereof to the Committee on Energy and Commerce of the House of Representatives and the Committee on Environment and Public Works of the Senate. SEC. 103. EMERGENCY AUTHORITY. (a) In General.--Notwithstanding sections 101 and 102, the Chairman is authorized to exercise emergency authority described in paragraph (4), subject to the following limitations: (1) The Chairman may not exercise emergency authority unless and until the Chairman declares a specific emergency exists and, not later than 24 hours after such declaration, notifies-- (A) the Commission, the Committee on Energy and Commerce of the House of Representatives, and the Committee on Environment and Public Works of the Senate, in writing; and (B) the public. (2) The Chairman may only exercise emergency authority in response to-- (A) an imminent safety threat pertaining to a facility or materials licensed or regulated by the Commission; or (B) a determination by the Secretary of Homeland Security, the Secretary of Energy, the Secretary of Transportation, the Director of the Federal Bureau of Investigation, the Director of the Central Intelligence Agency, or the Director of National Intelligence of an imminent security threat to a facility or materials licensed or regulated by the Commission. Where authority is exercised pursuant to this section, public notification may be delayed provided that the Chairman determines that prior public disclosure would constitute a risk to public health and safety and so notifies the Commission, the Committee on Energy and Commerce of the House of Representatives, and the Committee on Environment and Public Works of the Senate. (3) The Chairman may only exercise emergency authority for the duration of the emergency or 30 days, whichever is less. The Commission may approve extensions of that time. Each extension is limited to 30 days and requires notification of the public, the Committee on Energy and Commerce of the House of Representatives, and the Committee on Environment and Public Works of the Senate. (4) The Chairman's emergency authority includes the functions of responding to, issuing orders respecting, advising United States civil authorities and the United States public about, and directing and coordinating actions relative to such emergency incident. (b) Delegation.--The Chairman may delegate the authority to perform such emergency functions, in whole or in part, to any of the other members of the Commission. Such authority may also be delegated or redelegated, in whole or in part, to the staff of the Commission. (c) Consultation.--To the extent practicable, the Chairman shall consult with the full Commission on any regulatory or policy actions to be taken under an emergency. Such consultations shall be exempt from the requirements of section 552b of title 5, United States Code (commonly referred to as the ``Government in the Sunshine Act''). (d) Guidelines and Notice.--In acting under this section, the Chairman, or other member of the Commission delegated authority under subsection (b), shall conform to the policy guidelines of the Commission. (e) Termination of Emergency.--Upon termination of the emergency, the Chairman shall immediately notify the Commission, the public, the Committee on Energy and Commerce of the House of Representatives, and the Committee on Environment and Public Works of the Senate. (f) Report.--Within 30 days following the conclusion of the emergency, the Chairman, or the member of the Commission or member of the staff delegated the emergency functions under subsection (b), shall render a complete report of all actions taken during the emergency, specifically delineating actions taken utilizing the authority provided in this section, to the Commission, the Committee on Energy and Commerce of the House of Representatives, and the Committee on Environment and Public Works of the Senate. (g) Commission Procedures.--Not later than 90 days after the date of enactment of this Act, the Commission shall revise its procedures to comply with the requirements of this section. Such revision shall define the roles of the Commissioners during an emergency, specifying-- (1) complete access to records and information relating to actions taken during the emergency; (2) complete access to Commission staff involved in the management of the emergency; (3) complete access to the location or locations where decisions are made during the emergency; and (4) participation in decisions that may affect Commission actions and policies beyond the response to a particular emergency to the extent practicable. SEC. 104. REPORTING. (a) Delegation; Direct Communication.--The Chairman may make such delegations and provide for such reporting as the Chairman deems necessary, subject to provisions of law. Any officer or employee under the Commission may communicate directly to the Commission, or to any member of the Commission, whenever in the view of such officer or employee a critical problem, or matter of public health and safety or common defense and security, is not being properly addressed. (b) Executive Director for Operations.--The Executive Director for Operations shall report for all matters to the Chairman. (c) Functions.--The Directors of Nuclear Reactor Regulations, Nuclear Material Safety and Safeguards, and Nuclear Regulatory Research shall report to the Executive Director for Operations. (d) Direct Reporting.--The heads of the Commission level offices or successor offices, of General Counsel, Secretary of the Commission, Commission Appellate Adjudication, Congressional Affairs, Public Affairs, International Programs, Atomic Safety and Licensing Board Panel, and Advisory Committee on Reactor Safeguards shall report directly to the Commission and the Commission shall receive such reports. SEC. 105. RESCISSION OF REORGANIZATION PLAN APPROVAL. Approval of Reorganization Plan No. 1 of 1980 (5 U.S.C. App. 1) is rescinded. TITLE II--MISCELLANEOUS SEC. 201. CERTIFICATION OF DOCUMENTS TRANSMITTED TO CONGRESS. A letter or other document transmitted by the Nuclear Regulatory Commission, on behalf of the full Commission, to a member of Congress in his or her capacity as chairman or ranking minority member of a Committee of Congress, shall include a certification that the letter or document is being sent to both the Chairman and ranking minority member of that Committee in accordance with established Commission procedures. SEC. 202. TIME LIMITS FOR COMMISSION REVIEW OF ATOMIC SAFETY AND LICENSING BOARD DECISIONS. When reviewing the decisions and actions of the Atomic Safety and Licensing Board, the Commission shall follow the following procedures: (1) Each Commissioner shall vote on the matter not later than 90 days after receipt of final briefs, after which time the Commission shall not further delay a decision. Once a majority position is established, the Secretary shall notify in writing any Commissioners who have not voted that a majority position has been established. Any Commissioners who have not yet voted shall vote within three days of the Secretary's notice or be considered by the Secretary as not participating. (2) Not later than 30 days after a majority position is established, the Commission shall publish any resulting decision, including adjudicatory orders and direction to agency staff. If a majority position is not established due to a tied vote, not later than 30 days after Commission voting is complete, the Commission shall publish any resulting decision, including adjudicatory orders and direction to agency staff. SEC. 203. ALLEGATIONS OF WRONGDOING. (a) Referral to Inspector General.--Not later than 90 days after the date of enactment of this Act, the Nuclear Regulatory Commission shall revise its procedures to ensure that any allegation of wrongdoing on the part of the Chairman of the Commission is immediately referred to the Inspector General of the Commission. (b) Supervision of Inspector General.--During the pendency of any investigation by the Inspector General of the Chairman with respect to an allegation described in subsection (a), the Chairman shall delegate responsibility for supervising the Inspector General to a member of the Commission other than the Chairman, consistent with the Inspector General Act of 1978. SEC. 204. APPROVAL OF COMMISSIONER TRAVEL. The Chairman of the Nuclear Regulatory Commission shall authorize all international travel requested by other members of the Commission for official business unless the Chairman submits a notice of disapproval to the full Commission specifying the basis for the disapproval. The notice of disapproval shall be submitted within 5 days after the travel is requested or the travel shall be deemed approved. SEC. 205. IMPLEMENTATION. Except as otherwise specified in this Act, the Commission shall revise its procedures to conform to this Act within 180 days of its date of enactment.

Nuclear Regulatory Commission Reorganization Plan Codification and Complements Act - Codifies and expands the Reorganization Plan No. 1 of 1980 governing the administration of the Nuclear Regulatory Commission (NRC). Identifies approval of the distribution of appropriated funds according to programs and purposes proposed by the Executive Director for Operations, in addition to functions concerned with policy formulation, rule making, and orders and adjudications, as functions that remain vested in the Commission. Revises provisions of such Reorganization Act relating to: (1) the appointment and replacement of NRC officers and employees, (2) the role of the NRC Chairman, (3) the scope of the emergency authority of the NRC Chairman, and (4) NRC reporting procedures. Sets forth NRC policy with respect to: (1) certification of documents transmitted to Congress, (2) time limits for review of Atomic Safety and Licensing Board decisions and actions, (3) allegations of wrongdoing on the part of the NRC Chairman, and (4) approval of international travel requests by NRC members.

billsum

Synaptic long-term potentiation (LTP) at spinal neurons directly communicating pain-specific inputs from the periphery to the brain has been proposed to serve as a trigger for pain hypersensitivity in pathological states. Previous studies have functionally implicated the NMDA receptor-NO pathway and the downstream second messenger, cGMP, in these processes. Because cGMP can broadly influence diverse ion-channels, kinases, and phosphodiesterases, pre- as well as post-synaptically, the precise identity of cGMP targets mediating spinal LTP, their mechanisms of action, and their locus in the spinal circuitry are still unclear. Here, we found that Protein Kinase G1 (PKG-I) localized presynaptically in nociceptor terminals plays an essential role in the expression of spinal LTP. Using the Cre-lox P system, we generated nociceptor-specific knockout mice lacking PKG-I specifically in presynaptic terminals of nociceptors in the spinal cord, but not in post-synaptic neurons or elsewhere (SNS-PKG-I−/− mice). Patch clamp recordings showed that activity-induced LTP at identified synapses between nociceptors and spinal neurons projecting to the periaqueductal grey (PAG) was completely abolished in SNS-PKG-I−/− mice, although basal synaptic transmission was not affected. Analyses of synaptic failure rates and paired-pulse ratios indicated a role for presynaptic PKG-I in regulating the probability of neurotransmitter release. Inositol 1,4, 5-triphosphate receptor 1 and myosin light chain kinase were recruited as key phosphorylation targets of presynaptic PKG-I in nociceptive neurons. Finally, behavioural analyses in vivo showed marked defects in SNS-PKG-I−/− mice in several models of activity-induced nociceptive hypersensitivity, and pharmacological studies identified a clear contribution of PKG-I expressed in spinal terminals of nociceptors. Our results thus indicate that presynaptic mechanisms involving an increase in release probability from nociceptors are operational in the expression of synaptic LTP on spinal-PAG projection neurons and that PKG-I localized in presynaptic nociceptor terminals plays an essential role in this process to regulate pain sensitivity. Plasticity in peripheral nociceptors and their synapses with spinal neurons has been proposed as a cellular basis for the development and maintenance of pain hypersensitivity following peripheral inflammation or nerve injury [1]–[3]. Activation of nociceptive nerve afferents at frequencies relevant to pathological pain states can trigger long-term potentiation (LTP) at spinal synapses between nociceptor terminals and spinal neurons projecting nociceptive information to the brain [4], [5]. Importantly, this form of synaptic plasticity can be evoked by asynchronous activation of nociceptors in vivo [5], occurs in humans [6], and is functionally associated with a sensation of exaggerated pain [5], [6]. Although there is evidence for a requirement of post-synaptic calcium-dependent mechanisms in the induction of LTP at this synapse [5], the precise mechanisms underlying the expression of spinal LTP are not entirely clear [7]. Synaptic LTP evoked by natural, asynchronous low-rate discharges in C-nociceptors on spino-PAG neurons was recently shown to constitute a very fitting correlate of spinal amplification phenomena underlying inflammatory pain [5], [7]. This form of synaptic change has been reported to involve activation of NMDA receptors, NO release, and synthesis of cGMP [5], [7]. However, which of the diverse targets of cGMP come into play at this synapse and how they mechanistically bring about long-lasting changes in the transfer of nociceptive information between the nociceptors and spinal neurons projecting to the brain is not understood so far. Furthermore, very little is known about exactly how neural circuits involved in pain processing are modulated by cGMP and which cellular and molecular processes underlie these changes. Studies on several different biological systems have shown that cGMP regulates multiple cellular targets, including diverse cGMP-gated ion channels, such as cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, the cGMP-dependent protein kinases, PKG-I/cGK-I and PKG-II/cGK-II, as well as diverse phosphodiesterases (PDEs) [8], [9]. Nearly all of these molecular targets of cGMP are expressed in nociceptive pathways and may potentially contribute to the key role of cGMP in synaptic potentiation in the spinal cord. Amongst these targets, PKG-I has emerged as a key mediator of cGMP functions in smooth muscle and platelet function [8]. The α-isoform of PKG-I has been reported to be expressed very highly in the primary sensory neurons in the dorsal root ganglia (DRG) over developmental [10] and adult stages [11], and several regions in the brain and the spinal cord also express PKG-I [12], [13]. Pharmacological and genetic studies in global, constitutive mutant mice have linked PKG-I to the development of the nociceptive circuitry as well as to spinal mechanisms of hyperalgesia [14]. Based upon this background, this study was designed with two goals in mind. First, it addressed the potential involvement of presynaptic mechanisms in the expression of synaptic potentiation on spinal projection neurons, which has not been explored or described previously. Second, it aimed to explore a potential role for PKG-I localized presynaptically in the spinal terminals of nociceptors in spinal potentiation and to clarify cellular and molecular mechanisms underlying these processes. We reasoned that the use of a conditional, region-specific gene deletion strategy to specifically manipulate presynaptic mechanisms might constitute an unambiguous approach towards addressing the above questions. Our results show that spinal synaptic potentiation triggered by nociceptor activation is associated with a long-lasting change in the probability of neurotransmitter release from spinal terminals of nociceptors. Using viable, developmentally normal transgenic mice lacking the PKG-I specifically in nociceptors with preserved expression in spinal neurons, brain, and all other organs, we demonstrate here that PKG-I localised in nociceptor terminals constitutes a key mediator of synaptic LTP and that its activation is functionally associated with pain hypersensitivity in vivo. Mice lacking PKG-I specifically in a primary nociceptor-specific manner (SNS-PKG-I−/−) were generated via Cre/loxP-mediated recombination by mating mice carrying the floxed prkg1 allele (PKG-Ifl/fl) [15] with a mouse line expressing Cre recombinase under control of the Nav1. 8 promoter (SNS-Cre) [16]. We have previously demonstrated that SNS-Cre mice enable gene recombination commencing at birth selectively in nociceptive (Nav1. 8-expressing) sensory neurons, without affecting gene expression in the spinal cord, brain, or any other organs in the body [16], [17]. An anti-PKG-I antibody [18] yielded specific staining in wild-type dorsal root ganglia (DRG), but not in those from global PKG-I−/− mice [19], thereby revealing Cre/loxP-mediated deletion of PKG-I in DRG of SNS-PKG-I−/− mice (Figure 1A). Quantitative size-frequency analysis revealed that a majority of DRG neurons expressing PKG-I in wild-type mice are small-diameter neurons, which show a near complete loss of PKG-I expression in SNS-PKG-I−/− mice (Figure 1B; p<0. 001). In contrast, a few large-diameter neurons showed low levels of anti-PKG-I immunoreactivity in DRGs of PKG-Ifl/fl, which was entirely retained in SNS-PKG-I−/− mice (Figure 1A and B). Confocal analysis of dual immunofluorescence experiments revealed PKG-I immunoreactivity in nearly all Isolectin-B4 (IB4) -labelled non-peptidergic nociceptors and substance P-expressing peptidergic nociceptors in PKG-Ifl/fl mice, both of which are selectively lost in SNS-PKG-I−/− mice (typical examples in Figure 1C and quantitative summary in Figure 1D). In contrast, large-diameter neurofilament-200-immunoreactive neurons entirely retained PKG-I expression in the SNS-PKG-I−/− mice (Figure 1C, D). Taken together, these results show that PKG-I is normally expressed in nearly all nociceptors and is selectively lost from these neurons, but not from tactile-responsive and proprioceptive DRG neurons, in SNS-PKG-I−/− mice. We found that PKG-I expression is entirely unaltered in the brains of SNS-PKG-I−/− mice (an example of expression in cerebellar purkinje neurons is shown in Figure 1E, right panel), whereas global PKG-I−/− mice demonstrated a complete loss of anti-PKG-I immunoreactivity (Figure 1E, right panel). In the spinal cord of SNS-PKG-I−/− mice, anti-PKG-I immunoreactivity was decreased selectively in the superficial dorsal laminae, which represent termination zones of the nociceptive afferents, as would be expected from SNS-Cre-mediated gene deletion in nociceptors (Figure 1E, left panel). In contrast, neurons in the spinal cord entirely maintained immunoreactivity for PKG-I and appeared particularly conspicuous (arrowheads in Figure 1E, left panel) due to the loss of PKG-I labelling in afferent terminals in SNS-PKG-I−/− mice. Furthermore, anti-Cre immunohistochemistry as well as Western blot analysis with anti-PKG-I antibody confirmed that SNS-PKG-I−/− mice show a DRG-specific loss of PKG-I while retaining expression in the spinal cord and brain (Figure S1). In contrast to global PKG-I−/− mice, which typically demonstrate lethality in the first few weeks of life, SNS-PKG-I−/− mice were normal, fertile, and showed a normal life expectancy. In contrast to defects reported in global PKG-I−/− mice [10], SNS-PKG-I−/− mice showed normal early targeting of TrkA-expressing primary afferents arising from the DRG (arrowheads in Figure 1F, upper panels) in the developing spinal dorsal horn at embryonic day 13 (E13). Unlike global PKG-I−/− mice [10], SNS-PKG-I−/− mice did not show defects in T-branching of DiI-labelled primary afferents in the spinal cord over embryonic developmental stages (arrows in Figure 1F, lower panels). Similarly, central and peripheral patterning of peptidergic or non-peptidergic nociceptors was normal in adult SNS-PKG-I−/−mice, as revealed by immunostaining for substance P and binding to IB4, respectively, in the spinal dorsal horns and skin (Figure S2A). Because peptidergic mechanisms have been suggested to play an important role in spinal LTP [5], we ascertained that SNS-PKG-I−/− mice are not different from control mice with respect to the abundance of substance P in the spinal circuitry. Control and knockout mice exhibited the same prevalence of substance P-immunoreactive cells within DRG (33%±5% versus 29%±3%, respectively), which were not significantly different from each other (p>0. 05, Student' s t test). Moreover, the level of substance P immunoreactivity was similar in the superficial spinal dorsal horn across genotypes (mean intensities in PKG-Ifl/fl mice and SNS-PKG-I−/− mice were 50±3 and 51±3 arbitrary units, respectively). Importantly, confocal microscopy revealed normal density of synapses between substance P-containing nociceptive afferents and PSD-95-positive puncta (representing postsynaptic aspects of glutamatergic synapses) in the spinal dorsal horns of SNS-PKG-I−/− mice as compared to PKG-Ifl/fl mice (examples and quantification in Figure S2B). Finally, we addressed the internalization of NK1 receptors on spinal lamina I neurons following peripheral nociceptive stimulation in vivo, which has been demonstrated to be a clear indicator of nociceptive activity-induced synaptic release of substance P [20]. As shown in Figure S2C, application of a 52°C heat stimulus for 20 s to the plantar paw surface led to internalization of NK1 receptors in lamina I neurons of L3/L4 segments to a similar extent in SNS-PKG-I−/− and PKG-Ifl/fl mice (quantification in Figure S2D). Unlike global PKG-I−/− mice [14], SNS-PKG-I−/− mice showed a normal lamination of the spinal cord over early postnatal stages (Figure S2E). Thus, the multiple developmental defects in the patterning of sensory afferents and spinal lamination that have been reported in global PKG-I−/− mice were not observed in SNS-PKG-I−/− mice. To address activity-dependent plasticity at spinal synapses, we recorded C-fiber-evoked synaptic LTP on spinal lamina I neurons projecting to the periaqueductal grey (PAG), which were retrogradely labelled upon stereotactic injection of DiI in the PAG (the experimental scheme is shown in Figure 2A and an example of a labelled cell is shown in Figure S3A) [5]. In spinal-PAG projection neurons of wild-type mice, a conditioning low frequency stimulation of 2 Hz for 2 min produced synaptic LTP of monosynaptic C-fiber evoked EPSCs by more than 200% at 30 min (Figure 2B). LTP at these synapses was preserved in the presence of strychnine and gabazine, which block glycinergic and GABAergic inhibitory neurotransmission, respectively (Figure 2B). Similar results were obtained upon using another standard blocker of GABAergic neurotransmission, bicuculline, in combination with strychnine (Figure S3B). Hence, LTP does not manifest due to primary afferent depolarization mediated by presynaptic GABA receptors or disinhibition of the postsynaptic neuron. To test whether LTP requires a postsynaptic function of PKG-I, we dialyzed standard PKG-I inhibitors, such as the non-permeant peptide inhibitor RKRARKE [21], [22] or KT5823 [23], into spinal neurons via the patch pipette. These manipulations did not affect the magnitude or duration of C-fiber-evoked LTP at spino-PAG synapses (Figure 2C and Figure S3C), suggesting that PKG-I localized postsynaptically in spino-PAG projection neurons does not play a role in LTP at this synapse. To assess the role of PKG-I localized presynaptically in spinal nociceptor terminals, we then analysed PKG-Ifl/fl mice and SNS-PKG-I−/− mice. In spinal-PAG projection neurons of PKG-Ifl/fl mice, a conditioning low frequency stimulation of 2 Hz for 2 min produced LTP with a magnitude of more than 200% at 30 min and more than 300% by 60 min (typical examples of time course and EPSC traces are given in Figure 2D, E). Prior to the conditioning stimulus, baseline values of C-fiber-evoked EPSCs stayed constant over the period of recording in both genotypes (Figure 2D, E). In striking contrast to PKG-Ifl/fl mice, the conditioning stimulus did not evoke LTP in spinal-PAG projection neurons in SNS-PKG-I−/− mice (Figure 2D, E; see Figure 2F and 2G for quantitative summary at 30 min post-conditioning stimulus; p<0. 001; at least 13 neurons from each genotype were tested). For a clear interpretation of these data, it is imperative to address how basal nociceptive transmission at these synapses is affected in SNS-PKG-I−/− mice. Analysis of EPSC magnitude evoked by the first and the last pulse of the conditioning train revealed short-term depression of evoked EPSCs during the conditioning train, which was equivalent in PKG-Ifl/fl mice and SNS-PKG-I−/− mice (Figure 3A; p = 0. 95), showing that the conditioning stimulus was equally effective in mice from both groups. Furthermore, basal C-fiber-evoked EPSCs were comparable between PKG-Ifl/fl and SNS-PKG-I−/− mice. We also established detailed input-output curves representing the relationship between the intensity of dorsal root stimulation and evoked EPSCs in the absence of a conditioning stimulus and found no differences between PKG-Ifl/fl and SNS-PKG-I−/− mice (Figure 3B; p = 0. 74). Furthermore, the intensity of dorsal root stimulation required to elicit an action potential in post-synaptic spinal-PAG projection neurons was identical in PKG-Ifl/fl and SNS-PKG-I−/− mice (an example is shown in Figure 3C). The intact nature of responsiveness in spinal-PAG projection neurons was demonstrated by similarities in their activation profiles upon current injection in SNS-PKG-I−/− mice and their PKG-Ifl/fl littermates (an example is shown in Figure 3D). In all of the above experiments, quantitative analyses revealed that resting membrane potential, action potential width, delay after stimulation artefact, action potential threshold, delay for generation of first action potential (latency to first AP), as well as amplitude of after hyperpolarisation (AHP) were similar in SNS-PKG-I−/− mice and PKG-Ifl/fl mice (Figure 3E; p>0. 05 in all cases, Student' s t test). Finally, as an additional indicator of the number of fibers activated during electrical stimulation, we recorded fiber volleys in input/output measurements. Recording C-fiber volleys in the L4 and L5 dorsal roots derived from PKG-Ifl/fl and SNS-PKG-I−/− mice revealed typical responses, which increased in amplitude with increasing stimulus intensity (representative traces are shown in Figure 3F). The amplitudes of C-fiber volley responses were not significantly different between PKG-Ifl/fl and SNS-PKG-I−/− mice (stimulus-response curves are shown in Figure 3G; p>0. 05; n = 16 per genotype), demonstrating directly that the number of fibers activated upon electrical stimulation was comparable between genotypes and could therefore not explain the failure to evoke synaptic potentiation in SNS-PKG-I−/− mice. These comprehensive analyses show that a presynaptic loss of PKG-I was specifically linked to a failure of activity-dependent potentiation of transmission at synapses between nociceptors and spinal-PAG projection neurons, but not to modulation of basal synaptic transmission. Following our observation that a specific presynaptic alteration in PKG-I expression perturbed spinal LTP, we then addressed potential contributions of presynaptic mechanisms at synapses between nociceptors and spinal projection neurons [7], [24]. By recording miniature EPSCs in spinal-PAG projection neurons of control mice, we observed that quantal content varies largely, which is expected since these spinal projection neurons receive multisynaptic inputs from primary afferents as well as spinal interneurons. Furthermore, inputs arising from spinal interneurons are not expected to change in SNS-PKG-I−/− mice since the molecular perturbation is specific to nociceptor terminals. Thus, the net contribution of C-fibers to the population of mEPSCs is difficult to assess because it is unclear which fraction of mEPSCs can be attributed to C-fibers, which then makes detection of potentially small presynaptic changes highly unlikely when performing mini-analysis. To study synaptic events which could be clearly assigned to activation of presynaptic primary afferent fibers alone, we employed a protocol of minimal stimulation, setting the dorsal root stimulation parameters such that a synaptic failure rate of approximately 60% was achieved in recording solution containing 1 mM Ca2+ and 5 mM Mg2+. The failure rate remained constant over a period of at least 30 min upon repetitive test stimulation in the absence of a conditioning stimulus (55. 9%±3. 9% pre- and 57. 1%±8. 1% at 30 min). However, upon application of the conditioning stimulus, minimal stimulation using the same parameters evoked a decrease in the frequency of synaptic failures within a few minutes in slices derived from PKG-Ifl/fl mice, indicating a change in the probability of neurotransmitter release; decrease in synaptic failures was accompanied by a corresponding rise in the magnitude of C-fiber-evoked EPSCs (see Figure 4A for typical example and Figure 4C for quantitative summary of C-fiber-evoked EPSCs recorded every 15 s; n = 5; p = 0. 04). In contrast, the rate of synaptic failures did not change significantly following conditioning stimulus in slices derived from SNS-PKG-I−/− mice (see Figure 4B and 4C; n = 7; p = 0. 68). EPSC values at the end of the recording were not significantly elevated as compared to basal values in SNS-PKG-I−/− mice (25±1. 7 pA before and 21. 1±1. 2 pA at 30 min after the conditioning stimulus; p = 0. 14). In analogy to studies on hippocampal circuits, although the above evidence for a decrease in failure rate is indicative of increased presynaptic release probability, it has also been linked to unsilencing of silent synapses [25]. Therefore, to further consolidate presynaptic mechanisms, we focussed on the analysis of paired-pulse facilitation (PPF), which represents a short-lasting increase in the second evoked EPSP when it follows shortly after the first and is well accepted as an indication of presynaptic mechanisms of long-term potentiation in the hippocampus [26]. In hippocampal CA1 neurons, PPF can increase as well as decrease in conjunction with LTP in a manner inversely proportional to the PPF prior to the conditioning stimulus [26]. Indeed, we obtained similar results in recordings at spinal synapses between C-fibers and spinal-PAG projection neurons. In spinal slices derived from mice of both genotypes, we found evidence for PPF as well as paired-pulse depression (PPD) prior to the LTP-inducing conditioning stimulus (typical traces are shown in Figure 4D). Whereas a majority of neurons derived from PKG-Ifl/fl mice demonstrated a clear change in PPF or PPD following conditioning stimulus, neurons derived from SNS-PKG-I−/− mice did not (see examples in Figure 4D). We then plotted the paired-pulse ratio (PPR) of the entire cohort of recorded neurons at 30 min after conditioning stimulation as a function of the basal PPR recorded prior to the conditioning stimulus (Figure 4E, F). This analysis revealed that neurons in PKG-Ifl/fl mice with larger basal values of PPR prior to the conditioning stimulus (indicated by filled round symbols in Figure 4E) consistently showed a decrease in PPR after the conditioning stimulus (Figure 4D), which is indicative of an increase in the probability of release (PR, Figure 4E). This drop in PPF following conditioning stimulation did not come about or was reduced in neurons from SNS-PKG-I−/− mice (filled round symbols in Figure 4F). A smaller cohort of synapses in PKG-Ifl/fl mice showed an increase in PPF after conditioning stimulation, but this was restricted to neurons with a low magnitude of PPF prior to the conditioning stimulus (i. e., a PPR of about 1. 1–1. 2, filled square symbols in Figure 4E) and a low expression of LTP (Figure S4A). Again, this change was not observed in the corresponding cohort of neurons in SNS-PKG-I−/− mice (filled square symbols in Figure 4F, Figure S4B). Conversely, in PKG-Ifl/flmice, higher magnitudes of LTP (i. e., between 150% and 350%, indicated by black frame in Figure S4A) were consistently associated with a decrease in PPR, which is indicative of an increase in release probability. Neither LTP nor consistent changes in PPR were observed in SNS-PKG-I−/− mice (Figure S4B). In conclusion, the failure rate analysis and PPR analysis strongly support the inference that the expression of LTP at spino-PAG synapses comes about via presynaptic mechanisms involving an increase in release probability via PKG-I. In an effort to understand the underlying molecular mechanisms, we then addressed potential substrates for the kinase activity of PKG-I in nociceptors. In particular, we reviewed known substrates of PKG-I in other biological systems and focussed on those for which we hypothesized a role in synaptic transmission. We first set up an assay system for testing involvement of PKG-I substrates in the DRG selectively upon persistent nociceptive stimulation in vivo, using Vasodilator-stimulated phosphoprotein (VASP), a classical target of PKG-I, as an indicator of PKG-I activity [8], [27]. Lysates of L4-L5 DRGs from naïve PKG-Ifl/fl and SNS-PKG-I−/− mice showed comparable levels of VASP expression (Figure S5A, basal). Within minutes after persistent nociceptive stimulation via injection of formalin in the hindpaw, L4-L5 DRGs from PKG-Ifl/fl mice showed a striking phosphorylation of VASP at Serine 239 (typical examples in Figure S5A and summary in Figure S5B, C; see Text S1 for details; p = 0. 03 as compared to basal). This was markedly reduced in formalin-injected SNS-PKG-I−/− mice (Figure S5A, B; p = 0. 18 as compared to basal SNS-PKG-I−/− mice and 0. 03 as compared to formalin-injected PKG-Ifl/fl mice). These results show that persistent activation of nociceptors leads to rapid signalling via PKG-I in DRG neurons in vivo, which is lost in SNS-PKG-I−/− mice, as expected. Using this assay system, we then addressed another key target of PKG-I, which has been mainly studied so far mechanistically in smooth muscle cells. Dephosphorylation of myosin light chains (MLC) [28] via PKG-I-dependent phosphorylation and activation of myosin light chain phosphatase in smooth muscle cells is a decisive mechanism underlying NO-mediated vasodilation [8]. Following formalin injection in the paw, we observed a strong phosphorylation of MLC in L4-L5 DRGs from PKG-Ifl/fl mice, which was found to be lacking in formalin-treated SNS-PKG-I−/− mice (Figure 5A and 5B). These differences did not arise due to differences in expression levels of MLC between SNS-PKG-I−/− mice and PKG-Ifl/fl mice (Figure 5B; Figure S5C). This finding was unexpected because it suggests a role for PKG-I in increasing MLC phosphorylation in DRG neurons, which is contrary to the classical role ascribed to PKG-I in MLC dephosphorylation. We reasoned that if our findings hold true, synthesis of cGMP ought to be a critical intermediate step in activity-dependent MLC phosphorylation in DRG neurons. Indeed, in mice pre-treated with an inhibitor of the soluble guanylyl cyclase, ODQ, and a pan-inhibitor of membrane-bound guanylyl cyclases, LY83583, via intrathecal application, formalin-induced MLC phosphorylation in L4-L5 DRGs was strongly reduced (see examples in Figure 5C; quantitative summary from three experiments is given below the Western blot). Immunohistochemistry revealed that nociceptor activation-induced increase in MLC phosphorylation occurred in the spinal termination zone of nociceptors (lamina I and II) as well as in spinal neurons (Figure 5D). Interestingly, synaptic potentiation induced by a conditioning stimulus on spinal-PAG projection neurons was abolished in the presence of ML-7, an inhibitor of MLCK (Figure 5E and Figure 5F; p = 0. 004 as compared to vehicle-treated control slices). Furthermore, consistent with our observations in SNS-PKG-I−/− mice, inhibition of MLC phosphorylation did not affect basal transmission at this synapse (Figure 5G; p = 0. 761). Thus, the PKG-I target, pMLC, is functionally linked to potentiation of synaptic transmission in nociceptive laminae. Ikeda et al. [5] have reported that inhibition of IP3R activation blocks conditioning stimulus-induced synaptic potentiation at synapses between nociceptors and spinal-PAG projection neurons. This is particularly interesting because IP3R1 contains a PKG-I-recognition motif at serine 1755 and has been reported to be phosphorylated by PKG-I in vitro, putatively leading to gain of function [29], [30]. We observed that IP3R1 is indeed a target of PKG-I in nociceptors and is functionally associated with modulation of calcium release from intracellular stores. In immunoprecipitation experiments from L4-L5 DRGs, formalin-injected PKG-Ifl/fl mice demonstrated highly enhanced serine 1755 phosphorylation of IP3R1 over the basal state; this effect was markedly reduced in DRGs obtained from formalin-injected SNS-PKG-I−/− mice (Figure 6A, B), although expression levels of IP3R1 were comparable between SNS-PKG-I−/− mice and PKG-Ifl/fl mice (Figure S5D). PKG-I-mediated phosphorylation of serine 1755 in IP3R1 has been suggested to positively modulate IP3R1 activity in heterologous test systems [30]. We observed that this function of PKG-I indeed plays an important role in modulating calcium release from intracellular stores in nociceptive neurons of the DRG. We performed Fura-2-based calcium imaging on dissociated DRG neurons derived from PKG-Ifl/fl and SNS-PKG-I−/− mice using Fluro488-conjugated Isolectin B4 (IB4-Fluro488) for live identification of small-diameter nociceptive neurons (neurons dually labelled with Fura-2 and IB4-Fluor488 are indicated by arrowheads in Figure 6C). The baseline values of the Fura2 ratios (F340/F380) were not significantly different between control mice (1. 049±0. 010) and SNS-PKG-I−/− mice (1. 040±0. 008) (p>0. 05; Student' s t test). Stimulation of calcium release via activation of Gq/11-phospholipase C-IP3R1 pathway by addition of ligands, such as bradykinin (BK) and the P2Y-receptor ligand, UTP, led to typical increases in the ratio of Fura-2 fluorescence at 340/380 nm in neurons from PKG-Ifl/fl mice, which were markedly reduced in neurons from SNS-PKG-I−/− mice (see Figure 6D for typical examples and Figure 6E for quantitative summary; p<0. 001 with respect to BK and UTP). In contrast, Fura2-labelled neurons with large-diameter somata, which were IB4-negative, did not show differences in calcium responses between PKG-Ifl/fl mice and SNS-PKG-I−/− mice (an example is indicated by arrow in Figure 6C and quantitative summary is given in Figure 6E). In contrast, rapid calcium influx caused by KCl-induced depolarisation or capsaicin-evoked influx of calcium via TRPV channels was comparable in DRG neurons derived from SNS-PKG-I−/− mice and PKG-Ifl/fl mice (Figure 6D, E; p>0. 05), showing thereby that a loss of PKG-I in nociceptive neurons is specifically linked to defects in IP3R-mediated calcium release from intracellular stores. Taken together, these biochemical and functional experiments suggest that following persistent nociceptive stimulation, PKG-I mediates potentiation of IP3R1 activity and MLC phosphorylation in sensory neurons, which is functionally linked to synaptic LTP at synapses between C-nociceptors and spinal-PAG projection neurons. We then went on to address whether these findings bear relevance to pain-related behaviour in vivo and found a functional role for PKG-I and its substrates in behavioural paradigms for spinal sensitization. As a test system, we studied the Phase II of formalin-induced nocifensive behavioural responses, which are manifest at 10–60 min after intraplantar formalin injection, for two reasons: one, this represents a widely used paradigm for studying central changes in pain processing caused by a persistent activation of nociceptors [31], and two, intraplantar formalin induces synaptic LTP on spinal projection neurons with a matching time-course [5]. Formalin-induced phase II responses were significantly reduced upon intrathecal pretreatment with 2-APB or ML-7 to the lumbar spinal cord (Figure 7A; p<0. 01 for 2-APB and ML-7 in comparison to vehicle control, respectively), implicating involvement of IP3R function and MLC phosphorylation, respectively. Similarly, SNS-PKG-I−/− mice showed markedly reduced phase II responses than PKG-Ifl/fl mice (Figure 7B; p<0. 001 as compared to PKG-Ifl/fl mice). Basal withdrawal thresholds and response latencies to acute application of paw pressure (e. g., as tested with a dynamic aesthesiometer) (Figure S6A, left panel) or thermal stimuli (e. g., a radiant infrared heat ramp) (Figure S6A, right panel), respectively, to the paw surface were found to be similar across SNS-PKG-I−/− mice and their control littermates (p>0. 05). Furthermore, motor performance on a Rotarod was unaffected in SNS-PKG-I−/− mice (Figure S6B; p = 0. 20). We have previously shown in details that SNS-Cre mice show no alterations in the processing of acute pain or chronic inflammatory or neuropathic pain [6], [32]. In the context of studying disease-induced pain hypersensitivity, we first focussed on a model of inflammatory pain which is associated with primary hyperalgesia in the inflamed area and ongoing nociceptive inputs from the periphery throughout the time of testing, namely unilateral hindpaw inflammation induced by injection of Complete Freund' s Adjuvant (CFA) [32], [33]. CFA injection produced similar levels of edema in SNS-PKG-I−/− and PKG-Ifl/fl mice (Figure S6C) and hypersensitivity to graded von Frey mechanical stimuli (Figure 7C, D) or to plantar heat (Figure 7E) applied to the ipsilateral paw was assessed at 6,12,24,48, and 96 h thereafter. Following inflammation, PKG-Ifl/fl mice demonstrated the characteristic leftward and upward shift in the stimulus-response curve over basal curves reflecting mechanical hypersensitivity (black squares in Figure 7C). In contrast, SNS-PKG-I−/− mice demonstrated a less marked deviation from baseline behaviour upon CFA-induced inflammation (red squares in Figure 7C). Furthermore, the relative drop in response thresholds to von Frey hairs (defined here as minimum force required to elicit 40% response frequency) in the inflamed state over basal (pre-CFA) state occurred to a significantly lesser extent in SNS-PKG-I−/− mice as compared to PKG-Ifl/fl mice (left panel in Figure 7D; p<0. 05 at all time points tested). Finally, SNS-PKG-I−/− mice showed a significantly lower magnitude of thermal hyperalgesia than PKG-Ifl/fl mice at 6 h after CFA and did not show hyperalgesia at all from 12 h onwards after CFA injection, whereas PKG-Ifl/fl mice continued to show thermal hyperalgesia all the way up to the latest time point tested, namely 96 h following CFA injection (Figure 7E; p<0. 01 between PKG-Ifl/fl and SNS-PKG-I−/− mice at all time points tested). We infer from the above that the development of primary hyperalgesia and mechanical allodynia following somatic inflammation is impaired by a loss of PKG-I in nociceptors. Although perturbation of spinal LTP may have contributed to the above phenotype in SNS-PKG-I−/− mice, it is conceivable that a peripheral role for PKG-I in nociceptors may at least partially account for changes in primary hyperalgesia. To address functional changes in nociceptor sensitivity in the inflamed tissue, we utilised the skin-nerve preparation [34] to study the electrophysiological properties of identified polymodal C-fibres and Aδ-mechanoceptors (AM) in the saphenous nerve. The excitability of mechanoreceptive C-fibers and AM-fibers showed a small, but significant, increase following paw inflammation in PKG-Ifl/fl mice (see Figure 7F for typical examples), but not in SNS-PKG-I−/− mice (Figure 7F). These data indicate defects in the development of peripheral sensitization in nociceptors of SNS-PKG-I−/− mice, which could contribute to a reduction in primary hyperalgesia; however, they are unlikely to account for the marked defects in mechanical allodynia observed following inflammation in SNS-PKG-I−/− mice. To explore central contributions, we utilised two models of aberrant pain which are triggered initially by peripheral inputs but do not require ongoing nociceptor activity in the periphery for maintenance. For example, capsaicin injection in the skin activates C-fibers and evokes hyperalgesia in the area of the flare (primary hyperalegsia) as well as outside of the flare (secondary hyperalgesia). In PKG-Ifl/fl mice, we observed that injection of capsaicin in the skin of the lower thigh produced a marked allodynia at the hindpaw plantar surface, which was clearly excluded from the area capsaicin-induced flare (see shift in von Frey response frequency in Figure 8A; black symbols). SNS-PGK-I−/− mice showed markedly reduced secondary hypersensitivity with capsaicin as compared to PKG-Ifl/fl mice (red symbols in Figure 8A). Moreover, a capsaicin-induced drop in mechanical threshold (allodynia) was markedly reduced in SNS-PGK-I−/− mice as compared to PKG-Ifl/fl mice (Figure 8B). It is well accepted that capsaicin-induced secondary mechanical hypersensitivity reflects C-fiber-evoked central amplification processes and can last for several hours, long after nociceptor responses to capsaicin have ceased owing to desensitisation of TRP channels [35]. Nevertheless, to rule out a potential contribution of ongoing peripheral inputs to the above-described phenotypic differences, we performed experiments in which nerve conduction was blocked with lidocaine in the peripheral dermatome in which capsaicin was injected in wild-type mice. As expected, lidocaine-induced nerve blockade prior to capsaicin injection blocked the induction of capsaicin-induced mechanical hypersensitivity (Figure 8C); in contrast, when lidocaine was injected 15 min after capsaicin, mechanical hypersensitivity developed normally (Figure 8C), indicating that beyond the initial trigger, capsaicin-induced mechanical hypersensitivity is independent of ongoing input from peripheral nociceptors. In further experiments, we addressed the peripheral and central contributions of PKG-I. Pharmacological inhibition of PKG-I with KT5823 injected prior to injection of capsaicin in the same dermatome in wild-type mice did not block the development of capsaicin-induced mechanical hypersensitivity (Figure 8D); in contrast, when KT5823 was injected intrathecally prior to peripheral capsaicin injection, the induction of mechanical hypersensitivity was markedly inhibited (Figure 8E), indicating a role for central PKG-I, but not peripherally expressed PKG-I. To further delineate the origin of the central (spinal) locus of PKG-I function, we undertook similar experiments in PKG-Ifl/fl mice and SNS-PGK-I−/− mice. Interestingly, intrathecally administered PKG-I inhibitor blocked the development of capsaicin-induced mechanical hypersensitivity in PKG-Ifl/fl mice and did not lower mechanical sensitivity any further in SNS-PGK-I−/− mice (Figure 8F), demonstrating thereby the presynaptic locus of its action. In the muscle pain model by Sluka and colleagues [36], two consecutive injections of dilute acidic saline in the flank muscle lead to secondary mechanical hypersensitivity in the ipsilateral and contralateral paws, which lasts for several weeks. The initial peripheral insult (i. e., flank muscle) is spatially distinct from the area of application of nociceptive stimuli (paw surface), ruling out a contribution of peripheral paw sensitization to the behavioural phenotype. Secondly, it has been shown in details previously that the secondary hyperalgesia in the paw lasts for several months after muscle injection, is not associated with any persistent inflammation or injury to the muscle tissue, is independent of peripheral inputs, and is thus central in origin [36]. Upon testing at 24 h after the induction of muscle pain, PKG-Ifl/fl mice demonstrated a pronounced leftward and upward shift in the stimulus-response curve to von Frey hairs applied to the plantar paw surface (black squares in Figure 9A, middle panel), which was still evident 3 wk later in the ipsilateral (Figure 9A, right panel); this changes come about in the paw ipsilateral to the injected flank muscle (upper panels in Figure 9A) as well as in the contralateral paw (lower panels in Figure 9A). In contrast, SNS-PKG-I−/− mice did not show significant deviations (red squares in Figure 9A). Analysis of paw-withdrawal thresholds to graded pressure also consistently revealed that muscle injection-induced drop in paw mechanical thresholds at the paws was significantly lesser in SNS-PKG-I−/− mice than in control littermates at all time points tested (Figure 9B). In conclusion, these analyses support an essential role for presynaptic PKG-I in nociceptor terminals in central mechanisms of secondary mechanical hypersensitivity. Finally, we asked whether PKG-I expressed in nociceptors constitutes an important target of the NMDA-NOS-cGMP pathway. Consistent with previous studies [37], intrathecally administered NMDA produced a rapid facilitation of the paw withdrawal reflex (Figure 10A). Importantly, in striking contrast to PKG-Ifl/fl mice (black symbols), SNS-PKG-I−/− mice completely failed to develop hyperalgesia following intrathecal NMDA delivery (red symbols, upper panel in Figure 10A). Similar results were obtained upon delivery of an NO donor, NOC-12, to the spinal cord via intrathecal catheters (upper panel in Figure 10B). Furthermore, intrathecal delivery of NMDA and NOC-12 produced a facilitation of the tail flick reflex in PKG-Ifl/fl mice, but not in SNS-PKG-I−/− mice (lower panels in Figure 10A, B), showing thereby that PKG-I is critically required for the pro-nociceptive functions of the NMDA and NO. Because soluble guanylyl cyclases (sGC) represent a key molecular link between NO and activation of PKG-I, the above results imply that NO activates sGC in spinal presynaptic terminals of nociceptors. While some studies report a lack of sGC expression in DRG neurons [38], others reported expression in a population of small diameter DRG neurons and in primary afferents [39], [40]. Here, we carried out mRNA in situ hybridisation using riboprobes recognising the beta subunit of sGC on mouse DRG sections and observed distinct, specific signals over the soma of several large and small-diameter DRG neurons (arrowheads and arrows in Figure 10C, respectively). Furthermore, the satellite cells surrounding DRG neurons showed dense signals (red arrows in Figure 10C). Sense control probes did not yield any appreciable signals (Figure 10C). These results indicate that sGC mRNA is expressed in sensory neurons of the DRG. In addition to sGC enzymes, which are directly activated upon NO, the membrane-bound guanylyl cyclases (mGC; Npr family) also contribute to cGMP production in some organs (e. g., in the cardiovascular system) [41]. Stimulated by recent reports on expression of mGCs in DRG neurons [42], we administered a cocktail of natriuretic peptides (ANP, BNP, and CNP) intrathecally and observed marked hyperalgesia within 15 min after delivery, which lasted for about 45–50 min in wild-type mice (unpublished data) and PKG-Ifl/fl mice (Figure 10D). Interestingly, natriuretic peptide-induced hyperalgesia was also entirely abrogated in SNS-PKG-I−/− mice (Figure 10D). These results suggest that the NMDA-NOS-soluble guanylyl cyclase-cGMP pathway as well as the natriuretic peptide-mGC-cGMP pathways converge upon PKG-I expressed in spinal terminals of nociceptors to modulate nociceptive processing in the spinal cord. Finally, we undertook experiments to test whether PKG-I expression alone or some downstream factor perturbed by an early loss of PKG-I is responsible for the deficits in pain hypersensitivity in SNS-PKG-I−/− mice. We constructed chimeric Adeno-associated virions of the serotypes AAV1 and AAV2 expressing an C-terminally GFP-tagged version of the murine PKG-I cDNA [43]. Injection in unilateral L3 and L4 DRGs in vivo led to a broad expression in the DRG. AAV1/2 chimeric virions expressing GFP alone served as controls. PKG-Ifl/fl mice and SNS-PKG-I−/− mice expressing GFP-tagged PKG-I or GFP alone showed normal basal sensitivity to graded von Frey stimuli (Figure 11B). Upon peripheral injection of capsaicin, PKG-Ifl/fl mice expressing GFP-tagged PKG-I showed a small increase in mechanical hypersensitivity than PKG-Ifl/fl mice expressing GFP, which was only statistically significant at some intensities of mechanical stimuli (Figure 11C). As expected, SNS-PKG-I−/− mice overexpressing GFP in DRG showed markedly reduced mechanical hypersensitivity with capsaicin than PKG-Ifl/fl mice overexpressing GFP. Importantly, overexpression of GFP-tagged PKG-I fully restored mechanical hypersensitivity in SNS-PKG-I−/− mice (Figure 11D). This indicates that expression of PKG-I is both necessary and sufficient for inducing centrally maintained hypersensitivity upon persistent peripheral activation of C-fibers. In contrast to the intensively studied forms of LTP in the hippocampus, very few studies have addressed cellular and molecular mechanisms of LTP at spinal synapses regulating the flow of nociceptive information from the periphery towards the brain [7], [24]. Here we observed that nociceptive activity-driven LTP at synapses between nociceptive terminals and spinal neurons projecting nociceptive inputs to the PAG requires presynaptic mechanisms for its full expression. Furthermore, our results indicate that this function is mediated by cGMP acting via PKG-I. We base our inferences on three main observations: (1) A specific loss of PKG-I in presynaptic, but not post-synaptic, compartments of this synapse abolished C-fiber-evoked LTP without altering basal neurotransmission at this synapse; (2) LTP was temporally accompanied by a decrease in the rate of synaptic failures in a presynaptic-PKG-I-dependent manner; and (3) LTP was associated with a change in the PPR, which did not take place when PKG-I was deleted presynaptically in nociceptor terminals. Importantly, higher magnitudes of LTP were consistently associated with a decrease in PPF, and thereby with an increase in release probability. Previous studies have shown that the NMDA receptor-NO-cGMP pathway is important in the induction of spinal LTP [4], [5], and it has been assumed that this pathway comes into play in the post-synaptic compartment. However, all of the above signal transducers are also expressed presynaptically in afferent terminals in the spinal dorsal horn [44]. Thus, pre- and post-synaptic contributions to spinal LTP have not been worked out so far. There is evidence for a requirement for post-synaptic Ca2+ change for the induction of the LTP (i. e., in experiments with BAPTA in recording pipette; [5]). Taken together with our results, this suggests that a calcium-dependent postsynaptic mechanism may be required for the induction of LTP (e. g., via NMDA receptor-dependent generation of NO); in contrast, a presynaptic change involving cGMP- and PKG-I-dependent increase in neurotransmitter release may mediate the expression of LTP at synapses between nociceptors and spinal-PAG projection neurons. Mechanistically, this may come about via involvement of multiple phosphorylation targets of PKG-I. While some targets have been identified in heterologous systems, very little is known about the nature and functional role of PKG-I targets in vivo. Here, we identified and validated two primary targets in DRG neurons, namely the IP3R1 and MLC, and observed that PKG-I modulates intracellular calcium release as well as MLC phosphorylation differently in DRG neurons as compared to other biological systems, such as the smooth muscle. For example, in some biological systems, PKG-I has been reported to negatively modulate calcium signals via its interaction with IRAG [18]. However, IRAG interacts selectively with the beta-isoform of PKG-I, which is barely expressed in the DRG, but not with PKG-I-alpha, the predominant form found in DRG neurons [10]. Furthermore, our observations that PKG-I potentiates calcium release induced by typical mediators of nociceptive sensitization, such as bradykinin, in identified nociceptive neurons and that repetitive activation of nociceptors in vivo leads to PKG-I-mediated phosphorylation of IP3R1 at serine 1755, a site associated with positive functional modulation, implicate PKG-I as a positive modulator of calcium signalling in nociceptive neurons. In light of electrophysiological analyses reported here, this raises the possibility that calcium released from IP3R1-gated stores may participate in modulating presynaptic function. Although a few studies at hippocampal synapses have proposed an involvement of calcium stores in modulation of presynaptic release [45], [46], underlying cellular mechanisms are not known. Results of this study suggest that activation of presynaptic PKG-I may constitute the molecular link between synaptic activity and the elevation of resting levels of calcium in presynaptic terminals, thereby potentiating synaptic transmission via an increase in release probability. Furthermore, we observed that MLC was phosphorylated in a nociceptive activity-dependent manner and that PKG-I is required for MLC phosphorylation in the DRG. Although our electrophysiological data implicate phosphorylated MLC in LTP at spinal synapses, not much can be inferred about downstream mechanisms at this stage. At central synapses, MLC phosphorylation was initially implicated in vesicle transport and in regulation of vesicular pools [47]; however, these inferences could not be corroborated in detailed subsequent analyses [48]. Taken together, more detailed analyses overcoming current technical hindrances in studying mobilization of vesicular pools in the complex circuitry of the DRG and spinal dorsal horn will be required to understand mechanisms underlying PKG-I-mediated modulation of release probability at this synapse. The possibility of functionally linking synaptic changes described here to changes in nociceptive behaviour simultaneously represents a good opportunity and a major challenge. As the first parameter to test this relationship, we focused on the phase II responses in the intraplantar formalin test, which has been attributed to spinal nociceptive sensitization triggered by an initial barrage of C-fiber inputs [31]. Indeed, we observed that presynaptic loss of PKG-I as well as functional perturbation of MLCK and IP3R, its substrates involved in LTP, inhibited phase II behavioural responses. However, a contribution of central mechanisms could not be inferred from the formalin data due to several reasons: Although MLCK/IP3R inhibitors were administered spinally, the genetically induced loss of PKG-I in SNS mice occurred throughout the nociceptor, including peripheral terminals. Moreover, there is still some ongoing activation of peripheral nociceptors in the phase II of the formalin response [31]. Indeed, our electrophysiological analyses in the CFA inflammatory pain model suggested that peripheral PKG-I may contribute to primary hyperalgesia. Therefore, we focused on pain models in which nociceptive hypersensitivity is triggered by peripheral nociceptors, but maintained via central mechanisms that outlast and are independent of peripheral inputs. One of these is the chronic muscle pain model in which injections of dilute acidic saline in the gastronemius muscle evokes a long-lasting secondary mechanical hyperalgesia in the ipsilateral and contralateral paws, which lasts for several weeks, is unrelated to muscle damage and is not maintained by continued primary afferent input from the site of injury, as shown by experiments involving dorsal rhizotomy and lidocaine injections in the muscle [36]. Similarly, we addressed capsaicin-induced secondary mechanical hyperalgesia outside of the primary flare, which albeit triggered by C-fiber inputs, is maintained via mechanisms of central origin as indicated by previous studies [35] and our analyses. In both models, we found marked defects in central hypersensitivity in SNS-PKG-I−/− mice. Our results indicated that capsaicin-evoked mechanical hypersensitivity is neither dependent on peripheral PKG-I function nor does it require ongoing peripheral nociceptor sensitisation. Moreover, they revealed that PKG-I expressed in central terminals of nociceptors plays a decisive role in the induction of mechanical hypersensitivity after persistent C-fiber stimulation via capsaicin. Finally, reinstating PKG-I expression in the DRG in adult SNS-PKG mice fully restored capsaicin-evoked mechanical hypersensitivity, indicating that PKG-I directly, and not some factor affected by a genetic loss of PKG-I, is a functional determinant of C-fiber-evoked mechanical hypersensitivity. In summary, this study shows that PKG-I expressed in nociceptors terminals is the principal target of cGMP at nociceptive synapses. Furthermore, it suggests that PKG-I-mediated presynaptic facilitation and LTP in spinal projection neurons is functionally involved in activity-dependent centrally mediated nociceptive hypersensitivity. Homozygous mice carrying the flox allele of the mouse prkg1 gene, which encodes the cGMP dependent kinase 1 (PKG-Ifl/fl) [15], have been described previously in detail. PKG-Ifl/fl mice were crossed with SNS-Cre mice [16] to obtain PKG-Ifl/fl; SNS-Cre+ mice (referred to as SNS-PKG-I−/− mice in this article) and PKG-Ifl/fl mice (control littermates). Mice were crossed into the C57BL6 background for more than 8 generations. Mice lacking PKG-I globally (PKG-I−/− mice) have been described before. Only littermates were used in all experiments to control for background effects. Mice (14–18 d old) were anesthetized with a mixture of Dormitor, Dormicum, and Fentanyl, and stereotactic injections of DiI into the PAG were carried out (see Text S1 for details). After 2 to 3 d, transverse 350–450 µm thick spinal cord slices with dorsal roots attached were obtained and whole cell patch clamp recordings of identified DiI-positive neurons were performed as described in Text S1. Test pulses of 0. 1 ms with intensity of 3 mA were given at 30 s intervals to the dorsal root via a suction electrode. For studying the site of expression of synaptic potentiation, we used minimal stimulation in conditions of low release probability (in mM: NaCl 127; KCl, 1. 8; KH2PO4,1. 2; Ca2+ 1. 0; Mg2+, 5; NaHCO3,26; glucose, 15; oxygenated with 95% O2,5% CO2; pH 7. 4). Dorsal root was stimulated at intensity of threshold to evoke EPSCs on DiI-labelled spino-PAG projection neuron. Under these conditions, the failure rate was 60. 9%±6. 3% (n = 10). To induce synaptic potentiation, low frequency stimulation (conditioning stimulus, 2 Hz for 2 min) was applied to dorsal root as a conditioning stimulus with the same intensity as the test stimulus [5]. The recording mode during conditioning stimulation was the same as that before and after conditioning stimulation. Neurons are voltage clamped at −70 mV. Because a suction electrode was utilized to stimulate the whole root, a suprathreshold stimulus was required to fully recruit C-fibers in the root [5]. Synaptic strength was quantified by assessing the peak amplitudes of EPSCs. The mean amplitude of 4–5 EPSCs evoked by test stimuli prior to conditioning stimulation served as a control. Significant changes from control were assessed by measuring the peak amplitudes of five consecutive EPSCs every 5 min after conditioning stimulation. Additional details are given in Text S1. In some experiments, blockers of inhibitory neurotransmission, such as Gabazine (10 µM) and Strychnine (1 µM), were added to the bath. In a subset of animals, paired-pulse stimuli with an inter-stimulus interval of 110 ms (0. 1 ms pulse duration, 3 mA intensity, every 30 s) were used (see Text S1 for details). Paired-pulse ratio (facilitation or depression) of C-fiber-evoked EPSC was calculated as the amplitude of the second C-eEPSC divided by that of the first C-eEPSC in a pair. In a subset of experiments, PKG-I inhibitors such as KT5823 (10 µM) or RKRARKE (250 µM) were infused post-synaptically via the patch pipette. The following antibodies were used for Western blots and biochemical analyses: anti-IP3R1, anti-pS1755 IP3R1 (kind gift from Prof. Richard Wojcikiewicz), anti-VASP (Alexis Biochemical), anti-MLC, anti-alpha tubulin (Sigma), anti-pSer239 VASP, anti-pThr18/pSer19 MLC (Cell Signaling technology), anti-PKG-I antibody [18], secondary HRP labelled anti-rabbit (Sigma Aldrich), or anti-mouse (GK Healthcare UK Ltd.). The following antibodies were used for immunohistochemistry: phospho-ERK1/2 antibody (Cell signalling), anti-Fos antibody (Chemicon), anti-Isolectin B4 antibody (vector laboratories), anti-Calcitonin gene related peptide antibody (Immunostar), anti-Neurofilament 200 antibody (Chemicon), anti-Substance P antibody (Chemicon), anti-PKG-I antibody [18], anti-PSD-95 antibody (a gift from M. Watanabe) and anti-TrkA antibody (a kind gift from Prof. L. F. Reichardt), and anti-cre antibody (Novagen). The soluble guanylyl cyclase inhibitor, ODQ (25 mg/kg body weight; sigma Aldrich), and the membrane guanylyl cyclase inhibitor, LY83583 (12. 5 mg/kg body weight; sigma Aldrich), were dissolved in 50% DMSO and injected in a volume of 250 µl intraperitoneally. The following drugs were administered intrathecally in vivo: an inhibitor of MLCK (ML-7; 15 nmol Alexis Biochemical, dissolved in 5% DMSO), an inhibitor of IP3R (2-APB; 2 nmol; Calbiochem), NMDA (100 fmol; Sigma Aldrich), the NO donor, NOC-12 (17 nmol; Sigma Aldrich), atrial natriuretic peptide, brain natriuretic peptide and c-type natriuretic peptide (rANP1-28, mBNP45, and hCNP1-22; 330 pmol of each natriuretic peptide; American peptide company, Inc., USA), and the PKG-I inhibitor KT5283 (200 pmoles). See Text S1 for details on intrathecal delivery. Mice were allowed to recover for 2 d after surgery, and only animals showing complete lack of motor abnormalities were used for further experiments. 5 µl of drugs were applied followed by flushing of the catheter with 10 µl of 0. 9% saline. The following drugs were administered peripherally in the vicinity of the paw in experiments pertaining to capsaicin-induced mechanical hypersensitivity: KT5823 (200 pmoles) and lidocaine (10 µl of 2%). A total of 17 PKG-Ifl/fl and 15 SNS-PKG-I−/− mice were used in the electrophysiological recordings of nerve activity. An ex vivo skin-nerve preparation was used to study the properties of mechanosensitive C- and A-δ afferent fibres which innervate the skin in the inflamed area 24 h following CFA inoculation (20 µl) as described previously (see Text S1 for details). All animal use procedures were in accordance with ethical guidelines imposed by the local governing body (Regierungspräsidium Karlsruhe, Germany). All behavioural measurements were done in awake, unrestrained, age-matched mice of both sexes that were more than 3 mo old by individuals who were blinded to the genotype of the mice being analyzed (see Text S1 for details). The open reading frame of mouse PKG-I fused C-terminally with GFP [43] or EGFP alone was cloned in an AAV expression construct, and chimeric AAV1/2 virions were generated using standard protocols. Virions were diluted 1∶2 with 20% mannitol and injected unilaterally into L3 and L4 DRGs (1 µl per DRG, or approx. 107 transfection units per DRG) in deeply anesthetized mice as described in detail previously [49]. Mice were tested in behavioural tests 2 wk after injection. At the end of the experiment, mice were perfused as described above and expression of GFP was confirmed via fluorescence analysis. All data are presented as mean ± standard error of the mean (S. E. M.). For comparisons of multiple groups, analysis of variance (ANOVA) for random measures was performed followed by post hoc Fisher' s test to determine statistically significant differences. When comparing two groups that were studied in parallel, Student' s t test was employed. Unless otherwise specified, the p values shown in the figures and text are derived from ANOVA and post hoc Fisher' s test. p<0. 05 was considered significant.

Pain is an important physiological function that protects our body from harm. Pain-sensing neurons, called nociceptors, transduce harmful stimuli into electrical signals and transmit this information to the brain via the spinal cord. When nociceptors are persistently activated, such as after injury, the connections they make with neurons in the spinal cord are altered in a process called synaptic long-term potentiation (LTP). In this study, we examine the molecular and cellular mechanisms of LTP at synapses from nociceptors onto spinal neurons. We use multiple experimental approaches in mice, from genetic to behavioural, to show that this form of LTP involves presynaptic events that unfold in nociceptors when they are repetitively activated. In particular, an enzyme activated by the second messenger cGMP, referred to as Protein Kinase G-I, phosphorylates presynaptic proteins and increases the release of neurotransmitters from nociceptor endings in the spinal cord. When we genetically silence Protein Kinase G-I or block its activation in nociceptors, inflammatory pain is markedly reduced at the behavioural level. These results clarify basic mechanisms of pathological pain and pave the way for new therapeutic approaches.

lay_plos

(CNN) Every detail of this indiscriminate mass murder seemed meticulously planned. The selection of a hotel room overlooking a music festival, days before the attack. The cache of 23 weapons inside the gunman's Las Vegas suite. And thousands of rounds of ammunition -- plus an ingredient used in explosives -- inside the killer's home and car. The latest revelation came Tuesday afternoon when police said gunman Stephen Paddock set up cameras inside his hotel suite and in the hallway. Police are not aware whether the devices were transmitting -- the FBI is investigating their use -- but the Clark County sheriff told reporters he thinks the shooter might have used them to watch for people approaching his room. One camera looked out the peephole on the suite's door. The Bureau of Alcohol, Tobacco, Firearms and Explosives disclosed that Paddock had outfitted 12 of his rifles with a legal device called a bump-fire stock, which enables a shooter to fire bullets rapidly, similar to an automatic rifle. Authorities released the first body camera footage of police responding to the shooting. It captured the rapid staccato of the gunfire at a fairly close range. Officers were seen hunkering down behind a wall. "Go that way, get out of here! There's gunshots coming from over there," one officer is heard yelling at civilians. At one point, they were next to a patrol vehicle on Las Vegas Boulevard, where one officer was shot, Undersheriff Kevin McMahill said. No one knows why Paddock morphed from a retired accountant to the deadliest mass shooter in modern US history. His relentless gunfire -- police say he fired for nine to 11 minutes after the first 911 call -- on country music fans at an outdoor concert left 58 people dead. Another 500 people are still trying to recover from injuries -- everything from gunshot wounds to stampede injuries suffered when 22,000 people tried to flee the gunman's aim. So far, police believe Paddock acted alone -- which could make the motive harder to determine. Photos published by the Daily Mail of the United Kingdom show a body inside Stephen Paddock's room at the Mandalay Bay. Latest developments Stephen Paddock -- Paddock's girlfriend, Marilou Danley arrived at LAX on Tuesday night, and is being accompanied by the FBI in Los Angeles, a law enforcement source told CNN. Danley flew from Manila, said Maria Antoinette Mangrobang, a spokeswoman for the Bureau of Immigration in the Philippines. Danley had entered the Philippines in September 15, and again on September 25, traveling on her Australian passport, she said. There has been communication between authorities in the Philippines, the FBI and US Department of Homeland Security, Mangrobang said. -- Clark County Coroner John Fudenberg said that 58 people were killed. Authorities had previously said 59 were killed in the shooting, but on Tuesday clarified that number included the shooter. -- The Daily Mail newspaper of the UK has published several photos taken in Paddock's room after the shooting. In one photo, the legs of a dead shooter can be seen on the floor. The photos show semiautomatic assault-style rifles on the floor and on furniture. Stacks of ammunition magazines used in rifles can also be seen. -- Paddock wired $100,000 to the Philippines, a law enforcement source said. However, officials haven't able to see yet precisely when the wire happened or who was the recipient. The FBI is working with Filipino authorities to determine details. -- President Donald Trump tweeted: "It is a'miracle' how fast the Las Vegas Metropolitan Police were able to find the demented shooter and stop him from even more killing!" -- Five handguns, two shotguns and a "plethora" of ammunition were found in Paddock's Verdi, Nevada, property, police said. Authorities previously found 42 guns in Paddock's hotel room and at his Mesquite, Nevada, home. A photo published by the Daily Mail shows long guns, a hammer and a stack of magazines for rifles. 'I felt him get shot in the back' JUST WATCHED Shooter's angle prevented people from escaping Replay More Videos... MUST WATCH Shooter's angle prevented people from escaping 02:06 Heather Melton heard the noise interrupting the concert and told her husband, Sonny, she thought it might be gunfire. He, like most people, thought it was fireworks. Then the bullets started ricocheting off the ground around the Tennessee couple. She wanted to get low; he said, no, we'll get trampled. So they ran, away from the gunfire, Sonny just behind Heather, until he was felled by a bullet. "I felt him get shot in the back," she told "Anderson Cooper 360." There were bodies all over the ground. Several rifles are on two chairs pushed together while several lie on the floor, a photo obtained by the Daily Mail shows. "I was trying to talk to him and he wasn't responding," said Heather, an orthopedic surgeon. She said she got over him and started doing CPR. People said to get down. Sonny, a registered nurse, was bleeding from the mouth. "If he was one of the people who had survived,... he would've been one those people running back in" - Wife of shooting victim Sonny Melton pic.twitter.com/fX0m7ksNjG — Anderson Cooper 360° (@AC360) October 4, 2017 Heather Melton said she knew he probably was gone, but she wanted to hope. Sonny was declared dead at the hospital. "He was the most selfless person that I ever met, and even until his last breath he proved that," Heather said. The investigation Paddock's violent transformation has mystified everyone -- his brother, investigators and the families he victimized. Police had no prior knowledge of the gunman before the attack. "I don't know how it could have been prevented," Lombardo said. The massacre has no known link to overseas terrorism or terror groups, a US official with knowledge of the case said. And authorities say it's too early to tell whether the killings were an act of domestic terrorism. "We have to establish what his motivation was first," Lombardo said. For an act to be considered terrorism, it must appear that it was intended to intimidate or coerce a civilian population, or try to influence political change. The gunman's brother, Eric Paddock, said he was "completely befuddled" by his brother's actions. He said Stephen Paddock was an avid gambler who had "no history of violence. No history of anything -- couldn't give a s*** less about politics, religion, pointy hatted people, etc, etc. He just wanted to get a freaking royal flush." On Tuesday, Paddock's family sent condolences to the victims. "There are no words to describe the sadness we feel for those who lost their lives in this tragic event. Please know that you are in our prayers and that our hearts are heavy for the families who have been left heartbroken and without answers," the family said in a statement. Running back into danger The massacre started at about 10 p.m. Sunday at the Route 91 Harvest festival, Sheriff Lombardo said. Country singer Jason Aldean was on stage when bullets started raining onto the crowd. Photos: Mass shooting at Las Vegas music festival Debris is scattered on the ground Monday, October 2, at the site of a country music festival held this past weekend in Las Vegas. Dozens of people were killed and hundreds were injured Sunday when a gunman opened fire on the crowd. Police said the gunman fired from the Mandalay Bay hotel, several hundred feet southwest of the concert grounds. It is the deadliest mass shooting in modern US history. Hide Caption 1 of 30 Photos: Mass shooting at Las Vegas music festival Broken windows of the Mandalay Bay are seen early in Las Vegas on Monday. Police said the gunman fired on the crowd from the 32nd floor of the hotel. Hide Caption 2 of 30 Photos: Mass shooting at Las Vegas music festival People cross a street near the Las Vegas Strip just after sunrise on Monday. Thousands were attending the music festival, Route 91 Harvest, when the shooting started. Hide Caption 3 of 30 Photos: Mass shooting at Las Vegas music festival People embrace outside the Thomas & Mack Center after the shooting. Hide Caption 4 of 30 Photos: Mass shooting at Las Vegas music festival Police arrive at the Sands Corporation plane hangar where some people ran to safety after the shooting. Hide Caption 5 of 30 Photos: Mass shooting at Las Vegas music festival A woman cries while hiding inside the Sands Corporation plane hangar. Hide Caption 6 of 30 Photos: Mass shooting at Las Vegas music festival Concertgoers dive over a fence to take cover from gunfire on Sunday night. Hide Caption 7 of 30 Photos: Mass shooting at Las Vegas music festival Police take position outside the Mandalay Bay. Hide Caption 8 of 30 Photos: Mass shooting at Las Vegas music festival A man lays on top of a woman as others flee the festival grounds. The woman reportedly got up from the scene. Hide Caption 9 of 30 Photos: Mass shooting at Las Vegas music festival Hide Caption 10 of 30 Photos: Mass shooting at Las Vegas music festival People are seen on the ground after the gunman opened fire. Hide Caption 11 of 30 Photos: Mass shooting at Las Vegas music festival People run from the festival grounds. Hide Caption 12 of 30 Photos: Mass shooting at Las Vegas music festival A woman is moved outside the Las Vegas Tropicana resort. Multiple victims were being transported to hospitals in the aftermath of the shooting. Hide Caption 13 of 30 Photos: Mass shooting at Las Vegas music festival People are searched by police at the Tropicana. Hide Caption 14 of 30 Photos: Mass shooting at Las Vegas music festival An ambulance leaves the intersection of Las Vegas Boulevard and Tropicana Avenue. Hide Caption 15 of 30 Photos: Mass shooting at Las Vegas music festival A man in a wheelchair is evacuated from the festival after gunfire was heard. Hide Caption 16 of 30 Photos: Mass shooting at Las Vegas music festival Victims of the shooting are tended to in the street. Hide Caption 17 of 30 Photos: Mass shooting at Las Vegas music festival Concertgoers help an injured person at the scene. Hide Caption 18 of 30 Photos: Mass shooting at Las Vegas music festival People gather around a victim outside the festival grounds. Hide Caption 19 of 30 Photos: Mass shooting at Las Vegas music festival A couple huddles after shots rang out at the festival. Hide Caption 20 of 30 Photos: Mass shooting at Las Vegas music festival An injured woman is helped at the Tropicana. Hide Caption 21 of 30 Photos: Mass shooting at Las Vegas music festival Police and emergency responders gather at the intersection of Las Vegas Boulevard and Tropicana Avenue. Hide Caption 22 of 30 Photos: Mass shooting at Las Vegas music festival A police officer takes position behind a truck. Hide Caption 23 of 30 Photos: Mass shooting at Las Vegas music festival A crowd takes cover at the festival grounds. Hide Caption 24 of 30 Photos: Mass shooting at Las Vegas music festival Police officers advise people to take cover in the wake of the shooting. Hide Caption 25 of 30 Photos: Mass shooting at Las Vegas music festival People tend to a victim at the festival grounds. Hide Caption 26 of 30 Photos: Mass shooting at Las Vegas music festival Police stand at the scene of the shooting. Hide Caption 27 of 30 Photos: Mass shooting at Las Vegas music festival A woman sits on a curb at the scene of the shooting. Hide Caption 28 of 30 Photos: Mass shooting at Las Vegas music festival Police are deployed to the scene. Hide Caption 29 of 30 Photos: Mass shooting at Las Vegas music festival A man makes a phone call as people run from the festival grounds. Hide Caption 30 of 30 "On the main floor... there was no cover -- they were all exposed," survivor Rusty Dees said. "So you didn't know if somebody was shot or if they were laying down." Frantic concertgoers piled on top of each other, trying to get out of the line of fire. But an off-duty nurse ran back into the danger to help those who had been shot. "We went back because I'm a nurse and I felt I had to," she told CNN affiliate KTNV. "I went to three different scenes, and by the time I got to the third one, there was just dead bodies." The nurse said she was far from alone. "There was so many people, just normal citizens, doctors, cops, paramedics, nurses, just off-duty. Everyone was just communicating and working together." JUST WATCHED Random acts of heroism save lives in Las Vegas Replay More Videos... MUST WATCH Random acts of heroism save lives in Las Vegas 02:04 Corrine Lomas recalled the heroism of fellow concertgoers. "A lot of really good people (were) holding people's wounds shut, trying to help them while everybody was just ducked down," she said. $8.1 million raised Countless strangers have rallied to support victims, donating blood, money and supplies. By Tuesday evening, a GoFundMe page started by a Clark County commissioner had raised more than $8.1 million. "Funds will be used to provide relief and financial support to the victims and families of the horrific Las Vegas mass shooting," county commission chair Steve Sisolak wrote. Throngs of blood donors lined up for hours to help the wounded. JUST WATCHED Survivor held stranger's hand as he died Replay More Videos... MUST WATCH Survivor held stranger's hand as he died 02:00 "This is America -- people coming together, helping out." Hector Salas tweeted. "Likely more than 1000 people waiting in line to donate blood. Strangers also donated flights, housing, food and transportation to victims' relatives coming to Las Vegas, Clark County Fire Chief Greg Cassell said. "It takes the worst of America to also see the best of America," said Dees, who survived the gunfire. "Everybody was helping each other." Correction: An earlier version of this story incorrectly identified the name of survivor Rusty Dees. Breaking News Emails Get breaking news alerts and special reports. The news and stories that matter, delivered weekday mornings. / Updated By Dartunorro Clark In the midst of gunfire, chaos and carnage, Sonny Melton — a nurse from Tennessee — died shielding his wife from the bullets raining down on them. Mike Cronk, a retired teacher from Alaska, used his pickup truck as a makeshift ambulance to transport wounded concertgoers. Sonny Melton. Courtesy Heather Melton And Mike McGarry, a financial adviser from Philadelphia, threw himself on top of his kids as he heard hundreds of bullets ring out. These are some of the acts of heroism that have emerged out of the bloodshed created by Stephen Craig Paddock, 64, a lone gunman firing a barrage of bullets from his hotel room down at a Las Vegas music festival. White House Press Secretary Sarah Huckabee Sanders was on the verge of tears Monday as she recounted the heroes born out of the heartbreak. “What these people did for each other says far more about who we are as Americans than the cowardly acts of a killer ever could,” Sanders said, fighting back tears during the daily news briefing. Sanders placed a spotlight on Melton and McGarry, among others, including stories from witnesses who described local police officers shielding victims with their bodies or cars. McGarry told Reuters that his first instinct was to protect his children after the bullets began flying. “It was crazy," he said. "I laid on top of the kids. They're 20. I'm 53. I lived a good life." Jolene Bennett described how she drove a gunshot victim to safety. "As we are driving, people are running, screaming, wanting in the car," she told MSNBC. "We opened up the door, three people jumped in the back. And then as we were trying to take off we see a lady who had been shot and I open the front seat and she climbed in my lap. She was bleeding. Just pouring blood out of her arm." Cronk told NBC News' Lester Holt that his friend was shot three times and he and several others immediately started providing first aid. Cronk said they then carried him to a pickup truck where other wounded fans were waiting and then helped transfer the injured to an ambulance. For one of them, help came too late. "He was not by himself, he was always with somebody," Cronk said. Sanders said those courageous feats, and others, are an "eternal reminder that the American spirit cannot and will not be broken." “The memories of those who displayed the ultimate expression of love in the midst of an unmanageable act of hate will never fade," she added. That is also the hope of Heather Melton, Sonny Melton’s wife. Sonny Melton with his wife Heather Melton. Courtesy Heather Melton She said that as they rushed through the panicked crowd, he grabbed her from behind to shield her and then a bullet struck him in the back. Although she lost her husband, she said she wanted his act of bravery to be an indelible part of his legacy. "He saved my life," she told WSMV, an NBC affiliate in their home state of Tennessee. "I want everyone to know what a kindhearted, loving man he was, but at this point, I can barely breathe." "Can't describe in words how thankful and grateful I am to have you show me what a real true gentleman you are," Christine Young said of Murfitt on Facebook. "I'll keep the advice you gave me and I promise to take it as I go through life moving forward … you'll be kept in a special place in my heart." Students from University of Nevada Las Vegas hold a vigil Monday, Oct. 2, 2017, in Las Vegas. A gunman on the 32nd floor of the Mandalay Bay casino hotel rained automatic weapons fire down on the crowd... (Associated Press) LAS VEGAS (AP) — Rob Ledbetter's battlefield instincts kicked in quickly as bullets rained overhead. The 42-year-old U.S. Army veteran who served as a sniper in Iraq immediately began tending to the wounded, one of several heroes to emerge from the deadliest mass shooting in modern U.S. history. Amid the massacre in Las Vegas, which left 59 people dead and more than 500 injured, there were acts of compassion and countless heroics that officials say saved scores of lives. There was a man one survivor knows only as Zach who herded people to a safe place. There was a registered nurse from Tennessee who died shielding his wife. Like many people in the crowd of some 22,000 country music fans Sunday night, Ledbetter heard the pop-pop-popping noise and figured it was fireworks. Then he saw people dropping to the ground. When more booms echoed in the night air, he recognized the sound of automatic weapons fire. The gunman, identified as Stephen Craig Paddock, a 64-year-old retired accountant from Mesquite, Nevada, created his own sniper's perch inside the 32nd floor room at the Mandalay Bay casino hotel, across from the concert grounds. He appeared to fire unhindered for more than 10 minutes, according to radio traffic, and then killed himself before officers stormed in and found 23 firearms. "The echo, it sounded like it was coming from everywhere and you didn't know which way to run," said Ledbetter, who was at the concert with seven people including his brother, who was shot and injured, and his wife. They found cover in a VIP area of the concert. Once out of harm's way, he turned to injured strangers. Thanks to a man who took the flannel shirt off his back, Ledbetter says he put a makeshift tourniquet on a wounded teenage girl, whose face was covered with blood. "Some random guy, I said, 'I need your shirt,' "said Ledbetter, who is now a mortgage broker and a resident of Las Vegas. "He just gave me the flannel off his back." Ledbetter said he compressed someone else's shoulder wound, and he fashioned a bandage for a man whose leg was shot through by a bullet. "There was a guy that looked like he had a through and through on his leg, that we just put a T-shirt around and just did a bandanna tie," said Ledbetter, who was outside University Medical Center on Monday, where his brother was being treated for a gunshot that went through his arm and into his chest. He is expected to survive. Ledbetter and others grabbed the injured man, carried him out to Las Vegas Boulevard, put him in the back of a utility truck with five to 10 other people that was headed to the hospital. Ledbetter said he would have helped more people but couldn't clear the barrage of gunfire. "I'm saving people, or trying to do my best. But it got to the point, I saw people all over, laying where we used to be standing... just laying there and nobody getting to them and I couldn't get out there. The shots just kept coming in and bouncing. I would have been in harm's way," he said. He worries that those unfamiliar with battlefields will suffer what they have survived. "Everybody there is going to have emotional problems. I know that. There was blood everywhere I went: Excalibur, Luxor, on the Strip, on the street," Ledbetter said. "All these people are going to have PTSD. I feel bad for all of them." Another concertgoer, Anna Kupchyan, credits a man she knows only as Zach for saving her life and about nine others when he herded them into an outdoor trailer serving as a restroom. Kupchyan, a 27-year-old law student from Los Angeles, said bullets were raining down on the crowd as she and a horde of others began running in search of a way out of the outdoor venue. The man, Zach, opened a door and ordered people inside and then joined them and shut the door, Kupchyan said. They stayed inside as the shooting continued, everyone paralyzed in fear, she said. "Then security came and they shouted for us to get out, to run," she recalled. Outside the trailer, dead bodies were sprawled on the ground, including a man who had been shot in the head, she said. She and her best friend Leslie Aguilar, a 26-year-old therapist, eventually jumped in a cab that was driving by and befriended two other women survivors who let them stay in their hotel room until the danger subsided. Not all of Sunday night's heroes survived. Sonny Melton, a registered nurse, died in the shooting, according to The Henry County Medical Center in Paris, Tennessee, where he worked. His wife, Dr. Heather Melton, an orthopedic surgeon who was with him when shots were fired, survived. She told WZTV in Nashville, Tennessee, that her husband "saved my life and lost his." She said her husband was the most kind-hearted, loving man she ever met. ___ Associated Press Writers Jocelyn Gecker in San Francisco and Anita Snow in Las Vegas contributed to this report. Candles lit in memory of those who died in Las Vegas shooting Residents, faith leaders and lawmakers gathered on the front steps of City Hall on Monday evening, where a candle was lit for each of the 59 lives lost. The crowd began singing as the candles were lit at the end of the event, and attendees stayed afterward to continue singing on the front steps of City Hall. Pastors offered prayers, often mentioning unity and togetherness. Mayor Carolyn G. Goodman and Rep. Dina Titus, D-Nev., spoke during the vigil. “What has come about is beyond heartbreaking,” Goodman said. Titus, whose district includes the Las Vegas Strip, talked about the efforts of officials and residents to respond to the shooting. “We want to thank everybody who has put their heart and soul into trying to grieve today and then after today we will move forward and start talking about why we don’t need one more moment of silence in Congress for victims of gun violence,” Titus said. Henderson-based Community Ambulance Paramedic Melanie Bangle said the organization runs 911 calls in Clark County. She said Community Ambulance, as part of its contract, had stationed crews at the concert to provide medical care “just in case anything happened.” “Our special events director, (Glen Simpson) was on one side of the event and I know he ran across gunfire to get back to the medical tent to make sure his crews were safe,” she said. “I heard from crew members that our management was willing to risk their life to go make sure that our crew members, who are our family, were safe.” She said for an event that size, which had about 22,000 attendees, there are typically 10 to 12 paramedics on-site. The company was working from a medical tent and a couple of other areas at the event. “They were actually there and able to respond right away,” she said. “We were very fortunate to have civilians help us — off-duty firefighters, police officers, paramedics, veterans. It was fortunate for us that we were able to work with all the civilians and the other agencies in conjunction to make everything run as smoothly as possible, to transport as many victims as possible.” She said almost all of the company’s 250 employees stepped in after the shooting. Bangle said certified counselors were at the station Monday morning to help deal with any trauma that employees experienced, including those who were there during the gunfire, staff members who transported people, and dispatchers who were taking calls. “They’re grateful that they were there to be able to help,” Bangle said of the employees. “It is very traumatic though, trying to help people and sometimes you’re not able to. The crew members that were there, they could have been killed. But out of all the crew members I’ve talked to, a lot of them were glad they were able to treat the people that they did.” Bangle said her role has been a supportive one, making sure employees have everything they need. “We came in to relieve those that were on all night and to kind of pick up the pieces where they left off and continue going for the rest of the day,” she said. “This happened, but 911 calls don’t stop throughout the rest of the valley. We had people who stayed all night and continued on into the morning running 911 calls. As partners with our other crew members, when they need to leave, we pick up and we take over.” Las Vegas resident Natalie Haag was at the vigil and said her cousin Eugenia Arce-Alandia works at the Tropicana. “She basically saw outside just like a rainfall of bullets,” she said. “So she hid.” Haag also had an aunt who works at Mandalay Bay. She said her mom was finally able to reach her aunt after her call went unanswered, and all of her family members got home safe. She said she’d had a personally hard year already before starting to cry. “This was just another thing that I was freaked out that my family was going to be involved in,” she said. Sara Nelson, a Las Vegas resident since 1987, said she doesn’t know anyone personally who’s been affected. She said she attended the vigil to be around her fellow community members. “My parents actually called me earlier this morning at about 6 o’clock to make sure I wasn’t there,” she said. “And that’s how I found out.” She said Vegas is a big city, but it’s still a small town where everybody knows everybody, one way or another. “I never thought something like this would happen here,” she said. Get daily updates directly to your inbox + Subscribe Thank you for subscribing! Could not subscribe, try again later Invalid Email British troops enjoying time off from duty rushed to the scene of the Las Vegas casino shooting giving life-saving first aid to those wounded. Soldiers from the 1st Queen's Dragoon Guards, known as the Welsh Cavalry, were enjoying a rest and recuperation break after gruelling training at the Fort Irwin US Army base in California. A source said: “Due to their experience, the guys who heard the noise knew instantly it was gunfire. “Their training immediately kicked in and they rushed to the festival to help. “All soldiers in the cavalry are given medical training designed to preserve life on the battlefield. "The injuries they were seeing were the ones they are well versed in treating. (Image: Getty) (Image: REX/Shutterstock) (Image: Getty) (Image: Getty) “They paid no respect for their own safety and did whatever they could to help.” An Army spokesman said: “We can confirm that a number of serving personnel from 1 The Queen’s Dragoon Guards provided assistance to the wounded following the heinous shooting in Las Vegas. "Our thoughts go out to those affected by this terrible act.” (Image: Getty Images Europe) The soldiers had previously been posted to the Mojave Desert to take part in Exercise Diamondback to assist a US Brigade in its pre-deployment training. The Queen's Dragoon Guards is the Cavalry Regiment of Wales and Border Counties (Shropshire, Herefordshire and Cheshire. QDG are one of the most experienced regiments in the Army having seen action as the reconnaissance force during the Gulf War, Iraq War and most recently during three deployments to Afghanistan. The gunman killed at least 58 people and injured more than 515 others last night after opening fire on a packed outdoor music festival on the Las Vegas Strip. (Image: Getty) (Image: REUTERS) (Image: Getty Images North America) (Image: REUTERS) Stephen Paddock wielded a high-powered assault rifle and rapidly sprayed bullets at revellers at the Route 91 Harvest country music festival from a nearby hotel. Perched on the 32nd floor of the Mandalay Bay Hotel and Casino, he fired the gun for several minutes, before cops stormed his room and shot him dead. Police believe Paddock acted alone in the deadliest mass shooting in US history. The shooter died in his hotel room, police confirmed. He is believed to have released more than 200 rounds into the 22,000-strong crowd. (Image: Twitter) (Image: Getty Images North America) (Image: Getty Images North America) (Image: REX/Shutterstock) The shooting started towards the end of Jason Aldean's set and police responded at about 10pm local time. Video clips posted online captured what sounded like automatic weapons as panicked concert-goers fled and dropped to the ground, screaming. Witnesses later described how they saw victims fleeing as shots rained down. Some later huddled in the basement of the nearby Tropicana hotel-casino. Video Loading Video Unavailable Click to play Tap to play The video will start in 8 Cancel Play now Pictures of the scene showed many victims lying on the ground, covered in blood. Others captured people, both young and old, running for their lives. After quickly arriving at the scene, some police officers were seen taking cover behind their vehicles, while others carrying assault rifles ran into the hotel. ISIS has claimed responsibility for the shooting. The terror group claims Paddock converted to Islam months ago but police said they had no information about Paddock's motive, that he had no criminal record and was not believed to be connected to any militant group.

As investigators try to piece together a motive behind Sunday night's massacre in Las Vegas, stories of heroism are emerging. Rusty Dees says that after gunman Stephen Paddock opened fire on the Route 91 Harvest country music festival, concertgoers tried to take care of each other amid the panic and bloodshed. "It takes the worst of America to also see the best of America. Everybody was helping each other," he tells CNN. Other concertgoers say that as they desperately tried to get out of the gunman's line of fire, strangers tried to pull others to safety and to hold injured people's wounds closed. More: Lawmakers and faith leaders were among those at a vigil outside Las Vegas City Hall on Monday evening, where 59 candles were lit-one for each of the people killed in the rampage, the Las Vegas Sun report. "What has come about is beyond heartbreaking," said Las Vegas Mayor Carolyn Goodman. Officials say acts of heroism probably saved scores of lives during the massacre, the AP reports. Rob Ledbetter, a 42-year-old former Army sniper, says his battlefield instincts kicked in and after making his way to safety, he performed first aid on multiple victims with bullet wounds. The New York Times looks at what it calls the "controlled chaos" at trauma centers after victims started flooding in on Sunday, and at the horrific conditions emergency workers faced. "They were exposed to significant trauma, things that are very, very unusual-a lot of deaths, a lot of injury, a lot of hysteria in one place, a lot of tragedy, so quite frankly many of them will probably be dealing with this for the rest of their lives," says deputy fire chief Jeff Buchanan. British Prime Minister Theresa May has praised a group of British soldiers trained in treating battlefield injuries who rushed to help victims. They had been on R&R after training with American troops in California. "Due to their experience, the guys who heard the noise knew instantly it was gunfire," a source tells the Daily Mirror. "Their training immediately kicked in and they rushed to the festival to help." The Los Angeles Times has stories of some of the victims, including a commercial fisherman and a Disneyland worker. Emergency medical technician Travis Phippen carried his injured father to safety despite having been shot in the arm. John Phippen later died from his injuries. "He was my best friend," Travis says. "He never did anything wrong to anybody. He was always kind and gentle. He was the biggest teddy bear I knew." One of the heroes whose stories White House Press Secretary Sarah Huckabee Sanders recounted Monday was Mike McGarry, who jumped on top of his children when the firing started, NBC reports. "They're 20. I'm 53. I lived a good life," he later said. Sanders also mentioned Sonny Melton, a nurse who died saving his wife.

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Vascular network density determines the amount of oxygen and nutrients delivered to host tissues, but how the vast diversity of densities is generated is unknown. Reiterations of endothelial-tip-cell selection, sprout extension and anastomosis are the basis for vascular network generation, a process governed by the VEGF/Notch feedback loop. Here, we find that temporal regulation of this feedback loop, a previously unexplored dimension, is the key mechanism to determine vascular density. Iterating between computational modeling and in vivo live imaging, we demonstrate that the rate of tip-cell selection determines the length of linear sprout extension at the expense of branching, dictating network density. We provide the first example of a host tissue-derived signal (Semaphorin3E-Plexin-D1) that accelerates tip cell selection rate, yielding a dense network. We propose that temporal regulation of this critical, iterative aspect of network formation could be a general mechanism, and additional temporal regulators may exist to sculpt vascular topology. The unique vascular topology of different organs exists for organ-specific functions. Different vascular network densities determine the specific amount of oxygen and nutrients to be delivered to each host tissue. Development of new vascular networks depends upon two types of specialized endothelial cells that work together: (1) The endothelial' tip cell', which is located at the front of a growing vessel and guides its extension by sensing and responding to environmental cues, analogous to the axonal growth cone (Gerhardt et al., 2003; Kurz et al., 1996). (2)' Stalk cells', which trail behind the tip cell and elongate the sprout. Tip and stalk cell identities are primarily controlled by the Dll4-Notch lateral inhibition pathway, which is activated in endothelial cells in response to VEGF from the local environment. VEGF-induced Dll4 activates Notch1 on the neighboring cell, leading to the down-regulation of VEGF receptor levels (Figure 1B) (Hellström et al., 2007; Leslie et al., 2007; Lobov et al., 2007; Suchting et al., 2007), (Benedito et al., 2009; Phng et al., 2009; Roca and Adams, 2007). Thus, lateral inhibition between endothelial cells generates an alternating pattern of active tip cells (Dll4 high) and inhibited stalk cells (Dll4 low). Moreover, tip cell selection is a dynamic process, and tip cell identity is transient (Arima et al., 2011; Jakobsson et al., 2010). Reiteration of tip cell selection, sprout extension, and connection of neighboring sprouts (anastomosis) is the basis for building a sophisticated vascular network (Adams and Eichmann, 2010; Carmeliet, 2000). Although this VEGF/Notch signaling pathway has been well studied and found to be conserved among different vascular beds and species, how this central pattern generator is modified by various target tissue-specific signals to yield diverse network topologies is not known. 10. 7554/eLife. 13212. 003Figure 1. Calibrated computational model predicts delayed tip cell selection in the absence of Sema3E-Plexin-D1 signaling. (A) Whole-mount vascular staining (Isolectin B4) of retinas from Sema3e-/- and wildtype littermates at P4. The mutant vasculature exhibits a reduced number of tip cells and branching points (asterisks) and an uneven growth front (arrows and arrowheads). Scale bar: 500 μm. (B) Feedback between the VEGF/Notch and Sema3E-Plexin-D1 signaling pathways included in the extended agent-based computational model of tip cell selection. D1-D4: transcriptional delays. r1-r3: recovery delays representing degradation. δ, s, σ: change in expression levels in response to receptor activation. (C) Simulated tip cell selection. Colors represent Dll4 levels on a continuum from purple (low) to green (high). The red boxes highlight a time frame in which a salt and pepper pattern has formed in the control vessel, while in the absence of Sema3E-Plexin-D1 signaling, only few early tip cells have been selected. (D) Average number of selected tip cells in simulated vessels. At a timepoint where the simulated control vessel (black line) already exhibits an alternating pattern of tip and stalk cells, the simulated vessel lacking Sema3E-Plexin-D1 signaling (blue line, for a given set of parameter values: δ =5, s=3) shows a 50% reduction in tip cells. Thin lines: standard deviation. n=50. (E) In silico Dll4 levels in single endothelial cells during simulated tip cell selection. In the control situation (top), Dll4 levels quickly stabilize. In the absence of Sema3E-Plexin-D1 signaling (bottom) Dll4 levels fluctuate in near synchrony before they finally stabilize. DOI: http: //dx. doi. org/10. 7554/eLife. 13212. 003 Here, we propose a general principle of how collective cell behavior determines the diverse densities of different networks: the generation of vascular topologies depends heavily on the temporal regulation of tip cell selection. Integrated simulations predict that as cell neighborhoods change, due to anastomosis or cell rearrangement events, lateral inhibition patterns will necessarily be disrupted, requiring continual re-selection of new tip cells (Bentley et al., 2009; 2014a). In fact, mouse genetics experiments demonstrated that tip cell numbers are positively correlated with the branching points of the network (Hellström et al., 2007; Kim et al., 2011). Therefore, the length of time it takes to establish (and re-establish) the alternating pattern of tip and stalk cells may be a missing, critical determinant of vascular topology (Bentley et al., 2014b; 2014c). Here, we took an integrated approach combining computational modeling, mouse genetics, and in vivo endothelial cell tracking to determine whether tip/stalk patterning can be temporally modulated to generate different topologies. We hypothesize that the frequency of tip cell selection determines the length of linear extension vs. branching, thus dictating the density of the network. To begin to test this hypothesis, it is crucial to analyze dynamic single cell behavior and collective movement in the context of network formation (Arima et al., 2011; Jakobsson et al., 2010). Previously, we used static analyses of the postnatal mouse retina as a model to understand how neural signals shape vascular topology (Kim et al., 2011). We discovered that retina ganglion cell-derived Semaphorin3E (Sema3E) and its receptor Plexin-D1, which is expressed in endothelial cells at the front of actively sprouting blood vessels, control the VEGF/Notch pathway via a feedback mechanism. Mice lacking either Sema3E or Plexin-D1 exhibited an uneven vascular growth front and a reduction of tip cells that resulted in a less branched network compared to their wildtype littermate controls (Kim et al., 2011) (Figure 1A). However, it is not clear how this phenotype is generated: specifically, how the Sema3E-Plexin-D1 feedback mechanism regulates VEGF/Notch signaling at a dynamic cellular level, and whether changes in temporal modulation of this pathway lead to the overall vascular topology phenotype. To begin to understand how Sema3E-Plexin-D1 signaling modifies vascular topology formation in a dynamic, spatiotemporal manner, we took advantage of an existing agent-based computational model (the' MemAgent-Spring Model' or MSM) that simulates the cellular processes during tip cell selection making explicit the time it takes for gene expression (e. g. transcription/translation) changes to occur (Figure 1B, C – note time delay parameters D1 and D2) (Bentley et al., 2008; 2009). The MSM has been tested against numerous independent experimental data sets and validated as predictive of new mechanisms in vivo/in vitro (Bentley et al., 2014a; Guarani et al., 2011; Jakobsson et al., 2010). To now simulate tip cell selection in the context of Sema3E-Plexin-D1 crosstalk signaling with VEGF/Notch signaling (Fukushima et al., 2011; Kim et al., 2011) the MSM model was extended by adding four new parameters (Figure 1B, Video 1–5), with sensitivity analyses and calibration simulations performed, which include modulation of the existing parameter (δ) representing the induction level of Dll4 by VEGFR-2 activation (See methods section). These four new parameters represent the time delay for induction of Plexin-D1 by VEGF (D3), the time delay (D4) and strength (s) of the reduction of Dll4 levels in response to Sema3E-Plexin-D1 signaling, based on the experimental data previously shown (Kim et al., 2011), as well as the degradation rate of Plexin-D1 (r3). Loss of Sema3E-PlexinD1 signaling was simulated by setting all Plexin-D1 levels to zero. Loss of function simulations recapitulated the two prominent features of Plxnd1-/- and Sema3e-/- mutant retinal vasculature remarkably closely: In vivo, mutant vascular networks exhibit fewer tip cells and 1. 5–2 fold reduction in branching points, as well as an uneven growth front (compare Figure 1A with 1C [red boxes]) (Kim et al., 2011). Furthermore, the dynamic nature of the simulations provided a novel insight to explain how this phenotype is generated. Simulations predict that higher Dll4 levels in the cells generates a shift towards synchronized Dll4 fluctuations overtime in contiguous cells, as they collectively battle more strongly via lateral inhibition negative feedback, causing an overall delay in amplification of differences needed to select the alternating pattern of tip and stalk cells (Figure 1C–E, Video 6,7). Therefore, computational simulations suggest that Sema3E-Plexin-D1 signaling enhances the speed and frequency of tip cell selection and thus increases the density of retinal vascular networks. 10. 7554/eLife. 13212. 004Video 1. Calibration of time delay parameters (induction of Plexin-D1, reduction of Dll4 levels). Simulation of tip cell selection with Sema3E-Plexin-D1 related regulatory time delays equal to the time it takes for pVEGFR-2 to up-regulate Dll4 (D3+D4=D1). Color indicates cell Dll4 levels (green = high, purple = low). DOI: http: //dx. doi. org/10. 7554/eLife. 13212. 00410. 7554/eLife. 13212. 005Video 2. Calibration of time delay parameters (induction of Plexin-D1, reduction of Dll4 levels). Simulation of tip cell selection with Sema3E-Plexin-D1 related regulatory time delays slightly slower than the time it takes for pVEGFR-2 to up-regulate Dll4 (D3+D4=D1+1). Dll4 levels flash irregularly between very high and very low with no stable selection possible. Color indicates cell Dll4 levels (green = high, purple = low). DOI: http: //dx. doi. org/10. 7554/eLife. 13212. 00510. 7554/eLife. 13212. 006Video 3. Calibration of time delay parameters (induction of Plexin-D1, reduction of Dll4 levels). Simulation of tip cell selection with Sema3E-Plexin-D1 related regulatory time delays slightly faster than the time it takes for pVEGFR-2 to up-regulate Dll4 (D3+D4=D1-1). Dll4 levels flash irregularly between very high and very low with no stable selection possible. Color indicates cell Dll4 levels (green = high, purple = low). DOI: http: //dx. doi. org/10. 7554/eLife. 13212. 00610. 7554/eLife. 13212. 007Video 4. Calibration of Plexin-D1 degradation rate. Simulation of tip cell selection with slowed degradation of Plexin-D1 allowing it to affect transcription of Dll4 for one timestep (12 s) longer. Dll4 levels flash irregularly between very high and very low with no stable selection possible. Color indicates cell Dll4 levels (green = high, purple = low). DOI: http: //dx. doi. org/10. 7554/eLife. 13212. 00710. 7554/eLife. 13212. 008Video 5. Calibration of Plexin-D1 degradation rate. Simulation of tip cell selection with slowed degradation of Plexin-D1 allowing it to affect transcription of Dll4 for two timesteps (30 s) longer. Dll4 levels flash irregularly between very high and very low with no stable selection possible. Color indicates cell Dll4 levels (green = high, purple = low). DOI: http: //dx. doi. org/10. 7554/eLife. 13212. 00810. 7554/eLife. 13212. 009Video 6. Simulation of a vessel with 20 endothelial cells in the absence of Sema3E-Plexin-D1 signaling. Extended regions occur with no sprouting as cells battle for longer undergoing fluctuations as the Dll4 up-regulation is higher. These eventually resolve and tip cells are selected across the whole region. DOI: http: //dx. doi. org/10. 7554/eLife. 13212. 00910. 7554/eLife. 13212. 010Video 7. Simulation of a wildtype vessel for comparison with Video 6. The selection occurs much faster and more regularly than in the vessel without Sema3E-Plexin-D1 signaling. DOI: http: //dx. doi. org/10. 7554/eLife. 13212. 010 We next examined single cell behavior in a mosaic environment, because lateral inhibition requires collective coordination, and the relative, not absolute, levels of Dll4 expression among neighboring cells determine the outcome of the tip cell selection process (Jakobsson et al., 2010). A Plxnd1-/- cell will behave differently in competition with a second Plxnd1-/- cell than in competition with a wildtype cell. Thus, we investigated whether the cell autonomous function of Sema3E-Plexin-D1 signaling in the direct competition process between two neighboring endothelial cells can drive changes to the tip cell selection dynamics. In a simulated mosaic vessel, where cells lacking Sema3E-Plexin-D1 signaling are intermingled with normal cells, the contribution of cells lacking Sema3E-Plexin-D1 signaling to the tip cell population was predicted to increase to 68–74% (robustly across a range of parameter values: δ =4–6, s=2–4) in comparison to control cells (Figure 2A, B, Video 8), with an increased speed of patterning compared to a vessel entirely lacking Sema3E-Plexin-D1 signaling. This result suggests that normally, Plexin-D1 cell-autonomously suppresses the tip cell phenotype. 10. 7554/eLife. 13212. 011Figure 2. Sema3E-PlexinD1 signaling is cell-autonomously required to suppress tip cell identity. (A) Simulated tip cell selection in a mosaic vessel with 50% mutant cells placed randomly. Cells without Sema3E-Plexin-D1 signaling are indicated by bright pink color, which turns to yellow if Dll4 levels increase. For wildtype cells: purple = low Dll4, pink = high Dll4. (B) Comparison of simulated and in vivo contribution to the tip cell population by cells lacking Sema3E-Plexin-D1 signaling at 45% mosaicism. A range of δ values simulates different strengths of loss of Sema3E-Plexin-D1 signaling. Simulations, n=50, in vivo, n=6. Data is represented as mean +/- SEM. (C) Analysis of the occupation of tip or stalk cell position by Plxnd1 expressing wildtype cells in control retina (left), and Plxnd1-/- cells (middle) or GFP+ cells (right) in mosaic retinas at P5. Red: vascular membrane staining (Isolectin B4), blue: vascular nuclear staining (α-ERG), green: in situhybridization (left, middle), α-GFP staining (right). (D) Quantification of c. n. s. = not significant, **p=0. 0033. WT retinas, n=6; Plxnd1-/- mosaic retinas, n=6; GFP+ mosaic retinas, n=4. Scale bar: 50 μm. Data is represented as mean +/- SEM. DOI: http: //dx. doi. org/10. 7554/eLife. 13212. 01110. 7554/eLife. 13212. 012Video 8. Simulated mosaic vessel with 50% mutant cells placed randomly. Cells without Sema3E-Plexin-D1 signaling are indicated by bright pink color, which turns to yellow if Dll4 levels increase. For wildtype cells: purple = low Dll4, pink = high Dll4 as before. This chimera achieves a 75% contribution of mutant cells to the tip. DOI: http: //dx. doi. org/10. 7554/eLife. 13212. 012 To test the direct competition of Plxnd1-/- cells and wildtype cells in vivo, we performed mosaic analysis using mice with tamoxifen inducible loss of Plxnd1 expression in the vasculature (Cdh5-Cre-ERT2; Plxnd1flox/flox). In the sprouting front of the wildtype retina, cells expressing Plexin-D1 were equally distributed at both tip cell and the adjacent stalk cell positions (Figure 2C, left panel, Figure 2D), showing they have no preference for either position. However, at approximately 45% of Plxnd1-/- mosaicism, 75% of the Plxnd1-/- cells became tip cells (Figure 2C, middle panel, Figure 2D) indicating that these cells do have a competitive advantage over Plexin-D1 expressing wildtype cells during tip cell selection. In contrast, in control experiments using mice with tamoxifen inducible expression of GFP (Cdh5-Cre-ERT2; Z/EG+), GFP positive cells showed no preference for either position (Figure 2C, right panel, Figure 2D), demonstrating that preferential tip cell occupancy of mutant cells is due to lack of Sema3E-Plexin-D1 signaling. Taken together, in silico prediction and in vivo mosaic retinal analyses demonstrate that Sema3E-Plexin-D1 signaling suppresses the tip cell phenotype in a cell-autonomous manner. Interestingly, this cell-autonomous effect at single cell level contrasts with the effect of Sema3E-Plexin-D1 signaling at the collective level, where more new tip cells are selected in the presence than in the absence of Sema3E-Plexin-D1 signaling (Figure 1). This finding highlights the differences between collective and cell autonomous behaviors, and the challenge of intuiting one from the other. Having confirmed that the calibrated set of parameters is valid to model contributions of the Sema3E-Plexin-D1 pathway to VEGF/Notch patterning and to predict new in vivo data of a static nature, we next employed the model to predict the effect of Sema3E-Plexin-D1 signaling on the rate of tip cell selection during the fuller dynamic network formation processes in which tip and stalk cell identities are constantly re-defined during cell rearrangement/position switching. Here, we used a new' MSM-CPM' model that has been previously extended, parameterized and experimentally validated to simulate tip/stalk patterning together with cellular rearrangements within a vascular sprout (Bentley et al., 2014a). In this model, a cell can move within the adhered collective of the sprout powered by multiple local junctional movements. By incorporating the newly calibrated Sema3E-PlexinD1 signaling extension the MSM-CPM model we simulated cell rearrangements in vascular sprouts in the presence or absence of Sema3E-Plexin-D1 signaling (Figure 3A, B, Video 9,10). Simulations predicted that in the presence of Sema3E-Plexin-D1 signaling, the tip cell is repeatedly overtaken by another cell. In contrast, in the absence of Sema3E-Plexin-D1 signaling, tip cell overtaking frequency is reduced by factor of 1. 26; a given tip cell occupies the tip cell position for a longer time (Figure 3C). Given the MSM is a qualitative not quantitative model we also performed simulations over a range of parameters and found that if the strength of Dll4 upregulation by VEGF (δ) is increased then this delay in overtaking is further exaggerated; a given tip cell occupies the tip cell position longer (Figure 3C). 10. 7554/eLife. 13212. 013Figure 3. Computational simulation predicts that lack of Sema3E-Plexin-D1 signaling leads to prolonged tip cell occupancy and reduced tip cell overtaking frequency. (A) Single frames of cell rearrangements in simulated sprouts of 10 cells. VEGF gradient extends in direction of vessel. (B) Kymograph plots of cell rearrangements in simulated sprouts. Each line represents one endothelial cell. Arrows indicate overtaking events at the tip cell position in a and b. (C) Quantification of overtaking events at the tip cell position in the presence and absence of Sema3E-Plexin-D1 signaling. A range of δ values simulates different strengths of loss of Sema3E-Plexin-D1 signaling. The setting δ=5, which matched loss of Sema3E-Plexin-D1 signaling in other conditions in the paper exhibits a 1. 26 slower tip cell overtaking frequency (events/hour.) As δ increases there is a clear trend towards slower tip cell overtaking across δ values. Data is presented as mean +/- SD, n=50. DOI: http: //dx. doi. org/10. 7554/eLife. 13212. 01310. 7554/eLife. 13212. 014Video 9. Simulation of normal cell rearrangement and tip cell overtaking in a sprout consisting of ten cells, two per vessel cross section. VEGF gradient extends in the direction of the sprout. Each cell indicated by a different color. DOI: http: //dx. doi. org/10. 7554/eLife. 13212. 01410. 7554/eLife. 13212. 015Video 10. Simulation of cell rearrangement in a sprout, in the absence of Sema3E-Plexin-D1, consisting of ten cells, two per vessel cross-section. VEGF gradient extends in the direction of the sprout. Each cell indicated by a different color. DOI: http: //dx. doi. org/10. 7554/eLife. 13212. 015 To test experimentally if tip cell selection and overtaking rates are slowed down in the absence of Sema3E-Plexin-D1 signaling, we next performed live endothelial cell tracking to follow the behavior of individual cells in an actively forming vascular network. Previously, live imaging with single cell resolution has mainly been described in sprouting assays using aortic rings (Arima et al., 2011) or embryoid bodies (EBs) generated from ES cells (Jakobsson et al., 2010). However, those assays primarily give rise to simple linear sprouts with less frequent branching than observed during retinal angiogenesis. To better mimic in vivo temporal and spatial events within a highly branched network, we developed a new ex vivo system, using explants from embryonic lungs. Whole embryonic lungs (E12. 0) were embedded within a collagen matrix in a tissue culture dish containing medium supplemented with recombinant human VEGF. After one day, endothelial sprouts grow out of the explant (Figure 4—figure supplement 1A) with tip cells extending filopodia into the collagen matrix (Figure 4—figure supplement 1B). We observed branching events at various positions of the sprouts (Figure 4B), as well as anastomosis (Figure 4—figure supplement 1C), showing that all steps of the in vivo angiogenic sprouting process are recapitulated in our system even though there is no gradient of VEGF signaling. Lung explants endogenously express Sema3e and Plxnd1 (Figure 4—figure supplement 1D). We could also detect Plexin-D1 and Dll4 in sprouting endothelial cells (Figure 4—figure supplement 1E, F). We next performed long-term live imaging of wildtype and Plxnd1-/- explants and analyzed tip cell selection frequency. In contrast to the computational model where a tip cell selection event can only occur via a switch between two cells at the tip of the sprout (Figure 3A), in the live imaging assay, tip cell selection events occur in the following distinct categories (Figure 4A, B): a positional switch between a tip and a stalk cell in a linear sprout (switch), the selection of a new tip cell at the front of the sprout (branch, type I) or at more proximal sites (branch, type II). Single cells were tracked manually using a nuclear live stain. Consistent with the computational model prediction, in vivo live imaging data indeed showed a significant delay in the selection of new tip cells in Plxnd1-/- vascular sprouts compared to wildtype sprouts. The overall appearance of new tip cells, i. e. the tip cell selection frequency, was slowed down by factor 1. 5 in the mutants (Figure 4C, D, Video 11,12). When analyzing the different categories separately, the number of events per total imaging time was significantly reduced in the categories' switch' and' branch, type II' in the absence of Sema3E-Plexin-D1 signaling (Figure 4E). 10. 7554/eLife. 13212. 016Figure 4. Endothelial cell live tracking in ex vivo lung explants reveals a reduction in tip cell selection frequency and a less branched lung vascular network in the absence of Sema3E-Plexin-D1 signaling. (A) Different types of tip cell selection events observed during live imaging. (B) Single frames from live imaging experiments illustrating the different types of tip cell selection events. Arrowheads point out the newly selected tip cells. Nuclei: blue. Scale bar: 50 μm. (C) Long-term live imaging experiments of vascular sprouts from wildtype and Plxnd1-/- lung explants. Single planes from z-stacks are shown. Arrowheads indicate a tip cell selection event. Nuclei: blue (top and middle), white (bottom). Individual nuclei are outlined by different colors in the middle panel. (D) Quantification of tip cell selection frequency calculated as events per hour. Tip cell selection frequency is reduced by factor 1. 5 in sprouts from Plxnd1-/- explants. WT, n=24 sprouts from 12 explants. Plxnd1-/-, n=30 sprouts from 11 explants. Data is represented as mean +/- SEM. (E) Quantification of tip cell selection frequency calculated as incidence of events in each category as illustrated in (A) during total imaging time. **p=0. 013 (D), *p=0. 029 (e ‘switch’), 0. 041 (e ‘branch, type II’), permutation test with shuffled genotypes. (F) Vascular sprouts originating from Plxnd1+/- and Plxnd1-/- lung explants on day 3. Left: whole-mount vascular staining (green, PECAM), right: reconstructed/skeletonized network, Scale bar: 250 μm. (G) Quantification of branching points per area. The number of branching points is significantly reduced in Plxnd1-/- lung explants. Data is represented as mean +/- SEM. Plxnd1+/-, n=7 explants; Plxnd1-/-, n=6 explants. ***p=0. 0005, permutation test with shuffled genotypes. DOI: http: //dx. doi. org/10. 7554/eLife. 13212. 01610. 7554/eLife. 13212. 017Figure 4—figure supplement 1. The ex vivo sprouting assay. (A) Sprouts originating from the lung explant express endothelial specific marker PECAM. Explants at day 1 (left) and day 3 (right) are shown. Scale bar: 300 μm. (B) Tip cells extend filopodia (arrows) into the collagen matrix. Scale bar: 50 μm. (C) Anastomosis: a new connection (arrow) is formed between neighboring sprouts. Scale bar: 50 μm (D) Sema3e and Plxnd1 are expressed by embryonic lung explants as shown by RT PCR. (E) Sprouting endothelial cells express Plexin-D1 (green). PECAM staining shown in red. Scale bar: 10 µm. (F) In wildtype endothelial sprout Dll4 (green) localized in the tip cells (arrow). Dll4-positive area is increased in Plxnd1-/- explants. Scale bar: 10 µm. DOI: http: //dx. doi. org/10. 7554/eLife. 13212. 01710. 7554/eLife. 13212. 018Figure 4—figure supplement 2. Computational method used for vascular network analysis. The region of interest (ROI) was defined as the vascular network originating from the lung explants. Vasculature inside the lung tissue was excluded from the analysis. Stacks of confocal images were binarized and then skeletonized by thinning. Branching points were identified as skeleton pixels with at least three neighbors and quantified to characterize the vascular network. The 2D images shown are the maximum intensity z-projections of the original 3D stacks, except for the binary image, which is an intensity sum z-projection. DOI: http: //dx. doi. org/10. 7554/eLife. 13212. 01810. 7554/eLife. 13212. 019Figure 4—figure supplement 3. Model: Modifications of the central pattern generator lead to the formation of diverse vascular topologies. The unique topologies of organ-specific vascular networks are dependent on the tight temporal control of the tip cell selection process. The VEGF-Notch lateral inhibition pathway is the central pattern generator of tip cell selection. We suggest that in different tissues, specific target-derived signals functioning as' molecular metronomes' regulate the pace of the pattern generator to ensure the formation of networks that cater each tissue’s specific need. A' fast molecular metronome' (e. g. Sema3E-Plexin-D1 signaling) will speed up the Dll4/Notch feedback loop and increase the frequency of tip cell selection, leading to the formation of a dense network with a small pore size, while a' slow molecular metronome' will slow down the Dll4/Notch feedback loop and decrease the tip cell selection frequency, resulting in a network with larger pore sizes. DOI: http: //dx. doi. org/10. 7554/eLife. 13212. 01910. 7554/eLife. 13212. 020Video 11. Wildtype sprout in ex vivo endothelial cell tracking assay. The movie represents 9 hr of live imaging and corresponds to Figure 4C, left panel. Merge of fluorescent channel (nuclear live stain) and brightfield channel. Arrows indicate newly selected tip cells that give rise to a new sprout. Arrowhead indicates a switching event. Single planes from z-stacks are shown. DOI: http: //dx. doi. org/10. 7554/eLife. 13212. 02010. 7554/eLife. 13212. 021Video 12. Plxnd1-/- sprout in ex vivo endothelial cell tracking assay. The movie represents 9 hr of live imaging and corresponds to Figure 4C, right panel. Merge of fluorescent channel (nuclear live stain) and brightfield channel. Arrows indicate newly selected tip cells that give rise to a new sprout. Single planes from z-stacks are shown. DOI: http: //dx. doi. org/10. 7554/eLife. 13212. 021 Finally, to directly test experimentally whether the reduced tip cell selection rate observed by live imaging in our Plxnd1-/- lung explant indeed leads to a less branched network over time, we analyzed the topology of the lung explant under the same culture conditions as the live imaging paradigm. Using a computational method (Figure 4—figure supplement 2 and detailed description in the material and methods section) to analyze the number of branching points in an unbiased way we found a significant reduction in branching points of the network (Figure 4F, G). These data further demonstrate that a slowed tip cell selection rate results in a less branched vascular network. Together, the ex vivo and in silico results demonstrate that Sema3E-Plexin-D1 signaling modulate the pace of tip cell selection. In the absence of Sema3E-Plexin-D1 signaling, the rate of tip cell selection is reduced, which overall leads to longer linear sprout extension with less frequent branching substantially influencing the architecture of the growing vasculature and resulting in a less dense network, as seen in the Plxnd1 and Sema3e mutant lung explant as well as in retina (Kim et al., 2011). During angiogenesis, the topology of the network is shaped essentially through the dynamic process of stalk cells turning into a new tip cell (tip cell selection), which is dependent on the Delta-Notch lateral inhibition pathway, a widely used machinery to regulate aspects of development that require temporal control by a' molecular clock'. For example, during somitogenesis, Delta-Notch oscillations determine the frequency of new somite formation (' the somite clock' ) (Aulehla and Pourquié, 2008). In endothelial cells, we propose that modulation of any of the components of the central pattern generator will result in an altered pace of Delta-notch oscillations and an altered vascular patterning. Deceleration of the Delta-notch feedback loop (by a' slow molecular metronome' ) will lead to the selection of fewer tip cells within a certain time frame and thus to the formation of a less dense network with bigger pore sizes. Acceleration of the selection process (by a' fast molecular metronome' ) would result in an overly dense network (Figure 4—figure supplement 3). However, complex nonlinear feedback dynamics are often hard to intuit, and further careful simulation integrated with experimentation will be required to fully elucidate the temporal modulations and topological outcomes possible. In this work, we describe how Sema3E-Plexin-D1 signaling can modify vascular density by impinging on the central pattern generator VEGF/Notch signaling. Our computational modeling predictions, mouse genetics mosaic analysis, and live imaging of individual cell dynamics in actively forming blood vessel networks and computational quantification of branching points show that the lack of Sema3E-Plexin-D1 signaling slows down the rate of tip cell selection and rearrangement, resulting in a less branched vascular network. Therefore, the Sema3E-Plexin-D1 pathway represents a' faster molecular metronome' that results in a relatively dense network. These data suggest that temporal regulation of this critical, iterative aspect of network formation could be a general mechanism, and additional temporal regulators with varying pace (fast vs. slow) may exist to sculpt vascular topology in different tissues. Furthermore, our findings may provide insights into our understanding of morphogenesis in general, and aid in efforts to develop therapeutic approaches for tissue engineering and control of tumor progression and vascular diseases. Plxnd1flox/floxmice (Zhang et al., 2009), Plxnd1+/- mice (Gu et al., 2005) and Cdh5-Cre-ERT2 mice (Monvoisin et al., 2006) were maintained on a C57Bl/6 background. Z/EG+ reporter (Novak et al., 2000) mice were maintained on a 129P3J; C57Bl/6 mixed background. Pregnant mice were obtained following overnight mating (day of vaginal plug was defined as embryonic day 0. 5). All animals were treated according to institutional and US National Institutes of Health (NIH) guidelines approved by the Institutional Animal Care and Use Committee (IACUC) at Harvard Medical School. Cre-mediated recombination was induced by intraperitoneal injection of tamoxifen (T5648, Sigma-Aldrich) dissolved in safflower oil on postnatal day (P) 4. Due to the different Cre sensitivities at Plxnd1 and Z/EG locus, we experimentally determined the dosage of tamoxifen necessary for 45% of mosaicism. 25 pg of tamoxifen was injected in Cdh5-Cre-ERT2; Plxnd1flox/flox pups, and 10 ng of tamoxifen was injected in Cdh5-Cre-ERT2; Z/EG+ pups. Mice were sacrificed at P5 and retinas were isolated for analysis. In situ hybridization, Isolectin B4 staining and ERG immunohistochemistry (1: 200; SC353, Santa Cruz) were performed as described previously. Images of flat mounted retinas were taken at 40x magnification using Zeiss LSM 510 META confocal microscope. Images were processed using Adobe Photoshop and Image J (National Institutes of Health). Mosaic recombination in Cdh5-Cre-ERT2; Plxnd1flox/flox or Z/EG; Cdh5-Cre-ERT2 was analyzed by in situ signal or GFP positivity, respectively, in combination with Isolectin B4 and endothelial nuclear ERG staining. The tip cells were determined as blind-ended endothelial cells that are associated with filopodia protrusions at the sprouting front. Endothelial cells (either tip or stalk cells) were counted by combination of Erg positive staining and morphological definition. 84 endothelial cells from 3 Cdh5-Cre-ERT2; Plxnd1flox/flox animals and 64 endothelial cells from 3 wildtype animals were counted for tip cells and stalk cells that were either Plxnd1 positive or negative. 37 endothelial cells from 2 Z/EG; Cdh5-Cre-ERT2 animals were counted for tip cells and stalk cells that were either GFP positive or negative. Statistical significance was tested using two-way Anova. Total RNA was extracted from collagen embedded lung explants, or whole retina using the RNeasy Micro Plus RNA extraction kit (Qiagen) according to manufacturer’s instructions. cDNA was generated from 100 ng total RNA using the Superscript III reverse transcription kit (Invitrogen). The following primers were used to detect Plxnd1 and Sema3e transcripts: Sema3e forward 5’-aggctacgcctgtcacataaa-3’, Sema3e reverse 5’-ccgttcttgatactcatccagc-3’; Plxnd1 forward 5’-gctgactgtagcctatgggga-3’, Plxnd1 reverse 5’- gccatctggtgggatgtcat-3’. Lungs were dissected from E12. 0 embryos in ice-cold dissecting medium (DMEM + Penicillin/Streptomycin), divided into single lobes, washed in dissecting medium (30 min, 4°C) and embedded in a glass-bottom dish (Mattek) between two layers of polymerized collagen IA gel (1. 5 mg/ml, Cellmatrix) prepared as previously described (Jakobsson et al., 2006). After solidification of collagen (30 min, 37°C), imaging medium was added (DMEM without phenol red, 15% ES cell grade FBS, 30 ng/ml rhVEGF (R&D systems), antibiotics). Explants were grown overnight at 37°C, 5% CO2. Next day, nuclear live stain solution (NucBlue, Life Technologies, Carlsbad, CA) was added (2 drops/ml for 45 min). Imaging medium was replaced and explants were imaged immediately using a Leica SP8 confocal microscope equipped with a Tokai Hit chamber (37°C, 5% CO2). 70 μm stacks were acquired at 20x magnification every 10 min. Single cells were tracked manually using a combination of nuclear staining and bright-field images of the sprouts. Per explant, 1–4 sprouts were analyzed for 9–20 hr. Tip cell selection frequency was calculated as selection events observed per hour live imaging. All 3 types of tip cell selection events were considered for quantification of selection frequency. Statistical significance was tested using a permutation test with shuffled genotypes. Explants were dissected at E12. 0 or E13. 5, prepared as described above, and fixed on day 2 or 3 in 4% PFA for 1 hr at RT. Then they were washed in PBS (3 x 10 min), permeabilized in blocking solution (PBS + 2% BSA + 0. 2% Triton) for 1 hr at RT (2 x 30 min) and incubated with PECAM (1: 300, BD), Plexin-D1 (abcam) or Dll4 (R&D) primary antibody in PBS + 0. 2% Triton overnight at 4°C and additional 6 hrs at RT. Explants were then washed in PBS + 0. 1% Triton (6x 30 min), incubated with secondary antibody in PBS + 0. 1% Triton overnight at 4°C and washed in PBS + 0. 1% Triton (2 x 30 min). If HRP-conjugated secondary antibody was used, explants were washed longer (3 x 30 min in PBS) and stained with DAB staining solution (1 mg/ml Diaminobenzindine tetrahydrochloride in PBS, Sigma) for 3 min. For network topology analysis, Alexa488 conjugated secondary antibody was used and explants were imaged at a Leica SP8 confocal microscope at 10x magnification. In the previously established' MemAgent-Spring Model' (MSM) each endothelial cell is comprised of multiple smaller computational elements (' agents' ) that represent local sections of cell membrane and actin tension beneath. The memAgents have dynamic internal levels of proteins, which enable them to sense protein levels in the local extracellular environment (primarily VEGF ligands). The endothelial cell integrates this local spatial information to determine its behavior (e. g. extension of filopodia) and perform genetic regulatory processes (e. g. Dll4-Notch) after a time delay representing the processes of transcription and translation (parameters D1 and D2 in Figure 1B). As the time delay for Notch/VEGF gene expression is not currently known in the mouse, the model was calibrated to match the known Notch periodicity (30 min) of the zebrafish somite clock (Guidicelli and Lewis, 2004) so D1=D2=28 time steps (representing 7 min of real time) as it was previously shown that, consistent with period/delay relations in the Notch somite clock models (Guidicelli and Lewis, 2004), the periodicity of Notch/VEGF signaling in the MSM model = 2 x (D1+D2+R1+R2), where R1 and R2 are the recovery delays, representing degradation rates, which were both set to 1 (Bentley et. al., 2008). To include Sema3E-Plexin-D1 interactions to the existing MSM model of VEGF/Notch/Dll4 signaling in endothelial cells during tip/stalk selection (full model described in [Bentley et al., 2008; 2009]), four new parameters were included. Two new time delay parameters: D3 controls how long it takes for VEGFR-2 to increase Plexin-D1 protein levels at the membrane and D4 determines the time it takes for an active Plexin-D1 receptor to lower Dll4 expression. Additionally s was added to determine the strength of Plexin-D1 down-regulation of Dll4 (specifically how many fewer Dll4 are produced for one active Plexin-D1 receptor) and r3, which controls how long the down regulation effect lasts for, encompassing the factors such as Plexin-D1 degradation rate, see Figure 1B for schematic. As Sema3E is assumed to be uniformly present around the cell based on experimental data in the mouse retina (Kim et al., 2011), activation of Plexin-D1 by Sema3E is not directly modeled, but simply assumed to occur at a constant level. As the exact number of Plexin-D1 receptors on the cell surface is also not known we assume Plexin-D1 receptors vary within the same range as VEGFR-2 receptors (see (Bentley et al., 2008) for details), and are instantly activated by Sema3E when present. These assumptions produce the most parsimonious model possible, permitting Plexin-D1 levels to be controlled by just the D3 time delay parameter, and Sema3E-Plexin-D1 signaling strength to be determined by the modulation of a single parameter s, which varies the strength of effect of the signaling on Dll4 up-regulation specifically. Dll4 levels were then determined as follows: Dll4t+1=Dll4t+V''δ−P''s where V''is the number of active VEGFR-2 receptors by VEGF after time delay D1 has been applied, representing the current active VEGFR-2 level affecting gene expression in the nucleus. Likewise P''is the number of Plexin-D1 receptors (assumed activated by Sema3E) able to affect gene expression after time delay D4. Previously δ, which represents the up-regulation strength of Dll4 by VEGF-VEGFR-2 signaling, was calibrated to 2 to generate matching tip/stalk pattern selection and sprouting behavior in vivo under different conditions (Bentley et al., 2008; 2009). So now with a balancing inhibition term s representing reduction in Dll4 via Plexin-D1, we know that δ - s = 2 is required for normal sprouting. Any combination of δ and s values such that this relation held true would give normal sprouting. To simulate loss of Sema3E-Plexin-D1 signaling s was set to zero. Thus the value for δ chosen ultimately determines the strength of the simulated Sema3E-Plexin-D1 mutant phenotype, hence results are shown throughout across a range of δ values when s = 0. For control simulations s = δ-2. Experiments indicate that the rate of Plexin-D1 up-regulation by pVEGFR-2 is fast compared to pVEGFR-2 up-regulation of Dll4, indicating that together the delays D3+D4 >=D1. To investigate the effects of varying the temporal regulation of Plexin-D1 on tip cell selection a sensitivity analysis was performed simulating with different delay settings for the new parameters D3 and D4 (a full analysis of varying delays D1 and D2 is given in [Bentley et al., 2008]). It was found that the lateral inhibition mechanism is strictly sensitive to the values of these new delay parameters relative to the existing delays D1 and D1. In the model, only a setting of D3+D4 =D1 would allow for normal tip cell selection in control conditions (Video 1). Even a delay with D3 or D4 = ± 1 timestep in the model (representing 15 s) would disrupt the process and tip cells could not be selected and the system falls into unrealistic' flashing' oscillations as the cells instantly raise and then lower dll4 each time step through the Notch/VEGF negative feedback loop resulting in a counter intuitive hypersprouting rather than inhibited phenotype as no cell is under the inhibition long enough to become a stalk cell (Video 2,3). Interestingly if all delay parameters are set to zero, representing the null hypothesis that no time delays are required to explain the phenotype, the same system behavior occurs as in Video 2 and 3 illustrating the importance of explicitly representing the amount of time that gene expression takes in computational models. Disrupting the degradation rate r3 of Plexin-D1 was also found to have drastic affects on the ability of the system to select tip cells. The r3 parameter was required to satisfy: r3 = r2 = r1 = 1 timestep (representing 15 s). Any increase led to similar irregular flashing oscillations and abrogated tip cell selection as seen with increases to the delays D3 or D4 (Video 4 and 5). Thus for all simulations D3+D4 =D1, where D3= 1 and D4 = 27 timesteps. Mosaic vessels follow the same simulation method as simulations of a fully wildtype or mutant vessel, except that at the start of the simulation each cell is randomly assigned a wildtype or mutant setting of the δ and s parameters (Video 8). 45% mosaicism was calculated as average of simulations with 40% and 50% mosaicism. Results were averaged over 50 runs. In this model a cell can move within the adhered collective of the sprout powered by multiple local junctional adhesion movements (based on the Cellular Potts Model' CPM' of differential adhesion [Graner and Glazier, 1992]), which are regulated by VEGF/Notch signaling. The stacks were processed and analyzed in 3D using the following Python 2. 7 modules: Numpy, Scipy, Matplotlib, Opencv2, Igraph and Networkx. The first step of the algorithm was to apply a Gaussian smoothing filter to the stacks. A standard deviation of 5 µm was used for the Gaussian kernel. Next, the moment-preserving threshold technique (Tsai, 1985) was used in order to find a proper threshold for stack binarization. Pixels having intensity values larger than the calculated threshold were classified as belonging to a vessel. Remaining image components (Stockman, and Shapiro, 2001) smaller than 20000 µm3 were considered background noise and removed from the binary image. A thinning procedure (Palagyi and Kuba, 1998) was then applied to the binary image, resulting in what we call the skeleton (Costa and Cesar, 2009) of the blood vessels. The skeleton tends to present some sets of connected pixels having more than two neighbors each. Such sets were erased from the skeleton and represented as a single pixel at the center of mass of the set. The remaining skeleton pixels having at least three neighbors were classified as branching points, while pixels having one neighbor were considered a termination point. Spurious skeleton segments were removed by an iterative algorithm. First, termination segments smaller than 20 µm were erased, where a termination segment is defined as a segment having one termination point. After erasing such segments, new small termination segments might appear. They were iteratively erased until no new termination segments smaller than 20 µm remained. The sample was then characterized by quantification of the remaining branching points. Finally, in order to validate the analysis, we created images containing both the original image and the final skeletons and verified that the obtained skeletons were accurately representing the original blood vessel structure. Statistical significance was tested using a permutation test with shuffled genotypes.

Many animals have a network of blood vessels that supplies oxygen and nutrients to every part of the body. Each organ contains a unique pattern of blood vessels; some have lots of densely packed vessels, while others have fewer vessels that are more widely spaced. New blood vessels typically form by sprouting from the side of pre-existing vessels. This involves the endothelial cells that line the inner wall of blood vessels moving outwards to create a sprout that is made up of 'tip cells' and'stalk cells'. Tip cells are found at the front of the growing vessels and encourage the formation of new sprouts, while the stalk cells trail behind and elongate the sprout. Two signaling pathways that involve two proteins called VEGF and Notch interact with each other to control which cells become tip cells and which become stalk cells. Cells with higher levels of VEGF signaling will become tip cells. These cells also activate Notch signaling, which in turn blocks VEGF signaling in their neighboring cells. This feedback mechanism enables a new sprout to form by forcing cells present around a newly formed tip cell to become stalk cells. However, it was still not understood how the different organs develop blood vessel networks with different densities. In 2011, researchers revealed that two other proteins, Semaphorin3E and its receptor Plexin-D1, are expressed in tip cells in the back of the eye in mice and control the VEGF/Notch signaling pathway. Now Kur et al. - including some of the researchers involved in the 2011 work - have used a combination of predictive computer simulations and experimental approaches to understand this interaction in more detail. The analysis showed that Semaphorin3E and Plexin-D1 speed up VEGF/Notch signaling, which causes new tip cells to form at a faster rate, and results in a more densely packed network of blood vessels. For example, in mice that lack Semaphorin3E and Plexin-D1, VEGF/Notch signaling was slower and new tip cells formed more slowly, which resulted in the blood vessel network at the back of the mice's eyes being less dense. Kur et al. propose that different organs have different'molecular metronomes' that control the pace of VEGF/Notch signaling. A fast acting metronome would yield a dense network, while a slower one would form a less dense network. This helps to explain how diverse densities of blood vessel networks are formed in different organs. This work may aid efforts to develop therapeutic approaches for controlling the development of new blood vessels in cancers and other diseases.

lay_elife

Summarize the discussion about specific details connected with market interests. Project Manager: So we come to the third meetings. I have {gap} good. Industrial Designer: {vocalsound} Project Manager: Um so in the last meeting we have discussed the functional design and now we will talk about the conceptual design. So we will talk about some specific details. Industrial Designer: Okay so I think I will do my presentation on the components concept so can you please uh open uh {disfmarker} I'm participant two. Project Manager: This {disfmarker} Industrial Designer: Components design. Project Manager: {gap} Industrial Designer: Okay so uh the first thing uh I have done is to to made a review together with the uh manufactural uh department and have which components was uh available to build a remote control. So for energy sources we have we have to choose between the solar energy, hand dynamo and uh kinetic um well uh kinetic uh technique {vocalsound} to to store the energy. User Interface: {vocalsound} Marketing: {vocalsound} Industrial Designer: We also um {vocalsound} we also can put a regular battery in the in the remote control. Now {disfmarker} Project Manager: Uh this is what we have decided in the last meeting. But if we use battery {disfmarker} Industrial Designer: Yeah b uh f well uh I meant uh by by battery I meant uh I will not have a uh a wire between the remote control and the energy source but uh I didn't fou we didn't decide yet which kind of battery we will put inside the the remote. So uh it's a point to discuss. Then uh the case material we have uh uh also several choices, like wood, rubber, titanium or latex. {vocalsound} But uh well it's not a a re uh well a real issue for the {vocalsound} {disfmarker} from the technical uh point of view. Concerning the interface uh we can we can put mm just simple buttons or scrolls or buttons uh much more complicated, but it also requires that the chip to process the button is more complicated so. User Interface: Mm. Industrial Designer: {vocalsound} And uh this is the last point, the choice of chips. So what I have f found is that I think basic battery or kinetic uh energy uh collection is the is the better way to provide energy because I think solar energy wi won't work {vocalsound} in a cluttered uh {vocalsound} uh environment. User Interface: Mm. Industrial Designer: So um so I think we can start with these two main things. For the case uh well uh I think that uh titanium is um is a good choice because it's trendy and it's uh it's uh well it's modern and uh user are are are {disfmarker} mm will be uh very happy to have a {vocalsound} a a nice remote. For the interface uh I think that we can ach achieve uh all the desired functionalities by s just uh using uh rubber buttons, simple buttons and th thus this allow to use a regular chip {vocalsound} that are uh well cheaper. {vocalsound} User Interface: Mm. Industrial Designer: And s so uh we can move to the next slide. User Interface: Sorry. Industrial Designer: Yeah. User Interface: What is this single curved {disfmarker} what does it mean? Industrial Designer: Well uh uh i i it's uh it's the the shape of the um of the remote. User Interface: So it's it's not {disfmarker} Industrial Designer: You you will have the {disfmarker} well um the the curve will fit into your hand when you grab the {disfmarker} User Interface: Yo l yeah. When you hold on it, it is comfortable to hold. Industrial Designer: Yeah. It's more confog f comfortable that if these uh it's completely flat. User Interface: Okay. Yeah. And the battery, is it kind of a rechargeable or it doesn't matter? Industrial Designer: Yeah the um that's the point. The kinetic one is uh y you can recharge uh by the um {disfmarker} User Interface: That that's what it means by kinetic. Industrial Designer: {vocalsound} Yeah and by {disfmarker} well by just by moving the ar uh your arm the mm well the remote will uh accumulate energy. User Interface: Okay. Mm-hmm. Okay. Industrial Designer: But I d I don't know it's {disfmarker} if it is feasible because I don't know if yet if if the user will move enough to provide the remote um all the necessary energy. User Interface: Mm. {vocalsound} Mm. Okay. Yeah. Yeah. We we might check with our R_ and D_ department to see if they have this product {vocalsound} ready for market. {vocalsound} Industrial Designer: Yeah. {vocalsound} And {disfmarker} yeah and so can you go to the next slide please. So and uh that's uh that summarize well what I have said. User Interface: Mm mm. {vocalsound} {vocalsound} Wha Industrial Designer: So uh you're right we can uh see in our uh R_ and D_ uh {vocalsound} if the kinetic metal is sufficient to provide enough energy. User Interface: Ah the department. Mm. Industrial Designer: {vocalsound} That's it. User Interface: Uh {disfmarker} So I um keep in touch with the R_ and D_ department. Industrial Designer: Oh yeah User Interface: {vocalsound} Industrial Designer: I take care, it's all right. User Interface: {vocalsound} So the titanium case is the normal case that {disfmarker} I'll show you some pictures that I have and you tell me whether they are titanium case or not. Industrial Designer: {vocalsound} All right. Yeah. User Interface:'Cause I am not very sure, plastic, titanium or whatever. There's another point I want to make, is that the uh {disfmarker} well you've seen them I le na my presentation that um I point out some {disfmarker} why buttons are not the mm not the only ways you can {vocalsound} use {disfmarker} Yeah. Yeah, maybe n Industrial Designer: {vocalsound} Project Manager: We will, okay. User Interface: {vocalsound} Project Manager: Three. User Interface: Yeah. {vocalsound} So the user interface is uh i it uses the aspect uh of a computer system, a programme which can be seen or heard or otherwise perceived by the human user Project Manager: {gap} User Interface: and the commands and mechanism the user uses to control its operation and input data. So you s this gives you the ways to input data and we have uh {disfmarker} we are more {disfmarker} we emphasise more on the graphical user interface here. The idea is to represent buttons as figures, diagrams, symbols and on so you you can easily when you look at the symbols you understand what it is doing. Project Manager: What's the function of this button. User Interface: Yeah. Yeah. So. Project Manager: I think it makes the the interface really {disfmarker} User Interface: Ea easy to use. So next one. Project Manager: Graphical user interface {gap}. User Interface: {gap} function five. So I can use the button, the mouse maybe. Project Manager: A graphical user interface emphasise the use of pictures. User Interface: Yeah. So next line. So the {disfmarker} here are some examples. So they cluster the buttons together. They group them into col they colour them and uh they have different forms as well. Mm but this interface are kind of confusing. Uh basically there are too many buttons. Right. Next one. Industrial Designer: Yeah. User Interface: So some people are propose voice recognition and so {disfmarker} ah by the way I receive an email from the from one our departments saying that the voice recognition has been used in the coffee machine {vocalsound} for this by a company Industrial Designer: {vocalsound} User Interface: when you tell the {disfmarker} you say good morning coffee machine and the machine are reply to you. So I just got an email saying that. Industrial Designer: Mm-hmm mm mm. User Interface: And it seems like this voice recognition technology is ready to be used so we might consider that, supposedly. Industrial Designer: Yeah fine. User Interface: {vocalsound} The next one. Mm so somebody {disfmarker} some people use uh some people use a spinning wheel th with the L_C_ display so instead of using the mm buttons you have a L_C_D_ screen and then there you can u you can use that as buttons, you can use that as real {disfmarker} so so that could be an option as well. Touch screen, I mean. Project Manager: Yeah. User Interface: Next one. And some people propose a scroll button. Integrated with push buttons or you may have scroll button instead of p just the push button. Like the one we have here. Uh, next one. So mm Project Manager: Mm-hmm. User Interface: so there are a few aspects that I collected here. So s basically this deals with special users, children, handicapped people, old people, and uh mm and prog basically they are programmable, specially for children. And uh mm {disfmarker} yeah yeah. And then they also secure uh covers, to protect uh secure and hidden programming and battery covers that will protect your settings. So {disfmarker} But we don't have to integrate all these complicated features. I'm just saying that the {disfmarker} currently in the market there are there are control there are remote controllers f {gap} customisable for different people. Yeah, so that's the point. The next one. And uh you see this is the one where you have the protection cover. Mm maybe useful for children, they migh you you they only see the buttons outside. And for adults wh where you have more control you can see the one inside. So the adults might wanna have a key to lock that to pr so children will not touch the button inside. Industrial Designer: Yeah. User Interface: Yeah. Industrial Designer: S a good idea. User Interface: The next one. Industrial Designer: {vocalsound} User Interface: So this guy {disfmarker} this is another company that provides big buttons. At {disfmarker} I see that that is useful for old people and then you don't get it lost. But for our product we don't need a big one because you have voice recognition e eventually with use. Industrial Designer: Yeah. User Interface: And you can call your remote controller if you don't know where it is. Industrial Designer: {vocalsound} User Interface: {vocalsound} T_V_ remote controller where are you? Project Manager: {vocalsound} User Interface: And then, he will beeps and to say that I am here, {vocalsound} for example. Industrial Designer: {vocalsound} Project Manager: {gap} We should include speech synthesis in this case, no? User Interface: Is it possible? Industrial Designer: Yeah. Marketing: Yeah. User Interface: Uh? {vocalsound} Industrial Designer: Yeah but uh as Norman say if uh there is uh already a commercial product available who t who do this we we can check uh to integrate it i into our uh new remote control. Marketing: Yeah. User Interface: Yeah. Yeah. Yeah. Yeah. And uh, this is another one where you can uh {disfmarker} the the the part that's a V_ standing for the volume. So there's a up arrow and a down arrow. But you the see that in the V_, the V_ appears to be the down arrow on the top {disfmarker} on the top up arrow {disfmarker} {vocalsound} if you {disfmarker} Project Manager: Mm-hmm. User Interface: up arrow there's a V_ like as as if it's turning down so it's confusing interface, so I wanna avoid this kind of thing in the design. Industrial Designer: Yeah yeah. User Interface: And here are {disfmarker} is uh here is a s short summary that I summary that I compiled after the findings I found. Big buttons are convenient, voice recognition helps, push buttons, scroll buttons, spinning wheels can be used as navigation tools. And uh user customisable is important and finally simplicity simplicity is the key. Yeah. So {vocalsound} we have many concepts there Industrial Designer: Hmm. {vocalsound} User Interface: but we have to choose later on which ones are important to be used. Industrial Designer: Yeah. User Interface: And basically uh {disfmarker} Industrial Designer: Well I {vocalsound} I think you it's it's it's fine you have uh reviewed all all the possibilities User Interface: Yeah. Industrial Designer: but uh uh well uh i if we consider that uh the user interface is displayed on the T_V_ screen I don't think we nee uh we need much buttons in the remote Project Manager: {gap} User Interface: Mm-hmm. Industrial Designer: since we we just have to navigate and to have a okay or enter key or things like that, User Interface: Yeah. Mm. Industrial Designer: because uh adding wheels or scrolls uh makes the thing more complicated and more expensive also, so. User Interface: Mm. Okay. Project Manager: Or maybe we can include the user manual in the in the remote control {gap} and we should have just a button like help and you say uh and you ik you press the button help and maybe you see the the user m might in the in the T_V_. Industrial Designer: Yeah. That's a good idea. Marketing: {vocalsound} Industrial Designer: To have a help button. User Interface: A help button. Project Manager: Yeah. User Interface: So you are display on the screen. Industrial Designer: Yeah. Project Manager: On T_V_ T_V_ screen. User Interface: So {disfmarker} on the T_V_ screen. Industrial Designer: On the T_V_ screen. On the T_V_ screen the uh how to use your remote. Project Manager: So just you push the button User Interface: Okay. Okay. Okay. Project Manager: and we will {disfmarker} Marketing: Oh. User Interface: So that eliminates all the complicated documentation {gap}, Project Manager: Yeah. User Interface: okay. So wi Marketing: But people are often enough looking at the help, Project Manager: If the if {disfmarker} Marketing: once they see the help button they say oh this is a complicated stuff. Project Manager: {vocalsound} No {vocalsound} In the case where they need help, in the case where they need help. Industrial Designer: Uh yeah. {vocalsound} Marketing: {vocalsound} It's a psychology. Industrial Designer: {vocalsound} In a marketing point of view. Marketing: {vocalsound} Okay. User Interface: Yeah. Marketing: And let us see what the market demands. Project Manager: Yeah. Marketing: We could just go to my presentation. Industrial Designer: But {disfmarker} uh wel well I think {disfmarker} Project Manager: It's just for user customizable, for kids or old people. Marketing: Yeah that's right. User Interface: Mm. Project Manager: So {disfmarker} Marketing: I mean it just showed us the remote with an cap which could be used for kids and if you remove the {disfmarker} Project Manager: So it's the same {disfmarker} Marketing: Same remote with some {disfmarker} Project Manager: Can be used by both kids and old people. Marketing: Both yeah. User Interface: Mm. {vocalsound} Well uh what I s propose is that uh you know a remote controller, i {vocalsound} it could be a cube, Industrial Designer: {vocalsound} User Interface: is uh a small device that uh looks like a cube and maybe you can just change the {vocalsound} um the buttons, if you ch turn one side you get one one buttons, you turn the other side you get the other buttons, so for maybe new generation people who get used to the computer they want lots of controls. {vocalsound} Project Manager: Maybe for kids, kids they like uh t no l they like to {disfmarker} User Interface: Small {disfmarker} Industrial Designer: Uh well. User Interface: Yeah. Industrial Designer: So le le let's see what uh what {vocalsound} people want. User Interface: Let's see the market demand. Project Manager: {vocalsound} Marketing: And then we can decide what what we can {disfmarker} yeah. User Interface: What what {gap} market {disfmarker} yes yes. Project Manager: {gap} Marketing: So we just made an marketing survey of what people need from our remotes and how it could be special from the other remotes. And we got the best on the responses from the questionnaires. Uh we also have some prizes for the most creative solutions. And we found the following solutions which we could {disfmarker} which would be helpful for our design. So seventy percent of the users, they find their remote controls very ugly, they don't find it pleasant to use in the size or usage or anything. And eighty percent of the people they are always l I mean they are willing to spend more money if the remote control would look fancy. And the current remote controls do not match well the operating behaviour of the user. And seventy five percent of the users said they zap a lot. Project Manager: Yeah. {vocalsound} Marketing: And fifty percent say they use only ten percent of the buttons, Project Manager: Mm-hmm. Industrial Designer: Yeah. Marketing: so the rest of the ninety percent of the buttons they're not used most of the times. User Interface: Yeah. Yes. Marketing: So this were the findings which we found. And also they cited frustrations with the present remote controls. Most of {disfmarker} fifty percent of the time the remote controls are lost somewhere in the room and people are always searching for them {vocalsound} rather than watching the T_V_. User Interface: Yeah. Marketing: And by the time they found the remote control the program is finished. Industrial Designer: Yeah. Marketing: So {vocalsound} they're frustrated a lot {vocalsound} User Interface: {vocalsound} Marketing: And um if the remote control is too complicated it takes much time to learn the functionality of it. User Interface: Mm. Industrial Designer: Mm, the functionalities yeah. Marketing: So you can just see the percentage, fifty percent people they responded that they always lose their remotes and thirty four percent they say that it's quite difficult to learn if it's too complex. Industrial Designer: {vocalsound} Yeah. Marketing: So keeping in view all these findings and the frustrations I think this should be the solution for them. We should have an L_C_D_ on the rem remote control. User Interface: Oh. Industrial Designer: Well mm w well I I I don't really see the advantage of having uh L_C_D_ on the on the remote control if we have a a a big screen and uh display on the screen. User Interface: Big screen. Industrial Designer: It's {disfmarker} Marketing: Mm-hmm? Industrial Designer: yeah of course it's fancy trendy and so on but it's it's expensive to produce {vocalsound} and it's not really {disfmarker} Marketing: I mean as our survey says that people are willing to pay more if their remotes are fancy. Industrial Designer: Yeah. Marketing: So if we have a L_C_D_ on the remote, rather than looking onto the T_V_ you just look into a remote and navigate it. It's the same menu as we have saw that iPod remote control. User Interface: Mm yeah. Industrial Designer: {vocalsound} Yeah yeah. User Interface: Mm. The thing {vocalsound} {disfmarker} Marketing: We just {vocalsound} play around Industrial Designer: Yeah but when you play with the iPod you don't have {vocalsound} a big screen in front of you, s Project Manager: You can use this screen instead of the big se screen, User Interface: Yeah. Yeah. Marketing: Yeah. User Interface: Yeah. Project Manager: instead of use the {disfmarker} yeah. User Interface: If you re-use the existing screen, we element {disfmarker} eliminate the L_C_D_, after all the L_C_D_ just to display Project Manager: Hmm. User Interface: and if you have the colourful screen you can make the display colourful, fancy, as fancy as the one on the L_C_D_, Marketing: Yeah. User Interface: maybe even better. Industrial Designer: Yeah. User Interface: So {disfmarker} Marketing: I mean this were the points which we got from the market demands. User Interface: Yeah. Industrial Designer: Yeah. Yeah yeah. User Interface: Yeah. Marketing: So Industrial Designer: So I th I I {vocalsound} well I think we we can focus on the uh on the fancy look on the uh User Interface: Yeah. More on a fancy design. Marketing: {vocalsound} Yeah that's fine. Industrial Designer: on the speech recognition if the technology is available Marketing: Yeah. I mean that's {disfmarker} User Interface: {vocalsound} Yeah. Industrial Designer: but well I think L_C_D_ will uh will uh make us spend a lot of money for not so big results. User Interface: Mm. Remember we have a s budget for the cost of producing the remote controller. Marketing: Mm-hmm. Project Manager: But {disfmarker} Yeah. User Interface: Yeah. So i is {disfmarker} Marketing: Uh yeah we have uh {disfmarker} Project Manager: {gap} User Interface: so the thing is you can find out how much an L_C_D_ will cost and then we'll decide again. {vocalsound} Marketing: {vocalsound} I mean that should be found out by the Industrial Designers. {vocalsound} Project Manager: {vocalsound} Industrial Designer: {vocalsound} User Interface: Uh maybe you can find out the price and tell us next time {gap}. Is i if i Industrial Designer: So price of uh L_C_D_ display. User Interface: Yeah. Industrial Designer: And {disfmarker} Marketing: And it's always good to have an voice recognition for the remote controls. User Interface: Yeah. And also the cost for the speech recognition. Project Manager: Mm. It's for {disfmarker} User Interface: Ask our R_ and D_ department. Industrial Designer: Yeah. Project Manager: it's just for small vocabulary. We {disfmarker} it's not {disfmarker} User Interface: Yeah. Marketing: Yeah it's o only for a limited vocabulary, Project Manager: yeah. User Interface: Yeah. And ho Marketing: say eighty commands or so. Project Manager: Yeah. User Interface: Yeah. Industrial Designer: Yeah okay. User Interface: And also the scroller button, how much will it cost. {vocalsound} Industrial Designer: And {disfmarker} Well uh compared to the to s the simpl simpler simplest button. Project Manager: {vocalsound} Yeah. Push push {gap}. Marketing: Mm, the scroll button, {gap} from the survey we never see that people would like to have some scrolling button. Industrial Designer: Yeah. Yeah I think that {disfmarker} Marketing: Because they they just they're just frightened to use the scrollings or {vocalsound} help button. User Interface: Yeah. Industrial Designer: Yeah I I I think that uh well uh as we have seen in the in the presentation uh well uh about uh uh fifty percent of the of the percent n choose the button User Interface: Don't use the buttons. Industrial Designer: so uh I think to have uh five uh simple button is sufficient for our functionality. User Interface: Yeah. Yeah. Yeah. Yeah. Project Manager: It doesn't mean that the other buttons are not necessary or important. Marketing: Yeah. {vocalsound} User Interface: Important. Industrial Designer: Yeah. But {disfmarker} Project Manager: But they are just less used compar Marketing: They're not used much. Project Manager: yeah. Industrial Designer: But the uh the thing is is i is that we can add a functionality on the on the T_V_ screen User Interface: Yeah. Industrial Designer: like uh a a list of function User Interface: Yeah. Mm. Industrial Designer: and then you choose with the with the button to {disfmarker} well you navigate User Interface: Yeah, yeah. So so the at most {disfmarker} more power uh. Industrial Designer: and you {disfmarker} Project Manager: Or maybe we can u uh or maybe we can uh make this the ten percent of button more bigger than the others. Industrial Designer: Yeah. Project Manager: So. Marketing: {vocalsound} Industrial Designer: But if i i if we if we could have a a a display uh g a user interface that is very complete on the T_V_ screen {vocalsound} I think that just five buttons are sufficient, User Interface: Yep. Industrial Designer: one to go up left right down and uh enter User Interface: Yeah. Industrial Designer: and you you you just select the functionality you want to access or things like that. User Interface: Mm. Mm. Yeah. Industrial Designer: You don't have to to switch to a channel to another uh {disfmarker} Project Manager: Mm. User Interface: Yeah. Marketing: Or it could be like this, as the people say, if they have a L_C_D_ on the remote not on the television. Because when you have the L_C_D_ onto the television screen you miss the picture in the background, we are most focused on the commands. Industrial Designer: Yeah but {disfmarker} Marketing: So if you have then L_C_D_ in the remote, you just have a menu, and increasing and lower these signs here to change the programs and this menu when you press the menu, in the L_C_D_ displays as you go on pressing the menu it faster displays volume, then the program, then the brightness, contrast and all the stuff. User Interface: Mm. Industrial Designer: {vocalsound} {gap} Yeah but if you look at the L_C_D_ you you don't look at the T_V_ screen Marketing: And accordingly you can just increase or decrease. Project Manager: It's {disfmarker} User Interface: Mm. Mm. Industrial Designer: so {vocalsound} i i it's not really worth to get {disfmarker} to have the image if you don't look at, so. User Interface: I if {disfmarker} Mm. Mm. Project Manager: And I think it's increases the cost of the the remote control if you use L_C_D_. I {disfmarker} User Interface: Yeah. Industrial Designer: Yeah. Marketing: Yeah that has to be checked out. User Interface: I think that there's no contradiction here, because if there are few buttons, you don't have to look at your your controller any more because you know where the buttons are, so if you wanna control the screen d sh sharpness you just say sharpness Industrial Designer: Yeah. User Interface: and then you t turn {disfmarker} you just press lef increase or decrease button Industrial Designer: {vocalsound} User Interface: and the same for the volume and the channel, {vocalsound} Marketing: Yeah. User Interface: if you had the speech recognition there you just shout your channel, just tell your channel and then you don't even have to look at the butto at the controller so finally that wil eliminates the the need for L_C_D_, Marketing: Okay. User Interface: with the help of speech recogniser you can {disfmarker} Marketing: I mean, {gap} better if we could just check all the cost with L_C_D_ User Interface: Yeah. Marketing: and also with the speech recognition. User Interface: Mm. Industrial Designer: Yeah. Marketing: And then we could find which would would be a more suitable in this case. User Interface: Mm. Mm. {vocalsound} Yeah. A and {disfmarker} Marketing: And the third problem was to find the remote control. Always, so fifty percent of the people say they lose the remotes. Industrial Designer: Well so we we can think about a well a a vocal command like uh find User Interface: Mm. Industrial Designer: and {vocalsound} when the remote control uh hears fine well yeah just uh to make him beep or t Project Manager: You will listen to a peep, {vocalsound} special peep. User Interface: Where {gap}, yeah. Marketing: {vocalsound} Yeah that's right, that's exactly what I mean by voice commander. Industrial Designer: Yeah. Marketing: Or it could be also something like this, User Interface: Yeah. Marketing: uh it's always boring to change the batteries of the remotes control, User Interface: Mm. Industrial Designer: Yeah. Marketing: so we have some one charger there and whenever we don't use the remote control we put it in the charger. User Interface: Put it back at the charge. Industrial Designer: Put {disfmarker} User Interface: Yeah. Marketing: And when we're using that t remote and if we misplace somewhere, in the charger we have a small button, and just by pressing the button in the charger the uh remote control beeps, wherever it is. Project Manager: Okay. User Interface: Yeah. Industrial Designer: Yeah. And that's a good idea, that's simple, like in phones. Project Manager: Yeah. User Interface: Mm. Marketing: I mean it doe it also doesn't require a voice command, Project Manager: But you don't you don't have to move the the charger. Industrial Designer: Yeah. Marketing: because there are problems with a voice command. User Interface: Hmm. Mm. {vocalsound} Th yeah. Mm yeah. Yeah. Mm. Marketing: Yeah, yeah, yeah. Project Manager: You have to keep it {gap}. Marketing: I mean charger would be fixed Industrial Designer: Yeah. Marketing: because it's always with electricity plugged. Industrial Designer: Yeah if there if there uh there is nuff not enough battery. Also and uh uh the remote is lost. Project Manager: Okay. User Interface: Mm. There's {disfmarker} mm. Mm. Marketing: {vocalsound} User Interface: Yeah. Marketing: Yeah that's right. User Interface: That {disfmarker} we can {disfmarker} what we can do is we can program a function whereby when you press the switch off T_V_ button, the off button, the remote there be s uh instruction on the screen, please charge charge me. You never get it lost Industrial Designer: Yeah. User Interface: because uh every time you're off the computer {vocalsound} {disfmarker} the T_V_ you are asked the the command the T_V_ com remote controller would tell you to put it back to where {disfmarker} to the charger. Marketing: It's an good reminder, User Interface: Yeah. So you will never get lost {gap} {disfmarker} yeah. Marketing: yeah that's right. Industrial Designer: Okay. Project Manager: Maybe for some people {gap} {vocalsound} lazy people. User Interface: Yeah. Yeah because everything is programmed inside. Project Manager: Yeah yeah. User Interface: So it's it's uh it's all about strategy, y Marketing: And of course the final point is a fancy look. User Interface: Mm. Marketing: As we have seen earlier the remotes which were displayed by Norman they weren't fancy, User Interface: Mm. Industrial Designer: They were ugly. User Interface: {vocalsound} Yeah, yeah. Marketing: I mean mm very big or something with lot of buttons. Industrial Designer: {vocalsound} They {disfmarker} Project Manager: Mm. Marketing: I think we should have something {disfmarker} it {disfmarker} Industrial Designer: Well the last one with the um {vocalsound} yeah with the two parts was uh {gap} original, so {disfmarker} User Interface: With uh two two two parts controller. Marketing: I mean {gap} uh I mean uh I mean uh you see if it's like that even a kid who wants to have a control he could just plug it and {vocalsound} use it, you can't avoid him. Industrial Designer: {vocalsound} Yeah. Marketing: But you can have an button for child lock. User Interface: Yeah. Industrial Designer: Yeah. User Interface: Mm. Marketing: So just by pressing the button with some code, you t you put a lock onto the remote, so that he can't use even {gap} {disfmarker} User Interface: Mm. Mm. Mm. Mm. Industrial Designer: Well we can think about uh having uh on the on the on the user interface when you switch on the T_V_ you can uh well write a code or choose a category, if it is kids, uh things like that. Marketing: Mm uh {disfmarker} User Interface: Mm. Project Manager: Or {disfmarker} User Interface: Mm. Yeah. Mm. Marketing: That's right. Project Manager: Or maybe you have to to show some specific programmes for kids and then just just {disfmarker} yeah just push uh kids button so it's automatically User Interface: Mm. {gap} these are probl yeah. Mm. Mm. Mm. Project Manager: {gap}. So if he {gap}. Marketing: I think these other four points they're the market demands and so it's for the user interface design and industrial design to just think {disfmarker} Industrial Designer: So for mm {disfmarker} Project Manager: Yeah. User Interface: Mm. Industrial Designer: yeah. So for my part I will check the prices the um the prices difference uh of what to use, where to use, and s uh and so on. Marketing: Yeah I think it should be clearer for us in the next meeting that th uh these {gap} could be included. Industrial Designer: Yeah. User Interface: Mm. {vocalsound} I think we need to define also a s the set of vocabularies for the speech recogniser Industrial Designer: Yeah. User Interface: because uh if you want {gap} uh say we can sort by channels or sort by T_V_ programs, you have to decide a category of vocabularies for them. If numbers, they're easy, Project Manager: Mm-hmm. User Interface: but if {gap} name the channel by by name {disfmarker} Industrial Designer: Well I think we can we can have just numbers for channels and you can say to your remote control like uh sports and then on the T_V_ you have a list with with uh uh well with sports program playing now User Interface: Mm. Industrial Designer: and and uh {disfmarker} Marketing: No, we have a problem there. You see uh if you have a voice commands and you are s you are watching a score on uh {disfmarker} basketball score or something, and if the score comes twenty four thirty five, you've just say twenty five Project Manager: Yeah it's {disfmarker} yeah. Marketing: and suddenly {vocalsound} the screen the channel goes to twenty five. User Interface: Mm. Industrial Designer: That's right, yeah, yeah. Marketing: So I think there should be a prefix to some numbers {disfmarker} Industrial Designer: Well but well e every possible word uh has a probability to come about of the T_V_ so. {vocalsound} User Interface: Yeah. Marketing: I mean the the {disfmarker} you just check all the probability that saying T_V_ twenty five and just ordinary twenty five. User Interface: Mm. Industrial Designer: Yeah yeah. Marketing: Ordinary twenty five you almost there's a probability of being said around sixty seventy percent User Interface: Mm. Industrial Designer: Yeah but well {disfmarker} okay. Marketing: and T_V_ twenty five I dunno it will be round about one or two percent. User Interface: Mm. Project Manager: Mm-hmm. User Interface: Mm. Marketing: So it's better to have some prefix {gap} before the number. User Interface: But I I I think that the user would like wou would like to associate the channel or call the channel rather than than the numbers. Marketing: Yeah something, some code. User Interface: You say numbe channel number five of the T_V_ correspond to something else in the channel. Industrial Designer: Yeah yeah. User Interface: So some people may want to say, I want to see this channel. Industrial Designer: Mm mm. Well I {disfmarker} Marketing: That will be too big. Project Manager: Or just {disfmarker} Marketing: And it will be difficult for the vocabulary also. User Interface: Yeah. Check with the v R_ and D_ department the capability of recogniser. Project Manager: It's difficult to to just say the the name of the channel. It will be difficult to say just the name of the channel. User Interface: {vocalsound} Uh? Project Manager: Because you have to s t uh a ch User Interface: Well, it's convenient for the user. Project Manager: yeah but you have to to have all the name of the channel in your vocabulary. Marketing: Als might be you just forgot the channel name, you kno only know the number. Project Manager: Or maybe {disfmarker} Industrial Designer: Yeah. Marketing: Then {disfmarker} Project Manager: Or maybe the user can create his own vocabulary, User Interface: The {disfmarker} uh uh mm. {vocalsound} Mm. Project Manager: just pronouncing the the name of channels and include in the vocabulary. User Interface: I I think that I have {disfmarker} mm mm {vocalsound} I think there's another way you can do is that uh you can uh {vocalsound} if {disfmarker} when the user ch press a button to choose the channel for example, then what you can do is that the {disfmarker} you can make the T_V_ screen to split them into small little little squares of images where you you you have a snapshot of every channel, so let's say it's a four by four matrix of the images, so now what you do is f looking at the all the sixteen channels available at one time, you just use the control button uh, you just you you just choose the the option you want Marketing: Yeah, the {gap}. User Interface: and then you just hit the button and then you go to that channel. So {disfmarker} Project Manager: Or lets the user create his own vocabulary of channel. User Interface: Mm. So you you don't use the speech recogniser in that way. Project Manager: No. Just you have uh in the beginning you have uh t you have to train {disfmarker} you have to create the vocabulary by yourself. User Interface: Oh, okay. Yeah. Industrial Designer: Well I uh I also {disfmarker} Project Manager: By associating each channel with the name or {disfmarker} Industrial Designer: I I also think about uh another problem, if if there is uh more than one person who is watching T_V_ {vocalsound} Project Manager: And for each one has his own. {vocalsound} User Interface: {vocalsound} Industrial Designer: the s well the the speech uh r recogniser should be able to distinguish between the two. Marketing: Yeah yeah, {gap}. Industrial Designer: Because uh I remember when I was a {vocalsound} young child with my sister we {disfmarker} yeah we always want to w to watch different emission at the at the same time User Interface: Mm. Project Manager: Or you have to s {vocalsound} User Interface: Yeah, I wanna watch this, I wanna watch that. Marketing: Yeah. Industrial Designer: so it's a pr it could be a problem if uh if well if someone passes by when you are watching T_V_ and say oh T_V_ thirty {vocalsound} and just run, s User Interface: Mm. Marketing: A and in the same lines we would have a {disfmarker} one more problem. If we are using the television in the different environments, say in the factories or in the shops where there is a lot of noise and this voice commands if they fail to work, that would bring a bad name {disfmarker} bad uh reputation for our company. Industrial Designer: Yeah, yeah. Project Manager: Yeah. User Interface: Mm. Project Manager: Mm. Marketing: So I think we should specify some pre-requirements if we want to use a voice commands, say that it should be used in an silent homely environment or s something. Industrial Designer: {vocalsound} Yeah. Project Manager: Or we can switch on or switch off the the special {gap}, yeah. Marketing: Yeah that's right that should be an option. User Interface: Mm. Industrial Designer: Yeah that's {disfmarker} well I think that's a good uh a good option because it's simple and uh simple to implement also, so. User Interface: Hmm. Marketing: I think these are the practical problems. So we need to take care of them in the design. Project Manager: Maybe we'll discuss them in next meeting. Industrial Designer: {vocalsound} Project Manager: It's time to close this meeting. User Interface: Well, you you stay a five minutes. {vocalsound} Industrial Designer: Okay. Marketing: Okay. Project Manager: {vocalsound} Yeah {gap}. {vocalsound} Okay. Industrial Designer: {vocalsound} Marketing: {vocalsound} Oh it came there on three minutes back so we are {vocalsound} {disfmarker} I think we can just press the okay. {vocalsound} Industrial Designer: Okay. Project Manager: Thanks for your collaborations. Industrial Designer: So I see ya. Marketing: Okay. See you another thirty minutes.

The team discussed a marketing survey about remote controls. They agreed to design remote controls with more fancy outlook and less buttons. The remote control should be easier to find and less complicated so users take less time to learn the functionality of it and the team discussed how to implement this function. They also talked about voice recognition and the scroller button. Then they agreed to design a child lock. Users could open child lock by pressing the button with some code. At last they discussed the set of vocabularies for the speech recogniser. They decided to put numbers and words in the vocabulary. But they realized that it would be a challenge to make the speech recogniser distinguish between different voices and deal with noises and they would keep on discussing it.

qmsum

Identifying functionally critical regions of the malaria antigen AMA1 (apical membrane antigen 1) is necessary to understand the significance of the polymorphisms within this antigen for vaccine development. The crystal structure of AMA1 in complex with the Fab fragment of inhibitory monoclonal antibody 1F9 reveals that 1F9 binds to the AMA1 solvent-exposed hydrophobic trough, confirming its importance. 1F9 uses the heavy and light chain complementarity-determining regions (CDRs) to wrap around the polymorphic loops adjacent to the trough, but uses a ridge of framework residues to bind to the hydrophobic trough. The resulting 1F9-AMA1–combined buried surface of 2,470 Å2 is considerably larger than previously reported Fab–antigen interfaces. Mutations of polymorphic AMA1 residues within the 1F9 epitope disrupt 1F9 binding and dramatically reduce the binding of affinity-purified human antibodies. Moreover, 1F9 binding to AMA1 is competed by naturally acquired human antibodies, confirming that the 1F9 epitope is a frequent target of immunological attack. Malaria is a global health problem that results in up to 3 million deaths annually [1,2]. Most at risk are young children living in malaria-endemic regions. Older children develop immunity to the parasite such that there is a reduction in parasite densities and the associated morbidity and mortality [3]. Studies demonstrating protection from passive immunization suggest that a significant component of acquired protective immunity is antibody-mediated [4–6]. Identifying the antigens recognized by protective immune responses induced by malaria has been difficult, but many proteins associated with the merozoite surface or apical organelles are targets of antibodies that block merozoite invasion. One such antigen that shows promise as a vaccine candidate is apical membrane antigen 1 (AMA1). AMA1 is a type I integral membrane protein with a 55–amino acid cytoplasmic segment and a 550–amino acid extracellular region that can be divided into three domains on the basis of intradomain disulphide bonds [7]. Recombinant AMA1 ectodomain is highly effective at inducing protection in animal models of human malaria [8,9]. Protection by passive transfer in mice [10] and the absence of protection in B cell–deficient mice [11] suggest an important role for the humoral immune response. Refolded recombinant AMA1 induces protection, whereas no protection is induced by reduced and alkylated AMA1 [9,10]. Thus, protection induced by AMA1 is mediated by antibodies that recognize conformational epitopes on the surface of the protein. Rabbit and human anti-AMA1 antibodies have also been shown to efficiently inhibit parasite invasion of erythrocytes in vitro [12]. Sequencing of P. falciparum AMA1 from laboratory and field strains has produced over 130 non-redundant AMA1 sequences. These sequences result from an assortment of polymorphisms located throughout the molecule, but concentrated in domain I. The population distribution of these polymorphisms suggests that they have arisen due to diversifying selection, most likely to avoid the binding of inhibitory antibodies [13–15]. Consistent with this, protective responses induced by AMA1 have been shown to be strain-specific. Immunization of mice with recombinant P. chabaudi strain DS AMA1 conferred almost complete protection to homologous challenge, but little protection to challenge with the heterologous strain 556KA [9]. Similarly, in in vitro growth-inhibition studies, P. falciparum strain 3D7 was efficiently inhibited by polyclonal serum elicited by 3D7 AMA1, but the HB3 and W2mef strains were less efficiently inhibited by the same reagent [16]. Kennedy et al. showed that anti-AMA1 antibodies raised in rabbits inhibited merozoite invasion by heterologous parasite strains, but there was an inverse correlation between the degree of inhibition and the mutational distance of the strains studied [17]. Thus, inhibitory anti-AMA1 antibodies appear to recognize both polymorphic and conserved epitopes. AMA1 is currently being tested in several early clinical trials, and in one of these trials a combination of 3D7 and FVO AMA1 is being assessed in an attempt to overcome the problem of polymorphisms in this antigen [18,19]. Although AMA1 has been studied extensively, its biological function is still unknown. AMA1 is an unusual malaria vaccine candidate in that it is required for both merozoite invasion of erythrocytes [20] and sporozoite invasion of hepatocytes [21]. AMA1 is targeted to the micronemes of developing merozoites and is initially expressed as an 83 kDa precursor protein [22]. N-terminal processing produces a 66 kDa product that is released onto the surface of the free merozoite [23,24]. At the time of invasion, AMA1 is cleaved by a membrane-bound subtilisin-like protease, PfSUB2, resulting in the shedding of a 48 kDa fragment such that only the cytoplasmic, transmembrane, and a 29 residue membrane-adjacent fragment can be detected in ring-stage parasites [25,26]. The importance of the shedding process is not clear, but growth inhibitory anti-AMA1 polyclonal sera interfere with AMA1 shedding such that aberrantly processed forms of AMA1 are detected [26,27]. AMA1 has been shown not to contribute to the primary weak interaction between the merozoite and erythrocyte, but it is involved in secondary adhesion events which are thought to lead to tight junction formation immediately prior to host cell invasion [28,29]. Moreover, Toxoplasma gondii AMA1 forms a complex with proteins, including TgRON4, associated with the tight or moving junction that propels the parasite into the host cell [30], and P. falciparum AMA1 has recently been shown to interact with PfRON4 [31]. The effect of the immune response on this interaction is unknown. Crystal structures of AMA1 have revealed that the antigen contains a pair of closely associated PAN domains [32,33]. Seven loops extend from the PAN scaffold and surround a long hydrophobic trough [33], which we have speculated is a ligand-binding pocket. Orthologs of AMA1 have been identified in all apicomplexan parasites, with the hydrophobic trough conserved across the phylum, including in T. gondii [34]. Therefore, the evolutionary acquisition of the loops onto the PAN scaffold that gave rise to the hydrophobic trough resulted from ancient events that preceded apicomplexan parasite divergence. In more recent times, Plasmodium species have incorporated numerous polymorphisms into some of these loops. The most highly polymorphic region of AMA1 surrounds one end of the hydrophobic trough in domain I, but dimorphic residues extend down one side of the protein surface into domains II and III [33,35]. This suggests that the hydrophobic trough is a major target of protective antibodies, but it is clear that epitopes in other regions of AMA1 are also recognized by inhibitory antibodies [27,36,37]. Given that AMA1 is in clinical trials, it is highly desirable to know more about the antigenic characteristics of the protein. In particular, information regarding the number and location of epitopes recognized by inhibitory monoclonal antibodies will facilitate vaccine development. Two growth-inhibitory monoclonal antibodies (mAbs) have been characterized: 1F9, which recognizes a polymorphic epitope on domain I [38,39], and 4G2, which recognizes a conserved region of domain II [40]. Mutagenesis studies indicate that their respective epitopes are located on loops close to but at opposite ends of the hydrophobic trough [39,40]. Here, by determining the crystal structure of the complex, we provide a detailed picture of the 1F9–AMA1 interaction. 1F9 has a very large footprint on AMA1, which includes hydrophobic trough residues as well as residues from the surrounding loops. We also show that mutagenesis of key residues on the loops surrounding the hydrophobic trough is sufficient to interfere with mAb 1F9 binding, and that the binding of both 1F9 and 4G2 to AMA1 is competed by naturally acquired human antibodies. These observations provide evidence in support of the hypothesis that the hydrophobic trough is an AMA1 ligand-binding site and a major target of protective immunity. AMA1 domains I+II in complex with 1F9 Fab crystallized under identical conditions into two crystal forms: crystal form 1 and crystal form 2, which have been solved to 2. 4 and 2. 3 Å, respectively (Table 1). An overview of crystal form 1, the more complete of the two crystal structures, is shown in Figure 1A. 1F9 interacts exclusively with domain I of AMA1. The area of interaction encompasses one end of the group of solvent exposed hydrophobic residues that form part of the hydrophobic trough (coloured green in Figure 1A), which may be a ligand-binding site. The total buried surface area at the 1F9–AMA1 interface is 2,470 Å2, with 1,250 Å2 buried on the AMA1 surface and 1,220 Å2 buried on the 1F9 surface. This is considerably larger than previously reported Fab–antigen buried surfaces, which typically range from 600 to 900 Å2. CDR loops of both the heavy and light chains of 1F9 interact with AMA1 (Figure 1A and 1B). In addition to the CDR loops, 1F9 contacts AMA1 using a large area of framework residues, which contact the hydrophobic trough (Figure 1B). There is, therefore, an interesting reciprocity to the structure in that the less variable framework ridge interacts with the conserved hydrophobic trough, and the variable antibody CDR loops interact with the polymorphic loops that surround the trough. 1F9 covers one-half of the hydrophobic trough and surrounding loops on the surface of AMA1 (Figure 2A). Three of the domain I loops (Ic, Id, and Ie) comprise ∼90% of the buried surface on AMA1. The largest interaction is made by loop Id, which contributes 48% of the 1F9-covered area. The four residues that make the largest interactions, E197, H200, F201, and D204, each contributing ∼100 Å2, are all in the Id loop (Figure 2B and 2C). All four of these residues are polymorphic; residues 197 and 200 are highly polymorphic, residue 201 is less polymorphic, and residue 204 is strictly dimorphic (Table S1). Other loop residues that make a significant contribution to the 1F9 interface (∼80 Å2) are dimorphic residue 225 and nonpolymorphic P188 (Figure 2B and 2C). The structure of the 1F9–AMA1 complex is consistent with point mutants studied previously. Any substitution of E197 abrogated 1F9 binding, whereas mutating several other polymorphic sites (196,230,243, or 244) had no effect on 1F9 binding [39]. The crystal structure shows that residues 243 and 244 do not contact 1F9, whereas 196 and 230 are at the periphery of the interface where they are solvent-exposed such that mutations are accommodated (Figure 3A). A further series of point mutations have been generated in AMA1 domain I expressed on phage. Mutations were introduced at residues 200,201, and 204 within loop Id, and residues 225 and 228 within loop Ie; all are sites of frequently occurring polymorphisms. The residues in 3D7 AMA1 were substituted with residues occurring in other AMA1 alleles, except for residue 225, where a conservative I-L mutation was analysed. Mutations at residues 200,204, and 225 all abrogated binding (Figure 3). Substitution of valine for phenylalanine at position 201 also abrogated binding, whereas the substitution by leucine at this position only partially abrogated binding. Similarly, substituting lysine for aspartic acid at position 228 only partially abrogated binding (Figure 3). Deletion of residues in loop Ic had no effect on 1F9 binding [38]. This result was surprising because the deleted residues constitute 19% of the AMA1 interaction area. Presumably, because this area of interaction is at the periphery of the interface, deletions here can be accommodated. Overall, the mutational data highlighted the importance of polymorphic residues 197,200,201,204, and 225, and these data were consistent with the crystal structures in that these residues all present large 1F9-interacting areas. The principal residues on 1F9 that interact with AMA1 are derived from light chain CDRs 2 and 3, heavy chain CDR3, and heavy chain FR1 framework residues, with a minor contribution from heavy chain CDR1 (Figure 4A). The 1F9 heavy chain CDR3 loop is very short and consists of three residues (S99, H100, and F101, which form part of a tight type II' beta turn), all of which contact AMA1. Heavy chain FR1 framework residues E1 and V2, and G26, F27, and K28, form a cluster that, together with neighbouring framework residues, forms a contact surface area of 490 Å2 (Figure 4A). This accounts for >50% of the heavy chain interacting surface, and approximately 40% of the total 1F9 contact area. AMA1 therefore makes contact with a large region on the antibody surface outside the CDRs that is conserved in most human and most mouse antibodies. One half of the 1F9–AMA1 interface is largely polar, whereas the other half of the interface is hydrophobic. Hydrogen bond interactions observed in both crystal forms are shown in Figure 4B. Most hydrogen bonds extend from residues on loop Id. For example, E197 forms hydrogen bonds with T56 in the light chain CDR2, H200 forms hydrogen bonds with H100 in the heavy chain CDR3, and D204 forms a salt bridge with R96 in the light chain CDR3. Mutations at these three sites (197,200,204) disrupted 1F9–AMA1 binding (Figure 3B), consistent with the importance of these hydrogen bond interactions at the interface. AMA1 residue N223, which extends from the domain I PAN helix, hydrogen bonds to D31 in the heavy chain CDR1. The other half of the AMA1-1F9 interface consists of a large cluster of hydrophobic side chains (Figure 4C). 1F9 residues in the hydrophobic half of the interface are all part of the heavy chain. Heavy chain framework residues V2, L4, and F27 contribute 71,16, and 86 Å2, respectively (total 173 Å2), to the interface. Heavy chain residues T32, L98, and F101 extend from antibody variable regions and contribute 24,10, and 80 Å2, respectively (total 104 Å2), to the AMA1 interface. Thus, although conserved framework residues make the largest contribution to the hydrophobic cluster, variable residues, in particular F101 in heavy chain CDR3, also contribute. AMA1 residues that contribute to the hydrophobic interface cluster include M224, M190, and Y202 from the hydrophobic trough (green, Figures 4C and 5A), P188 from loop Ic, M193 extending from the central PAN beta sheet, and F201 in loop Id. F201 contributes the largest surface area to the 1F9 interaction (101 Å2), and P188 also has a large interaction (87 Å2). Hydrophobic trough residues, M190, Y202, and M224 present smaller surface areas to 1F9 of 40,24, and 24 Å2, respectively, whereas M193 plays a minor role, presenting a 12 Å2 surface to 1F9. Consistent with its position at the centre of the hydrophobic cluster, mutations at polymorphic residue 201, were critical for 1F9 binding. Mutating residue 201 to leucine, 201FL, partially reduced binding, whereas a smaller subsitution to valine, 201FV, totally disrupted binding (Figure 3B). All previously determined structures of AMA1 are incomplete, particularly in the loops that surround the hydrophobic trough. The structure of AMA1-1F9 crystal form 1 is the only AMA1 structure describing loop If. In addition, the region of loop II adjacent to the hydrophobic trough is visible, making this the first AMA1 structure that provides a complete view of the hydrophobic trough (Figure 5A). Previously, nine hydrophobic trough residues were defined on the basis of sequence and surface exposure in the 1Z40 structure [33]. In crystal form 1, three additional residues have been identified as components of the trough. The 12 residues are hydrophobic in all Plasmodium AMA1 sequences, have hydrophobic side chains solvent exposed by 7 Å2 or more, and form part of a continuous surface on AMA1. The 12 hydrophobic residues are V169 and L176 on either side of loop Ib, F183 and M190 either side of loop Ic, Y202 and V208 within loop Id, M224 N-terminal to loop Ie, Y251 and I252 at the centre of the trough that extends from a loop following PAN beta strand 4 in domain I, M273 C-terminal side to loop If, and L357 and F367 within loop II (Figure 5A). A plot of the distance between alpha carbons in the overlaid AMA1 structures 1Z40 (AMA1 alone) and AMA1-1F9 crystal form 1 reveals that the overall trajectory of the AMA1 backbone in the two structures is very similar, with root mean square deviations (rmsds) of less than 1 Å (Figure 5B). The regions where the rmsd exceeds 1 Å all correspond to the loops surrounding the hydrophobic trough. The deviations of loops Ia, Ic, Id, and Ie are not large and rmsds do not exceed 2 Å. The largest variations in the main-chain conformations of AMA1 among the 1Z40 and AMA1-1F9 crystal form 1 and 2 structures were observed in loop If and loop II. Loops If and II were largely invisible in AMA1-1F9 crystal form 2, but where main-chain density was observed, the loop trajectories differed by 4–8 Å (Figure 5C and 5D). In the 1Z40 structure, loop II extended up domain I to form a helix and beta-hairpin adjacent to the hydrophobic trough. Although sections of loop II were invisible in 1F9–AMA1 crystal form 1, the conformation of the helix-beta-hairpin was identical to that observed in the 1Z40 structure, with rmsds not exceeding 1 Å. Therefore, although loop II can form multiple conformations, when the loop II helix and beta-hairpin is observed it forms a precise conformation that packs against domain I to form part of the hydrophobic trough. Loops Ic and Id have identical conformations in crystal forms 1 and 2 (Figure 5D), presumably because these loops form a major part of the AMA1-1F9 interface. In contrast, within loop Ie, residue 225 interacts with 1F9 in a consistent manner, whereas the remainder of the loop adopts a different conformation in the two crystal forms. Polymorphism-scanning mutagenesis and competition ELISA approaches were used to determine whether the human immune response to P. falciparum parasites involves the production of antibodies that resemble 1F9 in their binding properties. 3D7 AMA1–purified antibodies were adsorbed onto plastic and M13 phage expressing 3D7 domain I were allowed to bind. The conservative substitution 197E-Q, which had previously been shown to reduce but not ablate 1F9 binding [39], had no discernable effect on the binding of polyclonal human antibodies (Figure 6A). In contrast, more radical substitutions (197E-V and 197E-H) dramatically reduced AMA1 binding to 1F9 and to human antibodies. E197 hydrogen bonds with T56 in the 1F9 light chain (see Figure 4B) and the E-Q mutation could preserve these hydrogen bonds. However, these hydrogen bonds would not be preserved with valine or histidine substituting for E197. Also, because of the hydrophobicity of valine and the size and charge of histidine, these mutations may cause significant pertubations of the surface topology in this region of AMA1. Mutating polymorphic residues 230 or 243, at either side of the 1F9 epitope (see Figure 3A), had little effect on the binding of human antibodies to AMA1 domain I (Figure 6A). The parallels between the binding of human antibodies and 1F9 to various forms of AMA1 indicate that antibodies targeting the 1F9 epitope are a component of the human antibody response to AMA1. In order to confirm the presence of 1F9-like antibodies in malaria-infected human plasma, the ability of individual human plasma to compete with 1F9 for AMA1 binding was also tested. Plasma (from Papua New Guinean blood donors) were pre-screened for their ability to recognize recombinant full-length 3D7 AMA1 ectodomain [12]. Plasma that reacted strongly with 3D7 AMA1 were tested for their ability to compete for AMA1 binding with 1F9 and with two other anti-AMA1 mAbs, 5G8 and 4G2. All six Papua New Guinean plasma samples reduced the binding of 1F9 to 3D7 AMA1 to some degree, whereas plasma from individuals with no exposure to malaria did not inhibit 1F9 binding (Figure 6B). The Papua New Guinean plasma samples also contained antibodies to the conserved epitope recognized by the inhibitory antibody 4G2. Only one of the six plasma contained antibodies to the epitope recognized by the non-inhibitory mAb 5G8. 5G8 recognizes a linear epitope at the N-terminus of AMA1, which is not present on the 66 kDa processed form of the antigen [22,38,39]. The reverse competition experiment, in which the binding of human antibodies to AMA1 was competed by excess 1F9, indicated that human antibodies recognizing the 1F9 epitope are relatively abundant (up to 40% of the total AMA1 reactivity) in the plasma of some individuals (Figure S1). This result is consistent with the AMA1 domain 1 mutagenesis experiment, suggesting that the 1F9 epitope is an important target of the human anti-AMA1 antibody response in individuals exposed to malaria. The crystal structures described here show that mAb 1F9, which inhibits merozoite invasion, interacts with an epitope on AMA1 that includes residues in the hydrophobic trough and on loops surrounding the trough. This is consistent with the hypothesis that the hydrophobic trough is a ligand-binding pocket that plays an essential role in merozoite attachment and/or invasion. The residues presenting the largest surface area to 1F9 are located on the loops surrounding the hydrophobic trough. Most of these residues are polymorphic, and mutagenesis confirmed that they are critical to 1F9 binding, consistent with the parasite strain specificity observed for 1F9-mediated inhibition [39]. This contrasts with the epitope of the non-inhibitory mAb F8. 12. 19, which is located in domain III and conserved in several plasmodial species [41]. The 1F9–AMA1 interface is unusual in that the buried surface area is very large and antibody framework residues make extensive contact with the antigen. These framework residues form a ridge that protrudes into the hydrophobic trough, giving complementary surfaces unlike most antibody/protein interfaces, which are typically flat [42]. Exceptions include antibodies with long heavy chain CDR3 loops such as mAb b12, where the CDR3 protrudes into the CD4 binding site of HIV gp120 [43]. The 1F9 heavy chain CDR3 loop is very short and, as a result, recessed such that neighbouring framework residues are exposed. These residues from heavy chain FR1 are commonly found in mouse and human antibodies apart from residue 26, which appears to have resulted from an N to K somatic mutation (Figure S2). The ridge framework residues, together with a short CDR3 loop, may allow antibodies to interact with antigens possessing clefts. Consistent with this, framework residues of the neutralizing mAb 17-IA make important contacts with a receptor-binding canyon on human rhinovirus 14 [44]. Serological studies indicate that a significant proportion of the human antibody response to AMA1 is directed towards polymorphic epitopes [45,46]. The 1F9 epitope encompasses the most polymorphic surface region of AMA1, and human antibodies compete with 1F9 for binding to full-length recombinant AMA1. These observations suggest that that the region containing the 1F9 epitope may be an antigenic hot-spot. Residue 197, on loop Id, is the most polymorphic site in AMA1 and appears to be a critical residue in this dominant epitope, as mutation of this residue not only ablates 1F9 binding, but also markedly reduces the binding of human antibodies. In an initial attempt to quantify the antigenicity of the 1F9 epitope, the ability of 1F9 to compete for human plasma was tested. These preliminary experiments suggest that in some, but not all, individuals, a significant fraction of anti-AMA1 antibodies bind to the 1F9 epitope region (Figure S1). It is not suprising that the unusual AMA1 loops are targets of the antibody binding given their surface exposure and flexibility [47]. The loops surrounding the hydrophobic trough in the region of the 1F9 epitope are moderately flexible, whereas loops II and If are considerably more flexible. It would be anticipated that antibodies binding to the region of the trough surrounded by the more flexible loops would be parasite-growth inhibitory, and, indeed, inhibitory mAb 4G2 is known to bind to loop II [40]. In contrast to the loops contributing to the 1F9 epitope, loops II and If contain very few polymorphisms. Loop II has no polymorphisms and loop If only has two polymorphic sites: 267, with conservative substitutions (glutamate or glutamine), and residue 273, which is mostly lysine with isoleucine occurring at a low frequency. Thus, there is a striking difference in the abundance of polymorphisms in the various loops surrounding the hydrophobic trough, and this is inversely correlated with loop flexibility. It is not clear why the more flexible loops are less polymorphic, but they border the non-polymorphic face of AMA1, and this face may be partially hidden on the parasite surface. Consistent with this, 4G2 is known to be a more effective inhibitor as the Fab fragment [27]. Flexibility itself may offer partial antigenic “protection, ” or there may be other fitness constraints that prevent mutations within the flexible loops. Antibodies to AMA1 could inhibit merozoite invasion by several mechanisms. For example, it has been reported that inhibitory antibodies block AMA1 processing events [27]. On the basis of the information presented here, a reasonable conclusion is that 1F9 inhibits merozoite invasion by blocking the access of a ligand to the hydrophobic trough. Although the 4G2 epitope has been mapped to the opposite face of AMA1, its epitope also lies adjacent to the hydrophobic trough. It is therefore likely that these different monoclonal antibodies inhibit merozoite invasion through a common mechanism. A hydrophobic trough-binding ligand has not been identified, but AMA1 is known to be associated with a rhoptry neck protein RON4 that is part of the moving junction between the surface of the invading merozoite and the erythrocyte membrane [30,31]. The nature of the interaction between RON4 and AMA1 is not understood; however, if the hydrophobic trough is involved in binding, it is unclear how antibodies would interfere with the AMA1/RON4 association if surface exposure of the complex is limited to the moving junction. The polymorphic nature of merozoite surface proteins is a major problem confronting the development of a malaria vaccine incorporating one or more of these antigens. In a vaccine trial in Papua New Guinea, a three-component vaccine containing one form of MSP2 reduced parasite densities, but the effect was restricted to parasites expressing MSP2 genotypes of the same dimorphic form as the vaccine component [48]. AMA1 lacks the highly polymorphic repetitive sequences seen in MSP2, but close to 10% of the residues in the ectodomain are polymorphic. Consequently, large numbers of AMA1 haplotypes occur in endemic areas [13–15], and a vaccine incorporating a single allelic form of AMA1 is also likely to induce protection to only a subset of P. falciparum genotypes. Given the number of alternative amino acid residues that are found in the 1F9-binding region, it seems unlikely that the strategy of combining two forms of AMA1 (3D7 and FVO) [19] will result in a vaccine that, acting alone, induces effective titres of antibodies to this polymorphic site on the majority of P. falciparum genotypes. In the context of endemic malaria, such a vaccine may still achieve a satisfactory level of efficacy because many infections are avirulent, and these have the potential to broaden the specificity of the immune response primed by the vaccine. These considerations underline the importance of analyzing the genotypes of breakthrough parasitemias in vaccine trials. If an AMA1 vaccine containing one or two forms of the antigen only protects against a minority of parasite genotypes, an alternative strategy may be to generate a form of AMA1 that targets the immune response to conserved regions of the molecule, including the “non-polymorphic” end of the hydrophobic trough. A potential strategy to achieve this may be to construct a form of AMA1 in which the polymorphic loops are removed or truncated. AMA1 domains I+II were expressed in E. coli and refolded and purified using procedures described previously [49]. Mouse mAb 1F9 (isotype IgG2b with a Kappa light chain) was produced in hybridoma cell cultures and purified by protein G affinity chromatography. 10 mg of 1F9 was digested with 2. 25 mg papain (Sigma) in 26 mM tris (pH 7. 5), 4 mM EDTA, 4 mM 2-mercaptoethanol for 3 h at 20 °C. Proteolysis was terminated by adding 100 mM iodoacetic acid for 1 h at 4 °C. The 1F9 Fab fragment was purified by cation exchange chromatography (Mono S) in 20 mM acetic acid–NaOH (pH 5. 0), eluting at 100 mM NaCl. The fragment was further purified by size exclusion chromatography (Hi Prep sephacryl S-200) in 20 mM acetic acid–NaOH (pH 5. 0). The purified protein was dialysed into water, concentrated to 10 mg/ml, and stored at 4 °C in 0. 02% sodium azide. For crystallization, AMA1 domains I+II and 1F9 Fab were mixed (with the Fab in slight excess) and added to well solution (20 mM MES [pH 6. 0], 10 mM MnCl2,6% PEG 3350). Crystallization was carried by vapour diffusion in hanging drops and yielded two crystal forms that grew within 5 days. Both crystal forms were stabilized in 20 mM MES (pH 6. 0), 10 mM MnCl2,10% PEG 3350 for manipulations, and momentarily suspended in stabilization solution plus 25% methylpetanediol or glycerol for cryofreezing. The crystals were maintained at 100 K and data collected in-house using a rotating anode generator and image plate detector. 180° of data was collected in 0. 5° sections and indexed using D*TREK [50]. 1F9 heavy and light chain variable domain sequences were determined following the method of Gilliland et al. [51]. Sequences are shown alongside the closest matching mouse genomic sequences in Figures S2 and S3. The structures were solved by molecular replacement with PHASER [52] using P. falciparum AMA1 structure 1Z40 [33] and mouse IgG2b/Kappa Fab structure 2CGR [53]. AMA1-1F9 complex structures were refined using REFMAC5 [54] and model building carried out using O [55]. The structures were divided into 5 TLS groups: AMA1, the light variable domain, the light constant domain, the heavy variable domain, and the heavy constant domain. In crystal form 2, the domain II loop is disordered and AMA1 residues A355–A387 were not observed in the electron density. Similarly, the domain I loop, If, was disordered (AMA1 residues A264–A272). There was one gap in the heavy chain constant domain between residues H128 and H132. In crystal form 1, there were two gaps in the AMA1 structure. Both of these gaps were in the domain II loop: A351–A353 and A377–A389. In addition, there were two electron density gaps in the heavy chain constant domain: residues H128–H131 and H155–H159. AMA1 mutations were generated using the Kunkel method as described previously [39]. Mutated fragments were inserted into phagemid vector pHENH6 and AMA1-expressing phage were generated by inoculating transformants with M13KO7 helper phage. Phage preparations were normalized for AMA1 expression by performing ELISAs to examine the binding of mAb 9E10 to a C-terminal c-Myc epitope. 1F9 binding was examined by binding phage to immobilized 1F9 and detected using peroxidise conjugated anti-M13 mAb followed by colourimetric assay and measurement of light absorbance at 450 nm [39]. 100 μl of 0. 3 μg/ml of full-length AMA1 ectodomain was immobilized on plastic. 100 μl dilutions of human plasma from Papua New Guinean blood donors (previously selected for reactivity to a series of malaria antigens, including full-length 3D7 AMA1 ectodomain) was added followed by 10 μl of 3. 0 μg/ml mAb (either 1F9,5G8, or 4G2). The ELISA plate was washed, peroxidase-conjugated anti-mouse antibodies added, and the assay developed. For reverse competition experiments, plasma was added prior to mAbs, and peroxidase-conjugated anti-human antibodies added to the washed plate. The Protein Data Bank (http: //www. rcsb. org/pdb/) ID numbers for the structures discussed in this paper are crystal form 1 (2Q8A) and crystal form 2 (2Q8B).

Malaria caused by Plasmodium falciparum causes more than 1 million deaths annually, and the development of a vaccine against this parasite is a major public health priority. Development of a vaccine is considered feasible because infection with malaria parasites induces protective immune responses, which include antibodies to a range of proteins on the parasite surface. Antigenic diversity allows the parasite to evade protective responses, and this may make it difficult to develop a vaccine that is effective against most infections. To facilitate the design of an effective vaccine, a more detailed understanding of how antibodies interact with their target parasite antigens is required. Here, we provide a detailed structural picture of the interaction between a growth-inhibitory monoclonal antibody and the leading vaccine candidate, AMA1. The results provide important insights into why some antibodies are inhibitory and why antigenic diversity in AMA1 enables the parasite to evade protective antibody responses.

lay_plos

Calcium/calmodulin-dependent protein kinase II (CAMK2) plays fundamental roles in synaptic plasticity that underlies learning and memory. Here, we describe a new recessive neurodevelopmental syndrome with global developmental delay, seizures and intellectual disability. Using linkage analysis and exome sequencing, we found that this disease maps to chromosome 5q31. 1-q34 and is caused by a biallelic germline mutation in CAMK2A. The missense mutation, p. His477Tyr is located in the CAMK2A association domain that is critical for its function and localization. Biochemically, the p. His477Tyr mutant is defective in self-oligomerization and unable to assemble into the multimeric holoenzyme. In vivo, CAMK2AH477Y failed to rescue neuronal defects in C. elegans lacking unc-43, the ortholog of human CAMK2A. In vitro, neurons derived from patient iPSCs displayed profound synaptic defects. Together, our data demonstrate that a recessive germline mutation in CAMK2A leads to neurodevelopmental defects in humans and suggest that dysfunctional CAMK2 paralogs may contribute to other neurological disorders. Calcium/calmodulin-dependent protein kinase II (CAMK2) is a calcium-activated serine/threonine kinase that is extremely abundant in the brain, comprising as much as 0. 3% of the total brain protein content (Bennett et al., 1983). CAMK2 is highly enriched at the synapses and is necessary for the process of long-term potentiation (LTP), the activity-dependent strengthening and modulation of synaptic activity that is thought to be the molecular basis of some forms of learning and memory (Kandel et al., 2014; Lisman et al., 2002). In humans, there are four genes encoding distinct CAMK2 iso-enzymes. CAMK2A and CAMK2B are the predominant isoforms in the nervous system, with CAMK2A being expressed 3–4 times higher than CAMK2B (Hanson and Schulman, 1992). Each enzyme comprises a kinase domain, a regulatory domain and an association domain. Structurally CAMK2 holoenzymes are homo- or hetero-oligomers, consisting of 12 or 14 CAMK2 subunits (Hudmon and Schulman, 2002b). The holoenzyme assembly requires the carboxy-terminal association domain, which forms stacked pairs of hexameric or heptameric rings with the regulatory and kinase domain projecting radially to interact with other essential proteins for CAMK2 function and localization (Bhattacharyya et al., 2016). In the absence of calcium signalling, CAMK2 is inactive, as the regulatory domain inhibits kinase function (Yang and Schulman, 1999). This auto-inhibition is relieved when calcium-loaded calmodulin binds to the regulatory domain, thereby exposing the kinase domain and allowing it to phosphorylate target substrates (Meador et al., 1993). Ca2+-Calmodulin binding also exposes Thr286 on the regulatory domain of CAMK2A, which becomes phosphorylated in trans by adjacent subunits. Once this residue is phosphorylated, the enzyme is persistently active, even in the absence of continual Ca2+-Calmodulin signaling (Rich and Schulman, 1998; Stratton et al., 2013). This switch from auto-inhibition to autonomous, persistent activity is thought to constitute a biochemical form of memory, which marks the neuron for having experienced a previous calcium influx (Bhattacharyya et al., 2016; Stratton et al., 2014; Stratton et al., 2013). Mice that are homozygous null for CAMK2A are viable and display impaired spatial memory and reduced LTP in the hippocampus (Silva et al., 1992a, 1992b). The heterozygous mutant (Camk2a+/-) show a significant deficit in spatial working memory and contextual fear memory (Frankland et al., 2001; Matsuo et al., 2009). More recently, it was demonstrated that mice with neuron-specific conditional knock-out of CAMK2A similarly displayed learning deficits and defects in LTP that were comparable to the complete knockout mice (Achterberg et al., 2014). These findings suggest that neuron-intrinsic CAMK2A function is indispensable during the period of learning for memory formation. CAMK2 is conserved in invertebrates, such as D. melanogaster and C. elegans, where the kinase also plays critical roles in behavioral and cognitive traits (Cho et al., 1991; Hudmon and Schulman, 2002a; Reiner et al., 1999; Rongo and Kaplan, 1999). In C. elegans, the only CAMK2 is encoded by the unc-43 gene, which is essential for synaptic function (Rongo and Kaplan, 1999). Loss of unc-43 causes worms to have flaccid muscle tone, locomotion defects and spontaneous body contractions that resemble seizures (Reiner et al., 1999). In pediatric care, global developmental delay in infants is defined as a significant functional delay in two or more developmental domains including gross and fine motor function, speech and language, cognition, social development and personal skills (Quality Standards Subcommittee of the American Academy of Neurology et al., 2003). These defects are detected at an early age in children age five years or under, and can persist throughout life (Shevell, 2008). About 25–50% of identified case are caused by germline genetic changes, including chromosomal abnormalities, copy number variants and monogenic mutations (Srour and Shevell, 2014; van Bokhoven, 2011). For many patients with global neuro-developmental delay, the genetic etiology remains unknown. Here, we report the identification of a consanguineous family from Jordan with two affected children manifesting global neuro-developmental delay with frequent seizures and convulsions. The two affected siblings had no dysmorphic features but failed to develop the ability to walk or speak (Figure 1A, B, Figure 1—figure supplement 1A). They displayed progressive psychomotor retardation with hypotonic muscles (Supplemental Material, Videos 1 and 2). Electroencephalogram (EEG) analysis revealed abnormal epileptiform transients (Figure 1C, Figure 1—figure supplement 1B), consistent with frequent myoclonic seizures. Magnetic resonance imaging (MRI) scan showed no major structural defects in the brain of proband II: 4 (Figure 1—figure supplement 1C). Serum metabolite levels were normal, ruling out potential lysosomal storage disorders. Assuming autosomal recessive inheritance, we performed identity-by-descent (IBD) homozygosity mapping using genomic DNA from both parents, two affected probands and three healthy siblings. Only one candidate locus greater than 5 cM on chromosome 5 spanning 28 Mb was delineated (Figure 1D, Figure 1—figure supplement 1D). Whole-exome sequencing was subsequently performed on index case II: 1. After filtering for variants with low quality and low sequencing coverage, 72 homozygous variants were identified, out of which four lie within the Chr. 5 IBD region (Table 1). Three of the homozygous variants had been previously annotated as known polymorphisms with minor allele frequencies > 0. 0001. In addition, healthy individuals who are homozygous for the minor alleles had been identified in genomic sequencing databases such as dbSNP and gnomAD (Figure 1—figure supplement 1E). We therefore filtered out these variants as non-pathogenic. The fourth homozygous variant within the IBD region mapped to the CAMK2A gene (MIM: 114078), resulting in a missense mutation p. His477Tyr that has never been observed in previous large-scale sequencing databases and our in-house ethnically-matched cohort (Figure 1E, Figure 1—figure supplement 1E). Using Sanger sequencing, this private mutation was confirmed to segregate with the disease in all seven family members (Figure 1A, D). CAMK2A is a neuron-specific, highly abundant serine/threonine kinase that plays critical roles in synaptic plasticity. To understand how neuronal function is affected due to the mutation in CAMK2A, we reprogrammed primary dermal fibroblasts from patient II. 4 into iPSCs, differentiated them into neurons and measured population-level neuronal activity using a multi-electrode array (MEA) system (Figure 2A). Compared to H1 embryonic stem cell derived neurons and an unrelated CAMK2A wild-type iPSC control, the patient’s iPSCs were equally efficient in differentiating into mature neurons expressing neuronal markers TUJ1 and MAP2 after 21 days of in vitro differentiation (Figure 2B). When these differentiated neurons were plated on MEA plates to measure spontaneous action potentials, we observed a significant reduction in both the total number of spontaneous spikes (Figure 2C, left) and the mean firing rate (Figure 2C, right) in the patient-derived neurons harboring the p. H477Y mutation compared to the wild-type controls, suggesting that CAMK2AH477Y causes a profound functional defect in cultured neurons. The CAMK2A enzyme consists of an N-terminal catalytic kinase domain, a Ca2+-calmodulin-binding regulatory domain and a C-terminal association domain (AD) that is necessary for the assembly of the 12- or 14-subunit holoenzyme. The identified mutation, p. H477Y is located in the association domain (Figure 1E) and affects a histidine residue that is invariant across all vertebrate and invertebrate CAMK2A homologues. It is also conserved in other human CAMK2 paralogs with a similar association domain such as CAMK2B, CAMK2D and CAMK2G (Figure 3A, Figure 3—figure supplement 1A). In addition, this mutation is predicted to be deleterious by both SIFT, PolyPhen and MCAP algorithms. Structurally, His477 is located at the dimeric interface that forms part of the extensive interaction surface at the ‘equatorial’ plane of the CAMK2A holoenzyme (Figure 3B). Based on prior findings that CAMK2A oligomerization through its association domain is indispensable for substrate phosphorylation and synaptic localization (Bhattacharyya et al., 2016), we hypothesize that the p. H477Y allele is hypomorphic and that biallelic loss-of-function in CAMK2A is the direct cause for the neurodevelopmental phenotypes in the two probands. To measure the oligomerization potential of the CAMK2AH477Y mutant in cells, we transiently expressed FLAG-tagged wild-type CAMK2A and CAMK2AH477Y mutant in 293 T cells, which do not express endogenous CAMK2A. A third mutant CAMK2AH477X, which lacks amino acids 477–489 and thus encodes a truncated association domain, was used as an additional control. Using native lysis conditions that preserve non-covalent macromolecular interactions, we found that wild-type CAMK2A forms a prominent complex with an apparent molecular weight ~1 MDa (Figure 3C, Native-PAGE, lane 2), which is consistent with the 12- or 14- subunit CAMK2A holoenzyme (Bhattacharyya et al., 2016). As compared to wild-type CAMK2A, the ~1 MDa, putatively oligomeric species was drastically reduced for the p. H477Y mutant and was undetectable for the p. H477X mutant (Figure 3C, Native-PAGE, lane 3 and 4 vs. lane 2). Next, we examined the ability of in vitro translated CAMK2A to self-oligomerize in a cell-free system. In contrast to the negative control protein GFP, wild-type FLAG-CAMK2A efficiently co-immunoprecipitated with HA-CAMK2A (Figure 3D, Lane 10 vs 11). This self-association was preserved between CAMK2AWT and CAMK2AH477Y (Figure 3D, lanes 12 and 15), but was completely lost between wild-type CAMK2A and the p. H477X mutant (Figure 3D, lane 13). In contrast, we could not detect any self-association between FLAG- CAMK2AH477Y and HA- CAMK2AH477Y (Figure 3D, Lane 16). Taken together, these results suggest that the missense p. H477Y partially disrupts the self-association between identical CAMK2A molecules, which had been shown to be required for holoenzyme assembly. The partial loss of function of the p. H477Y mutant, as compared to a more severe mutation p. H477X, is consistent with the observed autosomal recessive inheritance of the disease in the family, where the heterozygous carriers do not display apparent neuro-developmental symptoms. During the course of this analysis, we noticed that both p. H477Y and p. H477X mutants had reduced protein abundance. This effect on the p. H477Y mutant was, however, subtle and could not readily explain the difference in the CAMK2A oligomer observed in the native gel (Figure 3C, SDS-PAGE). As it is known that in general fully assembled oligomeric complexes have enhanced stability in vivo compared to partially assembled complexes with disrupted folding like CAMK2AH477Y (Lord, 1996; Oromendia et al., 2012), we hypothesized that the p. H477Y mutant may exhibit reduced stability and undergo proteasomal degradation. To test this, 293T cells were transfected with reporter constructs that encode GFP tagged wild-type and mutant CAMK2A followed by a self-cleaving peptide T2A and mCherry (Figure 3E). The intensity of GFP fluorescence was used as a direct quantitative measure of CAMK2A stability with the mCherry as an internal control for transfection and translational efficiency. We observed a significant reduction in GFP intensity in cells expressing CAMK2AH477Y and CAMK2AH477X as compared to wild-type CAMK2A or GFP alone (Figure 3E) despite comparable mCherry fluorescence levels. This reduction was rescued when we treated the cells with MG132, which blocked proteasomal degradation. MG132 treamentled to enhanced accumulation of the p. H477Y and p. H477X mutant, with a greater effect on p. H477X (Figure 3F, lane 3,4 vs lane 7,8, Figure 3—figure supplement 1B). By contrast, the level of the wild-type protein was reduced, likely due to the toxic effects of MG132 (Figure 3F, lane 2 vs. lane 6). These results suggest that in addition to causing reduced holoenzyme assembly, the p. H477Y mutation might also directly or indirectly reduce the overall CAMK2A levels by compromising its half-life. To unequivocally demonstrate the pathogenicity of the p. H477Y allele in vivo, we performed rescue experiments using C. elegans. A single ortholog of CAMK2, encoded by unc-43, is present in the worm genome and its functions in the nervous system are welldocumented (Reiner et al., 1999). To study the neuronal defects caused by loss of unc-43, we focused on motor neuron, DA9, which has its cell body located in the pre-anal ganglion with a dendrite that extends anteriorly and a posteriorly oriented axon extending via a commissure into the dorsal nerve cord, where it proceeds anteriorly to form approximately 25 en passant synapses onto body wall muscles and reciprocal inhibitory neurons (Figure 4A) (Klassen and Shen, 2007; Maeder et al., 2014). Using mCherry-tagged UNC-43, we observed that UNC-43 could be found along the entire DA9 neuron but concentrated at synaptic boutons (Figure 4B). The homologous patient-specific mutation p. H466Y in UNC-43 (homologous to p. H477Y in human CAMK2A) significantly disrupted the synaptic localization of UNC-43 and caused the protein to be dispersed throughout the entire axon (Figure 4B). To test the consequence of UNC-43 mutation on DA9 synaptic function, we used the itr-1 pB promoter to express the fluorescently conjugated synaptic vesicle marker RAB-3 (RAB3: : GFP) within DA9 in both the wild-type and unc-43 mutant background. In wild-type animals, RAB-3: : GFP accumulated in discrete puncta along the axon of DA9 at stereotyped synaptic locations. In the canonical unc-43 (e408) loss-of-function mutant, we observed a reduction in individual pre-synaptic puncta fluorescence intensity as compared to wild-type animals. This phenotype could be rescued by cell-autonomous expression of wild-type unc-43 in DA9. However, expression of unc-43 harboring the homologous patient mutation UNC-43H466Y failed to rescue this defect (Figure 4C, D). In addition, transgenic expression of human wild-type CAMK2A fully rescued this defect, confirming the high degree of functional conservation between CAMK2A homologs, while the patient-derived human CAMK2AH477Y failed to do so (Figure 4C, bottom panels). These results suggest that the p. H477Y mutation is defective in supporting pre-synaptic function in C. elegans. Immediately posterior to the stretch of presynaptic puncta is an asynaptic domain within the DA9 axon that is devoid of any RAB-3: : GFP fluorescence in wild-type animals (Figure 4E, top panel). Loss of unc-43 results in the mislocalization of the synaptic marker RAB3: : GFP into this asynaptic region (Figure 4E, bottom panel). This defect could also be rescued by cell autonomous expression of either unc-43 or human CAMK2A. Patient derived mutation CAMK2AH477Y or the worm homologous mutation UNC-43H466Y both failed to rescue this phenotype (Figure 4E). In addition, expression of UNC-43H466Y in wild-type animals did not cause any synaptic defect or mislocalizaton of RAB3: : GFP, suggesting that the mutation does not have dominant negative effects (Figure 4F). We further tested if the patient derived mutation in CAMK2A impacted worm locomotor behavior. Null mutants for unc-43 are flaccid in posture and move with a flattened uncoordinated waveform. The animals are variably convulsive, often spontaneously contracting and relaxing their body-wall muscles in brief repeating bursts that resemble seizures (Reiner et al., 1999). We expressed either wild-type UNC-43 or UNC-43H466Y in the muscles and neurons of unc-43 (e408) mutant worms and scored the behavior of young adults in a double-blind experiment. Only the wildtype UNC-43 was able to rescue the movement defects in unc-43 (e408) animals, but not UNC-43H466Y, suggesting that UNC-43H466Y is not functional (Figure 4G). Together, the data show that CAMK2AH477Y is a loss-of-function mutation which fails to support synaptic function in vivo. In summary, we have identified an autosomal recessive neurodevelopmental syndrome characterized by growth delay, frequent seizures and severe intellectual disability that is caused by a biallelic germline loss-of-function mutation in CAMK2A. Mechanistically this mutation disrupts CAMK2A self-oligomerization and holoenzyme assembly via its association domain. Our functional results are consistent with the high degree of evolutionary conservation of the affected residue H477 in CAMK2A orthologs, as well as previous structural data demonstrating that His477 is located in the interface between two stacked CAMK2A subunits, which together form the basic repeat unit of the ring-shaped holoenzyme (Bhattacharyya et al., 2016; Stratton et al., 2014). The pathogenicity of the biallelic p. H477Y mutation is furthered highlighted by rescue experiments in C. elegans, where in contrast to wild-type human CAMK2A the p. H477Y mutant failed to rescue the neuronal and behavioral defect in unc-43 (CAMK2 ortholog) null worms. Together, these data demonstrate that the loss of function of CAMK2A is the most plausible genetic cause for the neurodevelopmental defects observed in the two affected siblings. CAMK2 plays important and evolutionary conserved roles in synaptic plasticity, neuronal transmission and cognition in near all model organisms examined, and several groups have shown that somatic mutations in human CAMK2 isoforms may contribute to neurological disorders (Ghosh and Giese, 2015; Robison, 2014; Takemoto-Kimura et al., 2017). Notably, a de novo p. E183V mutation in the CAMK2A catalytic domain was shown to cause autism spectrum disorder (Stephenson et al., 2017). This mutation was shown to act in a dominant-negative manner to reduce wild-type CAMK2A auto-phosphorylation and localization to dendritic spines. While this manuscript was under revision, S. Küry et al. reported multiple families with intellectual disability caused by de novo, heterozygous mutations in both CAMK2A and CAMK2B kinase and auto-regulatory domains, which disrupted CAMK2 phosphorylation and caused neuronal migratory defects in murine models (Küry et al., 2017). Our discovery of a novel neuro-developmental syndrome caused by biallelic CAMK2A mutations further broadens the spectrum of human neurological disorders caused by the CAMK2 family of kinases. To the best of our knowledge, this represents the first Mendelian human disease caused by biallelic CAMK2A mutations. Our functional characterization of the novel mutation p. H477Y in vitro and in vivo also reveal novel insights on how the CAMK2A holoenzyme regulates neuronal function. In contrast to all previously reported mutations in CAMK2A in intellectual disability syndromes, the p. H477Y is located within the C-terminal association domain and results in a partial but significant disruption of self-oligomerization, suggesting that the assembly of CAMK2A oligomers, in addition to its kinase function is required for neuronal function. Interestingly, the CAMK2AH477Y mutant retains the ability to interact with wild-type CAMK2A but not with itself (Figure 3D). CAMK2A displays very specific cellular and subcellular expression patterns that is important for regulating substrate phosphorylation in cells (Liu and Murray, 2012; Tsui et al., 2005). The p. H477Y missense affects the subcellular localization in neurons and this may affect its ability to function efficiently. These biochemical results provide a mechanistic basis for the autosomal recessive nature of the disease in our family: the p. H477Y allele is hypomorphic and becomes pathogenic when recessively inherited in the homozygous state. We speculate that heterozygous carriers retain sufficient CAMK2A activity for proper neuronal function. As compared to the milder disease phenotypes reported by Kury et. al., the symptoms afflicting the p. H477Y patients likely represent the most severe manifestation of the CAMK2A dysfunction in humans. Due to the high degree of conservation of CAMK2A across evolution, we employed the established C. elegans unc-43 mutant to prove the pathogenicity of the p. H477Y mutation. UNC-43 is the worm homologue of vertebrate CAMK2. We demonstrated wild-type human CAMK2A could rescue the locomotive defects of the unc-43 mutants, while the p. H477Y mutant failed to do so, likely due to its inability to localize into neuronal synapses (Figure 4). We anticipate this in vivo functional assay in C. elegans to be widely applicable to assess the pathogenicity of newly discovered CAMK2 alleles found in human diseases. Patients were identified and diagnosed by clinical geneticists at King Hussein Medical Centre, Amman, Jordan. Informed consent was obtained from the families for genetic testing in accordance with approved institutional ethical guidelines (Institute of Medical Biology, Singapore, A*STAR, Singapore, NUS-IRB reference code 10–051). For patients in Figure 1, informed consent to publish photographs and videos was obtained from parents. Genomic DNA samples were isolated from saliva using the Oragene DNA collection kit (OG-500, DNAGenotek) and a punch skin biopsy was taken from patient II. 4. Whole exome sequencing of proband II-1 was performed using the Illumina TruSeq Exome Enrichment Kit for exome capture using 1 ug of genomic DNA. Illumina HiSeq2000 High-output mode was used for sequencing as 100 bp paired-end runs at the UCLA Clinical Genomics Centre and at the UCLA Broad Stem Cell Research Centre as previously described (Hu et al., 2014). An average coverage of 34X was achieved across the exome with 87% of these bases covered at ≥10X. After filtering, a total of 73 homozygous, 125 compound heterozygous and 493 heterozygous variants were protein-changing variants with population minor allele frequencies < 1%. Both parents and their five children were genotyped using Illumina Humancore-12v1 BeadChips following manufacturer’s instructions. Call rates were above 99%. Gender and relationships were verified using Illumina BeadStudio. Mapping was performed by searching for shared regions that are homozygous and identical-by-descent (IBD) in the two affected children using custom programs written in the Mathematica (Wofram Research, Inc.) data analysis software (Source code file 1 - IBD linkage program). Candidate regions were further refined by exclusion of common homozygous segments with any unaffected family members. The confidence criteria to identify IBD blocks were a minimum of 5 cM. We identified one shared candidate loci on chromosome 5. HEK 293 T cells (ATCC Cat# CRL-3216, RRID: CVCL_0063, from Lab Stock) were cultured in DMEM media (Gibco) supplemented with 10% FBS. Cell line identity was authenticated by commercial human STR profiling with ATCC (ATCC, #135-XV). All cell lines used in this paper tested negative for mycoplasma using Lonza MycoAlert (Lonza LT07). For transient transfection, 6 × 105 cells per well were seeded in 6 well plates 24 hr before being transfected with Lipofectamine 2000 (ThermoFisher) -complexed plasmids in OPTIMEM (ThermoFisher). To construct the CAMK2A expression plasmids, human CAMK2A cDNA was PCR-amplified from ImageClone with AscI and PacI restriction sites and cloned into pCDNA3. 1 with an N-terminal 3xFLAG or 3xHA tag. All CAMK2A mutants were generated using QuikChange XL (Agilent). Cells were treated with MG132 (Sigma, M8699) at 5 µM to block proteasome degradation. iPSCs-derived NPCs were differentiated into neurons for 21 days using a previously published protocol (Xu et al., 2017a). Briefly, NPCs were plated at a density of 50,000 cells/cm2 in a poly-L-ornithine and laminin-coated plates, cultured in N2B27 medium supplemented BDNF (20 ng/ml), GDNF (20 ng/ml), cAMP (N6,2’-O-dibutyryladenosine 3’, 5’-cyclic monophosphate; Sigma; 0. 3 mM) and ascorbic acid (0. 2 mM). H1 embryonic stem cells (Gift from Lawrence W. Stanton, WiCell, RRID: CVCL_C813) were also differentiated into neurons to use as controls. H1 embryonic stem cells were verified by karyotyping (Cytogenetics Laboratory, KK Women' s and Children' s Hospital, Singapore) and checked for pluripotency by differentiation into the three germ layers marked by Nestin (ectoderm), AFP (endoderm) and ASM-1 (mesoderm). For the immunofluorescence staining, neurons were fixed for 15 min in ice cold 4% (w/v) paraformaldehyde. Permeabilization using 0. 3% (v/v) Triton-X in 1X PBS was performed for 10 min then incubated with 1: 1000 mouse anti-Tuj1 (Covance Research Products Inc Cat# MMS-435P, RRID: AB_2313773), 1: 1500 guinea pig anti-MAP2 (Synaptic Systems Cat# 188 004, RRID: AB_2138181) overnight at 4°C in 5% (w/v) BSA diluted with 1X PBS. For visualization, 1: 1000 secondary antibody conjugated to Alexa Fluor 594 (Thermo Fisher Scientific Cat# A-11076, RRID: AB_2534120) or Alexa Fluor 488 (Thermo Fisher Scientific Cat# A-11001, RRID: AB_2534069) was applied. Counter staining for nuclei were performed using Dapi. Images were captured using the FV3000 Olympus confocal. For the multi-electrode array (MEA) recordings, neurons on day 21 were dissociated and replated on 0. 1 polyethylenimine (Sigma) -coated 48 well MEA plates (Axion Biosystems) in BrainPhys media supplemented with BDNF, GDNF, cAMP and ascorbic acid as previously described (Xu et al., 2017b). Spontaneous neuronal activity was observed and recorded at 37°C for 5 min every 2–3 days using the Maestro MEA System (Axion Biosystem). Independent measurements were taken from seven wells for each condition (technical replicates). CAMK2A proteins were synthesized in vitro using TNT T7 Quick Coupled Transcription/Translation (Promega) with 1 μg of plasmids in 20 μl reaction volumes for 90 mins at 30°C. For co-immunoprecipitation, the reactions were diluted 10x in TBS (100 mM Tris-HCl, pH = 8,150 mM NaCl) with 1% Nonidet P40 (NP40) and incubated with 10 μl anti-FLAG M2 agarose beads (Sigma) at 4°C overnight. Proteins were eluted with 1x Laemmli buffer after 3 washes in the 1xTBS with 1% NP40. Total protein lysate was quantified using a standard Bradford assay and 10 μg of lysate was used for immunoblotting experiments. For Blue Native PAGE, cells were lysed in 1x Sample Preparation buffer (ThermoFisher) containing 1% digitonin. 1% SDS was supplemented for SDS-PAGE. All proteins were transferred to PVDF membranes using TurboBlot (Bio-rad) at 2. 5 mA for seven mins. The primary antibodies used were anti-FLAG (M2, Sigma-Aldrich Cat# F3165, RRID: AB_259529), anti-GAPDH (Santa Cruz Biotechnology Cat# sc-47724, RRID: AB_627678) and anti-HA (Y-11, Santa Cruz Biotechnology Cat# sc-805, RRID: AB_631618). The secondary antibodies were anti-rabbit IgG-HRP (Jackson ImmunoResearch Labs Cat# 111-035-003, RRID: AB_2313567), anti-mouse IgG-HRP (light chain specific) (Jackson ImmunoResearch Labs Cat# 205-032-176, RRID: AB_2339056) and anti-mouse IgG-HRP (Jackson ImmunoResearch Labs Cat# 115-035-003, RRID: AB_10015289). All strains were maintained at 20°C on OP50 E. coli nematode growth medium plates as described (Brenner, 1974). N2 Bristol strain worms (WB Cat# CB4852, RRID: WB-STRAIN: CB4852) were used as the WT reference, and the unc-43 (e408) mutant was used. To visualize synaptic vesicles in DA9 neuron, wyIs85 [Pitr-1: : GPF: : RAB-3] was used (Klassen and Shen, 2007). OTL70 wyIs85[Podr-1: : dsred, Pitr-1: : gfp: : rab-3]; jpnEx40[Podr-1: : gfp, Pmig-13: : unc-43] OTL71 wyIs85; jpnEx41[Podr-1: : gfp, Pmig-13: : unc-43] OTL72 unc-43 (e408); wyIs85; jpnEx44[Podr-1: : gfp, Pmig-13: : unc-43 (H466Y) ] OTL73 unc-43 (e408); wyIs85; jpnEx45[Podr-1: : gfp, Pmig-13: : unc-43 (H466Y) ] OTL74 unc-43 (e408); wyIs85; jpnEx42[Podr-1: : gfp, Pmig-13: : unc-43] OTL75 unc-43 (e408); wyIs85; jpnEx43[Podr-1: : gfp, Pmig-13: : unc-43] OTL76 unc-43 (e408); wyIs85; jpnEx47[Podr-1: : gfp, Pmig-13: : CAMK2A] OTL77 unc-43 (e408); wyIs85; jpnEx48[Podr-1: : gfp, Pmig-13: : CAMK2A] OTL78 unc-43 (e408); wyIs85; jpnEx49[Podr-1: : gfp, Pmig-13: : CAMK2AH477Y] OTL79 unc-43 (e408); wyIs85; jpnEx50[Podr-1: : gfp, Pmig-13: : CAMK2AH477Y]] OTL82: jpnEx54[Podr-1: : gfp, Pmig-13: : mcheery: : unc-43] OTL83: jpnEx55[Podr-1: : gfp, Pmig-13: : mcherry: : unc-43] OTL84: jpnEx56[Podr-1: : gfp, Pmig-13: : mcherry: : unc-43 (H466Y) ] OTL85: jpnEx57[Podr-1: : gfp, Pmig-13: : mcherry: : unc-43 (H466Y) ] OTL86: unc-43 (e408); jpnEx58[Punc-122: : dsred, Punc-104: : mcherry: : unc-43, Phlh-1: : mcherry: : unc-43] OTL87: unc-43 (e408); jpnEx59[Punc-122: : dsred, Punc-104: : mcherry: : unc-43, Phlh-1: : mcherry: : unc-43] OTL88: unc-43 (e408); jpnEx60[Punc-122: : dsred, Punc-104: : mcherry: : unc-43 (H466Y), Phlh-1: : mcherry: : unc-43 (H466Y) ] OTL89: unc-43 (e408); jpnEx61[Punc-122: : dsred, Punc-104: : mcherry: : unc-43 (H466Y), Phlh-1: : mcherry: : unc-43 (H466Y) ] Expression plasmids for transgenic worm lines were made using the pSM vector (C. Bargmann), a derivative of pPD49. 26 (A. Fire). The mig-13 promoter was cloned between SphI/AscI, and C. elegans unc-43 isoform d or human CAMK2α was cloned between NheI/KpnI or AscI/NheI, respectively. P. H466Y and p. H477Y mutations were introduced by PCR-based mutagenesis using KOD-plus- high fidelity DNA polymerase (TOYOBO, Tokyo, Japan). Transgenic worms were generated as described (Mello, 1995). Plasmids were injected into animals at 10 ng/μl (in the case of Pmig-13: : unc-43) and 50 ng/μl (in the case of Pmig-13: : CAMK2α) together with coinjection markers at 90 ng/μl. All fluorescence images of DA9 were taken in live worms immobilized with 5% agar pad, 10 μM levamisol (Sigma) and 0. 1 mm polystylene beads (Polysciences, Inc., Warrington, PA, USA) with a 63×/1. 4 NA objective on a Zeiss Axioplan 2 Imaging System or a Plan-Apochromat 63×/1. 3 objective on a Zeiss LSM710 confocal microscope using similar imaging parameters for the same marker across different genotypes. Fluorescence quantification was determined using Image J software (ImageJ, RRID: SCR_003070). mcherry: : unc-43 or mcherry: : unc-43 (H466Y) were expressed in unc-43 (e403) mutant worms using both unc-104 promoter (neuron-specific promoter) and hlh-1 promoter (muscle-specific promoter). Two independent lines were established and analyzed. The behavioral phenotype of the transgenic worms was scored in a double-blind manner using a stereo microscope (SZX16, Olumpus, Tokyo, Japan). From the movement behavior on OP50 feeder NGM plates, each worm was classified to behave like ‘wild-type’ or ‘unc-43’. 100 worms were observed for each genotype on the same day from two independently derived transgenic lines.

Each year, some children are born with developmental disorders and intellectual disabilities. These conditions are often caused by mutations in specific genes. Sometimes both copies of a gene - one inherited from each parent - need to be mutated for the symptoms to develop. These mutations are known as recessive mutations. Here, Chia, Zhong, Niwa et al. diagnosed two siblings in their clinical care with a new form of neurological disease that affects the development of the brain and leads to frequent seizures. To test whether the young patients shared a genetic mutation that could explain their condition, the researchers analyzed the DNA of the children and compared the results with the DNA from their parents and healthy siblings. The results showed that the two children with the condition had inherited a recessive mutation in a gene called CAMK2A. The protein this gene encodes helps nerve cells to form connections and communicate with each other, and it has been shown to be essential for learning and memory. The CAMK2A enzyme is made up of several identical subunits that form a complex. Chia et al. discovered that the mutation prevented these subunits from joining together properly, resulting in a faulty protein. CAMK2A and other related proteins are crucial for the health of the brain in a wide range of animals. Indeed, experiments in Caenorhabditis elegans, a roundworm commonly used to study neurons, confirmed that the mutation inherited by the children indeed caused similar neurological defects in the worms. Taken together, these experiments suggest that the children's condition is caused by the mutation in both copies of the CAMK2A gene. For patients born with inherited diseases, it is often difficult to pinpoint exactly which mutation is responsible for the specific disorder. These findings could therefore help pediatric geneticists recognize this newly defined syndrome and reach the correct diagnoses. These results could also be the starting point for studies that look into restoring the activity of the defective CAMK2A protein. More broadly, identifying genes that are critical for the healthy development of the brain could shed light on common neurological conditions, such as epilepsy and autism.

lay_elife

(Open: Hotel - Laundry Area. There are gigantic washing machines and dryers all around. Workers are doing laundry. A man is walking around the floor with a woman.) HOTEL MANAGER: They put me in charge of all of this. I'm like one of those big shots on Wall Street only I deal with laundry, instead of money. WOMAN: Wow. HOTEL MANAGER: Not many people know it, but this is the nerve center of the entire hotel. WOMAN: Well, I would have thought it would be room service. HOTEL MANAGER: No way! Proper washing, at this volume, depends on water quality, bleach concentration, time and of course agitation and heat. WOMAN: Agitation and heat, yeah, I definitely got that. We're gonna be late for dinner. HOTEL MANAGER: I'll push our reservations. Check this baby out. (They stop in front of a large washing machine) Energy plus. That means, not am I only washing away the filth and the fluids that people get on your hotel sheets; I'm also the front line on the fight against global warming. (She hears a noise) WOMAN: What was that? HOTEL MANAGER: Foreign object in the wash barrel. It happens. WOMAN: It looks gooey. Won't it gum up your machine? HOTEL MANAGER: Whatever it is, this baby can handle it. (The woman leans in closer and sees a human skull and screams) (Cut to: Hotel - Laundry Area. Hours later. Booth and Brennan arrive and are going to meet Cam at the washing machine.) BOOTH: So, the only way the body could have gotten down here is if someone threw it down the laundry shoot. BRENNAN: That would seem to suggest homicide. BOOTH: Yes it would. (Booth rubs his hand down his tie - Brennan notices.) BRENNAN: Is that a new tie? BOOTH: Yeah, yeah. Yes, it is. You like it? BRENNAN: Well, I'm not sure why you'd want to wear frolicking cetaceans around your neck. BOOTH: Well, 'cause Catherine got it for me. Look, they're dolphins! BRENNAN: The marine biologist? BOOTH: Yeah. Yeah, it was a present. BRENNAN: Interesting. BOOTH: Wh-what do you mean, "Interesting"? (They arrive at the washer) CAM: Check this out. Good luck on the I.D. Too bad they didn't do it on the delicate cycle, huh? BRENNAN: Well, obviously the manufacturers didn't anticipate human remains. BOOTH: Well, according to the laundry guy, the body could have been sitting in a pile of dirty sheets for 2 days before it got thrown into the wash. CAM: Well, the heat in here would have sped up decomp. (She exhales) Look out, I'm going in. (Cam climbs into the washer as Brennan starts to examine the skeleton.) BRENNAN: Judging by the concavity of the rib end, the victim was middle age. Dismemberment occurred post-mortem; most likely during the spin cycle. BOOTH: Hey, what's so interesting about my tie. BRENNAN: Well, a gift is a social contract - a basic anthropological construct. By giving you a tie, Catherine has entered into a social contract with you. BOOTH: Really? (Brennan nods.) CAM: Fabric softeners permeated the soft tissue. Everything's swollen. Speaking of social contracts, do you like your gynecologist? BRENNAN: She's extremely competent, yes, but I don't think she's accepting new patients. I thought you were happy with Dr. Oxenburg? BOOTH: Alright, can you two just save the lady part discussion for when I'm not here? CAM: Dr. Oxenburg moved to California and I am looking for a doctor for Michelle. She's at that age, ya know? BOOTH: No. No, no. No. We're not going to be discussing your daughter's s*x life. Because A, she's a good girl; she doesn't have s*x and B, you're touching a dead body. BRENNAN: I don't follow your logic. CAM: I'm always touching a dead body, Seeley. If I let that be a variant of conversation, I wouldn't- (She's cut off by some of the remains falling from the washer and hitting her arm and rolls over and stops in front of Booth.) BOOTH: Oh, oh, ho. Okay. Can someone just please remove the eyeball. CAM: (walking over) Ooh. This is not an eyeball. (she picks it up and holds it out in front of her) BOOTH: What is it? CAM: I'll put it this way, our victim was male. BRENNAN: (smiling) Would you rather us go back to talking about lady parts? [OPENING CREDITS] (Cut to: Medico Legal Lab - Forensics Platform. Brennan, Arastoo and Hodgins are examining the skeleton) BRENNAN: The skeleton appears to have suffered a great deal of damage in the washer. HODGINS: Well, that wins the understatement award for today. BRENNAN: It's going to be very difficult to find cause of death. HODGINS: Well, if the 3lbs of muck I got here in the catch is any indication, then that washing machine must have been a beast. ARASTOO: Dr. Brennan, I found something. Right here, the junction of the maxilla and palatine. HODGINS: Bullet wound? He was shot? ARASTOO: It was a result of surgery, not a gunshot. Most likely from oral cancer. BRENNAN: There's also bone degeneration on the mandible. Our victim was probably a cigar smoker. Anything else? HODGINS: Condom! Unused. Never mind. ARASTOO: I also found a number of fully remodeled fractures. Here's one on the right hamate bone. BRENNAN: It's approximately 3 years old. ARASTOO: An injury like this is mostly typical for professional baseball players. The great Tony Pena suffered a similar fracture.. HODGINS: 0 for 11 as a utility infielder before being traded to the Royals. ARASTOO: To the White Sox, after a successful surgery to repair his hamate. HODGINS: Oh, and are all American Muslim drives in a run! Nice one, Arastoo! ARASTOO: Thank you. (Brennan clears her throat as a hint to get back to work.) ARASTOO: Uh, I through our victim played baseball but then I saw this. A number of mostly repaired impression fractures in the tibia and tarsus. BRENNAN: An injury pattern most commonly sustained by rock climbers. And these fractures occurred about a year after the hamate fracture. ARASTOO: And here's where I go for the triple because...another remodeled fracture. About a year old and to the hyoid. BRENNAN: With an accompanying micro-fracture on the parietal, almost certainly indicating our victim was involved in a high speed collison while wearing a helmet. What does this mean? HODGINS: Rock climbing, baseball playing, crash test dummy? (Cam enters.) CAM: Is there a testicle up here? HODGINS: Four that I know of... BRENNAN: I thought you located them at the scene? CAM: Just the one that scared Booth. I've cataloged the loose tissue and all the victims organs and glands are accounted for but the other testicle seems to have vanished. BRENNAN: Well, it's not here but it does appear our victim was in the habit of injuring himself, annually, in a variety of risky behaviors. CAM: So the guy with one gonad actually had balls. (Hodgins starts laughing, Brennan doesn't laugh) CAM: Well, if you'll excuse me, I've got an appointment. (Cut to: Royal Diner. Booth, Brennan and Sweets are seated at the counter.) BOOTH: So you're telling me I should be looking for a cigar smoking, thrill seeker missing one of the family jewels. BRENNAN: That's correct. SWEETS: You know, stereotypical male behavior, such as cigar smoking and risk taking, could be a form of overcompensation for a man who was born with only one testicle. I could write up a profile. BRENNAN: There's no reason too, Sweets. born without one. He could just have easily of lost it in one of his dangerous pursuits. If a testicle is severely twisted or degloved, it almost always necessitates removal. In the case of penetrating trauma.. BOOTH: No. No. Just- do you think we can go 20 minutes on this case without talking about testicles? SWEETS: Please. BRENNAN: Okay. (she pauses) Booth has made a social contract with the marine biologist. SWEETS: Sorry? BOOTH: (laughing) It's amazing - you go from injured testicles to the woman I'm dating. And you? You're supposed to say "That's interesting." in a very annoying way. BRENNAN: It was a logical transition. SWEETS: But it is very interesting. BRENNAN: Booth and I are friends. Catherine is an intelligent, attractive woman and I'm intrigued by their developing relationship. BOOTH: That's nice...I think. SWEETS: Yeah. I think it is nice. BOOTH: Thank you, Bones. SWEETS: Wow. You two seem to be handling dating very well. I'm impressed. BRENNAN: Well, you've known me for 2 years, Sweets. You should expect to be impressed by me. (Cut to: The Medical Plaza. Dr. Paul Lidner's Office. Cam is sitting with a file folder on her lap.) CAM: You were board certified in '99? DR. LIDNER: That is correct. CAM: And you did a fellowship at Vanderbilt in - DR. LIDNER: Reproductive Endocrinology. Boy, you really did your research. CAM: You were recommended by Dr. Oxenburg but I wanted to make sure this is the right fit. DR. LIDNER: Oh, yeah. Oh, of course. CAM: Do you consider yourself easy to talk to? Especially about delicate topics - like, someone's first time having s*x. DR. LIDNER: Um, their first time with a new partner? CAM: No. No. Their first time. Like in losing virginity. DR. LIDNER: Oh. Well, uh, yes. Of course. I-I'd be, uh, very sensitive with that topic. Um, especially if the delay in sexual maturity was due to some sort of trauma or negative conditioning. CAM: Trauma? Oh, my god. Why would you bring up trauma. DR. LIDNER: Well, when an adult woman, such as yourself, forgoes sexual activity, there are often deeper issues at play. (Cam starts to laugh) What? CAM: There's been a misunderstanding here. I'm looking for a gynecologist for my daughter, who's 16. DR. LIDNER: Oh, great. I mean, yeah. That just makes more sense. Um, I deal with a lot of teenagers and uh, they feel very comfortable with me because they know they're free to speak about anything and it will remain confidential. CAM: Excellent. Well, obviously, you're respectful and highly qualified. I think this will work. DR. LIDNER: I'm glad. You and your husband can feel confident that I will treat your daughter with the utmost care and consideration. CAM: (reaching out her hand) Thank you. (she turns to leave, but then turns back) Acutally, I uh, don't have a husband. Michelle's my adoptive daughter. DR. LIDNER: Oh, I see. CAM: So, thank you, Dr. Lidner. DR. LIDNER: Uh, Paul. Please. Just call me Paul. CAM: Paul. (She nods and leaves.) (Cut to: Medico Legal Lab - Ookay Room. Cam enters) CAM: You have something for me? HODGINS: Yup. The fingernails. CAM: I don't think they're gonna help with the I.D. HODGINS: I also have clothing remnants, mostly denim. A chain, also mostly likely part of the victims apparel. Some pieces of molded silicone elastomer.. CAM: (picking up the petri dish) The missing testicle. HODGINS: Oh, god. CAM: It's a prosthetic one. They're made of a silicone shell and filled with saline. Now, obviously, this has suffered a lot of damage in the wash, but if we can get a serial number off this? We can get an I.D. (Hodgins makes a face. He is not thrilled about the fake testicle) (Cut to: Medico Legal Lab - Angela's Office. Angela has the silicone pieces on the big screen.) ANGELA: Okay. I photographed each piece of the silicone elastomer and I used the photos to reassemble a virtual prosthetic. CAM: Well, the serial numbers were pretty warn from the washing machine. ANGELA: Yeah. Well, hopefully by looking at the image under different color filters we can at least get some of the digits. You know, I have to say. This whole "finding I.D. by testicle" definitely beats facial reconstructions. CAM: Does that prosthetic seem overly large to you? ANGELA: Well, it isn't' to scale, Cam. CAM: Guess it's been a while. ANGELA: Okay, I got the serial number for you. CAM: I'll get it to Booth. (Cam leaves) ANGELA: (looking at the screen) It's pretty big. (Cut to: FBI Headquarters - Booth's Office. Booth and Brennan are looking at a folder.) BOOTH: Richard Cole. 42. Single. Commercial Real Estate Developer. Left his entire fortune to the American Cancer Society. So that rules out, uh, financial motive. BRENNAN: Researchers can be ruthless. BOOTH: Yeah. Look at this. This article a cigar connoisseur. BRENNAN: He had oral cancer, he shouldn't have smoked. BOOTH: That is not his biggest problem right now. He talks about going to fantasy camps every year for his birthday. BRENNAN: Fantasy camps? BOOTH: They're expensive camps where grown-ups get to pretend to be, you know, race car drivers, uh, professional ball players - pretty much anything. BRENNAN: That would explain the yearly injuries. You could easily crush a testicle at a rodeo camp. BOOTH: Look what's going on this week at the hotel. God, I wish I had enough money for this one. BRENNAN: Why? What is it? (Booth plays a video) SIMON GRAHAM: (online) Did you ever dream about jamming with your favorite musical heros or playing gigs in front of throgs of screaming fans? Well, please join us at the one and only fantasy camp created by legendary music manager and promoter - me. Simon Graham. BRENNAN: Music camp. BOOTH: That's not music. That's Rock 'n Roll, Baby. Yeah! (Cut to: Hotel - Hallway.) BOOTH: According to Cole's lawyer, he was trying to buy a property that Simon Graham didn't want him to have. BRENNAN: You think Graham murdered Cole to safeguard some property? BOOTH: It's possible. Don't say that Cole is dead. I don't want anybody panicking or trying to flee the scene. BRENNAN: I was a very big fan of Toad the Wet Sprocket. BOOTH: You might want to keep that to yourself, alright. Now, don't get overwhelmed. It's going to be very loud in here and.. (They enter the room to find people waiting on line.) GUY: So, are you going to the seminar on string height? GIRL: I have a class on tone control then. GUY: Oh, that's great. BOOTH: They're all in line. They're not even pushing. This is not my rock and roll fantasy. (Cut to: BOOTH: No, no. This can't be right. Rock and roll is not about seminars. Come on, people. Does anyone remember Laughter? GINO: Hey, I remember. Zeppelin, man! CAMPER #1: Dude, you could lose the tie around here. BRENNAN: Well, he-he likes it. It's a gift from a woman. BOOTH: (holding up his badge) FBI Special Agent Seeley Booth. (to Brennan) Hacker get you anything? BRENNA: A subscription to Lapham's Quarterly. BOOTH: Sexy. GINO: Can I help you guys with something 'cause I really don't want to be late for class. I paid a fortune to be at this camp. BOOTH: Yeah, We're looking for Simon Graham. BEBE: I think he's near the stage. Walk this way. BOOTH: Aerosmith. GINO: Hey! You know you're music. This is Bebe. I'm Gino. CAMPER #1: Is something wrong? BRENNAN: It's about one of your camp mates. Richard Cole. BEBE: Cole? He hasn't been around for a couple of days. GINO: He probably got tired of fighting with Simon. SIMON GRAHAM (onstage) Boys and girls, Erik Dalton! GINO: Simon's in there. Erik Dalton is starting his workshop. BOOTH: What? Erik Dalton is here? (Erik starts playing his guitar. Booth starts heading to the stage. ) Bones! This is more like it! BRENNAN: (following him) Booth... BOOTH: Just gimme a minute, huh? BRENNAN: Booth. Booth. (Booth starts to jump around and play air guitar) BOOTH: Yeah! BRENNAN: (shouting over the noise) Shouldn't we try to talk to Simon? (Booth continues to play air guitar, ignoring her.) BRENNAN: Booth! (Brennan's had enough. She goes up to the stage and unplugs the guitar from the amp.) BOOTH: Whoa, whoa. ERIK DALTON: Who unplugged me?! No one unplugs me! BOOTH: (to Brennan) He's right. No one unplugs Erik Dalton. BRENNAN: Well, apparently, I do. It was the only way to get your attention. ERIK: Get her out of here, Simon. BRENNAN: (to Erik) You're- you're yelling unnecessarily, probably due to hearing loss from the music you play. SIMON: What do you think you're doing? BOOTH: Okay, uh, listen. Simon Graham, (Booth holds up his badge) FBI. We just have to ask you a few questions about Richard Cole. SIMON: Richard Cole? What the hell is this? I'm running a business here. BOOTH: I understand. It's not gonna take long. (to Erik - plugging his guitar back into the amp) Dude, I'm sorry. (Erik starts playing again - Booth holds up his finger to Simon and signals "one minute". He'll talk to him after he's done.) (Cut to: Medico Legal Lab - Angela's Office.) ARASTOO: I've identified 83 injuries to the skeleton that occurred either at the time of death or in the washing machine. I can't tell which. HODGINS: So, no cause of death. ARASTOO: Not without evidence from hemorrhagic staining and these remains went through a wash cycle with industrial strength bleach. I was pitching a no hitter and now I can't find the plate. HODGINS: This baseball thing? You allowed to play? ARASTOO: No. The Qur'an strictly forbids baseball. lacrosse, of course, and board games with hungry hippos. HODGINS: That's a yes, with an additional comment on my ignorance. ARASTOO: I was a state All-Star in high school. I even got scouted by a couple of farm teams HODGINS: No way. ARASTOO: Yeah. I still play on the weekends. My mosque is in a league. We play against churches and synagogues. HODGINS: Wow. ARASTOO: You should join us sometime. HODGINS: Oh, come on. I can't be on an all-Muslim team. I'm a lapsed Episcopalian. ARASTOO: No, every team has a few ringers. The Jews have a Unitarian batting 400. HODGINS: Really? Huh. Never tried to beat the infidels before. ARASTOO: As long as you find something in your washer goop that helps me determine cause of death, you can play short stop. HODGINS: You're on. (Cut to: FBI Headquarters. Sweets meets Booth as he's getting off the elevator) SWEETS: Hey, is it true that Simon Graham's here? BOOTH: Yeah. He's in the conference room. SWEETS: Okay. I can provide a valuable insight, Agent Booth. The man practically invented a rock sub-culture. You need me. BOOTH: You just want to meet him. SWEETS: There's a little of that, yes, but I'll be professional. You know I'm a good profiler. BOOTH: Okay. Professional. SWEETS: Yes. Sure. Okay. Do you know how many seminal rock concert tours he's managed? BOOTH: The guy's a god but may be a murdering god. So, use your ears, not your mouth. Just listen. You understand? SWEETS: Yeah. (Cut to: FBI Headquaters - Conference Room.) SIMON: My camp is for people who love music. Not wannabes in designer jeans and fancy guitars they never touch. BOOTH: You describing Richard Cole? SIMON: Well, yeah, he's the idiot that wanted me to turn him into Jimmy Page when the only guitar that he ever played came from a video game, I mean SWEETS: That would offend you. That's a personal affront as someone who's dedicated their life to nurturing real musicians. SIMON: Well, shouldn't it? BOOTH: What can you tell me about The Stock Yard. SWEETS: Oh, it's a famous rock club in downtown Baltimore. All the greats used to play there. Mr. Graham used to run it in the 80's. You weren't asking me. Sorry. BOOTH: Once Cole's deal went through, he was gonna tear it down, wasn't he? SWEETS: That's why you and Cole were fighting, right? SIMON: Yeah, we settled that though. BOOTH: What are you talking about? SIMON: Well, Cole said that he'd leave the club alone if I let him do one song with Erik Dalton at the end of camp night. SWEETS: Erik Dalton was one of your guests? Erik Dalton's one of your guest stars? BOOTH: He blackmailed you. So did you agree to let Cole play with Dalton? SIMON: Well, yeah. This is The Stock Yard we're talking about. BOOTH: How did Dalton feel about that? SIMON: How do you think he'd feel? Listen, guys, do you mind if I go back to work? BOOTH: Sure. SWEETS: Yeah. One-one quick question. Bar fight: who wins? Prince and the New Power Generation or Korn. SIMON: (standing) Never mess with Prince. (He leaves.) SWEETS: (to Booth) Never mess with Prince. (He exhales and raises his hand as to say - slow down there, kiddo) (Cut to: Royal Diner. Michelle and Cam are at the counter eating lunch.) CAM: Honestly, I think you'll like Dr. Lidner and you should have someone else to talk to..especially if there's anything - anything you prefer I didn't know about. MICHELLE: I told you, Perry and I are not having s*x. CAM: I know and I believe you but you're growing up and your body - it's a woman's body now, not a child's and you should treat it like a woman and I'm gonna stop talking now. MICHELLE: It's no big deal, Cam. It's just a doctor. CAM: I know, it just means your growing up, for real. MICHELLE: And you don't quite know how to handle that. CAM: What? No...yeah, kinda but I will, we will, right? I mean, you don't know how to handle me, either. Do you? Because that would be embarrassing. MICHELLE: We're fine, Cam. CAM: Because you and Perry aren't having s*x, right? Okay, okay. Fry? (Cut to: Medico Legal Lab - Bone Room. Arastoo is still looking over the skeleton when Hodgins and Angela enter.) HODGINS: How's it going? ARASTOO: Dr. Brennan's waiting for cause of death. At this rate, I'm gonna be John Gochnaur. ANGELA: Okay, boys, I'll bite. Who is John Gochnaur? ARASTOO: Worst Major League Baseball player ever. HODGINS: Cleveland Indians. 187 batting average. Zero home runs and 146 errors. ANGELA: Well, is that bad? HODGINS: Yeah. It's incomprehensibly bad. (handing a tray to Asastoo.) Here, this might help. If found it in the washing machine cache. ARASTOO: What is it? HODGINS: Well, it's bone, so that's your department. ARASTOO: (looks into the microscope) Cross-hatching. May be bone, but it isn't human. HODGINS: What is it? ARASTOO: It can't be... HODGINS: What can't it be. ARASTOO: It's a piece of bone - tusk, actually - from a Wooly Mammoth HODGINS: There was a prehistoric elephant in the washer with him? What are you, nuts? ARASTOO: No. It has schreger lines on the grain - it's a Wooly Mammoth but no help to me. ANGELA: I wouldn't be so sure about that. (Cut to: Medico Legal Lab - Angela's Office. Angela is showing Brennan, Hodgins and Arastoo something on the big screen) ANGELA: See how this top edge curves? Now, if the curved continued - and then down here, before it got worn away in the washer - there was a point.. BRENNAN: A guitar pick. ANGELA: Exactly. BRENNAN: Wait. Why would someone want a guitar pick made of extinct prehistoric mammal. ANGELA: Well, according to my dad, guitarists have this thing about their picks: different materials make different sounds. HODGINS: Brian May uses and English penny. ANGELA: My dad uses a Nicaraguan Cordoba. Some guys use tortoise shells or uh, a sharks tooth. BRENNAN: Do you know of any famous guitarist who use Wooly Mammoth picks? ANGELA: Erik Dalton. ARASTOO: Mr. Dalton's pick winded up in the washing machine with a dead body. That poses some serious questions, don't you think? BRENNAN: Yes, I do. ANGELA: And Dalton isn't exactly for keeping his cool. Check out this video from a concert in Australia, two years ago. (She plays the video. Dalton is onstage, playing when a fan jumps on stage and knocks him over. He gets up, punches the guy in the face, throws his guitar on the floor and starts to take more swings at the guy until security comes and breaks up the fight) HODGINS: This guy has got a seriously short fuse. (Cut to: Hotel - Erik Dalton's Room. Booth and Brennan are there to talk to him. He's sitting down, strumming an acoustic guitar.) BOOTH: So Richard Cole stopped showing up for his private rehearsals with you and you don't notice? ERIK DALTON: Are you kidding me? I was thrilled. I was sick of kissing that guys ass (to Brennan) Hey, what are ya doing, baby? Can I help you with something? BRENNAN: You have no expertise that would be of value to me. ERIK: I wouldn't be so sure. Why don't you come sit next to me. BOOTH: Excuse me, I really like your music, doesn't mean I'm not gonna clock ya, alright? Let's just focus. So, Simon Graham - he pays you a boatload of money and you still treat the campers like crap? ERIK: Guys a poser, dude. I got stuck with an ass-hat who couldn't even play rhythm for Toad the Wet Sprocket. BRENNAN: Oh, I love them. ERIK: Hmm. Suddenly, the inside of my pants isn't such a happy place. BRENNAN: Personally, I find your music discordant and irritating - rather reminiscent of Muruwari death wailing in its capacity to annoy. BOOTH: So maybe Cole pisses you off. Throw a little coke into the mix. You lose control - I mean, it's not like it hasn't happened before. ERIK: Hey, man. I've been clean for two years. I even do yoga - and that hurts. BRENNAN: Booth? BOOTH: Yeah? BRENNAN: This splintering is fresh. Something hit the side of this table quite hard and there are scuff marks on the wall beside it that could indicate a struggle. BOOTH: Did you have a fight in this room? ERIK: A party. I had some campers over Tuesday night- gives them a story to tell their friends. (Brennan examines the carpet with a UV light. There are blood stains.) BRENNAN: There's blood on the carpet. ERIK: Ah, I don't know anything about that. When things got wild, I left. Caught a cab across town, spent some quality time with a girlfriend. BOOTH: Well, I'm gonna need that girlfriends name and number. BRENNAN: And I'm gonna need this carpet. ERIK: Sure. [SCENE_BREAK] (Cut to: The Medical Plaza - Waiting Room. Michelle comes out of a door and meets up with Cam.) CAM: Hey, how did it go? MICHELLE: Fine. You really didn't need to come with me. CAM: I just wanted to make sure you were comfortable. MICHELLE: Sure. He's cool. Easy to talk to. CAM: Good. That's excellent. DR. LIDNER: Uh, excuse me, Dr. Saroyan. Um, do you have a minute? A couple of insurance questions is all. CAM: Sure. Be back in a minute. (Cut to: The Medical Center - Dr. Lidner's Office.) CAM: Is there a problem with Michelle? DR. LIDNER: Oh, no, no, no, no. She's-she's great. In perfect health. CAM: And she spoke to you..about things. DR. LIDNER: Confidential things. Yeah. I can tell you she's a wonderful girl. But that's not why I wanted to talk to you. CAM: Right. Insurance. DR. LIDNER: Uh, not about insurance, either, no. CAM: Now I'm stumped. DR. LIDNER: Um, I just thought - Would it be weird if I asked you to, uh, go out with me sometime? CAM: Ye-Yes, that-that, um...would be weird. DR. LIDNER: Of course. Uh, very weird. CAM: Yeah, right? It-it is weird. DR. LIDNER: Totally. Totally weird. CAM: But, um, I would say yes. DR. LIDNER: Really? (Cam nods) That's... great. CAM: Does that mean you're asking? (Cam's phone rings) Oh. I'm sorry. Excuse me. (she checks her phone) Work. I've got to go. DR. LIDNER: Oh, I-I am, though. CAM: What? DR. LIDNER: Asking. I'll call you? CAM: I would like that. (Cut to: Medico Legal Lab - ANgela's Office. Arastoo is showing Brennan and Angela micro-fractures.) ARASTOO: So we know that the micro-fractures radiate out of each trauma site. These micro-fractures expand until they run into other radiating fractures, which were created by earlier trauma wounds. ANGELA: Now, the trick is to separate out those points of trauma where the radiating micro-fractures just keep going because there were no earlier fractures to cross paths with. ARASTOO: In this way, we can identify the perimortem fractures, and therefore, determine that cause of death was trauma to the skull and chest cavity. BRENNAN: Cole was beaten to death. Excellent work. ARASTOO: Thank you. And Angela, of course. Double play, right? (They high five.) ANGELA: Yeah. BRENANN: I assume you were talking about baseball again, although I have no idea why. ANGELA: Well, it's baseball season, sweetie. This is when boys like to hit balls with sticks when the snow melts. I don't know why. BRENNAN: Oh. Well, what about the murder weapon? ARASTOO: I'm going to make castings of the pertinent injuries. ANGELA: I'm making him 3- D scans so he can focus on all the un-remodeled fractures. BRENNAN: Sweets would probably say that the need to hit balls with a large stick shows that you're insecure with your manhood. ARASTOO: I can assure you... BRENNAN: I think it's probably just enjoyable to hit things. (Brennan leaves. Angela laughs.) (Cut to: Medico Legal Lab - Cam's Office. Cam is talking to Brennan.) CAM: Erik Dalton's hotel room's a good bet for the murder scene. We found two sets of DNA. BRENNAN: So, two people bled on this carpet? CAM: Yes. The first set of DNA belonged to our victim. BRENNAN: Perhaps the second set belongs to our killer. CAM: I found traces of Klonopin in the mystery blood, which means, whoever it belongs to either has a panic disorder or epilepsy. BRENNAN: We should cross-reference the list of campers with the prescription drug database. (Brennan starts to walk towards the door but then turns back to Cam.) BRENNAN: Booth seems to like Catherine, don't you think? CAM: I do. I'm glad. It's been a long time since he's dated anyone. BRENNAN: I know. It's important for Booth to share his life. I prefer being alone. CAM: But you're seeing Hacker. BRENNAN: Yes, and I like him, but not like Booth. I mean not like Booth wants to like someone. CAM: All organisms evolve and develop along patterns only recognized in retrospect. Your life doesn't exist outside the laws of nature. BRENNAN: Then, in ignorance, I await my own surprise. Although the odds of it involving a commitment to another person are remote. CAM: I never thought I'd be dating now; yet I am. BRENNAN: You met someone. CAM: I think so. We're going to have lunch. BRENNAN: It's been quite a while for you. CAM: And thanks for pointing that out. (Brennan's receives a text message.) BRENNAN: Oh, Booth wants me to meet him at the hotel. CAM: Go. I'll call you if I get a hit on the Klonopin. (Cut to: Hotel - Stage Area. Students are practicing while Simon is giving some of them instructions.) SIMON: (O.S.) All right, now reverse on the chords. (Brennan enters the stage area. Booth is sitting on the Amp pretending to drum, his tie is now around his head, while the students are playing around him.) BRENNAN: Booth? Booth, I'm not sure this is a worthwhile use of our time. BOOTH: Why? We're still waiting for an I.D. on the blood, right? I mean, Come on. We've got a few minutes to spare. BRENNAN: This is pathetic, Booth, pretending to be something you're not. It's dilettante camp. BOOTH: Okay, listen. What if this was, like, anthropology fantasy camp, and you got to meet...I don't know, uh, Margaret Mead? BRENNAN: She's dead. BOOTH: Well, who would you want to meet? BRENNAN: Me. BOOTH: You? BRENNAN: Yes, if I were an anthropology enthusiast, I'd want to go to fantasy camp to meet me. BOOTH: Ah, come on, Bones. Play along. (The musicians start to play the opening notes to "Hot Blooded" by Foreigner.) It's rock and roll fantasy camp. It's cool, right? You hear that? That is our song. Remember "Hot Blooded"? BRENNAN: The last time we sang this song, Booth, someone tried to kill you. BOOTH: Yeah, but it was fun up until the blast, right? Come on. (BOoth jumps on the stage and starts singing into the microphone) BOOTH: (singing) I'm hot blooded, check it and see... (he notices Brennan strapping on a guitar) Wait a second. You play the guitar? BRENNAN: Well, I play the akonting, the folk lute of the Jola tribe, but the guitar is not dissimilar. (Brennan joins him on stage and starts to stum the guitar. They both start to sing.) BOOTH & BRENNAN: (singing) Well, I'm Hot blooded. Check it and see. I've got a fever of a hundred and three. Come on, baby, you can do more than dance. I'm hot blooded, hot blooded! (Brennan rocks on the guitar solo but receives a text message so she stops and checks her phone while Booth keeps rocking out) BRENNAN: Oh. (to Booth) Cam got an I.D. the other blood found in the hotel room. Fred Keaton. He's also registered here as a camper. BOOTH: Alright. (excited) One more verse. (Brennan smiles and starts playing the guitar again, they start singing. They're having a blast) BOOTH & BRENNAN: (singing) Well, I'm Hot blooded. Check it and see. I've got a fever of a hundred and three. Come on, baby, you can do more than dance. Hot blooded, hot blooded! BOOTH: Woo! (Cut to: FBI Headquarters - Interrogation Room. Booth is talking with Fred Keaton.) FRED KEATON: I didn't do anything. BOOTH: You took off from fantasy camp. You disappear and you end up hiding out in some cheap motel? FRED KEATON: I wasn't hiding. BRENNAN: We know you and Richard Cole had a fight in Erik Dalton's hotel room. Your blood is on the carpet with his. FRED KEATON: He had it coming, okay? BOOTH: Well, what was the fight about? FRED KEATON: I told him I was still hoping to be discovered. I know it's ridiculous, but I've spent years in my garage playing and I'm good. It could happen, right? BOOTH: Just keep going. FRED KEATON: Last Monday, we had a jam night at camp. This guy came up to me, told me he's with Rolling Stone. Told me my guitar playing is amazing; he's gonna include me in an article called "The 100 Best Guitarists You've Never Heard Of. " BRENNAN: Oh, that sounds like a good thing. BOOTH: Sounds too good, I'm guessing. FRED KEATON: Then these girls come up, told me I'm going to be a star. Asked if they could keep me company for a few hours. BRENNAN: What-what for? BOOTH: s*x, Bones. BRENNAN; Oh. Quite a lucky night for you. BOOTH: It was a prank. See, he was messing with you, wasn't he? FRED KEATON: How was I supposed to know? I called my wife, told her I wanted to take a break. Then at Erik Dalton's party, Cole starts laughing. Tells me the journalist was an actor. And the groupies were... BOOTH: Professionals. FRED KEATON: Rich b*st*rd ruined my life. BRENNAN: Excellent motive for murder. FRED KEATON: What are you talking about? BOOTH: Richard Cole is dead and I'm thinking you killed him and took off. FRED KEATON: No. I left to do damage control. My wife won't even let me in the front door. She wouldn't even talk to me. Look, I have no idea who killed Cole but what he did to me, he deserved it. (Cut to: Medico Legal Lab - Angela's Office. Brennan, Angela, Hodgins and Arastoo are looking at the big screen.) ARASTOO: Angela scanned the castings I made of the fatal injuries so we can get a better sense of the murder weapon. ANGELA: Yeah and I cleaned up the edges and these are the shapes that caused the injuries. HODGINS: Cam said the blood spatter analysis didn't show any drag marks on the carpet from Dalton's hotel room. ANGELA: Hmm. Which means that Cole wasn't killed there. BRENNAN: No two of the injuries share the same impact surface area. ARASTOO: I know. So it appears the guy was hit with various weapons that were each used once. BRENNAN: Or he was hit multiple times by a weapon with a variety of surface shapes. ANGELA: Well, you know, this could be a tailpiece. BRENNAN: A what? ANGELA: And-and that could be a tremolo arm, which means that those lines are from strings. HODGINS: Wait a minute. Out victim was beaten to death with a guitar? BRENNAN: I've actually seen this before. Solid body guitars can prove quite lethal. ARASTOO: There have to be over 50 guitars at the fantasy camp. Without cause, we can't get warrants for all of them. ANGELA: Yeah, but that shape. I mean, the bottom is too curved to be a Tele or a Strat, but...it's not an SG because the tailpiece wraps around and then there's the distance to the jack plate. You know, I think we're looking for a '57 Gibson Les Paul. HODGINS: That is so hot that you know that. Interesting. It's-that's interesting. ANGELA: Well, it's not like I know every guitar, but I do know the expensive ones. BRENNAN: How expensive? ANGELA: I'd say our victim was beaten to death by about a quarter of a million dollars. (Cut to: Park. Cam and Dr. are sitting on a bench, having lunch with Dr. Paul Lidner.) CAM: And then after a perfectly nice evening, I could tell he didn't even want to shake my hand. He looked positively pained. He's a science professor. No. Associate professor. Oh. people get weird when they find out that I handle dead people all day. Now I just say I'm an insurance underwriter. DR. LIDNER: Oh. Good one. CAM: Mm-hmm. DR. LIDNER: I'm an accountant. CAM: No. DR. LIDNER: Oh, yeah. No one wants to talk about work with an accountant. Or an insurance underwriter. CAM: Except other accountants or underwriters. DR. LIDNER: Mm. (he laughs) CAM: So, do we share any other great deceptions? DR. LIDNER: Um... I can make a coin disappear and come out of your ear. CAM: Ooh. I hate magic. I'm sorry. DR. LIDNER: Ah. Yeah. Me too. But it always worked for my Uncle Dave. Of course he was in a nursing home. CAM: Am I smiling like a fool? 'Cause that would be embarrassing. DR. LIDNER: Ah, well, you look beautiful embarrassed. CAM: Then I'll keep smiling. DR. LIDNER: I should get back to the office. CAM: And I have a murder to solve. DR. LIDNER: Oh, right. CAM: Um, what do you say we catch a movie on the weekend? DR. LIDNER: Yeah. CAM: I'll see what my parental duties are and give you a call? DR. LIDNER: Sounds good. Okay. (Cam leaves) (Cut to: Royal Diner. Booth, Brennan and Sweets are having lunch.) BOOTH: Do you remember when Simon told us that Cole showed up at camp with a fancy guitar? SWEETS: Yeah, he wasn't kidding. A '57 Gibson Les Paul? BOOTH: Yeah, well, it disappeared at the same time that Cole did. SWEETS: You think he was killed for his guitar? BRENANN: All we know is, he was killed with his guitar. SWEETS: With a '57 Gibson Les Paul. That's like whacking someone with the Mona Lisa. BOOTH: I got agents checking out dealers in the area. See if anyone tried to sell it. BRENNAN: Well, unless the killer destroyed it. BOOTH: Killing something like that would be like killing Altamont or, uh, "Smells Like Teen Spirit. " SWEETS: You know, the guitar has long been recognized as an unconscious symbol of copulation. The, uh, head and the shaft are phallic, the body feminine. Maybe our killer was acting out of sexual confusion. BOOTH: Or maybe someone just wanted the guitar. BRENNAN: Mm-hmm. BOOTH: Wouldn't you? SWEETS: Yeah. BOOTH: Yeah. (he gets a text) Oh, look at this; I got a hit. Dealer in DC got the guitar; said it was brought in by a woman with a pierced eyebrow. (Cut to: Hotel - Lobby. Bebe is seated, discussing music with ) GINO: Whatever. Jimi Hendrix choked to death on his own vomit. BEBE: The autopsy was inconsistent and Hendrix's manager confessed to shoving pills and red wine down his throat, hoping to cash in on an insurance policy. BOOTH: Wow. You really know your rock and roll deaths. BEBE: Oh, hi again. Yeah, I guess I do. BRENNAN: We need you to come with us. BEBE: What for? BOOTH: We have some unanswered questions about Kurt Cobain's death. We thought maybe you could give us some insight. BEBE: Seriously?! BRENNAN: Well, I believe he was being ironic but if you do have information about this Cobain person, I'm sure the FBI would appreciate that too. BOOTH: Thanks, Bones. (Cut to: FBI Headquarters - Interrogation Room. Booth is talking to Bebe) BEBE: Cole is dead. I thought he was just missing. BOOTH: That why you thought you'd get away with selling his quarter-of-a-million- dollar guitar? BEBE: How much? That dealer totally ripped me off. BOOTH: Stay on topic, all right? BEBE: Okay. After Dalton's party, I snuck into Cole's room and took the guitar. It was sitting on the stand all polished. He usual kept it filthy. I don't know why he suddenly gave a crap about it. BOOTH: How did you get in? BEBE: I had a key. We hooked up the first night of camp. You know, after all the loser musicians I dated, I thought I finally found a decent guy, but... BOOTH: What happened? BEBE: Cole told me that he was planning on sleeping with every woman at camp as part of his own rock and roll fantasy. He thanked me for being such low-hanging fruit. BOOTH: So you killed him and you stole his guitar. BEBE: He was a poseur; he didn't deserve that guitar. Look, I know it was wrong to take it, but I swear to God, I didn't kill him. (Cut to: FBI Headquarters - Observation Room.) SWEETS: I believe her. BOOTH: Me, too. Too bad she's going to prison for grand theft. BRENNAN: Then who killed Cole? SWEETS: Well, everything that you've learned about the victim, uh, indicates that he was only interested in the external signifiers of the rock and roll lifestyle, correct? BRENNAN: Yes, the clothes, the instruments, the groupers. BOOTH: Groupies, Bones. BRENNAN: Well, groupers would be more logical. Male groupers have harems of multiple females. If you enter into a social contract with a marine biologist, you should know these things. BOOTH: Yeah, well, a fish can't play the guitar. BRENNAN: Well, apparently, neither could Cole. BOOTH: You don't have to keep bringing up Catherine. BRENNAN: Well, you're welcome to bring up Andrew. SWEETS: I have an opinion on motive, if anyone's interested. BOOTH: Right. SWEETS: Okay. To a true music fan, the fact that Richard Cole was playing the ultimate guitar would be sacrilegious, profane, even. And the fact that the killer put it back in Cole's room, rather than destroy it, further demonstrates his reverence for rock and roll. BOOTH: So, you're saying that the music is the motive. SWEETS: I know it's wrong, but I am liking our killer better than our victim. (Cut to: Medico Legal Lab. The guitar is sitting on the stand.) ANGELA: Oh, wow. This is gorgeous. I wish my dad was here. CAM: No prints other than those of the dealer. HODGINS: The killer did an excellent cleaning job. CAM: Take it apart. See if he missed anything. ANGELA: Okay, um... I'm not going to watch that. HODGINS: I'll do it in the Ookey Room. Arastoo, little help here? (Hodgins grabs the guitar, Arastoo grabs the stand and they leave. Cam sees Michelle.) CAM: (to Angela) Will you excuse me? (Cut to: Medico Legal Lab - Hallway. Cam and Michelle are walking to her office.) MICHELLE: Dr. Lidner left a message at the house. CAM: Was there anything wrong with your tests? MICHELLE: No. He was confirming your date for Saturday night. CAM: Oh. That. Yes. I was going to tell you... MICHELLE: You're dating my gynecologist!? CAM: It wasn't my fault. It just happened. MICHELLE: What? Think about what you'd say if I said that to you. CAM: I'd ground you. I'm...sorry. We had lunch. We liked each other. That's all. And that was wrong. Very, very wrong. MICHELLE: Is that why you sent me to him? So you could get a date? I know you haven't seen anyone since I've been living with you. CAM: No. He's a good doctor, that's all. MICHELLE: Who just happens to be cute. CAM: Yes. No. God. MICHELLE: Why didn't you tell me? Don't you trust me? You have to sneak around behind my back? CAM: No. Wait. How did we switch roles here? MICHELLE: I can't go back to him now. That would be extremely skeevy. CAM: We just had lunch. I swear. I will never see him again. I promise. MICHELLE: Yes, you will. CAM: What? MICHELLE: You've been so focused on being a good mother that you've totally ignored yourself. Do you know what kind of pressure that puts on me? CAM: No, I... I didn't realize. MICHELLE: You need a life... for my sake. But don't sneak around behind my back. You two seem like a good fit. Have a little fun but don't go too fast. You're out of practice. CAM: Excuse me? MICHELLE: I thought you believed in honesty. CAM: Oh... all right. I'll go slow. Very, very slow. MICHELLE: And find me a new doctor. A woman. CAM: Sure. Right away. MICHELLE: I've got to get back to school. CAM: Okay. MICHELLE: Love you. CAM: Love you, too. (Cut to: Medico Legal Lab - Ookey Room.) ARASTOO: What do you have, Dr. Hodgins? Anything? A walk? A single? Don't strike out, please. BRENNAN: Mr. Vaziri, your obsession with sport will no doubt diminish your mental acuity. ARASTOO: Oh, on the contrary, Dr. Brennan, baseballs a game built on mathematical certainty, the physics of force and velocity, as well as its anthropological significance as being one of the unifying cultural traditions for Americans. Hmm. Intellectually, it's quite stimulating, and, uh, I like swinging a bat. HODGINS: Okay, this fret is cracked. BRENNAN: What, from impact? HODGINS: No, no. The cracks are tiny. Yeah, you know, every fret in the lower register is cracked. ARASTOO: The guitar is old. HODGINS: Yeah, but that doesn't matter. This is from chlorine. See, the frets are made out of nickel and nickel reacts like this when exposed to chlorine. BRENNAN: There's a rooftop pool at the hotel. If Cole was murdered there, the killer might have cleaned the guitar with pure chlorine. HODGINS: That's why it was so clean when Bebe stole it. ARASTOO: Well, the strings are made out of nickel, too, aren't they? HODGINS: Yeah, but they're brand-new. I mean, there's no cracking. ARASTOO: The killer must have known he couldn't get all the blood off, so he didn't even try. HODGINS: Well, if the killer restrung the guitar, then maybe he left us a little present. Bingo. BRENNAN: Is that a hair? HODGINS: Yes. Yes, it is. Purple. BRENNAN: I think I know whose that is. Someone who would kill to play with Erik Dalton. I believe we would call this a home run. (Cut to: Hotel Lobby. Gino is up on stage playing the final song of camp with Erik Dalton. All the campers are rocking out. Booth and Brennan make their way through the crowd.) BOOTH: Guess what, uh, kind of pick Bebe said Gino uses. BRENNAN: Woolly mammoth? (They get to the front. Booth sees Gino and hold up his handcuffs to let him know that they're there for him - but they let him finish playing the song.) CROWD: Whoo! BRENNAN: Whoo! GINO: Whoo! (He jumps off stage and goes over to Booth and Brennan) GINO: I couldn't let that poser play with it. Go ahead, man. It was worth it. BOOTH: Yeah, I'm sure it was. Come on. (He cuffs Gino and leads him away) GINO: WOO! (Cut to: Founding Fathers. Booth and Brennan are sitting at the bar, having a beer.) BOOTH: You know, our killer plays a mean guitar. I'm sure that they have, you know, a band in prison for him. BRENNAN: You're a very good singer. BOOTH: Thank you, Bones. And you-you play the guitar in a very interesting fashion. BRENNAN: I know. Does Catherine play? BOOTH: I don't know. I've only been out with her twice, Bones. BRENNAN: Last night, Andrew gave me a CD with music he likes. BOOTH: Mix-tape, huh? Talk about a social contract. BRENNAN: That's what I surmised. (she pauses) Our partnership is still important to me. You know that, right? BOOTH: Sure. Yeah. Die for your, partner. That's the way I look at it. (After a few seconds..) BRENNAN: I liked Andrew's taste in music except for a band called Led Zeppelin. BOOTH: Except for a band called Led Zeppelin? BRENNAN: (hesitantly) Yes. BOOTH: What? You kidding me? Led Zeppelin is, like, the best rock and roll band ever. I mean, they had a reunion tour in '07 in London. I would have killed for those tickets. BRENNAN: Really? My publisher offered me tickets, but when I heard "Zeppelin," I thought it was for some sort of air show. BOOTH: Air show? You turned down what probably was the last concert that Zeppelin would ever play? BRENNAN: Are you going to kill me? BOOTH: You're unbelievable! BRENNAN: Well, it's just a band, Booth. BOOTH: It's not just a band, okay? This is Led Zeppelin. You know what? I am your partner. You offer your partner those kind of things. BRENNAN: I didn't know that! BOOTH: Offer your partner the tickets. END.

When the body of a rich adventure-seeker is found in a hotel laundry machine, Booth and Brennan are led to the last place he was seen: a Rock 'n Roll Fantasy Camp. While Brennan and Booth lead the investigation outside the lab, Hodgins, Angela and intern Arastoo use a fragment of prehistoric wooly mammoth tusk - found with the remains - and Angela's knowledge of the music industry to connect the evidence to potential suspects. Meanwhile, Cam looks for a doctor for her daughter Michelle.

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The infection cycle of viruses creates many opportunities for the exchange of genetic material with the host. Many viruses integrate their sequences into the genome of their host for replication. These processes may lead to the virus acquisition of host sequences. Such sequences are prone to accumulation of mutations and deletions. However, in rare instances, sequences acquired from a host become beneficial for the virus. We searched for unexpected sequence similarity among the 900,000 viral proteins and all proteins from cellular organisms. Here, we focus on viruses that infect metazoa. The high-conservation analysis yielded 187 instances of highly similar viral-host sequences. Only a small number of them represent viruses that hijacked host sequences. The low-conservation sequence analysis utilizes the Pfam family collection. About 5% of the 12,000 statistical models archived in Pfam are composed of viral-metazoan proteins. In about half of Pfam families, we provide indirect support for the directionality from the host to the virus. The other families are either wrongly annotated or reflect an extensive sequence exchange between the viruses and their hosts. In about 75% of cross-taxa Pfam families, the viral proteins are significantly shorter than their metazoan counterparts. The tendency for shorter viral proteins relative to their related host proteins accounts for the acquisition of only a fragment of the host gene, the elimination of an internal domain and shortening of the linkers between domains. We conclude that, along viral evolution, the host-originated sequences accommodate simplified domain compositions. We postulate that the trimmed proteins act by interfering with the fundamental function of the host including intracellular signaling, post-translational modification, protein-protein interaction networks and cellular trafficking. We compiled a collection of hijacked protein sequences. These sequences are attractive targets for manipulation of viral infection. Many studies, mainly from bacteria and unicellular eukaryotes, focus on the exchange of genetic material between viruses and cellular hosts. Sequences are best studied through their structural and functional domains [1], [2], [3], [4], [5]. The evolution of domains is a significant force for shaping the proteins along the tree of life. Sequence exchange between genomes within and between superkingdoms is evident from the appearance of a domain in a particular phylogenetic branch [6]. The contribution of horizontal gene transfer is not limited to bacteria but has occurred across distant species [3]. For example, some signaling domains in bacteria are the consequence of a horizontal gene transfer [7]. The viruses are parasitic agents that maintain an intimacy with their host cells. Consequently, an extensive horizontal evolution [8] is associated with the viral life cycle. The lack of similarity of viral proteins (e. g., capsid proteins) with any cellular organisms is in accord with their early and unique origin [8], [9]. Most likely, the modern viruses originated at the early RNA world of the primordial genetic pool. With the increasing numbers of sequenced viruses, similarity among seemingly unrelated viruses was reported. A role of the hosts as vehicles for such cases is proposed. For example, the structural similarities observed between bacterial viruses (PRD1, Bam35), Chlorella virus (PBCV-1) and adenovirus in the coat proteins, led to the proposal that all viruses are old, probably preceding the cellular life. Furthermore, it is compatible with polyphyletic virus origins, as opposed to the monophyletic origin of cellular life [10]. Still, assignment of viruses to the phylogenetic tree of life remains unresolved [11]. Notably, viruses as vectors (mainly RNA viruses) have the potential to rearrange the genomic material, and thus, to change the domain architecture [12], [13], [14]. Studies on horizontal gene transfer focused primarily on viruses infecting bacteria and archaea (e. g., bacteriophages) [15], [16]. The co-evolution of viruses toward their hosts indicates an active crosstalk on an evolutionary time scale [17], [18], [19]. Several studies reported on a handful of cases of functional mimicry by viral proteins [20]. In few cases, evidence for gene transfer from the host to the virus is obvious. For example, the photosynthetic efficiency in cyanobacteria (Synechococcus and Prochlorococcus) relies on components of the photosystem II. These critical components express in the respective phages [21]. In the case of the phytoplankton–virus system, the DNA virus EhV that infects the microalge (Emiliania huxleyi), contains a complete metabolic pathway as a result of a horizontal gene transfer [22]. A similar case is demonstrated for the dUTPase genes (Dut) that are necessary for regulating the cellular levels of dUTP. Phylogenetic analysis revealed the origin of the viral Dut sequence in a monophyletic cluster of DNA viruses with eukaryotic hosts [23]. The Acanthamoeba polyphaga Mimivirus and the family Phycodnaviridae [24], contain many genes that are found in cellular organisms. For example, the giant virus Cafeteria roenbergensis virus (CroV) includes numerous eukaryotic-like genes for translation factors, ubiquitin pathway components, intein elements, histone acetyltransferase and more [25]. These are extremely large viruses of aqueous environments that infect bacteria, animals and protists [26]. A search for similarities between viral and host proteins has largely been focused on herpesviruses [27], Hepadnaviridae [28] and others. However, the high mutation rate of RNA viruses [29] and the coexistence between viruses and their hosts for millions of years has most likely blurred the sequence similarity. Recently, several studies challenged the origin of ancient viral segments in metazoan genomes. These sequences that are called EVE (for endogenous viral element) encompass all virus-derived genomic loci [30]. In this paper, we present a coherent survey on protein sequences that are shared between viruses and their hosts. We assess the scale of the phenomenon by focusing on the viral-related protein sequences that appear in metazoa. We have used the current archive of all proteins [31] as the basis for identifying sequences with a potentially common origin. Presumably, their appearance in the virus reflects virus-acquired sequences. Of about 190 instances of highly similar viral-eukaryotes sequences, we recognize that only a small number originated from a host origin. We extended the collection of viral proteins that have a host origin by investigating the eukaryotes-viruses Pfam families [32]. We focused on the 670 Pfam cross-taxa families that contain viruses and metazoa. A careful examination reveals that these instances reflect either missed annotations or the remnants of sequence exchange by virus infection. To distinguish these possibilities, we constructed sequence alignment trees for all 670 Pfam families. From the properties of the trees, we focused on 335 families that most likely contain viruses that hijacked sequences from their host. We found that most of the viral proteins in the orthologous families are much shorter and composed of simpler domain architectures. In almost all cases, the number of domains and the sequence of the tails and the inter-domain linkers are considerably shorter in the viral proteins relative to their counterpart host proteins. We discuss the potential of such short viral proteins to interfere with critical cellular functions and thus are candidates for manipulation strategies in defeating viral infection. Figure 1A shows two over-simplified scenarios in support of a genetic exchange from the virus to the host genome and in the reverse direction, from the host to the viral genome. In the first scenario, a viral sequence is detected in the host (e. g., human) but not in the rest of the phylogenetic branch. The following scenario accounts for viral sequences acquired from the host (Figure 1A, right). Under this scenario, the viral gene sequence is identified in a broad group of organisms that belong to a phylogenetic tree that includes the host (human). Therefore, the sequence in the virus is most likely a reflection of a hijacking event, according to an argument of maximum parsimony. Supporting evidence for the directionality of the genetic exchange of viral and cellular organisms relies on a detailed phylogenetic analysis. The topology of the reconstructed tree is used to support the most parsimonious scenario (see Materials and Methods). The simplified illustrations in Figure 1A do not address the more complicated, realistic instances in which different viruses carry sequences that resemble various organisms. An additional criterion used in supporting the occurrence of sequence acquisition by viruses is the presence of a sequence resemblance in the known host. The origin of viruses is probably preceding the cellular life [8], [10]. Thus, the ancient events in which viral sequences were incorporated into an ancestor eukaryote cannot be traced by their sequence similarity. Still, a conserved functional or structural similarity could expose such early events [33]. In this study, we have not attempted to date the horizontal transfer event. Furthermore, we will not discuss the events of genetic material exchange (see discussion in [34]), but limit our study to the acquisition of coding sequences in viruses and metazoa. There are about one million viral proteins in the UniProt database (990,049, August 2010) that represent about 66,000 viral strains. This is a highly redundant resource and about half of it composed of medically relevant strains including Hepatitis B viruses (HBV) and Human immunodeficiency virus (HIV). We took advantage of a reliable source of UniRef [35] that unifies sequences according to their identity level along the sequence length. We used UniRef90 classification (see Materials and Methods). There are >165,000 UniRef90 clusters that contain at least one viral protein (Figure 1B). However, from this set, we only considered 262 instances that contain at least two proteins, where one of them must be a eukaryote (Figure 1B). Of the 5,482 cross-taxa clusters that contain sequences from viruses and cellular organisms, 95% are sequences of bacteriophages and plasmids confined to the bacteria [36]. We will not further discuss the events that are confined to bacteria and archaea. A taxonomical view shows the diversity of the organisms that share the UniRef90 clusters with viral proteins (Figure 1C). It shows that the eukaryotes are the most diverse group with 106 species that share their homologues with viral proteins. This result suggests that the phenomenon of shared sequences is quite broad, and many eukaryotes have been subjected to a genetic material exchange. Among the UniRef90 clusters that contain viruses and eukaryotes (Figure 1B), ∼70% are from tetrapoda, 13% plants, 13% arthropoda, 4% fungi and only a smaller percentage of other taxa. We focused on the cross-taxa clusters of viruses and mammals (118 clusters include ∼2,200 proteins, Figure 1D). Viruses from Class I (dsDNA viruses with no RNA stage) and class VI (Retro-transcribing ssRNA, Plus strand) are prevalent among those that infect mammals [17]. The dominating Class VI viruses are characterized by their ability to integrate sequences into the host (Figure 1A, left). Table 1 lists the Class I viral proteins that share sequences with mammals (23 clusters, Figure 1D). In Class I viruses, the virus enters the nucleus before its replication (with the exception of Poxvirus family) and its infectivity is strongly dependent on the host cell division. Discriminating whether a sequence has originated from the virus or the host is not straightforward. We generated for each cluster a phylogenetic dendogram and analyzed the connectivity of the viral protein in view of its neighboring sequences. Often the analyzed cluster is too small. In such cases, we expanded the cluster to the relaxed UniRef50 classification. We applied additional criteria in support of a virus having acquired protein sequences from the host: (i) The tested sequence appears in several organisms (≥2) on the same evolutionary branch (as in Figure 1A, right); (ii) The tested sequence is not associated with viral contamination. Most analyzed cross-taxa clusters derive from the contamination by viral proteins following integration of the virus to a mammalian genome. Several instances were contaminated by the extensive use of viral vectors as vehicles in variety of molecular manipulations (e. g., Adenoviruses, Table 1). Another source of contamination is from cancerous cells infected by viruses (e. g., human papilloma virus). In such instances, some sequences that are assigned as ‘human’ are incorrectly annotated. In these instances that reflect the incorporation of the virus to the host, different protein sequences from the same virus are identified which are best explained as a result of infection or an integration event. For example, the proteins in UniRef90 clusters P06426, P06463, P21735, P06788, P36741 and P21736 belong to Human papilloma virus (Table 1). Studies on the viral sequences that were integrated into the vertebrate germ line and hence shaped the vertebrate genetic heritage were reported [37], [38], [39]. Herein, we only consider the protein sequences that are shared by viruses and their metazoan hosts. The principal virus families that infect multicellular eukaryotes are listed in Supportive data Table S1. For few instances, a support exists for viruses that hijacked sequences from the host. Among Class I viruses (Table 1) the shared functions include interlukin-10 (IL-10) (Figure 2), beta-1,6-N-acetylglucosaminyltransferase (β1,6GnT) (Figure S1) and Ubiquitin. The β1,6GnT and IL-10 are found exclusively in metazoa and the multicellular eukaryotic branch. The key features and the functional amino acids are conserved in the viral and the corresponding mammalian proteins (Figure 2A, Figure S1). Indeed, in human cells lacking β1,6GnT gene, the Bovine herpesvirus 4 (BoHV-4) sequence fully recovered the missing enzymatic activity [40]. Resolving the evolution of the Ubiquitin in the genome of Pestivirus suggested that the virus hijacked Ubiquitin-related sequences in two consecutive events [41]. A browsable table is available at www. protonet. cs. huji. ac. il/virost/tables/UniRef90-Class1. html. Figure 2 shows a prototypic case of viral proteins that resemble the host protein. Interleukin 10 (IL-10) inhibits the induction of pro-inflammatory cytokines. IL-10 was found in many viruses including Epstein–Barr virus (EBV), equine herpesvirus (EHV) and cytomegalovirus (CMV) [42]. Presumably, the gene product protects the infected cells from the host defense mechanism. An extended cluster of IL-10 (Table 1) covers 20 viruses and 96 cellular organisms (UniRef50_P22301). Representatives of viral and metazoan proteins are shown by the multiple sequence alignment (MSA) (Figure 2B). Most of the variations in the viral and metazoan protein reside in the sequence of the N-terminal that covers the signal peptide (Figure 2B). Traces of a genomic organization of the host in the viral genome were reported. For example, IL-10 like sequence from the gammaherpesvirus ovine herpesvirus 2 includes 5 exons and 4 introns [43]. Inspecting the UniRef90 clusters that contain proteins from viruses and metazoa (187 clusters) shows a wide variation in the distribution of protein lengths (Supportive data Table S2). Viruses tend to reduce their production load by deleting and reducing the unessential genetic material [44]. While this length reduction is an absolute necessity for most viruses, some giant viruses (e. g., Mimivirus, Chlorovirus, and Cafeteria roenbergensis virus) include ∼1000 proteins [24], [25]. The evolution origin of proteins from the Giant viruses remains unknown [45]. Still, 12% of the Acanthamoeba polyphaga mimivirus (APMV) proteins constitute a large number of host related sequences. The average length of this subset of the Mimivirus proteins (523 amino acids) is similar to the length of their homologous sequences. The small numbers of cases of viral acquired sequences (Table 1, Supportive data Table S2) may indicate the sequence divergence that had occurred throughout evolution. We therefore expanded the analysis for remote homologous. We questioned whether the viral protein sequences that were already substantially diverged due to a rapid evolution rate, or a long evolutionary history still maintain the host protein' s functional domain. The Pfam provides a comprehensive resource of functional and structural families and domains. Each Pfam entry represents a statistical model with an average sequence identity of 30–40% among the members of the family. Currently, Pfam covers 11,912 families, where 1,165 families include at least a viral protein and a eukaryotic protein representative. Some Pfam families are extremely large. Among families that contain metazoa and viral proteins are ‘Helix-loop-helix DNA-binding domain’ (∼6000 proteins) and ‘Sugar transporter’ (∼12,000 proteins). Contamination of viral proteins in metazoan proteomes (e. g., Capsid, Env, Tat) occurs mainly as a result of viral vector manipulations in cell lines, leading to incorrect assignment as a viral-eukaryotic cross-taxa family. An example is the GFP family (PF01353) that we have manually removed from the analysis. To reduce such sporadic instances, we considered Pfam families having at least two metazoan proteins, resulting in a list of 667 Pfam families. Supportive data Table S3 lists the species, composition of the domains and the proteins' length. The relatively small numbers of cases of viral acquired sequences (Table 1, Supportive data Table S2) may indicate the sequence divergence that had occurred throughout evolution. Therefore, we expanded the analysis for remote homologous. We questioned whether the viral protein sequences that were already substantially diverged due to a rapid evolution rate, or a long evolutionary history still maintain the host protein' s functional domain. Over 300 cross-taxa Pfam families (virus-metazoa) are best explained by a viral acquisition of host sequences. Instances of lateral gene transfer between bacteria and their bacteriophages dominate many of the cross-taxa Pfam families. Other families contain genuine viral proteins contaminated by metazoan proteins. In order to justify the directionality of sequences from the hosts to the virus, we constructed for each of the 667 Pfam families a sequence-based tree (MSA based on the domain and not the full length sequence). We considered Pfam families in which only 1–2 viral proteins are included in the family, and families in which the percentage of the virus proteins in the family is small (<5%, Figure 3A). The vast majority (547 families, 82%) of the analyzed Pfam families fulfilled these criteria (Figure 3A, blue). We also requested that the viral proteins are clustered in sub-trees within the family tree. We counted the number of viral proteins spreading within the sequence alignment tree. We suggest that viral proteins that are clustered in a defined sub-tree (called Viral Cluster, VC) are likely to represent a single episode of acquired sequence from the host. Consequently, only a limited diversity among the closely related viruses is expected in view of the rest of the tree. 64% of the 547 Pfam families from the previous selection fulfill the requirement for clustered viral proteins. These are the Pfam families that contain ≤2 viral clusters (60%), and other families (4%) that are specified by a high degree of condensation (i. e. the ratio of the viral proteins to the number of VCs is ≥3). These filtration steps further reduced the list of relevant Pfam families to 335 (Figure 3B). We show a tree constructed for one of the 335 families. The IL-6 (PF00489) family contains 10 viral proteins (Figure 3C, blue) that are split to two sub-trees of viral clusters (VC) and other 136 Metazoan proteins (marked as collapsed sub-trees). The maximal depth in this tree is 19 (included in the collapsed sub-tree, red triangle). The deepest viral protein in the tree is of depth = 9, and its normalized depth is 9/19 = 0. 474. The depth of the viral cluster (VC, the maximal sub-tree which contains all viral proteins) is therefore, 4/19 = 0. 211. The normalized depth for all the proteins in the 335 Pfam families (Figure 3D, top) is analyzed in view of the distribution of the normalized depth of the viral proteins within these families (Figure 3D, bottom). It seems that the two distributions are remarkably different (Figure 3D) which is in accord with the notion that the viral proteins are relatively isolated subsets among the proteins from the cellular organisms in the relevant Pfam families. Table 2 shows a sample of these families along with the cellular process and the protein function in the viral life cycle. A full list of the 667 Pfam families with the analyzed properties of their alignment trees is provided in Supportive data Table S3. One of the families that exemplified the trend found in virus-metazoa Pfam families is the PAAD/DAPIN/Pyrin family (PAAD_DAPIN, PF02758). This domain family is a diverse family (26% average sequence identity) that includes 34 cellular species and 5 dsDNA viruses that belong to the Poxviridae. The PAAD domain is at the N-terminal regions of proteins. This domain occurs in several multicellular organisms, in the context of inflammation, signaling and apoptosis (Figure 4). Several observations could be extracted for the PAAD domain: (i) Based on a multiple sequence alignment (MSA) of the PAAD domain sequences it is evident that the 5 viral proteins were diverged significantly (Figure 4B). All 5 viral proteins reside in one cluster, in the phylogenetic tree, together with other mammals as their sibling in the tree (Figure 4A, blue font). The domain architecture within the protein of the family is best explained by an initial extensive duplication of the PAAD domain (Figure 4A, green symbol). At present we identified ∼50 such proteins in human and mouse (Figure 4A); (ii) Most members of the PAAD family contain additional domains (in 158/177 occurrences). For example, the combination of PAAD and NACHT domains (Figure 4A, red symbol) are in 93 proteins, and PAAD, NACHT and LRR are in 2 proteins; (iii) The majority of the other domains (e. g., HIN-200, CARD) function in the regulation of apoptosis; (iv) All 5 viral proteins are single-domain proteins with PAAD domain. There are other 19 cases of the single domain proteins (Figure 4A, red font). Note that these proteins spread throughout the sequence-based tree. Presumably, it is a reflection of a domain loss event. Some of these proteins are fragments (e. g., Q5T3V8_HUMAN), and others include less characterized PfamB domains [32] (e. g., IFI4L_MOUSE, Q3UPZ5_MOUSE). The initial tests on UniRef90 covered 14,000 proteins in relatively small clusters (<90 proteins on average, Supportive data Table S2). In contrast, the collection of the cross-taxa Pfam families (Table S3) covers 161,000 viral proteins and 400,000 metazoan proteins. Therefore, focusing on the cross-taxa Pfam families provides an opportunity to increase the statistical power of the tests. Several statistical observations regarding the sequences among the cross-taxa families of viruses and multicellular organisms can be made: (i) The average length of the metazoan proteins is 507 amino acids, while the average length for the viral proteins in these families is only 396 amino acids (P-value of <1. 0e-17 by the KS-test, Figure 5A). (ii) For 73% of all families, the viral proteins are shorter than the length of the average metazoan proteins in the family (P-value<1. 0e-13 by the Hypergeometric test). (iii) In 67% of the families, the number of Pfam domain appearances (including several repeats of the same domain or different ones, Figure 5B) is smaller in the viral proteins relative to the metazoan proteins in the family (P-value<1. 0e-40 by the KS test). (iv) In 62% of the families, the number of different Pfam domains is higher in the metazoan proteins relative to the viral proteins. (v) For the discussed families, the median number of Pfam domains is 1. 06 while, for the metazoan proteins, this value is 1. 7 (P-value<1. 0e-32 by KS test). Many metazoan proteins are multi-domain (colored rectangle, Figure 5B). We tested whether the viral acquired sequences that belong to multi-domain proteins displayed a stronger tendency for a size reduction (see scheme, Figure 5B). A reduction in length of viral proteins may be a reflection of reducing the number of domains (Figure 5B, b–c), shortening the length of the linker sequences (Figure 5B, a) or even the trimming of the length of the domain itself. Among the 667 analyzed Pfam families, in 103 of them, the metazoan proteins contain at least 3 Pfam domains. In 85% of this set (88 families), the viral proteins are shorter (Figure 5B, virus). Remarkably, the average length of these 103 metazoan proteins families is 912 amino acids relative to 503 amino acids for the viral proteins that belong to these families. Similarly, in this set of multi-domain proteins the viral proteins have an average of 2. 9 domains, while the metazoan proteins have 4. 6 domains on average (paired t-test, p-value of 1. 0e-11). This shows that the tendency to reduce the protein length and the number of domains is stronger when the number of Pfam occurrence in the original host protein is higher. In order to reduce the risk of misclassification, we further restricted the analysis to Pfam families of viruses-metazoa (with ≥3 Pfam domains) that contain at least 2 viral proteins (total of 50 families). The length of the viral proteins is significantly reduced. For 90% of these families (above the reference line, Figure 5C), the viral proteins are shorter than their matched metazoan proteins. In order to determine whether the reduction in length is due to a reduction in the number or the properties of the domains, we repeated the analysis for the ratio of the number of distinct domains (depicted by the different colored rectangles, Figure 5B) in the viral and their relevant metazoan sequences (Figure 5D). For 80% of the families (families above the reference line), number of different Pfam domains that are associated with viral proteins is reduced. Note that by this measure, a short viral protein (Figure 5B a–b) still has a ratio of 1. 0. We show that the viral proteins are not only significantly shortened, but have also converged to a simpler domain composition. The length of the individual domains between the viral and the metazoan host proteins is identical (Supportive data Figure S2). Recall, that this observation may be mainly due to the definition of belonging to a Pfam domain family. The high statistical significance of these trends is consistent with a possibility that the short viral proteins have resulted from the acquisition of fragments from the host protein. Alternatively, it can be the result of a refinement of the acquired sequences during viral evolution. We separated each protein into three segments: (i) The Pfam domain (s); (ii) The tail linker (TAIL) that combines the amino acid extension towards the N- and the C-termini of the protein, beyond the boundary of the domain (s); (iii) The internal domain linker (IDOL) that comprises the sum of the amino acid spacers between domains. Clearly a single domain protein lacks IDOL. We performed a separate analysis for the TAIL and the IDOL sequences (Figures 6A–6D). The study was performed on all the families that have at least 2 Pfam domains (unique or repeated). The average TAIL in viral proteins is 14 amino acids while the metazoan protein TAIL length is 85 amino acids (p-value<1. 0e-150, Figures 6A–6B). Trimming of protein tails at both termini often leads to a loss of cellular localization signals (e. g., KDEL, PDZ binding sites are found at C-termini) [17]. Importantly, the average IDOL length of the viral proteins in the Pfam families is 30 amino acids, while, for the metazoan equivalent proteins, the length is 67 amino acids (p-value<1. 0e-150, Figures 6C–6D). While a short TAIL may be explained by the viruses having acquired a fragmented sequence from their hosts, the same trend was found for the IDOL. Figure 6C shows that while only 54% of the metazoa IDOL have a length of <40 amino acids, in the viral proteins from the same Pfam families, 96% of the proteins have IDOL that are shorter than 40 amino acids. These results are consistent with an active trimming and refinement process throughout viral evolution. Short IDOL length is advantageous in suppressing protein misfolding, and hence, improving translation effectiveness [46]. Similarly to the finding of short IDOL sequences in viral proteins, we identified instances of an internal domain which is missing in the viral protein while the flanking domains are maintained in the same order in the eukaryotic homologous protein (Figure 6E). The viral putative phosphatidylinositol kinase L615 (UniProt: Q5UR69) is a 701 amino acid protein from the Acanthamoeba polyphaga Mimivirus (APMV) that infects Amoeba. It has two Pfam domains: FYVE (PF01363) followed by PI3_PI4_kinase (PF00454). There are no other known proteins with identical domain architecture in the Amoebozoa kingdom (there are 7 such proteins in other kingdom, e. g., Stramenopiles and Excavata). However, there are 3 proteins from the genus Dictyosteliida (slime molds) that do belong to the Amoebozoa kingdom (UniProt D3BQ22, Q54UU9 and EGC34678). In all 3 of these proteins, the architecture is composed of FYVE domain followed by PI3Ka and PI3_PI4_kinase. The missing domain of PI3Ka in the Mimivirus (APMV) provides an evidence for an active elimination of an internal domain based on parsimonious argument. The findings of shorter IDOL (Figures 6C–6D) or absence of internal domains (Figure 6E) are probably the result of the trimming and shortening of the sequences after their acquisition by the virus. The possibility of a domain insertion in eukaryotes cannot be excluded. The exhaustive search for sequences that were hijacked by viruses from their host allowed us to speculate on the underlying modes of mimicry. It was shown that once a mimicry function by a virus is established, the corresponding functional partner protein of the host undergoes a fast positive selection to overcome the deleterious effect of the viral mimicry [20]. According to these findings, the viral proteins that originated from the hosts are short versions of the full-length host proteins (Figures 5–6, Supplemental Figures S2, S4). Furthermore, these proteins are characterized by a substantial reduction in the architectures of the domains (Figure 5) and the protein linkers (Figure 6). We classified these proteins into distinct (yet not exclusive) modes of action. For simplicity, we unified the viral acquired sequences from the cross-taxa families to 5 strategy modes (Figure 7). Mode A depicts a competition on a receptor binding by a viral ligand that replaces the natural one. Examples for this mode are the expression of the secreted IL-10 (Figure 2), IL-8 (UniProt: Q98158, Q98314, D2E2Z5) and PDGF (UniProtKB Q80GE8, Q2F842 and D0VXD7). These secreted mitogens are identified in class I and class VI viruses (Table 2). Viral proteins participate in a rich protein-protein interaction (PPI) network [47]. Mode B illustrates PPI, where the virus uses an acquired sequence for replacing a host partner protein or for interacting with a preexisting protein complex. The result is an alteration of the cells' function. Examples for viral proteins that interfere with the host PPI are the anti-apoptotic Bcl-2 sequences and Profilin (Table 2, for example UniProt: Q5IXM3, P33828, P68695). Mammalian Semaphorins (Sema7) and the Smallpox virus A39R protein (Table 2, UniProt: Q775N9, B7SV99, Q0N658, A0ES13) share identical binding modes with a cross-reactivity towards common receptors [48]. Mode C depicts the role of protein modifications (e. g., phosphorylation). A viral protein can either mimic the host modifications (Figure 7, marked C1). Alternatively, a modification occurs by a viral enzyme (Figure 7, marked C3). Such mimicry can lead to a modification of the original site or at an entirely new site (Figure 7, marked C2). Apparently, there are instances in which both the modifying enzyme and the target proteins are both sequences that were acquired from the host (Figure 7, marked C4). This mode is dependent on the presence of active kinases (or phosphatases). For example, human cytomegalovirus (HCMV) kinase introduces phosphorylation sites that perfectly mimic the function of the cellular CDK2 (cyclin dependent kinase) [49]. An evolutionary tree alignment for viral B1R protein kinase (Supplemental Figure S3) supports the functional overlap and mimicry with the closely related cellular kinases. Mode D depicts the importance of nucleic acid regulation of transcription. In this mode, a viral protein mimics the host regulation by either competing for an existing transcription factor (Figure 7, marked D1), or by modifying the transcription program following a DNA/RNA binding (Figure 7, marked D2). For example, the Epstein-Barr virus (EBV) encodes an activator protein that is similar to Fos/Jun family (bZIP_1, PF00170. For example, UniProt: Q80GR6, Q8QQX9, Q6USE5, D2Y5S7). The difference in specificity and the dimerization properties of the EBV activator allows the activation of an alternative transcription program [50]. Mode E collectively points to the generic strategies for damaging and deactivating the host proteins. It could be achieved by protein tagging (i. e., SUMO, ubiquitin), or the activation of viral proteases. Among the cross-taxa Pfam families, some families are associated with specialized proteases (Table S3). Mode E shows the various routes by which acquired sequences alter key cellular processes. Molecular mimicry in trafficking and the subcellular localization is common to many viruses. For example, Soluble N-ethylmaleimide sensitive factor Attachment Protein (α-SNAP) is a conserved protein among all eukaryotes. It was also found in Canarypox and Fowlpox viruses [51]. These proteins may alter the balance of the vesicular trafficking, docking and the membrane fusion machinery. In autophagy, viral proteins exploit processes such as membrane fusion and protein folding for the benefit of their replication [52]. We limit the discussion to the modes by which the shorter versions of the viral acquired proteins exhibit their impact on some cellular functions. The described modes (A–E) are effective in additional instances of molecular and functional mimicry [53], [54]. Inspecting the viral proteome is challenging, as the majority of viral sequences are redundant and poorly annotated. Importantly, the rapid evolution and the high mutation rate in some viral classes often leads to the loss of a detectable sequence similarity and, therefore, additional cases of virus hijacking events cannot be detected based on sequence similarity search methods. Despite these drawbacks, we have traced hundreds of viral proteins with respect to their hosts. Only a small fraction of them shows high sequence similarity with corresponding host proteins. For the majority of the cases, the origin of the viral sequences and possible derivations from the host call for applying powerful models for remote homologues. We provided analysis for 670 homologous families (according to the Pfam definition). For half of these families we provided support for sequence acquisition by the viruses from their hosts. The candidate sequences for a host to viral acquisition are useful in exploring the mechanisms by which viruses hijack and refine sequences. We found that most of the viral proteins that potentially originated from host sequences are significantly shorter and contain fewer domains. Furthermore, we propose that the sequence refinement by the virus is a dynamic process. The inter-domain linkers (e. g., sequences connecting domains, but excluding the amino- and carboxyl tails) are significantly short, relative to other related proteins (Figure 6). The viral proteins act in the cell according to a finite number of strategies. The simpler domain composition of these viral proteins is sufficient for the utilization of functional mimicry. Currently, we are expanding the analysis by identifying short peptides in viral proteomes that serve as competition agents for neutralizing critical cellular functions. The collections of 187 UniRef90 clusters and the 667 Pfam cross-taxa families are available as interactive tables. These tables are available at: www. protonet. cs. huji. ac. il/virost/tables/UniRef90. html www. protonet. cs. huji. ac. il/virost/tables/Pfam. html UniProKB includes 990,049 sequences (taxonomy-viruses). The viral proteins include ∼15,000 reviewed proteins (UniProt/SwissProt). The rest of the proteins are from UniProt/TrEMBL. There are 430. 6 K sequences after removal of HIV and HBV sequences. Only 241. 8 K are full-length (56. 1%), while the rest are denoted as ‘fragments’. The percentage of full-length proteins in metazoa is 54% (1. 191 M/2. 2051 M). The pre-calculated classifications of UniRef90 (i. e., identity of >90% at the amino acid level) reduce the UniProKB set to 175,236 clusters. Additional steps of filtrations are: (i) Considering only clusters with a minimal size of 2 proteins (62,129 clusters); (ii) Clusters that also include the metazoan proteins (187 clusters). ViralZone is a database that manually assigns host-virus pairs (http: //www. expasy. ch/viralzone, coordinated by UniProt/SwissProt). ViralZone holds reference strains viruses that belong to 83 families and 330 genera. This is a high quality collection of ‘complete proteome’. All viruses are classified into 7 disjoint classes (Baltimore classification index): (I) Double stranded DNA viruses; (II) Single stranded DNA viruses; (III) Double-stranded RNA and Single-stranded RNA viruses with positive and negative sense, respectively (IV, V); (VI) Positive sense single stranded RNA viruses that replicate through a DNA intermediate; (VII) Double-stranded DNA viruses that replicate via a single-stranded RNA intermediate. Major families of viruses infecting vertebrates are listed in Supporting information, Table S1. Pfam 24. 0 (11,912 families) [32] is a high quality resource for domains and families. A valid cross-taxa list was generated. Eukaryotes and viruses cross-taxa resulted in 1,165 Pfam entries. The following filtration steps were applied: (i) Pfam families with at least one viral protein and at least one metazoan protein (taxid: 33208), total of 859 Pfam families. (ii) Restricting the Pfam to families that have at least one metazoan protein and at least one metazoan-infecting virus resulted in 796 Pfam families. (iii) Pfam families with >95% viral proteins for structural element of the virus (e. g., Env, Coat, Capsid). (iv) Enzymes of the replication system were excluded, as these genes are the outcome of several events of genetic exchange [55]. Specifically, we excluded families of RNA/DNA polymerases (39 families), Exo/Endonuclease (16 families), Helicase (15 families), tRNA synthetase (8 families) and Primase (8 families). We also manually eliminated the cluster represented by the GFP (PF01353) that reflects the inevitable contamination from the extensive use of GFP as vectors in many molecular biology techniques. The filtered list includes 667 protein Pfam families (Supplemental data Table S3). We define linker sequences as TAILs (Tail Linkers) and IDOLs (Inter Domain Linkers). The TAILs are all sequences at the two terminals external to the first and last domain in the protein. Each protein provides two entries. The IDOL is a collection of all inter-domain sequences (excluding TAIL). Protein TAIL' s length was defined as the mean of the two tail segments. In the same way, IDOL length was defined as the mean of the lengths of the inter domains linkers. We collected the Pfam data for all proteins having at least 2 domains (i. e., having at least one IDOL) and one of the domains belong to the 667 Pfam domain families (Table S3). There are ∼57,000 such viral proteins and ∼98,000 metazoan proteins. Statistical tests were applied for the set of viral proteins in view of the host cellular protein for each cluster (or Pfam family collection). We applied statistical confidence tests (P-values) based on the non-parametric Kolmogorov-Smirnov (KS), Student t-test and the hypergeometric distribution tests. The KS test is based on the maximum distance between the two cumulative curves based on the separated viral and host proteins and viral and metazoan for the TAILs and IDOLs. Multiple sequence alignments (MSA) by ClustalW were used for constructing the Phylogenetic trees. Local alignment searches are from NCBI-BLAST. BLAST was activated with a ‘gap costs’ for Existence: 10 and for Extension: 1. The resetting of the BLAST parameters was needed for systematic identification of missing domains detection scheme. The phylogenetic trees were built using the iTol [56].

Many studies focused on the exchange of genetic material between viruses and cellular hosts. The diversity of viruses argues that, along the evolutionary history, viruses have shaped the host genomes. While most viruses have many opportunities to exchange genetic material with their hosts, tracing such events is challenging as the origin of the sequences is masked by the high mutation rate of many viruses. On the other end, for completing a successful infection cycle the viruses must cope with the cell machinery for entry, replication and translation while hiding from the host immune system. We collected evidence for instances of viral protein sequences that were most probably "stolen" from the hosts. Additionally, a shared ancestry with metazoa is associated with 670 Pfam domain families. For half of these families, the origin of the viral proteins from its host is supported. For about 75% of the cross virus-metazoa families, the viral proteins are significantly shorter than their counterpart host proteins. Most of these cross-taxa viral proteins are single domain proteins and proteins with a simple domain composition relative to the proteins of their hosts. These viral proteins provide insights on the overlooked intimacy of viruses and their multicellular hosts.

lay_plos

The accumulation of heteroplasmic mitochondrial DNA (mtDNA) deletions and single nucleotide variants (SNVs) is a well-accepted facet of the biology of aging, yet comprehensive mutation spectra have not been described. To address this, we have used next generation sequencing of mtDNA-enriched libraries (Mito-Seq) to investigate mtDNA mutation spectra of putamen from young and aged donors. Frequencies of the “common” deletion and other “major arc” deletions were significantly increased in the aged cohort with the fold increase in the frequency of the common deletion exceeding that of major arc deletions. SNVs also increased with age with the highest rate of accumulation in the non-coding control region which contains elements necessary for translation and replication. Examination of predicted amino acid changes revealed a skew towards pathogenic SNVs in the coding region driven by mutation bias. Levels of the pathogenic m. 3243A>G tRNA mutation were also found to increase with age. Novel multimeric tandem duplications that resemble murine control region multimers and yeast ρ− mtDNAs, were identified in both young and aged specimens. Clonal ∼50 bp deletions in the control region were found at high frequencies in aged specimens. Our results reveal the complex manner in which the mitochondrial genome alters with age and provides a foundation for studies of other tissues and disease states. The accumulation of heteroplasmic mitochondrial DNA (mtDNA) mutations is a well-accepted facet of the biology of aging [1]. Heteroplasmic single nucleotide variants (SNVs) which are predominately transitions have been identified in all regions of mtDNA in aged tissues. Heteroplasmic deletions that fall in the major arc between the origins of replication also accumulate with age. These so-called “major arc” deletions are generally associated with pairs of direct repeats that flank the deleted region [2]. At the tissue level major arc deletions tend to be heterogeneous and of low clonality. A key exception is the clonal “common” deletion [3], that occurs between two 13 bp direct repeats and is tightly associated with aging in brain [4]. mtDNA deletions accumulate to higher levels in post-mitotic tissues such as brain, heart and muscle [5]. Within brain the distribution of somatic deletions [6] appears to correlate with regional differences in mitochondrial oxidative phosphorylation activity [7]. At the cellular level, somatic mtDNA mutations accumulate stochastically to very high levels in a minority of cells [8], [9] through clonal expansion [10] of both de novo and inherited variants [11], [12]. These mechanisms dictate that specific mtDNA variants are present at very low levels within a tissue [13]. As a result, most of our understanding of somatic mtDNA mutation has come from the investigation of single mutations or single classes of mutation. To provide a more comprehensive picture of somatic changes to mtDNA, we have used next generation sequencing (NGS) of mtDNA-enriched DNA (Mito-Seq) to investigate mtDNA from putamen of young and aged donors at high coverage (Sample details provided in Table S1). Breakpoints indicative of mtDNA rearrangements such as deletions, were detected by BLAST alignment. The common deletion, m. 8483_13459del4977, was easily identifiable as a pair of clonal breakpoints in the coding region of aged samples (Figs. 1A–B). In agreement with other studies [6], [14], frequencies were significantly higher in the aged cohort than in the young cohort (P = 0. 0087, Fig. 2A) and ranged from 8. 4×10−4 to 3. 6×10−3 mtDNA−1. The eldest specimen in the young cohort, Y12 (34 yrs), carried the deletion at 1. 2×10−3 mtDNA−1 in line with observations that some individuals accumulate deletions from the third decade of life [6], [15]. Additional clonal and non-clonal deletions in the major arc between the mtDNA origins of replication are also associated with aging [16]. Dot-plots revealed “major arc” deletions as a consistent cloud of canonical breakpoints in aged specimens (Figs. 1D–E). The distribution of breakpoints in each sample matched pooled data from multiple studies of clonal deletions [17], demonstrating the extreme heterogeneity of breakpoints within individual tissue specimens. Cumulative frequencies were significantly higher in aged putamen than young (P = 0. 0152) and ranged from 0. 8×10−2 to 2. 6×10−2 mtDNA−1 (Fig. 2B). As with the common deletion, Y12 carried levels of major arc deletions within the aged cohort range. Assuming a simple linear model for the accumulation of deletions, our data showed that major arc deletions accumulated faster than the common deletion (Table 1). Levels of the common deletion increased 12. 5-fold and major arc deletions 3. 6-fold between 25 and 80 years of age. There appeared to be a close relationship between the frequencies of the common deletion and other major arc deletions (Fig. 2E). The proportion of total major arc deletion load accounted for by the common deletion appeared to be biphasic, increasing to a plateau at about age 40 and then increasing again beyond age 80 (Fig. 2F). It is possible this pattern reflects differences in the contribution of non-clonal de novo deletion and clonal expansion of the common deletion, to the total mtDNA deletion load with age. Two novel rearrangements were detected in the control region. The first, m. (16508_16544) _ (16565_57) dup (Fig. 1C) was present at up to 2. 5×10−2 mtDNA−1, a similar range to that of major arc deletions, although there was not a significant difference in frequency between cohorts nor association between frequency and age (Fig. 2C). These breakpoints resemble mtDNA control region multimers (CRMs) we previously identified in brain and heart of the progeroid PolgD257A/D257A mtDNA mutator mouse [18]. CRMs are large species composed of multimeric tandem duplications of part of the control region with very little or no other mtDNA sequence. This sequence structure, composed of multiple short tandem repeats, is very similar to that of ρ− mtDNAs in yeast [19]. We speculate that given their large size, any potential pathology associated with CRMs would likely be due to perturbation of nucleoid distribution. In PolgD257A/D257A mice, CRM repeat units have a mean length of 566 bp and a range from ∼200–800 bp. Repeat units in human putamen were shorter with a mean length of 81 bp and ranging from 44–87 bp with the most prominent form being m. 16509_22dup. Similar to CRMs in PolgD257A/D257A mice, direct repeats of 3 bp or larger were present in only 4% of CRM breakpoints suggesting they arise through a form of non-homologous end joining (NHEJ). This contrasts with major arc deletions which predominantly occur between direct repeats, inferring a role for homologous recombination [20], [21], and the present study where 83% of canonical breakpoints in the coding region involved direct repeats of 3 bp or longer. The presence of CRMs in our original putamen DNA samples was verified using inverted primer PCR and as seen in PolgD257A/D257A mice [18] this resulted in large heterogeneous amplicons (Fig. 1F). Applying the same PCR to DNA from cerebellum of the cases under study, we were unable to amplify CRM products (Fig. S2). Thus CRMs may be localized to regions that are sensitive to mitochondrial dysfunction [7] and accumulate higher levels of mtDNA damage [14]. The physiological impact of CRMs remains to be determined. Levels in putamen are ∼200-fold lower than in brain from PolgD257A/D257A mice [18] where CRMs were associated with a 45% depletion in mtDNA and an increase in mtDNA-encoded mRNAs of ∼3-fold. We also identified a cluster of clonal control region deletions (CRDs), m. (244_309) _ (311_489) del, present at frequencies of up to 1. 3×10−2 mtDNA−1, similar to that of major arc deletions in aged samples (Figs. 1B–C). These deletions disrupt conserved sequence block II (CSBII) involved in mtDNA replication and transcription termination [22]. Differences in the frequency of CRDs between the young and aged cohorts was significant (P = 0. 0043, Fig. 2D). 90% of CRDs were 50 bp long and the most abundant form was m. 307_356del50 which occurred between a pair of 9 bp direct repeats. The 5′ and 3′ flanking direct repeats and the resulting breakpoint encompass copies of an 11 bp degenerate sequence motif recently found to be over represented within 5 bp of mtDNA deletion breakpoints, including the flanking direct repeats of the common deletion [17]. The biological basis for the association of this motif with deletion breakpoints remains undetermined. The m. 307_356del50 deletion has been reported as a somatic mutation in cancers [23] and at high levels in the saliva, blood and hair follicles from a healthy Chinese family where it was shown to elicit no effect on mtDNA levels in blood [24]. PCR of original DNA samples verified the presence of CRDs in putamen (Fig. 1F). Unlike CRMs, PCR of DNA from cerebellum revealed CRDs in three aged specimens, one of which did not carry the deletion in putamen (Fig. S2). These findings define CRDs as both transmissible and somatic mtDNA mutations and indicate that they are a more prevalent feature of mtDNA mutation spectra than previously recognized. We did not find any relationship between the levels of CRMs and CRDs, nor either of these species with the levels of major arc or common deletions. Both CRMs and CRDs appear to be distinct from previously reported tandem duplications in the control region [25], [26]. To focus on somatic variation and reduce the confounding effects of inherited high frequency heteroplasmy, only SNVs with frequencies <0. 01 bp−1 were considered for analysis (Fig. S5). Analysis of errors in NGS has revealed nucleotide incorporation errors during library synthesis create false SNV calls that cannot be screened using quality filtering [27]. In particular, frequencies of G>T and C>A transversions are erroneously increased due to the presence of endogenous and/or exogenously-generated 8-oxoguanine. While inherent error limited the accuracy of absolute quantitation, the high level of sequencing coverage attained in our study (mean coverage 126,538 Table S1) enabled examination of differences in SNV frequencies and rates of SNV accumulation. In line with the consensus in the field (reviewed in [1] and [5]), the average frequency of total SNVs called in each alignment was significantly higher in mtDNA from aged putamen than young (P = 0. 0079; Fig. 3A). Assuming simple linear models for SNV accumulation, we determined rates of accumulation for SNVs (Table 1). Total SNVs accumulated at 4. 02±1. 81×10−7 per base pair per year (bp−1yr−1; ±95% CI), corresponding to an increase of 2. 6-fold between 25 and 80 years of age when adjusted to a baseline SNV load of 0. 37±0. 09×10−5 at less than one year of age determined for human forebrain [28]. Transitions account for about 90% of heteroplasmic SNVs [9] and are subject to significantly lower levels of NGS library-error than transversions [27]. Correspondingly, data for transitions were tighter than for total SNVs (Fig. 3B), providing a much more accurate picture of the SNV spectrum. In our putamen samples, transitions accumulated at a rate of 2. 22±0. 42×10−7 bp−1yr−1 across the entire mitochondrial genome, corresponding to a 2. 3-fold increase from 25 to 80 years of age. These rates place the SNV loads for human putamen at 80 years of age (Table 1) in good agreement with published values for aged forebrain [27], [28] and human colonic crypts [9] which range from ∼2. 2–3. 5×10−5 bp−1. Both of the above somatic SNV mutation rates are an order of magnitude higher than mutation rates for germline mtDNA haplotypes calculated from phylogenic studies [29], [30], likely reflecting the influence of purifying selection on the fixation of germline variants [31]. As seen in phylogenic [29], [30] and pedigree studies [32] of germline mtDNA, and in somatic SNV analysis [27] the most abundant SNVs clustered in the control region in both young and aged samples (data not shown). Although the average frequency of control region transitions remained significantly higher in the aged cohort than the young (P = 0. 0079). A plausible explanation for the clustering of SNVs in the control region is that a significant proportion of variance in this region is inherited. In addition, given the role of the control region in mtDNA replication and maintenance [33], expansion of variant mtDNA clones may drive increased somatic variance in this region as opposed to de novo mutation. Alternatively, as the control region is the most variable region of mtDNA [34] and this may simply reflect tolerance of sequence variation in this region. The rate of accumulation of transitions in the control region was 5-fold higher than the rate in the coding region (8. 82±3. 5×10−7 bp−1yr−1 and 1. 77±0. 4×10−7 bp−1yr−1 respectively, Fig. 3F). Again both values are an order of magnitude higher than germline mutation rates for these regions calculated from phylogenic data [29], [30]. However, in alignment with the determination of more rapid substitution rates when calculated over shorter timescales [35], they are very close to germline mutation rates calculated from pedigree analysis by Howell and coworkers [32]. In this study analysis of blood from multi-generational pedigrees combined with information from similar studies revealed mutation rates of 9. 5×10−7 bp−1yr−1 in the control region and 1. 5×10−7 bp−1yr−1 in the coding region (5. 3–15. 7×10−7 bp−1yr−1 and 0. 2–4. 9×10−7 bp−1yr−1 respectively at 99. 5% CI). While more work is necessary, this raises the intriguing possibility that apparent mtDNA SNV mutation rates may be similar in somatic and germline tissues. As there is clear evidence for purifying selection of germline mtDNA [36], which should lower the germline mutation rate, the similarity may reflect the antagonistic effect of the rapid expansion of permissive germline variants at replication bottlenecks during germ cell development [37]. When corrected for the difference in size of the coding and control regions, the rate of accumulation of mutations within each of these regions per mtDNA was 2. 7-fold higher for the coding region than the control region. This demonstrates that coding region mutations still constitute the major burden of somatic variance per mtDNA despite lower rates of accumulation per base pair (Fig. 3F). Within the coding region there was no notable difference in the rates of accumulation of transitions between RNA and protein coding genes (Fig. 3G). There did not appear to be any relationship between levels of SNVs and mtDNA rearrangements that could not be accounted for by corresponding relationships to age. The heteroplasmic transition m. 3243A>G in the MT-TL1 tRNA gene is likely the most prevalent pathogenic mtDNA mutation [38] and is primarily associated with MELAS and MIDD syndromes [39]. The region surrounding m. 3243 is an etiologic hotspot for mutations [40] although there have been conflicting reports as to whether m. 3243A>G accumulates in normal aging [41], [42]. We observed a distinct hotspot of SNV abundance spanning m. 3243 in aged samples (Figs. 3D–E) with a significantly higher average frequency for SNVs through m. 3242_3244 than young samples (P = 0. 0079 for each, <0. 0001 overall). In all aged samples the most abundant SNVs called at m. 3242_3244 were the transitions, m. 3242G>A, m. 3243A>G and m. 3244G>A, all of which have been associated with mitochondrial diseases [39], [43]. There is some evidence of association between detectable levels of m. 3243A>G in hair follicle DNA and age related hearing loss [38], implying this finding may have consequences for the biology of aging. Applying duplex sequencing to mtDNA from human forebrain, Kennedy and coworkers have recently described a novel strand bias for somatic transitions in the mtDNA coding region, detected as increased G>A versus C>T and T>C versus A>G transitions in the reference strand (L-strand) [28]. The G>A versus C>T mutation bias is proposed to be caused by cytosine deamination (C>U) on the H-strand, potentially occurring during replication while the H-strand is exposed. As the mtDNA reference strand is the opposing L-strand the bias is manifest as an increase in the frequencies of G>A relative to C>T transitions. Dissection of SNV spectra replicated this finding in our putamen specimens. Significant differences in the frequencies of both G>A versus C>T, and T>C versus A>G transitions were observed in the coding region of young and aged samples (P = 0. 0079 for each, Fig. 4A). The median bias in the G>A and C>T frequencies ([G>A]-[C>T]) in the coding region was 2. 56×10−5 bp−1 in the young cohort and 5. 18×10−5 bp−1 in the aged cohort, with a significant difference between cohorts (P = 0. 0079, Fig. 4C). For [T>C]-[A>G] bias in the coding region, median magnitudes were lower (Fig. 4D) and differences between cohorts were not significant (P = 0. 0556). In the control region, G>A versus C>T frequencies showed a similar difference in young samples as seen in the coding region (P = 0. 0079, Fig. 4B). However, in aged samples no difference in G>A versus C>T frequencies was observed. In addition, significantly lower magnitudes of [G>A]-[C>T] bias were seen within samples compared to the young cohort (P = 0. 0079, Fig. 4E), indicative of an age-related switch in [G>A]-[C>T] bias, contrary to the coding region where bias appears to increase with age. Examination of SNV frequencies revealed troughs in [G>A]-[C>T] bias in the control region (Fig. S7) driven by the accumulation of high levels of m. 64C>T and m. 16148C>T in all aged samples (Fig. 4G). Exclusion of these variants from analysis did not recapitulate the positive [G>A]-[T>C] bias seen in the coding region in the aged cohort. Both variants occur as haplotype polymorphisms [30] (MitoMaster GenBank frequencies 2. 6% and 1. 7% respectively [44]) and m. 64C>T has previously been noted in aged brain specimens [45]. Predicting either the consequence or origin of the accumulation of these variants is difficult as neither falls within a known mtDNA control element. While their accumulation may reflect the expansion of low consequence variants under a lack of mutational bias, it may also be that they represent unknown functional elements in the control region. Bias in control region T>C versus A>G frequencies shifted in the same direction as the coding region and was significant in both young and aged samples (P = 0. 0159 and 0. 0079 respectively) although differences in the magnitude of [T>C]-[A>G] bias between cohorts were not significant and the relationship with age was weak (Fig. 4F). As mentioned above, high G>T and C>A transversion frequencies, stemming from library synthesis base incorporation errors at 8-oxoguanine [27], were noted in the coding and control regions of all samples (Fig. 4A–B). Recent work has confirmed that in vivo there is no evidence for accumulation of G>T and C>A transversions with age in brain [28]. Analysis of “Mutpred” predicted pathogenicity scores [46] for germline mtDNA variants has demonstrated that variants with high predicted pathogenicity scores (>0. 6), are selected against [31]. In contrast, we found that transitions with high pathogenicity scores had higher average frequencies than those with lower ones in both the young and aged cohorts (Fig. 3C), in agreement with studies of single cells from colonic crypts of aged donors [9]. The skew in pathogenicity most likely reflects the combination of mutational strand bias described above and skewed base distribution at different pathogenicity scores due codon composition (Fig. S8). However, the apparently localized increases in frequencies of SNVs at m. 3242_3244, m. 64C>T and m. 16148C>T (Figs. 3D–E & 4G) suggests there may also be some modification of SNV spectra beyond strand bias. Transitions with pathogenicity scores >0. 667 accounted for 37% of the increase in transition SNV burden at protein coding bases and 20% across all bases. At an SNV load of 0. 15 mtDNA−1 at 25 years of age (Table 1) these percentages translate to pathogenic SNV burdens of 0. 03–0. 06 mtDNA−1, raising to 0. 07–0. 13 mtDNA−1 at age 80. Pathogenic mtDNA mutations have threshold mutation loads in tissues of 0. 80–0. 90 mtDNA−1 [47]. While it is uncertain whether heterogeneous mutations can have additive effects, this indicates that the steady-state pathogenic somatic mtDNA burden in normal putamen at 80 years of age is about 6–12-fold lower than that of a patient with a mitochondrial disorder. Nevertheless, as the etiology of the stoichiometric accumulation of somatic mtDNA mutations in aging is distinct from the inheritance of mtDNA mutations in patients with mitochondrial disorders [13], these estimates may still reflect a considerable stress. To examine the influence of nuclear mtDNA sequences (numts) [48] on our analysis we carried out identical sequencing of total DNA from human 143B. 206 ρ0 cells that do not have mtDNA [49] and subjected the resulting “pseudo”-mtDNA alignment to identical analysis (For alignment details see Table S1). Only 20 breakpoints were called in this alignment compared to the 4,183–13,877 identified in putamen specimens (median 11,009). These 20 included a single call for the common deletion and a single call for a CRM. Given the hundreds of hits for verifiable species like the common deletion and CRMs in our mtDNA-enriched samples, we determined that numts had negligible influence on analysis of rearrangements. In turn, the identification of the common deletion and a CRM breakpoint in an ostensibly nuclear DNA sample implies these are evolutionarily persistent mutations. With respect to SNVs the influence of numts in determining control region clustering can be excluded as this was not observed in our ρ0 alignment (Fig. S9C). In addition, no SNVs were reported in our ρ0 cell alignment between m. 3100 and m. 3300, ruling out an influence of numts in relation to increased SNV frequencies spanning m. 3243 (Fig. S9C). Interestingly an opposing skew in pathogenicity, towards higher average frequencies for SNVs with low predicted pathogenicity, was seen in the ρ0 alignment (Fig. S9B). This skew matches that seen in phylogenic studies of pathogenicity and higher mutation rates for 3rd base positions in studies of mtDNA haplotype variation [29], [30]. As the ρ0 alignment represents numts, this skew is in agreement with the concept of nuclear transfer of evolutionarily stable mtDNA variants that predominantly have low pathogenicity scores [31]. The data presented above represent the steady state somatic mutation spectra of tissue specimens. They are likely the product of opposing biological forces that act to increase or decrease mutation loads and result in the maintenance of somatic mutation burdens at tolerable levels. Dissecting the contribution of specific factors such as de novo mutation or clonal expansion is not possible from this data. Considering the relatively small samples size, the similarities between the mutation spectra in each cohort underlines the consistency which the mitochondrial genome alters with age in putamen. Of note, the rearrangements identified in the control region warrant further study given their frequency and undetermined biological impact. It is hoped these data will provide useful comparative benchmarks for studies of somatic mtDNA mutation in other tissues and in disease states. DNA extraction and sequencing. mtDNA-enriched total DNA extraction was based on our previously described approach [18] with minor alterations. Putamen samples were obtained from neurologically normal fresh frozen specimens at the University of Miami brain Endowment Bank (Table S1). All donors were Caucasian males. 0. 20–0. 35 g tissue punches were rapidly thawed at room temperature in 4 ml of homogenization buffer (200 mM mannitol, 50 mM sucrose, 10 mM HEPES (pH 7. 0), 1 mM ETDA) and homogenized using 30 strokes of a Teflon-glass Dounce homogenizer on ice. Crude mitochondrial fractions were harvested from homogenates by differential centrifugation at 600 g to clear debris and 9000 g to collect mitochondrial pellets. mtDNA-enriched DNA was obtained by resuspension in 1 mL extraction buffer (33 mM TRIS pH 8. 3,10 mM EDTA, 10 mM NaCl). To which SDS was added to 1% w/v and 3 mAU proteinase K solution (Qiagen) was added followed by incubation at 56°C for 4 hrs. Total nucleic acids were extracted twice using 25∶24∶1 phenol∶cholorfom∶isoamyl-alcohol (v/v/v) followed by two extractions with 24∶1 cholorfom∶isoamyl-alcohol (v/v). Nucleic acids were precipitated by ethanol/NaAc precipitation and resuspended in 55 uL 10 mM TRIS pH 8. 5. RNA was then digested with 0. 07 U RNAse A (Qiagen) and 300–500 ng dsDNA by Qubit (Invitrogen) analysis submitted for library synthesis. Libraries were prepared using Illumina Truseq PE V3-cBot-HS cluster kits and sequenced on the Illumina HiSeq 2000 platform at 5–8 libraries per lane with image processing using CASAVA V1. 7/1. 8 as 2×100 bp paired-end reads. Each sequencing run contained specimens from both young and aged cohorts with similar age distributions (Table S1). Alignment. Bioinformatic analysis was done with Genomics Work Bench V4. 7-5. 5. 2 (CLCBio). Reads were quality trimmed with an average post-trim read length >95 bp. Initial alignments were made against the revised Cambridge reference sequence (CRS) mtDNA reference sequence (NC_012920), using low stringency local alignment with a cutoff of 80% similarity over 50% length to collect mtDNA-like reads and reduce datasets. Aligned reads and sample-specific consensus sequences were extracted from these assemblies. Reads were then assembled back against respective sample-specific consensus sequences using high stringency local alignment with a cutoff of 90% similarity over 95% length. Reads that aligned at low stringency but not high stringency (generally <0. 7% aligned reads) were collected for detection of rearrangement breakpoints. mtDNA haplotyping was done using MitoTool 1. 1a [50]. Analysis of rearrangements. Breakpoints were identified using BLAST to align reads against NC_012920 with alternate “murine” numbering to provide a contiguous control region and a first base position at the start of TRNF (m. 577). This alternate reference sequence enabled detailed examination of recombination involving the control region, in particular rearrangement spanning m. 16569_1. To streamline output, a word length of 15 was used with open gap cost of 5 and extension cost 2. Data was parsed to collect reads with two segments in the same sense and collectively extending the full length of the read, neither of which was fully internal. Between 4,183–13,877 breakpoints were sequenced per alignment. The common deletion was quantified by counting: m. (8477_8483) _ (13262_13452) del; the cumulative burden of major arc deletions was determined by counting m. (5576_15976) del>320 excluding the common deletion and corrected for putative chimeras by subtracting m. (15976_5576) del>320 (Fig. S1C); CRMs were quantified by counting m. (16492_59) _ (16492_59) del>137; and CRDs m. (244_494) del (each described here with CRS numbering). The frequency of each type of rearrangement per mtDNA equivalent (mtDNA−1) was determined by normalizing to average coverage and assumes a single rearrangement per full length mtDNA. Data used for breakpoint dot-plots was corrected for coverage by reducing the volume of data plotted by the ratio of the average coverage of the alignment to the lowest coverage alignment, using cluster coordinates as a means to randomly shuffle reads. To reduce over interpretation of outliers and to provide conservative estimates of significance all tests of significance are two tailed Mann-Whitney rank tests. Analysis of SNVs. High stringency assemblies were used for SNV detection using CLCBio quality-based SNP detection algorithm. The algorithm filters variant calls on the basis of quality scores for the central base (>Q33), the average quality of neighborhood window (radius ±5 bp, >Q30) and the presence of other mismatches or gaps (< = 2) within the window. Significance filtering, i. e. limits on coverage or absolute counts, were not applied as a very low counts are biologically valid in a genetically heterogeneous system especially when considering the sampling effect of cluster generation. To exclude reads from chimeric fragments [51], all reads in broken pairs were excluded from analysis. Taken together these parameters excluded a significant amount of sequencing data and reduce effective sequencing coverage by 25–30% for SNV detection. SNV tables recorded frequencies for all four possible alleles at each base. To focus on somatic variation and avoid confounding effects of inherited high frequency heteroplasmy which is common, only SNVs with frequencies <0. 01 bp−1 were considered for analysis. Data from two sequencing runs were normalized by correcting linearly for the difference between mean SNV frequency of each run (Fig. S6). At the levels of coverage attained, the highly consistent nature of NGS sequencing error enabled detailed analysis of relative SNV frequencies but over-estimated absolute SNV levels due to incorporation errors. For examination of different classes of SNV frequencies, data was normalized by correcting linearly for the difference between means for each specific base change within the coding region across all samples between runs. These tables were used for calculation of mutation rates within different mtDNA coding and control regions, Mutpred-grouped protein coding bases and the m. 3242_3244 triplet. For analysis of coding regions, SNV data from all alignments was put in phase by aligning to m. 577G, there were no insertions/deletions in the coding regions of any consensus sequence. Control region length varied from −1 to +5 bp of CRS. Mutpred data tables for transitions in the CRS were taken from Pereira et al [31]. Fourteen codons in MT-ATP6 where bases overlap MT-ATP8 were considered only for MT-ATP8. Predicted pathogenicity scores for transitions were split into three groups, those with scores of >0. 667 and non-sense mutations (3468 bp), those with scores of 0. 666-0. 100 (3465 bp) and synonymous mutations plus the small number of bases with scores of <0. 100 (4386 bp). Transition frequencies for each base position were determined against sample-specific consensus sequences and aligned with mutpred scores calculated from CRS sequence for each base. The maximum sequence divergence between sample coding region sequence and CRS coding region sequence was 28 bp out of 15,447 bp. To reduce over interpretation of outliers and to provide conservative estimates of significance all tests of significance are two tailed Mann-Whitney rank tests. PCR. For detection of CRMs, m. (16508_16544) _ (16565_57) dup, inverted primer PCR was carried out using Kapa HiFi 2× master mix (Kapa Biosciences) containing a proofreading polymerase under manufacturers standard reaction conditions with a Tm of 62. 5°C and 60 s extension time. CRM Primers: 16562-F: TCACGATGGATCACAGGTCTAT 16540-B GTGGGCTATTTAGGCTTTATGACC For detection of the m. 307_356del50 CRD we used touchdown PCR with standard non-proofreading Taq polymerase (Bioline), reaction buffer (Bioline Mango) and conditions with Tm dropping 68-61°C over the first 10 cycles, followed by another 30 cycles at a Tm of 61°C and an extension time 90 s throughout. CRD Primers: CRD1-F: AAAAATTTCCACCAAACCCCAAAA CRD2-F: AAAAATTTTCACCAAACCCCAAAA 1421-B CACCTTCGACCCTTAAGTTTCATA. CRD forward primers span the m. 307_356del50 breakpoint. CRD2-F contains the m. 295C>T polymorphism and was used for the Haplogroup J samples (Y03 and A19, Table S1).

Mitochondria are unique among animal organelles in that they contain their own multi-copy genome (mtDNA). For the past 20 years it has been known that tissues like brain and muscle accumulate somatic mtDNA mutations with age. Because individual mtDNA mutations are present at very low levels, few details are known about the spectrum of mutations associated with aging. Advances in sequencing technology now permit the examination of mtDNA mutations at high resolution. We have examined the spectrum of mtDNA mutations present in putamen, a brain region prone to the accumulation of somatic mtDNA mutations. We were able to quantify the accumulation of clonal and non-clonal deletions in the mtDNA coding region which are known to have a strong association with aging. Partial deletions and novel duplications of the mtDNA control region were also identified, and appear to be more prevalent than previously recognized, but levels showed weaker associations with age than coding region deletions. Single nucleotide variants accumulate fastest in the control region, with a skew towards the accumulation of pathogenic mutations in the coding region. Understanding how the mitochondrial genome alters with age provides a benchmark for studies of somatic mtDNA mutations and dissection of the role they play in normal aging and degenerative diseases.

lay_plos

Complex tissues, such as the brain, are composed of multiple different cell types, each of which have distinct and important roles, for example in neural function. Moreover, it has recently been appreciated that the cells that make up these sub-cell types themselves harbour significant cell-to-cell heterogeneity, in particular at the level of gene expression. The ability to study this heterogeneity has been revolutionised by advances in experimental technology, such as Wholemount in Situ Hybridizations (WiSH) and single-cell RNA-sequencing. Consequently, it is now possible to study gene expression levels in thousands of cells from the same tissue type. After generating such data one of the key goals is to cluster the cells into groups that correspond to both known and putatively novel cell types. Whilst many clustering algorithms exist, they are typically unable to incorporate information about the spatial dependence between cells within the tissue under study. When such information exists it provides important insights that should be directly included in the clustering scheme. To this end we have developed a clustering method that uses a Hidden Markov Random Field (HMRF) model to exploit both quantitative measures of expression and spatial information. To accurately reflect the underlying biology, we extend current HMRF approaches by allowing the degree of spatial coherency to differ between clusters. We demonstrate the utility of our method using simulated data before applying it to cluster single cell gene expression data generated by applying WiSH to study expression patterns in the brain of the marine annelid Platynereis dumereilii. Our approach allows known cell types to be identified as well as revealing new, previously unexplored cell types within the brain of this important model system. Complex organisms are heterogeneous at several levels. For example, one can divide the body into functional organs: the skin, the brain, the liver and so on. This anatomical and functional classification implies that distinct organs are composed of different cell types. Interestingly, these functional building blocks are also not composed of homogeneous cell types. Indeed, they are composed of several tissues that together make up a complex organ. For example, the skin of mammals can be described as the superposition of the Epidermis, Dermis and Hypodermis [1]. However, even with this more precise description, each of these tissues will be heterogeneous. For instance in the Dermis, the cells making up the sweat glands will not be the same as the cells in the hair follicles. Additionally, this heterogeneity does not stop at this sub-sub classification: heterogeneity is still present and, with fine enough measurements methods, this remains true to the single cell level [2]. When reducing the scale of study, the classification of cells into distinct groups ceases to be anatomical. Instead, molecular biology has allowed scientists to define molecular characteristics that distinguish individual cells. The most widely used characteristic is mRNA expression, and gene expression signatures are now commonly used to define cell types [3], [4]. Conceptually, if a set of cells have similar expression profiles, this information can be used to gather these cells into a specific cell type; we focus on this, molecular, definition of a cell type in the remainder of this manuscript. To do this, gene expression measurements at the single cell level within the tissues under study are necessary. Recent technological developments have facilitated this shift from tissue to single cell resolution: in-situ hybridization [5] in a few organisms including P. dumerilii and single cell RNA sequencing assays [6] are amongst a number of methods that allow gene expression to be measured at the single cell level [7]. Given this, one key challenge is to develop computational methods that use the expression data to cluster single cells into robust groups, which can then be examined to determine their likely functional roles. Many popular clustering methods (e. g., hierarchical clustering, k-means and independent mixture models) exist and can be applied to address this problem [8]–[10]. However, these methods fail to take into account the spatial location of each cell within the tissue under study — when such information is available [3], [11], [12], it is extremely useful and should be incorporated into the downstream analysis. Specifically, we can hypothesise that cells that are close together are more likely to belong to the same cell type. In other words, if a cell has a" slightly" more" similar" expression profile to a typical cell in cell type b than in cell type a but all the surrounding cells have been classified as belonging to cell type a it seems sensible to assign this cell to cell type a. However, it is also important to note that cell migration, which takes place during the development of complex tissues, can lead to isolated cells with very different expression profiles than their neighbours, which also needs to be accounted for. To address these problems and, in particular, to utilise both the spatial and the quantitative information, we extended a graph theoretical approach developed for image segmentation to reconstruct noisy or blurred images [13], a method that finds its roots in the field of statistical mechanics as the Ising model [14] and its generalization, the Potts model [15]. The core concept of this method (Figure 1) is to estimate the parameters of a Markov Random Field based model using mean-field approximations to estimate intractable values as described in [16]. We use an Expectation-Maximization (EM) procedure to maximize the parameters as described in [13], [16]. To the best of our knowledge, such methods have not previously been applied to 3-Dimensional gene expression data. Additionally, from a theoretical perspective, we extended current models by allowing the degree of spatial cohesion per cluster to differ, thus allowing for the possibility that some cell types are more spatially coherent than others. After validating our approach using simulated data, we demonstrated its utility by applying it to data generated using methods described in Tomer et al. [3] who were interested in studying the ancestral bilaterian brain. Tomer et al. [3] used Wholemount in Situ Hybridisation (WiSH) to study the spatial expression pattern of a subset of genes, at single cell resolution, in the brain of the marine annelid Platynereis dumerilii 48 hours post fertilization (hpf). P. dumerilii, is an interesting biological model, sometimes considered a" living fossil" as it is a slow-evolving protostome that has been shown to possess ancestral cell types, and thus may provide a better comparison with vertebrates than fast evolving species like Drosophila and nematodes where derived features can obscure evolutionary signal [17], [18]. Wholemount in-Situ hybridization (WiSH) is an experimental technique where the practitioner uses labelled probes designed to be specific to a given mRNA to determine in which cells of the tissue under study that message is expressed. For a small organism like Platynereis, the staining can be applied to the whole animal and a 3-Dimensional representation of the expression pattern of a gene can be deduced using confocal microscopy to study the patterns of gene expression slice by slice. In practice, following the staining, imaging and alignment, the brain volume was partitioned into 32,203 3 voxels. The 3 volume was chosen to be slightly smaller than the average cell in Platynereis' s brain but it is possible to consider this grid as a simple cellular model where each voxel roughly corresponds to a cell in the brain. Within each voxel, the light emission (assumed to be correlated to the gene' s expression level) was measured (Figure 2). Theoretically, this luminescence data is quantitative but, on such a small scale, light contamination between voxels means that the quantitative measurements have to be interpreted with caution (Figure 3). Additionally, the light efficiency of probes can differ leading to a high experiment-to-experiment variability. Consequently, we binarized the dataset by setting the value of expression within a voxel to 1 or 0, depending upon whether the gene was or was not expressed, respectively (see Discussion). By repeating this process with different probes, expression patterns for 86 genes of interest were mapped. Importantly, due to the stereotypic nature of early Platynereis development [17], the expression patterns can be overlaid, meaning that for each 3 voxel it is possible to determine which subset of the 86 genes is expressed. We can represent this information in a matrix of binary gene expression, where the location of each voxel, roughly representing a cell within the brain, is referenced in a 3D coordinate system. Given this coordinate system, we can create a neighbouring graph representation, where each node in the equivalent undirected graph corresponds to a voxel in the in-situ data. The edges of the graph were computed following a simple neighbouring system taking only the 6 closest neighbours, one in each direction of the 3D space. Markov random fields (MRF) are statistical models that provide a way of modeling entities composed of multiple discrete sites such as images where each site is a pixel or, in our case, a biological tissue where each site is a single voxel roughly corresponding to a cell, in a context-dependent manner [19]. MRF based methods find their roots in the field of statistical mechanics as the Ising model [14] and its generalization, the Potts model [15]. Since then, they have been and are still mainly used in the field of image analysis, and the literature about them is ever growing [20]–[22]. More specifically, MRF methods are found in a wide range of applications such as image restoration and segmentation [23], surface reconstruction [24], edge detection [25], texture analysis [26], optical flow [27], active contours [28], deformable templates [29], data fusion [30] and perceptual grouping [31]. MRFs have also been used in a variety of biological applications from analysing medical imaging data [23], [32], [33] to analysing networks of genomic data [34]. Additionally, the Cellular Potts Model [35] has been used to model tissue development at a sub-cellular resolution. Mathematically, MRF models are built around two complementary sub-models. The field represents the sites and their spatial structure. The Hammersley-Clifford (1971) theorem states that the probability distribution of the Markov field can be represented as a Gibbs measure, which incorporates an energy function into which the spatial coherency parameters of the model are incorporated. Some critical choices in terms of the modeling framework are the structure of the neighbourhood system and the energy function. The emission model is used to describe the underlying data (gene expression measurements in our case) and it is necessary to make some assumptions about its form depending upon the underlying data. In our study the goal is to allocate the voxels described above into clusters, where is unknown, using the binarised matrix of gene expression measurements,. To incorporate spatial information into our clustering scheme, we assume that, the (latent) vector of length that describes the allocation of voxels to clusters, satisfies a first-order Markov Random Field (MRF), where the probability that a voxel is allocated to a given state depends only upon the states of its immediate neighbours. Additionally, within cluster, we assume that the expression of gene follows a Bernoulli distribution with parameter; we denote the full set of Bernoulli parameters using the matrix. In a typical MRF, the degree of spatial cohesion is determined by a single parameter, which is assumed to be constant for all clusters [36], [37]. However, in the context of tissue organisation, it is reasonable to expect that the degree of spatial cohesion will differ between clusters; consequently, we estimate a separate value of for each of the clusters (Methods). To estimate the parameters we use a fully-factorized variational expectation maximization (EM) approach in conjunction with mean-field approximations to infer intractable values [16]. To choose the optimal number of clusters,, we use the Bayesian Information Criterion (BIC). Simulating data with a spatial component is a non-trivial problem. Existing methods rely on MCMC approaches as described in [38]. However, in our case with a relatively large number of nodes in the graph (), this is computationally expensive. To overcome this problem, we exploited the fact that the Platynereis dataset already possesses a spatial structure, and use this as a synthetic example on which to base our simulations. As outlined in Figure 4, we start by clustering the gene expression data using different values of and store the corresponding parameter estimates. Subsequently, the estimated Bernoulli parameters,, were used to simulate binarised gene expression data from clusters where, for each voxel contained within cluster, the expression of gene is simulated from a Bernoulli distribution with parameter (Figure 4). Next, each simulated voxel was assigned to the same spatial location as the corresponding voxel in the biological dataset. As a result, the simulated and the biological datasets have the same neighbouring graph. We can then cluster these simulated datasets using the method outlined above and determine how accurately we can estimate the parameters () and choose the correct number of clusters,. The most important criterion for assessing the efficacy of our approach is the similarity between the inferred and true clusters. This also implicitly assesses the accuracy of the estimation of: if the inferred and true clusters are identical, the estimates of must be equal to the true values. In practice, we used the Jaccard coefficient to compare the inferred and the true clusters (Methods), where a Jaccard coefficient of 1 implies perfect agreement. To benchmark our approach' s performance, we also assessed the ability of two other models to cluster the simulated data: hierarchical clustering (hClust), a very widely used approach in genomics and elsewhere, and an independent mixture model, which allows the relative improvement in performance added by the spatial component to be studied. Additionally, the likelihood function that needs to be maximised possesses many stationary points of different natures. Thus, convergence to the global maximum with the Expectation-maximisation algorithm (see Methods section), depends strongly on the parameter initialisation. To overcome this problem, different initialisation strategies have been proposed and investigated (see for instance [39]–[41]). Herein, we compare a random initialisation scheme with an initialisation based upon the solution obtained by applying hClust. The results of these experiments are shown in Figure 5 for. Our method, when used with a random initialization scheme (Methods), has an average Jaccard coefficient of, and clearly demonstrates better performance than the other methods. The second best performing method is the independent mixture model with a random initialization, which has an average Jaccard coefficient of 0. 7. Since the independent mixture approach is equivalent to the MRF with all the parameters set equal to 0 (i. e., without a spatial component) this suggests that accounting for the spatial aspect yields improved results. Given this, it is perhaps unsurprising that hClust also performs relatively poorly. Additionally, we note that initializing the MRF with the hClust output yields results that are superior to those generated by hClust but that are still poorer than either the randomly initialized independent mixture model or the MRF approach. This is likely explained by noting that, depending upon the initialization, the EM algorithm might converge to a local maximum. Consequently, for the rest of this study we use the random initialization strategy to initialize the EM algorithm. As well as directly comparing the clusters, we can also determine how accurately the parameters are estimated. To this end, in Figure 6 we compare the true and inferred mean values of for different values of. The values of increase with, which is to be expected since more clusters implies the existence of more transition areas, thus making an increase of necessary to maintain the optimal spatial coherency of the model. Figure 6 also shows a slight but consistent underestimation of. This can be explained by noting that the simulation scheme used may reduce the spatial coherency within clusters. Specifically, as illustrated in Figure 7, clusters may not display homogeneous expression of a given gene: instead, depending upon the value of, a gene will be expressed only in a fraction of voxels. In reality, the voxels in which such genes are expressed may have a coherent spatial structure within the cluster that is lost in the simulation, thus explaining the consistently smaller values for that are estimated. To confirm this, we performed a second simulation using the parameter values estimated from the first simulation as a reference. In this context we did not expect any further loss of spatial coherency, which was indeed confirmed as shown by the blue curve in Figure 6. To validate further our estimation of, we randomized the coordinates of the voxels to lose any spatial component before re-clustering the data. As expected, we observed that the estimates of were very close to for all clusters (Figure 6), as well as there being very similar Jaccard coefficient values (relative to the true values) for the independent mixture and the MRF model. Both of these observations provide confidence in our assertion that the spatial component plays an important role in the fit. Finally, we assessed the ability of the model to choose the correct number of clusters,. To do this, we noted the" true" number of clusters underlying the simulated data and compared this with the chosen value,. The results for two representative choices of are shown in Figure 8 and demonstrate that our clustering approach, in conjunction with the BIC, is able to accurately determine the optimal number of clusters. After validating our method using simulated data, we next studied the biological meaning of each of the clusters generated by applying the HMRF model to the real data. To do this, we combined each cluster' s spatial location with its corresponding expression parameter. The latter parameter allows a stereotypical expression" fingerprint" to be associated with every cluster. In practice, not all of the 86 genes will provide insight into the biological function of a given cluster. For instance, in the case of a ubiquitously expressed gene,, the value of will be high for all clusters. To overcome this problem, we developed a score,, for each gene, and each cluster, where: For each gene,, and cluster,, is large if gene is specific to cluster. Consequently, the top scoring 3 or 4 genes for each cluster will represent a specific stereotypical expression pattern that will help us infer or confirm the identity of the functional tissue represented by each cluster. To provide confidence in our approach, we first considered well characterised regions within the Platynereis brain. Arguably the best-studied regions of the brain in Platynereis are the eyes: the brain has 4 eyes, two larval and two adult, and their locations and expression fingerprints are well known. As shown in Figure 9A, our approach generates two spatially coherent clusters that correspond to each of these regions. Importantly, the genes that best characterise these clusters are biologically meaningful: rOpsin and rOpsin3, both members of the well-described opsin family of photosensitive molecules [42], [43], best distinguish the adult eye and larval eyes respectively, consistent with the in-situ data images shown in Figure 10. As well as the eyes, a second region of the Platynereis brain, the mushroom bodies (which corresponds to the pallium, layers of neurons that cover the upper surface of the cerebrum in vertebrates [3]), are also clearly identified by our approach (Figure 9B). As well as identifying clusters corresponding to known cell types, we also identified clusters that might correspond to less well studied subtypes with specific biological functions. In Figure 11, the green cluster defines a region on the basal side of the larvae that can be associated both by its localization and by its most representative genes (MyoD [44], [45] and LDB3 [46], [47]) with the starting point of the developing muscles of the adult animal. Indeed, MyoD has been shown to play a key role in the differentiation of muscles during development in vertebrates and invertebrates [44], [45] and LDB3 codes for the protein LDB3, which interacts with the myozenin gene family that has been implicated in muscle development in vertebrates [47]. Given the location of the eyes and the developing muscles, the location of the pink cluster in Figure 11 is interesting. This cluster surrounds the larval eyes, the adult eyes and reaches the hypothetically developing muscles described above. Looking at the most representative genes for this pink cluster, it is interesting to note the presence of Phox2, a homeodomain protein that has been shown to be necessary for the generation of visceral motor-neurons (neurons of the central nervous system that project their axons to directly or indirectly control muscles) as described generally in [48] and in Drosophila [49]. The second most representative gene, COE, has also been shown to play a role in Platynereis and Drosophila neural tissue development [50]. In this context, although we lack biological validation, we can hypothesise that the cells within this particular cluster could be developing neurons that link the eyes to the muscles of Platynereis. Although this hypothesis remains purely speculative and would need validation in the laboratory, we believe this example is an interesting proof-of-concept that our clustering method can prove useful for hypothesis generation. Indeed, the analysis of the parameter values and the spatial localization attached to the clusters has allowed us to place with a reasonable level of confidence a functional hypothesis about a tissue that was not clearly defined either spatially or functionally. It is also interesting to note that hClust does not separate either putative region when clustering the same data with the same number of clusters. When we used an independent mixture model approach (i. e., with no spatial component) to cluster the data the results were more comparable to those obtained when using the HMRF strategy. However, as can be observed when comparing Figures 12 and 11, the clusters generated via the independent EM approach are considerably noisier and, as expected, less spatially coherent than those generated by the HMRF model. Further, for the developing muscle region, this noise is linked to biological imprecisions. When compared to in situ data generated by Fischer et al. [17], who used a phalloidin in situ stain to investigate the location of the muscles at this developmental stage, it can be observed that the muscles are restricted to regions located away from the axes of symmetry, more consistent with the HMRF clustering output. Similarly, the independent mixture model method associates to the hypothesized region of developing neurons around the eyes, some ventral areas that seem unlikely to belong to the same sub tissue. Consequently, it seems likely that the HMRF not only performs better than the independent mixture model on simulated data but also better reflects the underlying biology. As shown in Figure 3, we overcame problems linked to light contamination by binarizing the" quantitative" luminiscence information. To do this, it is necessary to specify a threshold above which a gene is considered expressed. Ideally the same threshold would be applied to all genes — however, when we examined the density plots of light intensities for each gene we observed significant differences that rendered such an approach impossible. Specifically, for some genes, the density of intensities clearly separated the voxels into two groups, corresponding to those where the gene is expressed and unexpressed, respectively (Figure 13 (left) ). For the remaining genes, however, the density plot was diffuse, with no clear separation of the voxels into expressed and unexpressed clusters (Figure 13 (right) ). Consequently, we binarized each gene manually by choosing an optimal threshold based upon inspection of the raw fluorescent microscopy images. This is possible since the number of genes under study is relatively small. However, as the number of genes for which data is available increases (as will be the case, for example, with single-cell RNA-sequencing studies), an automated method, perhaps based upon mixtures of Gaussians in the context of the WiSH data, will be required. Importantly, if the noise level in single cell expression datasets decreases to the extent where we can safely consider the results as quantitative, our method can easily be transformed to take this feature into account. The general outline of the model will stay exactly the same, the change will occur in the emission distribution. Instead of representing a Bernoulli parameter for each gene and each cluster, each could instead represent the parameter of a Poisson distribution. In our model we assume that, conditional upon the allocation of a voxels to a cluster, the gene expression levels can be described by independent Bernoulli distributions. This is a reasonable assumption in the context of the 86 genes chosen by Tomer et al. [3], since they were selected to have largely orthogonal expression profiles. In other words, they were chosen since they were known to correspond to distinct and potentially interesting regions of the Platynereis brain. However, in many other settings a larger number of genes, many with correlated expression profiles (i. e., genes in the same regulatory network) will be profiled and this assumption will be invalid. Consequently, extending the model to allow for dependence structure in the emission distributions will be a critical challenge. Additionally, as the number of genes increases, our approach for choosing the most specific genes will become less practical. Instead, entropy based approaches, such as the Kullback-Leibler divergence, might be more suitable. In summary, we have illustrated, using both simulations and real data, that accounting for spatial information significantly improves our ability to cluster voxels roughly representing brain cells into coherent and biologically relevant sets. While our approach converges very quickly (on the order of minutes) for the motivating dataset described herein, as the volume of data increases (i. e., by assaying the expression levels of thousands of genes in each cell using single-cell RNA-sequencing) it will be important to carefully investigate how easily our model scales. Nevertheless, we anticipate that our method will play an important part in facilitating interpretation of single-cell resolution data, which will be an increasingly important challenge over the next few years. To select the optimal number of clusters we used the BIC [45], which finds the optimal number of clusters,, by selecting the value of that minimises its value. However, due to the symmetry of the brain we used a slightly different approach. As shown in Figure 14 (blue dots), the BIC does not reach a clear minimum when applied to all voxels in the brain but instead reaches a plateau after a given number of clusters. This is most likely due to the highly, but not perfectly symmetrical nature of the brain: with a small, the same" tissue" on both the left and the right hand side of the brain will belong to the same cluster. However, because the two sides of the brain are not perfectly symmetrical, as increases the left and right part of the same" tissue" will be clustered separately. As a result, the likelihood continues to increase sufficiently to explain the flattened BIC curve. Moreover, this hypothesis seems to be confirmed by the fact that when computing the BIC on the right and left side of the brain separately, the curve has in both cases a clear minimum as shown in Figure 14 (red and green dots). Given this, we opted to choose as the point where the BIC curve reaches a plateau. The data are available as a binarized datset of single cell gene expression data for the 86 genes in the brain of Platynereis dumerilii. An implementation of the EM algorithm in the C programming language is also available on the Github page of the project [52].

Tissues within complex multi-cellular organisms have historically been defined in terms of their anatomy and function. More recently, experimental approaches have shown that different tissues express distinct batteries of genes, thus providing an additional metric for characterising them. These experiments have been performed at the whole tissue level, with gene expression measurements being "averaged" over millions of cells within a tissue. However, it is becoming apparent that even within putatively homogeneous tissues there exists significant variation in gene expression levels between cells, suggesting that additional cell subtypes, defined by distinct expression profiles, might be obscured by "bulk" experimental approaches. Herein, we develop a computational approach, based upon Markov Random Field models, for clustering cells into cell types by exploiting their gene expression profiles and location within the tissue under study. We demonstrate the efficacy of our approach using simulations, before applying it to identify known and putatively novel cell types within the brain of the ragworm, Platynereis dumerilii, an important model for understanding how the Bilaterian brain evolved.

lay_plos

A fundamental observation of comparative genomics is that the distribution of evolution rates across the complete sets of orthologous genes in pairs of related genomes remains virtually unchanged throughout the evolution of life, from bacteria to mammals. The most straightforward explanation for the conservation of this distribution appears to be that the relative evolution rates of all genes remain nearly constant, or in other words, that evolutionary rates of different genes are strongly correlated within each evolving genome. This correlation could be explained by a model that we denoted Universal PaceMaker (UPM) of genome evolution. The UPM model posits that the rate of evolution changes synchronously across genome-wide sets of genes in all evolving lineages. Alternatively, however, the correlation between the evolutionary rates of genes could be a simple consequence of molecular clock (MC). We sought to differentiate between the MC and UPM models by fitting thousands of phylogenetic trees for bacterial and archaeal genes to supertrees that reflect the dominant trend of vertical descent in the evolution of archaea and bacteria and that were constrained according to the two models. The goodness of fit for the UPM model was better than the fit for the MC model, with overwhelming statistical significance, although similarly to the MC, the UPM is strongly overdispersed. Thus, the results of this analysis reveal a universal, genome-wide pacemaker of evolution that could have been in operation throughout the history of life. Genome-wide analysis of distances between orthologous genes in pairs of organisms from a broad range of taxa belonging to all three domains of life (bacteria, archaea and eukaryotes) revealed striking similarity between the distributions of these distances. All these distributions are approximately lognormal, span a range of three to four order of magnitude and are nearly identical in shape, up to a scaling factor [1]–[3]. Although many different explanations are possible of this remarkable conservation of evolutionary rate distribution across the entire spectrum of life, the simplest underlying model is that all genes evolve at approximately constant rates relative to each other, i. e. the changes in the gene-specific rates of evolution are strongly correlated genome-wide. This general model of evolution can be denoted Universal PaceMaker (UPM) of genome evolution: all genes in evolving genomes, in each evolving lineage, change their evolutionary rate (approximately) in unison although the pacemakers of different lineages need not to be synchronized. The existence of UPM is compatible with the considerable amount of available data on fast-evolving and slow-evolving organismal lineages, primarily different groups of mammals [4], [5]. Conceivably, lineage-specific accelerations and decelerations of evolution can be caused by changes in the effective population size, and such rate changes are indeed expected to equally affect all genes in evolving genomes. The evolutionary rate has also been linked with other biological features of animals that are collectively denoted life history [5]. For instance, a genome-wide comparison of the evolutionary rates in the human and mouse lineages has shown that the number of fixed mutations per unit time is about twofold greater in rodents than it is in primates, with the implication that a lineage-specific, genome-wide change of evolutionary rate occurred after the separation of these lineages [6]. In the same vein, a genome-wide analysis of ratios between the evolutionary rates of orthologous genes in triplets of related bacterial, archaeal and mammalian species revealed near constancy of these ratios, with only a small percentage of gene-specific deviations that were attributed to functional diversification of individual genes [7]. A systematic study of densely populated phylogenetic trees for 44 mammalian genes has demonstrated clade-specific slowdown of evolution occurring independently in several orders including primates and whales [8]. Multiple studies of mitochondrial DNA evolution that used extensive samples from numerous taxa also detected consistent lineage-specific rates that differed by as much as an order of magnitude between animal taxa [9], [10]. However, in other analyses, striking differences between lineages in the relative rates of evolution of different genes have been discovered, casting doubt on the universality of lineage-specific rates, leading to the idea of ‘erratic evolution’ [11], [12]. The plausibility of the UPM notwithstanding, the genome-wide correlations between the evolutionary rates of individual genes also could be explained within the concept of molecular clock which is one of the central tenets of molecular evolution. In 1962 Zuckerkandl and Pauling discovered that the number of differences between homologous proteins is roughly proportional to the divergence time separating the corresponding species [13], [14]. This phenomenon became known as Molecular Clock (MC) and has been validated by multiple independent observations [15]–[18]. The MC is the basis of molecular dating whereby the age of an evolutionary event, usually the split between lineages (such as for example humans and chimpanzee), is estimated from the sequence divergence using calibration with dates known from fossil record [19]–[22]. From the phylogenetic point of view, when genes evolve along a rooted tree under the MC, branch lengths are proportional to the time between speciation (or duplication) events and the distances from each internal tree node to all descendant leaves are the same (ultrametric tree) up to the precision of the estimation (the latter being determined by sampling error which is inevitable in comparison of finite-length sequences). Over the 50 years that elapsed since the seminal finding of Zuckerkandl and Pauling, the MC has been shown to be substantially overdispersed, i. e. the differences between the root to tip distances in many or most subtrees of a given tree usually greatly exceed the expectation from sampling error, under the assumption of a Poisson mutational process [23]–[26]. Notably, the overdispersion of the MC has been shown to be lineage-specific: the MC in lineages with large effective population sizes is overdispersed to a greater extent than the MC in lineages with small populations implying that deviations from the MC are controlled by selection [27]. The demonstration of the overdispersion of the MC inspired the relaxed MC model which is a compromise between an unconstrained tree with arbitrary branch lengths and an MC tree [28], [29]. Under the relaxed MC, the evolutionary rate is allowed to change from branch to branch but this change is presumed to be gradual so that related lineages evolve at similar rates. The relaxed MC model underlies most of the modern methods of molecular dating. The strict MC implies that all orthologous genes present in a group of organisms and sharing the same evolutionary history evolve in a fully coherent manner even if at different rates. Indeed, if the divergence between gene sequences is solely determined by the divergence time and gene-specific evolution rate, phylogenetic trees reconstructed from different genes will have the same topology and nearly identical branch lengths up to a scaling factor which is equal to the relative evolution rate. Under the MC model, the differences between the corresponding branch lengths in different gene trees are due solely to the sampling error which arises from stochastic factors and is expected to be uncorrelated between trees. The relaxed MC model allows greater, non-random deviations in the lengths of corresponding branches but to our knowledge, the possibility that these evolution rate changes are correlated between genes has not been explicitly considered. The MC implies the constancy of gene-specific relative evolution rates, with deviations caused by overdispersion. However, the inverse is not true: the deviations of the absolute evolution rates from the clock could be arbitrarily high (hence no MC) but, if they apply to all genes in the genome to the same degree, the relative evolutionary rates would remain approximately the same throughout the entire course of evolution and in all lineages. In other words, the conservation of the evolutionary rate distribution follows from a model of evolution that is more general and less constrained than the MC, namely the UPM model. Here we sought to determine which of the two models of gene evolution, the MC and or the UPM, better fits the empirical data. To this end, we performed comparative analysis of phylogenetic trees for a genome-wide set of prokaryotic gene families and compared the goodness of fit for the two models. The results show that the UPM model is a better fit than the MC model for the evolution of prokaryotes. These findings are compatible with the previously observed accelerations and decelerations of evolution in individual evolving lineages. However, we show that synchronous, genome-wide change of evolutionary rates is a universal trend of genome evolution that appears to pervade the entire history of life. Our data set consisted of the “forest” of phylogenetic trees reconstructed for 6901 orthologous gene families representing 41 archaeal and 59 bacterial genomes [30] (see Supporting Text S1). Although horizontal gene transfer is widespread in the evolution of prokaryotes [31], [32], the tree-like statistical trend is detectable in the genome-wide data set and moreover dominates the evolution of (nearly) ubiquitous gene families [30], [33]. We encapsulate this trend in a rooted supertree (ST) that reflects the prevalent vertical descent in the evolution of archaea and bacteria (see Supporting Text S1). Each individual original gene tree (GT) is compared to the ST and reduced to the maximum agreement subtree (MAST), i. e. the largest set of leaves whose phylogeny fits the ST topology. Removal of discordant nodes and edges leads to collapse of several edges of the original GT into a single edge (Figure 1); then, the length of the newly created GT edge is the sum of the original contributing GT edges. Likewise, when a GT is mapped to the ST, several adjacent ST edges could correspond to a single edge in the reduced GT, forming a composite edge. Under both the MC and the UPM models, we assume that the lengths of the ST edges determine the expected lengths of the corresponding GT edges. For the MC model, edge lengths correspond to time intervals between speciation events, the ST is strictly ultrametric, and gene-specific evolutionary rates are measured in substitutions per site per time unit. Under the UPM model, edge lengths represent arbitrarily defined “ticks” of the universal pacemaker (internal time), and gene-specific evolutionary rates are measured in substitutions per site per pacemaker unit of internal time. Formally: where li, k is the length of the i-th edge of the k-th GT, tj is length of the j-th (possibly composite) ST edge corresponding to the i-th edge of the k-th GT, rk is the gene-specific evolution rate, and εi, k is the multiplicative error factor for the given edge. We further assume that the error is random, independent for branches both within and between GTs, and comes from a lognormal distribution with the mean of 1 and an arbitrary variance, translating to a model with an additive normally distributed deviation in the logarithmic scale. Because the distributions of evolutionary rates tend to follow symmetric bell-shaped curves in log scale [3], [34], the assumption of a multiplicative, log-normally distributed deviation seems natural. First, we seek to find the set of ST edge lengths t and gene rates r that provides the best fit to the entire set of GTs. Under the assumption of a normally distributed deviation, the likelihood function for the set of GTs given t and r iswhere n is the total number of edges in the set of GTs and E2 is the sum of squares of deviations between the expected and observed edge lengths in the logarithmic scale: where the summation for i is done over the edges of a given GT and the summation for k is done over all GTs (see Supporting Text S2). Thus, finding the maximum likelihood solution for {t, r} is equivalent to finding the minimum of E2. For the MC model, the ST edge lengths t are constrained by the ultrametricity requirement, whereas for the UPM model, ST edge lengths are unconstrained. For the analyzed set of 100 genomes, there is a choice of several possible ST topologies, produced using different methods (see Methods and Supporting Figure S1). We mapped all original GTs onto each of these STs and obtained reduced GTs that corresponded to the respective MASTs. The GTs that yielded MASTs with fewer than 10 leaves were discarded. The ST topology derived from the concatenated alignments of ribosomal proteins provided the maximum total number of leaves in the resulting set of reduced GTs and accordingly was chosen for further analysis. Altogether, we obtained 2294 reduced GTs with MAST size greater or equal to 10 species including 44,889 leaves and 82,896 edges. This set of trees was fit to an ultrametricity-constrained ST (MC model) and an unconstrained ST (UPM model) (Table 1, see Supporting Text S3 for details). We then compared the MC and UPM models in terms of the goodness of fit to the data. Obviously, the residual sum of squares is lower for the UPM model because it involves independent optimization of all 198 ST edge lengths, whereas under the MC model the edge lengths are subject to 99 ultrametricity constraints. To account for the difference in the numbers of degrees of freedom, we employed the Akaike Information Criterion (AIC) and the Bayesian Information Criterion (BIC) to compare the MC and UPM models. Under the assumption of normally distributed deviations: andwhere E2MC and E2UPM are the residual sums of squares for the MC and UPM models, respectively, n is the total number of GT edges and Δd is the difference in the number of parameters optimized in the process of fitting (in our case Δd = −99). Because lower AIC values correspond to better quality of fit, negative ΔAIC would indicate preference for the MC model whereas a positive ΔAIC would indicate support for the UPM model. The relative likelihood weight of the suboptimal model can be estimated as 1/exp (|ΔAIC|/2). The same calculations were repeated for smaller, more conservative subsets of gene families with MAST>20 and MAST>30 and also using BIC to compare the fit to the UPM and MC models (Table 1). Overall, the results presented in Table 1 reveal overwhelming support of the UPM model over the MC model. The only exception is the ΔBIC value for MAST>30 that weakly supports the MC model. This outcome is predictable given the much larger number of parameters in the UPM model, the small number of trees in this subset and the heavier penalty that BIC imposes on parameter-rich models [35]. Thus, the results show that the evolutionary rates tend to change synchronously for the majority (if not all) of the genes in evolving genomes although the rate of the UPM relative to the astronomical time differs for different lineages. The results of this analysis show that the apparent genome-wide constancy of the relative rates of gene evolution across vast spans of life' s history (Figure 2A) is not a trivial consequence of MC but at least in part results from a distinct, fundamental evolutionary phenomenon, the UPM (Figure 2B). The difference between the UPM and MC models is highly significant but small in magnitude. Root mean square deviation (r. m. s. d.) of GT edges from the expectations derived from UMP ST is large (a factor of 2. 45) and only slightly less that the r. m. s. d for the MC ST (a factor of 2. 48). Thus, similar to MC, the UPM appears to be substantially overdispersed. To assess the robustness of the finding that UPM fits the GTs better than MC, we isolated the contributions of individual trees to the E2MC and E2UPM (E2MC, k and E2UPM, k respectively), took 1000 bootstrap samples of the set of GTs and computed ΔAIC values for each sample. All 1000 ΔAIC values obtained for the resampled sets were positive (in the range of 1511 to 2147), providing 100% support to the superiority of the UPM model and ensuring that this result is consistent for the majority of the GTs and is not determined by a small number of strongly biased trees (see Supporting Text S3 and Supporting Figure S2 for details). The distribution of the E2MC, k/E2UPM, k ratios (Figure 3) shows a strong bias toward values greater than unity (73% of the GTs), supporting the robustness of this result. The E2MC, k/E2UPM, k ratio characterizes the degree to which the k-th GT favors the UPM model. Linear model analysis shows that this value is significantly and independently influenced by the average goodness of fit to the ST (p-value ≪0. 001; Figure 4), the fraction of the original GT leaves remaining in the MAST with ST (p-value ≪0. 001; Supporting Figure S3) and the number of the original GT leaves (p-value ≪0. 001; Supporting Figure S3). Thus, the GTs that retain a greater number of leaves in the MAST, fit the ST better and are wider distributed among prokaryotes, typically show the strongest preference for the UPM model over the MC model. These three factors together explain ∼9% of the variance in ln (E2MC, k/E2UPM, k). Neither the relative evolution rate nor the functional class of the gene significantly impact the degree of preference of UPM over MC (see Supporting Text S3 and Supporting Figure S3 for details). Interpreting these findings in terms closer to biology, widely-distributed genes that are subject to relatively little horizontal transfer or sporadic changes of evolution rate that reduce the fit to ST appear to make the greatest contribution to the UPM. These observations imply that the UPM is indeed a fundamental feature of genome evolution, at least in prokaryotes. The distribution of estimated relative evolution rates (Figure 5) spans values within a range slightly greater than an order of magnitude (0. 26 to 4. 58). This range is considerably more narrow than the range of rates measured over short evolutionary distances [3], [34]. Accelerations and decelerations of the UPM are likely to average out over long intervals of evolution, reducing the observed differences between genes. A logical extension of the UPM is a Multiple PaceMakers (MPM) whereby a number of uncorrelated pacemakers ‘guide’ their own sets of trees. In the extreme case, the number of PMs is equal to the number of GTs so that the individual GTs would be completely uncorrelated. We sought to explore this case in order to determine how well such a degenerate MPM (dMPM) model fits the data compared to the UPM and MC. Formally, under the basic assumptions of this work, the log likelihood of dMPM is infinite because the E2 value is estimated as the sum of squared differences between the observed and the expected edge lengths. Under dMPM, each edge is equal to its own expectation sothat E2 = 0. However, this logic assumes that the tree edge length is measured precisely and is not subject to any error, whereas the E2 value is dominated by deviations of individual GTs from the universal standard (MC or UPM). This assumption is obviously unrealistic, so to assess the likelihood of the dMPM, one needs to introduce the edge length estimate error explicitly. To obtain the lower limit on the E2 value induced by the inherent sampling fluctuations, one should note that the sum of the lengths of the 49,981 edges in 967 trees (MAST size ≥20) is 13,018. 5 (substitutions per site), on average 0. 26 per edge. With the typical prokaryotic protein length being ∼200 amino acids [36], this translates into the average of ∼52 substitutions per tree branch. Assuming that substitutions are generated by a Poisson-type random process, one expects the standard deviation of approximately and the “mean” error of the observed value on the order of (52+) /52 = 1. 14 or 0. 13 log units per branch. Multiplying the square of this value by 49,981 edges, we obtain the E2 value estimate of 843. 0, much lower than 35065. 0 for UPM. It should be noted that the use of the average gene length and the average number of substitutions per branch comprises the ‘best-case scenario’ because variations in both would necessarily introduce larger deviations which would increase the E2 value. To calculate the ΔAIC value, one needs to obtain the difference in the degrees of freedom between the UPM and dMPM models. The UPM model uses the estimates of 198 individual edge lengths in one UPM tree plus 967 GT rates; the dMPM model requires 967±198 edge length estimates and no GT rates, yielding Δd = −190,301. Plugging these values into the equation for ΔAIC, one gets the difference of −194,269 in the UPM-dMPM comparison. Thus, the dMPM model is less likely than the UPM model by 83,370 orders of magnitude, an obvious indication that the assumption of completely uncorrelated rate changes does not fit the data. More specifically, the data would support no more than 476 pacemakers for 967 GTs under ideal conditions (each GT follows its PM perfectly, so the E2 value remains to be solely determined by sampling fluctuations). Thus, the actual number of distinct pacemakers is expected to be much lower. The results of the genome-wide comparison of phylogenetic trees of prokaryote genes described here show that the UPM model fits the data substantially better than the MC model. These findings have no bearing on the validity of the MC but show that a more general conservation principle (the UPM) is sufficient to explain the observed correlations between gene-specific evolutionary rates. It seems a natural possibility that UPM is instigated by shifts in population dynamics of evolving lineages, with changes affecting all genes in the same direction and to a similar degree. In principle, UPM reflects the well-known phenomenon of lineage-specific acceleration-deceleration of evolution. However, to our knowledge, the previous studies on this phenomenon have focused primarily on mammals and to a lesser extent other vertebrates [4], [5]. Here we show that the UPM can explain the correlations between the evolutionary rates of prokaryote genes on the whole genome scale and over time intervals that span effectively the entire history of life on earth. The discovery of the UPM opens up several areas of further inquiry. We show here that an unconstrained model of evolution (dMPM) does not fit the data but it remains to be determined whether or not distinct pacemakers govern the evolution of different classes of genes. The biological connotations of the UPM are of major interest. Mapping UPM shifts to specific stages of the evolution of life, changes in the life style and population structure of organisms as well as to the geological record could become an important direction of future research. Three distinct supertrees (STs) were tested for the purpose of representing the vertical inheritance trend in the analyzed set of GTs. The first supertree (ST1) was from [30] (originally computed using the CLANN program [37]; the second supertree (ST2) was computed using the quartet supertree method [38] for all species quartets in the complete set of GTs the third supertree (ST3) was derived from a tree of concatenated sequences of (nearly) universal ribosomal proteins [39]. Maximum Agreement Subtrees (MAST) between the supertree (ST) and any given gene tree (GT) were computed using the agree program of the PAUP* package [40]. The set of MASTs with the analyzed GTs was computed for each of these STs, yielding a total of 43,068 MAST leaves for ST1,43,411 MAST leaves for ST2 and 44,889 MAST leaves for ST3 (MAST ≥10 for each ST). Accordingly, ST3 was used for all further analyses as the topology that best represented the entire set of GTs. To perform the LS optimization of the ST edge lengths and the GT relative evolution rates, we used the function fmin_slsqp () that is part of the scipy. optimize package of Python which minimizes a function using sequential least squares programming. The function also adopts a set of constraints that are necessary for the calculation. In both the MC and the UPM models, both the ST edges and the GT rates were constrained to positive values. For the UPM model, the distances from a node to any leaf in a subtree under that node were set equal for all subtrees. It can be shown by induction that this constraint implies an ultrametric tree. Thus, we have a constraint for every internal node; in a rooted binary tree with m leaves, there are m−1 such nodes. Consider a rooted supertree (ST) with a fixed topology. The ST encompasses a set of edges e defined by the ST topology and a set of unknown edge lengths t. Consider a set of unrooted GTs reduced to MAST with the given ST. Each GT encompasses a set of edges with known edge lengths and an unknown gene-specific evolution rate (bk, lk and rk for the k-th GT, respectively). Each edge of each GT uniquely maps to an ST path ej, that is a subset of adjacent edges in the ST (bk, i≡ej where ej⊆e for the i-th edge of the k-th GT). Let be the length of the path ej. We assume that the length of the i-th edge of the k-th GT is related to the length of the corresponding ST path ej: where εi, k is the multiplicative deviation factor for the given edge. We further assume that the deviation is random, independent for branches both within and between GTs, and comes from a lognormal distribution with the mean of 1 and an arbitrary variance, translating to a model with an additive normally distributed deviation in the logarithmic scale (i. e. ln εi, k∼N (0, σ2) ). Given t and r, the expectation for the logarithm of the length of the i-th edge of the k-th GT is: and the likelihood of observing the length li, k is: where E2i, k = (ln li, k−ln tj−ln rk) 2. For all observed edge lengths in all GTs (l), the likelihood function isIn the logarithmic scale: where n is the total number of GT edges (). Designating the residual sum of squares and substituting the estimate for σ2for large n, we obtain: Because n is constant for a given data set, finding the maximum of L (l | t, r) is equivalent to finding the minimum of E2. Least Squares (LS) is called linear if the residuals are linear for all unknowns. Linear LS can be represented in a matrix format which has a closed form solution (given that the columns of the matrix are linearly independent). However, our formulation requires taking logs over sums of unknowns in the case where a GT edge corresponds to a path in ST (). Then, the problem becomes non-linear with respect to LS and can be solved only using numerical algorithms where the solution is obtained by iteratively refining the parameter values. This approach requires supplying initial values for the parameters. The goodness of the initial value estimation is critical for the convergence time of the iterative method and the risk of being trapped in local maximum points. We employed the following strategy for determining the initial values: For each ST edge, we computed the mean value of the sum over all GT edges that uniquely correspond to the given edge. Therefore, if we assign one gene a specific rate value (e. g. the length of some edge), we obtain initial rate values for all genes. It can be easily shown that, if there are no errors in rates (i. e. σ2 = 0), the above procedure yields the accurate (ML) values for all unknowns.

A central concept of evolution is Molecular Clock according to which each gene evolves at a characteristic, near constant rate. Numerous studies support the Molecular Clock hypothesis in principle but also show that the clock is indeed very approximate. Genome-wide comparative analysis of phylogenetic trees described here reveals a distinct, more general feature of genome evolution that we called Universal Pacemaker. Under this model, when the rate of evolution changes, the change occurs synchronously in many if not all genes in the evolving genome. In other words, the relative rates of gene evolution remain constant across long evolutionary spans: if a gene is slow relative to the rest of the genes in the given lineage, it is always slow, and if it evolves fast, it is always fast. We show here that the Universal Pacemaker model fits the available data much better than the traditional Molecular Clock model. These findings are compatible with the previously observed accelerations and decelerations of evolution in individual lineages but we show that synchronous, genome-wide change of evolutionary rates is a global feature of genome evolution that appears to pervade the entire history of life.

lay_plos

ACT V. SCENE 2. The Grecian camp. Before CALCHAS' tent Enter DIOMEDES DIOMEDES. What, are you up here, ho? Speak. CALCHAS. [Within] Who calls? DIOMEDES. Diomed. Calchas, I think. Where's your daughter? CALCHAS. [Within] She comes to you. Enter TROILUS and ULYSSES, at a distance; after them THERSITES ULYSSES. Stand where the torch may not discover us. Enter CRESSIDA TROILUS. Cressid comes forth to him. DIOMEDES. How now, my charge! CRESSIDA. Now, my sweet guardian! Hark, a word with you. [Whispers] TROILUS. Yea, so familiar! ULYSSES. She will sing any man at first sight. THERSITES. And any man may sing her, if he can take her cliff; she's noted. DIOMEDES. Will you remember? CRESSIDA. Remember? Yes. DIOMEDES. Nay, but do, then; And let your mind be coupled with your words. TROILUS. What shall she remember? ULYSSES. List! CRESSIDA. Sweet honey Greek, tempt me no more to folly. THERSITES. Roguery! DIOMEDES. Nay, then- CRESSIDA. I'll tell you what- DIOMEDES. Fo, fo! come, tell a pin; you are a forsworn- CRESSIDA. In faith, I cannot. What would you have me do? THERSITES. A juggling trick, to be secretly open. DIOMEDES. What did you swear you would bestow on me? CRESSIDA. I prithee, do not hold me to mine oath; Bid me do anything but that, sweet Greek. DIOMEDES. Good night. TROILUS. Hold, patience! ULYSSES. How now, Troyan! CRESSIDA. Diomed! DIOMEDES. No, no, good night; I'll be your fool no more. TROILUS. Thy better must. CRESSIDA. Hark! a word in your ear. TROILUS. O plague and madness! ULYSSES. You are moved, Prince; let us depart, I pray, Lest your displeasure should enlarge itself To wrathful terms. This place is dangerous; The time right deadly; I beseech you, go. TROILUS. Behold, I pray you. ULYSSES. Nay, good my lord, go off; You flow to great distraction; come, my lord. TROILUS. I prithee stay. ULYSSES. You have not patience; come. TROILUS. I pray you, stay; by hell and all hell's torments, I will not speak a word. DIOMEDES. And so, good night. CRESSIDA. Nay, but you part in anger. TROILUS. Doth that grieve thee? O withered truth! ULYSSES. How now, my lord? TROILUS. By Jove, I will be patient. CRESSIDA. Guardian! Why, Greek! DIOMEDES. Fo, fo! adieu! you palter. CRESSIDA. In faith, I do not. Come hither once again. ULYSSES. You shake, my lord, at something; will you go? You will break out. TROILUS. She strokes his cheek. ULYSSES. Come, come. TROILUS. Nay, stay; by Jove, I will not speak a word: There is between my will and all offences A guard of patience. Stay a little while. THERSITES. How the devil luxury, with his fat rump and potato finger, tickles these together! Fry, lechery, fry! DIOMEDES. But will you, then? CRESSIDA. In faith, I will, lo; never trust me else. DIOMEDES. Give me some token for the surety of it. CRESSIDA. I'll fetch you one. Exit ULYSSES. You have sworn patience. TROILUS. Fear me not, my lord; I will not be myself, nor have cognition Of what I feel. I am all patience. Re-enter CRESSIDA THERSITES. Now the pledge; now, now, now! CRESSIDA. Here, Diomed, keep this sleeve. TROILUS. O beauty! where is thy faith? ULYSSES. My lord! TROILUS. I will be patient; outwardly I will. CRESSIDA. You look upon that sleeve; behold it well. He lov'd me-O false wench!-Give't me again. DIOMEDES. Whose was't? CRESSIDA. It is no matter, now I ha't again. I will not meet with you to-morrow night. I prithee, Diomed, visit me no more. THERSITES. Now she sharpens. Well said, whetstone. DIOMEDES. I shall have it. CRESSIDA. What, this? DIOMEDES. Ay, that. CRESSIDA. O all you gods! O pretty, pretty pledge! Thy master now lies thinking on his bed Of thee and me, and sighs, and takes my glove, And gives memorial dainty kisses to it, As I kiss thee. Nay, do not snatch it from me; He that takes that doth take my heart withal. DIOMEDES. I had your heart before; this follows it. TROILUS. I did swear patience. CRESSIDA. You shall not have it, Diomed; faith, you shall not; I'll give you something else. DIOMEDES. I will have this. Whose was it? CRESSIDA. It is no matter. DIOMEDES. Come, tell me whose it was. CRESSIDA. 'Twas one's that lov'd me better than you will. But, now you have it, take it. DIOMEDES. Whose was it? CRESSIDA. By all Diana's waiting women yond, And by herself, I will not tell you whose. DIOMEDES. To-morrow will I wear it on my helm, And grieve his spirit that dares not challenge it. TROILUS. Wert thou the devil and wor'st it on thy horn, It should be challeng'd. CRESSIDA. Well, well, 'tis done, 'tis past; and yet it is not; I will not keep my word. DIOMEDES. Why, then farewell; Thou never shalt mock Diomed again. CRESSIDA. You shall not go. One cannot speak a word But it straight starts you. DIOMEDES. I do not like this fooling. THERSITES. Nor I, by Pluto; but that that likes not you Pleases me best. DIOMEDES. What, shall I come? The hour- CRESSIDA. Ay, come-O Jove! Do come. I shall be plagu'd. DIOMEDES. Farewell till then. CRESSIDA. Good night. I prithee come. Exit DIOMEDES Troilus, farewell! One eye yet looks on thee; But with my heart the other eye doth see. Ah, poor our sex! this fault in us I find, The error of our eye directs our mind. What error leads must err; O, then conclude, Minds sway'd by eyes are full of turpitude. Exit THERSITES. A proof of strength she could not publish more, Unless she said 'My mind is now turn'd whore.' ULYSSES. All's done, my lord. TROILUS. It is. ULYSSES. Why stay we, then? TROILUS. To make a recordation to my soul Of every syllable that here was spoke. But if I tell how these two did coact, Shall I not lie in publishing a truth? Sith yet there is a credence in my heart, An esperance so obstinately strong, That doth invert th' attest of eyes and ears; As if those organs had deceptious functions Created only to calumniate. Was Cressid here? ULYSSES. I cannot conjure, Troyan. TROILUS. She was not, sure. ULYSSES. Most sure she was. TROILUS. Why, my negation hath no taste of madness. ULYSSES. Nor mine, my lord. Cressid was here but now. TROILUS. Let it not be believ'd for womanhood. Think, we had mothers; do not give advantage To stubborn critics, apt, without a theme, For depravation, to square the general sex By Cressid's rule. Rather think this not Cressid. ULYSSES. What hath she done, Prince, that can soil our mothers? TROILUS. Nothing at all, unless that this were she. THERSITES. Will 'a swagger himself out on's own eyes? TROILUS. This she? No; this is Diomed's Cressida. If beauty have a soul, this is not she; If souls guide vows, if vows be sanctimonies, If sanctimony be the god's delight, If there be rule in unity itself, This was not she. O madness of discourse, That cause sets up with and against itself! Bifold authority! where reason can revolt Without perdition, and loss assume all reason Without revolt: this is, and is not, Cressid. Within my soul there doth conduce a fight Of this strange nature, that a thing inseparate Divides more wider than the sky and earth; And yet the spacious breadth of this division Admits no orifex for a point as subtle As Ariachne's broken woof to enter. Instance, O instance! strong as Pluto's gates: Cressid is mine, tied with the bonds of heaven. Instance, O instance! strong as heaven itself: The bonds of heaven are slipp'd, dissolv'd, and loos'd; And with another knot, five-finger-tied, The fractions of her faith, orts of her love, The fragments, scraps, the bits, and greasy relics Of her o'er-eaten faith, are bound to Diomed. ULYSSES. May worthy Troilus be half-attach'd With that which here his passion doth express? TROILUS. Ay, Greek; and that shall be divulged well In characters as red as Mars his heart Inflam'd with Venus. Never did young man fancy With so eternal and so fix'd a soul. Hark, Greek: as much as I do Cressid love, So much by weight hate I her Diomed. That sleeve is mine that he'll bear on his helm; Were it a casque compos'd by Vulcan's skill My sword should bite it. Not the dreadful spout Which shipmen do the hurricano call, Constring'd in mass by the almighty sun, Shall dizzy with more clamour Neptune's ear In his descent than shall my prompted sword Falling on Diomed. THERSITES. He'll tickle it for his concupy. TROILUS. O Cressid! O false Cressid! false, false, false! Let all untruths stand by thy stained name, And they'll seem glorious. ULYSSES. O, contain yourself; Your passion draws ears hither. Enter AENEAS AENEAS. I have been seeking you this hour, my lord. Hector, by this, is arming him in Troy; Ajax, your guard, stays to conduct you home. TROILUS. Have with you, Prince. My courteous lord, adieu. Fairwell, revolted fair!-and, Diomed, Stand fast and wear a castle on thy head. ULYSSES. I'll bring you to the gates. TROILUS. Accept distracted thanks. Exeunt TROILUS, AENEAS. and ULYSSES THERSITES. Would I could meet that rogue Diomed! I would croak like a raven; I would bode, I would bode. Patroclus will give me anything for the intelligence of this whore; the parrot will not do more for an almond than he for a commodious drab. Lechery, lechery! Still wars and lechery! Nothing else holds fashion. A burning devil take them! Exit ACT V. SCENE 3. Troy. Before PRIAM'S palace Enter HECTOR and ANDROMACHE ANDROMACHE. When was my lord so much ungently temper'd To stop his ears against admonishment? Unarm, unarm, and do not fight to-day. HECTOR. You train me to offend you; get you in. By all the everlasting gods, I'll go. ANDROMACHE. My dreams will, sure, prove ominous to the day. HECTOR. No more, I say. Enter CASSANDRA CASSANDRA. Where is my brother Hector? ANDROMACHE. Here, sister, arm'd, and bloody in intent. Consort with me in loud and dear petition, Pursue we him on knees; for I have dreamt Of bloody turbulence, and this whole night Hath nothing been but shapes and forms of slaughter. CASSANDRA. O, 'tis true! HECTOR. Ho! bid my trumpet sound. CASSANDRA. No notes of sally, for the heavens, sweet brother! HECTOR. Be gone, I say. The gods have heard me swear. CASSANDRA. The gods are deaf to hot and peevish vows; They are polluted off'rings, more abhorr'd Than spotted livers in the sacrifice. ANDROMACHE. O, be persuaded! Do not count it holy To hurt by being just. It is as lawful, For we would give much, to use violent thefts And rob in the behalf of charity. CASSANDRA. It is the purpose that makes strong the vow; But vows to every purpose must not hold. Unarm, sweet Hector. HECTOR. Hold you still, I say. Mine honour keeps the weather of my fate. Life every man holds dear; but the dear man Holds honour far more precious dear than life. Enter TROILUS How now, young man! Mean'st thou to fight to-day? ANDROMACHE. Cassandra, call my father to persuade. Exit CASSANDRA HECTOR. No, faith, young Troilus; doff thy harness, youth; I am to-day i' th' vein of chivalry. Let grow thy sinews till their knots be strong, And tempt not yet the brushes of the war. Unarm thee, go; and doubt thou not, brave boy, I'll stand to-day for thee and me and Troy. TROILUS. Brother, you have a vice of mercy in you Which better fits a lion than a man. HECTOR. What vice is that, good Troilus? Chide me for it. TROILUS. When many times the captive Grecian falls, Even in the fan and wind of your fair sword, You bid them rise and live. HECTOR. O, 'tis fair play! TROILUS. Fool's play, by heaven, Hector. HECTOR. How now! how now! TROILUS. For th' love of all the gods, Let's leave the hermit Pity with our mother; And when we have our armours buckled on, The venom'd vengeance ride upon our swords, Spur them to ruthful work, rein them from ruth! HECTOR. Fie, savage, fie! TROILUS. Hector, then 'tis wars. HECTOR. Troilus, I would not have you fight to-day. TROILUS. Who should withhold me? Not fate, obedience, nor the hand of Mars Beck'ning with fiery truncheon my retire; Not Priamus and Hecuba on knees, Their eyes o'ergalled with recourse of tears; Nor you, my brother, with your true sword drawn, Oppos'd to hinder me, should stop my way, But by my ruin. Re-enter CASSANDRA, with PRIAM CASSANDRA. Lay hold upon him, Priam, hold him fast; He is thy crutch; now if thou lose thy stay, Thou on him leaning, and all Troy on thee, Fall all together. PRIAM. Come, Hector, come, go back. Thy wife hath dreamt; thy mother hath had visions; Cassandra doth foresee; and I myself Am like a prophet suddenly enrapt To tell thee that this day is ominous. Therefore, come back. HECTOR. Aeneas is a-field; And I do stand engag'd to many Greeks, Even in the faith of valour, to appear This morning to them. PRIAM. Ay, but thou shalt not go. HECTOR. I must not break my faith. You know me dutiful; therefore, dear sir, Let me not shame respect; but give me leave To take that course by your consent and voice Which you do here forbid me, royal Priam. CASSANDRA. O Priam, yield not to him! ANDROMACHE. Do not, dear father. HECTOR. Andromache, I am offended with you. Upon the love you bear me, get you in. Exit ANDROMACHE TROILUS. This foolish, dreaming, superstitious girl Makes all these bodements. CASSANDRA. O, farewell, dear Hector! Look how thou diest. Look how thy eye turns pale. Look how thy wounds do bleed at many vents. Hark how Troy roars; how Hecuba cries out; How poor Andromache shrills her dolours forth; Behold distraction, frenzy, and amazement, Like witless antics, one another meet, And all cry, Hector! Hector's dead! O Hector! TROILUS. Away, away! CASSANDRA. Farewell!-yet, soft! Hector, I take my leave. Thou dost thyself and all our Troy deceive. Exit HECTOR. You are amaz'd, my liege, at her exclaim. Go in, and cheer the town; we'll forth, and fight, Do deeds worth praise and tell you them at night. PRIAM. Farewell. The gods with safety stand about thee! Exeunt severally PRIAM and HECTOR. Alarums TROILUS. They are at it, hark! Proud Diomed, believe, I come to lose my arm or win my sleeve. Enter PANDARUS PANDARUS. Do you hear, my lord? Do you hear? TROILUS. What now? PANDARUS. Here's a letter come from yond poor girl. TROILUS. Let me read. PANDARUS. A whoreson tisick, a whoreson rascally tisick so troubles me, and the foolish fortune of this girl, and what one thing, what another, that I shall leave you one o' th's days; and I have a rheum in mine eyes too, and such an ache in my bones that unless a man were curs'd I cannot tell what to think on't. What says she there? TROILUS. Words, words, mere words, no matter from the heart; Th' effect doth operate another way. [Tearing the letter] Go, wind, to wind, there turn and change together. My love with words and errors still she feeds, But edifies another with her deeds. Exeunt severally ACT V. SCENE 4. The plain between Troy and the Grecian camp Enter THERSITES. Excursions THERSITES. Now they are clapper-clawing one another; I'll go look on. That dissembling abominable varlet, Diomed, has got that same scurvy doting foolish young knave's sleeve of Troy there in his helm. I would fain see them meet, that that same young Troyan ass that loves the whore there might send that Greekish whoremasterly villain with the sleeve back to the dissembling luxurious drab of a sleeve-less errand. A th' t'other side, the policy of those crafty swearing rascals-that stale old mouse-eaten dry cheese, Nestor, and that same dog-fox, Ulysses -is not prov'd worth a blackberry. They set me up, in policy, that mongrel cur, Ajax, against that dog of as bad a kind, Achilles; and now is the cur, Ajax prouder than the cur Achilles, and will not arm to-day; whereupon the Grecians begin to proclaim barbarism, and policy grows into an ill opinion. Enter DIOMEDES, TROILUS following Soft! here comes sleeve, and t'other. TROILUS. Fly not; for shouldst thou take the river Styx I would swim after. DIOMEDES. Thou dost miscall retire. I do not fly; but advantageous care Withdrew me from the odds of multitude. Have at thee. THERSITES. Hold thy whore, Grecian; now for thy whore, Troyan-now the sleeve, now the sleeve! Exeunt TROILUS and DIOMEDES fighting Enter HECTOR HECTOR. What art thou, Greek? Art thou for Hector's match? Art thou of blood and honour? THERSITES. No, no-I am a rascal; a scurvy railing knave; a very filthy rogue. HECTOR. I do believe thee. Live. Exit THERSITES. God-a-mercy, that thou wilt believe me; but a plague break thy neck for frighting me! What's become of the wenching rogues? I think they have swallowed one another. I would laugh at that miracle. Yet, in a sort, lechery eats itself. I'll seek them. Exit ACT V. SCENE 5. Another part of the plain Enter DIOMEDES and A SERVANT DIOMEDES. Go, go, my servant, take thou Troilus' horse; Present the fair steed to my lady Cressid. Fellow, commend my service to her beauty; Tell her I have chastis'd the amorous Troyan, And am her knight by proof. SERVANT. I go, my lord. Exit Enter AGAMEMNON AGAMEMNON. Renew, renew! The fierce Polydamus Hath beat down enon; bastard Margarelon Hath Doreus prisoner, And stands colossus-wise, waving his beam, Upon the pashed corses of the kings Epistrophus and Cedius. Polixenes is slain; Amphimacus and Thoas deadly hurt; Patroclus ta'en, or slain; and Palamedes Sore hurt and bruis'd. The dreadful Sagittary Appals our numbers. Haste we, Diomed, To reinforcement, or we perish all. Enter NESTOR NESTOR. Go, bear Patroclus' body to Achilles, And bid the snail-pac'd Ajax arm for shame. There is a thousand Hectors in the field; Now here he fights on Galathe his horse, And there lacks work; anon he's there afoot, And there they fly or die, like scaled sculls Before the belching whale; then is he yonder, And there the strawy Greeks, ripe for his edge, Fall down before him like the mower's swath. Here, there, and everywhere, he leaves and takes; Dexterity so obeying appetite That what he will he does, and does so much That proof is call'd impossibility. Enter ULYSSES ULYSSES. O, courage, courage, courage, Princes! Great Achilles Is arming, weeping, cursing, vowing vengeance. Patroclus' wounds have rous'd his drowsy blood, Together with his mangled Myrmidons, That noseless, handless, hack'd and chipp'd, come to him, Crying on Hector. Ajax hath lost a friend And foams at mouth, and he is arm'd and at it, Roaring for Troilus; who hath done to-day Mad and fantastic execution, Engaging and redeeming of himself With such a careless force and forceless care As if that luck, in very spite of cunning, Bade him win all. Enter AJAX AJAX. Troilus! thou coward Troilus! Exit DIOMEDES. Ay, there, there. NESTOR. So, so, we draw together. Exit Enter ACHILLES ACHILLES. Where is this Hector? Come, come, thou boy-queller, show thy face; Know what it is to meet Achilles angry. Hector! where's Hector? I will none but Hector. Exeunt ACT V. SCENE 6. Another part of the plain Enter AJAX AJAX. Troilus, thou coward Troilus, show thy head. Enter DIOMEDES DIOMEDES. Troilus, I say! Where's Troilus? AJAX. What wouldst thou? DIOMEDES. I would correct him. AJAX. Were I the general, thou shouldst have my office Ere that correction. Troilus, I say! What, Troilus! Enter TROILUS TROILUS. O traitor Diomed! Turn thy false face, thou traitor, And pay thy life thou owest me for my horse. DIOMEDES. Ha! art thou there? AJAX. I'll fight with him alone. Stand, Diomed. DIOMEDES. He is my prize. I will not look upon. TROILUS. Come, both, you cogging Greeks; have at you Exeunt fighting Enter HECTOR HECTOR. Yea, Troilus? O, well fought, my youngest brother! Enter ACHILLES ACHILLES. Now do I see thee, ha! Have at thee, Hector! HECTOR. Pause, if thou wilt. ACHILLES. I do disdain thy courtesy, proud Troyan. Be happy that my arms are out of use; My rest and negligence befriends thee now, But thou anon shalt hear of me again; Till when, go seek thy fortune. Exit HECTOR. Fare thee well. I would have been much more a fresher man, Had I expected thee. Re-enter TROILUS How now, my brother! TROILUS. Ajax hath ta'en Aeneas. Shall it be? No, by the flame of yonder glorious heaven, He shall not carry him; I'll be ta'en too, Or bring him off. Fate, hear me what I say: I reck not though thou end my life to-day. Exit Enter one in armour HECTOR. Stand, stand, thou Greek; thou art a goodly mark. No? wilt thou not? I like thy armour well; I'll frush it and unlock the rivets all But I'll be master of it. Wilt thou not, beast, abide? Why then, fly on; I'll hunt thee for thy hide. Exeunt ACT V. SCENE 7. Another part of the plain Enter ACHILLES, with Myrmidons ACHILLES. Come here about me, you my Myrmidons; Mark what I say. Attend me where I wheel; Strike not a stroke, but keep yourselves in breath; And when I have the bloody Hector found, Empale him with your weapons round about; In fellest manner execute your arms. Follow me, sirs, and my proceedings eye. It is decreed Hector the great must die. Exeunt Enter MENELAUS and PARIS, fighting; then THERSITES THERSITES. The cuckold and the cuckold-maker are at it. Now, bull! now, dog! 'Loo, Paris, 'loo! now my double-horn'd Spartan! 'loo, Paris, 'loo! The bull has the game. Ware horns, ho! Exeunt PARIS and MENELAUS Enter MARGARELON MARGARELON. Turn, slave, and fight. THERSITES. What art thou? MARGARELON. A bastard son of Priam's. THERSITES. I am a bastard too; I love bastards. I am a bastard begot, bastard instructed, bastard in mind, bastard in valour, in everything illegitimate. One bear will not bite another, and wherefore should one bastard? Take heed, the quarrel's most ominous to us: if the son of a whore fight for a whore, he tempts judgment. Farewell, bastard. Exit MARGARELON. The devil take thee, coward! Exit ACT V. SCENE 8. Another part of the plain Enter HECTOR HECTOR. Most putrified core so fair without, Thy goodly armour thus hath cost thy life. Now is my day's work done; I'll take good breath: Rest, sword; thou hast thy fill of blood and death! [Disarms] Enter ACHILLES and his Myrmidons ACHILLES. Look, Hector, how the sun begins to set; How ugly night comes breathing at his heels; Even with the vail and dark'ning of the sun, To close the day up, Hector's life is done. HECTOR. I am unarm'd; forego this vantage, Greek. ACHILLES. Strike, fellows, strike; this is the man I seek. [HECTOR falls] So, Ilion, fall thou next! Come, Troy, sink down; Here lies thy heart, thy sinews, and thy bone. On, Myrmidons, and cry you an amain 'Achilles hath the mighty Hector slain.' [A retreat sounded] Hark! a retire upon our Grecian part. MYRMIDON. The Troyan trumpets sound the like, my lord. ACHILLES. The dragon wing of night o'erspreads the earth And, stickler-like, the armies separates. My half-supp'd sword, that frankly would have fed, Pleas'd with this dainty bait, thus goes to bed. [Sheathes his sword] Come, tie his body to my horse's tail; Along the field I will the Troyan trail. Exeunt ACT V. SCENE 9. Another part of the plain Sound retreat. Shout. Enter AGAMEMNON, AJAX, MENELAUS, NESTOR, DIOMEDES, and the rest, marching AGAMEMNON. Hark! hark! what shout is this? NESTOR. Peace, drums! SOLDIERS. [Within] Achilles! Achilles! Hector's slain. Achilles! DIOMEDES. The bruit is Hector's slain, and by Achilles. AJAX. If it be so, yet bragless let it be; Great Hector was as good a man as he. AGAMEMNON. March patiently along. Let one be sent To pray Achilles see us at our tent. If in his death the gods have us befriended; Great Troy is ours, and our sharp wars are ended. Exeunt ACT V. SCENE 10. Another part of the plain Enter AENEAS, PARIS, ANTENOR, and DEIPHOBUS AENEAS. Stand, ho! yet are we masters of the field. Never go home; here starve we out the night. Enter TROILUS TROILUS. Hector is slain. ALL. Hector! The gods forbid! TROILUS. He's dead, and at the murderer's horse's tail, In beastly sort, dragg'd through the shameful field. Frown on, you heavens, effect your rage with speed. Sit, gods, upon your thrones, and smile at Troy. I say at once let your brief plagues be mercy, And linger not our sure destructions on. AENEAS. My lord, you do discomfort all the host. TROILUS. You understand me not that tell me so. I do not speak of flight, of fear of death, But dare all imminence that gods and men Address their dangers in. Hector is gone. Who shall tell Priam so, or Hecuba? Let him that will a screech-owl aye be call'd Go in to Troy, and say there 'Hector's dead.' There is a word will Priam turn to stone; Make wells and Niobes of the maids and wives, Cold statues of the youth; and, in a word, Scare Troy out of itself. But, march away; Hector is dead; there is no more to say. Stay yet. You vile abominable tents, Thus proudly pight upon our Phrygian plains, Let Titan rise as early as he dare, I'll through and through you. And, thou great-siz'd coward, No space of earth shall sunder our two hates; I'll haunt thee like a wicked conscience still, That mouldeth goblins swift as frenzy's thoughts. Strike a free march to Troy. With comfort go; Hope of revenge shall hide our inward woe. Enter PANDARUS PANDARUS. But hear you, hear you! TROILUS. Hence, broker-lackey. Ignominy and shame Pursue thy life and live aye with thy name! Exeunt all but PANDARUS PANDARUS. A goodly medicine for my aching bones! world! world! thus is the poor agent despis'd! traitors and bawds, how earnestly are you set a work, and how ill requited! Why should our endeavour be so lov'd, and the performance so loathed? What verse for it? What instance for it? Let me see- Full merrily the humble-bee doth sing Till he hath lost his honey and his sting; And being once subdu'd in armed trail, Sweet honey and sweet notes together fail. Good traders in the flesh, set this in your painted cloths. As many as be here of pander's hall, Your eyes, half out, weep out at Pandar's fall; Or, if you cannot weep, yet give some groans, Though not for me, yet for your aching bones. Brethren and sisters of the hold-door trade, Some two months hence my will shall here be made. It should be now, but that my fear is this, Some galled goose of Winchester would hiss. Till then I'll sweat and seek about for eases, And at that time bequeath you my diseases. Exit THE END

At Calchas's tent, Diomedes calls to Cressida. Her father fetches her, while Troilus and Ulysses watch from one hiding place and Thersites from another. With Thersites's profanity and Troilus's shock providing a counterpoint, Diomedes woos Cressida, who behaves reluctantly but coyly toward his advances, fending him off for a time but never allowing him to leave. Eventually, she gives him a sleeve that Troilus presented to her as a love-token--then she takes it back, and says that she never wants to see Diomedes again--then she softens, gives it to him once more, and promises to wait for him later, when he will come to sleep with her. When she is gone, and Diomedes too, Troilus is in agony, first denying the evidence seen with his own eyes, and then pledging to find Diomedes on the field of battle and kill him. Finally, as morning nears, Aeneas arrives to lead him back to Troy. In the city, Hector girds for battle, while the women--his wife Andromache and sister Cassandra--plead with him not to go. Both have had dreams that prophesy his death, but he dismisses their warnings. Troilus comes in and says that he will be fighting too; indeed, he chides Hector for having been too merciful to his enemies in the past, saying that today Troilus plans to slay as many men as he can. Cassandra leads Priam in, and the old king pleads with his son not to fight, saying that he too feels foreboding about this day, but Hector refuses to listen and goes out to the battlefield. Pandarus brings Troilus a letter from Cressida; Troilus tears it up and follows Hector out to the field. As the battle rages, Thersites wanders the field, escaping death by brazen cowardice. The Greeks are being driven back, and Patroclus is killed; Agamemnon orders his body brought to Achilles, who is roused to fury and joins the battle. He duels with Hector briefly, but tires and retreats; Hector continues slaying, while Achilles finds the Myrmidons, his men, and sets out to find Hector again. Eventually, as the battle nears its close, Achilles and his men find Hector, who has finished fighting and taken off his helmet. Surrounding the unarmed Trojan, they stab him to death and then tie his body to a chariot and drag it around the walls of Troy. The Trojan soldiers are grief-stricken, and Troilus leads them into the city to bring the heavy news. On the way, he encounters Pandarus, and curses him. Left alone on the stage, the unhappy Pandarus wonders why he should be so abused, when his services were so eagerly desired only a little while before.

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Mass drug administration (MDA) programs have dramatically reduced lymphatic filariasis (LF) incidence in many areas around the globe, including American Samoa. As infection rates decline and MDA programs end, efficient and sensitive methods for detecting infections are needed to monitor for recrudescence. Molecular methods, collectively termed ‘molecular xenomonitoring, ’ can identify parasite DNA or RNA in human blood-feeding mosquitoes. We tested mosquitoes trapped throughout the inhabited islands of American Samoa to identify areas of possible continuing LF transmission after completion of MDA. Mosquitoes were collected using BG Sentinel traps from most of the villages on American Samoa' s largest island, Tutuila, and all major villages on the smaller islands of Aunu' u, Ofu, Olosega, and Ta' u. Real-time PCR was used to detect Wuchereria bancrofti DNA in pools of ≤20 mosquitoes, and PoolScreen software was used to infer territory-wide prevalences of W. bancrofti DNA in the mosquitoes. Wuchereria bancrofti DNA was found in mosquitoes from 16 out of the 27 village areas sampled on Tutuila and Aunu' u islands but none of the five villages on the Manu' a islands of Ofu, Olosega, and Ta' u. The overall 95% confidence interval estimate for W. bancrofti DNA prevalence in the LF vector Ae. polynesiensis was 0. 20–0. 39%, and parasite DNA was also detected in pools of Culex quinquefasciatus, Aedes aegypti, and Aedes (Finlaya) spp. Our results suggest low but widespread prevalence of LF on Tutuila and Aunu' u where 98% of the population resides, but not Ofu, Olosega, and Ta' u islands. Molecular xenomonitoring can help identify areas of possible LF transmission, but its use in the LF elimination program in American Samoa is limited by the need for more efficient mosquito collection methods and a better understanding of the relationship between prevalence of W. bancrofti DNA in mosquitoes and infection and transmission rates in humans. Lymphatic filariasis (LF) caused by the diurnally subperiodic form of the mosquito-borne parasitic nematode Wuchereria bancrofti is endemic to American Samoa, a United States territory composed of the easternmost islands of the Samoan archipelago (Figure 1). LF is also endemic in the archipelago' s western islands which comprise the independent nation of Samoa [1], [2]. In the Samoan archipelago, Aedes (Stegomyia) polynesiensis Marks and Aedes (Finlaya) samoanus (Grünberg) are the major vectors of LF [3], [4]. Natural infections have also been detected in Aedes (Stegomyia) upolensis Marks and Aedes (Finlaya) tutuilae Ramalingam and Belkin, but these species are not considered to be as epidemiologically important due to their relatively low abundances in human landing catches [5], [6]. Aedes polynesiensis is widespread in the South Pacific, inhabiting islands south of the equator from Tuvalu and Fiji eastward to the Marquesas and Pitcairn Island [7]. It breeds in a wide range of natural and artificial containers [8], [9], [10] and feeds primarily in the daytime [11], [6]. Aedes polynesiensis is believed to be a weak disperser, rarely traveling as far as 92 m [12], [11]. Aedes samoanus occurs only in American Samoa and Samoa, breeding primarily in water collecting in leaf axils of the forest climber Freycinetia reineckei in American Samoa, and in axils of F. reineckei and Pandanus spp. in Samoa [5], [13]. Aedes samoanus females feed at night [5], [6]. The dispersal capabilities of Ae. samoanus have not been investigated. Other mosquito species abundant in Samoa and American Samoa are Culex (Culex) quinquefasciatus Say, Culex (Culex) annulirostris Skuse, Culex (Culex) sitiens Wiedemann, Aedes (Stegomyia) aegypti (L.), Aedes (Finlaya) oceanicus Belkin, and Aedes (Aedimorphus) nocturnus (Theobald) [7], [14]; however, none of these species have been found to play a significant role in LF transmission in the Samoan islands [12], [5], [14], [15]. During the years 2000–2010, the American Samoa Department of Health undertook a campaign to eliminate LF through annual mass drug administration (MDA) using diethylcarbamazine and albendazole [16]. The campaign ran in conjunction with similar campaigns in other South Pacific countries and territories, including neighboring Samoa, under the Pacific Programme to Eliminate Lymphatic Filariasis [17]. Population coverage by MDA was 24–52% in the first three years and improved to 65–71% in the subsequent four years [16]. Infection prevalence before, during, and after MDA has been monitored primarily by an immunochromatographic (ICT) test, which detects circulating filarial antigen (CFA) released into the blood by adult W. bancrofti [18]. The testing was done across all age groups. Prevalence of CFA in a baseline survey in 1999 was 16. 5% [19], and subsequent testing in four sentinel villages found CFA declining from 11. 5% in 2001 to 0. 95% in 2006 [20]. Prevalences in an additional four villages surveyed in 2006 were higher, ranging from 2. 1% to 4. 6% [20], [21], and a territory-wide serosurvey in 2007 found 2. 3% CFA prevalence. Additional MDA activities took place during 2007–2010, but the level of MDA coverage during those years is unclear. Testing the human population for CFA can provide information about prevalence of W. bancrofti infection, and antibody testing can provide a sensitive indicator of levels of exposure to W. bancrofti [22]. In addition, one can sample the human population indirectly by sampling mosquito species known to feed on human blood. Molecular xenomonitoring (MX), the detection of parasite DNA or RNA in mosquitoes using the polymerase chain reaction (PCR), allows the testing of pools of mosquitoes and can be more efficient and more sensitive than dissections, especially when large numbers must be examined to detect evidence of W. bancrofti when prevalence is low [23], [24], [25]. The ability to test large numbers of mosquitoes also depends on the availability of efficient collection methods for local species. The development of the BG Sentinel trapping system has, for the first time, made trapping large numbers of Ae. polynesiensis over large geographic areas feasible in American Samoa [26]. It is important to recognize that MX cannot provide a direct measurement of ongoing transmission unless the PCR method used specifically targets the infective third stage larva (L3) of W. bancrofti [27]. Instead, it provides an indirect assessment of human infection. Fischer et al. [28] and Erickson et al. [29], studying Brugia malayi, found that parasite DNA could be detected in both vector and non-vector mosquito species long after ingestion of microfilariae, even when those microfilariae did not survive in the mosquito. Workers wishing to assess transmission directly still need to measure vector biting rates and use dissection or reverse transcriptase-PCR to specifically detect L3 in the vector mosquitoes. In 2006, a pilot study evaluated the use of MX and traditional xenomonitoring concurrently with serological testing of humans in three villages in American Samoa. Trapped mosquitoes were examined by PCR or dissection, and village residents were tested for CFA and antifilarial antibody [21]. (The Bm14 antibody test used is an indicator of infection or exposure and may give a positive result prior to development of patent infections [30], [31], [32].) The serological tests found 3. 7–4. 6% of residents of the three villages were positive for CFA and 12. 5–14. 9% positive for antifilarial IgG4 antibody to the recombinant Bm14 antigen [21]. Dissection of approximately half of the Ae. polynesiensis catch found infection prevalences of 0–0. 23%, while PCR testing of the remainder gave estimates of 0. 52–0. 90% prevalence [25]. In summary, mosquito dissection proved relatively insensitive, while antigen and antibody testing and MX all gave similar results. All three indicated LF infections occurring at low levels in all three villages. In 2011, a territory-wide transmission assessment survey (TAS) was conducted according to the World Health Organization [18] guidelines for monitoring and assessment of MDA in LF elimination programs [33]. The TAS consisted of antigen and antibody testing of 6–7 year olds in the territory' s elementary schools. Overall CFA prevalence in the survey was below the threshold at which the guidelines would recommend additional MDA [33]. The TAS results provide guidance to determine whether or not to restart MDA at the territory level. But if LF infection is uneven across subpopulations or across geographic areas, then some groups or areas may require additional MDA even though aggregate LF prevalence is below a level deemed necessary to sustain the infection in the population. The limited dispersal ability of the major LF vector Ae. polynesiensis and its susceptibility to the BG Sentinel trap suggested that MX using mosquitoes trapped from throughout American Samoa may be a useful adjunct to the school-based TAS for detecting areas of possible continuing LF transmission. We here describe the results of PCR testing for W. bancrofti DNA in mosquitoes captured from villages throughout American Samoa. Results of the TAS will be described elsewhere. The mosquito collections were conducted on the islands of Tutuila, Aunu' u, Ofu, Olosega, and Ta' u (Figure 1). These are the only islands in American Samoa that have been continuously inhabited in recent years. The five islands are located between 14° 9′ and 14° 22′S and 169° 25′ and 170° 51′W. The largest, Tutuila Island, comprises 68% of the territory' s 199 km2 total land area and contains approximately 97% of its total population of 55,519 [34]. Aunu' u Island had 436 residents by the 2010 census [34]. Many of Aunu' u' s residents commute by boat to nearby Tutuila for work or school. The more distant Ofu, Olosega, and Ta' u Islands, which together comprise the Manu' a group, had 176,177, and 790 inhabitants, respectively, according to the 2010 census [34]. Much of the territory' s land is forested, steep, and rugged, with about half the area having 70% or greater slope and over half covered by rainforest [35], [36]. Human settlement is mostly along the coastlines, with the exception of the Tafuna-Leone plains and the Aoloau-Aasu uplands areas in the southwest portion of Tutuila Island. Trapping was conducted within residential areas of all major villages of the four smaller islands and 34 randomly selected villages out of the 67 on Tutuila. These randomly selected villages contained approximately 57% of Tutuila' s population and 52% of its land area [34]. In some cases, 2–4 adjacent selected villages on Tutuila Island were combined and treated as single village areas for trapping and analysis. In one case, leaders in a selected village were not available to assist during the trapping time, so a nearby village was used instead. In the TAS, only two children were identified as CFA positive [33]. These children both attended a school located in a village on Tutuila that was not among those randomly selected for mosquito trapping. As a result, additional trapping was conducted in and around the school grounds using the same procedures as in the selected villages. Because the school was not located in one of the selected villages, data from these traps were not included in the larger data set but are reported separately. In each village (or group of contiguous smaller villages) ten BG-Sentinel traps baited with BG Lure (Biogents AG, Regensburg, Germany) were placed throughout the village and operated for approximately 24 or 48 h, depending on catch rate. Exceptions occurred in the combined area of Alega and Avaio villages where only six traps were placed, and Amaua village where four traps were placed. Traps were removed after 24 h if it appeared that the catch had reached a target of 200 Ae. polynesiensis females. The traps were placed on the ground in locations protected from direct sunlight and rain, often under eaves of houses or outbuildings such as unused open-sided traditional cookhouses. Placements were determined in consultation with village leaders and individual families while attempting to spread the traps evenly throughout the residential area of each village. Although village lands may be extensive, often spanning areas from the coast to the interior ridgetops, in most cases the residential areas are largely confined to lands near the coast or near major roads. Mosquitoes were removed from the traps twice per day at approximately 10: 00 am and 6: 30 pm following peak feeding times of the major vector Ae. polynesiensis [11], [6]. In one village (Vatia) the second trap check scheduled for 10: 00 am had to be postponed to 4: 30 pm due to a tsunami warning and village evacuation, so the Vatia traps ran for approximately 30. 5 h rather than 24 or 48 h. Mosquitoes collected during the first day of trapping in Taputimu and Vailoatai villages were lost, so only the second day' s catch was used from these two villages. In the laboratory, the mosquitoes were anaesthetized with carbon dioxide and identified on a tray resting on an ice pack under a stereomicroscope using the taxonomic keys of Ramalingam [14] and Huang [37]. The few mosquitoes that could not be identified due to damage or that were missing substantial parts of the head, thorax, or abdomen were not included in the analysis. Female mosquitoes were placed in pools of ≤20 (range 1–20) into microcentrifuge tubes separated by species, trap, location, and collection date and time. After freezing to ensure all mosquitoes were dead, the tubes were left open in an oven to dry at 75°C overnight, then closed and stored in a sealed plastic box with dessicant at 23°C until they were shipped for PCR analysis at Smith College, Massachusetts, USA. Trapping was conducted February 21–April 8,2011 on Tutuila and Aunu' u and June 7–16,2011 on the more remote Ofu, Olosega, and Ta' u islands. DNA extraction was done using a modification of the commercial DNeasy kit protocol (Qiagen, Hilden, Germany) and methods adapted from Fischer et al. [38] and Laney et al. [27]. Briefly, a 4. 5 mm zinc-plated bead and 180 µl phosphate-buffered saline (pH 7. 2) were placed in each round-bottom 2-ml Eppendorf tube (Eppendorf North America, Hauppauge, NY, USA) containing up to 20 dried mosquitoes. The tube was capped and vortexed at high speed in a horizontal position for 15 min and again for an additional 5–10 min if necessary for complete maceration. The tube was centrifuged briefly before adding 20 µl proteinase K and 200 µl of Buffer AL. The mixture was vortexed gently for 3 sec, then incubated at 70°C for 10 min. After brief centrifugation, another 20 µl proteinase K was added and mixed with brief gentle vortexing before incubating for 1 h at 56°C. The mixture was then centrifuged at high speed, and the supernatant from each tube was added to a 1. 5-ml Eppendorf tube containing 200 µl of 95–98% ethanol and mixed using the pipet. The entire mixture from each tube was then applied to a DNeasy kit column and centrifuged at 8,000 g for 1 min. The column was transferred to another 1. 5-ml tube, and the DNA was washed twice with 500 µl of Buffer AW1, with each wash followed by a 1 min centrifugation at 8,000 g. The column was then transferred to another 1. 5 ml tube, 500 µl Buffer AW2 was added, and the tube spun at 8,000 g for 3 min. The waste solution was discarded, and the column spun an additional 3 min at maximum speed to dry the column. The column was then transferred to a 1. 5-ml microfuge tube and the DNA was eluted twice with 125 µl of Buffer AE followed by 2 min centrifugation, first at 8,000 g, and then at 10,000 g. The samples were held at 4°C until the qPCR was completed, then stored at −20°C. Real-time PCR was done using a 7300 Real-Time PCR System (Applied Biosystems, Foster City, California, USA). Each reaction contained 1 µl of template DNA and 24 µl of qPCR master mix including 10 µM each of forward and reverse primers and taqman probe. The primers were designed to amplify a fragment of the “long dispersed repeat” of W. bancrofti (LDR; GenBank accession no. AY297458) [39]. The sequence of the primers and probe were as follows [39]: forward primer (Wb-LDR1) 5′-ATTTTGATCATCTGGGAACGTTAATA-3′, reverse primer (Wb-LDR2) 5′-CGACTGTCTAATCCATTCAGAGTGA-3′, and probe (Wb-LDR) 6FAM-ATCTGCCCATAGAAATAACTACGGTGGATCTCTG-TAMRA. The cycling conditions were 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Four different controls were used: a negative extract control consisting of a DNA extract from 20 uninfected mosquitoes; positive PCR controls using 1 ng, 100 pg, or 10 pg DNA of W. bancrofti; a negative PCR control using the same ddH2O as used in the master mix; and a PCR inhibitor control comprised of 5 pg of W. bancrofti DNA added to 10 µl of negative extract control. The negative extract and PCR inhibitor controls were run periodically throughout the course of sample processing. Positive and negative PCR controls were run with every sample batch. Samples were run in duplicate, and qPCR results with Ct≥39 were checked by running two additional qPCR reactions on the same extract template. If the sample was positive at least once more, and all controls were as expected, then the sample was considered positive. If both verification reactions were negative, then the sample was considered negative. Geographic coordinates were recorded for each trap location using a Trimble GeoXT 2005 Series Pocket PC handheld global positioning system (GPS) device (Trimble Navigation Ltd., Sunnyvale, California, USA). For 16 out of the 310 trap locations, the Trimble device was unable to record the positions due to topography, tree cover, weather conditions or satellite positions at the time, so a Garmin GPSmap 60CSx (Garmin International, Inc., Olathe, Kansas, USA) device was used instead for those locations. The positions were mapped using ArcGIS 10. 1 software (Environmental Services Research Incorporated, Redlands, California, USA), and village boundaries were obtained from the 2010 U. S. Census Bureau' s TIGER/Line “Places” shapefile for American Samoa [40]. In a few cases, traps were placed in locations which were inside the village boundaries as indicated by village leaders, but which fell outside the boundaries on the Census Bureau map. Point estimates and 95% confidence intervals for the percentage of mosquitoes containing W. bancrofti DNA were calculated for each mosquito species for the overall sample and for the most abundant species, Ae. polynesiensis, within each of the villages. The program PoolScreen (version 2. 0. 3) was used to calculate maximum likelihood point estimates of prevalence, and confidence intervals were determined by the likelihood ratio method [41]. A total of 22,014 female mosquitoes were collected and sorted into 2,629 pools of ≤20 individuals each for PCR testing. PCR results for the most abundant species in the traps are shown in Table 1, and relative abundances of the three most numerous species having >1 positive pool are shown in Figure 2. Members of the Aedes (Finlaya) group of species occurring in American Samoa include Ae. oceanicus, Ae. samoanus, and Ae. tutuilae. They were difficult to distinguish due to their morphological similarity and the loss of scales in the traps, so were combined for PCR testing and analysis. Only one out of the 267 pools of Ae. (Finlaya) spp. was positive by PCR. Other species captured in lower numbers were Ae. nocturnus, Cx. annulirostris, and Cx sitiens. Wuchereria bancrofti DNA was not detected in these species (n = 68 pools). Aedes polynesiensis, Cx. quinquefasciatus, Ae. aegypti, and Ae. (Finlaya) group species all produced positive pools (Table 1). Estimated prevalence was highest in Ae. aegypti, although the 95% confidence interval for prevalence in this species overlapped with that for Ae. polynesiensis (Table 1). There were no positive pools of any species collected from the five major villages of the Manu' a Islands of Ofu, Olosega, and Ta' u. For Ae. polynesiensis, the most abundant species captured there, the upper limit for the one-sided 95% confidence interval estimate of prevalence across all three Manu' a Islands was 0. 066% (n = 212 pools). On Tutuila and Aunu' u islands, 38 out of 260 total trap placements produced at least one positive pool. Positive mosquitoes were detected in the majority (16 out of 27) of the village areas sampled on these two islands. Areas producing positive mosquitoes on Tutuila Island were widely distributed throughout the island (Figure 3). Aedes polynesiensis was by far the most abundant mosquito species trapped overall, and prevalence estimates for Ae. polynesiensis from the villages are depicted in Figure 4. There was no evidence of a positive relationship between prevalence estimate and number of Ae. polynesiensis females or mean pool size (Figure 5), suggesting that the number of mosquitoes collected affected the breadth of confidence intervals as evident in Figure 4, but not prevalence point estimates. Nine traps which produced no positive pools of Ae. polynesiensis did produce positive pools of Cx. quinquefasciatus (5 traps), Ae. aegypti (6 traps), or Ae. (Finlaya) spp. (1 trap). At the village level, two villages with no positive Ae. polynesiensis catches had positive Cx. quinquefasciatus (Onenoa and Vailoatai) or Ae. aegypti (Vailoatai) pools. Of the ten traps placed in and around the grounds of the elementary school attended by two children who tested positive for CFA in the TAS, five of the traps produced positive mosquito pools. Two of these traps had positive Ae. polynesiensis, two had positive Ae. aegypti, and one trap had both positive Ae. polynesiensis and positive Ae. aegypti. Prevalence estimates were 2. 8% with a 95% confidence interval of (0. 55–8. 0%) (n = 107 females) for Ae. polynesiensis and 8. 6% with a 95% confidence interval of (2. 2–20. 8%) (n = 55 females) for Ae. aegypti. Pools of the 84 Cx. quinquefasciatus and four Ae. (Finlaya) spp. females collected around the school were all negative. Molecular xenomonitoring of mosquitoes trapped from villages throughout American Samoa found evidence of low but widespread occurrence of W. bancrofti infections on Tutuila and Aunu' u islands which together are home to 98% of the territory' s population. The study did not find evidence of infections on Ofu, Olosega, and Ta' u islands. The ability to detect very low W. bancrofti prevalences was limited, however, due to the low numbers of mosquitoes collected in many of the villages. This lack of sensitivity was reflected in the wide confidence intervals on prevalence estimates for many of the villages (Figure 4). Mosquito collection efforts and the number of pools that could be tested were limited by the resources available for the project. The type of mosquito collection method used may also have affected the sensitivity of xenomonitoring [42]. Female mosquitoes can contain W. bancrofti DNA only after they have completed at least one blood meal. The BG Sentinel traps used in this study are designed to capture host-seeking females, many of which may be nullipars seeking their first blood meal. Collections with gravid traps targeting ovipositing females [43], [44] can help ensure that a larger portion of the mosquitoes captured will have had at least one blood meal, but currently available gravid traps catch few Ae. polynesiensis (MAS unpublished data). For endophagic species, collection of resting mosquitoes in houses can also produce larger proportions of previously blood-fed females [45], [24]. Gravid traps and collection of resting mosquitoes in houses have been effective for Cx. quinquefasciatus xenomonitoring in areas where that species is the major LF vector. Culex quinquefasciatus does not appear to be an important LF vector in the Samoan islands [15], but it was the second most abundant species in our BG Sentinel traps and an estimated 0. 11% contained W. bancrofti DNA. In villages where this species is abundant (Figure 2), use of gravid traps targeting Cx. quinquefasciatus in place of, or in addition to, BG Sentinel traps targeting Ae. polynesiensis might improve xenomonitoring efficiency by increasing both the capture rate and the proportion of the catch consisting of previously blood-fed individuals. This approach remains to be tested in American Samoa. The large proportion of traps which produced positive mosquitoes in the area of the school at which two children tested positive for CFA indicated possible ongoing transmission there. Examination of blood smears and PCR testing following the ICT failed to find evidence of microfilaremia in either child [33], suggesting they may not have been the sources of the W. bancrofti detected in the trapped mosquitoes. The two children came from different villages, and each lived approximately 1 km from the school. Because Ae. polynesiensis feeding times overlap with times when students are at school and at home [11], [6], transmission by this vector could occur in either setting. According to the 2010 census [46], approximately 21,196 of American Samoa' s population attended school (pre-kindergarten – college) and 12,070 of the territory' s 16,482 working population traveled more than 15 min from home to work. The mobility of the human population and the daytime feeding habits of Ae. polynesiensis suggest that W. bancrofti transmission likely occurs not only in residential areas of villages, but also at other locations, such as workplaces, bus stops, and schools. With the exception of the single school, this study did not sample these other potentially important locations. There were several similarities between the results of this study and the only other study to use MX in American Samoa [25]. Only one of the three villages sampled by Chambers et al. [25] was sampled again in the current study. Prevalence of W. bancrofti DNA in Ae. polynesiensis for Afao Village was estimated to be 0. 82% in the 2006 study and 0. 47% in the current one. The wide confidence intervals obtained in the two studies (Figure 4 here and Figure 4 of Chambers et al. [25]) indicate a much larger sample size would be required to evaluate the significance of a difference of this magnitude. The estimates for prevalence of W. bancrofti DNA in Ae. aegypti were higher than those for Ae. polynesiensis both in this study and in the 2006 study, although the 95% confidence intervals for the two species overlapped broadly in both cases. The high propensity of Ae. aegypti for feeding on human hosts is well documented (e. g., [47], [48]) and could result in a higher frequency of feeding on microfilaraemic individuals than would be the case for mosquito species with less affinity for humans. Aedes polynesiensis is known to feed on birds and mammals other than humans, but little is known about the frequency with which it feeds on the different hosts [11], [49], [5]. No W. bancrofti DNA was detected in the 262 Ae. upolensis collected from throughout the territory in the current study. A similar number of Ae. upolensis collected from three villages in the earlier study by Chambers et al. [25] produced one positive pool. The low incidence of W. bancrofti DNA in this species and the low numbers collected in villages support the suggestion that it is likely a minor vector of LF in American Samoa [14]. Positive PCR results for species not considered to be important LF vectors revealed evidence of W. bancrofti in some locations where results from Ae. polynesiensis collections did not. Only two of the six traps with positive pools of Ae. aegypti and only one of the five traps with positive Cx. quinquefasciatus also produced positive Ae. polynesiensis. At the village level, two villages (Onenoa and Vailoatai) produced positive Ae. aegypti or Cx. quinquefasciatus pools from multiple traps, but no positive Ae. polynesiensis pools. The discrepancies are likely due to behavioral differences and variation in relative abundance of the three species across trapping sites. Together they suggest that sampling multiple species—including non-vectors—with different feeding behaviors may provide a more complete assessment of W. bancrofti infections than sampling only a single important vector species. The three species exhibit important differences in feeding behavior [50], [7], [5]. Aedes aegypti, like Ae. polynesiensis, feeds primarily during the day, but is more endophilic than Ae. polynesiensis. Culex quinquefasciatus feeds mainly at night and feeds and rests both inside and outside houses. Differences in range of movement could also result in different exposures to W. bancrofti. Aedes aegypti and Ae. polynesiensis are believed to have limited dispersal ability [12], [11], [51], but Cx. quinquefasciatus may move longer distances [52], [53], [54], [55]. Finally, if multiple species are included in xenomonitoring, the reduced sensitivity resulting from a low catch rate for Ae. polynesiensis in some villages, as occurred in Vailoatai, might be partially compensated for by higher catches of other species (Figure 2). Xenomonitoring using multiple species, including non-vectors, is a departure from the approach of monitoring only a single vector species and comparing estimated prevalence in that species to model-based or empirical thresholds to assess progress in LF elimination programs [24], [42]. The latter approach is complicated in the Samoan islands due to the presence of an important secondary vector, Ae. samoanus, the lack of an effective trap for that species, and the difficulty in distinguishing it morphologically from a closely related non-vector species. Another complication is the spatial heterogeneity of LF prevalence and transmission [56], [57] which suggests that even when aggregate prevalence in mosquitoes captured over a large area may fall below a target threshold, some local prevalences may exceed it. In addition, earlier xenomonitoring efforts have revealed that W. bancrofti prevalence in Ae. polynesiensis collected at a single location can vary substantially over the course of a year or even between collection periods separated by as few as ten days [58], [25]. Together, these factors, along with the difficulty of collecting large numbers of vectors and the resulting wide confidence interval estimates, suggest that xenomonitoring currently has limited usefulness for quantifying the progress of LF elimination in American Samoa. Instead its operational value may lie in helping to map areas where human infections exist without the invasiveness of human blood collection. Even such presence-absence mapping, however, requires trapping sufficient mosquitoes at each location to provide a high probability of detecting positive mosquitoes in the locations where they occur—something that may be difficult to achieve in areas where prevalence and catch rates are low. In summary, the detection of W. bancrofti DNA in mosquitoes at many locations on Tutuila and Aunu' u islands suggests widespread occurrence of human infections on these islands, while the low overall prevalence estimate suggests a similarly low overall prevalence of human infections. But caution is required in making inferences about prevalence at more local levels due to small sample sizes in many villages. Currently xenomonitoring has little value for programmatic decision-making in American Samoa beyond its ability to identify areas where human infections may exist. Increasing its relevance to MDA decision-making will require additional research to develop more efficient mosquito collection methods and to improve understanding of the relationship between prevalence of W. bancrofti DNA in mosquitoes, infection rates in humans, and resulting transmission rates relative to critical thresholds.

Lymphatic filariasis (LF), a mosquito-borne parasitic disease, has been targeted for elimination in many countries since the introduction of mass drug administration (MDA) programs using two-drug combinations along with improved diagnostic methods. Sensitive molecular methods detecting parasite DNA in pools of mosquitoes, along with efficient mosquito collection methods, can help identify sites of continuing LF transmission that may require further treatment after MDA has eliminated transmission in most areas. We tested mosquitoes from villages throughout American Samoa after the conclusion of a series of annual MDAs. Widespread but low prevalence of parasite DNA in mosquitoes from two of the five islands suggested continued occurrence of LF. In this study, parasite DNA detection in mosquitoes helped identify areas where human infections exist and additional treatment may be needed. In the future, development of more efficient mosquito collection methods for local species would facilitate larger sample sizes and more precise estimates of prevalence. In addition, developing a better understanding of the epidemiological significance of parasite DNA prevalence in the local mosquitoes will increase the operational value of those estimates for LF elimination programs.

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Apple has been valued at more than $1tn – almost three times as much as Google, the US’s second most valuable company. Analysts at Cantor Fitzgerald on Monday said they thought Apple’s shares – which are currently trading at about $127, valuing the company at $733bn – could soon be worth $180 each, which would value the iPhone maker at $1.05tn. It is the first time any company has ever been valued at more than $1tn, and would make Apple more valuable than the gross domestic product (GDP) of Indonesia, the Netherlands or Saudi Arabia, according to World Bank statistics. It would also mean Apple would be worth 2.6 times as much as Google, the second most valuable company in the US, with a market valuation of $383bn. Cantor Fitzgerald analyst Brian White said he thought Apple was worth that much because of its continued strong performance in China, where sales are increasing by 70% year-on-year, and the introduction of the Apple Watch, its first new type of product in five years. “Next month, Apple will enter its first new product category in five years, while media reports over the past several weeks have highlighted potential new areas of future innovation,” White said in his note to clients. “Also, we believe Apple’s iPhone portfolio and position in China have never been stronger. Finally, Apple has shown its commitment to returning cash to shareholders, and we expect more in April. We believe the combination of these forces will drive the market to reward Apple’s stock.” Cantor Fitzgerald was already one of the most bullish analysts on Apple’s stock value potential. White’s previous price target was $160 a share. Morgan Stanley analysts also reckon Apple shares could be worth $160 in a year, but Berenberg Bank, the most bearish, predicts Apple’s shares might crash to $85. The average analyst share price forecast is $135, according to Forbes. White said he thought Apple may sell 25m Apple Watches in the 2016 financial year, making revenue of about $11.7bn. White also predicted that Apple’s much-talked-about electronic car project could be a more than $500m opportunity. Tim Cook assumed he was ready for the harsh glare that shines on Apple’s (AAPL) CEO. He had, after all, filled in for Jobs three times during the Apple founder’s medical leaves of absence. Cook ultimately became the company’s chief executive six weeks before Jobs died, in October 2011. What Cook found out instead is that there is no preparation for the scrutiny that comes with succeeding a legend. “I have thick skin,” he says, “but it got thicker. What I learned after Steve passed away, what I had known only at a theoretical level, an academic level maybe, was that he was an incredible heat shield for us, his executive team. None of us probably appreciated that enough because it’s not something we were fixated on. We were fixated on our products and running the business. But he really took any kind of spears that were thrown. He took the praise as well. But to be honest, the intensity was more than I would ever have expected.” Cook’s reflection on his trial by fire comes at an other-wise triumphant moment. On this sunny Sunday in March, he is taking a breather from rehearsals for the event the next day in which he will reveal details of the Apple Watch, the first all-new device of his tenure as CEO. Sitting under a canopy at an outdoor café in San Francisco, steps from the auditorium where Apple will put on the product-reveal spectacle, Cook, 54, nibbles on snacks as he reflects on the 31/2 years that he has run Apple. He’s heard the repeated refrains that “Apple can’t innovate under Tim,” that the company needed a low-cost iPhone to thwart the progress of Google’s Android, that Cook never could replicate the Jobs magic—and therefore that Apple never again would be “insanely great.” Cook taught himself, he says, to block out the noise. “I thought I was reasonable at that before, but I’ve had to become great at it. You pick up certain skills when the truck is running across your back. Maybe this will be something great that I’ll use in other aspects of my life over time.” Already there is tangible evidence that the tread marks left no permanent scars. No one will be able to say for quite a while whether the Apple Watch or new services like Apple Pay or the $3 billion acquisition of headphone maker and music service Beats last year will prove financially successful. What can be said is that each in its own way constitutes proof that Apple is moving forward under its first nonfounder CEO since Gil Amelio got the ax in 1997. What’s more, those moves and others speak volumes about Cook’s leadership, at least measured against the widespread assumption that he would simply mind the company Jobs left in his custody. Courtesy of Apple In fact, there’s little debate that the state of Apple under Cook is fundamentally sound. Its stock has soared from a split-adjusted $54 to a recent $126 since Jobs died, translating into a market capitalization well north of $700 billion, the first company to cross that level. Indeed, its market value is more than double that of either Exxon Mobil or Microsoft. At the same time, Apple’s cash hoard has tripled since 2010, to more than $150 billion. (That’s despite the fact that Apple has spent a total of $92.6 billion in dividends and buybacks under Cook, all the more noteworthy because Jobs frowned on distributing cash to shareholders.) Apple has defended its high-end turf in smartphones, especially in China, where it sold $38 billion of merchandise overall in 2014. Cook has handled the occasional product snafu—Apple Maps comes to mind—with candor and humility. As well, he largely has held together the long-tenured management team he inherited from Jobs, augmenting it with a few key players and owning up to the occasional hiring blunder. Remarkably, Cook has come into his own as a high-profile leader of Apple, not merely tolerating the spotlight but leaning into it to focus attention on issues of importance to him and his company. His decision last October to announce publicly that he is gay instantly made the onetime low-profile and exceedingly private executive a global role model. It also made him the only openly gay CEO in the Fortune 500. And Cook has used the pulpit provided him by Apple’s worldwide platform to opine on subjects as diverse as human rights, access to education, female representation on Wall Street, immigration reform, and privacy rights. He even ventured into the heart of the Deep South, to the capital of his home state of Alabama, to lament the sorry state of racial equality there. Cook has differentiated himself from Jobs in myriad ways, and not merely with his willingness to speak out on societal issues. Cook, who joined Apple from Compaq Computer in 1998, came from an operations background and had spent the formative years of his career at IBM. At Apple that means he’s not what company executives like to call a “subject-matter expert” on such critical areas as product development, design, and marketing. Consequently he behaves much more like a coach who trusts his players than the manipulative mastermind Jobs was. David Paul Morris—Getty Images The result has been a level of stability in the senior management ranks few expected. “He never tried to be Steve,” says Eddy Cue, senior vice president for Internet software and services, who joined Apple in 1989. “He tried to always be himself. He has been very good at letting us do our thing. He’s aware and involved at the high end, and he gets involved as needed. Steve got involved at the pixel level.” There is no model, of course, for following someone like Jobs, a founder-entrepreneur widely regarded for a ruthless impulsiveness that repeatedly resulted in greatness. What’s more, three-plus years of steady success is no guarantee for the future. Says Michael Useem, the prominent Wharton management professor and director of the school’s Center for Leadership and Change Management: “In my own small world the question of whether Cook can sustain Apple’s momentum comes up more often than just about any other question on top management these days.” For his part, Cook says he has grasped that more important than answering his critics is learning to ignore them. “I’m not running for office,” he says. “I don’t need your vote. I have to feel myself doing what’s right. If I’m the arbiter of that instead of letting the guy on TV be that or someone who doesn’t know me at all, then I think that’s a much better way to live.” Cook’s defiant, confident tone reflects the CEO he has become. No one guards Apple’s distinct corporate culture—a culture designed by Jobs—more fiercely than Cook. Yet he also is gradually tweaking Apple at its edges, leading the company where he wants to take it, adding his unique perspective, and subtly but clearly redefining Apple in his image. It isn’t clear if Jobs would have approved or disapproved. But the enigmatic founder himself, in his dying days, told Cook that he shouldn’t obsess over trying to channel Jobs when making decisions. Given that, the question of what Jobs would have thought of where Cook is leading the company is, in the end, beside the point. Moments of Truth Richard Tedlow taught the history of business at Harvard for 31 years. He developed an expertise on the technology industry, penning books about the Watson family’s turbulent stewardship of IBM and a biography of the mercurial CEO of Intel, Andy Grove. Today Tedlow teaches at Apple University, the in-house education unit Jobs established before his death. Apple University isn’t a typical managementtraining arm, and Tedlow calls it the “Think Different corporate university,” a nod to the famous advertising campaign Jobs orchestrated in the late 1990s. Its goal is to document for its employees Apple’s peculiar ways as well as to ensure that the company’s people consider non-Apple perspectives that will help them think critically and remain open to new ideas. Angela Ahrendts, Jony Ive, Eddy Cue, Jeff Williams, Lisa Jackson Courtesy of Apple Tedlow calls the school a “therapeutic alliance between technology and the liberal arts.” Its courses on topics seemingly far removed from the business of computers and gadgets unsubtly reinforce Apple’s view of itself. For example, the Stanford political philosopher Joshua Cohen has lectured about pianist Glenn Gould’s meticulous effort to record and then re-record the famous Bach Goldberg Variations. Jobs’ famous obsession with the perfect screws on the inside of the original Mac can’t be far from an Apple student’s mind. The course Tedlow has been teaching lately is called Moments of Truth. It features a discussion of Abraham Lincoln’s famous “with malice toward none” second inaugural address, which he made into “a moment not of retribution but of reconciliation,” says Tedlow. The 67-year-old former academic, who has remained almost completely out of the public’s eye since joining Apple, also includes Margaret Thatcher’s decision to commit to battle in the Falkland Islands and Johnson & Johnson CEO James Burke’s handling of the Tylenol bottle-tampering crisis. Tedlow draws a straight line from the moments of truth of Lincoln and others to the situation Cook faced when Jobs died. Sure, leading a beloved gadget maker isn’t quite on par with reuniting a great nation ripped asunder by a bloody civil war. But the emotional parallels resonate. “I certainly think he had to come onboard and take the weight of everybody’s expectations,” says Tedlow. At a memorial service for employees in the courtyard of Apple’s Cupertino, Calif., campus, Cook told the company, “Our best days are ahead of us”—a difficult message to deliver at that moment and one Tedlow compares to Lincoln’s trying to reassure a war-weary and deeply divided nation. Convincing anyone that the post-Jobs era held great promise was a tough sell for Cook. The company had few headline-level innovations on the near-term horizon. At a product launch the day before Jobs’ death, for example, the voice-recognition application Siri was one of the company’s few hints at something new. Yet Siri represented Apple playing catch-up to a feature Google’s Android already offered. Worse, it wasn’t particularly good. Siri quickly became the butt of jokes at Apple’s expense for its frequent inability to understand its user. A year later Apple was in hot water for another weak product, its version of mobile mapping. Apple had booted Google Maps from the prime position on the iPhone in favor of its own version. But Apple Maps was riddled with errors, comically leading users to wrong destinations. The product was so lame that Cook publicly told customers he was “extremely sorry” for the debacle. He recommended that they use Google Maps, among other products, instead. A short time later Cook fired Scott Forstall, the head of mobile software and a longtime Jobs acolyte. In early 2013, Cook confronted another senior management challenge. His highest-profile hire from outside Apple had been John Browett, the former head of the U.K.-based discount electronics chain Dixon’s. The chief of a low-end retailer was a curious choice for the head of Apple’s high-touch retail operation. (Ron Johnson, the former Target executive who had run the Apple Stores division since its inception, had by this time left Apple and later signed on as CEO of J.C. Penney.) Browett didn’t fit in at Apple—he angered store employees by changing scheduling practices, for example—and Cook dumped him in March 2013. (Browett later acknowledged in a speech that it was a shock to be let go not because he was incompetent but because he didn’t gel with the Apple culture. Reached for comment, he declined to elaborate.) Looking back, Cook sees the episode as part of his education as CEO. “That was a reminder to me of the critical importance of cultural fit, and that it takes some time to learn that,” he says. As CEO, “you’re engaged in so many things that each particular thing gets a little less attention. You need to be able to operate on shorter cycles, less data points, less knowledge, less facts. When you’re an engineer, you want to analyze things a lot. But if you believe that the most important data points are people, then you have to make conclusions in relatively short order. Because you want to push the people who are doing great. And you want to either develop the people who are not or, in a worst case, they need to be somewhere else.” Another challenge for Cook was figuring out how to deliver Apple’s message when its new products simply weren’t ready to be discussed. At a technology industry conference in mid-2013, for instance, Cook was so elusive that investors openly questioned whether he had a vision for the company. Apple’s stock by that time had fallen back to the level at which it had traded when Cook took over. Photograph by Joe Pugliese All the while, behind closed doors, Cook was shoring up his management team as the company was working on the new products the world so badly wanted. In late 2013 he lured the CEO of Burberry, Angela Ahrendts, to head Apple’s retail stores. A year later Apple launched the large-screen iPhone 6 and its even bigger iPhone 6 Plus. It also unveiled a new payment system, Apple Pay, and its soon-to-be-shipped Apple Watch. The new iPhones, more than anything else, put Apple back on its upward trajectory. It sold a stunning 74.5 million of them in the last quarter of 2014, as the company generated $18 billion in profits, sending the stock price on an upward tear. The success has allowed Cook to take mistakes in stride. In late 2014 a glassmaker called GT Advanced Technologies, which Apple had contracted with to make a next-generation screen for its devices, declared bankruptcy when Apple declined to use its product. GTAT, as it is known, filed suit against Apple, claiming that its finances were ruined because of investments it made to fulfill the Apple contract. Apple, for its part, said it was blindsided by GTAT’s bankruptcy. The two sides eventually settled, and Apple committed to building a data center and solar farm on the company’s former manufacturing site in Arizona. Apple also was forced to take a major write-off for the debacle—it won’t say how large—a painful screwup for a company that regularly commits billions of dollars to its manufacturing processes. Jeff Williams, senior vice president of operations and Cook’s successor in that role, says Cook told him three things after the lawsuit. “When I informed Tim of the problem, his response was, ‘Let’s see what we can learn from it. We’re not going to bat a thousand. And we’re going to keep betting on great technologies for our customers.’ ” Cook’s measured emotional approach in leading Apple is markedly different from his predecessor’s, but the focus on core products and a long-term orientation are exactly the same. In that context, it isn’t critical that Apple Pay or the new wristwatch amount to giant profit drivers. “I have a simplified view of Apple,” says Jean-Louis Gassée, an Apple executive in the 1980s who now writes a widely read weekly column on Apple in the email newsletter The Monday Note. “They have and always have had one business, personal computers. Now they make them in three sizes, small, medium, and large—the iPhone, the iPad, and notebook and desktop computers. Everything else, including Apple Watch in the case of the iPhone, exists to push up the margins of those products.” To Gassée’s way of thinking, Apple’s strategy under Cook resembles the digital-hub strategy Jobs put into place 15 years ago, where products like iTunes drove sales of the iPod and ultimately the Mac. “Tim is playing the long game in his own way,” he says. Cook frames the debate in terms of how investors ought to view Apple. “The kind of investors we seek are long term because that’s how we make our decisions,” he says. “If you’re a short-term investor, obviously you’ve got the right to buy the stock and trade it the way you want. It’s your decision. But I want everybody to know that’s not how we run the company.” Putting His Life in View In his early performances at the helm of Apple product launches and other public events, Tim Cook came off as wooden. But then, no matter how well he had done, he would have compared unfavorably with Jobs, a virtuoso of the keynote presentation. As time has passed, however, Cook has clearly settled into the role. At the watch event, he emceed with a natural smile on his face. When he hugged the fashion model and maternal-health advocate Christy Turlington Burns—who has been using an Apple Watch on long-distance runs—there was nothing awkward about their interaction. Cook even seems to be enjoying himself. The day after the product launch, he presided over Apple’s annual meeting in Cupertino, a tedious chore most CEOs endure rather than relish. Cook, on the other hand, visibly cottoned to the give-and-take with shareholders, answering questions in a folksy manner—and amiably avoiding the ones he didn’t want to address. After twice sidestepping questions about whether Apple would buy the much-admired and rather Apple-like automaker Tesla Motors, he playfully praised himself for artfully not taking the bait. “There’s some advantage to being CEO,” he quipped. While being CEO allows Cook the luxury of swatting away unwanted questions, it also gives him a powerful platform to address a host of other issues—even if they aren’t directly related to Apple. In late October his home state inducted him into its Alabama Academy of Honor. It chose Cook to represent his class—which included University of Alabama head football coach Nick Saban and Sen. Jeff Sessions—as the sole speaker at the ceremony, a decision some came to regret. Cook moved quickly beyond platitudes and used the occasion to lambaste Alabama for its slowness to act on racial equality, on educational opportunity, and on equality for gay, lesbian, bisexual, and transgender people. “This isn’t right,” he said. “It isn’t reflective of our values.” A local television station captured an awkward moment afterward between Cook and the Republican governor of Alabama, Robert Bentley, who audibly took umbrage at Cook’s comments. Don Logan, an Alabamian who is a former CEO of Time Inc. (Fortune’s owner), was in the audience at the state capitol in Montgomery. “Tim is a very courageous guy,” says Logan, a fellow Auburn University alum, who notes that the state legislature had only recently passed a bill to not allow gay marriage. “He knew he was speaking into the wind and that most people in the room didn’t agree with him.” A few days later Cook announced publicly, in an essay in Bloomberg Businessweek, that he is gay. With no further comment from him or Apple, the disclosure set off a media frenzy, most of it favorable. Looking back, he says that he primarily acted out of concern for kids who were bullied at school, some to the point of suicide, and because of the many states that still allow employers to fire workers over their sexual orientation. Also, whereas U.S. courts were moving surprisingly quickly on the issue, “I didn’t feel like business was exactly leading the way in the executive suite.” “I’m not running for office. I don’t need your vote. I have to feel myself doing what’s right. If I’m the arbiter of that,” says Cook, rather than worrying about what critics say about his decisions, “then I think that’s a much better way to live.” Courtesy of Apple Cook says that he’d come to the decision of coming out “quite some time ago” and that his announcement was viewed internally at Apple, where his sexual orientation was more or less well known, as a “yawner.” Speaking out so publicly was a big step for Cook, though, who has described himself as intensely private and who is rare among big-company CEOs for being genuinely ill at ease talking about himself. “To be honest, if I would not have come to the conclusion that it would likely help other people, I would have never done it,” he says. “There’s no joy in me putting my life in view.” Referencing the often-cited line that “to whom much is given, much is required,” Cook says, “I’ve certainly been given a lot.” The move made Cook famous for more than being the person running Steve Jobs’ company. Mike Sullivan is a San Francisco lawyer with the global law firm Pillsbury Winthrop Shaw Pittman who advises startup technology companies. Like Cook, he views his sexual orientation as a point of pride and affiliation but something that doesn’t define him professionally. “We have 500 CEOs in the Fortune 500 out there, and I can guarantee you some of them are gay,” he says. “The message Tim sent is, ‘It’s okay to be yourself. You don’t have to lead with it. But you don’t have to hide it either.’ ” Cook has become so ubiquitous that it’s tough to remember when he wasn’t so visible. On an early March trip to Europe he huddled in Berlin with German Chancellor Angela Merkel and in Brussels with Andrus Ansip, the former Prime Minister of Estonia and now the European Commission’s top regulator on digital issues. He is featured in a new book by the former Fortune journalists Brent Schlender and Rick Tetzeli, who report that Cook offered Jobs a piece of his healthy liver for a transplant. (Jobs turned him down.) In March, Cook even phoned in to a surprised—and delighted—Jim Cramer during a live airing of the 10th anniversary of the broadcaster’s financial shoutfest on CNBC. Representing their companies publicly is obligatory for CEOs, but Cook takes public stands on issues including stopping the transmission of AIDS, human rights, and immigration reform. He sees them as opportunities for leadership. “You want to be the pebble in the pond that creates the ripple for change,” he says, adding that Apple’s people have long cared about such issues even if they haven’t previously spoken so openly about them. To Cook, changing the world always has been higher on Apple’s agenda than making money. He plans to give away all his wealth, after providing for the college education of his 10-year-old nephew. There should be plenty left over to fund philanthropic projects. Cook’s net worth, based on his holdings of Apple stock, is currently about $120 million. He also holds restricted stock worth $665 million if it were to be fully vested. Cook says that he has already begun donating money quietly, but that he plans to take time to develop a systematic approach to philanthropy rather than simply writing checks. An irony of Cook’s Apple is that the company is becoming visibly more open under its guarded CEO than it was under the publicity-savvy demigod who ran Apple before him. Whereas Jobs severely restricted interactions between all his employees and the press, Cook has ushered in a period of glasnost with the news media. It is highly unlikely that Jobs would have tolerated, for example, The New Yorker’s recent 16,000-word profile of Jony Ive, Apple’s chief designer. Cook says such exposure is part of his plan. “My objective is to raise the public profile of several of the folks on the executive team, and others as well. Because I think that’s good for Apple at the end of the day.” The new openness serves two purposes. First, it ensures that the world continues to talk about Apple. Granting a longer leash to executives with healthy egos also is a valuable retention tool. “A true coach is happy with his star players getting media time,” says Gassée, the ex-Apple executive. “Tim Cook is a true impresario who takes care of his prime donne. As long as the box office is good, the impresario will do that.” Building for the Future Tim cook is standing atop a giant mountain of dirt. He has come to tour the construction site in Cupertino that by the end of 2016, if all goes as planned, will be Apple’s new corporate campus. The dirt has been excavated from the massive pit below, and the pile is just about eye level with where the rooftop will be over the four-story, ringed building that will soon rise here. The building’s doughnut-shaped design has sparked many comparisons to a spaceship. Looking down as trucks and workers scurry to and fro, Cook begins to talk about one of the subjects that really gets him going: where people work. It always amazes him, he says, how drab workspaces are in metropolitan office skyscrapers. Apple’s new home will not be like that. “It shouldn’t be a place that doesn’t turn on your creative juices,” he says, musing about how future college recruits will feel when they first visit. Visible in the distance are Apple’s existing Cupertino campus, downtown San Jose, and Levi’s Stadium, where the San Francisco 49ers play and which, incidentally, would fit into the 30-acre park that will be at the center of the main spaceship building. The building site of what will be Apple’s new corporate campus, photographed March 3, 2015. Cook calls the high-tech complex “the mother of all products.” Courtesy of Apple Steve Jobs spent a considerable amount of the last two years of his life planning the campus, including hiring the British architect Norman Foster to design it. Everything about the site is large scale, and Cook, a numbers man, can recite most of the figures by heart. The main building itself will be 2.8 million square feet and will house 13,000 employees. About 2,000 more workers will fill up adjacent buildings on the site, which will include a 100,000-square-foot fitness and wellness center, a café that will serve 15,000 lunches a day, and more than 8,000 trees, all native to the Santa Clara Valley. Cook visits the work site periodically —including twice already with Apple’s board—and he exhibits an engineer’s glee at watching the 22 construction cranes that dot the landscape. He says Apple hasn’t decided yet exactly what it will call “Apple Campus 2,” the current internal designation. Some naming element of the buildings or the entire locale will almost certainly include an homage to Jobs, depending on his family’s wishes, says Cook. On a 90-minute tour of the site, Cook dishes out details of the campus, which he calls “the mother of all products.” For instance, Apple is investing in cutting-edge technology to manage tasks as mundane as parking. A system of sensors and apps will play traffic cop for employees as they enter the facility, eliminating the fuel-wasting hunt for a parking place. Just as it has done for its retail stores, Apple has built entire mockups of wings of the building to see how they look—and then torn them down. As to why Apple isn’t building higher than four stories, the same height as its existing campus, Cook says, “When we mocked up five we didn’t like the looks of it.” He is particularly excited about the mostly below-ground, 1,000-seat auditorium in the southeast corner of the campus, which will be the company’s new site for all its public presentations other than its annual developers conference. “No more scheduling months ahead of time around other people’s schedules,” says Cook enthusiastically. In talking about the new campus, Cook is particularly ornery about one label for it. “I hate the word ‘headquarters,’ ” he says. “There’s real work going on here. It isn’t overhead, and we’re not bureaucrats.” Indeed, among Apple’s employees there is considerable speculation as to which groups will be assigned to the new building—and which will be relegated to the existing real estate. “We’ve decided three times,” says Cook. “And we’ll probably decide it three times more.” On the drive back to Cook’s current office at 1 Infinite Loop, his Apple Watch emits a chiming sound that sounds like the ding! from a symphonic triangle. Cook is wearing the entry-level Sport version of the watch, with a white plastic wristband. It’s the first time in nearly two hours that he’s received a notification, and he says it’s a text message from his assistant that Al Gore, an Apple board member, would like to speak with him. The electronic interruption doesn’t require Cook to extract his iPhone from his pocket, one of the key attributes Apple believes will drive adoption of the watch. It does give him an opportunity to show off some of the watch’s features, including the iconic Mickey Mouse watch face, cleverly updated so that the Disney mascot cheerfully taps his feet at the rate of one per second. A self-described fitness nut, Cook proudly shows off his daily physical activity as measured by the watch. So far he has clocked 50 minutes of exercise and has traveled 8,139 steps, or about four miles. An early riser, he has been on his feet for 12 hours, and it’s not quite 3:30 p.m. His workday, and his job leading Apple, are far from over. Check out our World’s 50 Greatest Leaders list here. This story is from the April 1, 2015 issue of Fortune. It originally stated that the Siri product launch occurred days after Steve Jobs died. The event was the day before his death.

Before Tim Cook kicks the bucket-and after he sets aside enough money for his 10-year-old nephew's education-he plans to leave all of his wealth to charity. In an interview with Fortune, the CEO of Apple, which could soon become the world's first company worth $1 trillion, says he has already begun donating quietly. Why? "You want to be the pebble in the pond that creates the ripples for change," says Cook, who claims $120 million in Apple stock, plus $665 million in restricted stock. No word on which specific charities he would support, but Cook "plans to take time to develop a systematic approach to philanthropy rather than simply writing checks," Fortune notes. He has previously expressed his support for human rights and equality and described a need to address climate change and HIV/AIDS. With Cook at the helm, Apple itself has been no stranger to philanthropy. In an early 2012 meeting, he reportedly touted the company's $50 million donation to AIDS/tuberculosis/malaria charity Product Red, as well as a donation in the same amount to Stanford hospitals, the Verge reported at the time. In the interview with Fortune, Cook, a fairly private guy, also touches on his decision to come out as gay last year, noting most people at Apple were aware of his sexuality and found the announcement to be a "yawner." But seeing kids bullied at school and employees fired over their sexual orientation tipped the scales. "To be honest, if I would not have come to the conclusion that it would likely help other people, I would have never done it," he says. "There's no joy in me putting my life in view."

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[Outside Babylon. Brian pounces on Justin.] Brian: I'm gonna f*ck you. I'm gonna f*ck you all night long. [ They make out.] Justin: Aren't you getting enough? Brian: Never, enough. Justin: Why we can't save it until the weekend? Brian: Why should we save it? So, we're get the car. Justin: I was thinking that we could go away, just we two of us. Snowboarding. Brian: Cool! Except there is no snow. Justin: In Vermont. Daphne was just there with her boyfriend. She said it was amazing. They stay in the awesome place. There was a Jacuzzi and a fireplace in every room. Brian: Did they have little mints on the pillows? Justin: I forgot. Brian Kinney doesn't do romance. Brian: I don't need any excuse to f*ck. Justin: You also doesn't need an excuse to turn me down. Why just you admit you don't wanna go away with me for the weekend? Brian: I don't wanna go with you for the weekend. Justin: No need to so open. Brian: I wanna go away with you the whole f*ckin' week. Justin: Right! Brian: Hey, if you're not interested I can find somebody who will. [Brian smarms up to a stranger.] Brian: A week with me in Vermont with Jacuzzis and fireplaces? I guess it's just the two of us. Justin: Are you serious? Brian: We're on your Spring Break and I'm make some partner which makes me more than entire week of snowboarding. [Justin jumps into Brian's arms and they kiss.] [Mel and Lindz. Mel wears a glas and read something.] Mel: Did you put Gus down? Lindsay: Yep, he's snuckling up in his bed. Now it's my turn. [Lindsay pounces the bed and rolls on Melanie's work. Mel takes off her glasses and pets Lindsay's head.] Mel: Baby, you look exhausted. Lindsay: So do you. Mel: Roll over. Lindsay: What for? Mel: Go ahead. Go on! [Mel sits on Lindsay's back and grabs a bottle of lotion.] Mel: You are goin' to love this. Lindsay: [moan] Mmmh, this feels good. Mel: Let all the tension go. [Just as Mel lifts Lindsay's shirt mostly up her back, Leda walks right into their bedroom.] Leda: I just finished the floors in green. Mel: That's nice. Leda: Night. Lindsay: Night. [Leda is away] Maybe we should I've should gone with wood instead of the pie. Mel: Don't think about it. Just relax. [Melanie makes Lindsay roll over so she can start massaging Lindsay's breasts.] Mel: This is a special form of massage. It's called foreplay. Lindsay: Oh, I've missed this. Mel: Oh, you're so hot. [Lindsay yawns.] Lindsay: Sorry. [Mel curls her hands around Lindsay's breasts and rests her head in between them. They're asleep.] [Liberty Diner. Emmett has decided to balance his checkbook.] Emmett: You know, the thing about checkbooks it allows you to take a trip down Purchase Lane. Ted: Let me see. In the future? Balance before you buy. How much cost me this time? Emmett: Mmmh, fifty? Close back to eighty if I want my phone back. Michael: Try a hundred if you had to pay your electric. [Some guy walks into the diner and makes incredibly obvious eyes at Brian, who watches him back.] Ted: So, is he good as he looks? And spare us your mindless saying answers. Brian: First I made him worship my cock for an hour. [Michael looks up.] Then I let him rim me for a good forty-five minutes. After that, I f*cked him so hard he passed out. I'm surprised he's up and walking around. Ted: Ups, cancel the s*x talk. Lesbian approaching. [Mel walks up with a to-go cup of coffee.] Michael: Are you kidding? Mel and Lindsay are a couple of s*x machines. They soak the sheets. Mel: I don't care to discuss my self life with a bunch of foul-mouthed fags. Emmett: Something is on the wax. Brian: Or maybe not any. Mel: You see how much you get when you have a family to support, a kid to raise and a major renovation under the roof but of course you never will since your only resonsiblity is to your dicks. Brian: That's why I'm smiling and you're not. Ted: Sounds to me like a case of LBD. Michael: What the hell is that? Mel: Lesbian Bed Death? Emmett: First it dries up. Then it shrivels up. Then it closes up. And then it disappears. Michael: How come I never heard of that? Mel: Because it only exists in the minds of cunty gay men. Ted: Don't worry Mel, there's research and new developments every day. Brian: There's going to be a telethon. Emmett: Yeah, we all can wear a little ribbons on our lapels. [Mel goes away.] [Ryder Advertising.] Cynthia: He wants you see you, right away. Brian: What's the rush? Cynthia: He's on the phone all morning with his attorney and that can only mean one thing. Brian: He's divorcing his wife? Cynthia: He's making you a partner. Brian: What are you so exciting about it? Cynthia: I'm happy for you. And a partner assistent gets to be a important bitch to everybody. Now get in there. And good luck, partner. [She opens the door to Ryder.] Ryder: Brian. Brian: What's up? Ryder: Big change up. Brian: How? Ryder: I sold the agency. Brian: What? Ryder: To Gardner Aven. It's been on my telephon for years finding me an offer that I couldn't refuse. Now I can buy a place in Bermuda for retire and play golf until I drop. Brian: Congratulations. Ryder: Thank you. Brian: What about me? You made me a promise if we had another 5 million place this year... Ryder: Look, I had no choice. I had to sell. Brian: You wouldn't have a company to buying without me. I brought you over 4th of your accounts. Ryder: And I made sure that Vance knows about that. You're are gonna be his guy. He needs you. I give you my word. [Brian smirks, pouts, and stomps off.] [The bank. At the ATM.] Emmett: With the money Teddy loaned me, my new balance should be $147.16. Michael: So, were your calculations accurate? Emmett: Just a switch off. This time I have $10,000,146.16. Michael: Huh? Obviously that's a mistake. Emmett: Not necessarily. This is just a borrowing account. Michael: What are you doing? Emmett: Only I way to find out. [He do it again. With an new coupon. He see it and he must laugh.] [Mel and Lindz. At the living room.] Leda: Lesbian Bed Death. What kind of bullshit is that? Mel: It must be something to it. We're not like other couples who stopped having s*x. Leda: Every couple goes through hot and cold spells. [Leda takes her clothes off in the living room. She's only in her wife-beater.] Leda: How long you've been together? Lindsay: Seven years. Leda: I consider yourself as lucky. Listen, I'm not an authority on long-term relationships, in fact I tried to avoid them. But I do know a thing or two about s*x. And I can tell you this. Your battery isn't dead, it just needs recharging. [she crouches down and holds a beer bottle between Lindz legs] What you need is shake things up, do something different, bein' spontaneous. Mel: Spontaneous? Leda: Yeah, a couple of fuckable babes like you? You shouldn't have any problem. [Leda gives her beer bottle head and then walks upstairs. Mel and Lindz sit there for a while. Lindsay's blushing, and Mel's neck is getting an erection.] [Woddy's. Justin, Michael, Ted and Brian are they] Justin: I found this gay B&B. Ted: Sounds suspiciously lesbian. Justin: It's the best snowboarding in Vermont. [to Brian] This is the first time we've ever gone away together. Ted: Is that such good idea when your company are taking over? Brian: They need me more than you know. Ted: I guess you got bigger balls than I do. Brian: Was that ever a question? [Emmett flits into his seat.] Emmett: What'll be boys? Drinks are on me. Ted: Is that what you're gonna do with the hundred bucks I gave you? [Emmett returns the money to Ted.] Emmett; I won't be needing any financial assistence. Michael: Someone accidentally deposited ten million dollars in his account. Justin: f*ck. Ted: I hope you didn't touch any of it! Emmett: Only a measly three hundred. Ted: Do you know what you've done? It's committed bank fraud, That's a federal offense. Emmett: Oh! Oh, my God! Michael: I've warned you. Emmett: What I'm gonna do? Ted: Tomorrow you gonna go back to the bank, return back every cent. Emmett: Do you mind if I borrow back the hundred. There is that fabulous Gucci belt. [Brian's at work. Now he works for Vanguard Advertising. Cynthia stands in front of him. Speachless. Brian goes in for his meeting.] Gardner: Gardner Vince. Brian: Brian Kinney. Gardner: Sit. Ryder tells me that you've the best account exec he's got. Brian: He's right. Gardner: That's why I'm fired everyone else. Brian: I've always hated those long lines at the water cooler. Gardner: He also telly me that you are arrogant, willful, and insubordinate. Brian: I try my best to live up my reputation. Gardner: Why you stop and telling me why I shouldn't fire you, to. Brian: For one thing, I know more about this company than you do. For another I had the relationship with the clients. If I go, they go. And finally we both know you get more from me than from talentless boys for a half the price. Gardner: Before I quit this I learned everything about it. Everything. I was contacted everyone of your clients and they agreed to stay with or without you. The results of my hiring some tody. At least they give me the one thing you're never will. They willty. Are they other reasons why I shouldn't fire you to? Brian: I can't think of one. [He stands up and try to leave.] Gardner: You've got a week prove to me that you're worth with.. Brian: That long? Gardner: Oh by the way rumor has it that you're gay. Brian: The rumor's right. But unless I'm f*cking you, it's none of your business. [He leaves with that.] [The bank. Emmett and Ted are groveling to a bank teller.] Ted: There is this slide misunderstanding. Emmett: Very, very slide. Tiny. Itsy-bitsy winsy. Ted: Yeah, seems Mr.Honeycut unintentionally drew money that didn't belong to him. [The teller reads from her monitor.] Emmett: But here is the money. I'll have to cash like never happen. Have a nice day. Teller: Wait! Right there. Emmett: Oh my god, they're arrest me. Ted: Just calm down. You're gonna play dumb. Emmett: I can do that. Ted: You can say, you drew the money and never realising it wasn't yours which you come by and return it. Emmett: That's... that's perfect. They're sure believe me. Ted: But if they don't try a cell with Southern exposure. Bank manager: Which of you is Mr.Honeycut? Emmett: I...I...i... Ted: He is. Bank manager: Please come with me. [In the office.] Emmett: I did it. I stole the money. I'm guilty. I'm so sorry. [He sobs into his hand.] Ted: It was a mistake. Bank manager: Oh, that was no mistake. The money was correctly deposit in your account. All ten million dollars. Emmett: I... I need to consult with my accountant. [to Ted] What exactly did he mean by that? Ted: What exactly did you mean by that? Bank manager: The money was left to you by George Schickle. Emmett: George? [The manager pulls the Tape of Exposition out of his file cabinet and hands it over to Emmett.] Bank manager: He also wanted you to have this. [Emmett slowly, very slowly takes the tape.] [Vance add company. Brian and his assistant Cynthia in his office.] Cynthia: Find anything? Brian: Gardner Vance,41, gratuated Harvard business school. Start with his copyright for DBD in New York, then junior ad assec. Then Chicago. What about you? What you've find? Cynthia: Mostly the same as you plus feature article of one of the Chicago papers. Brian: Divorced twice. Boxing fanatic. Wow. Straight guy. Cynthia: Well, there is an intersting quote though, I circle it. Brian: Of one ask if there was an accounted he'd never able to win, Vince said, "Yeah, Brown Athletics." [he thinks over that.] [Brian stands up.] Cynthia: Where d'you goin'? Brian: See a few of my hottest tricks. Cynthia: You're going to the baths now. You'll f*ck us both out of a job. [A nasty, pay-by-the-hour motel.] Lindsay: This is the filthiest, the slieciest, the most disgusting motel I've ever seen! Mel: You expected a Hilton? Lindsay: Uh-huh. [she wants to go.] Mel: Hey, where'd you goin'? Lindsay: Home? Mel: We're just walked in! Lindsay: And now we're just walking out! Mel: But we already paid! Lindsay: By the hour. What kind of motel has hourly raise? Mel: That kind were you stick away for a quicky with some chickie and hope it doesn't find out. Take off your coat. We're stay a while. Lindsay: But they are stained on the bed spread and in the carpet. And it smells! Mel: Now it's sleazy, sponteanious. Lindsay: Can't we sponteanious in some place else like where they chance the sheets and they have room-service? Mel: This is the room service. A complete line of s*x toys including anyone flavors of lubrican in the front desk. [They kiss. Moaning. Groping. Pulling. Mel yanks Lindsay up as she opens Lindsay's shirt. Kissing. Moaning. Yanking. Mel takes off her shirt. Lindsay touches Mel's breasts. The girls stop making out when they hear someone else having s*x in another room. It's a woman's moaning.] Mel: I call the manager. Lindsay: No. We put the TV on instead to drown them out. [Every channel at the hotel is showing p0rn. Mel cups her own breasts into her hands.] Mel: Are those things real? Lindsay: I hope not. [They keep changing the channel until they find a cooking show.] Lindsay: Oh, the cooking show! He's preparing mandarine duck. I always wanted to know how to do it. [Emmett, Ted, and Michael are watching George's video.] George: "Hey, Emmett." Emmett: Hi, George. George: "I miss you." Emmett: I miss you, too. George: "Now, you must'n cry." Emmett: I'll try not to. George: "Let me see your smiling." Emmett: I didn't know if I can. George: "Emmett, you can do better." [He smiles a little.] George: "That's better. Well, did you received my little gift?" Emmett: A little? I couldn't believe it. Michael: Neither could we. George: "See, you always say, 'f*ck 'em all'. Well to do that you got have my f*cking all money." Emmett: What I'm gonna suppose to do with it? George: "Whatever you want. Whatever makes you happy." Emmett: You'll make me happy. George: "You too. More than you know." Ted: Ten mills are a clear indication. George: "Well, I... I guess that's it. You offered your adventure, I'm offer mine." [George blows Emmett a kiss.] [Brian's add fotoshooting. Many most naked men are there] Brian: [to the photographer] I need those in 3 hours. Photographer: I'm not a rusher. Brian: Please. Photographer: I've try. [He does. He takes picture of the guys in sport pose. Cynthia comes in.] Cynthia: Here are your tickets. I don't think you'll be needing it. I called Browns office. They said he doesn't accept unsolicited pitches. Do you want me to cancel? [Next scene. At Debbie at home. Emmett gives Lindsay a car key.] Lindsay: Oh Emmett! You can't do this! Mel: People don't get their friends a car! Michael: Ask this question in a game show! Emmett: It's not just a car - it's a Subaru SUV. I've read it that this is the number 1 choice for lesbians. Debbie: I'm sure Mr. Subaru would be thrilled! Emmett: Um, Justin this is for you. Justin: A trip to Italy? Emmett: Yeah, George and I were planing... never mind. You're goin' as art for centuries to the eternal city, to Florence, to Venice. It is there, that you've discover your soul. Ted: Then see what the statues of naked men too Emmett: When you and Brian are in Milan he can use this. Is a gift of the Armani fall collection. Justin: Oh, he love the new series. Emmett: The entire collection. Never wonder what you wear again. And Debbie this is for you. [Emmett hands Debbie a diamond tennis bracelet.] Debbie: Oh sh1t, you're gonna make me cry. No, Emmett, no. Emmett: Yeah! Now when you slinging that hash everyone will think you were a princess. Debbie: Thank you. Emmett: Michael. [he opens a envelope.] Michael: Jesus, Em. He paid the mortage on my comic book store for the next 5 years! You've changed my life! [Mel and Lindsay are kissing in the corner.] Ted: Oh, I guess there is nothing left in the bag for me. Emmett: What I really want for you I couldn't get. Someone wonderful to spend your life with. Ted : Well, there's always a golden retriever. [He looks at a paper, Emmett's givin' him.] Ted: Lifetime orchestra seats at the opera. Emmett: Two left seats. Cause I know you find someone. [Ted embrace his friend and starts to cry. Mel walks over and hugs Ted.] Mel: What about yourself, Emmett? Lindsay: I hope you got yourself something. Emmett: Oh no, no. It's much better to give than to receive. Except in bed. [Everyone laughs.] Emmett: There was a little something that catch my eyes. George wanted to be "f*ck 'em all" money. Well Georgie would love this! [Emmett jumps back into the room wearing a black velvet coat. Emmett dances in a circle.] [Lindsay walks downstairs in her darkened living room and bumps into Leda.] Lindsay: Oh, hey. I was just on my way to the kitchen to make myself a sandwhich. Leda: Come, try mine. Lindsay: I didn't expect you be here. I figured you out and painting the town red or whatever color they paint towns these days. Leda: All the hot gals are home with their wives and getting on. How about you guys? Lindsay: We'd tried going to a motel. Leda: [laughs] No sh1t! Did they have you hear screaming through the walls? Lindsay: That was someone else. We were the one who's watching the cooking show. Leda: When I've were there, I would have pinned you both to the bed and done a dozen dozen: twelve orgasms from twelve different positions. [Lindsay chokes on Leda's sandwich. Leda rubs Lindsay's shoulder.] Leda: You're okay? Lindsay: Just went down the wrong way. Leda: Christ, you're tight! We're need to loosing it up. Lindsay: No, no, no, no. It's okay. Leda: No, just let me wrap your shoulders. I get all the stress get out and then you and the missed can spontaneously can bust. You know what you need? Jujietsu. Or even better Tantra. Have you ever had a deep rectal massage? Lindsay: Not recently. Leda: You have no idea how much tension people carry in their sphincters. [Mel slowly walks down the stairs in the dark and watches Leda lying on top of Lindsay as they giggle and touch each other.] [Back in Brian's loft.] Justin: Emmett get everyone the coolest stuff. Cars, trips. He even get Debbie a diamond bracer. Brian: I really have hand it to him. He must givin' George a hell of a blowjob. Have you seen my Prota tie? Justin: f*ck the tie. You're got the entire Armani collection. Brian: Yeah, but what I'm supposed to wear until them? Justin: He got me a trip to Italy to see all the Masters. Brian: I've heard they have a big lether scene there. Justin: Why you are bringing a tie when we're snowboarding? Brian: I'm not going snowboarding. I'm going to Chicago. Justin: Chicago? What the hell is in Chicago? Brian: My new account. Justin: But we're going to Vermont. Brian: Some other time. Justin: C'mon, you're promised. Brian: It's business. Justin: f*ck business! Brian: That's exactly who you're f*cking. It's business. It's my business. It pays for this loft, it pays for snowboarding in Vermont, it pays for the Pittsburgh Institute of Fine Arts. Now, when I'm not get in this plane, I'm gonna be collecting unemployment and my f*cking Armani folk collection. [Justin is pissed off. Brian leaves.] [At the attic. Leda is finishing the floors. Mel has walked up.] Leda: Hey, sweets. What do you think? Mel: Looks great. Leda: Lindsay's all happy too. Mel: You're doin' an amazing job. Leda: Thanks. Mel: She entire eternally grateful. Leda: Anything for you guys, you know that. Mel: But, you know, it's not fair you asking to do all this by yourself. Leda: I'm loving at. Mel: No, really. It's too big a job. We can find a carpenter or handyman to coming in and finished up. Leda: Sugar, there is no handyman at the moment here than me. Mel: I know about your handy work. Like the job you're doin' on Lindsay last night. Leda: That was just a friendly little massage. Mel: Looks more than a little friendly for me. Leda: Jesus, is that the jealous wife routine? Mel: She fell asleep. You rubber and she purse. Leda: Oh so, now I'm to blamem for your lesbian bed death. Mel: All I know is everything was just fine before you moved in. Leda: Who the hell invited me? Mel: I did. And... now I'm asking you to leave. [Justin and Michael are having breakfast at Michael's place.] Michael: You want some? [he means cereal.] Justin: I'm not hungry. Michael: What are you trying to do, starve yourself to death? Justin: You sound like Deb. Michael: I wonder how that happened. C'mon, eat. Wanna hear my secret fantasy? Justin: I don't like to discuss kink on empty stomach. Michael: I see Rage on a box. Preferably on the Froot Loops. With action figur inside. Justin: Collect all three. Michael: It could happened. Anything is possible. Justin: Except Brian and I actually spending the time together. Michael: I'm sure he would rather gone with you. He had no choice. Justin: Do you always defend him? Michael: Look, if he doesn't make it now, he never will. Justin: He's already made it. He has money, success, a killer of loft what else would he want? Michael: To be the best. If he gets this account, he'll be a star. If he doesn't he'll be out of a job. It won't be that easy to starting over a new firm. You know, all those new guys, younger guys coming up all the time. Justin: So what about me? Where do I fit in? I don't want away. I want a boyfriend who only wants to be with me. Who wants to stay home every once in the while. At least gets jealous when some other guy sucking my dick right in front of him. Michael: That's not Brian. He never will be. [Brown Athletics. A young secretary in front of Brian.] Secretary: I'm sorry, Mr.Kinney. But he left work to your assistant that Mr.Brown wasn't interested in meeting with you. Brian: I'm all the way from Pittsburgh just to see him. Secretary: Unfortunately he's in closed door meeting and can't be disturb. Brian: Are we? Secretary: He's out of house. Brian: I'm sure if he saw my presentation... Secretary: Look, I'm trying to say that you're wasting your time. Brian: No, you're wasting his time because the sooner you get me in to see him the sooner I can show him these. [He's taking his pictures out from the shooting.] Secretary: Interesting. Brian: I can see you impressed. Maybe you like a copy for yourself? [SCENE_BREAK] [Cut to the copy room. Brian f*cks him at the copy machine.] Secretary: Mr.Brown eats lunch at the club. [Cut to the club. Brian goes right to him. He's siting at his table. He's reading the newspaper.] Mr.Brown: Who the hell are you? Brian: Brian Kinney. Vangard advertising. Your assistant said I've find you here. Mr.Brown: I thoughed I tell him to tell you that I wasn't interested in. Brian: Really? He was into it. Mr.Brown: [to the bartender] Herman, would you kind show this gentlemen out? Brian: Herman, would you kindly bring us some of dom. On Vengard of course. We're celebrating Brown new athletics ad campaign. Mr.Brown: If you're here to convince me to be more hip you're wasting your time. I'll be damn if I make a pair of sneakers that light up when you bounce. Brian: Leave tendious to the other guys. What you sell is good old american tradition. But you need something with more head. [Brian shows him a bunch of pictures] Mr.Brown: Young man, there is no heat in baseball caps and wind breakers. Brian: You're right, there is not. But it's my job to make them think that they're is. Mr.Brown: "The one thing to wear". Brian: We're running these in all the top magazines. We're appeal the women because they do the most spending AND to the new gay market. [He shows him a guy with a jock strap on. And he shows us his butt.] Brian: Herman, would you bring me a steak? Medium rare. [Liberty diner. Debbie pours water on Michaels glass.] Debbie: You've notices how I call the attention on it without following attention on it? It's called classy. Ted: You've got any ice? Debbie: Whaddaya call this? Ted: For my water! Debbie: Coming right up. Hey, Mr.Moneybags, what can I give for you? Emmett: Uh, a donut please. Debbie: You've got it. Michael: You're shopping? Ted: Of course he has! What else have a faggot of leisure to do all day except shop? Emmett: Too many people, too tiering, too cold. Ted: And complained? Emmett: Well, I've seen a few interesting sales but then I realised that I don't need balancing anymore and it all seem suddenly so empty. Debbie: One queer donut on the house. Emmett: Thanks, Deb. So, I'm begin to think. Ted: Oh, it's a dangerous sign. Emmett: I can more than waste some of money. I have a responsibility. I mean I broughed presents for everyone except for the one person who make that possible. George. Michael: Em, George is dead. Emmett: I know. I mean, I can't buy him a car or a watch but I can honor his memory. Michael: Well he's already have such of things to honor of him. Ted: Concert hall. Debbie: And the pickle. Emmett: I was thinking of more belong of lines "George Schickle Home for Gay and Lesbian Youth". Debbie: George sure knew what he's doin' when he's giving the money to you, honey. [Debbie kisses Emmett full on the mouth] Debbie: Hello. Get you anything? Man: Emmett Honeycutt? Emmett: I'm Emmett Honeycutt. But I'm not on the menu. Man: This is for you. [he gives him some papers and leaves] Michael: What is it? Emmett: Something about the Schickle heir. Ted: Let me see that. Yada yada. Debbie: Yada, yada what? Ted: The Schickle family is contesting Emmett's inheritance. Emmett: What? They can't do that. He left it to me. Ted: Well apperently they think he should left it to them. They frozen your account. Emmett: What is that all mean? Ted: That means maybe you should passed up those sales quite so quickly. [Back at Mel and Linds.] Lindsay: You told her to leaving without asking me?! Mel: You never wanted her in the first place! Lindsay: Well I like her when she's here now. Mel: I bet you do! Lindsay: She's doin' an amazing job! Mel: And not just in the attic! Lindsay: Excuse me? Mel: The reason that we're not feeling very sexual is because we're too tired, that we have to much in the lines when I realize that in your minds it was her! Lindsay: That is ridiculous! Mel: God! Don't lie to me! I live with you for seven years I know you. Lindsay: So maybe I do find her... attractiv. What about you? Mel: Me? That was years ago! Lindsay: Now, who's lieing? And then she's back in your life and you couldn't wait to jump on her back of her motocycle. Mel: All right. So she's hot. So what? [Leda knocks on their bedroom] Leda: You know, you two have a sound proof the boudoir can hear every word. Mel: Christ. Leda: I just wanted to say that I'm sorry for any grief. [Before Leda has walked away, Mel gets the timing to say something] Mel: Uh, Leda? I have the feeling that we've been friends for too long. Lindsay: We don't want you to leave like this. Leda: You're right. It's bullshit. [Girls start kissing, grabbing, groping. Leda pushes Mel and Lindsay's mouths together. The girls are kissing and making out. Leda's kissing Mel and has her thumb jammed into Lindsay's mouth. Everybody grabs a handful of buttons and gets to undressing.] [Gardner Vance ad agency.] Gardner: 200 dollars for lunch? What the hell we're doin' in Chicago? [Brian calls a number of his telephon.] Brian: Say hello to our newest client. Leo Brown. Mr.Brown: Hello? Gardner: Mr.Brown? Gardner Vince. I just wanted to welcome you upboard. Oh, yes. He... he suddenly is. Yes. Absolutely I will so. Thank you. [he hangs up] He sends his regards. I've been after him for years. How did you managed that? Brian: Did my homework. Gardner: Well, I suppose get calls for a prize. Brian: I think it calls more from that. Brown signs the two years commitment based on on conditioncy. Gardner: What's that? [Brian goes away and Gardner looks through the agreement.] >[Brian's loft. Brian's back and carries a bottle of champagne] Brian: Hey, Sunshine! Come gratulate me. Your partner just made partner. [The loft is empty.] [Babylon. At the entrance.] Emmett: Hey, can you loan me a ten? Ted: Oh, how the mighty have fallen. [The boys stand in a blue light, just as Brian appears.] Michael: Look who it is! Emmett: Hey, what are you're doin' here? Ted: The face looks familiar. Brian: Shut the f*ck up. Ted: Unfortunately the voice sounds the same. Michael: When you get back? Brian: A few hours ago. Where's Justin? Emmett: He's not here. Brian: Where is he? I have big news. Michael: He went to Vermont. Emmett: Snowboarding. Brian: Alone? Michael: Alone. Ted: So, what's the big news? Brian: Nothing. [Brian goes away. We watch lonely, dejected Brian walk away for a very long time down the very wide, crowded alley behind Babylon] Music: Queen #Good Old Fashioned Loverboy #Ooh love - Ooh Loverboy What're you doin' tonight, hey boy - Set my alarm, turn on my charm That's because I'm a good old-fashioned loverboy Ooh let me feel your heartbeat (grow faster, faster) Ooh Ooh let me feel your love heat Come on and sit on my hot-seat of love And tell me how do you feel right after-all I'd like for you and I to go romancing Say the word - your wish is my command Ooh love - Ooh loverboy What're you doin' tonight, hey boy Write my letter Feel much better I'll use my fancy patter on the telephone When I'm not with you I think of you always I miss you - (I miss those long hot summer nights) When I'm not with you Think of me always I love you - Love you Hey boy where do you get it from Hey boy where did you go? I learned my passion in the good old fashioned school of loverboys-#

Brian has to cancel plans with Justin to impress his new boss at work. Emmett receives a VERY generous gift from the late George. Melanie and Lindsay fight off the dreaded Lesbian Bed Death.

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Gram-positive bacteria, including Staphylococcus aureus are endemic in the U. S., which cause life-threatening necrotizing pneumonia. Neutrophils are known to be critical for clearance of S. aureus infection from the lungs and extrapulmonary organs. Therefore, we investigated whether the NLRP6 inflammasome regulates neutrophil-dependent host immunity during pulmonary S. aureus infection. Unlike their wild-type (WT) counterparts, NLRP6 knockout (KO) mice were protected against pulmonary S. aureus infection as evidenced by their higher survival rate and lower bacterial burden in the lungs and extrapulmonary organs. In addition, NLRP6 KO mice displayed increased neutrophil recruitment following infection, and when neutrophils were depleted the protective effect was lost. Furthermore, neutrophils from the KO mice demonstrated enhanced intracellular bacterial killing and increased NADPH oxidase-dependent ROS production. Intriguingly, we found higher NK cell-mediated IFN-γ production in KO mouse lungs, and treatment with IFN-γ was found to enhance the bactericidal ability of WT and KO neutrophils. The NLRP6 KO mice also displayed decreased pyroptosis and necroptosis in the lungs following infection. Blocking of pyroptosis and necroptosis in WT mice resulted in increased survival, reduced bacterial burden in the lungs, and attenuated cytokine production. Taken together, these novel findings show that NLRP6 serves as a negative regulator of neutrophil-mediated host defense during Gram-positive bacterial infection in the lungs through regulating both neutrophil influx and function. These results also suggest that blocking NLRP6 to augment neutrophil-associated bacterial clearance should be considered as a potential therapeutic intervention strategy for treatment of S. aureus pneumonia. Acute pneumonia is a leading cause of childhood mortality (<5 years of age) accounting for the death of 920,136 children annually [1], and methicillin-resistant Staphylococcus aureus (MRSA) has been implicated in severe life-threatening infections, including necrotizing pneumonia and sepsis [2]. In addition, S. aureus infection is also one of the major causes of secondary pneumonia following influenza infection [3]. Furthermore, S. aureus has developed resistance to multiple antibiotics and effective treatment strategies against this bacterium are limited [2,4]. Therefore, S. aureus is a serious threat to human health and novel therapeutic strategies are needed. The lung pathology induced by S. aureus is attributed to virulence factors, the intense inflammatory response, and evasion of host defense mechanisms, including neutrophil-mediated ROS-dependent bacterial killing [5,6]. Regarding innate immune responses, nucleotide-binding oligomerization domain-like receptor (NLR) pyrin domain-6 (NLRP6) is a recently identified NLR present in the cytosol of innate immune cells [7]. Co-transfection of plasmids containing NLRP6 and apoptosis-associated speck-like protein containing card (ASC) in 293T cells led to activation of NF-kB and in COS-7L cells to a synergistic increase in caspase-1 activation and IL-1β secretion [8]. Under homeostatic conditions Levy et al have demonstrated that NLRP6 co-localizes with ASC and caspase-1 to form a complex resulting in IL-18 secretion from intestinal epithelial cells, which is essential to prevent development of dysbiosis [9]. Together these results suggest that NLRP6 can co-localize with ASC for inflammasome formation and activation. However, the mechanisms of NLRP6 inflammasome activation and its role in host defense specifically during pulmonary microbial infection has not been explored. Moreover, it remains unknown whether the NLRP6 inflammasome is activated by microbial infections and whether NLRP6 co-localizes with ASC and caspase-1 during such infections to induce pyroptosis. In a mouse model of systemic infection, Anand et al [7] found that the NLRP6 negatively regulates host defense against Listeria and Salmonella infections as NLRP6 KO mice showed higher survival, decreased bacterial burden, and attenuated pathology compared to WT mice. In contrast, a study reported by Wlodarska et al [10] revealed a positive regulatory role of the NLRP6 inflammasome on immune function during enteric infection with Citrobacter rodentium. In this investigation, NLRP6 KO mice were shown to have an increased C. rodentium burden in the intestine, which correlated with extensive mucosal damage in the KO mice compared with WT controls. However, these results cannot be extrapolated to other bacterial infections in different organs, such as the lung, because Gram-positive bacteria have unique virulence factors and immune evasion strategies and the route of administration of bacteria also dictates host responses. Thus, the role of the NLRP6 inflammasome in pulmonary immunity against Gram-positive infections remains unclear. To this end, we have used NLRP6 KO mice to demonstrate how S. aureus exploits the NLRP6 inflammasome to dampen neutrophil function and enhance pyroptosis and necroptosis to increase mortality during acute bacterial pneumonia. Our results show NLRP6 as a potential therapeutic target for treatment of S. aureus-infected pneumonic patients. To determine whether NLRP6 is upregulated in the lungs of pneumonic patients, we stained human pneumonic and normal lung tissue sections with anti-NLRP6 antibody and found that NLPR6 was upregulated in key innate immune cells in the lungs, such as neutrophils (lipocalin-2+), macrophages (CD68+), and epithelial cells (CD326+) (Fig 1A). Next, we determined if NLRP6 expression is upregulated in S. aureus-induced pneumonia in mice. In line with the results seen in human pneumonic lung sections, NLRP6 was upregulated in neutrophils (Ly6G+), macrophages (F4/80+), and epithelial cells (CD 326+) of mouse lungs following S. aureus infection (Fig 1B). Consistent with the immunofluorescence results, the expression of NLRP6 was also increased in lung lysates of human pneumonic patients, S. aureus-infected human cell lines (THP-1 and HL-60), and mouse bone marrow-derived macrophages (BMDMs) (Fig 1C–1E). To investigate whether NLRP6 triggers inflammasome activation during S. aureus infection, we infected BMDMs from WT and NLRP6 KO (KO) mice with S. aureus (MOI: 20) and measured the extent of caspase-1 activation at 8 hours post-infection. Both cleaved caspase-1 (p-20) in macrophage lysates and IL-1β levels in culture supernatants following infection were attenuated in the NLRP6 KO samples compared to the WT control (Fig 1F and 1G) indicating the activation of the NLRP6 inflammasome by S. aureus. Similarly, IL-18 was also reduced in NLRP6 KO BMDMs after S. aureus infection (S1A Fig). Since IL-1β was sharply reduced in the KO BMDMs, we determined if NLRP3 inflammasome is still intact in these BMDMs. IL-1β production in NLRP6 KO BMDMs after treatment with specific NLRP3 agonists (ATP and Nigericin) was comparable with that of WT BMDMs suggesting that NLRP3 is indeed intact in NLRP6 KO macrophages (S1B Fig). S. aureus has several virulence components, such as α-hemolysin (hla), clumping factor B, β toxin, phenol-soluble modulins, and panton-valentine leukocidins [5] that could potentially activate the NLRP6 inflammasome. Because hla has been reported to activate the NLRP3 inflammasome in human and mouse monocytic cells under similar conditions [11,12], we investigated whether hla can also activate the NLRP6 inflammasome in BMDMs and found this to be the case (Fig 1E and 1F). Consistent with the in vitro results, NLRP6 KO mice showed lower levels of IL- 1β in bronchoalveolar lavage fluid (BALF) after infection with S. aureus, providing evidence of NLRP6 inflammasome activation in vivo (Fig 1H). However, there was no difference observed in the levels of IL-18 between WT and the KO mice (S1C Fig). ASC is known to be an integral part of NLRP3 inflammasome signaling although it is dispensable for NLRC4 inflammasome activation [13]. We infected BMDMs with S. aureus and performed immunofluorescence assay to observe whether NLRP6 co-localizes with ASC and caspase-1. We found that NLRP6 co-localized with ASC and caspase-1 during S. aureus infection (Fig 1I and 1J and S1D Fig). To assess the role of the NLRP6 inflammasome in pulmonary host defense against S. aureus, we infected WT, ASC KO, and NLRP6 KO mice intratracheally with a lethal dose of S. aureus (USA 300) (2X108 CFUs per mouse) and observed the survival patterns for 10 days. Although all WT mice died within 3 days, 70% of NLRP6 KO mice survived longer than 10-days post-infection (Fig 2A). Furthermore, ASC KO mice displayed a survival pattern similar to that of the NLRP6 KO mice (Fig 2A). To determine whether the difference in survival is due to differences in the bacterial burden in various organs, we measured the bacterial burden in the lung, BALF, and extra-pulmonary organs after infecting mice with a sub-lethal inoculum (5X107CFU) of S. aureus. As compared to NLRP6 KO mice, WT mice had higher bacterial burdens in the lungs, BALF, and liver at both 12 and 24-hours post-infection (Fig 2B–2D). Accordingly, the total protein in the BALF, which is a measure of pulmonary leakage, was higher in WT mice compared to their NLPR6 KO counterparts (Fig 2E). To determine whether the detrimental effects of NLRP6 are bacterial strain specific, we infected WT and KO mice with a methicillin-susceptible Staphylococcus aureus strain (MSSA, Newman strain) and measured the bacterial burden in lungs and BALF at 24-hours post-infection. Consistent with the results seen with the USA 300 strain, the bacterial burden of the methicillin-sensitive strain was also higher in the lungs and BALF of WT mice compared to that of KO mice (Fig 2F and 2G). NLRP6 inflammasome has been implicated in regulating intestinal microbiota [9,14]. To determine if observed difference in the phenotype between WT and NLRP6 KO mice is due to difference in gut microbiota, we co-housed WT and NLRP6 KO mice for 4 weeks and infected them with S. aureus. As observed with non-co-housed mice, co-housed NLRP6 KO mice had significantly less bacterial burden in the lungs and BALF as compared to co-housed WT mice (Fig 2H and 2I). These data suggest that NLRP6 regulates pulmonary S. aureus infection independent of microbiota composition. Since we found upregulation of NLRP6 in both hematopoietic (neutrophils and macrophages) and non-hematopoietic cells (epithelial cells) (Fig 1B), we sought to determine if NLRP6 from each of these compartments is detrimental to bacterial clearance of S. aureus-induced pneumonia. Using bone marrow chimeric mice, we found that WT mice that received KO bone marrow (KO→WT) had lower bacterial burdens in the lungs and BALF than WT mice that received WT bone marrow (WT→WT) (Fig 2J and 2K). However, KO mice that received WT bone marrow (WT→KO) showed no increase in bacterial burden in the lungs and BALF compared to KO mice that received KO bone marrow (KO→KO) (Fig 2J and 2K). Together, these results suggest that NLRP6 derived from both hematopoietic and non-hematopoietic compartments is detrimental for bacterial clearance during S. aureus pneumonia. Neutrophils have been shown to be essential for containing pulmonary Staphylococcal infections [15,16]. To determine if neutrophils confer augmented host protection in NLRP6 KO mice, we depleted neutrophils in the KO mice prior to infection with a lethal dose of S. aureus and observed survival. We found that depletion of neutrophils reversed the survival benefit observed in the NLRP6 KO mice suggesting that protection is neutrophil-dependent (Fig 3A). Since neutrophils are essential for survival, we investigated if disruption of NLRP6 affects recruitment of neutrophils into alveolar spaces during S. aureus pneumonia. Compared to WT mice, KO mice had more leukocytes, including neutrophils, and macrophages recruited into alveolar spaces (Fig 3B–3D). Furthermore, to measure neutrophil accumulation in the lung parenchyma, we performed a myeloperoxidase (MPO) assay and found that KO mice had more MPO activity than WT mice (Fig 3E). Since NLRP6 was found to be upregulated in neutrophils and macrophages in the lungs, we wanted to know whether deletion of NLRP6 affects the function of these cells. To this end, we compared the intracellular killing ability of bone marrow derived-neutrophils (BMDNs) and BMDMs isolated from both WT and NLRP6 KO mice following infection with S. aureus (MOI: 10). Our results indicate that neutrophils, but not macrophages, from NLRP6 KO mice had improved killing ability compared to WT cells as depicted by the reduction in intracellular CFUs in KO neutrophils (Fig 4A and S2A Fig). However, the rate of phagocytosis was similar in neutrophils from both groups (WT and KO) (S2B Fig). It is known that neutrophils use NADPH oxidase-dependent reactive oxygen species (ROS) to kill S. aureus intracellularly [17–19]. To determine if differences in killing ability of WT and NLRP6 KO neutrophils is due to an alteration in ROS production, we compared ROS production by neutrophils from WT and KO mice after infection with S. aureus and found that KO neutrophils produced more ROS than WT neutrophils (Fig 4B). To confirm these in vitro findings, we assessed the expression of NADPH oxidase components in lung homogenates from infected mice by western blotting. The expression of p47phox, p67phox, and gp91phox was increased in the lungs from NLRP6 KO mice compared to those from WT mice, supporting the finding of higher ROS production by KO neutrophils (Fig 4C). IFN-γ has been shown to activate phagocytic cells during intra-pulmonary S. aureus infection [20]. Therefore, we measured the levels of IFN-γ secreted in the BALF of WT and NLRP6 KO mice after infection with S. aureus. Interestingly, we found that IFN-γ production was higher in KO mice compared to that of WT mice (Fig 4D). The production of IFN-γ requires activation of MAPK pathways [21]. Accordingly, we found higher MAPK activity in NLRP6 KO lungs than in WT lungs (Fig 4E). IFN-γ has been shown to induce ROS production in human mast cells during Staphylococcal infection [22]. Based on this observation, we assessed if IFN-γ contributes to the enhanced bacterial killing by neutrophils through induction of ROS. To this end, BMDNs from WT and KO mice were pre-treated with IFN-γ and subsequently infected with S. aureus (MOI of 10) followed by assessment of ROS production. Treatment of neutrophils with IFN-γ increased NADPH oxidase activity and ROS production (Fig 4F and 4G). The activation of phagocytes by IFN-γ involves activation of signal transducer and activator of transcription (STAT) proteins [23]. Supporting this observation, higher expression of phospho-STAT3 was found in the lungs of KO mice compared to WT (Fig 4E). Collectively, these results suggest that genetic ablation of NLRP6 increases bacterial killing by neutrophils through increased IFN-γ and ROS production, which are associated with higher NADPH oxidase activity. Our in vivo experiments demonstrate that NLRP6 KO mice exhibit improved bacterial clearance (Fig 2B–2D), higher neutrophil accumulation, as well as enhanced IFN-γ production (Figs 3C and 4D). Moreover, our in vitro experiments using BMDNs demonstrate that IFN-γ enhance bacterial killing by neutrophils through increased ROS production (Fig 4G). Therefore, we examined if blocking IFN-γ hinders bacterial clearance in the KO mice following infection with S. aureus. In this context, we administered anti-mouse IFN-γ antibody (100 μg/mouse i. p.) to one group of the KO mice and a similar volume of isotype antibody to another group of mice 12 hours before infection with S. aureus. Blocking of IFN-γ in the KO mice increased the bacterial burden in the lungs and BALF suggesting that IFN-γ mediates bacterial clearance in the KO mice (Fig 5A and 5B). We next sought to identify the cellular sources of IFN-γ during S. aureus infection. To this end, we performed flow cytometric analysis of lung cells from WT and KO mice following infection with S. aureus and found that NLRP6 KO mice had more IFN-γ-positive NK and CD4+T cells (Fig 6A–6D). In this regard, Nguyen et al reported NK cells as the major source of IFN-γ during S. aureus infection [20]. We also found CD8+T cells and γδT cells produce IFN-γ, although there was no difference between WT and KO mice in the total number of IFN-γ-positive CD8 or γδT cells in the lungs (S3A–S3D Fig). To determine whether increased IFN-γ production in the KO mice is due to higher MAPK activity in NK and CD4 T cells, we isolated these cells from the lungs of WT and KO mice and treated them with MAPK inhibitor prior to infection with S. aureus. However, blocking MAPK did not reduce IFN-γ secretion by NK and CD4 T cells (S3E Fig and S3F Fig). Furthermore, we measured the number of NK and CD4 T cells in the lungs after infection through flow cytometry and found that the KO mice had more NK and CD4 T cells accumulated in the lungs compared to that of WT mice (Fig 6E). These results together suggest that increased IFN-γ observed in the KO mice is due to enhanced numbers of NK and CD4 T cells recruited during infection. Since we found decreased neutrophil recruitment and increased protein leakage in the lungs of WT mice compared to KO mice (Figs 3C and 2E), we hypothesized that cell death in the lungs may be responsible for these results during S. aureus infection. In this regard, LDH, IL-1α, and HMGB-1 are intracellular molecules released exclusively after cell death and are thus termed alarmins [24,25]. Therefore, we measured the levels of these alarmins in an in vivo setting and found their expression to be increased in WT mice compared to NLRP6 KO mice. This suggests that NLRP6 enhances inflammatory modes of cell death during S. aureus-induced pneumonia (Fig 7A–7C). We also measured the extent of cell death in BMDNs following infection with S. aureus. Neutrophils from WT mice exhibited increased cell death, as seen by increased cytotoxicity (LDH release), compared to that of KO neutrophils (Fig 7D). Next, to determine the nature of cell death, BMDNs from WT and KO mice were pre-treated with either Ac-YVAD-CMK (Caspase-1 inhibitor) or Necrostatin-1s (Nec-1s, necroptosis inhibitor) and infected with S. aureus. Pre-treatment of neutrophils with Ac-YVAD-CMK or Nec-1s reduced the cell death in WT neutrophils suggesting that the nature of cell death is both pyroptosis and necroptosis (Fig 7D). In this regard, it is reported that caspase-1 and gasdermin-D mediate pyroptosis during bacterial infection [26–28]. To validate that NLRP6 induces pyroptosis, we assessed the expression of caspase-1 and gasdermin-D in BMDMs from WT and NLRP6 KO mice after infection with S. aureus (MOI of 20) for 8 hours. Both cleaved caspase-1 (Fig 1F) and gasdermin-D (Fig 7E) expression were higher in WT mice compared to KO mice. Also, we performed immunofluorescence microscopy on BMDMs to quantify caspase-1 and gasdermin-D expression and found increased caspase-1 and gasdermin-D- positive cells in BMDMs from WT mice compared to NLRP6 KO mice (Fig 7F and 7G; S4A and S4B Fig). It has been reported that S. aureus induces pathology in the lungs by a distinct cell death mechanism known as necroptosis [29]. Receptor-interacting-serine-threonine kinase-1 (RIP1), RIP3, and mixed lineage kinase-domain like protein (MLKL) are the core protein kinases that initiate and execute necroptosis [29–31]. Recently, caspase-8 has been shown to negatively regulate necroptosis during Salmonella infection in an enteritis model [32]. Thus, to explore whether NLRP6 can enhance necroptosis in the lungs during S. aureus infection, we assessed the expression of phospho-MLKL RIP1, RIP-3, and caspase-8 in lung homogenates obtained from S. aureus-infected WT and NLRP6 KO mice through western blotting. Interestingly, both phospho-MLKL and RIP-3 were higher in the lungs of WT mice compared to NLRP6 KO mice (Fig 7H). To further confirm this finding at the cellular level, we infected both WT and NLRP6 KO BMDMs with S. aureus for 8 hours and quantified necroptosis using immunofluorescence microscopy. The immunofluorescence assay also revealed more phospho-MLKL and RIP-3-positive cells in WT macrophages compared to NLRP6 KO macrophages after S. aureus infection, confirming that NLRP6 increases necroptosis (Fig 7I and S4C Fig). Since S. aureus has been shown to induce necroptosis in human cells, such as THP-1 cell lines [29], we examined if activation of the necroptosis pathway occurs in lungs of human patients during pneumonia. Immunofluorescence microscopy conducted on lung sections obtained from pneumonia patients displayed more necroptosis, as evidenced by increased phospho-MLKL and RIP-3 expression, compared to that of healthy control lungs (Fig 7J). We found that NLRP6 enhances both pyroptosis and necroptosis pathways during pulmonary S. aureus infection (Fig 7A–7I). Because pyroptosis has been found to be beneficial for bacterial clearance during intracellular bacterial infections [28], we examined whether pyroptosis is advantageous during S. aureus infection. Blockade of pyroptosis in mice by intra-peritoneal administration of Ac-YVAD-CMK (caspase-1 inhibitor) 12 hours prior to infection with S. aureus resulted in reduced pulmonary leakage, LDH release, and bacterial burden in lungs and BALF (Fig 8A–8D). In addition, blockade of pyroptosis suppressed cytokine secretion and enhanced survival of WT mice (Fig 8E–8H), confirming that pyroptosis is detrimental during S. aureus infection. It should be noted that the bacterial burden in WT mice receiving Ac-YVAD-CMK was still high when compared with NLRP6 KO mice (Fig 8C), suggesting that an NLRP6-dependent, but caspase-1-independent, mechanism also exists to increase susceptibility to S. aureus infection. Next, to confirm that NLRP6-mediated necroptosis is detrimental for host defense, we blocked necroptosis using an MLKL inhibitor (GW806742X) 12 hours before infection with S. aureus. There was a decrease in total protein and LDH release in the BALF of WT mice treated with GW806742X, which was comparable to that seen in NRLP6 KO mice (Fig 9A and 9B). Moreover, blocking necroptosis reduced the bacterial burden in lungs and BALF and ameliorated the inflammatory cytokine levels in WT mice (Fig 9C–9G). Also, leukocyte recruitment (neutrophils and macrophages) and survival were also increased in WT mice treated with necroptosis inhibitor (Fig 9H–9K). Inhibition of necroptosis using a RIP-1 inhibitor (Nec-1s) also reduced total protein leakage and LDH release in the BALF (S5 Fig). Collectively, these results reveal that NLRP6-mediated pyroptosis and necroptosis are detrimental to bacterial clearance and host survival during pulmonary S. aureus infection. Lung diseases induced by Gram-positive pathogens are an important cause of morbidity and mortality in both immunocompetent and immunocompromised individuals [13,33]. Although antibiotics decrease the mortality rates of bacterial pneumonia, the efficacy is somewhat limited due to the substantial number of immunocompromised individuals, growing number of elderly patients, and the rise of multi-antibiotic resistant bacterial strains. Thus, alternative therapeutic approaches, including the manipulation of host signaling events, are needed. However, detailed understanding of the host innate immune response is critical for the design of potential therapeutic interventions. Because the lung is continuously exposed to pathogens and their virulence factors, this organ possesses a multifaceted host defense system. Moreover, a successful immune response in the lung is critical for efficient clearance of microbial pathogens and therefore, the innate immune system possess germline-encoded pattern-recognition receptors. The NOD-like receptors (NLRs), including inflammasomes, are specialized cytosolic pattern-recognition receptors/sensors necessary for clearance of invading cytosolic pathogens. Under normal homeostatic conditions, the NLRP6 inflammasome is highly expressed in intestinal epithelial cells [9,14,34,35] where it co-localizes with ASC and caspase-1 [9]. It is also expressed in immune cells including neutrophils, T-cells, macrophages, and dendritic cells [7]. Despite high expression of NLRP6 in the lower respiratory tract, the role of NLRP6 in lung inflammation has not previously been explored. In the current study, we demonstrate that NLRP6 is upregulated in neutrophils, macrophages, and epithelial cells in the lungs of human pneumonia patients. Furthermore, NLRP6 is upregulated in myeloid and non-myeloid cells in the lungs and co-localizes with ASC in a mouse model of pulmonary S. aureus infection. We also found that the important virulence factor, α-hemolysin, can activate the NLRP6 inflammasome. The immune response to S. aureus is manifested by vascular leakage, neutrophil recruitment into the alveolar space, and upregulation of cytokines and chemokines [5,6]. The current study demonstrates that the NLRP6 inflammasome increases susceptibility to S. aureus-induced lung infection. Delving into the mechanisms underlying this, we found that NLRP6 dampens NK cell-mediated IFN-γ secretion thereby hindering ROS-dependent bacterial clearance by neutrophils. Moreover, our study identifies NK cells and CD4-T cells as the primary source of IFN-γ during acute pulmonary S. aureus infection. In agreement with these findings, studies of Listeria and Salmonella infections [7] have also shown that NLRP6 signaling is detrimental to host defense. Nonetheless, in a non-infectious model, the NLRP6 inflammasome was found to be important for epithelial self-renewal, proliferation, and mucus secretion, which were essential for protection against chemical-induced colitis [9,34]. Studies from different groups have shown that NLRP6 inflammasome regulates gut microbiota composition [7,9, 14]. NLRP6 KO mice were found to have different microbiota configuration which make them more susceptible to chemical-induced colitis compared to the WT mice [14]. In contrast, recent studies have demonstrated that NLRP6 and ASC-related inflammasome do not regulate gut microbiota composition [36,37]. Although it remains debatable whether the NLRP6 inflammasome influence gut microbiome, the reported difference in microflora composition in the KO mice can be nullified by co-housing the mice together with WT for 4 weeks [7]. In addition to colitis, microbiota have been shown to influence various disease conditions such as rheumatoid arthritis [38], diabetes [39], inflammatory bowel disease [40], and colorectal cancer [41]. In our study, however, co-housing of WT with NLRP6 KO mice did not change the resistant phenotype of the KO mice against S. aureus. In this context, similar report has been demonstrated by Anand et al, showing that co-housing does not alter NLRP6 mediated immune mechanism during Salmonella and Listeria infection [7]. It is widely accepted that hematopoietic and non-hematopoietic (stromal) cells in the lung produce numerous proinflammatory mediators, including cytokines and chemokines. Although hematopoietic cells secrete chemokines or neutrophil chemo-attractants, including CXCL1/KC and CXCL2/MIP-2, the stromal cells (alveolar epithelial cells) secrete other neutrophil chemo-attractants, such as CXCL5/LIX and CXCL15/lungkine [42]. Our observations indicate that NLRP6 from both cell types contributes to the enhanced susceptibility to S. aureus-induced pneumonia. These conclusions are consistent with previous studies of the role of hematopoietic and non-hematopoietic cells in the context of bacterial infections in the lungs. In this context, NLRP6 in both hematopoietic and non-hematopoietic cells increases susceptibility to Listeria and Salmonella infections [7]. Similarly, CXCL1/KC secreted by both hematopoietic and stromal cells was found to be crucial for bacterial elimination and neutrophil accumulation in the lungs following Klebsiella pneumoniae infection [43]. Nevertheless, it is clear from this investigation that neutrophil accumulation and function are critical for host protection against S. aureus. Pyroptosis and necroptosis are two distinct inflammatory modes of cell death. While pyroptosis is mediated by caspase-1 [26–28] and executed by gasdermin-D [26,27], necroptosis is regulated by RIP1, RIP3, and MLKL (caspase-1 independent) [29,30]. Pyroptosis has been shown to play an essential role in limiting several intracellular bacterial infections such Salmonella typhimurium, Legionella pneumophila, and Burkholderia thailandensis [28]. However, extensive caspase-1 activation and subsequent pyroptosis have also been associated with the severity of several diseases such as myocardial infarction [44], inflammatory bowel disease [45], and endotoxic shock [46]. Pertaining to these observations, we report that during S. aureus infection, NLRP6-mediated pyroptosis is detrimental for host survival. Furthermore, blocking pyroptosis reduced the hyper-inflammatory milieu and subsequently increased survival suggesting that pyroptosis triggers exaggerated inflammation during S. aureus infection. This difference in the role of pyroptosis can be attributed to differences in pathogenic properties and life styles of bacterial pathogens. While S. aureus is predominantly an extracellular pathogen, studies have shown that it can also survive intracellularly [47] and can resist anaerobic conditions [48]. Necroptosis induced by S. aureus is responsible for pathology in the lung [29]; however, its relationship with inflammasomes was previously unknown. Although ASC and NLRP3 have been linked with pore-forming toxin-induced necroptosis [31], the precise role of inflammasomes in the induction of necroptosis is not clear. In this study, we used both in vivo and in vitro experiments to show that NLRP6 mediates necroptosis of immune cells during acute pulmonary S. aureus infection. Moreover, S. aureus exploits NLRP6 to drive necroptosis, which is accompanied by an intense inflammatory response and loss of macrophages and neutrophils. It is possible that reduced cell death in NLPR6 KO mice attributed to higher leukocyte accumulation in the lungs of these mice. Since TNF-α has been shown to induce necroptosis [49,50], the reduction of TNF-α found in the NLRP6 KO mice suggests that NLRP6 can trigger necroptosis via the TNF-α pathway. However, more comprehensive future studies will be needed to identify the detailed molecular mechanisms underlying NLRP6-mediated necroptosis. Future studies are also needed to determine whether other toxins or virulence factors produced by S. aureus can also activate the NLRP6 inflammasome. In conclusion, the present study reveals the detrimental role of NLRP6 during S. aureus pneumonia (S6 Fig). Furthermore, NLRP6 in both hematopoietic and resident lung compartments contributes to S. aureus-induced lung inflammation. Not only does NLRP6 subdue neutrophil function by dampening IFN-γ and ROS production, it also triggers pyroptosis and necroptosis in the lungs that may lead to hyper-inflammation, loss of neutrophils, and mortality. However, future studies are essential to determine whether NLRP6 interacts with other inflammasomes such as NLRP3 and/or NLRC4 to induce pyroptosis and necroptosis during S. aureus infection. Comprehensive studies using specific double- or triple-KO mice would be useful to delineate these interactions in a conclusive manner. Furthermore, extending upon our findings, we propose that functional single nucleotide polymorphisms in human NLRP6 may have effects on host defense mechanisms against gram-positive microbes. Mouse experiments were conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Louisiana State University (protocol number 16–072). All animal experiments were performed in a manner to ensure minimal pain and distress. Nlrp6-/-, Asc-/-, and Caspase-1/11-/- were obtained from the Millennium Pharmaceuticals (Cambridge, MA) whereas C57Bl/6 mice were obtained from Taconic (Rensselaer, NY) and Jackson (Bar Harbor, ME) Laboratories. THP-1 (human monocytic) cells and HL-60 (human neutrophil-like) cells were purchased from ATCC (Manassas, VA). Lysates of human healthy control tissue and pneumonic lung tissue were obtained from Novus Biologicals, CO. Immunofluorescence microscopy of lung sections was performed as described previously [51]. Human lung sections from lungs without evidence of infection or injury (control) or from patients who died due to ALI/ARDs (pneumonic) were used from BioChain Institute Inc. (Newark, CA). Mouse lung sections were from saline-challenged or S. aureus-infected mice. Lung sections were incubated with anti-NLRP6 (Abgent, CA) and antibodies for surface markers including anti-lipocalin Ab for neutrophils (R&D Systems, MN), or anti-CD68 Ab for macrophages (BioLegend, CA), and anti-CD326 Ab for alveolar epithelial cells (BioLegend, CA). For mouse lung sections, we used antibodies for surface markers including anti-Ly6G for PMNs (BioLegend, CA), anti-F4/80 for macrophages (BioLegend, CA), and anti-CD326 for alveolar epithelial cells along with anti-NLRP6 Ab (Sigma, MO). Appropriate Alexa-conjugated secondary antibodies (Invitrogen, CA) were used. For detection of necroptosis and pyroptosis through an immunofluorescence assay, antibodies against mouse NLRP6 and ASC (Sigma, MO), p-MLKL, (Abcam, MA), RIP3 (Cell signaling, MA), caspase-1 (Adipogen, CA), and gasdermin-D (Santa cruz, CA) were used. Excess antibodies were washed off, and the cells were labeled with secondary antibodies, such as mouse IgG/IgM (H+L) Alexa fluor 488,568 (Invitrogen, CA). Images were obtained using an Axiocam digital camera (Zeiss, NY) connected to a Zeiss Axioskop 2 Plus microscope. BMDMs or lungs were harvested at designated time points and homogenized in PBS containing 0. 1% Triton X-100 (phosphatase and protease inhibitor cocktail added). After centrifugation the supernatants were used for immunoblotting. Total protein content in the supernatant was measured using a BCA protein assay kit (Thermofisher, NY) to ensure that equal amounts of proteins were loaded onto 10% SDS-PAGE gels. Proteins were transferred to polyvinylidene fluoride membrane according to the protocol provided by Bio-Rad. Appropriate primary antibodies against mouse NLRP6 (Sigma, MO), phospho-MLKL (Abcam, MA), RIP3, RIP1, P47phox, P67phox, gp91phox, phospho-p38 MAPK, phospho-JNK, phospho-Stat3, caspase-8, GAPDH (Cell Signaling, MA), caspase-1 (Adipogen), and gasdermin-D (Santa Cruz, CA) were added to the membrane and incubated overnight at 40 C. Appropriate secondary antibodies were used, and the films were developed using ECL plus western blot detection system (ThermoFisher, NY). IL-1β, TNF-α, IFN-γ, IL-1α, and IL-6 were measured in BALF supernatants by ELISA according to the manufacturer’s protocol (eBioscience, CA). Eight to twelve-week-old WT mice (C57BL/6 background) were used. Equal age- and gender-matched NLRP6 KO, ASC KO, and Caspase-1/11 DKO mice on the C57BL/6 background were used throughout the experiments. Mice were kept on a 12: 12 hour light/dark cycle under specific pathogen-free condition with free access to food and water. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Louisiana State University. To induce pneumonia, mice were anesthetized using ketamine (100 mg/kg) and xylazine (5 mg/kg) prior to intratracheal inoculation of S. aureus (USA 300 strain). A small midline incision was made on the ventral aspect of the neck and excess fat was separated to expose the trachea. Fifty microliters of bacterial suspension containing 5X107 CFU of log phase S. aureus in isotonic saline was injected into the lungs by piercing trachea using a 28. 5-gauge needle. At 12- and 24-hours post-infection, mice were euthanized to collect BALF, lungs, and liver for quantification of bacterial burden and leukocyte recruitment. BALF and homogenized organs were serially diluted and plated onto Tryptic soy agar (TSA) plates for bacterial enumeration. For survival experiments, we used 2 X 108 CFUs/mouse of S. aureus and observed survival for 12 days post-infection. BALF was collected as described in our previous publication [51]. Briefly, after specific time points, mice were humanly euthanized, and the trachea was exposed. Using a 20-gauge catheter, 0. 8 ml of PBS (heparin and dextrose added) was instilled inside the lungs and collected in a clean tube. The process was repeated a total of four times so that a minimum of 2. 8–3 ml of BALF was collected from each mouse. Total leukocyte count was performed in a hemocytometer using 10 μl of BALF and the differential count was done under light microscopy using cytospin slides stained with DiffQuik reagent. The remaining cell-free BALF was preserved at -80o C for cytokine analysis. Co-housing experiments were performed as described by Anand et al [7]. In brief, age and sex matched WT and NLRP6 KO mice were co-housed together in 1: 1 ratio for 4 weeks. After co-housing, WT and the KO mice were infected with S. aureus (i. t.) and euthanized 24 hours post-infection to measure the bacterial burden. Bone marrow chimeras were generated as described previously [51,52]. In brief, the recipient mice were lethally irradiated with a 1000-rad inoculum from a cesium source. Bone marrow cells collected from healthy donor mice were injected into recipient mice via tail vein (8 million cells per mouse). The chimeric mice were kept under 0. 2% neomycin sulfate treatment for 15 days after transplantation. After 8 weeks of transplantation, the chimeric mice were infected with 5X 107 CFU of S. aureus. The mice were euthanized at 24-hour post-infection to estimate cellular recruitment and bacterial burden in the lungs. For neutrophil depletion, mice were treated with 500 μg of anti-Ly6G antibody (clone 1A8, BioLegend, CA) [53] intraperitoneally 24 and 2 hours prior to infection with lethal inoculum of S. aureus (2 X 108 CFUs/mouse). For IFN-γ inhibition, mice were treated with 100μg of IFN-γ antibody (BioXCell, NH) 12 hours prior to infection with S. aureus. For caspase-1 inhibition, mice were treated with 100 μg of caspase-1 inhibitor (Ac-YVAD-CMK, Cayman chemical, MI) 12 hours prior to infection with S. aureus. For MLKL inhibition, 100 μl of 100 μM MLKL inhibitor (GW806742X, Adipogen, CA) [31] was injected into each mouse i. p. 12 hours prior to infection with S. aureus. For RIP1 inhibition, mice were treated with 300 μg of necroptosis inhibitor (Nec-1/necrostatin-1s, Calbiochem, MA) [29] 18 hours before and at the time of bacterial infection, as published elsewhere [29]. Mice were euthanized to collect BALF and organs 24 hours post infection. For MPO activity assay, lungs obtained from WT and KO mice after infection were homogenized. The supernatants obtained after centrifugation were mixed with 50 mM potassium phosphate buffer (with 0. 5% hexadecyltrimethylammonium bromide) in 1: 5 ratio and then centrifuged again. The supernatants were transferred to a 96-well plate. Absorbance was measured using a spectrophotometer at 460 nm after adding hydrogen peroxide/O-dianisidine hydrochloride buffer. The intracellular killing assay was performed as described [54] with slight modification. Briefly, bone marrow neutrophils from NLRP6 KO and WT mice were isolated, infected with S. aureus (MOI: 10), and treated with gentamicin for 30 minutes at designated time points post-infection (30 min, 60 min, 90 min, and 120 min) to kill extracellular bacteria. Cells were washed several times with sterile PBS to remove excess gentamicin and were then lysed with 0. 1% triton X to release intracellular bacteria. The lysate was serially diluted with PBS, plated onto the TSA, and incubated at 370 C overnight for bacterial load estimation. Total neutrophils, isolated from bone marrow of WT and NLRP6 KO mice using a magnetic negative selection cell isolation kit (STEMCELL Technologies, Vancouver, Canada), were infected with S. aureus (MOI 10). Total intracellular ROS production was quantified using a Fluorometric kit (AA Bioquest, CA). The effect of IFN-γ on ROS production was determined by pretreating neutrophils with either 20 ng/ml of recombinant mouse IFN-γ (R&D Systems, MN) or an equal volume of PBS for 30 minutes before infection with S. aureus. The total ROS production was quantified after 30 minutes of infection using a spectrophotometer. BMDNs were isolated from WT and NLRP6 KO mice and pretreated with Nec-1s (300 μM) or Ac-YVAD-CMK (100 μg/ml) or DMSO for 30 minutes before infection with S. aureus (MOI: 20). The percentage of cytotoxicity in BMDNs and LDH release into the alveolar space after S. aureus infection were measured using the Cytotox-ONETM homogenous membrane integrity assay kit (Promega, WI). HMGB-1 was measured using a commercially available kit from Chondrex Inc, WA. Data are represented as mean ± SEM. The Mann-Whitney test was used to compare the bacterial burden between two groups. Student’s t-test was used whenever the data were parametric in nature. We used one-way ANOVA followed by Bonferroni’s multiple comparison test wherever more than two groups were involved. All statistical analyses were performed using GraphPad Prism 7 software. The survival curve was analyzed using Log-rank (Mantel Cox) test. All experiments were performed thrice. Significant differences are indicated by * (p<0. 05), ** (p<0. 01), *** (p<0. 001), and **** (p<0. 0001).

Gram-positive bacteria, including Staphylococcus aureus remain a major cause of acute pneumonia worldwide. Due to emergence of multidrug-resistant strains, alternative strategies for treatment of S. aureus pneumonia are needed. To this end, it may be possible to harness host defenses to eradicate the infection instead of directly targeting the bacteria. Neutrophils are a crucial innate immune cell type and serve as a first line of defense against bacterial lung infection. NLRP6 is a recently identified member of Nod-like receptor family. Nonetheless, the molecular and cellular immunological mechanisms by which the NLRP6 regulates neutrophil-mediated host immunity during acute S. aureus pneumonia remain elusive. We found that NLRP6 gene-deficient/knockout (KO) mice demonstrate increased survival and lower bacterial burden in the lungs along with enhanced neutrophil recruitment during acute S. aureus pneumonia. Moreover, neutrophils from NLRP6 KO mice showed increased bactericidal ability compared to those from controls. Similarly, NLRP6 KO mice demonstrated decreased cell death through pyroptosis and necroptosis following infection. Blocking of these cell death mechanisms in WT mice resulted in increased survival and decreased bacterial burden in the lungs following infection. Therefore, our study provides novel insights into the novel mechanisms mediated by NLRP6, which serves as a negative regulator of neutrophil-mediated host defense during Gram-positive pneumonia.

lay_plos

Telomerase is expressed in early human development and then becomes silenced in most normal tissues. Because ~90% of primary human tumors express telomerase and generally maintain very short telomeres, telomerase is carefully regulated, particularly in large, long-lived mammals. In the current report, we provide substantial evidence for a new regulatory control mechanism of the rate limiting catalytic protein component of telomerase (hTERT) that is determined by the length of telomeres. We document that normal, young human cells with long telomeres have a repressed hTERT epigenetic status (chromatin and DNA methylation), but the epigenetic status is altered when telomeres become short. The change in epigenetic status correlates with altered expression of TERT and genes near to TERT, indicating a change in chromatin. Furthermore, we identified a chromosome 5p telomere loop to a region near TERT in human cells with long telomeres that is disengaged with increased cell divisions as telomeres progressively shorten. Finally, we provide support for a role of the TRF2 protein, and possibly TERRA, in the telomere looping maintenance mechanism through interactions with interstitial TTAGGG repeats. This provides new insights into how the changes in genome structure during replicative aging result in an increased susceptibility to age-related diseases and cancer prior to the initiation of a DNA damage signal. All mammalian telomeres (the ends of linear chromosomes) are composed of large tracts of repeated 5ʹ-TTAGGG sequences. Telomeres are well-conserved DNA end structures from yeast to mammals, and it is believed that the primary role of telomeres, in combination with shelterin proteins, is to provide protection of the linear chromosome ends from being recognized as damaged or broken DNA [1] and to facilitate the completion of DNA replication each cell cycle. Telomeres prevent DNA end-joining, DNA recombination, and loss of essential genetic information during DNA replication. Telomeres are maintained by many essential genes, including the six-component shelterin (TRF1, TRF2, POT1, TIN2, RAP1, and TPP1) and the CST (CTC1-STN1-TEN1) complexes [1,2]. Impairment of these genes is closely associated with age-related clinical pathology and defects in germ cell and stem cell maintenance [3–5]. It is well established that hTERT, the catalytic core reverse transcriptase component, protein levels are rate-limiting for telomerase activity and telomere length homeostasis [6]. Human embryonic stem cells and transit amplifying adult progenitor stem-like cells express hTERT and have active/functional telomerase that can fully or partially maintain telomeres during the substantial number of cell divisions required in fetal development [7]. While telomerase is present from the blastocyst stage in early human development, at approximately 16–18 wk of gestation, telomerase activity is silenced in the vast majority of somatic cells [8]. The molecular mechanisms (i. e., transcriptional regulation, alternative splicing changes, epigenetic modifications, or other regulatory processes) that trigger the silencing of telomerase at specific times during human development remain uncertain. Irrespective, telomerase largely remains silent throughout adult life except for tumor development. In ~90% of human tumors, telomerase is upregulated or reactivated for the maintenance of telomeres during the numerous rounds of cell divisions required for the emergence of malignant and metastatic disease [9]. Thus, tight regulation of telomerase and progressive telomere shortening are thought to be an initial barrier to the early onset of cancer. High resolution mapping of the three-dimensional chromatin interactome addresses many unanswered questions about the cis-regulatory long-range looping interactions important in gene regulation. The human genome is composed of continuous chromosome loops and TADs (topologically associating domains), forming gene territories [10,11]. Distal enhancers and/or insulators are believed to be responsible for the regulation of genes along the genome via chromatin folding. Recently, we demonstrated that telomeres also loop to specific loci to regulate gene expression, which we have termed TPE-OLD (telomere position effect—over long distance) [12–14]. In the examples characterized so far, genes close to telomeres are silenced in young cells (with long telomeres) and become expressed when telomeres are short. Importantly, re-elongation of cells with short telomeres by exogenous expression of the hTERT gene (active telomerase) results in expression patterns that mirror the expression of these genes in cells with long telomeres [12–15]. As we have observed genes between the TPE-OLD regulated genes that are not regulated by TPE-OLD, this mechanism is clearly distinct from classic TPE, which regulates genes proportional to the proximity to the telomeric repeats [15]. In the present study, we show that the expression of the hTERT gene itself is also regulated by TPE-OLD. The ability to regulate genes by telomere length without induction of a DNA damage signal from a single or a few critically short telomeres has potential explanatory value for what regulates the maximum length of human telomeres during fetal development and ways to regulate major age-associated transitions as well as to activate or repress genes as part of normal aging without requiring a DNA damage signal. Long-ranged genomic interactions between telomeres and distal loci may play important roles in the regulation of gene expression, a phenomenon that we previously referred to as TPE-OLD [12,13]. Through previous microarray analyses [12], we identified the human CLPTM1L (cleft lip and palate-associated transmembrane protein 1-like) gene that is ~1. 3 mega bases apart from the chromosome 5p telomere as a putative TPE-OLD candidate gene. CLPTM1L is frequently upregulated in cancer cells [16] and shows preserved colocalization with the TERT locus for a shared synteny in many species (Fig 1A). We analyzed mRNA expression of the genes at this locus, including CLPTM1L and hTERT, in BJ human fibroblast clones with long and short telomeres, to determine if the expression of this locus is regulated by TPE-OLD. CLPTM1L was expressed in normal young passaged cells but showed increased gene expression with progressive telomere shortening (S1A Fig). Historically, it is generally believed that hTERT is not actively transcribed in normal telomerase silent cells; however, expression of hTERT splice variants does occur [17]. The reason for this misconception is that most investigators use primer pairs designed to measure transcripts containing only the RT domain of TERT (exons 5–10), while exons outside of the RT domain are not measured (i. e., exons 1–4 and 11–16). It is now known that hTERT transcripts can be detected in a variety of telomerase-negative cells and tissues, but the mRNA produced is not full-length mRNA capable of producing active telomerase [17]. To test if replicative age or telomere length influenced hTERT expression, we measured hTERT gene expression using a primer pair targeting the 5ʹUTR to exon 1 of hTERT. We observed that hTERT is expressed at higher levels in two human fibroblast strains with short telomeres compared to the same cells with long telomeres (Fig 1B, S1 Fig). As previously described, we did not detect any transcripts that contain the RT domain of hTERT (Fig 1B); thus, transcripts that could code for active telomerase were not observed. We also analyzed protein expression of CLPTM1L (S1B Fig) and observed that the expression of CLPTM1L protein significantly increased during progressive telomere shortening, but the expression was greatly decreased when we re-introduced hTERT in old BJ cells and re-elongated telomeres (S1B Fig). We also examined mRNA expression of genes located between the 5p telomere and the hTERT-CLPTM1L locus (S1A Fig) in young and old BJ cells. The expression of the intermediate genes on chromosome 5p showed no significant increase in BJ cells with short telomeres (S1A Fig). We explored if telomere repeat containing RNA, TERRA, was also altered and potentially important in TPE-OLD. Consistent with previous reports [18,19] we observed an increase in TERRA expression from three subsets of chromosomes (1q-21q, 5p, and 9p-15q-Xq-Yq; Fig 1C) when telomeres were short compared to long. The TERRA data support our observations that the chromatin environment surrounding chromosome 5p and hTERT change when telomeres are short. Overall, this implies that the hTERT locus may be influenced by the length of telomeres through long-ranged chromatin interactions. Perhaps not surprising, but potentially significant, is that the location of the TERT gene is also evolutionarily conserved (Fig 1D). TERT genes are located at the very end of their chromosomes, near the telomere, in higher primates including humans and most other large long-lived mammals. However, the location of the TERT gene in rodents and many other smaller shorter-lived mammals is non-telomeric. The local genome structure around the TERT locus in rodents is quite different from primates, implying they may have developed different strategies to regulate telomerase expression [20,21]. Based on these observations, we decided to test if there is a functional role for TERT being localized at the end of human chromosome 5p. As the distance between the hTERT locus and the telomere is only ~1. 3 mega bases, we postulated that hTERT might also be regulated in part by a long-ranged telomere looping mechanism in human cells. We designed two specific BAC probes to visualize the hTERT locus and the sub-telomeric 5p region for three-dimensional fluorescence in situ hybridization (3D-FISH) (Fig 2A). We measured the distance between the hTERT locus and the sub-telomeric 5p region, and the pairs of alleles were divided into adjacent to (A) or separated (S) by the three-dimensional location (S2A and S2B Fig). We first stained the sub-telomeric BAC region, the hTERT locus and telomeres in old BJ cells, with short telomeres (Fig 2B). The telomere staining was detected at the hTERT locus with sub-telomere 5p in the adjacent allele pair. However, we observed at least one hTERT allele that was spatially separated from the sub-telomere 5p probe in old BJ cells without telomere staining. We measured the distance between the hTERT locus and the closest telomere (Fig 2C). The results showed that the hTERT locus colocalized with the telomere when it is adjacent to the sub-telomeric 5p region (Fig 2B and 2C). This implies that the telomere is likely to be adjacent to the hTERT locus for potential long-ranged looping interactions. We next tested if telomere looping close to the hTERT locus changes when telomeres became short. We measured and compared the distance between the hTERT locus and sub-telomeric 5p in young BJ fibroblasts at 20 population doublings (PDs) with long telomeres versus old BJ fibroblast at PD90 with short telomeres (Fig 2D). More than 70% of allele pairs were adjacent in BJ cells at PD20, implying that the telomeric heterochromatin might affect the expression of the hTERT locus in young BJ fibroblasts. BJ cells are telomerase-negative, but non-catalytic alternatively spliced variants are expressed, as shown in Fig 1 and as previously described [17]. This might explain why a small proportion of alleles is separated from the telomere in telomerase-negative young BJ cells with long telomeres, based on the assumption that the looping interactions suppress transcription. In old BJ cells at PD90, we found that the percentage of adjacent allele pairs was significantly reduced. Almost 60% of alleles were separated in the old cells with short telomeres, indicating that there is at least one hTERT locus spatially separated from the telomere in each cell. Importantly, we confirmed these 5p/TERT looping interactions in a second fibroblast cell strain, IMR90 (S2C and S2D Fig). We measured the number of separated and adjacent alleles in IMR90 cells young (PD 22) and old (PD 52) and show a shift from the majority of alleles being adjacent (76%) in young cells compared to the majority of alleles being separated (88%) in old cells. The looping data and the expression of hTERT are consistent. We suggest that old cells (with short telomeres) lose one control mechanism in regulating the hTERT locus (i. e., telomere chromatin looping) that helps repress the expression of hTERT. However, while we observed increased transcription of exon 1 of hTERT, there must be additional mechanisms preventing the inclusion of exons critical to produce active telomerase. There is substantial evidence that alternative splicing of hTERT may also have a major role in suppressing the production of active telomerase in old cells [22–24]. Furthermore, we performed 3D-FISH analysis in transformed SW26 and SW39 cells. SW cells are SV40 antigen expressing clones of IMR90 cells that have spontaneously immortalized using either telomerase (SW39) or an alternative lengthening of telomeres (ALT; SW26) mechanism to maintain telomeres (S2E Fig). In both cell lines, the majority of the alleles were separated (SW39 = 72%; SW26 = 66%), indicating that short telomeres due to replicative aging are likely responsible for the change in chromatin conformation and that a secondary change occurs to cause the production of full-length TERT or engage ALT. It has been suggested that hTERT shows mono-allelic expression in cancer, which is sufficient to preserve constant telomere length [25,26]. Our results support this assumption, as we observed that, on average, only one hTERT allele was generally in the open configuration during in vitro aging well before the onset of cancer. As controls for global conformational changes at chromosome 5p, we performed two additional FISH experiments. In the first experiment, we stained intermediate genomic region between the hTERT locus and the 5p telomere (S3A–S3C Fig). In addition, we also stained cells for two loci located 25. 5 MB and 30. 6 MB away from hTERT (S3D–S3F Fig). There were no changes in distances between the control loci in young and old cells, demonstrating that the conformation change occurs at the specific genomic region encompassing hTERT during in vitro aging, and this change is not due to classic TPE. To determine if we could artificially shorten telomeres and recapitulate the aging phenotype, we utilized CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat-associated 9) to remove a large portion of the telomere and subtelomere region from chromosome 5p. This experiment allows testing the role of chromosome 5p’s telomere in regulating the looping observed in cells with short and long telomeres. As illustrated in Fig 2D, we also infected young BJ cells with a lentivirus expressing Cas9 protein and single guide-RNA targeting the sub-telomeric region of 5p to specifically disturb telomeres at chromosome 5p for a short period of time [27]. We also added an NHEJ inhibitor, SCR7, simultaneously during the infection to suppress repair of the double strand breaks induced by the Cas9 protein [28]. The targeted cells showed an unstable end structure of chromosome 5p (S5 Fig), and the specific disturbance of the 5p telomere significantly diminished telomere looping at the end of the chromosome 5p. We further examined if the proposed mechanism was present in BJ cell clones in which both young and old cells were passaged the same amount of time in culture. This approach was necessary to eliminate the possibility that young and old cells that were in culture for vastly differing times could introduce artifacts. To accomplish this, we expressed a floxable hTERT in BJ clones, followed by excision at different time points in order to make isogenic cells with different length of telomeres but passaged similar times in cell culture [12,29]. Telomere length of the early-excision clone was 9 kb, and this was extended up to 13 kb in the late-excision clone. The telomere length (terminal restriction fragment [TRF]) results are presented in Figure 1B. Population doublings were evenly matched between clones (to avoid confounding effects of passage of time in culture), and we also analyzed telomere looping. Similar to our observations in normally passaged BJ cells, the isogenic clones also showed decreased levels of telomere looping with telomere shortening (Fig 2E). Importantly, there were only background levels of DNA damage signaling during telomere shortening (Fig 2F) indicating that the change in genome structure occurred before initiation of DNA damage responses from critically short telomeres. To ensure that our staining protocol was robust, we induced DNA damage (double strand breaks) by treating long and short telomere BJ cells with zeocin and assaying for DNA damage (S2F Fig). These data can be interpreted to indicate that our staining protocol is robust and that we are analyzing cells before telomere-DNA damage induced foci are present or significant DNA damage occurs in the cells. We next performed droplet digital 3C (chromatin conformation capture) to detect the genomic interactions between the 5p telomere and the hTERT locus in young and old BJ cells (Fig 2G, left side). The results showed that the hTERT locus has specific genomic interactions with the 5p telomere, and the interaction was reduced during in vitro aging and telomere shortening. A proximity control primer which is 10kb away from the fixed primer at the hTERT locus was selected for normalization of 3C results (Fig 2G, right side). Taken together, telomere looping exists between the hTERT locus and the sub-telomeric 5p in normal human cells, and this looping is greatly reduced by gradual telomere shortening. It has been shown that cis-elements upstream of the hTERT locus may play important roles in the tight regulation of human telomerase [30]. Thus, we decided to test if telomere looping could affect the epigenetic status of the hTERT proximal promoter region. We first analyzed DNA methylation of the region from -720bp to +90bp of the hTERT promoter in isogenic BJ cells with different lengths of telomeres but similar times in cell culture (Fig 3A). The relationship between DNA methylation and transcription in the hTERT promoter remains controversial in normal and cancer cells [31,32], but the transcription start site of hTERT retains little or no methylation in telomerase-active cancer cells for active transcription [33]. We found that the level of DNA methylation is significantly higher in BJ cells with long telomeres at several regions associated with hTERT and the hTERT region in comparison to cells with shorter telomeres. The largest differences were observed at -580bp, -250bp, -30bp, and +20bp of the hTERT promoter, including the E-box motif (a putative Myc binding sequence). It has also been reported that the proximal region of the hTERT promoter, including exon 1 and 2, regulates the activity of the hTERT promoter and that the methylation of this region is responsible for binding of several proteins [34,35]. Therefore, our results can be interpreted to indicate that telomere length-associated changes in methylation levels of the hTERT proximal promoter might affect transcriptional regulation of this locus. We next analyzed active and inactive histone marks on the hTERT proximal promoter using chromatin immunoprecipitation combined with droplet digital polymerase chain reaction (ChIP-ddPCR; [12]) (Fig 3B). We measured two histone marks associated with active chromatin H3K4 trimethylation (H3K4me3) and H3K9 acetylation (H3K4ac) and two histone marks associated with repressed chromatin H3K27 trimethylation (H3K27me3) and H3K9 trimethylation (H3K9me3), which have key roles in regulating gene expression [36]. We observed an increase in both H3K4me3 and H3K9ac across the TERT promoter in aged cells with short telomeres (Fig 3B). We also observed an increase in the repressive histone mark H3K27me3, but did not observe any significant differences in young or old BJ cells for the repressive histone mark H3K9me3. Collectively, this shows that the chromatin status of the hTERT promoter in old BJ cells with short telomeres is different and may be more transcriptionally permissive compared to young BJ cells with long telomeres. These data correlate well with the increased hTERT transcription we observed in cells with short telomeres. Furthermore, we analyzed chromatin at the promoters of three genes surrounding TERT that could also be affected by the altered chromatin environment with aging. We analyzed the proximal promoter regions of CLPTM1L, SCL6A18, and SCL6A19 for the same histone marks described above in the same cells and preparations used for TERT ChIP. At the CLPTM1L promoter we observed significant increases in histone marks indicating active transcription (Fig 3B). These data correlate well with an increase in CLPTM1L transcripts and protein levels (S1 Fig). We also observed significant changes in the chromatin surrounding the solute/amino acid transporter genes (SCL6A18 and SCL6A19), even though these genes are not expressed above basal/background levels in old/short telomere BJ cells. Specifically, we observed that both the repressive histone marks were increased in old cells (short telomeres) compared to young cells (long telomere). However, there was an increase in the activation marks as well. This indicates an intricate balance between chromatin modifications, methylation status, telomere length, and the expression of tissue-specific transcription and splicing factors that dictates the activation or repression of genes with replicative aging (telomere shortening—TPE-OLD). While we demonstrated that telomere shortening induced conformation changes between the hTERT locus and the sub-telomeric 5p resulting in up regulation of exon 1, presumably containing spliced hTERT transcripts in normal BJ cells (see Fig 1B), it did not result in full-length telomerase activity competent transcripts. Thus, we suggest that telomere shortening may render the hTERT locus more permissive and under oncogenic stress may lead to the production of full-length hTERT mRNA transcripts that could in turn produce telomerase activity. To test this, we simulated a step in spontaneous cancer transformation by knocking down p21 (CDKN1A) and analyzing mRNA expression level of hTERT (Fig 3C). The knockdown of p21 was previously shown to de-repress hTERT expression [37]. Thus, we tested if the knockdown of p21 would increase the expression of hTERT mRNAs and result in the inclusion of exons 7/8 in the short-telomere old BJ cells but not in the young BJ cells with long telomeres. We measured the expression level of hTERT transcripts in young and old BJ cells with and without p21 stable knockdown; mRNA containing exons 7/8 (exons coding for critical residues in the reverse transcriptase domain of TERT) and exon 15/16 (most splice variants of hTERT contain exons 15 and 16), responsible for putative active hTERT and total hTERT variants respectively. Both the active and the total hTERT transcript variants significantly increased with the knockdown of p21 in old BJ but not in young BJ cells; however, we did not detect telomerase activity (S6 Fig). While we observed an increased portion of transcripts that contain exons 7/8 of the TERT RT domain, other critical regions such as exon 2 may be spliced out [38]. Further work into the regulation of hTERT splicing is necessary to more fully understand the complex regulatory network surrounding hTERT and why the majority of transcripts are inactive splice variants as opposed to full length. While this result does not prove a causal role during cancer development, this series of experiments does demonstrate that telomere shortening in cells that bypass replicative senescence leads to the hTERT locus entering into a more permissive state (e. g., increased hTERT mRNA expression) in the presence of oncogenic stresses, consistent with the disengagement of telomere looping. Characterization of cis- or trans-acting factors responsible for telomere looping will be important to understand this novel mechanism for telomerase regulation. A recent report showed that TRF2 (telomeric repeat-binding factor 2) protein is essential for the functional organization of chromosome ends, including human fibroblasts [39,40]. There is also mounting evidence for off-telomere functions of the shelterin components [41]. While a recent whole genome sequencing study found 2,920 interstitial TTAGGG repeats throughout the human genome [39], we also found frequent internal (interstitial) telomeric sequences (ITS) near the TERT locus in higher primates but not in rodent cells (Fig 4A). Thus, we first checked for a putative role of TRF2 in telomere looping in BJ cells as a candidate approach. We knocked down TRF2 by siRNA and performed 3C to directly assess the genomic interactions between the telomere and the hTERT locus (Fig 4B). The knockdown of TRF2 significantly reduced the genomic interactions between the telomere and the hTERT locus in young PD30 BJ cells, implying TRF2 may have a role in telomere looping interaction on hTERT locus. As shown in Fig 4A, a region 100 kb downstream of the hTERT (Chr5: 1,154,047–1,154,347) contains a series of internal telomeric sequences that may recruit TRF2 shelterin protein (hereafter termed hTERT-ITS). Thus, we reasoned that this region would be a putative binding site for TRF2 and may be responsible for the telomere looping interaction between the telomere and the hTERT locus in cells with long but not short telomeres. ChIP-qPCR analysis showed that the TRF2 protein associates with the hTERT-ITS region in young and old BJ cells as proposed (Fig 4C). We next performed 3C to further clarify that hTERT-ITS interact with the hTERT promoter by genome folding to affect transcriptional permissiveness as shown in Figs 1 and 3. Within 200kb, we found more than 20 HindIII restriction enzyme sites were in the hTERT/CLPTM1L locus (Fig 4D). Droplet digital PCR (ddPCR) -mediated amplification showed specific interactions between the 5ʹ end of hTERT and the hTERT-ITS (Fig 4E). Moreover, the interaction was weakened in old BJ cells, implying there might be a transition from a more repressive state to a more active state of this TAD location during in vitro aging, consistent with the increased hTERT mRNA, altered methylation, and chromatin. This result also shows that there is an additional genome folding between the hTERT locus and the hTERT-ITS at an intermediate region between the SLC6A18 and SLC6A19 loci. The hTERT promoter is not close to the hTERT-ITS on a linear genome map, but the unique genome folding at this region potentially positions the hTERT promoter close to the ITS, followed by putative TRF2-mediated telomere recruitment to the hTERT promoter only in cells with long telomeres. In Fig 4F, we demonstrate that TRF2 protein is also enriched in the hTERT promoter region using ChIP-qPCR approaches. While TRF2 protein was enriched at proximal regions on the hTERT promoter, the interaction was significantly decreased in old BJ cells at the genomic regions containing -350bp to -50bp of the hTERT promoter. This shows TRF2 protein can occupy the hTERT promoter region, but the interaction is weakened during in vitro aging and telomere shortening. Together, we interpret these experiments to indicate that TRF2, and perhaps upregulated TERRA, may have at least a partial mechanistic role in telomere looping at the hTERT locus through interaction with the conserved interstitial telomeric repeats. Because we have shown the interaction between the 5p telomere and the hTERT locus, we modeled one possibility for the detailed local genome structure of this locus (Fig 4G). In this model, the hTERT promoter is close to the hTERT-ITS by genome folding in young cells with long telomeres. In addition, this model shows that TRF2 protein is recruited to hTERT locus and hTERT-ITS, which makes this interaction potentially dependent on telomere length. In summary, the hTERT promoter has specific interactions with the hTERT-ITS through gene looping, which may also recruit telomere length-dependent looping (TPE-OLD) mechanisms through TRF2 protein. We next performed 3D-FISH to visualize the genomic structure changes between the hTERT locus and the sub-telomeric 5p (Fig 4H). Control PD17 BJ cells showed that 89% of the hTERT and sub-telomeric 5p allele pairs were adjacent, but knockdown of TRF2 reduced this down to 34%. We also knocked down CTCF (CCCTC-binding factor) and LDB1 (LIM domain-binding protein 1), which are proposed to be essential proteins in global gene looping maintenance [42,43]. CTCF and LDB1 knockdown also significantly reduced the adjacent allele pairs, implying that the general gene looping mechanisms may also be involved in telomere looping. Western blotting was also performed to show knockdown efficiency (Fig 4I). Taken together, TRF2, part of the shelterin complex, may be mechanistically involved in the establishment of telomere looping near the hTERT locus through ITS together with general chromosome looping mechanisms. In almost all primary human cancers, telomere length is very short compared to adjacent normal tissues [44]. It is likely that short telomeres, in combination with oncogenic alterations, result in the hTERT gene becoming more permissive for protein expression and enzyme activity. Thus, we next investigated how telomere length affects hTERT expression in telomerase-active cancer cells. We first infected hTERT and hTR (hTERC) into the SW39 cell line (SV40 immortalized human telomerase expressing fibroblasts) and analyzed mRNA expression of the endogenous hTERT by examining the 3ʹ untranslated region. We observed that the extended telomere length reduced endogenous expression of hTERT mRNA in qPCR analysis (Fig 5A) implying TPE-OLD remains engaged at least in this tumor cell line. We further established isogenic HeLa cell clones with different telomere lengths by excising a floxable hTERT cDNA at different time points. We examined expression of splice variants of hTERT mRNA containing total, full-length (indicative of telomerase activity), and minus beta alternative spliced forms through ddPCR analysis (Fig 5B). All three splice variants showed significantly decreased expression in the long-telomere HeLa clone. We also performed 3D-FISH to analyze changes in genomic structure between the hTERT locus and the sub-telomeric 5p after the extension of telomeres in HeLa cells (Fig 5C). The long-telomere HeLa clone showed a higher percentage of adjacent allele pairs compared with the short-telomere HeLa clone. This indicates that the expression of hTERT may also be influenced by the length of telomeres through TPE-OLD in telomerase-positive cancer cells. The local genome structure around the hTERT locus may be important for the tight regulation of human telomerase. For example, introduction of proximal cis-elements of the hTERT promoter sufficiently inhibits the activity of the TERT promoter [45]. In addition, chemicals perturbing chromatin structure, including trichostatin A and 5-aza-2ʹ-deoxycytidine, induce changes in hTERT expression [46]. Moreover, chromosomal translocation and gene duplication of the hTERT locus can occur as part of the immortalization process in primary cultured cells [47,48]. Here, we reasoned that the hTERT locus might recruit telomeric heterochromatin to regulate its own gene expression, especially in large, long-lived mammals where tumor suppression mechanisms are perhaps more important. We showed that telomere looping exists in long-telomere young fibroblasts and that telomere looping was reduced by in vitro aging. This is one possible explanation for why higher primates preserved the location of the TERT gene at the end of one of their chromosomes. We speculate that, in addition to other conserved tumor suppressor mechanisms, higher primates also developed a mechanism to suppress the undesired expression of TERT. For example, it is well established that during human fetal development, full-length telomerase transcription is repressed and correlates with increases in nonfunctional alternative splicing changes in hTERT [8]. Thus, during early human development, when telomerase is active, telomeres elongate. Our current results are consistent with the idea that longer telomeres can fold back on the TERT locus and repress or significantly reduce transcription. Our results also show that replicative senescence, while initially a tumor suppressor mechanism, may paradoxically impinge on the predisposition to cancer through telomerase transcriptional de-repression. While still preliminary, the hTERT locus is arranged in a local chromatin domain that is regulated by telomere length and the interstitial telomere sequences in the vicinity of the hTERT locus. We showed that expression of the CLPTM1L gene (adjacent to hTERT) is also regulated by the length of telomeres and predicts transcriptional permissiveness of this locus. However, because hTERT re-activation is an extremely rare event, there may be additional levels of regulation. We propose that, upon telomere shortening, the hTERT region becomes permissive (as indicated by increased transcription of exon 1 containing RNAs), but this first step is not sufficient to support full-length hTERT transcripts at an adequate level to produce telomerase enzyme activity. We further propose that there is another biological role for telomere looping at this locus during development to repress telomerase when telomere length homoeostasis is reached (i. e., suggesting that having too-long telomeres may be detrimental). Here, we demonstrated a novel epigenetic mechanism regulating hTERT expression during in vitro aging (Fig 5D). Cells with long telomeres at the end of chromosome 5p in young passaged cells form a chromatin loop in the region of the hTERT locus. Importantly, we demonstrated that the chromatin loop is disengaged in cells with short telomeres, leading to partial increased expression of hTERT mRNA during in vitro aging and in response to p21 knockdown; however, telomerase activity was not detected, and, alternatively, spliced variants were likely produced [17,22–24,38]. Finally, we demonstrated that, in old cells with short telomeres, re-introduction of hTERT and elongation of telomeres results in a re-engagement of TPE-OLD. We found that DNA methylation and histone modifications in the hTERT promoter region showed significant changes as cells developed shorter telomeres, and that TRF2 and, perhaps, TERRA, may have important roles in these age-dependent genomic changes. These observations offer a model and a partial explanation for how age-dependent changes in the genome structure affect the regulation of hTERT without initiating a DNA damage response from a critically shortened telomere. BJ, SW39, HeLa, HEK293FT, IMR90, and Phoenix A cells were maintained in a 4: 1 ratio of Dulbecco’s modified Eagle’s medium to Medium 199 containing 10% of fetal bovine serum (Hyclone, Logan, UT, USA) under 5% CO2 in a humidified incubator. Retrovirus containing human TERT cDNA was infected into BJ cells and HeLa cells, followed by adenoviral infection for transient expression of Cre recombinase at different time points to produce cells with different lengths of telomeres that had been passaged in vitro for similar times [12,29]. Retrovirus was prepared by transfecting viral vectors into Phoenix A cells for 48 h. Medium containing virus was filtered through a 0. 45 μm pore and provided to cells in the presence of 2 μg/ml polybrene. Lentivirus was prepared by transfecting viral vectors into HEK293FT cells with two packaging vectors (pMD2 and psPAX2) for 48 h. Medium containing virus was filtered through a 0. 45 μm pore and cells exposed to lentivirus in the presence of 2 μg/ml polybrene. Selection for hygromycin was performed using 100 μg/ml and for puromycin using 1 μg/ml. CRISPR-Cas9 introduction for 5p genomic editing was performed by infecting cells with lentivirus carrying sgRNA target sequence of 5ʹ-GCCTCACTCCTTACGGAGTG-3ʹ. 3D-FISH was performed as described previously [12]. 104 BJ cells were seeded into 4-chambered slides. Cells on slides were fixed with 4% paraformaldehyde, followed by permeabilization with 0. 1% Triton X-100 in PBS. Repeated liquid nitrogen freezing-thawing cycles were performed for further permeabilization with preservation of intact nuclear structure under 20% glycerol in PBS. After 5 d of incubation of with 50% formamide in 2X SSC, cells were stained with indicated probes at 37°C for overnight. Slides were washed with 0. 1% SDS in 0. 5X SSC at 70°C for 5 min, followed by 2 rounds of PBST (Phosphate-buffered saline with Triton X-100) washing for 10 min. Images were acquired using a LSM780 confocal microscope (Carl Zeiss, Jena, Germany), and analyzed by Imaris deconvolution software (Bitplane, Zurich, Switzerland). The proximity of allele pairs was determined visually and quantitated. At least 30 nuclei were counted for the statistical analyses. We used the following criteria for the analyses: adjacent ~0. 5 μm space or less between probes, separated ~1. 0 μm between probes or more. The length was determined by calculating the 3D distance between each center of deconvolved fluorescent spots. Probes were prepared using nick translation kits (Abbott Laboratories, Abbott Park, IL, USA) from each BAC following manufacturer’s instructions. BAC plasmids were purchased from CHORI (Children’s Hospital Oakland Research Institute, Oakland, CA, USA); RP11-990A6 for hTERT locus staining and RP11-44H14 for sub-telomeric region 5p staining. Quality of probes was assessed by metaphase spread analyses and PCR. DdPCR and ddTRAP were performed as previously described [12,49]. Messenger RNA was prepared from RNeasy plus mini kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. 100 ng of RNA was reverse-transcribed from cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) by following the manufacturer’s instructions. Ten percent of synthesized cDNA was used for the ddPCR reaction. For ddTRAP, harvested cells were lysed in NP40 lysis buffer (1mM Tris-Cl pH8. 0,1mM MgCl2,1mM EDTA, 1% NP40,0. 25mM sodium deoxycholate, 10% glycerol, 0. 15M NaCl, 0. 05% 2-ME) for ddTRAP. Lysate was used for TS extension, and the extended products were analyzed with ddTRAP. Endogenous levels of 3ʹUTR were assessed with EvaGreen dye (Bio-Rad, Hercules, CA, USA). Probes were purchased from Roche (Basel, Switzerland), and the primer sequences are described below: 100 ng of gDNA was modified using the EpiTect Bisulfite kit by following the manufacturer’s instructions (Qiagen, Valencia, CA, USA). Modified DNA was PCR-amplified and cloned into the T vector system (Promega, Madison, WI, USA). 7~10 bacterial clones were sequenced for methylation analysis. Primers for the hTERT promoter region amplification were designed as previously described [33]. Chromatin conformation capture (3C) was performed as previously described [12]. Five million cells were washed with PBS and fixed with 25 ml of medium containing 1% formaldehyde for 10 min at room temperatures. To quench the crosslinking reaction, 1. 5 ml of 2. 5 M glycine was added and incubated for 10 min at room temperature, followed by an additional 15 min of incubation at 4°C. Cells were washed with PBS and harvested into 1 ml of cold-PBS with protease inhibitor. Cells were next lysed by homogenization, and the nuclear pellet was collected by centrifugation. The nuclear pellet was washed and resuspended in 500 μl of ice-cold NEBuffer 2 (NEB, Ipswich, MA, USA). 15 μl of 10% SDS was added and incubated at 37°C for 1 h, followed by addition of 46. 35 μl of 20% Triton X-100 for 1 h on a shaking incubator. HindIII (400U) was added and incubated overnight. Enzyme reaction was stopped by adding 88 μl of 10% SDS at 65°C for 20 min. Samples were next transferred to DNA ligation mix containing 50 mM Tris-Cl, pH 7. 5,10 mM MgCl2,1 mM ATP, 10 mM DTT, and 50 μg/ml BSA. 372 μl of 20% Triton X-100 was added and incubated at 37°C for 1 h. 2,000 U of ligase (NEB, Ipswich, MA, USA) was added and incubated for 5 h at 16°C. 40 μl of 20 mg/ml Proteinase K was added to the ligation mix at 65°C overnight. DNA extraction was performed by phenol-chloroform extraction and ethanol precipitation. Quality of the libraries were determined by checking for a single DNA band under agarose gel electrophoresis. Taq-man probe and 5ʹ primers were selected to amplify constant regions at the 5p telomere regardless of genome conformation. 3ʹ primers were selected to amplify the genomic interaction between 5p telomere and subtelomeric genes up to 1. 3 mega base pairs from 5p containing hTERT. Primer binding regions are 100 base pairs apart from a HindIII recognizing motif. Primer and probe sequences are described below; Chromatin immunoprecipitation was performed as previously described [12]. Antibodies against total histone H3 and a 1: 1 mixture of rabbit and mouse IgG isotypes were used as pulldown positive and negative controls of ChIP analyses, respectively. Relative occupancy was determined by first normalizing the target results with amplification signals from total H3 and then dividing by 1% input chromatin extracts. Antibodies against H3K4me3, H3K27me3, H3K9me3, H3K9ac, and LDB1 were purchased from Abcam (Cambridge, MA, USA). Antibody against TRF2 was purchased from Novus biologicals (Littleton, CO, USA). Antibody against CTCF and histone H3 was purchased from Cell signaling (Cell signaling technology, Danvers, MA, USA). Primers for hTERT promoter amplification were described in a previous study [33]. Primers for detection of CLPTML1, SLC6A18, CLC6A19, and the hTERT-ITS are described below; The TIF assay is based on the co-localization detection of DNA damage by an antibody against gamma-H2AX and telomeres using FITC-conjugated telomere sequence (TTAGGG) 3-specific peptide nucleic acid (PNA) probe. Briefly, BJ cells with long and short telomeres (100,000 cells) were seeded to four-well chamber slides, and, after the cells attached to the surface (next day), slides were rinsed twice with 1xPBS and fixed in 4% formaldehyde (ThermoScientific, IL) in PBS for 10 min. Then, cells were washed twice with PBS and permeabilized in 0. 5% Triton X-100 in PBS for 10 min. Following permeabilization, cells were washed three times with PBS. Cells were blocked with 10% goat serum in 0. 1% PBST (TritonX-100) for 1 h. Gamma-H2AX (mouse) (Millipore, Billerica, MA) was diluted 1: 1,000 in blocking solution and incubated on cells for 2 h. Following three washes with PBST (1x PBS in 0. 1% Triton) and three washes with PBS, cells were incubated with Alexaflour 568 conjugated goat anti mouse (1: 500) (Invitrogen, Grand Island, NY) for 40 min, then washed five times with 0. 1% PBST. Cells were fixed in 4% formaldehyde in PBS for 20 min at room temperature. The slides were sequentially dehydrated with 70%, 90%, and 100% ethanol. Following dehydration, denaturation was conducted with hybridization buffer containing FITC-conjugated telomere sequence (TTAGGG) 3-specific peptide nucleic acid (PNA) probe (PNA Bio, Thousand Oaks, CA), 70% formamide, 30% 2xSSC, 10% (w/v) MgCl2. 6*H20 (Fisher Sci), 0. 25% (w/v) blocking reagent for nucleic acid hybridization and detection (Roche) for 7 min at 80°C on heat block, followed by overnight incubation at room temperature. Slides were washed sequentially with 70% formamide (Ambion, Life Technologies, Grand Island, NY) / 0. 6 x SSC (Invitrogen) (2 x 1 h), 2 x SSC (1 x 15 min), PBS (1 x 5 min), and sequentially dehydrated with 70%, 90%, and 100% ethanol, then mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA). Images were captured with Deltavision wide-field microscope using the 60X objective. TIFs were quantified using Image J and representative pictures were prepared in Imaris software after deconvolution using Autoquant X3. The average length of telomeres (terminal restriction fragment lengths) was measured as described in [50] with the following modifications. DNA was transferred to Hybond-N+ membranes (GE Healthcare, Piscataway, NJ) using vacuum transfer. The membrane was air-dried and DNA was fixed by UV-crosslinking. Membranes were then probed for telomeres using a DIG-labeled telomere probe [51], detected with an HRP-linked anti-DIG antibody (Roche), and exposed with CDP-star (Roche).

Telomerase is very tightly regulated in large, long-lived species such as humans. Telomerase is expressed during early human fetal development, turned off in most adult tissues, and then becomes reactivated in most human cancers. However, the exact mechanism (s) regulating these switches in expression are not fully known. We recently described a phenomenon in which genes near chromosome ends (telomeres) are regulated by telomere length-dependent loops (telomere position effect-over long distances; TPE-OLD). Interestingly, the TERT gene is only a megabase from the human chromosome 5p end. We observed that when telomeres are long, TERT gene expression is repressed and the 5p sub-telomeric region and the TERT locus are spatially co-localized. When telomeres are short, at least one of the TERT alleles is spatially separated from the telomere, developing more active histone marks and changes in DNA methylation in the TERT promoter region. These findings have implications for how cells turn off telomerase when telomeres are long during human fetal development and how cancer cells reactivate telomerase in cells that have short telomeres. These studies add to the growing support for the role of telomeres in regulating gene expression via TPE-OLD. Furthermore, telomere length may be one of the mechanisms of how cells time changes in physiology without initiating a DNA damage response.

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Ted from 2030: Kids, one morning in 2010, I opened the newspaper only to discover an op-ed written by Zoey Pierson. You remember Zoey. [FLASHBACK] Zoey: Key Ted Mosby's car. [END OF FLASHBACK] Ted from 2030: In those four column inches, she railed against me and my company, GNB, for wanting to tear down a beautiful old building: The Arcadian. And as if that wasn't bad enough, the piece ran on a Saturday, which as you both know, is Dad's crossword day. Ted: She ruined crossword day! I can't believe this. She singles me out by name. Calls me a "fat cat." Me and my "fat-cat friends." We're not fat cats. Barney: Exactly. I say, Marshall, my good man, how's my bow tie? Marshall: Impeccable, old bean. To industry! Barney: Ah, bully! Ted from 2030: Okay, that night we weren't entirely un-fat-catty. You see, every year the Natural History Museum holds its Autumn Spectacular. It's attended by some of the most powerful and important people in New York, and, thanks to Goliath National Bank... us. Inside a cab Marshall: Look at us, huh? In tuxedos? Can you imagine if our college selves saw us like this? Ted: They'd pelt us with their Phish bootlegs. Marshall: Yeah, we were pretty anti-establishment back then. Oh, God, remember Russell? [FLASHBACK] (Marshall's college room) Marshall: Nice monkey suit, Russell. Russel: Come on, guys. Marshall: Oh, I'm sorry, I can't hear you with that corporate noose around your neck. And don't even try showing up to the drum circle this weekend. (Russel leaves the room, Ted enters) Ted: Oh, hey. You guys seen Russell? I'm supposed to drive him to his mom's funeral. [END OF FLASHBACK] Robin: I wish I knew you guys back then. You know why? Because you can't kick a story in the nuts. Lily: Hey, we're still those people. One of these days, Marshall's going to quit his job and go to work for the NRDC, and save the world, right, baby? Marshall: Absotively. But let's just remember, I mean, nobody's the same as they were in college. You know, it's like, I wear a suit to work every day. Lily: Well, yeah, but you wear it ironically, like Ted's fanny pack. Ted: Next time we go to Great Adventure, you're carrying your own sunblock. The museum Lily: Ooh! I love this exhibit. One time when I was a kid, this room was closed for cleaning, so I snuck under the rope. Everyone: Ooh. Barney: Wow, that's pretty cool. When I was a kid, I knocked down the blue whale. Marshall: Okay, the giant blue whale hanging from the ceiling? Barney: I was six. My uncle Jerry brought me here for the day. He said, "Don't touch anything". To a kid. That's like someone telling us "Don't look at that girl's perky and impossibly symmetrical knockers." Everyone: Ooh. Robin: Not bad. Barney: So, naturally, I snapped the rib off a triceratops, blahbity-blahbity-blue, I knocked down the whale. I'm surprised security didn't stop me on the way in. Robin: Well, I'm sure they don't remember. I mean, it's been like 30 years since that completely made-up story didn't happen. Barney: It happened. And these people don't forget. This is not the Natural Stuff That Happened No More Than Five Minutes Ago Museum. Huh? (Arthur comes over, with another man) Arthur: Marshall, Barney, there you are. I want you to meet an old friend of mine from Exeter, George Van Smoot. George: But you can, and should, call me The Captain. Marshall: The Captain? Barney: The Captain? Arthur: Back in school we met during a production of Guys and Dolls. The Captain was Nathan Detroit to my assistant stage manager. Marshall and Barney here, are the future of Goliath National Bank. George: Well, ahoy. Barney: Ahoy. Marshall: Ahoy, The Captain. Arthur: The Captain pretty much paid for this entire shindig. George: Please, enjoy yourselves, have fun, but don't touch anything. Marshall: Thank you, The Captain. Barney: Challenge accepted. Lily: Wow. "The future of Goliath National Bank"? Marshall: I know, it's so, uh... You know, I totally forgot to tell you, but, um, the other day, Arthur offered me a five-year contract. Lily: Oh, well, don't turn him down here in public. I broke up with Scooter at the prom. Right before the picture, too. Lily: So whatever you do, don't tell him here tonight, 'cause... Marshall: I think I'm going to say yes. (Barney slightly touches a statue) Barney: Ah, that's the stuff. [OPENING CREDITS] Robin: I didn't realize you were small potatoes. And to be clear, I am referring to your testicles. (Robin touches the same statue from head to arm) Barney: Impressive. Try this on for size. (He raises the statue's belt and shakes it) Robin: You want to dance? Let's dance. (Robin lays a hand on the statue, Barneys does the same) Barney: I live for the dance. Robin: Get... your other hand... off my ass. Barney: Sorry, sorry. Lily: What do you mean, you're going to say yes? Marshall: I-I want to keep working at GNB. Lily: But I thought that you... Ted: Guys, guys, guys? Architecture fun fact: If you stand right here, and you whisper, a person all the way across the other end of the room hears it like you're standing right next to them. It's one of the most sophisticated pieces of acoustical design in the world. Watch. (whispers): Diarrhea. Right? Right? Lily: But a five-year contract. I thought you hated GNB. Marshall: Look, I don't hate all of it. Tonight's fun. Take a look around. I mean, this is pretty high-class. Ted, whispering: Poo-poo. Poo-poo platter. (Ted spots Zoey in the crowd) Ted: Zoey? Well, well, well. Zoey: You have got to be kidding me. Ted: So, what are we protesting tonight? Rising cost of jet fuel? The government's oppressive top hat and monocle tax? Zoey: And what are you doing here? Oh, right. Beautiful old building... you're here to knock it down. Can I finish my drink first? George: Darling, there you are. Zoey: Ted, this is my husband. Ted: Yeah, old stuff's great. (Barney and Robin still have their hand onto the statue, Barney is trying to catch her glass) Robin: Mmm. Ah, this Scotch is good. How's your drink? Barney: This is ridiculous. We are two grown adults standing among the greatest collection of natural artifacts in the Western hemisphere, and look at what we're doing. Robin: You're right. Barney: Want to go touch a bunch of stuff? Robin: Yeah, I do. Ted: So, Captain. How'd you get that name, anyway? George: Gave it to myself. A real man chooses his own name. Ted: Well, pleased to meet you, Captain. I'm Galactic President Superstar McAwesomeville. Zoey: This is Ted. George: Capital. Honey, I may cut out early. I have to go check up on the boat. Ted: The boat? There's a boat? You must tell me about this boat, Captain. George: Well, she's an 85-foot sloop. Ted: She! George: Do you like boats? Does the sea call to you like it calls to me? Ted: Yes. The sea is all like, "Ted, come hang out." George: I like Galactic President Superstar McAwesomeville. You're coming on the boat sometime. Stepping off. Ted: Man, I wish me and my dad were as close as you guys are. Zoey: Oh. You want to make this personal? Okay. Destroy Ted Mosby. Now it's personal. Ted: No, if I wanted to make it personal, I'd call you a bored little trophy wife who likes to play activist when the shops on 5th Avenue are closed. Zoey: You're going down. Ted: Down where? To the yacht club? Oh! I would love to. W-w-wait. I'm half Jewish, will that be a problem? Lily: So what about becoming an environmental lawyer? What about saving the world? Ted, whispering: Wieners. Marshall: That was a great dream. But we have a mortgage, and we're trying to have kids. We're grown-ups now, Lily. Ted: Wieners and gonads. Lily: What would College You say if he heard what you were saying right now? Marshall: Honestly? Probably something pretentious, and pseudo-intellectual, like... Ted: Boogers. Marshall: We all change, Lily. You know, you don't spell "women" with a "Y" anymore. And I'm okay with that. And you need to be okay with the fact that I may never become an environmental lawyer. Lily: So how long have you felt this way? Marshall: Honestly? Since my first day at GNB. Ted: Hershey squirts. (Robin joins Barney who was going to touch a wall) Robin: Hey. How do you like my date's tux? Ooh! Uh, a-thank you! Oh, none for him. He's stuffed. Ted: Oh! Zoey! There you are. Oh, my God. You have a monocle. Is this real? Is this really happening? Zoey: Can you excuse us for a moment? Let's go for a walk. Ted: Good luck killing James Bond. (Zoey takes Ted away) Are we allowed in here? Zoey: What do you want from me? Ted: I want my crossword day back. Okay? Go live your perfect little life, and leave me the hell alone. Zoey: My life isn't perfect. Ted: Oh, please, what's your biggest problem? Having to sail back to the marina because the Captain's all out of white Zin? Oh. Great. Now you're crying. Like that's going to get my sympathy. Ted from 2030: It did. Lily: You've known about this for two and a half years? So every time you've talked about wanting to be an environmental lawyer since then, that was a lie. Marshall: Technically, I never lied. You asked me questions, and I responded with made-up words. Lily: What? [FLASHBACK] Lily: So, you'll probably quit GNB in a couple years, right? Marshall: Affirmatootly. Lily: And become an environmental lawyer? Marshall: Yepskerdoodles. Lily: Hey, by the way, do you like this scarf? Marshall: Posititochadochmecochepopocha. [END OF FLASHBACK] Marshall: Lawyered. Lily: Okay, that's also a made-up word. Marshall: Okay. Lily, what do you want from me? I want you to be the person I fell in love with. (Robin has a fur on her back and a javelin; Barney, dressed up as a Pharao, scares her) Barney: Niled it. Museum guard: Excuse me. Barney: Thank God you're here. She's been messing with the exhibits. Zoey: I got married when I was 22 to a man who calls himself The Captain. Ted: He seems like a good guy. He wears those red pants. Zoey: I hate boats, Ted. I do, I hate 'em. I can't be on them. I can't be near them. I can't even think about them without getting seasick. You want to know why I want to save that building? Because when I look up at The Arcadian, I see something big and solid, and right now everything else in my life just feels like I'm on a boat. I know it's crazy to care that much about a building. Ted: It's not crazy at all. I'm the same way. Look, Zoey, The Arcadian should be a landmark, it should. The lion head stonework is iconic. I hate that we have to tear it down. I hate working for GNB. They're a bunch of wieners and gonads. Zoey: Ted, that was... really easy. Ted: What? (Zoey puts out a recorder) Ted's voice (on a recording): They're a bunch of wieners and gonads. Zoey: This should be useful. Ted from 2030: And in that moment, another headline appeared before my eyes. Ted: You tricked me. Zoey: Well, it the bug room, Ted. Your ass just got bugged. Oh, the offer still stands. We simply must have you out on the boat sometime. [SCENE_BREAK] Barney and Robin are in the guard's office Guard: Well, aren't you two clever. Well, guess what, this museum has seen every kind of prank you can think of. Mummies playing poker, penguins sticking out of volcanoes, dinosaurs from the Cretaceous period hanging out with dinosaurs from the Jurassic period. One time a kid knocked down the blue whale. You name it... Robin: I'm sorry. Did you say someone knocked down the blue whale? Guard: Oh no, not just someone. A six-year-old. Barney: Oh, yeah, that story is legend... (phone ringing) Hold on....dary. And, um, would you happen to know what that young man's name was? Guard: No. But I could, uh, check the files. Barney: Thank you. George: Now, Arthur, your turn. I just sang three songs. Now you-you do your part from Guys and Dolls. Arthur: Take your seats, everyone. The show's about to start. Douche. (George spots Ted alone in a corner) George: So I hear my wife got you pretty good. Ted: She caught me on tape trashing GNB. George: Oh, that damn recorder. Try being married to that. "But you said you'd get the corgis neutered this weekend." "I said no such thing." "Oh, yeah?" Click. You're a good guy, Galactic President Superstar McAwesomeville. Tell you what, when Zoey goes to sleep, I'll find that tape and erase it for you. No hard feelings. Ted: Really? You'd do that to your own wife? George: Sure. Why not? I mean, I'm glad she has these little causes, they keep her out of troubles, but when she throws a temper tantrum and it gets in the way of someone doing their job, that's a problem. Ted: No, you know what? Don't erase the tape. And for what it's worth, I don't think she's throwing temper tantrums. I just think she's, you know, standing up for what she believes in. I respect that. George: Hey, what about this? I'll take you out on the boat sometime. You've got to see this boat. She's breathtaking. Ted from 2030: Kids, there's an amazing architectural phenomenon in the Natural History Museum. If you stand in the right spot, you can hear an entire conversation all the way across the room. The guard's office Guard: July 23, 1981, incident report. At approximately 1000 hours,...vandal dislodged rib from triceratops skeleton...and flung said rib at giant whale. Causing said giant whale to fall in a downward trajectory. And the vandal's name... Well, I'll be damned... Barney Stinson. Barney: Who's the master, Leroy? Guard: Stinson was reprimanded and returned to the custody of his father, Jerome Whittaker. Barney: Uh, no, uncle. Jerome Whittaker is my uncle. Guard: Uncle Jerry. Says father. Even signed it and checked the box for father and everything. Barney: Jerry's my uncle. College Marshall: Lily? Honey, what's wrong? You okay? Do you want a hit of this sandwich? Lily: I want you. College Marshall: Awesome. Let me just put a sock on the doorknob. Lily:No. I mean, I want you as opposed to who you've become. You've changed so much. College Marshall: What? How have I changed? Did I cheat on you? Lily: No. College Marshall: Did I stop writing poems for you? Lily: Yes, but I'm okay with that. College Marshall: Am I not as good at making the sweet, sweet love to you? Lily: Actually, you're way better now. You last, like, two, three times as long. College Marshall: You said that any longer would be too much. Lily: It's okay. College Lily thinks those are orgasms. No, it's... it's none of that. It's just this new Marshall... Corporate Marshall... he wears suits all the time. He doesn't care about saving the world. He's not you. I want you back. College Marshall: Well, you can't have me. Look at the sign. I'm extinct. I've gone the way of Jane's Addiction. Lily: Actually, Jane's Addiction got back together. College Marshall: They did? Lily: Yeah, they've done a few tours, they put out a new album. College Marshall: Are you serious?! That is awesome! Are they just as good? Lily: Sure. College Marshall: Look, I know that Corporate Marshall wears a tie and everything, but it sounds like he hasn't changed where it counts. (The Marshall from today arrives) Marshall: Hey. Lily: Hi. Marshall: Look, Lily, I know that you would have been okay if we were poor and I was trying to save the world, but will you still be okay if I make a lot of money and I spend all of it spoiling you and our kids? Lily: We'll make it work. (Lily and Marshall leave the museum) College Marshall: There he goes. The Marathon Man. Mr. Stamina himself. I can kiss better than that old man. (Robin and Barney are sitting at a table) Robin: So when was the last time you saw him? Barney: It was that day... July 23, 1981. My mom got pretty mad that he let me destroy a New York City landmark. Robin: Moms. Barney: He never came around anymore after that. Think he moved away. Robin: Well, maybe the security guy had it wrong. You never know... Barney: But you do know, you do know. That's the thing. You know. He's my dad. Robin: Barney, do you want...? Barney: I don't want to do anything. Don't tell anyone about this, okay? Ted: I'm serious. It's a great look. I think it could come back, but one question. Does it cost half as much as glasses? Zoey: Can I steal you for a second? You don't need to worry. I... What are you doing? Ted: Oh, I thought we were... Zoey: Fine. I erased the tape. Ted: What? Zoey: I don't need it. I'm going to beat you fair and square. Ted: Thanks. Zoey: But it's good to know how easily you can be manipulated by a woman. Ted: You look gross when you cry, you know that? Some women look cute. You look like a basset hound. Zoey: Oh, laugh it up now. Because starting Monday, I got you in my crosshairs. Ted: Bring it on, Princess. (Marshall is in his office when Arthur enters) Arthur: Eriksen... It's, uh, 3:00 a.m. You know what, you might as well not even go home. Ted from 2030: And so Marshall stayed right on at Goliath National Bank. Of course, it wouldn't last forever. But that's another story.

When the gang goes to a black-tie event at the Natural History Museum, Ted gets introduced to Zoey's husband, The Captain. Barney and Robin dare each other to break the laws of the museum by touching all of the exhibits.

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Orientia tsutsugamushi (Ots) frequently causes severe scrub typhus infections in the Asia-Pacific region. Korean investigators have demonstrated that Ots encodes five different autotransporter domain (ATD) proteins (ScaA-ScaE). ScaA functions as an adhesin and confers protective immunity in a lethal mouse model of Ots infection. Specific antibodies are detected against ScaA and ScaC in Korean scrub typhus patients. However, there is limited data on the distribution of the Sca protein genes in diverse isolates of Ots. By BLAST analysis with the conserved beta barrel autotransporter domain (ATD) regions of the sca proteins, we discovered a sixth gene scaF among 3 of 10 new partial Ots genome sequences available at NCBI GenBank (Sido, Karp, AFSC7). We designed two to seven specific TaqMan assays to detect the ATD for each of the six sca genes. The TaqMan assays among those for each sca gene which gave the greatest sensitivity and linearity with DNA log dilutions were then used for screening DNAs from Ots isolates grown in L929 mouse cells for sca genes. The sca prevalence survey was performed for all six sca genes with 178 DNAs from isolates from 12 countries. The survey results were confirmed by conventional PCR with primers from conserved regions of the passenger domains (PD) and ATD of the sca proteins. The ATD was highly conserved between the DNAs of different genotypes compared to the sca PD but each TaqMan assay was sca specific. The percentage positivity for 56 kDa and scaA genes in the 178 DNAs using Ha primers was 59. 6% and 62. 4%, respectively. Our scaA conventional ATD PCR assay was positive in 98. 3% but scaA was present in all 178 DNAs (100%) by ATD TaqMan. scaB, scaC, scaD, scaE and scaF were detected in 33. 7%, 97. 8%, 93. 8%, 97. 2% and 43. 3% isolates by ATD TaqMan, respectively. The ATDs of Ots sca genes are thus sufficiently conserved between different genotypes for molecular assay design. Four sca genes are widely distributed among diverse Ots isolates from diverse geographical areas. scaB and scaF were detected in fewer Ots isolates and absent from some available genome sequences. Whether the utility of the ScaA, ScaC, ScaD, and ScaE antigenic passenger protein domains exceeds that of the mixed 56 kDa type surface antigens of Ots now used in combination diagnostic assays needs to be determined before they can be considered as suitable alternative serological antigens for diagnosis of scrub typhus. Scrub typhus, caused by Orientia tsutsugamushi (Ots), is a common acute febrile illness in the Asia Pacific region; however, related agents with a few cases of associated disease have been described recently in new places in Africa, South America and related agents may occur in Europe [1–4]. Scrub typhus presents with fever, headache, chills, myalgia, and arthralgia, as is common with other tropical febrile illnesses, but may also present with a characteristic diagnostic eschar at the site (s) of the vector chigger bites [5] The severity of the disease can range from mild forms with low grade fever to severe fatal illnesses with high grade fever and complications involving multiple organ failure. The prevalence of disease has increased in recent years partly because of improved diagnostic methods and its recognition in many new foci; indeed, in many endemic regions it is the leading cause of treatable non malarial illness [6]. The public health impact of this disease is huge as at least a billion people are at risk of this disease with at least a million cases suspected annually [6,7]. Appropriate and timely antibiotic treatment with doxycycline or chloramphenicol generally leads to a very good prognosis [8,9]; however, recognizing the disease early and with certainty has been a significant public health issue, especially in the rural parts of the endemic world which lack access to specific diagnostic tests [10]. Since clinical diagnosis can be difficult due to overlapping signs and symptoms with other tropical febrile illnesses, confirmatory diagnosis is still largely based on non-specific Weil-Felix laboratory tests in many district hospitals [10]. Gram negative bacteria (GNB) including the Rickettsiales with two cell membranes (diderms) have complex cell envelopes with a cytoplasmic membrane, periplasmic space, outer membrane and frequently surface layer molecules that have roles in virulence. This presents a challenge for the transport of the surface and outer membrane proteins, lipopolysaccharides and capsule components across the cell envelope. [11]. Protein transport systems in GNB are termed as types I to IX [12]. The simplest secretion pathway was originally thought to be the intrinsic type V autotransporter (AT) secretion system which employs an N-terminal signal sequence recognized by the Sec machinery for passenger domain (PD) transport across the cytoplasmic membrane and the attached transmembrane beta barrel autotransporter domain (ATD) for outer membrane transport [13]. However, it is now recognized that beta-barrel assembly machinery (BAM) proteins and in some cases translocation and assembly module proteins (TAM) are essential for the translocation of ATD proteins [14]. The ATD proteins, many of which are cell surface proteins, often play an important role in GNB virulence functions such as adhesion, aggregation, invasion, biofilm production and toxicity and are now being exploited in bacteria for surface display of other protein moieties [15]. Among the sequenced Rickettsia genomes, a family of 17 ATD paralogous genes has been identified which are also called the surface cell antigen (sca) genes [16]. The major sca/ATD genes ompA, ompB, and sca4 are used for differentiating species of Rickettsia [17]. The roles in pathogenesis of five of these proteins (OmpA, OmpB, Sca1, Sca2, and Sca4 have been well studied in Rickettsia [16–22]. In particular, the OmpA and OmpB proteins are important antigens conferring protective immunity to infection and because they are immunodominant antigens, they are targets for serological diagnosis of infections due to Rickettsia [23]. In contrast to the significant amount of attention given the sca/ATD proteins of Rickettsia, the sca/ATD proteins of their nearest relatives in the genus Orientia have received scant attention. Korean investigators have demonstrated that Ots encodes five different autotransporter domain (ATD) proteins (ScaA-ScaE) [24] based on the two complete genomes of Orientia tsutsugamushi strains (Boryong, 2007 and Ikeda, 2008) then available [25,26]. ScaA of Boryong strain functions as an adhesin and confers protective immunity in a lethal mouse model of Ots infection [27]. Specific antibodies were also detected against ScaA and ScaC in Korean scrub typhus patients [28]. Ha et al. (2012) detected and sequenced 4 of the 5 Sca Genes in three other prototype isolates of Orientia (Gilliam, Karp, Kato) by use of conserved Boryong-Ikeda derived PCR primers [28]. However, there is only limited data available on the distribution of homologues of these Sca protein genes in antigenically and genetically diverse isolates of Orientia tsutsugamushi. Serological and molecular tests for scrub typhus are more commonly used than culture based assays, as these bacteria are obligately intracellular and thus require antibiotic-free cell cultures for cultivation. Furthermore, BSL-3 conditions are recommended for producing specific Orientia antigens and reagents; this is both costly and highly restricted to a few specialty laboratories. The most common gene target for both serological assays such as Dip-Stick, Flow Assays, and ELISA and molecular diagnosis has been the 56kDa scrub typhus type specific surface antigen (TSA) [29]. Since this gene exhibits substantial genetic variability, it is the target most commonly used for differentiation and typing of the infecting genotypes of O. tsutsugamushi [30]. However, because of this TSA antigenic diversity, matching a clinical antibody response to Ots to the particular antigenic type of Ots infecting a patient is desirable to assure high sensitivity of serological detection [31,32]. Consequently, while serological tests with mixed STA56 antigenic types have been employed to overcome this limitation or reliance on the presence of cross-reactive antibodies to single antigen types have been used, they may be inappropriate for geographic regions whose Ots isolate types are unknown or for antigenically distant infecting strains. Another recombinant protein antigen target for serological diagnosis of scrub typhus is the more conserved STA47 antigen gene of Ots but the antibody response for this target may be delayed in comparison to 56kDA TSA, especially in acute phase sera [33] In the present work, we have assessed the prevalence of O. tsutsugamushi sca protein genes in a large collection of isolates from the Asia-Pacific region by means of new specific TaqMan assays targeting conserved sites in each of the scaA-E autotransporter domains. We also describe the TaqMan detection of a new ATD protein gene, scaF. The assays were designed based on available sca gene data extracted from partial genome sequences of Ots available at NCBI in 2017 when the study was performed. Using tBLASTn (search translated nucleotide databases using a protein query) [https: //blast. ncbi. nlm. nih. gov/Blast. cgi? CMD=Web&PAGE_TYPE=BlastHome] with the five complete Boryong ScaA-ScaE protein sequences available from Ha et al. [24,27,28] we analyzed the two complete and 12 partial Ots genome sequences available in NCBI database (S1 Table) for the presence of different sca genes. The different sca protein genes we found were also classified based on their greatest protein (coverage and identity) tBLASTn sequence homologies with Boryong Sca proteins (S2 Table). An additional ScaF protein (645 aa) was detected in Sido, AFSC7, Karp2M and KarpIGS contigs and its similarity and distribution was further evaluated by tBLASTn of the genome sequences with the Karp ScaF sequence (S1 and S2 Tables). The presence and size of N-terminal putative signal sequences or transmembrane regions and ATD in these Sca protein genes was determined using SMART (simple modular architecture research tool [http: //smart. embl-heidelberg. de/]). Full sca gene and DNA sequences adjacent to the 5’ and 3’ ends of the complete sca genes were extracted in order to design primers suitable for amplifying all of the PD and ATD of the sca genes detected in Ots. In some cases where incomplete genes or adjacent fragments of ATD were annotated, frame shift errors were corrected (generally single base insertions or deletions) and overlapping or adjacent contig sequences were joined based on the closest sca genes available in each class (S1 Table);. tBLASTn and BLASTn were performed again on the genomes with the closest homologous sequences available for genomes in which only partial sequences (S1 Table) were initially detected to ensure they were not overlooked. The sca protein gene regions (PD and ATD) identified for O. tsutsugamushi were aligned using Muscle [34] as embedded in Geneious R 9. 1. 6 [35]. The ATD regions were identified with SMART and the signal peptide plus passenger domain (SP-PD) region was defined as the complete region 5’ or N-terminal to the consensus ATD region predicted by SMART. In some cases this included clearly predicted signal sequences and in others, only transmembrane domains at the amino terminus. The number of amino acid identities and percentage amino acid identifies for SP-PD and ATD were obtained from Geneious (S2 Table). Multiple sequence alignments for the SP-PD, and ATD sca nucleotide and Sca protein sequences were performed using MUSCLE [34]; subsequently, the phylogenetic trees were constructed using Maximum Likelihood (ML), and Neighbor Joining (NJ) approaches. Model JTT+G+I/GTR+G and JTT+G/GTR+G+I (JTT: Jones-Taylor-Thornton; GTR: General Times Reversible; I: Invariable sites, G: rate variation among sites, I+G: both) were identified as the best substitution models by the Model Test program for reconstructing the ML tree in MEGA7 with 100 bootstrap supports for SP-PD and ATD domain protein/gene sequences, respectively [36] (Fig 1). The NJ tree was constructed under p-distance [37] with 100 bootstrap supports (S1 Fig). Tandem repeats (TR) in the sca genes were identified using the Tandem Repeat Finder program (http: //tandem. bu. edu/trf/trf. basic. submit. html) to avoid including those repeated regions when primers and probes were designed [38]. None of the large tandem repeats were in the sca ATD. The ATD was highly conserved between the DNAs of different genotypes compared to the sca passenger domains (Fig 1, S1 Fig, S2 Table) but each Taqman assay (scaA-scaF) was sca specific by BLAST analysis of the primers and probes and differences in amplicon sizes and reactions with different DNAs (S1 Table). Multiple primers and probes were designed for conserved regions in the flanking 5’ and 3’ regions, SP-PD and ATD of scaA, scaB, scaC, scaD, scaE and newly identified scaF (S3 Table, Figs 2 and 3). The best primer pairs were selected by conventional PCR with prototype control Orientia DNAs (KarpPP, Boryong B, KatoPP, Gilliam PP, AFSC7, Calcutta) to identify those providing good amplification of sca gene regions without spurious bands, with minimal primer-dimer formation, and of the expected amplicon size. Control DNAs were serially diluted from 10–1 to 10–6 dilutions from working stock and the sensitivity of different probe/primer combinations were evaluated by performing TaqMan assays on control Orientia DNAs as described below (Fig 3). Optimal TaqMan primer and probe combinations were then selected for the sca gene survey of the Ots isolate DNAs (Fig 4). Gamma irradiated or cycloheximide treated (0. 5 μg/ml) L929 cell passaged well-characterized reference isolates (CDC collection) of Orientia tsutsugamushi from 12 different countries (Table 1) were extracted with single Qiagen spin columns as described previously [39]: Medium and cells harvested from one T150 flask with glass beads was centrifuged at 8000 rpm for 10 minutes and then washed in 1 ml of phosphate buffered saline; one half of the pellet was lysed and purified with the Qiagen spin column; the column DNA was then eluted into 0. 5 ml of Qiagen AE buffer and stored at 4°C [38]. Working stocks (1: 10 dilution in AE) of these master stocks of Ots DNAs were used for all molecular analyses. Conventional PCR was done in an Eppendorf master gradient cycler in a total reaction volume of 20 μl. The mixture consisted of 2 μl of DNA working stock template, 10 μl of 2x concentrated Qiagen PCR master mix, 0. 5 μl (20 μM) each of both forward and reverse primers and 7 μl of molecular grade water. The cycling conditions were initial denaturation at 95°C for 5 minutes followed by denaturation at 95°C for 1 minute, annealing at 57°C for 2 minutes and extension at 70°C for 2 minutes. The above cycle was repeated for 35 times followed by a final extension step at 70°C for 2 minutes. The PCR products were run on 1–3% agarose gels in Tris-borate buffer at 80 V based on the anticipated length of the PCR amplicon along with molecular markers (BioRad Laboratories EZ load 100 bp or 1 KB plus molecular rulers). Documentation of ethidium bromide stained gels post electrophoresis was done on a BioRad Gel Doc imager and stored for further size analysis. The sca ATD assays were conducted in a total volume of 20 μl of qPCR mixture in 96 well plates. The mixture consisted of 2 μl of DNA working stock template or no template control added to a master mix comprised of 10 μl of 2x concentrated iTaq universal probes super mix (Bio-Rad), 6. 8 μl H2O, 0. 5 μl (20 μM) of both forward and reverse primers and 0. 2 μl (10 μM) of each probe (all 5’-FAM, 3’ BHQ1 labeled) for each of the sca targets. The cycling conditions were initial denaturation at 95°C for 3 minutes followed by 40 cycles of two step amplification: denaturation at 94°C for 15 seconds and 60°C for 60 seconds on a Bio-Rad CFX 96 thermocycler. Plate reading for fluorescence was recorded during every thermal cycle at the annealing step and data was analyzed using Bio-Rad CFX software manager version 3. 0. To enable accurate direct comparison of the different primer probe combinations evaluated for each sca ATD target assay, they were all analyzed on the same microplate with aliquots of the same DNA dilution series. Recombinant pCR 2. 1 plasmids carrying the ATD regions of each of the 6 sca’s were generated following the TA cloning strategy (In Vitrogen, ThermoFisher Catalog K202020). Briefly, each of the sca ATD regions (S3D Table) were PCR amplified and checked for amplicon purity on 2% agarose gels. Amplicons ligated into pCR 2. 1 were transformed into One Shot Competent Cells (TOP 10) and screened for transformants by plating on LB agar plates with 50μg/mL of kanamycin, overlaid with 40μl of 40mg /mL X-Gal. Plates were Incubated overnight at 37ºC. Four well-spaced white colonies from each transformation plate were selected and colony PCR was performed to confirm successful transformation and size of the insert. Positive clones by colony PCR were cultured in 5 mL of 50μg/mL of kanamycin overnight and plasmids were purified using Qiagen plasmid mini kit. For sca qPCR specificity experiments, plasmid DNA quality and yield (Qubit Double Strand DNA assay) were determined and equal amounts of two dilutions of each plasmid were added as DNA templates for TaqMan reactions (S4 Table). Each of the 6 plasmids (two different amounts) were screened with all of the sca primer and probe combinations (9 ATD assays) along with positive Orientia DNA and no template controls. Six Sca gene proteins were detected by tBLASTn in the 14 Orientia genome sequences available (S1 Table, Fig 1). The gene clusters of the maximum likelihood (ML) and Neighbor Joining (NJ) phylogenetic trees matched those expected from the Boryong ScaA-E and Karp ScaF tBLASTn predictions (Fig 1, S1 Fig). The new type scaF was only found in three isolates (Karp, AFSC7, Sido) including both the independently obtained Karp2M and KarpIGS sequences. ScaB was also only present in 3 isolates but 2 identical copies were detected in the Boryong sequence. scaA, scaC, scaD, and scaE were present in all genomes except Sido which had the least genome coverage. However, except for Boryong and Ikeda, the other genome assemblies are incomplete and comprised of many contigs; consequently, it is not possible to know whether the scaB and scaF genes are really absent or just not found in the available incomplete assemblies. Several probable frame shift errors and truncations of sca protein genes were found and the reading frame (RF) corrected sequences are shown in S1 Table. scaB size was highly conserved with only insignificant size differences found among the two 1950 bp Boryong copies, and the TA716 (1995 bp) and Sido (1998 bp) sequences. The three scaF gene sequences were nearly identical in sequence and were identical in size (1938 bp). Among the other four sca genes, the scaC genes had the least size variation, varying between 1554 bp (UT76) and 1581 bp (Boryong) and contained two tandem repeats accounting for most of the small size differences. The scaD gene sizes were quite variable from 1998 bp (Kato) to 2997 bp (Karp) due in part to varying numbers of tandem repeats and some large INDELs. The scaA genes were the largest (4344–4722 bp) and varied in tandem repeat structure, length of an encoded polyQ region, and the type of INDELs present. UT76 had two copies of scaA with slightly different sizes, with different tandem repeats and sequence divergence. The scaE genes exhibited moderate size differences between 2238 bp (AFSC4) and 2283 (Boryong). The SP-PD domains were significantly more divergent than the ATD beta barrel for each sca gene (Fig 1, S1 Fig, S2 Table). Consequently, we decided to evaluate the conserved sites in sequence alignments of SP-PD and particularly, the ATD regions to develop sensitive surveillance methods for the sca genes in our collection of isolates of Ots. All four ML trees (amino acid and nucleotide of SP-PD and ATD domains) indicated the presence of two major sca gene clades, one consisting of two clusters (class-1), including scaA and scaC and another one consisting of four clusters (class-2), including scaB, scaD, scaE and scaF with 90–100% bootstrap support in the majority of the major nodes (Fig 1). Within each sca gene clade, the isolate branchings were similar for sca types with more sequences (scaA, scaC, scaD, scaE) and the same pattern was generally seen with the NJ trees as found by ML (S1 Fig). This indicates that the two classes of sca were closely related and likely diverging from a common ancestor. The overall topologies of ML and NJ trees were quite similar, however, some minor differences in branching order within the clusters and cluster arrangements, particularly in class-2 sca types were apparent. Overall, 181 non duplicate DNA samples were evaluated in the present study; 178 were from reference isolates of Ots and 3 were from uninfected cell cultures. These 3 uninfected DNAs served as negative controls in all the experiments. The 178 isolate DNAs originated from 12 different countries (Table 1) These isolates are all well characterized CDC reference strains which all grow and stain like Orientia and all contained the Orientia scaA gene (Table 2). The previously published 56 kDa TSA primers of Ha et al [28] amplified a 1344 bp product and Ha scaA passenger domain primers amplified an 1121 bp product (Fig 2). The Ha TSA and scaA primers were used as DNA quality controls to evaluate whether primers derived from more conserved sites on the scaA gene ATD alignment would be more efficient in detecting this gene (S3 Table, Fig 1D). After screening multiple scaA primers by PCR with DNAs from KarpPP, KatoPP and GilliamPP prototypes, the best primer pair that amplified a scaA product (586 bp) with a good intensity and without spurious bands was selected to screen all the DNAs (Table 2). The 56kDa Ha primers detected 106/178 DNAs (59. 6% positivity) and scaA Ha PD primers identified scaA in 111/178 DNA’s (62. 4% positivity). Our conventional scaA ATD PCR primers were much more sensitive in that they amplified scaA from 175 of 178 Ots DNAs with high sensitivity (98. 3%). This confirmed that the quality of the Ots DNAs we used was excellent and that these scaA gene ATD sites were indeed sufficiently conserved across a wide range of TSA genotypes of Ots isolates to be useful. To increase the likelihood of detection of sca genes with divergent passenger domain sequences (using the sequence alignments for all of the available sca genes and proteins), we selected unique conserved regions of the ATD domains for each of scaA-scaF genes (Fig 1, S1 Fig, S2 Table, S3 Table) to develop ATD TaqMan assays. Four such TaqMan assays were tested (two sites each with two probes each) (Fig 2 general design, S3 Table) for linearity and sensitivity of detection with serial tenfold dilutions of prototype Ots DNAs (Fig 3). We evaluated the sca specificity of each assay (lack of cross-over between sca targets) by testing the best assay against cloned ATD target plasmids for each sca gene (S4 Table). None of the assays showed cross-talk between sca genes and they all showed similar levels of sensitivity for the same control DNA and each of the sca plasmids. That assay for each target sca ATD was then used to survey all the Ots isolate DNAs in Table 1 for the presence of each sca gene (Table 2, Fig 4). Among the 178 Ots DNAs tested with the six ATD TaqMan assays, all the isolate DNAs tested were positive for scaA gene (Table 2) while the 3 control DNAs gave no signal. The scaC, scaD, and scaE ATD targets were also detected with very high prevalence rates (Table 2, Fig 4). Consistent with the available Ots genome sequence data, scaB and scaF were less prevalent but they were both detected at higher rates than the genome sequence data would suggest. This result could be due to the bias in the sequence data inherent in being derived from a high proportion of strains from Thailand. To examine this possibility, we also partitioned the prevalence of sca genes by country (S3D Table, Fig 4). The scaA ATD target was detected in all the 178 isolates tested. The scaB ATD target was detected in the least number of isolates tested. Except for the one isolate from Korea, which was positive, there were isolates negative for scaB from every other country. However, most of the Australian isolates (88. 9%) were positive for scaB. The scaC ATD was detected in 97. 8% of the DNA’s tested. Only 3 isolates from Australia and an isolate from Pakistan were scaC negative. The scaD ATD target was detected in 93. 8% of the DNAs tested. An isolate each from Japan and many isolates from Pakistan (9 isolates) were negative for scaD. The scaE ATD target was detected in 97. 2% of the DNAs tested. A single isolate from China, Malaysia, Thailand and 3 isolates from Pakistan were negative for scaE. The scaF ATD target was detected in 43. 3% of the DNAs tested and with all of the Solomon Islands and Vietnam DNAs positive. However, more than half (51. 2–71. 4%) of the isolate DNAs tested from Australia, Malaysia, Taiwan and Thailand were negative for scaF. The immunodominant major TSA of Ots is the most extensively studied serological and molecular target in scrub typhus diagnosis; however, it exhibits great antigenic diversity in each of its four variable domains [29]. Similarly, AT proteins comprise one of the largest and functionally diverse group of secreted and outer membrane proteins found in GNB and play an important role in their virulence [13,40]. From our work at least six sca AT paralogs are now known to be present in Ots and four of those are widely distributed throughout the endemic region for scrub typhus. scaA functions as an adhesion factor in Ots and anti scaA antibody significantly neutralized Ots infection of host cells [24,27]. Additionally, immunity to heterologous strains was observed for Ots when vaccination was performed with ScaA combined with 56kDa STA [27]. ScaA bound to zinc oxide nanoparticles also provided good homologous protective immunity [41]. Previous immunological work on Sca proteins used the Boryong genotype of Ots, the predominant endemic strain causing scrub typhus in Korea [42] but it is not the predominant strain in other endemic countries [43–46]. Indeed, the Boryong Sca genes were outliers phylogenetically so the probable immunological properties of other Sca proteins in other Ots isolates needs to be confirmed. Indeed, the presence and absence of antigenically different Sca proteins in different Ots isolates (S3D Table) may account for some of the clinical and epidemiological strain specific differences seen with this species. [29,42]. Using the previously published conventional PCR primers that amplified 56kDa TSA and scaA genes, [28], the lower percentage positivity for these genes was 106 (59. 6%) and 111 (62. 4%) of the Ots isolates, respectively. The significant proportion of DNAs negative for these PCR’s is likely due to the primer design because these researchers used the outlier Boryong sca sequences for their primer design. The demonstrated benefit of using conserved regions of more DNA sequences for scaA for both conventional PCR and TaqMan ATD assays allowed us to design highly effective assays for three of the other sca genes as well. However, the scaB and scaF primer designs are limited by the same lack of sequences faced by Ha et al. [28]. Since we have identified additional isolates with different sequences of these genes, we expect those assays can be improved to be more efficient with better primer selection. On the other hand, the sensitivity and efficiency of the other four sca ATD TaqMan assays (scaA, scaC, scaD, scaE) appear to approach that of other quantitative PCR assays for Orientia that are routinely used for detection in clinical, animal, and chigger samples based on our analysis of these same DNAs with other Ots TaqMan assays [39]. ScaA is the largest of the Ots sca’s and appears to be universally distributed based on our survey. This suggests it has an essential role in the pathogenesis of this agent but the extent to which its variability affects the clinical manifestations of different Ots isolates is unknown. ScaB seems to be the outlier among all the sca’s as it was detected in the smallest number of the strains tested despite being present in two copies in Boryong isolate. Its biological role and immunogenicity has not been studied. The scaC gene is the smallest Ots sca and is most conserved in size based on available the scaC sequences (S1 Table). Owing to these results, scaC seem to be a suitable candidate either individually or along with scaA for use in serodiagnostic assays and for evaluation as a vaccine candidate. Whether the apparent large size differences in scaD are due mostly to variable passenger domain tandem repeat differences and whether those will affect the biological properties and immunogenicity of Ots isolates is also unclear. The medium size scaE gene is also well conserved but this increase in size over scaC may make it more difficult to clone and purify for use in serodiagnostic assays and vaccines. The newly identified scaF was found in only 43. 3% of the tested isolates but, as noted above, this may be an artefact of the limited number of sequences available for primer design of the ATD-TaqMan assay. The percent identity matrix [47] for the complete proteins of the six sca types exhibited substantial intra cluster variability in both class-1 (scaA, 73. 29–100%; scaC, 86. 85–100%) and class-2 (scaB, 55. 99–100%; scaD, 76. 04–100%; scaE, 74. 49–100%; scaF, 99. 53–100%) sca types. How much this is biased by the limited number of sequences available at this point, remains to be determined so it is probably quite premature to try to assess whether there are stronger evolutionary pressures on any particular Sca protein category or either class of Sca. As the number of cases and outbreaks of scrub typhus has been continuously increasing in recent years, improved diagnostic assays based on the Orientia sca autotransporter proteins may contribute to earlier and more accurate diagnosis of scrub typhus. However, their primary importance may lie in the largely unexplored realm of their interactions with the host cells that are invaded by Orientia during scrub typhus infections. Whether they will be suitable vaccine candidates that can enhance TSA mediated immunity is also an important issue to resolve. In summary, we have shown that there is genetic diversity in the sequences and distribution of different sca genes among diverse isolates of Orientia tsutsugamushi and that these six paralogous AT genes have evolved independently, probably after early gene duplication events much as was detected for scaB in Boryong isolate. It is possible recombination between these proteins may occur but branching topology of each gene family does not yet support that possibility. As more genome sequences of diverse Ots isolates become available, it is quite possible that additional paralogous Ots sca genes may be identified that could rival the complexity of the sca gene family found in species of Rickettsia.

Orientia tsutsugamushi (Ots) frequently causes severe scrub typhus infections in the Asia-Pacific region. Korean investigators had previously demonstrated that Ots encodes five different cell surface (Sca) proteins which have functional regions that mediate their transport to the cell surface. One of the proteins (ScaA) is able to serve as a vaccine against a Korean strain of Ots. Several of the Sca proteins stimulate production of antibodies during scrub typhus infections in humans. However, very little was known about the distribution of the Sca protein genes in isolates of Ots in other countries in the Asia-Pacific region where scrub tyhus occurs. We discovered there is a sixth gene scaF in some Ots strains. We designed sensitive molecular assays from conserved regions of each protein to survey the presence of the six sca genes in 178 DNAs from isolates from 12 countries. Only four sca genes are widely distributed among diverse Ots isolates from diverse geographical areas. scaB and scaF were detected much less frequently in Ots isolates. Future studies will be required to determine whether the Sca proteins are suitable for improved diagnostic assays and vaccines for scrub typhus.

lay_plos

Police now outnumber protesters at Frank H. Ogawa Plaza. People have been in the plaza all day after police cleared more than 100 tents from the Occupy Oakland encampment. The 5 a.m. police action was peaceful, although 33 people were arrested for failing to disperse. Most have been released. Marchers gathered Monday afternoon and returned to the plaza, where police officials said they are free to rally, but camping or sleeping will not be tolerated. Meanwhile, about two dozen tents remain in Snow Park, near Lake Merritt. 11:05 p.m. Fewer than a dozen people left at Frank H. Ogawa The former Occupy Oakland camp is calm and quiet with fewer than a dozen people milling around the plaza. Police remain on scene, but the mood is peaceful. 10:31 p.m. Occupy Oakland camp is nearly clear Police officers now outnumber the 25 or so protesters at Frank H. Ogawa Plaza, the site of the former Occupy Oakland camp. A guy with a guitar is singing about freight trains as people mill around and talk. 10:12 p.m. About 80 remain at Frank H. Ogawa Plaza Only 80 people remain at the former Occupy Oakland encampment at the plaza in front of City Hall. Most are standing around, chatting. The plaza is slated to close at 10 p.m. and police are on the scene, but they are not asking people to leave. The crowd is peaceful. 9:59 p.m. General assembly has ended, plan to march to UC Berkeley Friday Advertisement The general assembly has ended at Frank H. Ogawa Plaza and for about 20 minutes people dancing to Michael Johnson. The sound guy killed the music and the crowd is slowly clearing. People are cleaning up the plaza and turning their focus on Tuesday's strike and teach-ins at UC Berkeley, an all-day event starting at 10 a.m. Protesters have said that when night falls, they plan to pitch tents on campus. Campus officials have said camping would not be allowed. 9:21 p.m. First interview with former Deputy Mayor Sharon Cornu Deputy Mayor Sharon Cornu has resigned, effective immediately, the second member of the mayor's team to submit her resignation Monday and the fourth in a month. She spoke with Oakland Tribune reporter Sean Maher about the decision: "I felt I wasn't being effective," Cornu said in an interview, adding that her work didn't seem to be meshing well with the style of Quan's existing team. Cornu said she made her decision last week. She had been in the post a little less than a year. In a statement, Mayor Jean Quan wrote, "Sharon has been a tremendous asset to my administration. We wish her well and I'm grateful for her contributions. I will be restructuring my administration and making additional personnel announcements in the coming days." Asked for more details, Quan's spokeswoman Sue Piper said the departure is a personnel issue. "It was clear that a more effective team needed to be put together," Cornu said. She declined to directly address Quan's reputation among opponents as being difficult to work with and self-absorbed, but said she respects and believes in the mayor. "Oakland really needs a political center, and I don't mean ideologically," Cornu added. "I mean a central place where people come to do the community's work and where people work together. That's going to be an enormous task, to build the relationships and create the trust that really launches Oakland into the future it can have. That's going to take a very different approach from everybody in City Hall, in labor and business and the community." Quan needs people "that can really help her project her voice. It's a unique voice among mayors, coming from where she comes from, and having led the fights she's led," Cornu said. Quan's longtime friend, confidant and legal adviser Dan Siegel also quit Monday, saying he couldn't support the mayor's choice to dismantle the camp. Police Chief Anthony Batts quit four weeks ago and public relations guru Nathan Ballard, a respected crisis communication expert hired by Quan in the wake Batts' departure, also quit. City Hall sources said City Administrator Deanna Santana was ready to resign over how Quan handled the Oct. 25 police raid on the camp, but was talked out of it by another City Hall leader. A Santana spokesperson said the two are working well together. 9:13 p.m. Protest planned for noon on Friday Occupy Oakland have called for a protest at 7th and Clay streets at noon Friday in support of a protester who was arrested early Monday and is facing deportation. Meanwhile, people continue to take turns speaking at the microphone at plaza, suggesting future actions for the movement. One speaker said Thanksgiving and Black Friday, traditionally the start of holiday shopping, are "awful traditions" and are "great opportunities" for action. 8:45 p.m. Sign hung on City Hall mocking mayor Someone has hung a sign on the main door of City Hall that reads, "There was a mayor named Jean Quan with a fetish for gas and batons. She was shaking her fists and looking quite pissed cause those kids wouldn't get off of her lawn." About 200 people remain at the plaza continuing to discuss future plans for the group. 8:28 p.m. General Assembly has reconvened at Frank Ogawa Plaza Between 200 and 300 people remain at the plaza after splitting into small groups to discuss future plans for Occupy Oakland. A proposal to conduct outreach to north, west and the east Oakland communities was shot down earlier. The general assembly has reconvened and is continuing to discuss what is next for the group. People are offering up different proposals, such as using the plaza for organizing during the day, but not camping there at night. Some say it's important to reoccupy the plaza, but no one is suggesting they do that tonight. Others say the Occupy movement needs a presence at the plaza 24 hours a day, 7 days a week. There was also a suggestion to set up "satellite camps" in various neighborhoods to draw more people into the movement. Meanwhile, Snow Park remains calm and quiet with about 25 tents and roughly a dozen people there. There is also a person camping, with an Occupy Oakland sign nearby, under a tarp at the Downtown Oakland Veterans Memorial building on Grand Avenue. 7:54 p.m. Tents occupying Snow Park Twenty-five tents and about a dozen people are enjoying a calm, cold evening at Snow Park near Lake Merritt. All is quiet at the camp, except a lone camper was seen coughing violently, shouting, "Don't leave me. Don't leave me." Interim Chief Howard Jordan has said that camp will also be cleared, but the timing is unknown. Some of the campers from Frank H. Ogawa Plaza have moved to Snow Park. 7:30 p.m. Another member of mayoral team resigns Deputy Mayor Sharon Cornu has resigned, effective immediately, the second member of the mayor's team to submit her resignation just today. Mayor Jean Quan's legal adviser and longtime friend, Dan Siegel, also resigned today over the mayor's handling of the Occupy encampment. In a statement, Mayor Jean Quan wrote, "Sharon has been a tremendous asset to my administration. We wish her well and I'm grateful for her contributions. I will be restructuring my administration and making additional personnel announcements in the coming days." Asked for more details, Quan's spokeswoman Sue Piper said in an email, "It's a personnel issue." Here's more about Cornu from her biography on the city website: "Sharon Cornu is a longtime political strategist, community organizer and former elected labor leader. She returned to Oakland after serving as national field director for the AFL-CIO in 2010. A magna cum laude graduate of Brown University, she holds a master's degree in human services from the University of Massachusetts. She serves on the Alameda County Democratic Central Committee and the East Bay Economic Development Alliance. She was recognized by East Bay Housing Organizations for her leadership on affordable housing in 2009 and was Assemblymember Sandre Swanson's Woman of the Year in 2007. Cornu first met Mayor Quan as a PTA activist at Laurel School, where her sons participated in our schools' award-winning music program." 7:20 p.m. Veteran tree sitter holds his perch Zachary Runningwolf, the only occupier who remains in Frank Ogawa Plaza, apparently plans to stay a while -- he has asked for supplies to be brought to his perch in a sycamore on 14th Street. Runningwolf has a storied history in trees. He was one of the original tree sitters at the University of California, Berkeley memorial grove. The group went into the trees to save them from being razed in 2007 to clear the way for a sports training center. The group came down in 2009 followed long negotiations with the university. The trees were cut minutes after the last protesters came down. Runningwolf also ran unsuccessfully for Berkeley city mayor. 6:55 p.m. General assembly discusses future plans About 600 people are gathered at Frank Ogawa Plaza, holding a general meeting to discuss future plans. The group is voting on a proposal to conduct outreach to north, west and the east Oakland communities. 6:40 p.m. ACLU and National Lawyers Guild file suit against Oakland Police Department Earlier today, the American Civil Liberties Union of Northern California and the National Lawyers' Guild filed a federal lawsuit against the Oakland Police Department seeking an emergency temporary restraining order to stop police violence against the protesters, according to a news release. The suit was urgent because another police encounter was imminent, following the removal of the Occupy Oakland camp this morning, the news release stated. "Excessive police force is never acceptable, especially when it's in response to political protest," said Linda Lye, staff attorney at the ACLU of Northern California, a prepared statement. The city must respond by 5 p.m. Tuesday, according to an order issued by United States District Court Judge Richard Seeborg. If police use excessive force, the city would need to justify those actions to the court, the news release states. The suit was brought on behalf of videographer Timothy Scott Campbell, who was shot with a bean bag projectile while filming police Nov. 2-3, according to the release. Additional plaintiffs are Kerie Campbell, Marc McKinnie, Michael Siegel and guild Legal Observer Marcus Kryshka. "I was filming police activity at Occupy Oakland because police should be accountable," Campbell said in a prepared statement. "Now I'm worried about my safety from police violence and about retaliation because I've been outspoken." Guild attorney Rachel Lederman called the police department's actions "wholesale and flagrant violations of Oakland's own Crowd Control Policy," in a prepared statement. The suit alleges that Oakland police and officers from other agencies "attacked" peaceful Occupy Oakland protesters on Oct. 25 and Nov. 2, indiscriminately shooting flash bang grenades, projectiles and excessive amounts of tear gas into crowds of people who were exercising their First Amendment rights, including some who were filming police. The suit alleges police violated the Fourth Amendment rights of protesters by using excessive force and violated their First Amendment rights to assemble and demonstrate. The suit also alleges that the recent actions violated the Oakland Police Department's Crowd Control Policy, which was adopted as part of a lawsuit settlement that resulted from a large 2003 protest. 6:30 p.m. Protesters plan Tuesday march to UC Berkeley Several hundred protesters gathered in Frank Ogawa Plaza are being urged to march at 2:30 p.m. Tuesday from 14th and Broadway in Oakland down Telegraph Avenue to Sproul Plaza at UC Berkeley to support the Occupy Berkeley movement. Jevon Cochrane, 21, a UC Berkeley student, reminded the crowd that university students joined the general strike in Oakland. "Just as Berkeley students marched down Telegraph Avenue five miles to the port to show solidarity with Occupy Oakland," he said, "the Oakland protesters on Tuesday are planning to show their support for the Berkeley protesters." "Students there have been upset at tuition increases and more to come," Cochrane said. "Their efforts to establish a tent city there last Wednesday were thwarted by police." But Cochrane said a new larger group on Tuesday could help students reestablish their encampment. "We need numbers to resist the police violence," Cochrane said. "The police have guns, but we have power -- social power." 6:20 p.m. City tallies cost of day's activities so far City officials have been issuing newsy and frequent news releases updating the public and media on the day's events. The latest provides a pretty comprehensive summary of the day: Update from the City of Oakland on Decampment of Frank Ogawa Plaza This morning at 4:30 a.m., the Oakland Police Department enforced the "Notice of Violations and Demand to Cease Violations" issued on Friday, November 11, to persons staying overnight in Frank Ogawa Plaza. The operation went smoothly and peacefully. Below is a summary of some operational details: Clean up Activities at Frank Ogawa Plaza * Public Works Agency staff members have completed debris removal at Frank Ogawa Plaza. More than 27.8 tons of debris and 8.2 tons of green waste for a total of more than 36 tons. * Frank Ogawa Plaza has been reopened to the public less than 12 hours after this morning's enforcement action. Any individual that decides to enter the Plaza will do so at their own risk given that the City cannot ensure that all biohazards have been cleared. * More information about the retrieval process for valuables left in Ogawa Plaza can be found on the City's homepage at www.oaklandnet.com, under Occupy Oakland updates. * Going forward, the Plaza will remain open for peaceful demonstrations and assemblies, but lodging will be strictly prohibited. Police Operations * The Oakland Police Department is monitoring the rally at the Oakland Public Library and march to Frank Ogawa Plaza. * The Oakland Police Department is prepared to facilitate a peaceful assembly and march. Temporary street closures may be required to accommodate the march's movement to Ogawa Plaza. * Should the crowd's actions warrant, OPD is prepared to take enforcement action to prevent the destruction of property and provide public safety. * Mutual aid is available to assist this evening, if necessary. The City will maintain a strong police presence at Frank Ogawa Plaza 24/7 and is pursuing other security alternatives for the future. * Activities at Snow Park are being monitored. There are approximately 25 tents. Demonstrators have been very peaceful. The Snow Park encampment will also be cleared in the near future. City Operations * Tomorrow, Tuesday, November 15, will be a regular business day for City of Oakland offices in Frank Ogawa Plaza. Homeless Services * The City of Oakland has made arrangements to open the Winter Shelter at the former Oakland Army Base today, a day earlier than originally announced. * Outreach teams from the City and Operation Dignity will be in Snow Park and Frank Ogawa Plaza this afternoon to provide homeless shelter vouchers and transportation for those in the encampments who require shelter. Cost Estimates * Preliminary City of Oakland costs spent on responding to Occupy Oakland events total is estimated at $2,402,400. * Personnel — $1,088,500 * Oakland Police Department: $1.04 million * Public Works Agency: $31,500 * Information Technology and KTOP: $17,000 * Other Costs — $1,313,900 * Oakland Police Deparment: $100,900 * Public Works Agency: $70,000 * Information Technology security upgrades: $100,000 * Early opening of Winter Shelter by one day: $3,000 * Mutual Aid for 11/14/11: $500,000 * VMA Security Contract for 30 days: $540,000 * These are preliminary costs and are subject to change. The personnel costs are mainly for overtime costs and do not reflect regular staff time. An estimated $100,000 in regular staff time that would have been devoted to other activities was spent addressing Occupy Oakland events. Recap of this Morning's Enforcement * There was no use of force and no injuries to citizens or officers. * There were a total of 33 arrests, 9 of whom are Oakland residents. * Mutual aid was provided by seven law enforcement agencies, including: Alameda County Sheriff's Office, Santa Clara County Sheriff's Office, San Mateo County Sheriff's Office, San Francisco Police Department, Hayward Police Department, Fremont Police Department and San Leandro Police Department. Although other cities' police departments were present, they were coordinated by the counties in which they are located. * City offices surrounding the Plaza (150 Frank Ogawa Plaza, 250 Frank Ogawa Plaza and City Hall) will resume normal business hours tomorrow. Media Parking * There will be no parking of media vehicles inside the Frank Ogawa Plaza area, and no parking in red zones in the vicinity of 14th and Broadway. 6 p.m.: Gas masks selling well A man selling gas masks at Frank Ogawa Plaza has cut his price to $5, after some said they couldn't afford $10, and he's enjoying a brisk business. Helicopters can be heard overheard and about 40 police, some in riot gear, are staging at the rear of the plaza, with helmets on and batons. About 1,000 people are now massed at the plaza. 5:34 p.m. General assembly about to begin in Frank Ogawa Plaza The crowd is sitting in an amphitheater at Frank Ogawa Plaza, preparing to begin their general assembly. As many as 500 people are reportedly crowding the plaza, and one person has been seen in a gas mask. Police have surrounded the area, but are watching from the outskirts. 5:25 Protesters have reached Frank Ogawa Plaza, say they're reoccupying it Hundreds of protesters are pouring into the plaza, after marching from the library. They are cheering and saying they are reoccupying it. The plaza is really muddy, but people are walking through the mud. A few are wearing black masks like those worn by violent protesters last week. Police on motorcycles escorted the protesters. So far, the protest is peaceful. 4:45 p.m. Plaza opens; protesters prepare for march and general assembly meeting to discuss three options Frank Ogawa Plaza is open. The barriers have been removed, so people can walk around freely, but will not be allowed to camp. One man is still in a tree. Police are standing by and have said they will not disturb the tree-sitter. Meanwhile, outside the library, hundreds of people have gathered and police have blocked off one block of 14th Street, between Madison Street and Lakeside Drive. A line of people is taking turns speaking during open mic. "This movement cannot end," said Jeremy Gameros, one of those arrested this morning. "The reason it cannot end, the reason it cannot be stopped, is because the conditions that created it still exist." About a half-dozen police officers, including a few on motorcycles, are blocking the street and monitoring activity. Some speakers want to walk to the plaza to hold a general assembly meeting at about 6 p.m. and discuss three possible options: reoccupying the plaza, occupying Snow Park or occupying vacant buildings in Oakland. The third option received loud applause from the crowd. 4:13 p.m. As rally begins, some say those arrested are being released About 100 people have gathered outside the library, although no one has emerged as a leader of the group. "This is an organic entity," said Kerie Campbell, 47, who has been a part of the encampment since it started. "It's not hierarchical. It's not structured. It just unfolds. You have to wait and see." One man shouted to the crowd that he had been released, after being arrested this morning. He said others were being released "as we speak." Protesters do not appear to be angry and seemed to believe their message had been heard because Oakland police had acted calmly in clearing out the camp this morning. "I lost my tent and my belongings," one woman said. "But I did not lose my teeth." Protesters have yet to make clear their intentions today. Several have said they would like to reoccupy City Hall. "What I'm hoping is we're going right back in the plaza," Campbell said. When asked about the requests from police and the mayor to not erect a tent city, Campbell said they are "clearly working for the moneyed interests and they are irrelevant to me." If police don't want tents, Campbell said, "it's winter, maybe people should donate garden sheds." Media helicopters are flying overhead, but only three police officers are visible, across the street. Some protesters are talking about marching back to the plaza. One man is selling respirators for $10 each. 3:39 p.m. Protesters assemble outside library About two dozen protesters have gathered in front of the Oakland public library, near the corner of 14th and Madison streets, waiting for a scheduled 4 p.m. rally and possible march back to the Frank Ogawa Plaza, which they expect to be reopened soon. Earlier today, Interim Oakland Police Chief Howard Jordan said officers would remove barriers between 4 and 6 p.m. to allow people to gather there. At this time, the barricades remain around the plaza, where the camp previously stood. 3 p.m. Small, but determined group remains at Frank Ogawa Plaza By Monday afternoon, the crowd of protesters had shrunk to a dozen or so outside barricades put up by police after they cleared the camp. A man was arrested after he threw down one of the barricades and spat at officers; he was taken to hospital for psychiatric observation. Paul Benton Sr., 53, came to Frank Ogawa Plaza wearing a hat with the message: "Leave my city in peace. Unoccupy Oakland." Benton, who has lived in the city for 35 years, said he agrees with the protesters' message but that their actions are damaging the city. "Their message is heartfelt," he said. "But the way they're going about it -- destroying what little is left in Oakland -- that makes me very sad." Brad Newsham had a different view. The 60-year-old Oakland resident has been attending Occupy Movement protests in Oakland and San Francisco since they began. He arrived at the plaza carrying a "Re-Occupy Oakland" sign. "The powers that be in these buildings around us would like nothing more than for us to go back to sleep so that the travesty can accelerate and continue," he said. Jack Radey, 64, took part in the Free Speech Movement and anti-Vietnam War protests in the 1960s. He now lives in Eugene, Ore., but was in Oakland for a visit and decided to check out what was going on at the plaza. "We're just getting started," Radey said. "I've seen a mass movement around Vietnam and civil rights -- and it's come again." 2:30 p.m. Snow Park encampment grows to at least 40 After protesters were ousted from Frank Ogawa Plaza early this morning, many headed to Snow Park near Lake Merritt to pitch their tents. Although Interim Oakland Police Chief Howard Jordan has said that plans were under way to clear the makeshift camp, no law enforcement officers were in sight by the early afternoon, when about 40 tents dotted the area. 1:30 p.m. Police plan to reopen plaza for 4 p.m. rally and to clear campers out of Snow Park Interim Oakland Police Chief Howard Jordan told a 1 p.m. news conference that the city will reopen Frank Ogawa Plaza for the planned rally at 4 p.m. at the library, in an effort to "reduce tensions." He said barriers would be removed and protesters would be allowed to enter the camp. When asked what would happen if people started setting up tents again, as they did in the past, Jordan said, "We will deal with the issue of lodging as it occurs. "We really don't have any reason to keep people out of the plaza," Jordan said. "It's a public space." Unlike the Nov. 2 general strike, there will be a heavy Oakland police presence at the 4 p.m. rally, Jordan said. The city is 80 percent finished with the cleanup the camp. A total of 33 arrests were made, he said. Although mutual aid for the raid cost the city between $300,000 and $500,000, Jordan said the city would not have to pay if additional mutual aid is needed later Monday. This is because the morning raid was planned, he said, whereas anything that happens in the evening would be considered an emergency. Mutual aid is on standby, he said. The money to pay for the aid is coming from the city's $30 million reserve fund, said City Administrator Deanna Santana. Jordan said there is a plan to remove about 26 campers who are currently at Snow Park near Lake Merritt, but he wouldn't say how or when that would be accomplished. Mayor Jean Quan was not at the news conference. 12:10 p.m. Some protesters remain while city workers throw away campers' belongings More than a dozen protesters continue to congregate at the corner of 14th and Broadway, standing in front of about a dozen police officers who are behind metal fences. The protesters are doing TV interviews and sitting in a prayer circle. A group of Hare Krishnas is playing drums and singing. Meanwhile, inside the camp, public works crews are slowly removing items using two dump trucks. It appears they are throwing everything in the garbage. 11 a.m. Teacher observes scene Also at the intersection of 14th and Broadway was a line of union workers, all wearing white armbands, who planned to help mediate if tensions erupted between police and protesters. "We are a peaceful observer presence, to make sure nobody gets hurt," said Janan Apaydin, 55, a 4th grade teacher at Oakland's Kaiser Jr. Elementary School. Like much of the crowd on Broadway, the union line found itself blocked off from the plaza when police swiftly overtook the camp area from multiple directions. 10:15 a.m. Police union thanks protesters, chief -- everyone but the mayor The Oakland Police Officers Association released a statement today thanking the protesters for peacefully leaving the tent city. The statement also praises Interim Chief Howard Jordan and City Administrator Deanna Santana for their leadership during the raid. But there's no mention of Mayor Jean Quan. The union has criticized her in recent days for her changing position on the Occupy Oakland camp. The statement reads "On behalf of the 645 Oakland police officers we represent, Oakland Police Officers' Association would like to thank our Police Chief Howard Jordan and City Administrator Deanna Santana for their leadership in the peaceful removal of the occupier encampment. We are also appreciative of the mutual aid provided by law enforcement agencies from throughout the Bay Area." To the protesters, the statement adds: "Thank you for your peaceful exit from Frank Ogawa Plaza -- it was greatly appreciated by all," said Sgt. Dom Arotzarena. "We respect your right to peaceful protest, and urge your continued willingness to abide by the law." 9 a.m. Mutual aid from police cost $300,000 to $500,000 Mutual aid from departments around the area today cost the city $300,000 to $500,000, Interim Chief Howard Jordan said. Departments that sent officers include the San Francisco Police Department, BART, Alameda County Sheriff's Department, Hayward, Fremont, Richmond, San Leandro and San Mateo. Jordan did not say how many officers were involved in the raid. Officers from the Santa Clara Sheriff's Department, and San Jose, Gilroy and Burlingame police departments were also seen downtown. The Hayward Police stayed after many others left. 8:30 a.m. 12th Street BART station open The 12th Street Oakland City Center Station is open. However, 14th Street and Ogawa Plaza entrance/exits are closed. All other entrances open, according to BART. 8:20 a.m. Mayor'relieved' that eviction was peaceful A tired-looking Oakland Mayor Jean Quan said Monday that she was "relieved" that the police raid on the camp was peaceful. She implored protesters not to be destructive. At a news conference, Quan said the camp had put the city's resources to the test and placed a "tremendous strain" on all departments. She said there were 179 calls to 911 and other calls for emergency service that didn't get answered because police were responding to demonstrators last week. Interim Chief Howard Jordan said there were no injuries reported by police or protesters. City spokeswoman Karen Boyd said the city is asking employees and businesses to delay work until 10 a.m. today. 8:10 a.m. Campers set up at Veterans Memorial Building The dust had barely settled after the raid at the Occupy Oakland encampment at Frank Ogawa Plaza on Monday morning when campers started to relocate to smaller sites on the edge of downtown. Evie McKnight is one of at least seven people who set up camp last week in front of the Veterans Memorial Building, at the intersection of Harrison Street and Grand Avenue. When told that the Ogawa Plaza encampment had been closed by police, McKnight said that was a bad idea. "We'll have to reopen her," she said. "It's a group effort; an ever-expanding group effort." Ben Hanks, a Redding resident, said he joined the Occupy movement last week after he visited family in the Bay Area last week. He has spent the past three nights in a sleeping bag in front of the Veterans Memorial Building. Hanks opposed the early morning raid, saying that a lot of people need places to sleep because their homes were foreclosed. "You can't have police coming in and telling people, who are just sitting around and talking about what's wrong with society, that they don't have the right to be," he said. "Not in America. Where are they supposed to sleep? They've lost their homes." He condemned any violence committed by protesters in previous Occupy-related incidents, but he also criticized the Monday morning raid, saying it was a symbol of what the Occupy movement is protesting. "That's about money and power," he said. "You have a small amount of people directly controlling a lot of people's lives." Now that the city's most populated Occupy encampment has been closed, some expect large numbers of campers to join the less populated camps in Snow Park, at 20th and Harrison streets, or the one inhabited by Hanks, McKnight and a few others. Hanks said he believes those areas will be raided soon. "Until then, I'll be right here," he said. 7:55 a.m. 32 arrested, including many clergy members Police said 32 were arrested and that nine of them were from Oakland. One came from France and many others from the East Coast. There were many clergy members among those arrested. Most were cited for failing to disperse. The mayor and police chief toured and surveyed the area for about 10 minutes, and then left for a media briefing. The area is sealed off. Police are planning to inventory everything, to make sure belongings are returned. A few dozen protesters are left at the barricaded intersection of 14th and Broadway, and streets are still blocked off in the immediate area. Three people remain in a tree, where police are keeping an eye on them. One tree is right off the 14th Street sidewalk. Protester Katia Ten of Oakland said she was glad police didn't lob tear gas canisters as they did the evening following the last raid, but she still would have preferred to see city leaders leave the camp alone or communicate better with the occupiers. "They're trying a different tactic but it's not much better," Ten said. "It's just a waste of everybody's time and money." 7:40 a.m. Snow Park camp remains Early Monday, about 20 tents remained loosely spread on the lawn near Lake Merritt. While there weren't any police to be seen around 7 a.m., camper Andre Little, 38, said he expected to be evicted soon. "But we'll come back," said Little, who has been with the Oakland movement since it began a month ago. Little said some of the people evicted from Frank Ogawa Plaza may show up at Snow Park, but he also expected them to regroup and recamp at the downtown location. Carly Jean, another Snow Park camper, said the raids were "inflicting fear" on people in the movement. "I don't think we're anti-organization," she said. "They just need to say, 'Let's talk.' It's not all about chaos." She said that in her mind, the movement is geared toward squatters' rights and the entitlement to occupy public spaces. "It's been a real awakening for a lot of people," she said. "Most people get it. And if they don't get it, they will. The truth is on our side." 7:20 a.m. Mayor's legal adviser resigns over raid Dan Siegel, Mayor Jean Quan's legal adviser, posted on Facebook that he has resigned over Monday's police raid of Occupy Oakland. His Facebook post: "No longer Mayor Quan's legal adviser. Resigned at 2 a.m. Support Occupy Oakland, not the 1 percent and its government facilitators." Siegel and Quan have been friends for decades, since they attended University of California, Berkeley together. Siegel was on Quan's transition team before she took office in January and stayed on as an adviser after that, drawing controversy when he openly opposed a gang injunction policy sought by the city attorney. 6:50 a.m. Police declare 'crime scene,' move out media The plaza has been cleared of media by police. A small group of protesters has taken up residence in a tree house in a sycamore along 14th Street. A van carrying Mayor Jean Quan, City Administrator Deanna Santana, Interim Police Chief Howard Jordan and a few other officials just left to tour the plaza. The crowd in the intersection of 14th and Broadway has thinned to about 100 protesters, facing down police on the west side of Broadway. 6:30 a.m. About 20 arrested so far Police have arrested about 20 protesters so far and have begun dismantling the camp. Hundreds of protesters remain on Broadway and 14th. While they can't return to the camp, police are not ordering them to leave. In the camp, police are taking down tents and making arrests. Everything remains peaceful. Police arrested 14 protesters who had been praying all night in the interfaith tent all night amid by candles. The protesters, who had planned to peacefully resist the raid, sang "We Shall Overcome" as the police arrested them. "Our plan is to remain here," said Kuhwald, a Unitarian Universalist minister, speaking as police began to surround the plaza. Nearby protesters yelled "The British are coming! The British are coming" as police officers marched up a wide alleyway, one of several that lead to the plaza from nearby streets, but the people inside the interfaith tent remained calm. The group planned on being arrested in a peaceful act of civil disobedience. Several individual protesters also chose to get arrested. Brandon Walsh, 33, an Oakland bike mechanic, said calmly that he was "passively occupying" despite police orders to leave the camp. The Oakland resident had not been living at the camp, but wanted to do something to support Occupy Oakland and its battle against economic disparities. "I have the privilege of having a voice and the luxury to do something with it," he said. "I'd prefer not to be, but I'm not going to leave." Protesters continue to affirm that the raid will not hinder the movement. "The campers are going to be back in a day or two," said engineering student Mark L., who identifies as a Republican. "Police brutality has galvanized the X-Box generation. If they'd just ignored it in Oakland or Zuccotti Park, it would have gone away." Many of the people who were the last holdouts in the plaza were calm because they were planning to get arrested, while others were battling anxiety about what could happen. "I'm trying to remove all emotion from my thinking, to just think really logically," said Hayward resident and Cal State East Bay student Kevin Shields, 18, as he stared up into the night sky. Like many campers, he eventually decided to leave as police ordered all occupiers to get out or be arrested. 6 a.m. Police start making arrests Police are starting to make arrests, of a group of interfaith protesters gathered in a circle near the plaza. The tent city has yet to be dismantled. On the other side of the plaza, in the amphitheater, Paul Bloom, of San Francisco is waiting to be arrested. He had been at the Wall Street Occupy Camp, but returned after the first Oakland raid. He has been camping for the last four days. "I want to get arrested," he said, sitting near three others who were meditating. "I feel sad that we haven't communicated what we wanted to communicate. "This is a movement for future generations" 5:25 a.m. Camp deserted as police walk through The Occupy Oakland camp is looking desolate, abandoned. A police helicopter is hovering overhead, shining a light and announcing over a speaker that anyone there must leave now. But there's no one in the tents, it seems empty. There are some people nearby, lingering near the police line on the plaza. It seems about 30-40 tents were taken down in anticipation of the raid. Police have surrounded plaza at this point. There have been no reports of violence or police skirmishes with protesters. By the time police actually starting dismantling the tents, dawn was breaking. The moon was still over City Hall, but the sky was rapidly getting brighter and a little bluer. Although the vast majority of the tents were empty, police found and arrested at least one tent occupant. 5:15 a.m. Police surround camp, start to walk through Police have peacefully surrounded both the camp and the plaza. A line of police officers have encircled the plaza, and some are walking through the camp. They've also surrounded the more than 500 protesters in the intersection. They have not made any arrests, or clashed with protesters. They are letting people out but not letting anyone in 5:05 a.m. Police march on camp A couple hundred police in riot gear are trying to surround the camp. They are forming a line at least three officers deep at Broadway, across 14th Street. They have sealed off the crowd from the camp. Police haven't entered the camp yet. 4:45 a.m. Police closing in Police in riot gear are very close to the encampment, next to the Rotunda building. The line of police are about 50 feet from the camp. Most of the occupiers have left the encampment and moved to 14th street and Broadway. There are about 50 or 75 people still in camp, but the majority are now waiting in the intersection. The 12th Street BART station is closed, according to a BART advisory. 4:30 a.m. Police assembling downtown There's a large contingent of Fremont police officers at 14th and Franklin streets. Broadway is being blocked off in both directions, keeping traffic away. Several Fremont police officers in riot gear are standing next to a Fremont PD SUV, and Hayward police also have a van here. Protesters have announced several times through mic checks that these forces are moving on the camp, only to correct themselves a few moments later. 4:20 a.m. Many campers packing up Although many tents are still standing, the camp has become a patchwork as protesters starting packing up. At least 20 tents are coming down. One man who wouldn't give his name looked regretful as he packed up his things. "We are going to lose a lot of resources out here." Lara Bitar, 28, was helping collapse three of the camp's four "intifada tents." "It feels pretty sad because we built a community here, and now they can just come and destroy it," she said. "At the same time, this movement is about more than just the space here." Bitar said the group, which is affiliated with the Palestinian Youth Movement and the anti-Zionist Network, was putting the tents in storage to protect their belongings. "We left one just as a statement," she said. Other occupiers are moving their tents to a smaller camp at Snow Park, at 19th and Harrison streets. Bitar, who works as a news producer, said that the raid and any police brutality would only encourage the protesters. Many protesters have masks or have covered their faces covered in rags. The song being chanted by protesters in the street is: "We are Occupy; We are never going to die; Every time you kick us out; We are going to multiply." Meanwhile, DeLauer's Super Newsstand is open and doing brisk business selling water, coffee and snacks to the protesters. At about 4 a.m., the first helicopter of the night flew over the camp. 3:45 a.m. Police assembling at Coliseum Public safety officers are setting up a command post and are assembling at the Coliseum, KCBS is reporting. Meanwhile, at the intersection of 14th and Broadway, Occupy Oakland camper Randy Peppers of Pt Richmond decided to be pragmatic and pull up stakes early. He was pushing a cart with his camping gear. His rationale is that he can't be arrested if he's mobile. Peppers had been staying at the camp since it was reoccupied after the Oct. 25 raid. "I can't reoccupy if they take my tent away." 3:40 a.m. Protesters are prepare for an imminent raid About 500 people have spilled into the intersection of Broadway and 14th Street and are playing bucket drums and chanting. The crowd is younger than usual, and some protesters are passing out vinegar-drenched rags for protection against tear gas. Many campers are sitting in front of their tents waiting. About a dozen are praying in the camp's interfaith tent amid a half circle of candles. Sunday, 11 p.m. General Assembly At their nightly assembly on Sunday in the amphitheater at Frank Ogawa Plaza, speakers at Occupy Oakland warned of an imminent police raid but also went about their regular business. They began with an indigenous prayer ceremony honoring the Ohlone people of the East Bay. The camp later voted 215 to 8, with 11 abstentions, to declare the camp a sanctuary for "all immigrants with or without papers." The occupiers found less consensus on their next item, a resolution to respect a "diversity of tactics" in the Occupy movement, part of a long-running debate within the camp on whether its members should publicly condemn protesters who use vandalism or violence. To publicly denounce certain protest tactics, said supporters of the measure, would help the media sow divisions in the movement. "We're not going to include words like violence and nonviolence. Those are loaded words," said one speaker at Sunday's general assembly outside Oakland City Hall. Another speaker said she thought protest actions "should be decided by the people who participate in them." Others in the camp strongly opposed that philosophy and wanted Occupy Oakland to more explicitly embrace nonviolence. They also said discouraging public opinions amounted to censorship or a "code of silence." "Occupy Oakland needs to denounce violence, and it needs to do it strongly tonight," said one speaker. He didn't get his wish, as 60 percent of the assembly voted to honor the "diversity of tactics" approach. When the nearly 4-hour meeting ended, and amid looming rumors of a raid, the occupiers launched an "emergency dance party" they called the Occupocalypse. Staff writers Robert Salonga, Theresa Harrington, Chris De Benedetti, Matt O'Brien, Paul Rosynsky, Robert Dennis, Julia Prodis Sulek, Dana Hull, Cecily Burt and Hannah Dreier contributed to this report. About 1,000 Occupy Oakland protesters returned Monday night to Frank Ogawa Plaza, 12 hours after police evicted the movement's tent city, and debated a range of reactions from re-establishing the encampment to refocusing on community organizing. The city reopened the newly cleared plaza outside City Hall around 5 p.m. and said protesters could gather there around the clock. However, police said they would prevent camping from now on, and as the night went on, there were no tents in evidence. A few dozen police officers mingled amiably with protesters conducting a peaceful general assembly in the plaza's amphitheater, where the main topics were denunciations of corporate greed and strategies for the coming days. Most agreed to delay until Wednesday any decision on establishing another encampment. In the meantime, several suggested branching out beyond downtown to enlist a wider pool of steady supporters. Others promised to join Occupy movement rallies at UC Berkeley this week. To the next level "A lot of the people who started the camp want to see this move on to the next level," said Anthony Owens, a 40-year-old sales consultant from Oakland who has been a key activist. He said he didn't see much reason to restart a tent city. "I'd like to see the tents replaced with booths, community resources, workshops, teach-ins, and maybe even a kitchen still serving the homeless," Owens said. Earlier in the day, the tone was more strident. "If they (police) take over the camp, we're going to reoccupy," Ronald "Rasta" Jones, 31, an Oakland resident who had lived in the Occupy Oakland camp since its first day, Oct. 10, said before officers moved in around 5 a.m. to evict people. "Our objective is for them to keep spending money.... We're not going to stop." His emotions were echoed in angry chants at times throughout the day, but those had disappeared by 10 p.m., when amphitheater discussions had ended and the crowd was down to a handful. Police stayed to keep an eye on the plaza throughout the night. Mayor Jean Quan, who ordered the eviction, said the city was prepared to clear camps no matter how many times it took. "If you look across the country, we even know that we may have to go and move the encampments again," Quan said. "This is an international and national movement. This is their tactic." Still determined Boots Riley, a key organizer of Occupy Oakland, said the raids have not dampened the determination of the core protesters. "Whatever they do, they're just going to make us keep going," Riley said of city officials. "They're in a lose-lose situation. We're putting out the idea that the working class can organize itself, can withhold its labor and can cause them to have to deal with us. That idea is not going to go away by evicting this camp." Hundreds of officers from Oakland and 13 other law enforcement agencies carried out the predawn sweep of the encampment outside City Hall at 14th Street and Broadway, arresting 32 people without incident. The eviction followed three days of city warnings, which cut the size of the encampment from 180 tents to about 60. Those arrested were booked on suspicion of illegal lodging and remaining at the scene of a riot. Public Works crews spent the day cleaning the plaza and hauling away 36 tons of debris. In the afternoon, activists headed to the main Oakland library at 14th and Madison streets for a planning session, and then at about 5:15 p.m. marched back to the plaza. The crowd included several middle-aged office workers and children - a more diverse mix than the City Hall tent city, which by Sunday consisted largely of longtime homeless people, hard-core activists and anarchists. Reoccupying debate

Police in riot gear moved to clear out the Occupy Oakland camp early today, arresting dozens as protesters watched. Hundreds of police moved in at about 5am, according to the San Francisco Chronicle. Protesters had broken into two groups, with some defending the tent city and hundreds of others standing, marching, and dancing outside in protest. So far, 25 people have been arrested. Many tents were already deserted when police moved in, according to the San Jose Mercury News. One that wasn't: An "interfaith tent," where police arrested 14 who had been praying all night. As they were handcuffed, they sang "We Shall Overcome." Protesters had ample warning of the raid, and some had removed their tents in anticipation. But many said they would soon return. "Whatever they do, they're going to just make us keep going," one protester said. "The camp is not going to go away."

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What’s new this flu season? A few things are new this season: Flu vaccines have been updated to better match circulating viruses [the B/Victoria component was changed and the influenza A(H3N2) component was updated]. For the 2018-2019 season, the nasal spray flu vaccine (live attenuated influenza vaccine or “LAIV”) is again a recommended option for influenza vaccination of persons for whom it is otherwise appropriate. The nasal spray is approved for use in non-pregnant individuals, 2 to 49 years old. There is a precaution against the use of LAIV for people with certain underlying medical conditions. All LAIV will be quadrivalent (four-component). Most regular-dose egg-based flu shots will be quadrivalent. All recombinant vaccine will be quadrivalent. (No trivalent recombinant vaccine will be available this season.) Cell-grown flu vaccine will be quadrivalent. For this vaccine, the influenza A(H3N2) and both influenza B reference viruses will be cell-derived, and the influenza A(H1N1) will be egg-derived. All these reference viruses will be grown in cells to produce the components of Flucelvax. No intradermal flu vaccine will be available. The age recommendation for “Fluarix Quadrivalent” was changed from 3 years and older to 6 months and older after the annual recommendations were published last season to be consistent with Food and Drug Administration (FDA)-approved labeling. The age recommendation for Afluria Quadrivalent was changed from 18 years and older to 5 years and older after the annual recommendations were published last season to be consistent with Food and Drug Administration (FDA)-approved labeling. Baloxavir marboxil (trade name Xofluza®) is a new influenza single-dose antiviral drug approved October 24, 2018 by the Food and Drug Administration (FDA). Baloxavir is approved for the treatment of acute uncomplicated flu in people 12 years and older who have had flu symptoms for less than 48 hours. More information is available: Influenza Antiviral Drug Baloxavir Marboxil. CDC is reporting in-season estimates of the cumulative number of flu illnesses, medical visits and hospitalizations in the U.S. These Preliminary In-Season Burden Estimates will be updated weekly over the course of the flu season. What flu vaccines are recommended this season? For the 2018-2019 flu season, providers may choose to administer any licensed, age-appropriate flu vaccine (IIV, RIV4, or LAIV4). Options this season include: There is a table showing all flu vaccines that are FDA-approved for use in the United States during the 2018-2019 season. What viruses will the 2018-2019 flu vaccines protect against? There are many different flu viruses and they are constantly changing. The composition of U.S. flu vaccines is reviewed annually and updated as needed to match circulating flu viruses. Flu vaccines protect against the three or four viruses (depending on vaccine) that research suggests will be most common. For 2018-2019, trivalent (three-component) vaccines are recommended to contain: A/Michigan/45/2015 (H1N1)pdm09-like virus A/Singapore/INFIMH-16-0019/2016 A(H3N2)-like virus (updated) B/Colorado/06/2017-like (Victoria lineage) virus (updated) Quadrivalent (four-component) vaccines, which protect against a second lineage of B viruses, are recommended to contain: the three recommended viruses above, plus B/Phuket/3073/2013-like (Yamagata lineage) virus When should I get vaccinated? You should get a flu vaccine before flu begins spreading in your community. It takes about two weeks after vaccination for antibodies that protect against flu to develop in the body, so make plans to get vaccinated early in fall, before flu season begins. CDC recommends that people get a flu vaccine by the end of October. Getting vaccinated later, however, can still be beneficial and vaccination should continue to be offered throughout flu season, even into January or later. Children who need two doses of vaccine to be protected should start the vaccination process sooner, because the two doses must be given at least four weeks apart. Can I get a flu vaccine if I am allergic to eggs? The recommendations for people with egg allergies are the same as last season. People who have experienced only hives after exposure to egg can get any licensed flu vaccine that is otherwise appropriate for their age and health. People who have symptoms other than hives after exposure to eggs, such as angioedema, respiratory distress, lightheadedness, or recurrent emesis; or who have needed epinephrine or another emergency medical intervention, can also get any licensed flu vaccine that is otherwise appropriate for their age and health, but the vaccine should be given in a medical setting and be supervised by a health care provider who is able to recognize and manage severe allergic conditions. (Settings include hospitals, clinics, health departments, and physician offices). People with egg allergies no longer have to wait 30 minutes after receiving their vaccine. Implications of Cell-Based Vaccines Why is it significant that cell-grown vaccine reference viruses are used to produce some components of one type of flu vaccine? Cell-grown reference viruses do not have the changes that are present in egg-grown reference viruses, so they should be more similar to circulating “wild-type” viruses. Vaccine effectiveness depends in part on the match between the vaccine virus and circulating flu viruses. Is flu vaccine made using a cell-grown reference virus and cell-based technology more effective than vaccine made using an egg-grown reference virus and egg-based technology? While the use of cell-grown reference viruses and cell-based technology may offer the potential for better protection over traditional, egg-based flu vaccines because they result in vaccine viruses that are more similar to flu viruses in circulation, there are no data yet to support this. There is no preferential recommendation for one injectable flu vaccine over another. Flu Activity What sort of flu season is expected this year? It is not possible to predict what this flu season will be like. While flu spreads every year, the timing, severity, and length of the season varies from one season to another. Will new flu viruses circulate this season? Flu viruses are constantly changing so it’s not unusual for new flu viruses to appear each year. More information about how flu viruses change is available. Will the United States have a flu epidemic? The United States experiences annual epidemics of seasonal flu. This time of year is called “flu season.” In the United States, flu viruses are most common during the fall and winter months. Influenza activity often begins to increase in October and November. Most of the time flu activity peaks between December and February, and it can last as late as May. CDC monitors certain key flu indicators (for example, outpatient visits of influenza-like illness (ILI), the results of laboratory testing and reports of flu hospitalizations and deaths). When these indicators rise and remain elevated for a number of consecutive weeks, “flu season” is said to have begun. Usually ILI increases first, followed by an increase in flu-associated hospitalizations, which is then followed by increases in flu-associated deaths. For the most current influenza surveillance information: Weekly U.S. Influenza Surveillance Report. When will flu activity begin and when will it peak? The timing of flu is unpredictable and can vary in different parts of the country and from season to season. Seasonal flu viruses can be detected year-round; however, seasonal flu activity often begins as early as October and November and can continue to occur as late as May. Flu activity most commonly peaks in the United States between December and February. How many people get sick with flu every year? CDC conducts surveillance for people who see their health care provider for flu-like illness through the Outpatient Influenza-like Illness Surveillance Network (ILINet); a network of thousands of health care providers who report the proportion of patients seeking care for flu-like illness weekly to CDC. This system allows CDC to track levels of medically attended flu-like illness over the course of the flu season. CDC does not know exactly how many people get sick with seasonal flu each year. There are several reasons for this including that ILINet does not include every health care provider and monitors flu-like illness, not laboratory-confirmed influenza cases. Also, flu illness is not a reportable disease and not everyone who gets sick with flu seeks medical care or gets tested. CDC uses mathematical modeling in combination with data from traditional flu surveillance systems to estimate the numbers of flu illnesses in the United States. CDC estimates that flu has resulted in between 9.3 million and 49 million illnesses each year in the United States since 2010. More information on these estimates is available on CDC’s Disease Burden of Influenza page. During the 2018-2019 flu season, CDC began reporting cumulative, in-season estimates of the disease burden of influenza, including estimates of the total number of flu illnesses in the United States. More information regarding these Preliminary In-Season Burden Estimates is available. For more information on CDC surveillance systems, see CDC’s Overview of Influenza Surveillance in the United States. How many people are hospitalized from flu every year? CDC conducts surveillance for flu-related hospitalizations through the Influenza Hospitalization Surveillance Network (FluSurv-NET), a collaboration between CDC, the Emerging Infections Program, and additional Influenza Hospitalization Surveillance Project (IHSP) states in 13 geographically distributed areas in the United States. The network includes hospitals that serve roughly 9 percent of the U.S. population. The data collected through FluSurv-NET allows CDC to calculate an overall hospitalization rate, as well as by age group, but this system does not provide the total number of flu hospitalizations that actually occur in the United States. Reported FluSurv-NET hospitalization rates are adjusted to correct for under-detection, which is calculated from the percent of persons hospitalized with respiratory illness who were tested for influenza and the average sensitivity of influenza tests used in the participating FluSurv-NET surveillance hospitals. CDC uses these methods to estimate the true burden of flu hospitalizations in the United States. Since 2010, CDC estimates that flu has resulted in between 140,000 and 960,000 hospitalizations each year. For more information on these estimates see CDC’s Disease Burden of Influenza page. During the 2018-2019 flu season, CDC began reporting cumulative, in-season estimates of disease burden of influenza, including estimates of the total number of people hospitalized in the United States from flu. More information regarding these Preliminary In-Season Burden Estimates is available. For more information on CDC surveillance systems, see CDC’s Overview of Influenza Surveillance in the United States. How many adults die from flu each year? Flu deaths in adults are not nationally notifiable. In order to monitor influenza related deaths in all age groups, CDC tracks pneumonia and influenza (P&I)–attributed deaths through the National Center for Health Statistics (NCHS) Mortality Reporting System. This system tracks the proportion of death certificates processed that list pneumonia or influenza as the underlying or contributing cause of death. This system provides an overall indication of whether flu-associated deaths are elevated, but does not provide an exact number of how many people died from flu. As it does for the numbers of flu cases, doctor’s visits and hospitalizations, CDC also estimates deaths in the United States using mathematical modeling. CDC estimates that from 2010-2011 to 2017-2018, influenza-associated deaths in the United States ranged from a low of 12,000 (during 2011-2012) to a high of 79,000 (during 2017-2018). The model used to estimate flu-associated deaths uses a ratio of deaths-to-hospitalizations in order to estimate the total flu-related deaths during a season. For more information: How CDC Estimates Burden. As data allow, CDC will begin reporting cumulative, in-season estimates of the influenza mortality burden. More information regarding these Preliminary In-Season Burden Estimates is available. For more information: Overview of Influenza Surveillance in the United States, “Mortality Surveillance.” How many children die from flu each year? Influenza-associated deaths in children (people younger than 18) became nationally reportable in 2004. Since that time the number of pediatric flu deaths reported to CDC each year has ranged from 37 (2011-2012 season) to 185 deaths (2017-2018 season). It’s important to note that the actual number of flu deaths in children is thought to be higher than what is reported by states to CDC because not all flu deaths in children are detected/reported. CDC also estimates the numbers of flu-related deaths using statistical models. Estimates of deaths in children since 2010 have ranged from 37 (2011-2012) to about 1,200 (2012-2013). CDC believes these estimated numbers of pediatric deaths are likely a better estimate of the number of pediatric flu deaths. For more information on these estimates see CDC’s Disease Burden of Influenza page. Why is it difficult to know exactly how many people die from flu? There are several factors that make it difficult to determine accurate numbers of deaths caused by flu regardless of reporting. Some of the challenges in counting flu associated deaths include the following: the sheer volume of deaths to be counted; the lack of testing (not everyone that dies with an influenza-like illness is tested for influenza); and the different coding of deaths (influenza-associated deaths often are a result of complications secondary to underlying medical problems, and this may be difficult to sort out). For more information: Estimating Seasonal Influenza-Associated Deaths in the United States. Protective Actions What should I do to protect myself from flu this season? CDC recommends a yearly flu vaccine for everyone 6 months of age and older as the first and most important step in protecting against this serious disease. In addition to getting a seasonal flu vaccine, you can take everyday preventive actions like staying away from sick people and washing your hands to reduce the spread of germs. If you are sick with flu, stay home from work or school to prevent spreading flu to others. In addition, there are prescription medications called antiviral drugs that can be used to treat influenza illness. What should I do to protect my loved ones from flu this season? Encourage your loved ones to get vaccinated. Vaccination is especially important for people at high risk for developing flu complications, and their close contacts. Also, if you have a loved one who is at high risk of flu complications and they develop flu symptoms, encourage them to get a medical evaluation for possible treatment with flu antiviral drugs. These drugs work best if given within 48 hours of when symptoms start. CDC recommends that people who are at high risk for serious flu complications and who get flu symptoms during flu season be treated with flu antiviral drugs as quickly as possible without waiting for confirmatory testing. People who are not at high risk for serious flu complications may also be treated with flu antiviral drugs, especially if treatment can begin within 48 hours. Do some children require two doses of flu vaccine? Yes. Some children 6 months through 8 years of age will require two doses of flu vaccine for adequate protection from flu. Children in this age group who are getting vaccinated for the first time will need two doses of flu vaccine, spaced at least 4 weeks apart. Children who have only received one dose in their lifetime also need two doses. Your child’s doctor or other health care professional can tell you if your child needs two doses of flu vaccine. What can I do to protect children who are too young to get vaccinated? Children younger than 6 months old are at high risk of serious flu complications, but are too young to get a flu vaccine. Because of this, safeguarding them from flu is especially important. If you live with or care for an infant younger than 6 months old, you should get a flu vaccine to help protect them from flu. Advice for Caregivers of Young Children is available for more information. Everyone else who is around the baby also should be vaccinated. Also, studies have shown that flu vaccination of the mother during pregnancy can protect the baby after birth from flu infection for several months. In addition to getting vaccinated, you and your loved ones can take everyday preventive actions like staying away from sick people and washing your hands to reduce the spread of germs. If you are sick with flu, stay home from work or school to prevent spreading flu to others. Vaccine and Vaccination How much flu vaccine will be available this season? Flu vaccine is produced by private manufacturers, so supply depends on manufacturers. For the 2018-2019 season, manufacturers projected they would provide between 163 million and 168 million doses of injectable vaccine for the U.S. market. (Projections may change as the season progresses.) Flu vaccine supply updates will be provided as they become available at Seasonal Influenza Vaccine & Total Doses Distributed. Are any of the available flu vaccines recommended over the others? For the 2018-2019 flu season, ACIP recommends annual influenza vaccination for everyone 6 months and older with any licensed, age-appropriate flu vaccine (IIV, RIV4, or LAIV4) with no preference expressed for any one vaccine over another. There are many vaccine options to choose from; the most important thing is for all people 6 months and older to get a flu vaccine every year. If you have questions about which vaccine is best for you, talk to your doctor or other health care professional. Why is the nasal spray being recommended as an option this year when it has been shown to not be effective in past flu seasons? While observational data from 2010-11 through 2015-16 flu seasons indicate that LAIV was not effective among 2 through 17-year-olds against H1N1pdm09 influenza viruses in the U.S., LAIV was effective against influenza B viruses, and was similarly effective to inactivated influenza vaccines against H3N2 viruses. Some data suggest that the new H1N1 vaccine virus included in the new LAIV vaccines will have improved effectiveness against circulating H1N1 viruses; however, no published effectiveness estimates are available yet. When should I get vaccinated? Getting vaccinated before flu activity begins helps protect you once flu season starts in your community. It takes about two weeks after vaccination for the body’s immune response to fully respond and for you to be protected, so make plans to get vaccinated. CDC recommends that people get a flu vaccine by the end of October. However, getting vaccinated later can still be beneficial. CDC recommends ongoing flu vaccination as long as influenza viruses are circulating, even into January or later. Children 6 months to 8 years old who need two doses of vaccine should get the first dose as soon after vaccine is available to allow time to get the second dose before the start of flu season. The two doses should be given at least 4 weeks apart. Where can I get a flu vaccine? Flu vaccines are offered by many doctor’s offices, clinics, health departments, pharmacies and college health centers, as well as by many employers, and even by some schools. Even if you don’t have a regular doctor or nurse, you can get a flu vaccine somewhere else, like a health department, pharmacy, urgent care clinic, and often your school, college health center, or work. The HealthMap Vaccine Finder helps you to locate where you can get a flu vaccine. What is flu vaccination using a jet injector? The U.S. Food and Drug Administration (FDA) has approved two influenza vaccines (Afluria and Afluria Quadrivalent) for administration by a jet injector device (the PharmaJet Stratis 0.5ml Needle-free Jet Injector) for people 18 through 64 years of age. These are the only two influenza vaccines approved for administration by jet injector. People aged 18 through 64 years may receive these vaccines either by jet injector or needle. A jet injector is a medical device used for vaccination that uses a high-pressure, narrow stream of fluid to penetrate the skin instead of a hypodermic needle. More information about Flu Vaccination by Jet Injector is available. What is adjuvanted flu vaccine? The U.S. Food and Drug Administration (FDA) licensed a seasonal influenza (flu) vaccine containing adjuvant for adults 65 years of age and older. An adjuvant is an ingredient added to a vaccine to create a stronger immune response to vaccination. FLUAD™[155 KB, 13 pages] was licensed in November 2015 and will be available during the 2018-2019 flu season. It contains the MF59 adjuvant, an oil-in-water emulsion of squalene oil. FLUAD™ is the first adjuvanted seasonal flu vaccine marketed in the United States. How long does a flu vaccine protect me from getting flu? Multiple studies conducted over different seasons and across flu vaccine types and influenza virus subtypes have shown that the body’s immunity to influenza viruses (acquired either through natural infection or vaccination) declines over time. The decline in antibodies is influenced by several factors, including the antigen used in the vaccine, the age of the person being vaccinated, and the person’s general health (for example, certain chronic health conditions may have an impact on immunity). Older people and others with weakened immune systems may not generate the same amount of antibodies after vaccination; further, their antibody levels may drop more quickly when compared to young, healthy people. Getting vaccinated each year provides the best protection against flu throughout flu season. It’s important to get a flu vaccine every season, even if you got vaccinated the season before and the viruses in the flu vaccine have not changed for the current season. Can I get vaccinated and still get the flu? Yes. It’s possible to get sick with flu even if you have been vaccinated (although you won’t know for sure unless you get a flu test). This is possible for the following reasons: You may be exposed to a flu virus shortly before getting vaccinated or during the period that it takes the body to gain protection after getting vaccinated. This exposure may result in you becoming ill with flu before the vaccine begins to protect you. (Antibodies that provide protection develop in the body about 2 weeks after vaccination.) You may be exposed to a flu virus that is not included in the seasonal flu vaccine. There are many different flu viruses that circulate every year. A flu vaccine is made to protect against the three or four flu viruses that research suggests will be most common. Unfortunately, some people can become infected with a flu virus a flu vaccine is designed to protect against, despite getting vaccinated. Protection provided by flu vaccination can vary widely, based in part on health and age factors of the person getting vaccinated. In general, a flu vaccine works best among healthy younger adults and older children. Some older people and people with certain chronic illnesses may develop less immunity after vaccination. Flu vaccination is not a perfect tool, but it is the best way to protect against flu infection. Flu Vaccine Effectiveness How effective will flu vaccines be this season? Influenza vaccine effectiveness (VE) can vary from season to season and among different age and risk groups and even by vaccine type. How well the vaccine works can depend in part on the match between the vaccine viruses used to produce vaccine and circulating viruses that season. It’s not possible to predict in advance what flu viruses will predominate. CDC monitors circulating viruses throughout the year and provides new and updated information about their similarity to flu vaccine viruses as it becomes available. Information is published weekly in FluView and summarized at intervals in the Morbidity and Mortality Weekly Report (MMWR). Vaccine effectiveness estimates are also provided when they become available. While vaccine effectiveness can vary, recent studies show vaccine reduces the risk of flu illness by about 40% to 60% among the overall population during seasons when most circulating flu viruses are like the vaccine viruses. Similar reductions against hospitalization have been observed too. More information about previous vaccine effectiveness, is available. Will this season’s flu vaccine be a good match for circulating viruses? It’s not possible to predict with certainty if a flu vaccine will be a good match for circulating flu viruses. A flu vaccine is made to protect against the flu viruses that research and surveillance indicate will likely be most common during the season. However, experts must pick which flu viruses to include in a flu vaccine many months in advance in order for flu vaccines to be produced and delivered on time. Also flu viruses change constantly (called “drift”). They can change from one season to the next or they can even change within the course of one flu season. Another factor that can impact vaccine effectiveness, especially against influenza A(H3N2) viruses, are changes that can occur in vaccine viruses as they are grown in eggs, which is the production method for most current flu vaccines. Because of these factors, there is always the possibility of a less than optimal match between circulating flu viruses and the viruses in a flu vaccine. Over the course of flu season, CDC studies samples of circulating flu viruses to evaluate how close a match there is between viruses used to make the flu vaccine and circulating flu viruses. One of the ways that helps CDC evaluate the match between flu vaccine viruses and circulating flu viruses is with a lab process called ‘genetic and antigenic characterization’. Results of genetic and antigenic characterization testing are published weekly in CDC’s FluView. Can a flu vaccine provide protection even if the flu vaccine is not a “good” match? Yes, antibodies made in response to vaccination with one flu virus can sometimes provide protection against different but related flu viruses. A less than ideal match may result in reduced vaccine effectiveness against the flu virus that is different from what is in the flu vaccine, but it might still provide some protection against flu illness. In addition, it’s important to remember that a flu vaccine contains three or four flu viruses (depending on the type of vaccine you receive) so that even when there is a less than ideal match or lower effectiveness against one virus, a flu vaccine may protect against the other flu viruses. For these reasons, even during seasons when there is a less than ideal match, CDC continues to recommend flu vaccination for everyone 6 months and older. Vaccination is particularly important for people at high risk for serious flu complications), and their close contacts. If You Get Sick What happens in the body when someone has flu? Influenza viruses usually infect the respiratory tract (i.e., the airways of the nose, throat and lungs). As the infection progresses, the body’s immune system responds to fight the virus. This results in inflammation that can trigger respiratory symptoms such as cough and sore throat. The immune system response can also trigger fever and cause muscle or body aches. When infected persons cough, sneeze, or talk, they can spread influenza viruses in respiratory droplets to people who are nearby. People might also get flu by touching a contaminated surface or object that has flu virus on it and then touching their own mouth or nose. Most people who become sick will recover in a few days to less than two weeks, but some people may become more severely ill. Following flu infection, moderate complications such as secondary ear and sinus infections can occur. Pneumonia is a serious flu complication that can result from either influenza virus infection alone or from co-infection of flu virus and bacteria. Other possible serious complications triggered by flu can include inflammation of the heart (myocarditis), brain (encephalitis) or muscle (myositis, rhabdomyolysis) tissues, and multi-organ failure (for example, respiratory and kidney failure). Severe complications can happen to anyone, but may be more likely to happen to people who have certain chronic medical conditions, or in elderly persons. What should I do if I get sick with flu? Most people with flu have mild illness and do not need medical care or antiviral drugs. If you get sick with flu symptoms, in most cases, you should stay home and avoid contact with other people except to get medical care. If, however, you have symptoms of flu and are at high risk of flu complications, or are very sick or concerned about your illness, contact your health care provider. There are drugs your doctor may prescribe for treating flu called antivirals. These drugs can make you better faster and may also prevent serious complications. Antiviral drugs are prescription drugs that can be used to treat flu illness. People at high risk of serious flu complications recommended for prompt antiviral treatment include children younger than 2 years of age (although all children younger than 5 years are considered at higher risk for complications from influenza, the highest risk is for those younger than 2 years of age), adults 65 years of age and older, pregnant women, people with certain long-term medical conditions, and residents of nursing homes and other long-term care facilities). Antiviral treatment as early as possible is also recommended for people who are very sick with flu (such as those with complicated, progressive illness or people hospitalized because of flu). Other people can be treated with antivirals at their health care professional’s discretion. Treating high risk people or people who are very sick with flu with antiviral drugs is very important. Studies show that prompt treatment with antiviral drugs can prevent serious flu complications. Prompt treatment can mean the difference between having a milder illness versus very serious illness that could result in a hospital stay. Treatment with antivirals works best when begun within 48 hours of getting sick, but can still be beneficial when given later in the course of illness. Antiviral drugs are effective across all age and risk groups. Studies show that antiviral drugs are under-prescribed for people who are at high risk of complications who get flu. Four FDA-approved antiviral medications are recommended for use during the 2018-2019 flu season: oseltamivir (available in generic versions and under the trade name Tamiflu®), zanamivir (Relenza®), peramivir (Rapivab®), and baloxavir marboxil (Xofluza®). More information about antiviral drugs can be found at Treatment – Antiviral Drugs. This guidance is consistent with the 2018-2019 flu season recommendations published by the Infectious Diseases Society of America (IDSA) on December 19 in Clinical Infectious Diseases. “The Flu: What To Do If You Get Sick” What is baloxavir marboxil? Baloxavir marboxil (trade name Xofluza®) is an influenza single-dose antiviral drug approved October 24, 2018 by the Food and Drug Administration (FDA). Baloxavir is approved for the treatment of acute uncomplicated flu in people 12 years and older who have had flu symptoms for less than 48 hours. In clinical randomized trials, baloxavir was similar to oseltamivir, a currently recommended flu antiviral drug, in alleviating flu symptoms. More information regarding baloxaviris available: Influenza Antiviral Drug Baloxavir Marboxil. Surveillance How does CDC track flu activity? The Epidemiology and Prevention Branch in the Influenza Division at CDC collects, compiles and analyzes information on flu activity year round in the United States and produces FluView, a weekly influenza surveillance report, and FluView Interactive, which allows for more in-depth exploration of influenza surveillance data. The U.S. influenza surveillance system is a collaborative effort between CDC and its many partners in state, local, and territorial health departments, public health and clinical laboratories, vital statistics offices, healthcare providers, clinics, and emergency departments. Information in five categories is collected from eight different data sources that allow CDC to: Find out when and where influenza activity is occurring Track influenza-related illness Determine what influenza viruses are circulating Detect changes in influenza viruses Measure the impact influenza is having on hospitalizations and deaths in the United States For more information, visit “Overview of Influenza Surveillance in the United States”. What will CDC do to monitor flu vaccine effectiveness for the 2018-2019 season? CDC collaborates with partners each season to assess how well the seasonal flu vaccines are working. During the 2018-2019 season, CDC is planning multiple studies on the effectiveness of flu shots. These studies measure vaccine effectiveness in preventing laboratory-confirmed influenza among persons 6 months of age and older. A summary of CDC’s latest vaccine effectiveness estimates is available at Seasonal Influenza Vaccine Effectiveness, 2005-2018. What is CDC doing to monitor antiviral resistance in the United States during the 2018-2019 season? Antiviral resistance means that a virus has changed in such a way that antiviral drugs are less effective or not effective at all in treating or preventing illnesses with that virus. CDC will continue to collect and monitor flu viruses for changes through an established network of domestic and global surveillance systems. CDC also is working with the state public health departments and the World Health Organization to collect additional information on antiviral resistance in the United States and worldwide. The information collected will assist in making informed recommendations regarding use of antiviral drugs to treat influenza. How does CDC estimate the burden of seasonal flu on the United States? The burden of influenza on the United States can vary widely from season to season and is determined by a number of factors including the characteristics of circulating viruses, the timing of the season, population immunity to circulating viruses, how well flu vaccines are working, and how many people have gotten vaccinated. While the impact of flu varies, it places a substantial burden on the health of people in the United States each year. CDC uses a model to estimate the numbers of influenza illnesses, medical visits, and hospitalizations in the United States (as well as the impact of influenza vaccination on these numbers). The same model is also extended to estimate flu-related deaths in the United States. This methodology has been used to retroactively calculate influenza burden, including deaths, going back to 2010. Starting with the 2018-2019 flu season, CDC will report cumulative, in-season estimates of the burden of influenza. These Preliminary In-Season Burden Estimates will be updated weekly over the course of the flu season. How does CDC classify flu season severity? In 2017, CDC adopted and outlined a new methodology for determining flu season severity. Based on data from past flu seasons, CDC researchers used key flu indicator data to develop intensity thresholds (ITs) to classify the severity of flu seasons. Based on the intensity thresholds, CDC researchers classified seasonal severity from 2003-2004 through the 2017-2018 flu seasons. Overall, four seasons were classified as low severity, seven as moderate, two as high, and none as very high. What is flu forecasting? Influenza (flu) places a significant disease burden on the U.S. population each year, but the magnitude and timing varies from season to season, making the annual impact difficult to predict at the beginning of each season. Flu forecasting can change that by predicting in advance when the start, peak, and increases in flu activity will occur. Unlike CDC’s traditional influenza surveillance systems, which measure influenza activity after it has occurred, flu forecasting offers the possibility to look into the future and better plan ahead, potentially reducing the impact of flu. A Florida child who hadn't received a flu vaccination died from the virus in late September or early October, according to the state's department of health. Interested in Flu Season? Add Flu Season as an interest to stay up to date on the latest Flu Season news, video, and analysis from ABC News. Add Interest It's the first reported pediatric death in Florida for the 2018-19 flu season. Vaccinations have been shown to reduce a child's likelihood of dying from the flu by 60 percent, health department officials said. Vaccines are recommended for anyone at least six months old. The unidentified child was otherwise healthy before getting the flu, according to the Florida Department of Health's Bureau of Epidemiology. The child tested positive for influenza B at a local health care provider. About 80,000 Americans, including 180 children, died last year in the deadliest flu season in more than 40 years, according to the Centers for Disease Control and Prevention. Approximately 80 percent of those who died were not vaccinated. A child in Florida has become the first person to die of the flu this season, according to state health officials. State epidemiologists say the child had not been vaccinated and was otherwise healthy before getting sick with the flu. The child, who tested positive for influenza B, died sometime during the week of Sept. 30, although privacy concerns prevent officials from saying exactly where, CBS affiliate WTSP-TV reports. Last flu season, 183 children in the U.S. died from flu or flu-related causes. That's the most since the Centers for Disease Control and Prevention (CDC) began keeping these records in 2004. Overall, an estimated 80,000 Americans died from flu last season. CBS News medical contributor Dr. Tara Narula says this latest news should be a wake-up call to parents to get their children vaccinated. "What this is a strong clear message to parents about the importance of vaccination," she told "CBS This Morning." "This vaccine is safe. It is the most effective tool we have. And we know of the pediatric deaths last year, 80 percent were in kids who were unvaccinated." A new survey suggests that many children may not be getting the potentially life-saving flu shot because of their parents' misconceptions about the safety and importance of vaccines. The survey by Orlando Health Arnold Palmer Hospital found: More than half of parents think that their child can get the flu from the flu shot. 30 percent of parents feel flu vaccines are a conspiracy. 28 percent of parents believe flu vaccines can cause autism. "None of these things are true. It's important that we deal with the science and the facts," Narula said. The CDC recommends everyone age 6 months and older get vaccinated against the flu every year. "Officials have said it's like wearing a seat belt," Narula said. "This is really a no-brainer for parents."

An unvaccinated child in Florida has become the first death of flu season, officials say. The child, whose age and hometown has not been disclosed, died between Sept. 30 and Oct. 6 of influenza B, ABC reports. Officials say the child was otherwise healthy, with no underlying conditions. According to the Centers for Disease Control and Prevention, some 80,000 Americans died during the brutal 2017-18 flu season, including 183 children who died from flu or flu-related causes, the highest number since the CDC starting keeping records on nationwide pediatric influenza deaths in 2004. Around 80% of the children who died had not received the vaccine. Health officials recommend flu vaccines for anybody at least 6 months old. The CDC's vaccination fact sheet suggests people get vaccinated in early fall. The death of the Florida child should be a wake-up call for parents, says CBS medical contributor Dr. Tara Narula. This "is a strong clear message to parents about the importance of vaccination," she says. Despite misconceptions, she says, "this vaccine is safe. It is the most effective tool we have. And we know of the pediatric deaths last year, 80% were in kids who were unvaccinated." A recent study found that more than 50% of parents believe that the vaccine can give their child influenza, and 28% believe it can lead to autism.

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Highly pathogenic H5N1 influenza A viruses have spread across Asia, Europe, and Africa. More than 500 cases of H5N1 virus infection in humans, with a high lethality rate, have been reported. To understand the molecular basis for the high virulence of H5N1 viruses in mammals, we tested the virulence in ferrets of several H5N1 viruses isolated from humans and found A/Vietnam/UT3062/04 (UT3062) to be the most virulent and A/Vietnam/UT3028/03 (UT3028) to be avirulent in this animal model. We then generated a series of reassortant viruses between the two viruses and assessed their virulence in ferrets. All of the viruses that possessed both the UT3062 hemagglutinin (HA) and nonstructural protein (NS) genes were highly virulent. By contrast, all those possessing the UT3028 HA or NS genes were attenuated in ferrets. These results demonstrate that the HA and NS genes are responsible for the difference in virulence in ferrets between the two viruses. Amino acid differences were identified at position 134 of HA, at positions 200 and 205 of NS1, and at positions 47 and 51 of NS2. We found that the residue at position 134 of HA alters the receptor-binding property of the virus, as measured by viral elution from erythrocytes. Further, both of the residues at positions 200 and 205 of NS1 contributed to enhanced type I interferon (IFN) antagonistic activity. These findings further our understanding of the determinants of pathogenicity of H5N1 viruses in mammals. In 1997, the first human case of influenza caused by an H5N1 virus occurred in Hong Kong [1], [2]. In 2003, a new outbreak of H5N1 virus was identified in Vietnam. Since then, H5N1 viruses have spread across Asia, Europe and Africa. As of July 22,2010,501 cases of H5N1 virus infections in humans have been reported by the World Health Organization (WHO; http: //www. who. int/en/), 297 of which were fatal. The mortality is, therefore, approximately 60%. H5N1 viruses have been characterized by using a variety of mammalian models [3]. In mice, enhanced HA cleavability, as well as lysine at position 627 of the polymerase subunit PB2, plays an important role in the virulence of H5N1 viruses [4]. Viruses possessing these properties replicate systemically and cause death in mice. Ferrets are considered suitable for evaluating infection of human influenza viruses because these viruses replicate in the upper respiratory tract without adaptation in ferrets, and some strains cause severe pneumonia in these animals. Some of the H5N1 viruses isolated from humans can kill ferrets, whereas H5N1 viruses isolated from birds tend to cause mild disease in this animal model [5], [6]. Systemic infection, high replication efficiencies, and neurovirulence are associated with the high lethality of human H5N1 viruses in ferrets. Salomon et al. [7] reported that the genes encoding the nonstructural proteins (NS) and polymerase complex are important for the lethality of the human H5N1 virus A/Vietnam/1203/04 in ferrets, compared with the avian H5N1 virus A/quail/Vietnam/36/04. However, the molecular bases for the high virulence of H5N1 viruses in ferrets are not fully understood. To advance our understanding of the pathogenicity of H5N1 viruses, we compared the virulence of H5N1 influenza viruses isolated from humans in a ferret model. By generating reassortant viruses between the most virulent A/Vietnam/UT3062/04 (UT3062) virus and the avirulent A/Vietnam/UT3028/03 (UT3028) virus, we identified the genes responsible for high virulence in ferrets. We also performed in vitro studies to determine the molecular mechanisms by which H5N1 viruses exhibit high virulence in mammals. To compare the virulence of H5N1 influenza viruses isolated from humans in ferrets, we intranasally inoculated 5- to 7-month-old male animals (n = 3) with 107 plaque-forming units (PFU) of virus and observed the lethality, changes in body weight and body temperature, clinical signs, and virus shedding in the upper respiratory tract of the virus-infected animals (Table 1). The UT3062, A/Vietnam/UT3040/04, A/Vietnam/UT3028II/03, A/Vietnam/UT30850/05, A/Vietnam/UT3030/03, A/Vietnam/UT3040II/04, and A/Vietnam/UT3047III/04 were virulent in ferrets, causing the deaths of the virus-infected animals. These virulent viruses, with the exception of A/Vietnam/UT3028II/03, caused mean maximum weight loss of 9. 1%–18. 4% and anorexia, consistent with previous studies [5], [6]. Systemic viral infection was observed in most of the fatally infected animals (Table S1). Notably, inoculation of animals with UT3062 resulted in 100% lethality with 15. 4±2. 7% mean maximum weight loss (Table 1). These results demonstrate that UT3062 is the most virulent in ferrets of the viruses we tested. By contrast, six other human H5N1 viruses, A/Vietnam/UT30259/04, A/Vietnam/UT3035/03, A/Indonesia/UT3006/05, UT3028, A/Vietnam/UT30408III/05, and A/Vietnam/UT30262III/04 did not kill any ferrets and all, except A/Vietnam/UT30259/04, caused limited body weight loss (1. 6%–6. 6% mean maximum weight loss, median 4. 2%) (Table 1), indicating that H5N1 viruses isolated from humans differ in their virulence in ferrets. Among the H5N1 viruses listed in Table 1, two viruses, A/Vietnam/UT3035/03 and A/Vietnam/UT30408III/05, which did not kill any ferrets, were isolated from patients who recovered from their H5N1 virus infections. The rest of the viruses used were isolated from patients who ultimately died. Of note, the virulence of test viruses in ferrets generally correlated with that in mice [8]. On days 3 and 6 post-infection (p. i.), we collected nasal washes from the virus-infected animals and titrated them in Madin-Darby canine kidney (MDCK) cells. On day 6 p. i., the virus titers in the nasal washes of animals infected with the virulent viruses (except for A/Vietnam/UT3028II/03) were generally higher than those of animals infected with viruses that were not lethal (Table 1). Sequence comparisons of the viruses used in this study revealed that the most virulent virus UT3062 was most closely related to an avirulent virus UT3028, with 18 amino acid differences in their 9 proteins (Table S2); there were no amino acid differences in the matrix (M) proteins of the two viruses. Therefore, we generated UT3062 and UT3028 by reverse genetics and confirmed their virulence by intranasally inoculating 5- to 6-month-old male ferrets with 107 PFU of the viruses. Animals infected with the UT3062 virus died on days 6–8, resulting in 67% lethality and showed −13. 4±3. 1% mean maximum weight loss (Figures 1 and 2). Conversely, all animals infected with the UT3028 virus survived and showed appreciably less mean maximum weight loss (−1. 1±0. 3%) (Figures 1 and 2). These results were similar to those obtained with the respective original viruses (Figure 2 and Table 1). To determine the molecular basis for the high virulence of UT3062, we generated reassortant viruses between the UT3062 and UT3028 viruses using reverse genetics and tested their virulence by intranasally inoculating 5- to 6-month-old male ferrets with 107 PFU of the viruses and observing them for 10 days for clinical manifestations. Since no amino acid differences were identified in M proteins of the two viruses, we used the UT3028 M gene for generating all reassortant viruses. The reassortants were named according to the origin of their UT3062 or UT3028 genes. For example, 3062 (Pol+NP+HA+NA) indicates a virus possessing the polymerase complex (PB1, PB2, and PA), nucleoprotein (NP), HA, and neuraminidase (NA) genes from UT3062 and the rest of its genes from UT3028 (Figure 2). Reassortant viruses possessing both the UT3062 HA and NS genes, 3062 (NP+HA+NA+NS), 3062 (Pol+HA+NA+NS), 3062 (HA+NA+NS), 3062 (HA+NS), and 3062 (NP+HA+NS), were lethal to animals (33%–100% lethality). By contrast, those possessing either the HA or NS genes of UT3028 were not lethal to any animals. Mean maximum body weight loss in the animals infected with the former viruses (8. 5%–14. 4%, median 11. 4%) tended to be greater than that in the animals infected with the latter viruses (0. 7%–8. 6%, median 4. 1%). Animals infected with 3062 (HA+NS) resulted in 83% lethality and 17. 8%±2. 7% mean maximum weight loss. These results show that the difference in virulence between UT3062 and UT3028 is mainly attributable to both of the HA and NS genes. Sequence comparison between UT3062 and UT3028 revealed only one amino acid difference in their HA proteins and four amino acid differences in their NS proteins, two in NS1 and two in NS2 (Table 2). Since NS1 and NS2 mRNAs are produced from the same gene segment, with the NS1 mRNA being unspliced and the NS2 mRNA being spliced, the nucleotide alterations can affect both proteins; i. e., the amino acid differences at positions 200 and 205 of NS1 were coupled to those at positions 47 and 51 of NS2, respectively. Therefore, it was impossible to substitute the amino acids only in the NS1 or the NS2 protein. Here, we generated two reassortant viruses with a mutation in the NS segment by reverse genetics. 3062 (HA) +NS1N200S possesses the UT3062 HA gene, a mutant NS segment encoding the UT3028 NS1 protein, which has an asparagine-to-serine substitution at position 200 (and encodes NS2 with a threonine-to-alanine substitution at position 47) and the rest of its genes from UT3028. 3062 (HA) +NS1G205R possesses the UT3062 HA gene, a mutant NS segment encoding the UT3028 NS1 protein, which has glycine-to-arginine substitution at position 205 (and encodes NS2 with a methionine-to-isoleucine substitution at position 51) and the rest of its genes from UT3028. We then tested their virulence in ferrets as described above. As shown in Figure 2, both of the reassortant viruses, 3062 (HA) +NS1N200S and 3062 (HA) +NS1G205R, were not lethal to any animals, with 4. 8%±1. 4% and 2. 3%±1. 4 mean maximum weight loss, respectively. These results suggest that all of the amino acids in HA and NS proteins contribute to the virulence in ferrets, although it is unclear whether changes in NS1, NS2, or both affect virulence. In addition, 3062 (HA+NA+NS) (33% lethality and 8. 5±3. 4% mean maximum body weight loss) was attenuated compared to 3062 (NP+HA+NA+NS) (67% lethality and 14. 4±4. 9% mean maximum body weight loss). Further, 3062 (NP+HA+NS) and 3062 (HA+NS) killed 100% and 83% of animals, respectively. These results suggest that the UT3062 NP gene may enhance virus virulence in ferrets. These results are consistent with previous findings that virulence of influenza virus is multigenic [7], [9], [10]. To understand the basis for the difference in virulence in ferrets among the viruses, we examined the in vitro and in vivo replication of the parental UT3062, UT3028, and the reassortant viruses. For in vitro testing, we compared their growth kinetics in mink lung epithelial (Mv1Lu) cells by infecting these cells with viruses at a multiplicity of infection (MOI) of 0. 001 and monitoring the growth kinetics for 48 h. All of the viruses replicated to more than 108 PFU/ml at 36 or 48 h p. i. and the differences in their viral titers were less than one log PFU/ml at each time point (Figure S1), indicating that there were no substantial differences in their replicative ability in these cells. To examine viral replication in ferrets, we infected animals with 107 PFU of the parental UT3062, UT3028, and selected reassortant viruses (3062 (HA+NS), 3062 (NP+HA+NS), and 3062 (HA+NA+NS) ). Virus titers in nasal and tracheal swabs, and organs were examined. On days 3 and 7 p. i., three animals from each infected group were sacrificed for virus titration. As shown in Table 3, UT3062,3062 (HA+NS), 3062 (NP+HA+NS), and 3062 (HA+NA+NS) were detected systemically on days 3 and 7 p. i., whereas UT3028 was detected mainly in the upper respiratory tracts of ferrets on day 3, but not 7, p. i. The differences in replicative ability of these viruses in ferrets thus correlate with lethality in this animal model. When we compared the histopathology between ferrets infected with UT3062 and those infected with UT3028, we found three major differences (Figures 3 and 4): (1) host reaction to viral exposure in the lungs on day 1 p. i., (2) viral infection in the tracheobronchial lymph node, and (3) distribution of viral antigens and the inflammatory reaction in the lungs on day 3 and beyond p. i. Firstly, cells infiltrating the lung lesions differed between animals infected with UT3062 and those infected with UT3028. Although substantial numbers of viral antigen-positive cells were detected in the lungs of ferrets infected UT3062 or UT3028, the lungs of ferrets infected with UT3062 had marked infiltration of eosinophils around/in the bronchi (Figure 3A). By contrast, the lung lesions of ferrets infected with UT3028 contained many neutrophils (Figure 3B). Secondly, viral infection in the tracheobronchial (pulmonary regional) lymph node at 1 day p. i. differed between the two viruses. Although we did not detect viral antigen in the tracheobronchial lymph node of ferrets infected with UT3028, we did find viral antigen at this site in all three ferrets infected with UT3062 (Figure 3D and E). Thirdly, in animals infected with UT3028, we did not detect viral antigen beyond 3 days p. i., with the exception of one ferret, which was euthanized at 5 days p. i. (Figure 4A). The numerous neutrophils observed on 1 day p. i. were replaced by lymphocytes, macrophages and regenerative epithelial cells during the course of infection (data not shown). On the other hand, in animals infected with UT3062, a substantial number of viral antigen-positive cells were detected in the lungs even 3 days p. i. and the areas in the lungs where the viral antigen-positive cells were detected expanded widely by 5 and 7 days p. i. in some ferrets (Figure 4B). Moreover, when compared to ferret lung lesions with less viral antigen-positive cells, the lesions of ferrets with extensive viral antigen-positive cells had fewer lymphocytes and substantial pulmonary edema, hemorrhaging and fibrinous exudates (data not shown). These findings indicate that there was a tendency for delay in viral clearance in UT3062-infected ferrets and consequently some animals progressed to death. The virus was, however, completely eliminated in some animals, presumably because of individual animal variability. Next, to evaluate the effect of the UT3062 HA and NS genes in vivo, we examined the pathogenicity of 3062 (HA+NS) virus, which possesses UT3062 HA and NS genes and its remaining genes from UT3028. When we examined ferrets infected with 3062 (HA+NS) on days 3 and 7 p. i., we found that they had pathological lesions that more closely resembled those of ferrets infected with UT3062 than those of ferrets infected with UT3028. Namely, the ferrets had viral infection in the tracheobronchial lymph node and widely distributed viral pneumonia by 3 days p. i. (Figure 3G to I). Pulmonary edema, hemorrhages and fibrinous exudates were obvious in the lung lesions rather than recruitment of lymphocytes and regenerative changes, which were characteristic of ferrets infected with UT3028. Therefore, the UT3062 HA and NS gene products play a critical role in viral pathogenicity in this ferret model. Viruses first replicated in the lungs (at the primary site of viral exposure), and infection then expanded into the tracheobronchial lymph node. Viral infection in the regional lymph node may negatively affect viral exclusion from the host, leading to continued viral replication. UT3062, like almost all other human H5N1 viruses, has alanine at position 134 of HA (H3 numbering). UT3028, however, has threonine at this position (Table 2). These findings suggest that a single substitution at position 134 (A134T) of HA affects virulence in ferrets. Previously, Auewarakul et al. [11] showed that substitutions at positions 129 and 134 (L129V/A134V) allow virus recognition of both sialic acid liked to galactose by α2,3 linkage (SAα2,3Gal) and SAα2,6Gal, unlike the parent virus, which recognizes only SAα2,3Gal. Yamada et al. [12], however, found that a single substitution at position 134 (A134T) did not change receptor-binding preference with the same sialylglycopolymers used by Auewarakul et al. [11]. We, therefore, performed virus elution assays using chicken and horse erythrocytes. From chicken erythrocytes, which express both SAα2,3Gal and SAα2,6Gal [13], UT3062 and a reassortant possessing the UT3062 HA were not eluted even after 20h of incubation at 37°C. By contrast, UT3028 and a reassortant possessing the UT3028 HA were gradually released from chicken erythrocytes (Figure 5). Since the viruses possessing the UT3028 HA were eluted regardless of the origin of the NAs (either UT3028 or UT3062), this difference in elution from erythrocytes is due to the difference in the amino acid residue at position 134 of HA. When we used horse erythrocytes, all of the viruses were more rapidly eluted from these erythrocytes than from chicken erythrocytes; however, UT3028 and a reassortant possessing the UT3028 HA were eluted more efficiently from horse erythrocytes than were UT3062 and reassortants possessing the UT3062 HA (data not shown). These results suggest that UT3062 HA differs from UT3028 HA in its receptor-binding property. NS1 mediates type I IFN antagonism and affects viral growth in cells. We, therefore, assessed the IFN antagonistic activity of these NS1s by using an IFN bioassay [14], [15], [16], [17], [18], [19], [20]. Briefly, Mv1Lu cells were infected with each virus at an MOI of 1. 25 and the supernatants were collected at 12–24 h p. i. H5N1 viruses in the supernatants were inactivated with UV and neutralizing antibody (A1A1, [21]) treatment. The supernatants were added to fresh Mv1Lu cells and cultured for 22 h, followed by infection with vesicular stomatitis virus (VSV) to determine VSV infectivity of the above-described supernatant-pretreated Mv1Lu cells. As a control, we used a recombinant influenza virus expressing an RNA-binding- and IFN-antagonism-defective NS1 protein within which two basic amino acids were substituted to alanines (R38A/K41A) on the UT3062 backbone [17], [22]. We also generated reassortant viruses possessing the mutant NS1 of UT3028 that has either the N200S or the G205R mutation, on the UT3028 backbone and designated them 3028NS1-N200S and 3028NS1-G205R, respectively. As described above, these viruses also possessed amino acid substitution in NS2, T47A, or M51I, respectively. At 18 h and 24 h p. i., the supernatant from Mv1Lu cells infected with UT3028 or a virus possessing the UT3028 NS gene and the remaining genes from UT3062 (i. e., 3028 (NS) ) inhibited VSV plaque formation more efficiently than did the supernatants from cells infected with UT3062 (statistically significant difference at P<0. 05, Tukey Honestly Significant Difference [HSD] test) (Figure 6). Furthermore, the supernatant of cells infected with either 3028NS1-N200S or 3028NS1-G205R inhibited VSV plaque formation more efficiently than did that from viruses possessing the UT3062 NS gene (i. e., UT3062 and 3062 (NS) ) (statistically significant difference, P<0. 05, Tukey HSD test), but less efficiently than that from viruses possessing the UT3028 NS gene (i. e., UT3028 and 3028 (NS) ) (Figure 6). These results indicate that both serine at position 200 and arginine at position 205 of NS1 contribute to the enhanced type I IFN antagonistic property of UT3062 NS1, which, in turn, leads to high virulence in ferrets. To further assess the IFN antagonistic property of NS1, we investigated the effects of the amino acids in NS1 on the expression of the firefly luciferase reporter gene under the control of an interferon-stimulated response element (ISRE) in 293 cells treated with IFNβ. Briefly, 293 cells were transfected with pISRE-Luc, pRL-TK, and pCAGGS NS1 or pCAGGS GFP (negative control). At 24 h post-transfection, the cells were treated with recombinant human IFNβ. At 30 h post-transfection, the cells were lysed and luciferase activities were measured by using the Dual-luciferase Reporter assay system. There were, however, no significant differences in expression from the ISRE between UT3062 NS1 and UT3028 NS1 (data not shown). We then investigated the effects of the amino acids in NS1 on the expression of the firefly luciferase reporter gene under the control of the IFNβ promoter in 293 cells treated with Sendai virus (SeV) as described previously [23]. Briefly, 293 cells were transfected with p125-Luc, pRL-TK, and pCAGGS NS1 or pCAGGS GFP (negative control). At 36 h post-transfection, the cells were treated with SeV (Cantell strain). At 48 h post-transfection, the cells were lysed and luciferase activities were measured by using the Dual-luciferase Reporter assay system. The results of this experiment also showed that there were no significant differences in expression from the IFNβ promoter between UT3062 NS1 and UT3028 NS1 (data not shown), indicating that other mechanisms affect the IFN antagonistic property. Here, using H5N1 viruses isolated from humans, we found that receptor-binding property and NS1 IFN antagonism play important roles in the high virulence of these viruses in ferrets. HA is a receptor-binding and fusion protein and, therefore, is required for virus entry. It is known to play a critical role in virulence [4], [24], [25], [26], [27]. In this study, we found that viruses possessing threonine at position 134 of HA were appreciably attenuated in ferrets compared to those possessing alanine. Although Yamada et al. [12] did not find differences in the receptor-binding preference between HAs with a single substitution at position 134 (134A or 134T) in a direct binding assay to sialylglycopolymers, we found that this substitution affected receptor-binding property as detected by a virus elution assay (Figure 5). Since the amino acid at position 134 is located near the receptor-binding pocket but does not directly interact with sialyloligosaccharides [11], the substitution at this residue may influence the receptor-binding property indirectly. Alanine at position 134 of HA is highly conserved in avian H5N1 viruses—only one virus is known to harbor serine at that position (the Influenza Sequence Database (ISD; https: //flu. lanl. gov/, registration system [28]) ). Similarly, most human H5N1 viruses also have alanine at this position; however, UT3028 and two other H5N1 viruses that we isolated from humans have threonine at this position (Y. Sakai-Tagawa and Y. Kawaoka, unpublished). Further, eleven H5N1 viruses isolated from humans have valine and one has serine at this position (ISD; https: //flu. lanl. gov/[28] and Y. Sakai-Tagawa and Y. Kawaoka, unpublished). These data indicate that an amino acid substitution at position 134 of HA is more frequently observed in human H5N1 viruses than in avian viruses, suggesting that viruses possessing a substitution at position 134 of HA may be selected during replication in humans. Although NS1 is a multifunctional protein, one of its main functions is to suppress type I IFN production [29]. Recent studies revealed that NS1 plays an important role (s) in antiviral responses via dsRNA-dependent protein kinase R (PKR) and 2′5′-oligoadenylate synthetase/RNase L [30], [31]. Here, using an IFN bioassay, we showed that both serine at position 200 and arginine at position 205 of NS1 contribute to the enhanced type I IFN antagonistic property of UT3062 that leads to high virulence in ferrets. However, we did not observe significant differences in IFNβ-stimulated expression from the ISRE or in SeV-stimulated expression from the IFNβ promoter in 293 cells between the UT3062 and UT3028 NS1 proteins. It may be that NS1 exhibits type I IFN antagonism by a mechanism other than tested in this study. Alternatively, the difference observed in the highly sensitive IFN bioassay using VSV is not detectable in other IFN assays. The amino acid residues at positions 200 and 205 of NS1 are not well conserved, although the residues at these positions in UT3062 have been observed in other human and avian H5N1 viruses (Table S3). These findings support the hypothesis that the amino acid residues determined to be important in this study are affected by the genetic background of the test viruses. Nonetheless, the HA amino acid at position 134 and the NS1 amino acids at positions 200 and 205 may now be included as virulence markers for H5N1 viruses. Our research protocol for the use of ferrets followed the University of Tokyo' s Regulations for Animal Care and Use, which was approved by the Animal Experiment Committee of the Institute of Medical Science, the University of Tokyo (approval number: 19–29). The committee acknowledged and accepted both the legal and ethical responsibility for the animals, as specified in the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, 2006. Madin-Darby canine kidney (MDCK) cells were maintained in minimal essential medium (MEM) with 5% newborn calf serum. Human embryonic kidney 293 and 293T cells were maintained in Dulbecco' s modified Eagle' s MEM (DMEM) with 10% fetal calf serum. Mink lung epithelial (Mv1Lu) cells were maintained in MEM with 10% fetal calf serum and 1% non-essential amino acids. All cells were grown at 37°C in 5% CO2. H5N1 viruses isolated from humans in Vietnam and Indonesia were used in this study (Table 1). Virus stocks were propagated through two passages in MDCK cells for 24–48 h at 37°C. The cell supernatants were harvested, clarified by centrifugation, aliquoted, and stored at −80°C. The frozen virus stocks were thawed and titrated for virus infectivity in MDCK cells by plaque assays. Virus titers were calculated as PFU/ml. All experiments were performed under biosafety level 3+ conditions. Viral RNA was extracted directly from the supernatants of H5N1 virus-infected MDCK cell cultures with a QIAamp Viral RNA Mini Kit (Qiagen, http: //www1. qiagen. com/). Complementary DNA was generated by SuperscriptIII (Invitrogen, http: //www. invitrogen. com/) with the universal primers for influenza A virus genes. The resulting products were PCR-amplified using PfuUltra High-Fidelity DNA polymerase (STRATAGENE, http: //www. stratagene. com/) with specific primers for each virus gene and cloned into a plasmid under the control of the human RNA polymerase I (PolI) promoter and the mouse RNA PolI terminator (PolI plasmids). We altered the NS1 gene sequence that encodes the RNA-binding site of UT3062 to create the RNA-binding defective sequence (R38A/K41A) as previously described [17]. All reassortant viruses and the parental UT3062 and UT3028 viruses were generated by plasmid-based reverse genetics, as described by Neumann et al. [32]. Briefly, PolI plasmids and protein expression plasmids were mixed with a transfection reagent, TransIT 293T (Mirus Bio, http: //www. mirusbio. com/); incubated at room temperature for 15 min; and then added to 293T cells. Transfected cells were incubated in OPTI-MEM I (Invitrogen) for 48 h. Supernatants containing infectious viruses were harvested and propagated in MDCK cells at 37°C for 48 h. The supernatants were harvested, aliquoted, and stored at −80°C. We used male ferrets, 5–7 months old (MarshallBioResources, http: //www. marshallbioresources. com/) in this study. All ferrets were inoculated intranasally with 107 PFU of infectious virus in 500 µl of phosphate-buffered saline (PBS) under anesthesia with ketamine (25 mg/kg) and xylazine (2 mg/kg). Clinical signs, body weights, and body temperatures were recorded daily for 10 days post-infections (p. i.). The percent changes in body weights were calculated by comparing the weights of each ferret at each time point to its initial weight on day 0. Body temperatures were measured using a rectal thermometer. Changes in body temperature were calculated by comparing the body temperatures of each ferret at each time point to its initial body temperature on day 0. All animals exhibiting more than 20% weight loss, hemorrhage from any body orifice, or inability to remain upright were euthanized. Surviving ferrets were euthanized under deep anesthesia at 3 weeks p. i. On days 3 and 6 p. i., nasal washes were collected from anesthetized ferrets and titrated for virus infectivity in MDCK cells by plaque assays. Ferrets infected with the parental UT3062 and UT3028 and selected reassortant viruses (Table 3) were euthanized with deep anesthesia and necropsied on days 3 and 7 p. i. Tissue samples of the brain, olfactory bulb, lungs, hilar lymph node, liver, kidney, spleen, duodenum, and descending colon were collected. A portion of each was stored at −80°C for virus titration and the rest were preserved in 10% neutral buffered-formalin for pathological examination. To prepare 10% tissue emulsions, frozen tissue samples were thawed, weighed, and homogenized in 10 volumes (w/v) of sterile PBS using a multi-beads shocker (Yasui Kikai, http: //www. yasuikikai. co. jp/). After centrifugation of the samples at 800× g for 5 min at 4°C, the supernatants were collected. In addition, swabs from nose and trachea were collected and suspended in 1 ml of sterile PBS containing 0. 3% BSA and penicillin (200 U/ml). After centrifugation at 800× g for 5 min at 4°C, the supernatants were collected and stored at −80°C. Virus in nasal washes, swabs and tissue samples was titrated for virus infectivity in MDCK cells by plaque assays. Virus titer was expressed as PFU/g for tissue samples and PFU/ml for nasal washes and swabs. The limitations of virus detection were 102. 0 PFU/g for tissue samples and 101. 0 PFU/ml for nasal washes and swabs. The formalin-fixed tissues were processed for routine paraffin embedding. The paraffin-embedded tissues were cut into 5 µm thick slices and stained using hematoxylin-and-eosin (H&E). Additional sections were cut for immunohistological staining with rabbit polyclonal antibodies against an H5N1 virus (A/Vietnam/1203/04). Specific antigen-antibody reactions were visualized by means of 3,3′ diaminobenzidine tetrahydrochloride and the Dako EnVision system (Dako, http: //www. dako. jp/). All animal experiments were approved by the Animal Research Committee of The University of Tokyo. The reassortant viruses and the parental UT3062 and UT3028 viruses were inoculated into Mv1Lu cell monolayers at an MOI of 0. 001 PFU with MEM containing 0. 3% bovine serum albumin, and incubated at 37°C. Cell supernatants were harvested at a given number of hours p. i. After centrifugation at 1,000× g for 5 min, samples were titrated for virus infectivity in Mv1Lu cells by plaque assays. The ability of viruses to be eluted from erythrocytes was assessed as previously described [33], [34], with some modifications. Briefly, virus stocks were diluted serially in calcium saline (0. 9 mM CaCl2-154 mM NaCl in 20 mM borate buffer, pH 7. 2) and 50 µl aliquots were incubated with 50 µl of 0. 55% chicken erythrocytes at 4°C for 1 h in microtiter plates. The plates were then transferred to 37°C and monitored periodically for 20 h. Levels of IFN secreted by virus-infected Mv1Lu cells were assessed as previously described [14], [15], [16], [17], [18], [19], [20], with some modifications. Briefly, Mv1Lu cells were infected with each virus at an MOI of 1. 25. Supernatants from infected cells were harvested 12–24 h p. i. To inactivate viral infectivity, the supernatants were treated with UV light for 20 min and then mixed with neutralizing α-VN1203HA monoclonal antibodies (A1A1, [21]). The supernatants were added to fresh Mv1Lu cells and incubated for 22 h. These pretreated Mv1Lu cells were then infected with VSV, and the VSV infectivity titers were determined. As a control, we used a recombinant influenza virus expressing an RNA-binding- and IFN antagonism-defective NS1 protein within which two basic amino acids were substituted to alanines (R38A/K41A). The experiments were carried out in triplicate and were independently repeated twice. 8×104 of 293 cells were transfected with 50 ng of pISRE-Luc (Clontech, http: //www. clontech. com/) and 50 ng of pRL-TK (Promega, http: //www. promega. com/) by using TransIT293 (Mirus, http: //www. mirusbio. com/). 100 ng of pCAGGS NS1 or pCAGGS GFP were co-transfected. Cells were incubated for 24 h and were treated with 100 units of recombinant human IFNβ, 1a (PBL interferonSource, http: //www. interferonsource. com/). At 30 h post-transfection, cells were lysed and luciferase activities were measured by using the Dual-luciferase Reporter assay system (Promega, http: //www. promega. com/) according to the protocol provided by the manufacturer. Firefly luciferase values were divided by Renilla luciferase values to normalize for transfection efficiency. 2×105 of 293 cells were transfected with 250 ng of p125-Luc (the reporter plasmid carrying the firefly luciferase gene under the control of the IFNβ promoter was kindly provided by Takashi Fujita) and 250 ng of pRL-TK (Promega, http: //www. promega. com/) by using TransIT293 (Mirus, http: //www. mirusbio. com/). 5–500 ng of pCAGGS NS1 or pCAGGS GFP were co-transfected. After incubation for 24 h, cells were stripped with trypsin and divided into two wells of a 12-well plate. At 36 h post-transfection, cells were treated with approximately 5×1010 focus-forming unit of SeV (Cantell strain). At 48 h post-transfection, cells were lysed and luciferase activities were measured by using the Dual-luciferase Reporter assay system (Promega, http: //www. promega. com/) according to the protocol provided by the manufacturer. Firefly luciferase values were divided by Renilla luciferase values to normalize for transfection efficiency.

Highly pathogenic H5N1 influenza A viruses have caused more than 500 human infections with approximately 60% lethality in 15 countries and continue to pose a pandemic threat. The recent worldwide spread of pandemic H1N1 influenza A viruses raises the concern of reassortment between the H5N1 viruses and other influenza viruses. However, the molecular determinants for high virulence of the H5N1 viruses in mammals are not fully understood. We, therefore, investigated their virulence in a ferret model, which is a widely accepted animal model for assessing human influenza virus replication. We identified an amino acid in hemagglutinin and four amino acids in nonstructural proteins that are associated with high virulence of a human H5N1 virus, A/Vietnam/UT3062/04. We also found that the amino acid in hemagglutinin changes its receptor-binding property and the amino acids in nonstructural protein 1 affect its interferon antagonistic ability. These findings provide insight into the pathogenesis of H5N1 viruses in mammals.

lay_plos

The visual world is complex and continuously changing. Yet, our brain transforms patterns of light falling on our retina into a coherent percept within a few hundred milliseconds. Possibly, low-level neural responses already carry substantial information to facilitate rapid characterization of the visual input. Here, we computationally estimated low-level contrast responses to computer-generated naturalistic images, and tested whether spatial pooling of these responses could predict image similarity at the neural and behavioral level. Using EEG, we show that statistics derived from pooled responses explain a large amount of variance between single-image evoked potentials (ERPs) in individual subjects. Dissimilarity analysis on multi-electrode ERPs demonstrated that large differences between images in pooled response statistics are predictive of more dissimilar patterns of evoked activity, whereas images with little difference in statistics give rise to highly similar evoked activity patterns. In a separate behavioral experiment, images with large differences in statistics were judged as different categories, whereas images with little differences were confused. These findings suggest that statistics derived from low-level contrast responses can be extracted in early visual processing and can be relevant for rapid judgment of visual similarity. We compared our results with two other, well- known contrast statistics: Fourier power spectra and higher-order properties of contrast distributions (skewness and kurtosis). Interestingly, whereas these statistics allow for accurate image categorization, they do not predict ERP response patterns or behavioral categorization confusions. These converging computational, neural and behavioral results suggest that statistics of pooled contrast responses contain information that corresponds with perceived visual similarity in a rapid, low-level categorization task. Complex natural images are categorized remarkably fast [1], [2], sometimes even faster than simple artificial stimuli [3]. For animal and non-animal scenes, differences in EEG responses are found within 150 ms [4] and a correct saccade is made within 120 ms [5]. This speed of processing is also found for other scene categories [6] and may require less attentional resources compared to artificial images [7], [8]. This suggests that relevant visual information is rapidly and efficiently extracted from early visual responses to natural scenes. However, the neural computations involved in this process are not known. Importantly, natural images differ from other image types such as white noise in low-level properties (e. g., sparseness), leading to the suggestion that the visual system has adapted to these low-level properties [9]. This idea paved the way for optimal coding models for natural images [10], [11] and successful predictions of response properties of visual neurons [12]. Recent work identified statistical properties that differ even within the class of natural images, e. g. between natural scene parts [13], [14] or natural image categories [15], showing that image statistics such as power spectra of spatial frequency content or distributions of local image features are informative about scene category. The fact that it is mathematically possible to distinguish categories based on image statistics, however, does not imply that they are used for categorization in the brain. Image statistics may not be sufficiently reliable, or their computation may not be suitable for neural implementation [12], [16]. We recently showed that statistics derived from the frequency histogram of local contrast – summarized by two parameters of a Weibull fit, Fig. 1A – explain up to 50% of the variance of event-related potentials (ERPs) recorded from visual cortex [17]. These parameters inform about the width and shape of the histogram, respectively, and appear to describe meaningful variability between images (Fig. 1B). Importantly, we found that these parameters can be reliably approximated by linear summation of the output of localized difference-of-Gaussians filters modeled after X- and Y-type LGN cells, suggesting that this global information may be available to visual cortex directly from its early low-level contrast responses [17]. Moreover, we found that output of contrast filters with a larger range of receptive field sizes captures additional image information [18]. This is not surprising since objects in natural scenes appear at many distances and hence spatial scales [19]. In the present implementation, the model first estimates at which scale relevant contrast information is present, as well as characteristics of the distribution of contrast strengths at those scales. This model, which approximates early visual population responses based on spatially pooled contrasts, was able to explain almost 80% of ERP variance to natural images [18]. These previous findings suggest that images with more similar contrast response statistics evoke more similar early visual activity. Could these responses already contain relevant information about the stimulus for rapid categorization? The two parameters appear to index meaningful information such as degree of clutter, depth and figure-ground segmentation [17], but how the two dimensions in Fig. 1B influence perception has not been examined. The goal of the current study was thus to explore what type of visual information is contained in the variance of the earliest visual contrast responses that is so well described by these two parameters. Specifically, we were interested in whether these parameters cannot only predict variance in visual activity, but also ‘variance in perception’. In other words, do images with more similar contrast statistics also lead to more similar perceptual representations, and perhaps ultimately, to similar images being considered a single category? We aimed to answer this question in a data-driven manner, by investigating 1) which images group by similarity early in visual processing and 2) whether this grouping matches with perceived similarity of those images. For the first part of this question, we obtained reliable evoked responses to individual images. The advantage of this approach relative to traditional ERP analysis (which is based on averaging many trials across individual images within an a priori determined condition) is that it provides much richer data [20]–[24] that can be used for model selection. We used these single-image evoked responses to compute dissimilarities in ‘neural space’, similar to the pattern analysis approach used in fMRI [25], [26]. This allowed us to track, over the course of the ERP, to what extent the representation of an image is (dis) similar to all images in the data set. For the second part of the question, we needed to obtain an image-specific behavioral judgment of perceived visual similarity. However, simply judging similarity of natural scenes is problematic, because these images obviously contain rich semantic content: there are many features of natural scenes that can be similar or dissimilar, which is likely to lead to different categorization strategies by different subjects. Also, it is uncertain to what extent specific semantic tags that are provided by the researcher (e. g. ‘openness’ or ‘naturalness’, [27]), can be uniformly interpreted as a relevant stimulus dimension that has a linear mapping to processing in early vision. Therefore, to explore the variance explained by contrast response statistics in a bottom-up way, we used stimuli that were simplified model images of natural scenes (‘dead leaves’, Fig. 2A), which have similar low-level structure as natural scenes (e. g. 1/f power spectra) but are devoid of semantic content. These images are created by filling a frame with objects - much like fallen leaves can fill a forest floor – and are used in computer vision to study, for example, how the appearance and the distribution of these objects influences the low-level structure of natural scenes [28]. By manipulating properties of the objects in a controlled manner, we created distinct image categories, and then tested whether differences between these categories in contrast statistics matched with behaviorally perceived similarity by letting human observers perform a same-different categorization task on all combinations of image categories. Specifically, we used the space formed by the two Weibull parameters to compute geometric distances between images in contrast statistics, and used these distances as quantitative predictors of dissimilarity [29]–[31]. We thus tested whether these parameters can predict the extent to which image categories induced dissimilar single-image EEG responses (experiment 1) and whether they match with perceptual categorization at the behavioral level (experiment 2). We predicted that images with very different Weibull statistics would appear less similar, i. e. be less often confused than images from categories with similar statistics. By using controlled images that we quantified using a model originally derived from contrast responses to natural images, we aim to build a bridge between findings obtained with systematic manipulation of artificial stimuli and those obtained with more data-driven natural scene studies. For purpose of comparison, and to better understand which statistical information is captured by the Weibull parameters, we also tested two other global contrast statistics (Fig. 2C). Following [32] we calculated the intercept and slope of the average power spectrum to parameterize spatial frequency information, a commonly used measure of low-level information in scene perception. In addition, we followed [33] to derive the skewness and kurtosis of the contrast distribution for a range of spatial scales: these higher-order properties of distributions have previously been suggested (e. g. [34], [35] to reflect low-level differences between images that are relevant for perceptual processing. We find that Weibull statistics explain substantial variance in evoked response amplitude to the dead leaves images, predicting clustering-by-category of occipital ERP patterns within 100 ms of visual processing. In addition, they correlate with human categorization behavior: specific confusions were made between categories with similar Weibull statistics. By comparison, Fourier power spectra and skewness and kurtosis can be used for accurate classification of image category, but fail to predict neural clustering and behavioral categorization. These convergent results provide evidence for relevance of pooled contrast response statistics in rapid neural computation of perceptual similarity. The experiments reported here were approved by the Ethical Committee of the Psychology Department at the University of Amsterdam; all participants gave written informed consent prior to participation and were rewarded with study credits or financial compensation (7 euro/hour). Gray-scale dead leaves images (512×512 pixels, bit depth 24) were generated using Matlab. Images contained randomly placed disks that were manipulated along 4 dimensions (opacity, depth, size and distribution) to create 16 categories. Disks were either opaque or transparent; intensity at the outer edges of the disk was either constant (leading to a 2D appearance) or decaying (3D appearance), and disk size was determined by drawing randomly from a range of small, medium or large diameters (exact settings as in [28]. Twelve categories were created by systematically varying these properties of power-law distributed disks. Four more categories were created using medium-diameter, exponentially distributed disks that could be 2D or 3D and opaque or transparent. For each category, 16 images were created using these category-specific settings: the random placement and use of ranges of diameter sizes ensured that each of these 16 images was unique. This procedure thus resulted in a total of unique 256 images, divided into 16 distinct categories, which were used for experimentation (Fig. 2A). If we set out all 256 dead leaves images against the three sets of image statistics (Weibull parameters, Fourier parameters and skewness/kurtosis), stimuli cluster by category in all cases, with Fourier parameters leading to the most separable clusters (Fig. 4A–C). There were considerable correlations between the various parameters (Fig. 4D; individual correlations plots in Fig. S2). Skewness and kurtosis correlated highly (ρ = 0. 91, p<0. 0001), but other significant correlations are observed as well, for example between Fourier slope and the Weibull beta parameter (ρ = 0. 57, p<0. 0001) and also between the two Weibull parameters (ρ = 0. 48, p<0. 001). A correlation of similar magnitude was also observed [17] for natural scenes, supporting the notion that the dead leaves stimuli used here have similar low-level structure as natural stimuli. Interestingly, however, the ‘similarity spaces’ formed by each set of parameters are quite different between the various models. If Weibull parameters determine the axes of the similarity space (Fig. 4A), highly cluttered images with many strong edges (e. g. 2D opaque stimuli with small disks) are located in the upper right corner (high gamma, high beta); images containing fewer edges (e. g. with larger disks) are found more on the left (low gamma); and most of the transparent stimuli, with weak edges, cluster together in the bottom of the space (low beta). For Fourier intercept and slope (Fig. 4B), transparent categories are highly separated across the space: however, most images with strong edges end up in a similar part of the space (low slope, high intercept). Based on either skewness or kurtosis (Fig. 4C), a few categories are distinct, but most tend to cluster together. These qualitative results suggest that all parameters are informative about clustering of image categories, but that they index different image properties. Importantly, they give rise to different predictions about which categories should lead to similar evoked responses based on overlapping parameter values. We tested these predictions using the single-image ERP data. Whether low-level statistics are indeed actively exploited during scene or object categorization is a topic of considerable debate. Whereas some studies report that manipulation of low-level properties influences rapid categorization accuracy [54], [55] as well as early EEG responses [56], [57], other studies have shown that not all early visual activity is obliterated by equation of those properties [58]–[60] and, conversely, that early sensitivity to diagnostic information is revealed in stimuli that do not differ in low-level statistics [20], [61]. We find that, at least for our set of simplified models of natural scene images, early differences in ERPs are correlated with low-level contrast statistics that are themselves also directly predictive of perceptual similarity. It is however likely that the degree to which low-level properties are relevant for processing of natural image categories is highly dependent on stimulus type and context, even within actual natural scene stimuli: for example, low-level information may influence rapid detection of faces to a larger extent than objects [22] and the effects of low-level statistics on animal detection may interact with scene category (man-made vs. natural) [62]. In addition, the present work is very different from these previous reports in that our experiments did not require formation of a high-level representation but only a same-different judgment. There are also notable differences between our ERP effects and those obtained with standardized object/scene categories: our maximum explained variance was found at around 100 ms, whereas those studies report sensitivity starting at 120 ms and onwards [63]–[66]. Maximal sensitivity of evoked activity to faces and objects is found at lateral-occipital and parietal electrodes (PO, e. g. [58]), whereas our correlations are clustered around occipital electrode Oz. This suggests that the dead leaves images may mostly engage mid-level areas of visual processing, such as those sensitive to textural information, e. g. V2 [24], [67]–[69]. Our results implicate that clustering of image similarities at this level of processing can, in principle, already predict perceptual similarity – in turn, these similarities can be derived from Weibull contrast statistics. Given that for natural scenes, the Weibull statistics explain similar amounts of variance in EEG activity as reported here, we can hypothesize that image similarities as predicted by Weibull statistics are also present in evoked activity to actual natural scenes. If Weibull statistics indeed approximate meaningful global information in natural images, which image features do they convey? By manipulating computational image categories in their perceptual appearance, we were able to get a better understanding of the information contained in the Weibull parameters. They appear to index the amount of clutter, i. e. are related to occlusion and object size. These properties may be relevant for natural scene categorization: a forest has a higher degree of clutter (high gamma) and lower mean edge strength (high beta) compared to a beach scene. An image containing a few strong edges (low beta) that are sparsely distributed (low gamma) has high probability of coinciding with a single salient object, for example a single bird against an empty sky, suggesting that these statistics may be relevant for object detection in natural scenes. Here, behavioral confusions (and corresponding dissimilarities in ERP signals) were found between stimuli without coherent edge information (transparent stimuli with either large or small disks), or that were highly cluttered (opaque stimuli with small disks) which were exactly the categories that overlapped in Weibull parameter values. For comparison, we computed Fourier power spectra and higher-order properties of the contrast distribution (skewness and kurtosis), two sets of statistics that each index different sources of information in natural images: spatial frequency content and central moments of the contrast distribution, respectively. Deviations in the power spectra of natural images inform about variations in contrast across spatial scales: the slope and intercept parameters describe the ‘spectral signature’ of images [32] which is diagnostic of scene category [15]. Skewness and kurtosis were proposed to be relevant for texture perception [35], [70] which in turn can be important for feature detection [53], [71] and the presence of featureless regions of images [34], [72]. Our results confirm that both frequency content and central moments of the contrast distribution inform about image properties: both lead to accurate image classification. However, in the present study they did not predict neural and behavioral categorization patterns, suggesting that these statistics may not be plausible computations involved in visual processing of the dead leaves images. Even though we used controlled, computationally defined image categories, it is still possible that an image property other that the contrast statistics tested here will provide a better prediction of the (neural and behavioral) data, for example one of the manipulations used to create the image categories (e. g., opacity). However, neither the observed clustering-by-category of ERPs in the RDM, nor the pattern of categorization errors in behavior mapped clearly onto one of the manipulations used to create the categories (e. g., opaque vs. transparent; as is visible in Fig. 7B, there are also differences within opaque and transparent categories, and this complex pattern of clustering is only predicted by Weibull statistics). Why is the Weibull model better than widely used contrast statistics in predicting early neural and perceptual similarity? Although higher order moments of distributions can be diagnostic of textural differences, they may in practice be difficult for the visual system to represent [35]. In addition, it has been suggested that rather than amplitude spectra, phase information derived from the Fourier transform [73], [74], or the interaction between these two [75], [76] contains diagnostic scene information. The reason that higher-order statistics derived from the phase spectrum may contain perceptually relevant information [77] is that they carry edge information. In the Weibull model, contrasts, i. e. non-oriented edges, are explicitly computed (as the response of LGN-type neurons) and evaluated at multiple spatial scales. The model may thus be able to capture information contained both in power spectra (scale statistics) as well as central moments (distribution statistics). The Weibull parameters appear to reflect different aspects of low-level information: the beta parameter varies with the range of contrast strengths present in the image, reflecting overall contrast energy, whereas the gamma parameter varies with the degree of correlation between local contrast values, reflecting clutter or spatial coherence. Obviously, the Weibull fit is still a mathematical construct. However, the two parameters can also be approximated in a more biologically plausible way: with our previous single-scale model [17], we demonstrated that simple summation of X- and Y-type LGN output corresponded strikingly well with the fitted Weibull parameters. Similarly, if the outputs of the multi-scale filter banks used here (reflecting the entire range of receptive field sizes of the LGN) are linearly summed, we again obtain values that correlate highly with the Weibull parameters obtained from the contrast histogram at minimal reliable scale (S. Ghebreab, H. S. Scholte, V. A. F. Lamme, A. W. M Smeulders, under review). This suggests that Weibull estimation can in fact be reduced to pooling of neuronal population responses by summation, which is a biologically realistic operation. Why would summation of contrast responses of low-level neurons convey the same information as the Weibull parameters? This is likely a result of the structure of the world itself: distributions of contrast in natural images tend to range between power-law and Gaussian, which is the family of distributions that the Weibull function can capture [78]. It appears that this statistic simply provides a good characterization of the dynamic range of the low-level input to the visual cortex when viewing natural images. Since our brain developed in a natural world, early visual processing may take advantage of this regularity in estimating global properties to arrive at a first impression of scene content. The present results extend our previous findings [17], [18] with natural images to other image types (computational categories) and to prediction of behavioral categorization. Interestingly, even though the subjects in experiment 1 (EEG) were not engaged in categorization of the dead leaves images, their results generalize to the behavioral categorization patterns that were found in experiment 2, suggesting that similarity of bottom-up responses measured in EEG - in a different person - can be predictive of the perceived similarity during categorization of these images. This observation is now restricted to computationally defined categories. An interesting question for future work is whether in construction of high-level categorical representations of natural stimuli - considered a computationally challenging task - the brain actively exploits the pattern of variability of the population response to low-level information, estimated from early receptive field output. Contrary to the classical view of the visual hierarchy (e. g., [79]) it has been proposed that a rapid, global percept of the input (gist) precedes a slow and detailed analysis of the scene [80]–[83]. Natural image statistics provide a pointer to information that could be relevant for such a global percept [84], [85]. However, the mechanism by which global information can be rapidly extracted from low-level properties is not directly evident from natural image statistics alone. As explained above, in our model, the statistics are derived from a biologically realistic substrate (the response of early visual contrast filters). We suggest that to build a realistic model of natural image categorization, it is essential to understand how statistics derived from very early, simple low-level responses can contribute to gist extraction. In conclusion, our findings suggest that global information based on low-level contrast can be available very early in visual processing and that this information can be relevant for judgment of perceptual similarity of controlled image categories.

Humans excel in rapid and accurate processing of visual scenes. However, it is unclear which computations allow the visual system to convert light hitting the retina into a coherent representation of visual input in a rapid and efficient way. Here we used simple, computer-generated image categories with similar low-level structure as natural scenes to test whether a model of early integration of low-level information can predict perceived category similarity. Specifically, we show that summarized (spatially pooled) responses of model neurons covering the entire visual field (the population response) to low-level properties of visual input (contrasts) can already be informative about differences in early visual evoked activity as well as behavioral confusions of these categories. These results suggest that low-level population responses can carry relevant information to estimate similarity of controlled images, and put forward the exciting hypothesis that the visual system may exploit these responses to rapidly process real natural scenes. We propose that the spatial pooling that allows for the extraction of this information may be a plausible first step in extracting scene gist to form a rapid impression of the visual input.

lay_plos

Dark spruce forest frowned on either side the frozen waterway. The trees had been stripped by a recent wind of their white covering of frost, and they seemed to lean towards each other, black and ominous, in the fading light. A vast silence reigned over the land. The land itself was a desolation, lifeless, without movement, so lone and cold that the spirit of it was not even that of sadness. There was a hint in it of laughter, but of a laughter more terrible than any sadness--a laughter that was mirthless as the smile of the sphinx, a laughter cold as the frost and partaking of the grimness of infallibility. It was the masterful and incommunicable wisdom of eternity laughing at the futility of life and the effort of life. It was the Wild, the savage, frozen-hearted Northland Wild. But there _was_ life, abroad in the land and defiant. Down the frozen waterway toiled a string of wolfish dogs. Their bristly fur was rimed with frost. Their breath froze in the air as it left their mouths, spouting forth in spumes of vapour that settled upon the hair of their bodies and formed into crystals of frost. Leather harness was on the dogs, and leather traces attached them to a sled which dragged along behind. The sled was without runners. It was made of stout birch-bark, and its full surface rested on the snow. The front end of the sled was turned up, like a scroll, in order to force down and under the bore of soft snow that surged like a wave before it. On the sled, securely lashed, was a long and narrow oblong box. There were other things on the sled--blankets, an axe, and a coffee-pot and frying-pan; but prominent, occupying most of the space, was the long and narrow oblong box. In advance of the dogs, on wide snowshoes, toiled a man. At the rear of the sled toiled a second man. On the sled, in the box, lay a third man whose toil was over,--a man whom the Wild had conquered and beaten down until he would never move nor struggle again. It is not the way of the Wild to like movement. Life is an offence to it, for life is movement; and the Wild aims always to destroy movement. It freezes the water to prevent it running to the sea; it drives the sap out of the trees till they are frozen to their mighty hearts; and most ferociously and terribly of all does the Wild harry and crush into submission man--man who is the most restless of life, ever in revolt against the dictum that all movement must in the end come to the cessation of movement. But at front and rear, unawed and indomitable, toiled the two men who were not yet dead. Their bodies were covered with fur and soft-tanned leather. Eyelashes and cheeks and lips were so coated with the crystals from their frozen breath that their faces were not discernible. This gave them the seeming of ghostly masques, undertakers in a spectral world at the funeral of some ghost. But under it all they were men, penetrating the land of desolation and mockery and silence, puny adventurers bent on colossal adventure, pitting themselves against the might of a world as remote and alien and pulseless as the abysses of space. They travelled on without speech, saving their breath for the work of their bodies. On every side was the silence, pressing upon them with a tangible presence. It affected their minds as the many atmospheres of deep water affect the body of the diver. It crushed them with the weight of unending vastness and unalterable decree. It crushed them into the remotest recesses of their own minds, pressing out of them, like juices from the grape, all the false ardours and exaltations and undue self-values of the human soul, until they perceived themselves finite and small, specks and motes, moving with weak cunning and little wisdom amidst the play and inter-play of the great blind elements and forces. An hour went by, and a second hour. The pale light of the short sunless day was beginning to fade, when a faint far cry arose on the still air. It soared upward with a swift rush, till it reached its topmost note, where it persisted, palpitant and tense, and then slowly died away. It might have been a lost soul wailing, had it not been invested with a certain sad fierceness and hungry eagerness. The front man turned his head until his eyes met the eyes of the man behind. And then, across the narrow oblong box, each nodded to the other. A second cry arose, piercing the silence with needle-like shrillness. Both men located the sound. It was to the rear, somewhere in the snow expanse they had just traversed. A third and answering cry arose, also to the rear and to the left of the second cry. "They're after us, Bill," said the man at the front. His voice sounded hoarse and unreal, and he had spoken with apparent effort. "Meat is scarce," answered his comrade. "I ain't seen a rabbit sign for days." Thereafter they spoke no more, though their ears were keen for the hunting-cries that continued to rise behind them. At the fall of darkness they swung the dogs into a cluster of spruce trees on the edge of the waterway and made a camp. The coffin, at the side of the fire, served for seat and table. The wolf-dogs, clustered on the far side of the fire, snarled and bickered among themselves, but evinced no inclination to stray off into the darkness. "Seems to me, Henry, they're stayin' remarkable close to camp," Bill commented. Henry, squatting over the fire and settling the pot of coffee with a piece of ice, nodded. Nor did he speak till he had taken his seat on the coffin and begun to eat. "They know where their hides is safe," he said. "They'd sooner eat grub than be grub. They're pretty wise, them dogs." Bill shook his head. "Oh, I don't know." His comrade looked at him curiously. "First time I ever heard you say anything about their not bein' wise." "Henry," said the other, munching with deliberation the beans he was eating, "did you happen to notice the way them dogs kicked up when I was a-feedin' 'em?" "They did cut up more'n usual," Henry acknowledged. "How many dogs've we got, Henry?" "Six." "Well, Henry... " Bill stopped for a moment, in order that his words might gain greater significance. "As I was sayin', Henry, we've got six dogs. I took six fish out of the bag. I gave one fish to each dog, an', Henry, I was one fish short." "You counted wrong." "We've got six dogs," the other reiterated dispassionately. "I took out six fish. One Ear didn't get no fish. I came back to the bag afterward an' got'm his fish." "We've only got six dogs," Henry said. "Henry," Bill went on. "I won't say they was all dogs, but there was seven of'm that got fish." Henry stopped eating to glance across the fire and count the dogs. "There's only six now," he said. "I saw the other one run off across the snow," Bill announced with cool positiveness. "I saw seven." Henry looked at him commiseratingly, and said, "I'll be almighty glad when this trip's over." "What d'ye mean by that?" Bill demanded. "I mean that this load of ourn is gettin' on your nerves, an' that you're beginnin' to see things." "I thought of that," Bill answered gravely. "An' so, when I saw it run off across the snow, I looked in the snow an' saw its tracks. Then I counted the dogs an' there was still six of 'em. The tracks is there in the snow now. D'ye want to look at 'em? I'll show 'em to you." Henry did not reply, but munched on in silence, until, the meal finished, he topped it with a final cup of coffee. He wiped his mouth with the back of his hand and said: "Then you're thinkin' as it was--" A long wailing cry, fiercely sad, from somewhere in the darkness, had interrupted him. He stopped to listen to it, then he finished his sentence with a wave of his hand toward the sound of the cry, "--one of them?" Bill nodded. "I'd a blame sight sooner think that than anything else. You noticed yourself the row the dogs made." Cry after cry, and answering cries, were turning the silence into a bedlam. From every side the cries arose, and the dogs betrayed their fear by huddling together and so close to the fire that their hair was scorched by the heat. Bill threw on more wood, before lighting his pipe. "I'm thinking you're down in the mouth some," Henry said. "Henry... " He sucked meditatively at his pipe for some time before he went on. "Henry, I was a-thinkin' what a blame sight luckier he is than you an' me'll ever be." He indicated the third person by a downward thrust of the thumb to the box on which they sat. "You an' me, Henry, when we die, we'll be lucky if we get enough stones over our carcases to keep the dogs off of us." "But we ain't got people an' money an' all the rest, like him," Henry rejoined. "Long-distance funerals is somethin' you an' me can't exactly afford." "What gets me, Henry, is what a chap like this, that's a lord or something in his own country, and that's never had to bother about grub nor blankets; why he comes a-buttin' round the Godforsaken ends of the earth--that's what I can't exactly see." "He might have lived to a ripe old age if he'd stayed at home," Henry agreed. Bill opened his mouth to speak, but changed his mind. Instead, he pointed towards the wall of darkness that pressed about them from every side. There was no suggestion of form in the utter blackness; only could be seen a pair of eyes gleaming like live coals. Henry indicated with his head a second pair, and a third. A circle of the gleaming eyes had drawn about their camp. Now and again a pair of eyes moved, or disappeared to appear again a moment later. The unrest of the dogs had been increasing, and they stampeded, in a surge of sudden fear, to the near side of the fire, cringing and crawling about the legs of the men. In the scramble one of the dogs had been overturned on the edge of the fire, and it had yelped with pain and fright as the smell of its singed coat possessed the air. The commotion caused the circle of eyes to shift restlessly for a moment and even to withdraw a bit, but it settled down again as the dogs became quiet. "Henry, it's a blame misfortune to be out of ammunition." Bill had finished his pipe and was helping his companion to spread the bed of fur and blanket upon the spruce boughs which he had laid over the snow before supper. Henry grunted, and began unlacing his moccasins. "How many cartridges did you say you had left?" he asked. "Three," came the answer. "An' I wisht 'twas three hundred. Then I'd show 'em what for, damn 'em!" He shook his fist angrily at the gleaming eyes, and began securely to prop his moccasins before the fire. "An' I wisht this cold snap'd break," he went on. "It's ben fifty below for two weeks now. An' I wisht I'd never started on this trip, Henry. I don't like the looks of it. I don't feel right, somehow. An' while I'm wishin', I wisht the trip was over an' done with, an' you an' me a-sittin' by the fire in Fort McGurry just about now an' playing cribbage--that's what I wisht." Henry grunted and crawled into bed. As he dozed off he was aroused by his comrade's voice. "Say, Henry, that other one that come in an' got a fish--why didn't the dogs pitch into it? That's what's botherin' me." "You're botherin' too much, Bill," came the sleepy response. "You was never like this before. You jes' shut up now, an' go to sleep, an' you'll be all hunkydory in the mornin'. Your stomach's sour, that's what's botherin' you." The men slept, breathing heavily, side by side, under the one covering. The fire died down, and the gleaming eyes drew closer the circle they had flung about the camp. The dogs clustered together in fear, now and again snarling menacingly as a pair of eyes drew close. Once their uproar became so loud that Bill woke up. He got out of bed carefully, so as not to disturb the sleep of his comrade, and threw more wood on the fire. As it began to flame up, the circle of eyes drew farther back. He glanced casually at the huddling dogs. He rubbed his eyes and looked at them more sharply. Then he crawled back into the blankets. "Henry," he said. "Oh, Henry." Henry groaned as he passed from sleep to waking, and demanded, "What's wrong now?" "Nothin'," came the answer; "only there's seven of 'em again. I just counted." Henry acknowledged receipt of the information with a grunt that slid into a snore as he drifted back into sleep. In the morning it was Henry who awoke first and routed his companion out of bed. Daylight was yet three hours away, though it was already six o'clock; and in the darkness Henry went about preparing breakfast, while Bill rolled the blankets and made the sled ready for lashing. "Say, Henry," he asked suddenly, "how many dogs did you say we had?" "Six." "Wrong," Bill proclaimed triumphantly. "Seven again?" Henry queried. "No, five; one's gone." "The hell!" Henry cried in wrath, leaving the cooking to come and count the dogs. "You're right, Bill," he concluded. "Fatty's gone." "An' he went like greased lightnin' once he got started. Couldn't've seen'm for smoke." "No chance at all," Henry concluded. "They jes' swallowed'm alive. I bet he was yelpin' as he went down their throats, damn 'em!" "He always was a fool dog," said Bill. "But no fool dog ought to be fool enough to go off an' commit suicide that way." He looked over the remainder of the team with a speculative eye that summed up instantly the salient traits of each animal. "I bet none of the others would do it." "Couldn't drive 'em away from the fire with a club," Bill agreed. "I always did think there was somethin' wrong with Fatty anyway." And this was the epitaph of a dead dog on the Northland trail--less scant than the epitaph of many another dog, of many a man. Breakfast eaten and the slim camp-outfit lashed to the sled, the men turned their backs on the cheery fire and launched out into the darkness. At once began to rise the cries that were fiercely sad--cries that called through the darkness and cold to one another and answered back. Conversation ceased. Daylight came at nine o'clock. At midday the sky to the south warmed to rose-colour, and marked where the bulge of the earth intervened between the meridian sun and the northern world. But the rose-colour swiftly faded. The grey light of day that remained lasted until three o'clock, when it, too, faded, and the pall of the Arctic night descended upon the lone and silent land. As darkness came on, the hunting-cries to right and left and rear drew closer--so close that more than once they sent surges of fear through the toiling dogs, throwing them into short-lived panics. At the conclusion of one such panic, when he and Henry had got the dogs back in the traces, Bill said: "I wisht they'd strike game somewheres, an' go away an' leave us alone." "They do get on the nerves horrible," Henry sympathised. They spoke no more until camp was made. Henry was bending over and adding ice to the babbling pot of beans when he was startled by the sound of a blow, an exclamation from Bill, and a sharp snarling cry of pain from among the dogs. He straightened up in time to see a dim form disappearing across the snow into the shelter of the dark. Then he saw Bill, standing amid the dogs, half triumphant, half crestfallen, in one hand a stout club, in the other the tail and part of the body of a sun-cured salmon. "It got half of it," he announced; "but I got a whack at it jes' the same. D'ye hear it squeal?" "What'd it look like?" Henry asked. "Couldn't see. But it had four legs an' a mouth an' hair an' looked like any dog." "Must be a tame wolf, I reckon." "It's damned tame, whatever it is, comin' in here at feedin' time an' gettin' its whack of fish." That night, when supper was finished and they sat on the oblong box and pulled at their pipes, the circle of gleaming eyes drew in even closer than before. "I wisht they'd spring up a bunch of moose or something, an' go away an' leave us alone," Bill said. Henry grunted with an intonation that was not all sympathy, and for a quarter of an hour they sat on in silence, Henry staring at the fire, and Bill at the circle of eyes that burned in the darkness just beyond the firelight. "I wisht we was pullin' into McGurry right now," he began again. "Shut up your wishin' and your croakin'," Henry burst out angrily. "Your stomach's sour. That's what's ailin' you. Swallow a spoonful of sody, an' you'll sweeten up wonderful an' be more pleasant company." In the morning Henry was aroused by fervid blasphemy that proceeded from the mouth of Bill. Henry propped himself up on an elbow and looked to see his comrade standing among the dogs beside the replenished fire, his arms raised in objurgation, his face distorted with passion. "Hello!" Henry called. "What's up now?" "Frog's gone," came the answer. "No." "I tell you yes." Henry leaped out of the blankets and to the dogs. He counted them with care, and then joined his partner in cursing the power of the Wild that had robbed them of another dog. "Frog was the strongest dog of the bunch," Bill pronounced finally. "An' he was no fool dog neither," Henry added. And so was recorded the second epitaph in two days. A gloomy breakfast was eaten, and the four remaining dogs were harnessed to the sled. The day was a repetition of the days that had gone before. The men toiled without speech across the face of the frozen world. The silence was unbroken save by the cries of their pursuers, that, unseen, hung upon their rear. With the coming of night in the mid-afternoon, the cries sounded closer as the pursuers drew in according to their custom; and the dogs grew excited and frightened, and were guilty of panics that tangled the traces and further depressed the two men. "There, that'll fix you fool critters," Bill said with satisfaction that night, standing erect at completion of his task. Henry left the cooking to come and see. Not only had his partner tied the dogs up, but he had tied them, after the Indian fashion, with sticks. About the neck of each dog he had fastened a leather thong. To this, and so close to the neck that the dog could not get his teeth to it, he had tied a stout stick four or five feet in length. The other end of the stick, in turn, was made fast to a stake in the ground by means of a leather thong. The dog was unable to gnaw through the leather at his own end of the stick. The stick prevented him from getting at the leather that fastened the other end. Henry nodded his head approvingly. "It's the only contraption that'll ever hold One Ear," he said. "He can gnaw through leather as clean as a knife an' jes' about half as quick. They all'll be here in the mornin' hunkydory." "You jes' bet they will," Bill affirmed. "If one of em' turns up missin', I'll go without my coffee." "They jes' know we ain't loaded to kill," Henry remarked at bed-time, indicating the gleaming circle that hemmed them in. "If we could put a couple of shots into 'em, they'd be more respectful. They come closer every night. Get the firelight out of your eyes an' look hard--there! Did you see that one?" For some time the two men amused themselves with watching the movement of vague forms on the edge of the firelight. By looking closely and steadily at where a pair of eyes burned in the darkness, the form of the animal would slowly take shape. They could even see these forms move at times. A sound among the dogs attracted the men's attention. One Ear was uttering quick, eager whines, lunging at the length of his stick toward the darkness, and desisting now and again in order to make frantic attacks on the stick with his teeth. "Look at that, Bill," Henry whispered. Full into the firelight, with a stealthy, sidelong movement, glided a doglike animal. It moved with commingled mistrust and daring, cautiously observing the men, its attention fixed on the dogs. One Ear strained the full length of the stick toward the intruder and whined with eagerness. "That fool One Ear don't seem scairt much," Bill said in a low tone. "It's a she-wolf," Henry whispered back, "an' that accounts for Fatty an' Frog. She's the decoy for the pack. She draws out the dog an' then all the rest pitches in an' eats'm up." The fire crackled. A log fell apart with a loud spluttering noise. At the sound of it the strange animal leaped back into the darkness. "Henry, I'm a-thinkin'," Bill announced. "Thinkin' what?" "I'm a-thinkin' that was the one I lambasted with the club." "Ain't the slightest doubt in the world," was Henry's response. "An' right here I want to remark," Bill went on, "that that animal's familyarity with campfires is suspicious an' immoral." "It knows for certain more'n a self-respectin' wolf ought to know," Henry agreed. "A wolf that knows enough to come in with the dogs at feedin' time has had experiences." "Ol' Villan had a dog once that run away with the wolves," Bill cogitates aloud. "I ought to know. I shot it out of the pack in a moose pasture over 'on Little Stick. An' Ol' Villan cried like a baby. Hadn't seen it for three years, he said. Ben with the wolves all that time." "I reckon you've called the turn, Bill. That wolf's a dog, an' it's eaten fish many's the time from the hand of man." "An if I get a chance at it, that wolf that's a dog'll be jes' meat," Bill declared. "We can't afford to lose no more animals." "But you've only got three cartridges," Henry objected. "I'll wait for a dead sure shot," was the reply. In the morning Henry renewed the fire and cooked breakfast to the accompaniment of his partner's snoring. "You was sleepin' jes' too comfortable for anything," Henry told him, as he routed him out for breakfast. "I hadn't the heart to rouse you." Bill began to eat sleepily. He noticed that his cup was empty and started to reach for the pot. But the pot was beyond arm's length and beside Henry. "Say, Henry," he chided gently, "ain't you forgot somethin'?" Henry looked about with great carefulness and shook his head. Bill held up the empty cup. "You don't get no coffee," Henry announced. "Ain't run out?" Bill asked anxiously. "Nope." "Ain't thinkin' it'll hurt my digestion?" "Nope." A flush of angry blood pervaded Bill's face. "Then it's jes' warm an' anxious I am to be hearin' you explain yourself," he said. "Spanker's gone," Henry answered. Without haste, with the air of one resigned to misfortune Bill turned his head, and from where he sat counted the dogs. "How'd it happen?" he asked apathetically. Henry shrugged his shoulders. "Don't know. Unless One Ear gnawed'm loose. He couldn't a-done it himself, that's sure." "The darned cuss." Bill spoke gravely and slowly, with no hint of the anger that was raging within. "Jes' because he couldn't chew himself loose, he chews Spanker loose." "Well, Spanker's troubles is over anyway; I guess he's digested by this time an' cavortin' over the landscape in the bellies of twenty different wolves," was Henry's epitaph on this, the latest lost dog. "Have some coffee, Bill." But Bill shook his head. "Go on," Henry pleaded, elevating the pot. Bill shoved his cup aside. "I'll be ding-dong-danged if I do. I said I wouldn't if ary dog turned up missin', an' I won't." "It's darn good coffee," Henry said enticingly. But Bill was stubborn, and he ate a dry breakfast washed down with mumbled curses at One Ear for the trick he had played. "I'll tie 'em up out of reach of each other to-night," Bill said, as they took the trail. They had travelled little more than a hundred yards, when Henry, who was in front, bent down and picked up something with which his snowshoe had collided. It was dark, and he could not see it, but he recognised it by the touch. He flung it back, so that it struck the sled and bounced along until it fetched up on Bill's snowshoes. "Mebbe you'll need that in your business," Henry said. Bill uttered an exclamation. It was all that was left of Spanker--the stick with which he had been tied. "They ate'm hide an' all," Bill announced. "The stick's as clean as a whistle. They've ate the leather offen both ends. They're damn hungry, Henry, an' they'll have you an' me guessin' before this trip's over." Henry laughed defiantly. "I ain't been trailed this way by wolves before, but I've gone through a whole lot worse an' kept my health. Takes more'n a handful of them pesky critters to do for yours truly, Bill, my son." "I don't know, I don't know," Bill muttered ominously. "Well, you'll know all right when we pull into McGurry." "I ain't feelin' special enthusiastic," Bill persisted. "You're off colour, that's what's the matter with you," Henry dogmatised. "What you need is quinine, an' I'm goin' to dose you up stiff as soon as we make McGurry." Bill grunted his disagreement with the diagnosis, and lapsed into silence. The day was like all the days. Light came at nine o'clock. At twelve o'clock the southern horizon was warmed by the unseen sun; and then began the cold grey of afternoon that would merge, three hours later, into night. It was just after the sun's futile effort to appear, that Bill slipped the rifle from under the sled-lashings and said: "You keep right on, Henry, I'm goin' to see what I can see." "You'd better stick by the sled," his partner protested. "You've only got three cartridges, an' there's no tellin' what might happen." "Who's croaking now?" Bill demanded triumphantly. Henry made no reply, and plodded on alone, though often he cast anxious glances back into the grey solitude where his partner had disappeared. An hour later, taking advantage of the cut-offs around which the sled had to go, Bill arrived. "They're scattered an' rangin' along wide," he said: "keeping up with us an' lookin' for game at the same time. You see, they're sure of us, only they know they've got to wait to get us. In the meantime they're willin' to pick up anything eatable that comes handy." "You mean they _think_ they're sure of us," Henry objected pointedly. But Bill ignored him. "I seen some of them. They're pretty thin. They ain't had a bite in weeks I reckon, outside of Fatty an' Frog an' Spanker; an' there's so many of 'em that that didn't go far. They're remarkable thin. Their ribs is like wash-boards, an' their stomachs is right up against their backbones. They're pretty desperate, I can tell you. They'll be goin' mad, yet, an' then watch out." A few minutes later, Henry, who was now travelling behind the sled, emitted a low, warning whistle. Bill turned and looked, then quietly stopped the dogs. To the rear, from around the last bend and plainly into view, on the very trail they had just covered, trotted a furry, slinking form. Its nose was to the trail, and it trotted with a peculiar, sliding, effortless gait. When they halted, it halted, throwing up its head and regarding them steadily with nostrils that twitched as it caught and studied the scent of them. "It's the she-wolf," Bill answered. The dogs had lain down in the snow, and he walked past them to join his partner in the sled. Together they watched the strange animal that had pursued them for days and that had already accomplished the destruction of half their dog-team. After a searching scrutiny, the animal trotted forward a few steps. This it repeated several times, till it was a short hundred yards away. It paused, head up, close by a clump of spruce trees, and with sight and scent studied the outfit of the watching men. It looked at them in a strangely wistful way, after the manner of a dog; but in its wistfulness there was none of the dog affection. It was a wistfulness bred of hunger, as cruel as its own fangs, as merciless as the frost itself. It was large for a wolf, its gaunt frame advertising the lines of an animal that was among the largest of its kind. "Stands pretty close to two feet an' a half at the shoulders," Henry commented. "An' I'll bet it ain't far from five feet long." "Kind of strange colour for a wolf," was Bill's criticism. "I never seen a red wolf before. Looks almost cinnamon to me." The animal was certainly not cinnamon-coloured. Its coat was the true wolf-coat. The dominant colour was grey, and yet there was to it a faint reddish hue--a hue that was baffling, that appeared and disappeared, that was more like an illusion of the vision, now grey, distinctly grey, and again giving hints and glints of a vague redness of colour not classifiable in terms of ordinary experience. "Looks for all the world like a big husky sled-dog," Bill said. "I wouldn't be s'prised to see it wag its tail." "Hello, you husky!" he called. "Come here, you whatever-your-name-is." "Ain't a bit scairt of you," Henry laughed. Bill waved his hand at it threateningly and shouted loudly; but the animal betrayed no fear. The only change in it that they could notice was an accession of alertness. It still regarded them with the merciless wistfulness of hunger. They were meat, and it was hungry; and it would like to go in and eat them if it dared. "Look here, Henry," Bill said, unconsciously lowering his voice to a whisper because of what he imitated. "We've got three cartridges. But it's a dead shot. Couldn't miss it. It's got away with three of our dogs, an' we oughter put a stop to it. What d'ye say?" Henry nodded his consent. Bill cautiously slipped the gun from under the sled-lashing. The gun was on the way to his shoulder, but it never got there. For in that instant the she-wolf leaped sidewise from the trail into the clump of spruce trees and disappeared. The two men looked at each other. Henry whistled long and comprehendingly. "I might have knowed it," Bill chided himself aloud as he replaced the gun. "Of course a wolf that knows enough to come in with the dogs at feedin' time, 'd know all about shooting-irons. I tell you right now, Henry, that critter's the cause of all our trouble. We'd have six dogs at the present time,'stead of three, if it wasn't for her. An' I tell you right now, Henry, I'm goin' to get her. She's too smart to be shot in the open. But I'm goin' to lay for her. I'll bushwhack her as sure as my name is Bill." "You needn't stray off too far in doin' it," his partner admonished. "If that pack ever starts to jump you, them three cartridges'd be wuth no more'n three whoops in hell. Them animals is damn hungry, an' once they start in, they'll sure get you, Bill." They camped early that night. Three dogs could not drag the sled so fast nor for so long hours as could six, and they were showing unmistakable signs of playing out. And the men went early to bed, Bill first seeing to it that the dogs were tied out of gnawing-reach of one another. But the wolves were growing bolder, and the men were aroused more than once from their sleep. So near did the wolves approach, that the dogs became frantic with terror, and it was necessary to replenish the fire from time to time in order to keep the adventurous marauders at safer distance. "I've hearn sailors talk of sharks followin' a ship," Bill remarked, as he crawled back into the blankets after one such replenishing of the fire. "Well, them wolves is land sharks. They know their business better'n we do, an' they ain't a-holdin' our trail this way for their health. They're goin' to get us. They're sure goin' to get us, Henry." "They've half got you a'ready, a-talkin' like that," Henry retorted sharply. "A man's half licked when he says he is. An' you're half eaten from the way you're goin' on about it." "They've got away with better men than you an' me," Bill answered. "Oh, shet up your croakin'. You make me all-fired tired." Henry rolled over angrily on his side, but was surprised that Bill made no similar display of temper. This was not Bill's way, for he was easily angered by sharp words. Henry thought long over it before he went to sleep, and as his eyelids fluttered down and he dozed off, the thought in his mind was: "There's no mistakin' it, Bill's almighty blue. I'll have to cheer him up to-morrow." The day began auspiciously. They had lost no dogs during the night, and they swung out upon the trail and into the silence, the darkness, and the cold with spirits that were fairly light. Bill seemed to have forgotten his forebodings of the previous night, and even waxed facetious with the dogs when, at midday, they overturned the sled on a bad piece of trail. It was an awkward mix-up. The sled was upside down and jammed between a tree-trunk and a huge rock, and they were forced to unharness the dogs in order to straighten out the tangle. The two men were bent over the sled and trying to right it, when Henry observed One Ear sidling away. "Here, you, One Ear!" he cried, straightening up and turning around on the dog. But One Ear broke into a run across the snow, his traces trailing behind him. And there, out in the snow of their back track, was the she-wolf waiting for him. As he neared her, he became suddenly cautious. He slowed down to an alert and mincing walk and then stopped. He regarded her carefully and dubiously, yet desirefully. She seemed to smile at him, showing her teeth in an ingratiating rather than a menacing way. She moved toward him a few steps, playfully, and then halted. One Ear drew near to her, still alert and cautious, his tail and ears in the air, his head held high. He tried to sniff noses with her, but she retreated playfully and coyly. Every advance on his part was accompanied by a corresponding retreat on her part. Step by step she was luring him away from the security of his human companionship. Once, as though a warning had in vague ways flitted through his intelligence, he turned his head and looked back at the overturned sled, at his team-mates, and at the two men who were calling to him. But whatever idea was forming in his mind, was dissipated by the she-wolf, who advanced upon him, sniffed noses with him for a fleeting instant, and then resumed her coy retreat before his renewed advances. In the meantime, Bill had bethought himself of the rifle. But it was jammed beneath the overturned sled, and by the time Henry had helped him to right the load, One Ear and the she-wolf were too close together and the distance too great to risk a shot. Too late One Ear learned his mistake. Before they saw the cause, the two men saw him turn and start to run back toward them. Then, approaching at right angles to the trail and cutting off his retreat they saw a dozen wolves, lean and grey, bounding across the snow. On the instant, the she- wolf's coyness and playfulness disappeared. With a snarl she sprang upon One Ear. He thrust her off with his shoulder, and, his retreat cut off and still intent on regaining the sled, he altered his course in an attempt to circle around to it. More wolves were appearing every moment and joining in the chase. The she-wolf was one leap behind One Ear and holding her own. "Where are you goin'?" Henry suddenly demanded, laying his hand on his partner's arm. Bill shook it off. "I won't stand it," he said. "They ain't a-goin' to get any more of our dogs if I can help it." Gun in hand, he plunged into the underbrush that lined the side of the trail. His intention was apparent enough. Taking the sled as the centre of the circle that One Ear was making, Bill planned to tap that circle at a point in advance of the pursuit. With his rifle, in the broad daylight, it might be possible for him to awe the wolves and save the dog. "Say, Bill!" Henry called after him. "Be careful! Don't take no chances!" Henry sat down on the sled and watched. There was nothing else for him to do. Bill had already gone from sight; but now and again, appearing and disappearing amongst the underbrush and the scattered clumps of spruce, could be seen One Ear. Henry judged his case to be hopeless. The dog was thoroughly alive to its danger, but it was running on the outer circle while the wolf-pack was running on the inner and shorter circle. It was vain to think of One Ear so outdistancing his pursuers as to be able to cut across their circle in advance of them and to regain the sled. The different lines were rapidly approaching a point. Somewhere out there in the snow, screened from his sight by trees and thickets, Henry knew that the wolf-pack, One Ear, and Bill were coming together. All too quickly, far more quickly than he had expected, it happened. He heard a shot, then two shots, in rapid succession, and he knew that Bill's ammunition was gone. Then he heard a great outcry of snarls and yelps. He recognised One Ear's yell of pain and terror, and he heard a wolf-cry that bespoke a stricken animal. And that was all. The snarls ceased. The yelping died away. Silence settled down again over the lonely land. He sat for a long while upon the sled. There was no need for him to go and see what had happened. He knew it as though it had taken place before his eyes. Once, he roused with a start and hastily got the axe out from underneath the lashings. But for some time longer he sat and brooded, the two remaining dogs crouching and trembling at his feet. At last he arose in a weary manner, as though all the resilience had gone out of his body, and proceeded to fasten the dogs to the sled. He passed a rope over his shoulder, a man-trace, and pulled with the dogs. He did not go far. At the first hint of darkness he hastened to make a camp, and he saw to it that he had a generous supply of firewood. He fed the dogs, cooked and ate his supper, and made his bed close to the fire. But he was not destined to enjoy that bed. Before his eyes closed the wolves had drawn too near for safety. It no longer required an effort of the vision to see them. They were all about him and the fire, in a narrow circle, and he could see them plainly in the firelight lying down, sitting up, crawling forward on their bellies, or slinking back and forth. They even slept. Here and there he could see one curled up in the snow like a dog, taking the sleep that was now denied himself. He kept the fire brightly blazing, for he knew that it alone intervened between the flesh of his body and their hungry fangs. His two dogs stayed close by him, one on either side, leaning against him for protection, crying and whimpering, and at times snarling desperately when a wolf approached a little closer than usual. At such moments, when his dogs snarled, the whole circle would be agitated, the wolves coming to their feet and pressing tentatively forward, a chorus of snarls and eager yelps rising about him. Then the circle would lie down again, and here and there a wolf would resume its broken nap. But this circle had a continuous tendency to draw in upon him. Bit by bit, an inch at a time, with here a wolf bellying forward, and there a wolf bellying forward, the circle would narrow until the brutes were almost within springing distance. Then he would seize brands from the fire and hurl them into the pack. A hasty drawing back always resulted, accompanied by angry yelps and frightened snarls when a well-aimed brand struck and scorched a too daring animal. Morning found the man haggard and worn, wide-eyed from want of sleep. He cooked breakfast in the darkness, and at nine o'clock, when, with the coming of daylight, the wolf-pack drew back, he set about the task he had planned through the long hours of the night. Chopping down young saplings, he made them cross-bars of a scaffold by lashing them high up to the trunks of standing trees. Using the sled-lashing for a heaving rope, and with the aid of the dogs, he hoisted the coffin to the top of the scaffold. "They got Bill, an' they may get me, but they'll sure never get you, young man," he said, addressing the dead body in its tree-sepulchre. Then he took the trail, the lightened sled bounding along behind the willing dogs; for they, too, knew that safety lay open in the gaining of Fort McGurry. The wolves were now more open in their pursuit, trotting sedately behind and ranging along on either side, their red tongues lolling out, their lean sides showing the undulating ribs with every movement. They were very lean, mere skin-bags stretched over bony frames, with strings for muscles--so lean that Henry found it in his mind to marvel that they still kept their feet and did not collapse forthright in the snow. He did not dare travel until dark. At midday, not only did the sun warm the southern horizon, but it even thrust its upper rim, pale and golden, above the sky-line. He received it as a sign. The days were growing longer. The sun was returning. But scarcely had the cheer of its light departed, than he went into camp. There were still several hours of grey daylight and sombre twilight, and he utilised them in chopping an enormous supply of fire-wood. With night came horror. Not only were the starving wolves growing bolder, but lack of sleep was telling upon Henry. He dozed despite himself, crouching by the fire, the blankets about his shoulders, the axe between his knees, and on either side a dog pressing close against him. He awoke once and saw in front of him, not a dozen feet away, a big grey wolf, one of the largest of the pack. And even as he looked, the brute deliberately stretched himself after the manner of a lazy dog, yawning full in his face and looking upon him with a possessive eye, as if, in truth, he were merely a delayed meal that was soon to be eaten. This certitude was shown by the whole pack. Fully a score he could count, staring hungrily at him or calmly sleeping in the snow. They reminded him of children gathered about a spread table and awaiting permission to begin to eat. And he was the food they were to eat! He wondered how and when the meal would begin. As he piled wood on the fire he discovered an appreciation of his own body which he had never felt before. He watched his moving muscles and was interested in the cunning mechanism of his fingers. By the light of the fire he crooked his fingers slowly and repeatedly now one at a time, now all together, spreading them wide or making quick gripping movements. He studied the nail-formation, and prodded the finger-tips, now sharply, and again softly, gauging the while the nerve-sensations produced. It fascinated him, and he grew suddenly fond of this subtle flesh of his that worked so beautifully and smoothly and delicately. Then he would cast a glance of fear at the wolf-circle drawn expectantly about him, and like a blow the realisation would strike him that this wonderful body of his, this living flesh, was no more than so much meat, a quest of ravenous animals, to be torn and slashed by their hungry fangs, to be sustenance to them as the moose and the rabbit had often been sustenance to him. He came out of a doze that was half nightmare, to see the red-hued she- wolf before him. She was not more than half a dozen feet away sitting in the snow and wistfully regarding him. The two dogs were whimpering and snarling at his feet, but she took no notice of them. She was looking at the man, and for some time he returned her look. There was nothing threatening about her. She looked at him merely with a great wistfulness, but he knew it to be the wistfulness of an equally great hunger. He was the food, and the sight of him excited in her the gustatory sensations. Her mouth opened, the saliva drooled forth, and she licked her chops with the pleasure of anticipation. A spasm of fear went through him. He reached hastily for a brand to throw at her. But even as he reached, and before his fingers had closed on the missile, she sprang back into safety; and he knew that she was used to having things thrown at her. She had snarled as she sprang away, baring her white fangs to their roots, all her wistfulness vanishing, being replaced by a carnivorous malignity that made him shudder. He glanced at the hand that held the brand, noticing the cunning delicacy of the fingers that gripped it, how they adjusted themselves to all the inequalities of the surface, curling over and under and about the rough wood, and one little finger, too close to the burning portion of the brand, sensitively and automatically writhing back from the hurtful heat to a cooler gripping-place; and in the same instant he seemed to see a vision of those same sensitive and delicate fingers being crushed and torn by the white teeth of the she-wolf. Never had he been so fond of this body of his as now when his tenure of it was so precarious. All night, with burning brands, he fought off the hungry pack. When he dozed despite himself, the whimpering and snarling of the dogs aroused him. Morning came, but for the first time the light of day failed to scatter the wolves. The man waited in vain for them to go. They remained in a circle about him and his fire, displaying an arrogance of possession that shook his courage born of the morning light. He made one desperate attempt to pull out on the trail. But the moment he left the protection of the fire, the boldest wolf leaped for him, but leaped short. He saved himself by springing back, the jaws snapping together a scant six inches from his thigh. The rest of the pack was now up and surging upon him, and a throwing of firebrands right and left was necessary to drive them back to a respectful distance. Even in the daylight he did not dare leave the fire to chop fresh wood. Twenty feet away towered a huge dead spruce. He spent half the day extending his campfire to the tree, at any moment a half dozen burning faggots ready at hand to fling at his enemies. Once at the tree, he studied the surrounding forest in order to fell the tree in the direction of the most firewood. The night was a repetition of the night before, save that the need for sleep was becoming overpowering. The snarling of his dogs was losing its efficacy. Besides, they were snarling all the time, and his benumbed and drowsy senses no longer took note of changing pitch and intensity. He awoke with a start. The she-wolf was less than a yard from him. Mechanically, at short range, without letting go of it, he thrust a brand full into her open and snarling mouth. She sprang away, yelling with pain, and while he took delight in the smell of burning flesh and hair, he watched her shaking her head and growling wrathfully a score of feet away. But this time, before he dozed again, he tied a burning pine-knot to his right hand. His eyes were closed but few minutes when the burn of the flame on his flesh awakened him. For several hours he adhered to this programme. Every time he was thus awakened he drove back the wolves with flying brands, replenished the fire, and rearranged the pine-knot on his hand. All worked well, but there came a time when he fastened the pine- knot insecurely. As his eyes closed it fell away from his hand. He dreamed. It seemed to him that he was in Fort McGurry. It was warm and comfortable, and he was playing cribbage with the Factor. Also, it seemed to him that the fort was besieged by wolves. They were howling at the very gates, and sometimes he and the Factor paused from the game to listen and laugh at the futile efforts of the wolves to get in. And then, so strange was the dream, there was a crash. The door was burst open. He could see the wolves flooding into the big living-room of the fort. They were leaping straight for him and the Factor. With the bursting open of the door, the noise of their howling had increased tremendously. This howling now bothered him. His dream was merging into something else--he knew not what; but through it all, following him, persisted the howling. And then he awoke to find the howling real. There was a great snarling and yelping. The wolves were rushing him. They were all about him and upon him. The teeth of one had closed upon his arm. Instinctively he leaped into the fire, and as he leaped, he felt the sharp slash of teeth that tore through the flesh of his leg. Then began a fire fight. His stout mittens temporarily protected his hands, and he scooped live coals into the air in all directions, until the campfire took on the semblance of a volcano. But it could not last long. His face was blistering in the heat, his eyebrows and lashes were singed off, and the heat was becoming unbearable to his feet. With a flaming brand in each hand, he sprang to the edge of the fire. The wolves had been driven back. On every side, wherever the live coals had fallen, the snow was sizzling, and every little while a retiring wolf, with wild leap and snort and snarl, announced that one such live coal had been stepped upon. Flinging his brands at the nearest of his enemies, the man thrust his smouldering mittens into the snow and stamped about to cool his feet. His two dogs were missing, and he well knew that they had served as a course in the protracted meal which had begun days before with Fatty, the last course of which would likely be himself in the days to follow. "You ain't got me yet!" he cried, savagely shaking his fist at the hungry beasts; and at the sound of his voice the whole circle was agitated, there was a general snarl, and the she-wolf slid up close to him across the snow and watched him with hungry wistfulness. He set to work to carry out a new idea that had come to him. He extended the fire into a large circle. Inside this circle he crouched, his sleeping outfit under him as a protection against the melting snow. When he had thus disappeared within his shelter of flame, the whole pack came curiously to the rim of the fire to see what had become of him. Hitherto they had been denied access to the fire, and they now settled down in a close-drawn circle, like so many dogs, blinking and yawning and stretching their lean bodies in the unaccustomed warmth. Then the she- wolf sat down, pointed her nose at a star, and began to howl. One by one the wolves joined her, till the whole pack, on haunches, with noses pointed skyward, was howling its hunger cry. Dawn came, and daylight. The fire was burning low. The fuel had run out, and there was need to get more. The man attempted to step out of his circle of flame, but the wolves surged to meet him. Burning brands made them spring aside, but they no longer sprang back. In vain he strove to drive them back. As he gave up and stumbled inside his circle, a wolf leaped for him, missed, and landed with all four feet in the coals. It cried out with terror, at the same time snarling, and scrambled back to cool its paws in the snow. The man sat down on his blankets in a crouching position. His body leaned forward from the hips. His shoulders, relaxed and drooping, and his head on his knees advertised that he had given up the struggle. Now and again he raised his head to note the dying down of the fire. The circle of flame and coals was breaking into segments with openings in between. These openings grew in size, the segments diminished. "I guess you can come an' get me any time," he mumbled. "Anyway, I'm goin' to sleep." Once he awakened, and in an opening in the circle, directly in front of him, he saw the she-wolf gazing at him. Again he awakened, a little later, though it seemed hours to him. A mysterious change had taken place--so mysterious a change that he was shocked wider awake. Something had happened. He could not understand at first. Then he discovered it. The wolves were gone. Remained only the trampled snow to show how closely they had pressed him. Sleep was welling up and gripping him again, his head was sinking down upon his knees, when he roused with a sudden start. There were cries of men, and churn of sleds, the creaking of harnesses, and the eager whimpering of straining dogs. Four sleds pulled in from the river bed to the camp among the trees. Half a dozen men were about the man who crouched in the centre of the dying fire. They were shaking and prodding him into consciousness. He looked at them like a drunken man and maundered in strange, sleepy speech. "Red she-wolf.... Come in with the dogs at feedin' time.... First she ate the dog-food.... Then she ate the dogs.... An' after that she ate Bill.... " "Where's Lord Alfred?" one of the men bellowed in his ear, shaking him roughly. He shook his head slowly. "No, she didn't eat him.... He's roostin' in a tree at the last camp." "Dead?" the man shouted. "An' in a box," Henry answered. He jerked his shoulder petulantly away from the grip of his questioner. "Say, you lemme alone.... I'm jes' plump tuckered out.... Goo' night, everybody." His eyes fluttered and went shut. His chin fell forward on his chest. And even as they eased him down upon the blankets his snores were rising on the frosty air. But there was another sound. Far and faint it was, in the remote distance, the cry of the hungry wolf-pack as it took the trail of other meat than the man it had just missed. PART II

Two men, Bill and Henry, apparently drew the short straw, and have to haul a dead body to Fort McGurry. Along the way, they are chased by a pack of hungry wolves. One clever wolf, a female with a red sheen to her coat, is sneaking into the camp and stealing fish. She cunningly makes the two men lose count of how many dogs are on their team. The she-wolf lures away one of the dogs, Fatty, with promises of canine seduction. Then her pack-mates eat him. Talk about a honey trap. The men are sad and worried that Fatty is gone, but press on their way. We get a lather, rinse and repeat of chapter 1, as the she-wolf lures away a second dog. "A gloomy breakfast" is consumed afterwards. Bill ties the remaining dogs up with leather thongs and sticks to keep them from chasing after the canine Mata Hari. It doesn't work: a third dog is lured away and eaten. The men find the stick they had tied them with... the only thing left of the dog. They push on, despite the fact that the sun doesn't come up until nine in the morning and Bill has only three rounds left in his gun. Yikes. They spot the she-wolf on the trail. She's unimpressed by their manly-man attempts to scare her. Bill vows a bloody, terrible revenge against her, which clearly ain't gonna happen. That night when they camp, the wolves draw in closer. They must keep the fire burning and their dulcet slumber is interrupted by seriously freaked out sled dogs. The trip continues, but the sled gets tangled between a rock and a tree. While the men try to get it unstuck, a dog named One Ear runs off with the she-wolf. The men get a front-row seat to his seduction and consumption by the wolf pack. Bill runs off to rescue the dog. Henry hears the gunshots, but Bill doesn't come back. It's wolves: 5, men: 0 going into the fourth quarter. Henry pushes on, but has to stop for the night... with a boatload of firewood and an axe to keep him company. It doesn't work. The wolves get even closer, despite the fact that he occasionally smites them with burning wood. The next morning, Henry builds a scaffold to keep the coffin safe. Because the most important thing in this scenario is the welfare of a man who is already dead. He pushes on, but he's getting awfully tired and the wolves are getting closer. Every time he dozes, he risks ending up a hors d'oeuvre for the pack. Henry starts thinking of his body in a disturbing way: as meat for the wolves. The next morning, he can't make it back to the trail, and the wolves are ready to end the charade once and for all. At night, he slips into a dream... about wolves. Gee, we wonder why? He wakes up to see the dream brought to glorious life. The wolves are trying to eat him. Wacky mayhem ensues. He fights off the wolves by pulling hot coals out of the fire and flinging them at the wolves. The last two dogs get eaten, but Henry lives to fling hot coals another day. He then sets the fire in a circle and crouches inside of it. It holds them off for a while, but eventually burns low. Henry finally gives up the fight and goes to sleep. He wakes up to see the she-wolf looking at him, then wakes up again. The wolves are gone and men have come to rescue him. Naturally, they're more interested in the body he left than his well being. At least he's dedicated.

booksum

The endosymbiotic bacteria Wolbachia pipientis (wMel strain) has been successfully established in several populations of Aedes aegypti, the primary dengue vector. The virulent Wolbachia strain wMelPop is known to cause several pathological impacts (increased egg mortality, life shortening, etc.) reducing overall fitness in the mosquito Ae. aegypti. Increased egg mortality could substantially reduce egg banks in areas with a lengthy monsoonal dry season, and be employed to eliminate local populations. We tested this application under semi-field cage conditions. First, we determined that wMelPop infection significantly reduced the survival of desiccation-resistant eggs of the dengue vector Ae. aegypti, with shade and temperature having a significant impact; nearly all wMelPop-infected eggs failed to hatch after 6 and 10 weeks in summer and winter conditions, respectively. In laboratory selection experiments we found that egg desiccation resistance can be increased by selection, and that this effect of wMelPop infection is due to the nuclear background of the host rather than Wolbachia. We then conducted an invasion of wMelPop within a semi-field cage using sustained weekly releases of wMelPop infected mosquitoes, with fixation achieved after 9 weeks. The egg populations wMelPop infected and an uninfected control were then subjected to a simulated prolonged monsoonal dry season (2. 5 months) before flooding to induce hatching. The wMelPop infected eggs suffered significantly greater mortality than the controls, with only 0. 67% and 4. 35% of respective infected and uninfected eggs held in 99% shade hatching after 80 days. These studies suggest that wMelPop could be used to locally eliminate populations of Ae. aegypti that are exposed to prolonged dry conditions, particularly if combined with vector control. Dengue is the leading cause of arboviral disease in humans. An estimated 390 million infections and 90 million clinical cases occur annually [1]. There is no commercially available vaccine, so vector control and mosquito avoidance are the only methods to limit transmission. Traditional control of the mosquito vector Aedes aegypti is increasingly hampered by physiological resistance against insecticides, especially synthetic pyrethroids [2–5]. Furthermore, the logistics of controlling a vector that exploits artificial containers that are often cryptic, subterranean and difficult to access (eg., elevated sites such as rain water tanks) makes effective control difficult to achieve [6–8]. The adult Ae. aegypti is endophilic, and is often found inside dark, quiet areas within premises [9]. These areas are not effectively targeted by insecticidal sprays applied by air or ground [9,10]. Collectively, vector control efforts to control dengue are generally not successful, and dengue continues to expand in breadth and reach globally. New vector population manipulation methods have been developed to overcome the issues of traditional vector control. The release of insects infected with a dominant lethal gene (RIDL) employs the release of male Ae. aegypti that induce sterility by passing a lethal gene to wild females they mate with [11]. Serial releases of these genetically modified male mosquitoes thus lead to collapse of the resident wild population [11,12]. The vector capacity of resident populations of Ae. aegypti can also be reduced to lessen dengue transmission. Strains of the endosymbiotic bacteria Wolbachia were transinfected into Ae. aegypti eggs, and are then passed on maternally [13]. Male Ae. aegypti infected with Wolbachia induce embryo death when mating with uninfected females [13,14]. This creates a powerful drive mechanism that allows Wolbachia to spread naturally within a population of uninfected Ae. aegypti, and to persist once fixed [15–17]. The presence of Wolbachia infection also interrupts dengue (and other arboviruses) replication in Ae. aegypti, interfering with transmission [18–20]. Two virus blocking strains of Wolbachia (wMel and wMelPop) have been established in Ae. aegypti. To date, populations of wMel-infected Ae. aegypti have been released and established in seven localities around Cairns, Australia. The more virulent wMelPop over-replicates inside the mosquito, inducing a variety of physiological manifestations. These include early death (life-shortening) [13], egg mortality [21,22], reduced blood feeding [23], delayed larval development [24] and reduced overall fitness [25]. The high density of wMelPop probably contributes to its almost complete blocking of dengue replication and transmission in Ae. aegypti [18], but efforts to introduce this infection into wild populations have proved challenging [25], although it has been accomplished in semi-field cages [19]. One implication of high mortality, especially reduced egg longevity, is that wMelPop could lead to reduced population size of Ae. aegypti [26]. In areas with a pronounced monsoonal dry season, mosquitoes survive for up to several months as desiccation resistant eggs [27]. If wMelPop can be established in these populations during the wet season, populations might naturally die off during the dry season. In this paper we measure the relative survival of wMelPop infected Ae. aegypti eggs under natural conditions. Because this strategy could be thwarted if there is evolution in wMelPop to counter any deleterious fitness effect, we also explore strain variation involved in egg survival. Finally, we simulate a wMelPop intervention from invasion to subsequent death of the eggs in a semi-field cage to test the concept that wMelPop could be used to crash and locally eliminate Ae. aegypti egg banks under a prolonged monsoonal dry season. A protective awning with three different shade regimes was built to house mosquito eggs in the survival study. The awning (17 m x 3. 3 m) was built between 2 semi-field cages [29] and divided into three sections that provided 30%, 70% and 99% shade (Fig 1). The awning roof consisted of waterproof translucent vinyl to protect eggs from rainfall. Two 5. 6 m sections of the vinyl had an interior piece of 40% and 95% shade cloth to provide additional shade to create the 70% and 99% shade; the section with vinyl alone provided 30% shade. Temperatures and relative humidity within each section were monitored using data loggers (Hygrochron iButton DS1923, Dallas Semiconductors) set atop and below the table, supplemented with data from the Bureau of Meteorology (BOM) site 15 km SE of the site when loggers failed. Preliminary trials indicated that ambient temperatures (under table) in the 70% and 99% shade (S1 Fig) were nearly identical to screen height temperatures at the Cairns Airport BOM site. However, loggers on the table that were exposed to sunlight (30% and 70% shade) experienced a spike in temperatures that exceeded 50°C and 60°C in the winter and summer, respectively (S2 Fig). Egg strips were obtained by exposing sandpaper strips to gravid mosquitoes. Eggs were allowed to incubate within the flooded oviposition cup for 3 days, then dried. Viable (non flattened) eggs on strips were counted and each strip labeled with egg count using masking tape on a bulldog clip). Eggs strips were attached vertically to the inside of a wire mesh basket that was set on a card table within each section (Fig 1). The legs of the table were set inside a saucer containing talcum powder to prevent ants from invading the table and predating eggs. Three egg strips were randomly picked and hatched in a dilute yeast solution at 0,1, 2,3, 4,6, 8,12 and 16 weeks post exposure. The experiment was conducted in the cool dry (10 May– 30 Aug. 2012) and warm dry season (11 Oct. 2012–3 Jan. 2013). Percent hatch was calculated as the number of hatched eggs (1st instar larvae) divided by total viable eggs. To assess changes in hatch rates across time in the summer and winter egg quiescence experiment, we considered rates observed in the first 6 weeks or less and then ran General Linear Models (GLMs) examining the impact of strain and shade on hatch rates, with time treated as a linear and quadratic variable. We were particularly interested in the interaction between time and strain reflecting changes in hatch rate over time. GLMs were run with and without angularly transforming hatch rates but only the latter are represented because the conclusions were identical. We conducted a trial to see if establishing a fixed population of wMelPop infected Ae. aegypti could be used to crash the viable egg bank during the dry season and potentially eliminate the local population under semi-field cage conditions. Two semi-field cages (4 x 7 x 4 m) were populated with wild (F1-2) Cairns Ae. aegypti. Each cage contained 4 10-L buckets and 4 potplants with flooded potplant base (PPB). Three times per week a volunteer sat in the cage for 10 min to bloodfed mosquitoes; 1 pellet of lucerne was added to each bucket and the potplant base was refilled with water. Buckets and PPBs were flooded weekly to hatch new eggs. The populations were introduced into the cage on 28 April 2013 and allowed to establish for 4 weeks. Then populations were crashed (simulated source reduction campaign) by emptying 3 of the 4 buckets and PPBs to kill immatures then spraying the inside of each with 4% sodium hypochlorite (commercial bleach) to kill eggs [30]. One week after population suppression, PPB and buckets were refilled with water. Mean pupal counts were used to estimate the adult population in both cages by integrating estimated adult populations for 0. 8 and 0. 9 daily survival over 14 days [31,32]. Single Biogents Sentinel traps (BGS) were run for 3 hr in each cage 2–3 times/week to monitor adult populations. We used the relative change in BGS captures during the 2 weeks before and after releases of wMelPop-infected mosquitoes to estimate the size of the released cohort [33]. On 6 June 2013 weekly release of wMelPop infected mosquitoes began in the treatment cage. A colony of AOMB wMelPop cultured at a constant temperature was the source of release material. Two cups of 50 (1: 1 male: female) adult Ae. aegypti infected with wMelPop were released into the north (treatment cage). This equated to a relative release ratio of 1. 4 and 2. 0 for female and male infected mosquitoes, respectively, based on the relative change in BGS captures from 2 weeks before and after release [33]. Mosquito populations were also monitored in both cages by counting pupae in buckets and PPBs once week. Mosquitoes in the control and treatment cage were monitored for Wolbachia infection by PCR analysis of sample of up to 30 larvae/week; larvae were reared to adults for PCR. We also conducted another source reduction (3/4 of buckets and PPBs treated as before) supplemented with removal of adults using a sweepnet on 5 July. Releases of Wolbachia infected mosquitoes continued until 100% of sampled mosquitoes in the treatment cage were infected with wMelPop for two successive weeks; the last release was on 26 Sept. 2013. We characterized the infection frequency in the cage by testing samples of 30–90 adults (collected as larvae) for the Wolbachia infection. DNA was extracted from adult mosquitoes by using a Chelex 100 resin (Bio-Rad Laboratories, Hercules, CA) extraction method. 38 mosquitoes were ground in 3 L of proteinase K (20 mg/mL) (Roche Diagnostics Australia Pty. Ltd., Castle Hill New South Wales, Australia) and 250 L of 5% Chelex solution, incubated at 65°C for 1 hour, followed by incubation for 10 minutes at 90°C and storage at -20°C. Wolbachia infection status was determined by using methods developed for a Roche LightCycler 480 [34] with a modification to the primers used as described elsewhere [25]. Quantitative real-time polymerase chain reaction (PCR) was used to amplify three markers with three primer sets: a mosquito primer set to detect mosquitoes from the Aedes genus, Ae. aegypti specific primers to differentiate Ae. aegypti from other Aedes species, and Wolbachia specific primers to determine Wolbachia infection status, as well as density. PCR conditions were as follows: 95°C for 10 minutes, 40 cycles of 95°C for 5 seconds, 58°C for 15 seconds and 72°C for 15 seconds, ending with a 95°C 1-minute heating followed by cool down to 40°C for 20 seconds before raising to 65°C. A melting curve analysis was performed via a gradual increase of temperature from 65°C to 95°C. Diagnosis was based on crossing-point values for the PCR and melting temperature from high resolution melt analysis. We tested the potential for wMelPop to eliminate the field cage population of Ae. aegypti. Mosquitoes in both the treatment and control cages were allowed oviposit for three weeks after the last release. To provide a substrate that could be easily divided into separate egg cohorts to measure survival, we placed a roughened plastic liner inside each bucket for oviposition [30]. Sections of each liner were then cut off with scissors, viable eggs counted microscopically, and placed within the 70% and 99% shade sections of the awning used to measure egg survival. Strips of wMelPop infected and uninfected eggs were hatched in dilute yeast solution after 29,59 and 80 days exposure (7 Nov, 16 Dec and 6 Jan). Strips were dried for 1 week then reflooded to hatch any viable eggs not initially hatched. Percent hatch was calculated as with the egg survival study. In the final hatch all potplant bases and buckets were flooded to see if any eggs that may have been laid on the container rather than the ovistrip survived. We compared the total percent hatch between treatment and control for exposure period using chi square. Selection experiments were conducted to examine if egg desiccation resistance was due to a nuclear or Wolbachia effect. Eggs were collected daily for four days using sandpaper ovistrips and were fully conditioned as per the standard protocol [22]. All ovistrips were placed into a desiccation chamber set at ~60% Relative Humidity (RH) using a saturated solution of sodium bromide [22]. RH was monitored using a hygrochron (1-wire, iButton. com). To select a wMelPop desiccation resistant line (AOMB-DS), we were aiming to obtain around 10% hatch rate over four rounds of selection. Previous experiments have shown that a 10% hatch rate occurred around Day 20–21. Therefore, the ovistrips were divided into batches and hatched over a range of days (18–26) to obtain a hatch rate close to 10%. Eggs were hatched using Reverse Osmosis (RO) water, Tetramin fish food and yeast [22]. Hatch rate was determined by (number of eggs/number of larvae) x 100. In each generation of selection, at least 100 eggs were used in the next generation. Each round of selection was followed by a non-selected generation to increase the number of adults for producing eggs available for selection in the next generation. There were four rounds of selection and the colony was then maintained in the laboratory over several generations. To determine if the selection response in AOMB-DS was due to a nuclear or Wolbachia effect, the selected line was crossed to unselected lines. Two new lines (AX1, AX2) were created by backcrossing AOMB-DS to males or females of the original AOMB line. Backcrossing was continued for three generations to place Wolbachia from the selected line onto the nuclear background of the infected (AX1) stock (Fig 2) or to place Wolbachia from the unselected line onto the nuclear background of the selected infected line (AX2). To test for viability, eggs were collected over four day periods from the AOMB-DS, AOMB, and C20 colonies as well as the backcrossed AX1 and AX2 lines. These were placed in the 60% desiccation chamber once conditioned. Three days later, eggs were cut into 10 batches of at least 25 eggs then counted and hatched into separate cups filled with 170 ml RO water and TetraMin fish food. Larvae were counted one week later as an estimate of viability. This process was carried out on eggs aged for 3 days and then repeated after 10,17,24,31,38,45,52,59 and 66 days [22]. Following selection, to compare the association between hatch rates and time in the different strains, we truncated the changes in hatch rates at 32 days (ie before all the unselected wMelPop eggs had become non-viable) and ran GLMs that included linear and quadratic terms for time as well as line type. These parameters provided an adequate fit to the data or each line (R2>0. 90). We then considered whether lines differed for estimated parameters in these models. Because multiple strains were compared we computed parameter estimates for the different treatments and interactions and tested their significance using bootstrapped confidence intervals estimated in IBM SPSS Statistics 22. To determine if the selection response in AOMB-DS was due to a nuclear or Wolbachia effect, the selected line was crossed to unselected lines. Two new lines (AX1, AX2) were created by backcrossing AOMB-DS to males or females of the original AOMB line. Backcrossing was continued for three generations to place Wolbachia from the selected line onto the nuclear background of the infected (AX1) stock (Fig 2) or to place Wolbachia from the unselected line onto the nuclear background of the selected infected line (AX2). To test for viability, eggs were collected over four day periods from the AOMB-DS, AOMB, and C20 colonies as well as the backcrossed AX1 and AX2 lines. These were placed in the 60% desiccation chamber once conditioned. Three days later, eggs were cut into 10 batches of at least 25 eggs then counted and hatched into separate cups filled with 170ml RO water and TetraMin fish food. Larvae were counted one week later as an estimate of viability. This process was carried out on eggs aged for 3 days and then repeated after 10,17,24,31,38,45,52,59 and 66 days [22]. All summary data can be accessed in Dryad Digital Repository at http: //dx. doi. org/10. 5061/dryad. 3vg33 [35]. The wMelPop infected Ae. aegypti eggs exposed to a range of shade (Fig 1) suffered higher mortality rates than uninfected eggs (Fig 3). Mortality (decrease in % hatch) was highest in summer and in sun exposed (30%, 70% shade) eggs. Indeed, mortality of wMelPop infected eggs was nearly 100% after 8 weeks in all shade groups in summer. Uninfected eggs also suffered high mortality, but only in the highly exposed 30% shade treatment. The GLM on data from the summer experiment indicated a significant effect of shade and strain overall as well as time as a linear and quadratic term (Table 1). Interactions between strain and the time variables were not significant, although the quadratic—strain interaction approached significant. Increased shade led to lower viability across time (Fig 3) and the parameter estimates indicated significant differences between the 99% shade and the 30% treatment, but there was no interaction between shade and strain or other interactions with shade (all P>0. 25). The wMelPop strain had lower viability overall and tended to show a more rapid decrease in viability over time. For the winter experiment, the strain-shade interaction effects were also non-significant (P > 0. 25). All other effects except for the main effect of strain were highly significant (Table 1). There were different patterns of changes in the hatch rates of strains across weeks, with linear and quadratic components. The wMelPop infection had a sharper drop in hatch rates compared to the uninfected line (Fig 3). The temperature in the winter trial ranged from ca. 15–28°C, with slightly higher temperatures in the 30% shade section (S1 Fig). In winter, temperatures recorded from loggers on tables exposed to direct sunlight (30% and 70% shade) peaked at 40–50°C in the afternoon (S2 Fig). Relative humidity was generally high, ranging from ca. 50% to nearly 100% at night, although the maximum values are likely too high (S3 Fig). In summer, temperature range increased to ca. 20–35°C (S4 Fig), with exposed loggers peaking at 40–60°C at mid day for 30% and 70% shade (S2 Fig). Relative humidity ranged from 50% to 90% (S3 Fig). Releases resulted in the wMelPop infection gradually moving to fixation within the semi-field cage. Two successive weeks of 100% wMelPop infection rates were achieved after 16 consecutive weekly releases and two source reduction/vector control interventions (Fig 4). The mean temperature in the 70% and 99% shade sections was 26. 2°C and 25. 4°C, respectively, and ranged from 20–35°C (S5 Fig). Relative humidity ranged from 50% to 95%. While we did not record temperatures in the exposed data loggers (set on tables), results from the earlier egg survival study indicate that temperatures of 50°C would have occurred in the 70% shade section for a few hours on sunny days (S2 Fig). Mean (± SE) pupal counts for individual PPB and buckets in the wMelPop and uninfected cages were 0. 62 ± 0. 96 and 1. 06 ± 1. 37; and 28. 9 ± 30. 6 and 50. 4 ± 27. 2, respectively. Collectively, the mean pupal production/day was 59 ± 49 and 103 ± 46 for the wMelPop and control cages, respectively. Based on a 0. 8 and 0. 9 adult daily survival (DS), this would equate to a mean adult population, using the integration method [30], of 280–451 and 490–790 adults for the wMelPop and uninfected cages, respectively. Eggs infected with wMelPop suffered significantly higher mortality than uninfected eggs for each exposure period except for 80 days at 70% shade, where no eggs hatched for either cohort (Fig 5). wMelPop infected egg survival dropped noticeably after 59 days, and only 3/443 (0. 67%) eggs hatched from the 99% shade cohort after 80 days. Mortality was also high in the uninfected eggs, but 7/154 (4. 4%) eggs hatched in the 99% shade cohort. No wMelPop infected eggs hatched from the PPB and buckets flooded at the end of the trial, while 9 hatched from a PPB with uninfected eggs. After four rounds of selection for desiccation resistant mosquitoes, there was an increase in the time required to produce a hatch rate of around 10% from around 17 days to almost 40 days (Table 2). This followed a rapid extension after two rounds of selection. In the backcrossed lines, the AOMB-DS line maintained a higher hatch rate in aged eggs compared to the AOMB line despite several generations without selection (Fig 6). The AX2 line showed a similar pattern to that of the AOMB-DS line. As expected, the uninfected C20 had the highest hatch rate. The unselected AOMB and AX1 lines had similar low hatch rates. The GLM showed that there was a significant effect of line on hatch rate (F (4,285) = 4. 097, P = 0. 003) and there were also significant interactions between line and the linear time component (F (4,285) = 6. 974, P<0. 001) and between line and the quadratic time component (F (4,285) = 5. 042, P = 0. 001). These analyses indicate differences among strains in hatch rate changes across time involving both a linear and non-linear component. The time effects point to an increase in the hatch rate (survival) of the selected line with the wMelPop infection associated with the nuclear background of the line rather than the Wolbachia background. The AX1 line and AOMB did not differ in any way and all parameter estimates overlapped for these strains (main effects of strain, interactions with linear and quadratic time components). This was also the case for AX2 and AOMB-DS. In contrast, the linear parameter for AX2 and week (-0. 103) fell outside the 95% confidence intervals for the equivalent value for AOMB (-0. 582, -0. 192) and this was also the case for the quadratic component (0. 001) which fell outside the 95% confidence intervals for AOMB (0. 015,0. 101). The quadratic component also differed between the selected and unselected AOMB lines (AOMB, 0. 058,95% CIs 0. 015,0. 101; AOMB-DS, -0. 031,95% -0. 074,0. 013) as did the linear component (AOMB, -0. 387,95% CIs -0. 582, -0. 192; AOMB-DS, 0. 051,95% -0. 144,0. 246). Aedes aegypti eggs infected with wMelPop suffer significantly higher mortality than uninfected eggs. Nearly all of the infected eggs failed to hatch after 2 months for all shade and temperature exposures (Fig 2). Our semi-field cage experiment indicted that if releases of wMelPop successfully establish fixation, the population will potentially collapse during prolonged dry periods. Indeed, the hatching rate (survival) of wMelPop infected eggs was significantly lower after 59 and 80 days exposure (Fig 3). This points to the feasibility of using releases of wMelPop infected Ae. aegypti to eliminate local populations of Ae. aegypti and confirms the potential of this approach as first proposed and modelled in Rasic et al. [26]. Nevertheless, using wMelPop releases for elimination of Ae. aegypti populations will be more difficult than invasions involving wMel releases. To obtain fixation in the semi-field cage, we conducted 16 weeks of releases and two rounds of vector control. This is considerably more difficult than the invasions of wMel that have successfully established in Cairns after 11 weeks of releases, and at a faster rate in semi-field cages [17,19]. Indeed, sustained releases of wMelPop-infected Ae. aegypti failed to establish fixation by wMelPop at two field sites in Cairns in 2012 [25]. We could have improved efficacy of the releases by increasing the number of infected mosquitoes released. At the start of releases, the population in the treatment cage, based on pupal integration, was ca. 200 adults. We released 100 per week, or a release rate equivalent to 50% of uninfected adult population, although BGS captures suggested the initial 2 weeks of release cohorts were 150% of the uninfected population. The successful wMel invasions in Cairns involved releases at numbers roughly equivalent to wild populations. Higher releases rates should have improved the speed at which wMelPop became fixed in our semi-field cage. The use of wMelPop to eliminate local populations of Ae. aegypti will likely be limited by several constraints. It took 16 weeks of releases, with 2 cycles of vector control, to fix wMelPop in a small semi-field cage. Thus, releases without vector control, population suppression or another mechanism to assist invasion like the use of insecticide resistance are likely to be challenging [36]. The site will likely need to be isolated to avoid rapid re-infestation from adjacent areas, and will likely need to be relatively small, such as an isolated township [26]. Also the locale must have a prolonged dry period to facilitate egg death, as modelled in Rasic et al. [26]. Indeed, there were 4 viable infected eggs (3 from a bucket, and 1 from a PPB) after 80 days in the full shade cohort, suggesting that wMelPop may persist at a very low level requiring additional vector control for elimination. Finally, candidate areas need to be focused on the elimination of Ae. aegypti, rather than simply a reduction of dengue risk, which will be more easily achieved by releases of wMel rather than wMelPop. The data generated from this study could be used to identify geographic areas through modelling where the use of virulent Wolbachia and vector control for population suppression would be most effective. Vector control and population manipulation can be used to facilitate wMelPop releases [36]. Improved vector control methods that target cryptic breeding sites, such as the use of auto-dissemination application of pyriproxyfen [37], could temporarily remove cryptic foci of uninfected mosquitoes, enhancing invasion. Other temporary vector control methods, such as use of bleach to treat larval habitat [30] and non-persistent adulticides such as metofluthrin [38], could provide a quick knockdown and avoid residual effects that would impact released mosquitoes (crash and release strategy). A novel approach would be to couple genes for pesticide resistance to wMelPop, in effect engendering protection of the wMelPop mosquitoes from insecticides [39], although this approach may be coupled with the fear of introducing resistant strains, and could be difficult to sell to the public, regulators and government officials. While the wMelPop infection has deleterious effects on egg viability when these are in a dried state, these effects are attenuated by strong selection. The crosses point to this attenuation being due to a nuclear background effect (Fig 6). Previous research has shown that the expression of Wolbachia effects on the host can be influenced by host background. Perhaps the most dramatic example of this is where Wolbachia causing cytoplasmic incompatibility or male killing in one host and then transfected into a different host species no longer expresses the same phenotype [40]. In addition, lifespan effects generated by Wolbachia in Drosophila melanogaster are altered through longevity selection and involve the nuclear background [41]. Once wMelPop have been successfully introduced into populations, evolution in Wolbachia may attenuate fitness effects, as recently documented in Drosophila [42] although this is clearly not always the case. Evolution seems likely to change the nuclear background and lead to attenuation, but this process may be countered by other selective pressures. Does use of a virulent Wolbachia strain such as wMelPop to eliminate local Ae. aegypti populations have advantages over traditional releases of sterile (sterile insect technique (SIT) ) or incompatible (incompatible insect technique (IIT) ) males? Certainly use of Wolbachia eliminates the need for costly irradiation equipment and any potential fitness cost associated with irradiating males. Reared Wolbachia-infected cohorts do not need to be sexed before release, and the relative numbers of released insects should be much lower that SIT programs where overwhelming release ratios of high quality males are required [43]. There is no sudden population “bounce back” if releases of Wolbachia infected mosquitoes stopped (assuming Wolbachia is nearly fixed in the population) unlike SIT/IIT where residual wild populations can rapidly rebound. Wolbachia is seen as a biological control program with generally strong community support [44], whereas some IIT programs involve genetically modified organisms, and may be relatively difficult to obtain public and regulatory support for. Disadvantages of use of virulent Wolbachia include the release of biting females, of the need to create a suitable infected line, and the potential requirement to conduct complimentary vector control. The survival of uninfected Ae. aegypti eggs exposed to direct sun (30 and 70% shade) deserves comment. While egg survival was highest in full shade, many eggs exposed to sunlight (30% and 70% shade) survived several weeks (Fig 2). Ae. aegypti eggs exposed to temperatures > 46°C suffer rapid mortality [45,46]. Our data loggers recorded mid day temperature spikes of up to 40°C to 60°C, 20–30°C higher than ambient temperature, lasting several hours (S2 Fig). We suspect that the loggers, oriented horizontally on top of the sample jar (Fig 1), were subject to high solar gain and heating, and thus misleadingly high temperature readings. The Aedes eggs, on the other hand, are oriented vertically, minimizing their silhouette area when the sun is overhead and thus reducing solar gain. The small size of eggs would also facilitate cooling by the air. In summary, we have established the effects of the wMelPop infection on egg mortality when these are maintained in a dried state under semi-field conditions. This provides the possibility of using wMelPop to suppress and even eliminate mosquito populations in relatively isolated populations particularly where there is a long dry season.

Dengue is a leading cause of morbidity in the tropics. As a commercial vaccine is not available, control or modification of the mosquito vectors is employed to prevent transmission. Strains of the endosymbiotic bacteria Wolbachia affect the survival and ability of the dengue vector Aedes aegypti to transmit dengue. The Wolbachia strain wMelPop over-replicates within Ae. aegypti, inducing strong dengue virus blocking and early mortality of both egg and adult mosquitoes. We investigated whether this life-shortening Wolbachia strain can be used to eliminate local populations of Ae. aegypti in a semi-field cage. Our results indicate that Ae. aegypti eggs infected with wMelPop died at a significantly higher rate than uninfected eggs, and were nearly eliminated during a simulated dry season of 2-3 months. This suggests that that releases of wMelPop could facilitate control and elimination of Ae. aegypti if used in concert with vector control.

lay_plos

These crawls are part of an effort to archive pages as they are created and archive the pages that they refer to. That way, as the pages that are referenced are changed or taken from the web, a link to the version that was live when the page was written will be preserved.Then the Internet Archive hopes that references to these archived pages will be put in place of a link that would be otherwise be broken, or a companion link to allow people to see what was originally intended by a page's authors.The goal is to fix all broken links on the web. Crawls of supported "No More 404" sites. I am not an animal rights activist by any stretch of the imagination. I wear leather and eat meat. But there is something very, very wrong with a new law being proposed by a republican legislator in Utah. He is sponsoring a bill that would legalize the shooting of all feral animals, on sight. It calls for: the humane shooting or killing of an animal if the person doing the shooting or killing has a reasonable belief that the animal is a feral animal. Reasonable belief? What exactly is that and who is regulating the guy on the corner to make sure he isn't just shooting his neighbors dog because it is off its leash? A feral animal is any domesticated animal that "has returned to live in wild conditions." So all of those homeless cats you see roaming around... or abandoned dogs... they are the animals Republican Representative Curt Oda is talking about. He says this is the cheapest and quickest way to get rid of, essentially, the homeless pet population in Utah. And he may be right, but there are advocate groups like No More Homeless Pets in Utah, that say he is wrong and have employed other methods to cope with the overpopulation of these animals. This proposed law is over-the-top, especially since the animal doesn't even have to be noticeably aggressive or sick to be shot. It just has to be considered, by the shooter, to be feral. It seems terribly dumb to give rights to every person in the state to shoot or kill these animals by way of clubbing, decapitation, or a bow and arrow. Yes, you read that correctly. I mean, if you can't afford a gun you should be able to participate too! The bill also includes the rights to kill rodents and other "pests" such as pigeons. Listen, I've never jumped on Bert's love for pigeons, but that doesn't mean they should suffer a clubbing. Are you as outraged by this as I am? Image via peyri/Flickr

Irritated by the proliferation of stray cats and dogs? Just move to Utah, where soon you'll be able to take care of the problem... by shooting them. The Stir points to a new law being proposed by Rep. Curt Oda, a Republican who wants to legalize the killing of all feral animals. Of course, in order for the killing to be legal, the person doing it must be reasonably certain the animal is truly wild, and not just someone's wandering pet. And if you don't have a gun, no worries: clubbing, decapitation, or use of a bow and arrow would also be perfectly okay. Oda justifies the law by claiming it's the cheapest and fastest solution to the homeless animal problem, but his idea has, not surprisingly, sparked a bit of an uproar amongst animal rights organizations, ksl.com reports. Click for more, including the other types of animals a person would be able to kill.

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The pathogenesis of bacteraemia after challenge with one million pneumococci of three isogenic variants was investigated. Sequential analyses of blood samples indicated that most episodes of bacteraemia were monoclonal events providing compelling evidence for a single bacterial cell bottleneck at the origin of invasive disease. With respect to host determinants, results identified novel properties of splenic macrophages and a role for neutrophils in early clearance of pneumococci. Concerning microbial factors, whole genome sequencing provided genetic evidence for the clonal origin of the bacteraemia and identified SNPs in distinct sub-units of F0/F1 ATPase in the majority of the ex vivo isolates. When compared to parental organisms of the inoculum, ex-vivo pneumococci with mutant alleles of the F0/F1 ATPase had acquired the capacity to grow at low pH at the cost of the capacity to grow at high pH. Although founded by a single cell, the genotypes of pneumococci in septicaemic mice indicate strong selective pressure for fitness, emphasising the within-host complexity of the pathogenesis of invasive disease. Streptococcus pneumoniae, one of the major human bacterial pathogens, is also part of the normal upper respiratory tract flora, where nasopharyngeal colonisation with one or more strains often lasts weeks to months with seasonal peaks in late winter [1], [2]. Carriage of S. pneumoniae (pneumococci) may result in disease as the consequence of contiguous spread from the nasopharynx to other sites in the upper or lower respiratory tract causing, for example, otitis media or pneumonia. More rarely, there is hematogenous dissemination of pneumococci resulting in septicaemia and metastatic disease such as meningitis [1], [3]–[5]. In experimental models of pneumococcal infection, the challenge dose required to induce disease is dependent on the route of infection, the genetic background of the host and the virulence of the infecting strain [6] and may vary from a very few to millions of organisms [7]. Following intravenous inoculation of mice with laboratory grown pneumococci, a hallmark of experimental bacteraemic infections is the rapid and efficient clearance of most of the inoculated bacteria [8]–[10]. In non-immune rodents, major factors mediating this clearance are splenic macrophages and complement mediated opsonisation [11]–[14]. A challenge dose of about one million virulent, encapsulated pneumococci is generally needed to induce bacteraemia in about half of challenged animals (the effective dose or ED50) and which is the dose generally used to address investigations into the early events shaping an infectious process. Most work on the pathogenesis of infectious disease focuses on specific virulence determinants which are generally presented as the cause, either alone or in combination with other factors, of the events leading to the infection of the host where the microbial population is considered to be a uniform entity. However, several investigations have addressed the within host population dynamics, especially on the early phases of host-pathogen interactions [15]. There are different models which address these early events that include: (i) the model of independent action, which postulates that at the LD50 (lethal dose for 50% for the hosts) the hosts develop infection “following the multiplication of only one of the inoculated bacteria, regardless of the total number of bacteria inoculated” [16], (ii) the hypothesis of synergy which “postulates that inoculated bacteria co-operate and that fatal infections will be initiated by more than one bacterium and that this will lead to the predominance of several variants” [16], and models which introduce time as a factor into the process and propose a two-stage model where a birth–death phase would be responsible for generation of the heterogeneity within the population later during the infection [17]. Both for viral and bacterial infections it has been shown that the effective number of infectious agents which actually start the disease is generally many orders of magnitude below the actual dose used for challenge [16], [16], [18]–[21]. In particular a series of reports, generally based on experimental challenge using an inoculum containing an approximately equal mixture of two isogenic variants at the LD50, has shown experimentally that systemic infections may be initiated by the multiplication of as few as a single organism [22]–[27]. Models differ widely, and due to the nature of pathogenesis of many infections, they rely on experimental challenge at a site different to that investigated for disease implying that multiple bottlenecks occur and that potentially a series of invasive events could be enucleated [16], [18]–[27]. We here have investigated the host and microbial determinants that underpin the occurrence of the single cell bottleneck in the pathogenesis of pneumococcal septicaemia following inoculation of mice with three isogenic variants by the intravenous route. This route has an advantage over other experimental infection models as there are less biological events between the initial challenge and full blown disease so that rigorous analysis of the events is facilitated. CD1 mice were inoculated intravenously (i. v.) with a mixture of pneumococci comprising approximately equal numbers of each of three isogenic TIGR4 mutants (FP122, FP321 and FP318) with different resistance markers (Table 1) [28]. Following inoculation, blood samples were collected at different times and spread on selective plates. Colony counts allowed quantitation of the distribution of the different mutants making up the pneumococcal population in the blood (Figure 1). Two hours after bacterial challenge, blood samples from all mice, with one exception, grew all three of the variants that had been included in the challenge dose. Samples of the second group of mice, sampled one hour thereafter, showed a mixed population of the variants: two in 6 mice, three in 17 mice and one mouse had negative blood culture (Figure 1A). At 7 h after challenge, this pattern was distinctly different: there were 10 positive and 2 negative blood cultures and the numbers of bacteria were significantly reduced. At 8–9 h post-infection, most blood cultures (25/29 mice) were negative. The remaining 4 mice had monoclonal blood cultures in that each grew colonies of only one mutant (Figure 1A). In the subsequent hours of infection, bacteria were detected in the blood at high concentrations (up to 1×106 CFU) in 17/55 (31%) mice at 24 h, 16/43 (37%) mice at 48 h and 15/24 (62%) mice at 72 h (Figure 1B). At all these time points, most blood samples yielded colonies of only one of the variants: 12 out of 17 blood cultures at 24 h, 12/16 at 48 h and 8/16 at 72 h. Among the 32 single-variant blood samples each variant was more or less equally represented as the progenitor, although owing to the smaller challenge dose of strain FP318 in one experiment, this strain was recovered at lower density from the blood and caused fewer single-variant infections (Table S1). In three of twelve mice for which three serial blood cultures were taken we observed an increase in the number of variants. One of these mice showed evidence of infection with 3 variants at 48 h after earlier having a monoclonal infection and a further two mice had three variants infection at 72 h after having previously had infection with only one or two variants respectively. Seven of the single-variant bacteraemia isolates were checked for colony morphology and each was found to have the opaque phenotype, in contrast to the challenge strains (not-mouse-passaged) that yielded a mixed population of about similar proportions of opaque and translucent colonies [29]. The bacterial counts from cultures of spleen tissue were assayed in twelve mice at each time point (Figure S1). All mice which had positive blood cultures showed bacterial counts also in splenic samples. In addition, small numbers of organisms were cultured from spleen tissue both at 24 and 48 h in two mice each which presented with sterile blood cultures. Similarly at 72 h, bacteria were only detected in the spleen in one mouse (Figure S1). These data show that infectious foci can be detected in the spleens of mice that have negative blood cultures, indicating that the spleen is the probable site where the infection originates. Our data indicate that the near totality of bacteria in the challenge with three isogenic pneumococcal variants is cleared by the immune system (predominantly by splenic macrophages; see below) and that few bacterial cells remain viable within a defined site of the host (i. e. the spleen). This small number of bacteria may start to grow and re-invade the host giving rise to bacteraemia. In our experimental infection most of the mice challenged were not bacteraemic and, of those becoming bacteraemic, most were infected by a single bacterial variant. In addition, we detected in some mice an increase in variants within blood cultures over time. These data indicate that over time more than one invasion event may occur. Theoretically bacteraemia may be generated in two ways: (i) by a single bacterium establishing a population in the blood in a single invasion event or several bacteria each independently establishing a population in distinct invasion events (independent action); (ii) by a defined number (more than one) of bacteria acting together to invade once or several times (co-operative synergism) [22]. We hypothesise that the former explanation pretains. To statistically evaluate the number of bacteria involved in founding the blood population in each invasion event, we construct a model that assumes that bacteria invade and establish a population in the blood at random (Supplementary text) [23]. In this model, the number of invasion events in each mouse is assumed to follow a Poisson distribution so the expected number can be estimated from the proportion of the non-bacteraemic mice. Then we determined the expected numbers of mice infected with one, two or three variants, assuming that the number of bacteria (w) responsible for establishing the blood population in each invasion event were 1,2, 3, etc (Table 2). Given that some mice were culled during the course of the experiment and some got multiple samplings, we limited the statistical analysis to the observations at the 24 h time point. In table 2, we report the comparison between the expected versus the observed numbers of the infected mice with one, two and three variants for different numbers of bacteria (w) potentially responsible for founding a population blood. The statistical analysis shows that the most probable number of bacteria responsible for establishing a blood population is 1 (Table 2). Note that the p-value was calculated by combining data for blood cultures with two or three variants because of the small expected frequencies in these two categories. Given that w is equal to one, we conclude that polyclonal blood infections are the result of more than one invasion event, each event founded by a single bacterium, consistent with the observed time-dependent increase in the frequency of polyclonal bacteremia over the 72 h of the experiment. Prior to the assessment of the impact of host factors in the control of bacteraemia, we compared bacterial counts in the blood of two mouse strains known to be resistant to pneumococcal infection (outbred CD1 mice and inbred BALB/c mice) and a susceptible mouse strain CBA/Ca [30]. The mice were inoculated i. v. with a mixture of three encapsulated pneumococcal strains of different serotypes, D39 (type 2), TIGR4 (type 4) and G54 (type 19F). Bacterial clearance in CD1 and BALB/c mice showed similar kinetics (Figure 2 A–B), while CBA/Ca mice were less able to reduce the initial number of bacteria (Figure 2 C). All mouse strains cleared G54 bacteria immediately (no positive blood culture 10 min after infection) and showed a first phase of rapid clearance also for both D39 and TIGR4. Only the two resistant mouse strains showed bacterial numbers in the blood that were less than the limit of detection. In contrast, bacterial numbers increased in the susceptible strain after the first phase (Figure 2 A–C). This indicates that, depending on which host-pathogen pair was investigated, the bottlenecks may vary considerably. Since BALB/c mice showed a more uniform clearance of bacteria, subsequent experiments were conducted with this mouse strain in order to keep experimental groups to a minimal size. To identify the host immune cells responsible for the initial clearance of bacteria from the blood, we performed a set of experiments in BALB/c mice depleted either of macrophages or neutrophils. Macrophage depletion was achieved by intraperitoneal (i. p.) injection of clodronate liposomes and neutrophil depletion by using anti-GR-1 monoclonal antibody [31]–[33]. Control groups were treated either with PBS-containing liposomes as control for clodronate experiments or with isotype-matched antibody in the case of experiments with anti-GR-1. The results obtained for the control groups were comparable to the untreated control mice and differed from the groups of mice treated with clodronate (Figure S3 A–C) or anti-GR-1 (Figure S3 E–G). To verify macrophage or neutrophils depletion, spleen samples were analyzed by flow cytometry. The reduction of macrophages in the spleen of clodronate-treated mice was 61%±14. 2 measure by anti-F4/80 and 47%±10. 4 by anti-CD11b compared to naïve mice. Similar results were obtained when liver samples were analyzed (Figure S3 D). In anti-GR-1-treated mice, the neutrophil number was reduced by 83%±2. 7 as compared to control mice (Figure S3 H). To check for anti-pneumococcal antibodies in naïve mice, we evaluated the reactivity of mouse serum towards whole pneumococcal cells. Mice had no detectable serum antibodies to any of the pneumococcal serotypes (Figure S3 I–K). A result supported by the observation that addition of type specific rabbit serum to the blood from naïve mice conferred specific bactericidal activity (P<0. 01). To analyse the role of macrophages and neutrophils, mice were divided into three groups: untreated (Figure 3 A1–A4), clodronate-treated (Figure 3 B1–B4), and anti-GR1-treated (Figure 3 C1–C4). After i. v. challenge with a mixture of four different strains (TIGR4, D39, DP1004 and G54), time course of bacterial counts was monitored by sampling blood, spleen, lung, liver and kidney (Figure 3 and S2). Analysis of control mice allowed categorisation of the pneumococcal strains into two groups: TIGR4 and D39, which were slowly cleared (virulent strains), and G54 and DP1004, which were cleared from the blood within minutes. The counts of TIGR4 and D39 were higher in the blood than in the spleen at 5 min and at 4 h compared to the other two strains (P<0. 05). Bacterial loads in the other organs were similar to those found in the spleen (Figure S2). In contrast, mice infected with strains DP1004 and G54 showed higher CFU counts in the spleen than in the blood and other organs (P<0. 05 at 5 min for both strains, P<0. 01 at 4 and 8 h for DP1004) (Figure 3 A3–4). The groups of mice depleted of macrophages showed significantly reduced ability to clear bacteria from the bloodstream. An increase in bacterial numbers in blood from 5 min to the later time points was observed in mice infected with TIGR4 (Figure 3 B1) and D39 (Figure 3 B2) (P<0. 01). Blood bacterial counts were significantly higher in the clodronate-treated mice than in the control group (P<0. 05 for all time points for both D39 and TIGR4). Bacterial counts of TIGR4 and D39 in liver and spleen were lower but with a similar trend, over time, to those in the blood (Figure 3 B1–B2 and Figure S2 B1–B2). In clodronate-treated mice, the numbers of non-virulent bacteria (strains G54 and DP1004) were higher in the blood than in the spleen (P<0. 05 at 5 min for DP1004 and P<0. 05 at 5 and 4 h for G54) and paralleled the trend observed for the virulent strains TIGR4 and D39 in untreated animals (Figure 3 B3–B4 compared to A1–A2). In neutrophil-depleted mice, bacterial counts of both TIGR4 (Figure 3 C1) and D39 (Figure 3 C2) in blood and spleen decreased in the first 4 h after challenge with a similar trend to that observed in untreated animals. Thereafter, blood and organ counts remained stable (Figure 3 C1–C2 and Figure S2 C1–C2). For both virulent strains, the number of bacteria were higher in the blood than in the spleen (P<0. 01 at 5 min). Interestingly, strain G54 had a peculiar behaviour in neutrophil-depleted mice, as it persisted in the spleen at high levels throughout the whole experiment despite being cleared from the blood within a few min of infection (P<0. 001 at 4 and 8 h), (Figure 3 C4). At 4 h post-infection G54 bacteria reappeared in the blood (Figure 3 C4). The experiment was repeated for the later time points, and the pattern of counts was identical. The rough DP1004 strain was cleared from each body site as well as in untreated mice (Figure 3 C3). To determine more precisely the early events occurring in the clearance of pneumococci, we have plotted separately the data on blood bacterial counts obtained 5 min and 30 min after challenge (Figure 2 D–E). In untreated mice, bacterial blood counts of the invasive strains D39 and TIGR4 were respectively 6. 2×104 and 5. 4×104 CFU/ml, while those of the non-invasive strains, DP1004 and G54, were three times lower (reduction of 60 to 75%). Differences between the virulent and non-virulent strains were statistically significant (P<0. 01). Given the key role of splenic macrophages in pneumococcal clearance, we evaluated the capacity of splenic macrophages to internalise pneumococci. Splenic BALB/c macrophages were grown as primary cell cultures, washed and re-cultured for seven days in M-CSF supplemented medium. Cytoflourimetric data showed expression of the characteristic markers of splenic macrophages, CD11b, CD11c, F4/80 and SIGLEC-1 (Figure 2 H) [34]. Adhesion was evaluated by counting pneumococci after 45 min and phagocytosed bacteria were enumerated by plating after a further 30 min of antibiotic treatment (viable intracellular bacteria). Despite similar values in adherent cells (Figure 2 F), our data show higher numbers of intracellular bacteria for the rough DP1004 and for the G54 strain and less for the virulent D39 and TIGR4 (Figure 2 G). Essentially identical data where obtained when performing the experiment with splenic macrophages from C57BL/6 mice, while in contrast bone marrow macrophages from BALB/c mice and RAW264. 7 macrophages showed different patterns of surface markers expression to the spleen macrophages and their phagocytosis of pneumococci showed no correlation to the extent of early clearance in the host (Figure S4). These data emphasise the importance in the choice of cell lines for performing phagocytosis assays in vitro to assess pneumococcal clearance in vivo. Pneumococci grown from the blood of 6 mice were subjected to whole genome sequencing (Table 3). In each case, the isolates had the identical antibiotic resistance phenotype and were therefore presumptively monoclonal. Two of the blood cultures were obtained from the same mouse, but at 24 or 48 h respectively, (mouse 3. 1. 5; Table 3). To identify possible mutations characterising the founding cell of the monoclonal blood culture, we searched for single nucleotide polymorphisms (SNP) present in all cells isolated from a given blood culture. Such a SNP would demonstrate that the re-expanded population arose from a single cell. We identified one or two SNPs in 100% of the bacterial populations from four out of six mice (3. 1. 5,4. 1. 4,4. 2. 2 and 4. 2. 6), when compared to bacteria from the challenge inoculum (Table 3). The identification of a SNP common to all bacteria of a given sample is conclusive genetic evidence that the pneumococcal populations were monoclonal. Further, the SNPs were either inter-genic, silent or in regions not predicted to be functional in pathogenesis. Thus, we conclude that these mutations were unlikely to be associated with changes in within-host fitness. This argues strongly that bacteremia was founded as the result of a stochastic process rather than the selection of fitter variants. However, further analysis identified a second set of SNPs in pneumococci of 5 of 6 blood cultures. Crucially, this second set of SNPs were only found in a proportion of the bacteria obtained from mouse blood and therefore must have occurred after the bottleneck. Further, these SNPs differed between isolates of different mice, but all were located within distinct sub-units of the pneumococcal F1/F0 ATPase operon (Table 3). In three bloods, more than one SNP was detected. To determine if more than one SNP in the ATPase operon occurred in a single cell, we sequenced single colony isolates of these populations. In all cases, where a multi-SNP profile would have been possible according to the genomic data, only clones with a single SNP within the F1/F0 ATPase operon were recovered. These isolates included FP490, a 3. 2. 4 derivative with a SNP in atpA, FP487, a 3. 1. 5 derivative with a SNP in atpC, and FP489 and FP498, two 4. 1. 6 derivatives with different SNPs in atpD (Table 1 and 3). In few cases subpopulations with mutations in other genes were detected (pilus sortase, potassium uptake protein, metE, and SP0760) (Table 3), but no confirmation by direct sequencing was performed for these genomic data and we do not think that these mutations are of major biological relevance. Phenotypic analysis of eight independent ex vivo blood isolates each having a mutation (SNP) in the ATPase (Table 1 and 3), showed normal colony morphology on agar plates and no significant change in their susceptibility to optochin. In liquid culture, the mutants showed normal or more efficient growth in Todd Hewitt Yeast Extract (Figure 4 A), but were unable to grow in other media (Tryptic Soy Broth) (Figure 4 B). Given that the F1/F0 ATPase is involved in multiple aspects of proton trafficking, we investigated the impact of pH, buffer composition and salt concentration on bacterial growth. Using a phenotype microarray for osmotic susceptibility using Biolog microtiter plates PM9, we compared the phenotype of parental strains derived from strain TIGR4 to the ex vivo mutants. The mutants had acquired a series of metabolic characteristics, also shared by strain D39 (Figure S5). Growth experiments performed in serial buffer and salt dilutions showed that TIGR4 and its isogenic derivatives used in the challenge experiments had a restricted pH optimum when compared to D39, which limited growth at potassium phosphate concentrations below 10 mM and pH below 6. 8 (Figure 4 D). Interestingly many of the mutants had gained this capacity, making them equally able to grow at low pH as D39. In contrast, high buffer concentrations (80 mM K2HPO4 and pH 8), inhibited growth of all mutants (Figure 4 C). To investigate effects on intracellular pH homeostasis of the ATPase mutations we transformed the frame-shift in the atpC gene into the non-encapsulated strain DP1004. Using in vivo NMR, the atpC mutant and its parental strain were both shown to have an identical intracellular pH of 6. 52 to 6. 56 during active metabolism of glucose. No differences in susceptibility to neutrophil killing were observed when mutants were assayed in an opsonophagocytosis assay in the presence of type specific antibodies (Figure 4 E). Also, data of macrophage phagocytosis were unaltered in primary cultures of splenic macrophages (Figure 4 F). To check for any fitness cost in vivo, the encapsulated atpC mutant was compared to the challenge strain FP321 in our i. v. mouse sepsis model. At early time points both strains showed comparable blood counts (data not shown). Also at 72 h post-challenge, bacterial counts in blood were similar, but bacterial spleen counts for the atpC mutant were significantly increased when compared to the wild-type (Figure 4 G). We have investigated the pathogenesis of pneumococcal bacteraemia following intravenous inoculation of mice with three isogenic clones (variants). In our model, the infection followed the classic, three phase pattern in which a majority of pneumococci are cleared in the first minutes post-challenge. This leads to an “eclipse phase” of several hours in which bacterial numbers decline further or are undetectable. This is followed by the emergence of sustained and high density bacteraemia in a proportion of the challenged animals [8], [10]. By analysing the survival in the blood of three isogenic variants of S. pneumoniae, we observed that the majority of blood cultures arose from only one of the three variants. We used a statistical model to characterise the infection dynamics in which the number of bacteria starting the infection in each invasion event is w and the number of times this happens is k [23]. From the model, we could infer that the number of bacteria at the origin of infection is below 2 (w = 1). Thus, it follows that bacteraemia was generated by either (a) a single bacterium establishing a population in the blood in a single invasion event or (b) several bacteria each of which independently established a population in distinct invasion events. The probability of (b) is small (about 5% in our data, because the probability of two or more invasion events occurring is about 5%). Genome sequencing provided genetic evidence in 4/6 cases that monoclonal bacteraemia did actually start from a single bacterial cell (w = 1) confirming the first statement. For the remaining 2/6 cases we could not determine w = 1 by genome sequencing as we could not distinguish several invasions of a single bacterial variant from one invasion of several cells of the same variant without any SNPs. More complex is the experimental observation of invasive events. For this we could document bacteraemia in mice with previous negative blood samples (k≥1) and in other mice the increase of variants in serial blood samples (k>1). Since after the first 24 h the observed numbers of both these types of invasion events are similar, this strongly favours the occurrence of polyclonal infections resulting from independent, not cooperative action. In the case of H. influenzae it had been hypothesised that the single cells giving rise to the monoclonal infection might be selected by within-host evolution [23]. Our work now tests this hypothesis by whole genome sequencing. The data show in two cases absence of any SNPs and in four cases SNPs that apparently do not indicate selection for virulence. Despite the low numbers, it suggests that the single cells at the origin of infection apparently have no advantage (higher virulence) over the other cells in the population. Such results show that the bacteria in the challenge dose act independently to give rise to infection, that each has a similar probability of causing infection and that a dose near the LD50, a single cell may initiate disease. These criteria satisfy the theory of independent action [16], [17], [22], [23]. As such our investigation provides strong evidence that the single founding cell of an invasive infection is the result of a stochastic event. However, it must be emphasised that epigenetic variations would have eluded our genetic and genomic analysis. Previously published studies have shown a major role for splenic macrophages in the initial clearance of pneumococci. In the seminal investigations of Brown et al. [35], they conclude: “… it appears that an anatomically normal spleen plays a unique role in the clearance of experimental pneumococcal bacteraemia, and that this role is of increasing importance as the pathogenicity of the invading organism increases”. Our data provide evidence that splenic macrophages have properties not found in those derived from other tissue sites, with respect to their efficiency to ingest and kill pneumococci. It is worth noting that the impressive efficiency of splenic clearance in vivo in the non-immune host is somewhat at odds with the relatively inefficient ingestion and killing of pneumococci in standard in vitro phagocytosis assays [34]. The innate host factors that result in the removal of the vast majority of bacteria within 45 minutes of challenge [11], [35], [36] deserve further attention. Despite the efficiency of splenic macrophages in clearance, sustained bacteraemia eventually occurs after an eclipse phase of several hours during which bacteria are largely undetectable in blood. Similar data were obtained in work based on intranasal inoculation of H. influenzae, where also mixed blood cultures were detected in the first minutes after challenge and before the eclipse period [25]. We propose for our intra venous injection model that during this time, a fraction of the inoculated bacteria are sequestered in extravascular tissues, most probably in the spleen, in accordance with our data on positivity of bacterial spleen counts also in mice with negative blood cultures (Figure S1). This emergence of a clone from the potential splenic focus into the “sterile” bloodstream can be viewed as equivalent to the “invasive events” described for models which consider more than one organ system [16], [18], [21], [23]–[25]. Sustained bacteraemia is initiated from replication of one bacterial cell, perhaps a stochastic event in which the first replicon to reach a threshold biomass sufficient to seed the blood “wins the day”. The exponential increase in the number of bacteria in the blood is consistent with contributions from both intravascular and extravascular replication of pneumococci. We favour a scenario in which, at a challenge dose below the LD50, the rate of replication occurring in the extra-vascular site, followed by seeding of bacteria to the blood, exceeds host clearance rates thereby resulting in progressively more severe bacteraemia. Our data do not infer that only one pneumoccocus survives the initial host clearance, but rather that from those that do survive; only one cell initiates bacteraemia. The observed increase of polyclonal infections over time, as predicted also by the independent action hypothesis, is in accordance with the doubling of the ratio of infected mice at those time points (0. 31 at 24 h to 0. 67 at 72 h) [22]. The strong positive selection which drives the emergence of the ATPase-SNP subclones during the later phase of the infection is novel with respect to previous models (i. e. the live-death model), which postulates a neutral selection during this phase [16], [17], [37]. The in depth genomic analysis, in contrast to previous works [16], [17], [37], shows evidence for a more dynamic behaviour of the infecting bacterial population with an increase in heterogeneity of the monoclonal population over time due to a strong positive selection after the single cell bottleneck. However, we observed added complexity; the residual, but inadequate, innate clearance mechanisms exert a selective pressure resulting in the emergence of adaptive mutants. Sequencing of bacteria from blood revealed that in most mice the bacterial clones had each acquired SNPs in different sub-units of the pneumococcal F1/F0 ATPase gene. This apparently high frequency of mutations, given the relatively small biomass of pneumococci in each animal, is consistent with the estimated mutation rates of up to 5×10−4 per genome described recently for pneumococci during one-cell bottleneck in vitro passages [38]. The selection for altered function of the ATPase, was found only in a proportion of the bacteria making up the population obtained from blood, compelling evidence that the ATPase mutations must have occurred after the single cell bottleneck. As stated above, the observation of subclones being selected during the bacteremic phase underlines a highly dynamic situation, which extends over the neutral two stage infection models [16], [17], [37]. In pneumococci, it has been recognised that ATPase mutations occur at high frequency during pneumococcal infection in humans, possibly in response to oxidative stress [39], and have been described both in vitro and in clinical isolates [40]–[44]. Polymorphisms in F0_atpA and F0_atpC (the trans-membrane part of the ATPase) were found to confer phenotypes of reduced susceptibility to optochin, quinine and mefloquine [40], [42], [43]. In particular, the detection of optochin resistant pneumococci in clinical samples is well described [44], as it has a practical impact on pneumococcal identification in the diagnostic laboratory [41]. None of the ex vivo ATPase mutants in our investigation were optochin resistant and the SNPs accordingly did not map to the optochin resistance conferring regions. The F1/F0 ATPase is encoded by a highly conserved eight-gene operon and, as in aero-tolerant anaerobes, it is involved in the maintenance of intracellular pH through the generation of a membrane proton gradient [45]. In some of the mutants we were able to identify a clear metabolic benefit of the mutations which enabled growth at pH lower than 6. 8, albeit all mutants showed that loss of capacity to grow at pH above 7. 8. Interestingly the phenotype of TIGR4 mutants recovered from blood was not different from other virulent pneumococcal stains, such as D39. The high frequency of mutation observed here, given by the many different sites mutated, strongly suggests within-host adaptation through selective pressure during sepsis. While in vitro susceptibility of the ATPase mutants to antibody mediated neutrophil killing and macrophage phagocytosis was essentially unaltered, the phenotypic consequence of the ATPase mutations may be linked to a gain in fitness related to the increased survival of bacteria within the splenic, extravascular focus that provides the source of pneumococci re-seeding the blood and sustaining the progressively escalating and ultimately lethal bacteraemia. In agreement with this hypothesis is the recent description of inhibition of the own F1F0 ATPase by both Salmonella enterica and Mycobacterium tuberculosis as strategy to withstand phagolysosomal activity [46]. In summary, we propose that after the majority of the bacteria of the challenge inoculum have been removed, a few bacteria survive the predominantly lethal activity of splenic macrophages and neutrophils. From these rare survivors, single pneumococcal cells may start to replicate and initiate seeding of the blood resulting in a steady state bacteraemia in which efficient host clearance is off-set by re-seeding from the original, persisting extravascular reservoir of bacteria. These extravascular bacteria are subjected to strong selection for adaptive mutations. Later during infection, selected subpopulations of the initial clone may become part of the bacterial population causing disease. These observations are in accordance with a two stage model of infection where independent action generating the initial stochastic event is followed by a dynamic birth-death phase which increases heterogeneity due to strong selection [16], [17], [24]. In the case of the model organism S. pneumoniae, our data show different selective pressures shaping the invasive bacterial population during different phases of infection [16]. Given the demonstration that pneumococci are independent in generating disease in our rodent model and that less than twenty per cent of human pneumonia cases are bacteraemic [3], we hypothesize that human pneumococcal bacteraemia is generally monoclonal originating from a single cell in analogy to the monoclonal meningitis case recently described [47]. Presentation of a model which foresees development of invasive disease from a single bacterium and strong selection during outgrowth represents an important example on which to model fitness selection during invasive infection. Three isogenic zmpC knock-out mutants of TIGR4 (FP122, FP318 and FP321) that differed only for the resistance marker, ermB (erythromycin resistance), aad9 (spectinomycin resistance) and aphIII (kanamycin resistance), respectively were constructed for co-infection studies with isogenic clones [28], [48]. The experiments with mice depleted of macrophages and neutrophils were done with four different strains: TIGR4 (serotype 4; strain FP321 zmpC: : aphIII), G54 (serotype 19F; erythromycin and tetracycline resistant) [49], [50], D39 (serotype 2; strain FP335 bglA: : aad9; gift of Hasan Yesilkaya, Leicester), and the streptomycin resistant non-encapsulated D39 derivative DP1004 [51], [52]. The transfer of the atpC frame-shift into DP1004 was performed by transformation of a marker flanked by two PCR fragments, one of which containing the frame-shift. This was possible since the atpC SNP is only 76 bp from the end of the operon. Two representative transformants FP499 and FP500 were confirmed by sequencing. The series of ATPase mutants isolated are described in Table 3, while all other strains are listed in Table 1. Strains were cultured in Tryptic Soy Broth (TSB, Liophilchem, Teramo) or Todd Hewitt (THY, Oxoid, Milano) supplemented with 0. 5% Yeast Extract (Liophilchem). Solid media were blood agar plates (Tryptic soy agar, Difco) supplemented with 3% horse blood (Biotech, Grosseto). The colony morphology was checked on Todd-Hewitt agar plates containing 200 units/ml of catalase (Sigma-Aldrich, Milano, Italy) [53], [54]. Antibiotics were used at the following concentrations: 1 mg/L erythromycin, 500 mg/L kanamycin, 100 mg/L spectinomycin and 500 mg/L streptomycin (all from Sigma-Aldrich). The intracellular pH was determined by Nuclear magnetic resonance (NMR). In brief, 400 ml of mid log pneumococcal cells grown in Todd Hewitt broth were pelleted and mixed with 1 ml of sodium alginate 6% (w/v 0. 9‰ NaCl). Mixture were extruded manually through 25G needle on a surface of 0. 25 M CaCl2 solution. The small drops were washed and transferred in the 10 mm NMR tube. NMR 31P spectra were recorded on a Bruker DRX 600 instrument operating at 242. 9 MHz. 31P spectra were recorded with a 1. 5 s repetition time and 45°flip angle. Line broadening of 10 Hz were applied before Fourier Transform. 31P chemical shift were determined by comparison with external standard Trisodium trimetaphospate at −20. 80 ppm. Intracellular pH was determinate by Pi (intracellular phosphate) chemical shift in phosphate-free perfusion model [55]. Active metabolism of pneumococci was confirmed by acidification of the extracellular medium during the experiment carried out at 37°C. Growth profiles of wild type strains and ATPase mutants were assayed both in standard laboratory media and in defined media. Standard laboratory media included TSB (Liophilchem) and Todd-Hewitt broth supplemented with Yeast Extract (0. 5%) (Oxoid). Defined media were prepared in CAT medium by adding serial concentration of potassium phosphate buffer with different range of pH (6 to 8) and by adding several concentration of K2HPO4 as source of salt. CAT medium was composed by: Casitone (10gr/l) (Becton Dickinson), Tryptone (10 gr/l) (Oxoid), Yeast Extract (1 gr/l) (Liophilchem), NaCl (5 gr/l) (Panreac, Milano, Italy), Catalase (200 U/ml) (Sigma-Aldrich) and Glucose (0. 2%) (J. T. Baker, Milano, Italy). Metabolism of pneumococcal strains including wild type and ATPase mutants were assayed by Phenotype MicroArray (PM) microplate PM9 containing a total of 96 different osmolyte sources. PM technology measures active metabolism by recording the irreversible reduction of tetrazolium violet to formazan as an indirect evidence for NADH production. PM procedures were carried out as previously described (Viti C 2009). Quantitative colour change were recorded automatically every 15 min for a period of 72 h. Analyses were performed by the Omnilog-PM Software (Biolog, inc.) and data were filtered using average height as a parameter. Animal experimentation in Italy is regulated by Decreto Legislativo 116/92 and Directive 210/63/EU. The animal protocol was approved by the “Comitato Etico Locale” of the Azienda Universitaria Ospedaliera Senese and received thereafter the relative project licence issued by the Italian Ministry of Health (193/2008-B). Six to seven-weeks old female CD1, BALB/c, and CBA/Ca mice were purchased from Charles River Italia (Lecco, Italy). For the bottleneck experiments, outbred CD1 mice were used, while BALB/c mice were chosen for both in vivo macrophage and neutrophil depletion and ex vivo experiments. CBA/Ca data are shown only for comparison of the dynamics of the early phases of infection. Animals were sacrificed by intraperitoneal (i. p.) injection of xylazine hydrochloride and zolazepam tiletamine cocktail (Xilor 2%, Bio 98 S. r. l., Bologna, Italy and Zoletil 20, Virbac S. r. l., Milano, Italy). Mice were kept at the animal facility of the LAMMB, University of Siena, according to its guidelines for the maintenance of laboratory animals [48], [56]–[58]. Blood samples from mice were collected by sub-mandibular vein or cardiac puncture under terminal anaesthesia. To prevent blood coagulation, 100 U/ml of heparin (MS Pharma, Milano, Italy) was added. All the collected organs (spleen, lung, liver and kidney) were homogenized in 1 ml of TSB, and then frozen at −80°C after making to 10% v/v of glycerol. Two series of experiments were performed in order to define the bottleneck for invasive pneumococcal infection with a total of 68 CD1 mice. Mice were challenged intravenously (i. v.) as described [48], [56]–[58] with a mixture of the three isogenic TIGR4 derivatives (FP122, FP318 and FP321) at 3. 3×105 CFU each/mouse. At pre-set time points blood samples were collected and selected groups were sacrificed for obtainment of spleen samples. Two blood samples from each animal, taken at different time points, are reported in Figure 1. Bacteria were enumerated by plating on selective media. The dose of the experiment was decided after having observed in a preliminary experiment 5/8 mixed and 3/8 monoclonal infections using two pneumococcal clones at a dose of 2×10∧6 (data not shown). A pilot experiment for comparison of virulence in CD1, BALB/c and CBA/Ca mice was carried out by infecting i. v. four mice each with a mixture of G54, D39 and TIGR4 (3×105 CFU each/mouse). Three blood samples per mouse were obtained. For depletion of macrophages, BALB/c mice were treated 24 h prior to challenge by i. p. injection with 750 µl of a suspension of clodronate (CL2MBP) liposomes. One control group received PBS-containing liposomes [31] and the other was untreated. Clodronate was encapsulated in liposomes, as described earlier [31] and was a gift of Roche Diagnostics (Mannheim, Germany). Neutrophil depletion was performed by a single i. p. injection of 150 µg/mouse of anti-GR-1 antibody (Ly6G and Ly6C, clone RB8-8C5; Becton Dickinson) 24 h prior to infection [32], [33]. Two control groups were either left untreated or administered with a rat isotype control antibody IgG2b K (kappa) (Becton Dickinson). Groups of mice depleted of macrophages or neutrophils were infected i. v. with 1×106 CFU/mouse containing 2. 5×105 CFU of each TIGR4, D39, DP1004 and G54. Bacterial viable counts were determined at preset time points. The virulence of the ATPase mutant FP487 (atpC mutant) was assayed in parallel with TIGR4. BALB/c mice (n = 6) were infected with 1×106 CFU/mouse i. v. and blood and spleen samples collected at 72 h. Spleen and bone marrow macrophages were isolated from mice using a modified protocol previously described [34]. Cells were cultured in medium supplemented with 25 ng/ml of recombinant M-CSF (Invitrogen) and re-seeded at day 7 at the concentration of 2×105 cell/ml. After 24 h, 0. 1 ml of pneumococci cultured to OD590 0. 25 were added. After 45 min plates were washed and reincubated with 10 mg/L of penicillin and 200 mg/L of gentamicin for 30 min. Intracellular bacteria were enumerated after lysis with saponin 1%. Phagocytosis of RAW264. 7 macrophages followed the same protocol, but in addition samples were reincubated after removal of the antibiotics for an additional hour in fresh medium. Flow cytometric analysis was conducted on bacteria suspended in 1% v/v paraformaldehyde in PBS on a FACScalibur machine (Becton Dickinson, California, USA). To verify macrophage and neutrophil depletion, homogenised organ samples were washed in DMEM (Sigma-Aldrich) and non-specific binding was blocked with FcR blocking agent [59]. Cells were incubated 30 min with 1 µg of specific fluorochrome-conjugated antibodies per 106 cells. Neutrophils were stained using a rat anti-GR-1 antibody (MACS, Bologna, Italy). Macrophages were detected with rat anti-F4/80 mAb (BM8 clone; Abcam, Milano, Italy), and a rat anti-mouse CD11b mAb (Becton-Dickinson). Surface markers of macrophages were analysed using the following antibodies: anti-F4/80 mAb, anti-mouse CD11b mAb, anti-CD11c mAb (eBioscience), anti-mouse SIGNR1/CD209b Ab, goat IgG control Ab, anti-mouse Siglec-1 mAb, rat IgG2A Isotype control Ab, anti-mouse MARCO mAb and rat IgG1 isotype control Ab (R&D Systems). To assay for the presence of anti-pneumococcal antibodies in mouse sera, the four pneumococcal strains TIGR4, G54, D39 and DP1004 were blocked in PBS-BSA 1% v/v for 30 min at 37°C and incubated for 1 h at 37°C with sera (1∶100) obtained from BALB/c mice and the positive anti-serotype 2 control serum (Staten Serum Institute, SSI, Copenhagen, DK). Samples were marked with anti-mouse IgG (1∶64) or anti-rabbit IgG (1∶160; Sigma-Aldrich). In order to evaluate the capacity of whole blood to kill or inhibit the multiplication of pneumococci and to investigate the effect of specific antibodies, ex vivo experiments were set up. Blood from BALB/c mice was collected into tubes containing heparin and infected with pneumococci. For the assay of opsono-phagocytosis of ATPase mutants 1×104 CFU/ml of parental and ATPase mutant were inoculated in blood and incubated in rotation. The anti-type 4 serum (SSI) was used at 1∶50 dilution. For the evaluation of growth of pneumococci in blood 3×105 CFU/ml of G54, D39 and TIGR4 were inoculated in rotating blood. The efficacy of type 2 anti-serum (SSI) on D39 and its non-encapsulated derivative DP1004 was assayed as above using a inoculum of 3×105 CFU/ml and a 1∶100 dilution of the serum. Chromosomal DNA was extracted using the High Pure PCR Template preparation kit (Roche). Whole genome sequencing was performed by the Institute of Applied Genomics and IGA Technology Services srl (University of Udine, Italy) using an Illumina (Solexa) Genome Analyzer II platform [60]. Reads of both, parent and mutant strains, were aligned to the reference genome of TIGR4 (accession NC_003028) using the Mosaik Assembler suite (The MarthLab, Boston College, Massachusetts, USA). Single nucleotide polymorphisms (SNPs), insertions and deletions (INDELs) were retrieved with VarScan software [61]. SNPs and INDELs of the challenge strains were subtracted from those found by aligning the blood isolates. All F1/F0 ATPase mutations were re-sequenced by the Sanger method and deposited in GenBank (accession KF705516 to KF705525). In order to evaluate the number of bacteria at the origin of blood infection, a model derived from that previously described [23], was developed. A full description of the statistical model is given in the supplementary materials. Statistical analysis of bacterial counts in blood and organs was performed by the Student' s t-test for data reported in Figures 3,4, S2 and S3. The analysis of different bacterial blood clearance at 5 and 30 min and the differences in bacterial phagocytosis and data of phenotype microarray were performed using Kruskal-Wallis and Dunn' s multiple comparison post test (Figure 2 C–F and S5). Values of P<0. 05 were considered statistically significant. The Fluorescence Index (Figure S3 K) was calculated by multiplying the percentage of positive events with the geometric mean fluorescence intensity (GeoMean).

Decades of research on bacterial sepsis have been devoted to analysing the steps that lead from a local event, either carriage or a localised infection, to systemic disease. Our work analyses in depth the events determining systemic infection by one of the main human pathogens, Streptococcus pneumoniae. Consistent with similar findings on the pathogenesis of bacteraemia due to other commensal pathogens, our results show that after an intravenous inoculum of a million pneumococci, the resulting septicaemia is often founded by a single bacterial cell. Investigation into the nature of this monoclonal infection identified strong within-host selective pressure for metabolic fitness during outgrowth of the bacterial population.

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Between March 2014 and July 2015 at least 10,500 Ebola cases including more than 4,800 deaths occurred in Liberia, the majority in Monrovia. However, official numbers may have underestimated the size of the outbreak. Closure of health facilities and mistrust in existing structures may have additionally impacted on all-cause morbidity and mortality. To quantify mortality and morbidity and describe health-seeking behaviour in Monrovia, Médecins sans Frontières (MSF) conducted a mobile phone survey from December 2014 to March 2015. We drew a random sample of households in Monrovia and conducted structured mobile phone interviews, covering morbidity, mortality and health-seeking behaviour from 14 May 2014 until the day of the survey. We defined an Ebola-related death as any death meeting the Liberian Ebola case definition. We calculated all-cause and Ebola-specific mortality rates. The sample consisted of 6,813 household members in 905 households. We estimated a crude mortality rate (CMR) of 0. 33/10,000 persons/day (95%CI: 0. 25–0. 43) and an Ebola-specific mortality rate of 0. 06/10,000 persons/day (95%-CI: 0. 03–0. 11). During the recall period, 17 Ebola cases were reported including those who died. In the 30 days prior to the survey 277 household members were reported sick; malaria accounted for 54% (150/277). Of the sick household members, 43% (122/276) did not visit any health care facility. The mobile phone-based survey was found to be a feasible and acceptable alternative method when data collection in the community is impossible. CMR was estimated well below the emergency threshold of 1/10,000 persons/day. Non-Ebola-related mortality in Monrovia was not higher than previous national estimates of mortality for Liberia. However, excess mortality directly resulting from Ebola did occur in the population. Importantly, the small proportion of sick household members presenting to official health facilities when sick might pose a challenge for future outbreak detection and mitigation. Substantial reported health-seeking behaviour outside of health facilities may also suggest the need for adapted health messaging and improved access to health care. Between March 2014 and July 2015, more than 10,500 Ebola virus disease (EVD) cases, including over 4,800 deaths, occurred in Liberia; the majority of these cases was identified in Montserrado County, where the capital city of Liberia, Monrovia, is located [1]. However, official reported numbers of EVD cases might underestimate the size of the outbreak for several reasons: i) during the intense phase of the outbreak in August 2014, Ebola treatment units (ETUs) were overwhelmed with patients, some of whom were turned away uncounted [2]; ii) the continued identification of cases that had not been registered as contacts of known EVD cases beforehand indicates that contact tracing remained incomplete throughout the outbreak and cases are likely to have been missed [3] and iii) communities hesitated to send sick members to ETUs [2,4]. A capture-recapture study conducted in August 2014 suggested that actual case numbers could be three times higher than the number of notified EVD cases [5]. Health care providers in the field have assumed that closure of health facilities and mistrust in existing structures resulted not only in EVD-related excess morbidity and mortality but also had an impact on non-EVD morbidity and mortality: In Monrovia—similar to what has been described in other affected countries and cities [6]–the capacity of health care facilities was greatly reduced in August 2014 as compared to prior to the epidemic [7–10]. Even in health facilities that continued to provide health care in Monrovia, the number of consultations was reduced by at least 40% compared to previous years [7]. In addition, the health care system in Liberia lost at least 178 health care workers to EVD from the beginning of the outbreak until the end of 2014 [11]. Fear of EVD and mistrust in existing health care structures led to underutilization of services [7,8, 12]. In 2013, crude mortality rates (CMR) in Liberia were reported between 0. 22/10,000 persons/day [13], 0. 25/10,000 persons/day [14] and 0. 27/10,000 persons/day [15]. To quantify mortality and morbidity and to describe health-seeking behaviour in Monrovia during the EVD outbreak, Médecins Sans Frontières (MSF) conducted a mobile phone survey in Monrovia. The specific objectives were to a) retrospectively estimate the all-cause-mortality rate (non-EVD and EVD) during the recall period, b) retrospectively estimate disease-specific attack rates for non-EVD and EVD during the 30 days preceding the survey, c) retrospectively estimate the EVD attack rate during the recall period and d) describe health-seeking behaviour. The study was conducted in Monrovia, Liberia, from December 2014 to March 2015. Classical survey designs, such as household visits with face-to-face interviews, were determined unfeasible due to the risk of EVD transmission given the contact and movement requirements to implement a community household survey. To minimize these risks we chose instead to conduct a mobile phone based survey. The survey covered a recall period from the 14th May 2014 (National Unification Day in Liberia) to the day of the survey. The study population included the whole population of greater Monrovia. Greater Monrovia is located at the northern portion of the Liberian coast at the mouth of the Mesurado River and extends across a series peninsulas and wetlands. Greater Monrovia consists of a population of about 1 million inhabitants (MSF operational data) and is the most urbanized area in Liberia, its capital and main port. Telecommunication using landlines is almost absent in most parts of Monrovia. The sampling frame therefore included households where at least one person in the household owned a Subscriber Identity Module (SIM) card from a selected large mobile phone network provider in Monrovia and had been connected to the network in Monrovia at some point in the 30 days prior to the date of the survey. The selected network provider had, according to the company, coverage of more than half of the population of Monrovia with customers in all age-groups that represented diverse socio-demographic strata. The calculation of the sample size is based on the crude mortality rate and the expected EVD attack rate. For an expected crude mortality rate of 0. 5/10,000/day based on doubling of the national baseline mortality [13–15], a precision of 0. 15 and a recall period of 255 days (from 15 May 2014 to mid-survey date, 24 January 2015), a total of 5,986 individuals in 1,197 households were needed in the sample, assuming an average household size of five household members. Assuming that real case numbers were threefold higher than those notified [5], and thus an expected attack rate of 0. 51% for EVD during the whole study period (based on notified cases in the Liberian EVD patient database up to 9 November 2014 according to WHO data packages) and a precision of 0. 15%, 8,660 individuals in 1,732 household were needed. Non-response was estimated at 50% as suggested by the telephone network provider, based on their usual response rates in previous telephone surveys. Therefore, 3,500 households were included in the sample. The network provider drew a simple random sample of telephone numbers. The network provider sent a text message to the selected customers informing them that they had been randomly selected to participate in an MSF survey. Participants sent a text message if they agreed to have their phone number forwarded to MSF and received one US Dollar of free airtime after providing this consent (regardless of whether they eventually completed the survey when contacted by the surveyors). Trained MSF surveyors made a maximum of three attempts to contact each telephone number. Respondents were only included in the survey if they were over 18 years of age, lived in greater Monrovia and consented to participation. Any sickness or death was counted as EVD-related if it fulfilled the Ebola case definition of the Liberian Ministry of Health (MOH) for suspect, probable or confirmed cases [16]. A household was defined as a group of people living under the same roof and sharing the same meal at least 3 times a week regardless of family ties. We trained 15 surveyors for three days to collect data using standardized questionnaires on tablets. Surveyors entered data directly into an Open Data Kit (ODK) form on the tablet during the telephone interview. At the end of each day the lead epidemiologist reviewed any recorded deaths and EVD cases together with the surveyors to ensure data quality. Each respondent was asked to answer the questions on behalf of the entire household. For each household, the number of members of the household was recorded. Respondents were asked about any sick person in the household within the past 30 days. For each EVD case according to the case definition, information about symptoms, history of contact, isolation of the cases and burial circumstances (where applicable) was obtained. We also inquired about non-EVD deaths among household members during the recall period, along with cause of death according to the respondent and the circumstances of burial. Reported EVD cases were compared to the case definition of the Liberian Ministry of Health. We calculated disease specific attack rates in the past 30 days–and for EVD for the whole recall period–using the sampled population at the beginning of the recall period as the denominator. Crude mortality rate (CMR) and EVD-related-mortality-rate per 10,000 population per day were calculated using the recall period as the denominator. Household members who left, arrived or were born during the recall period were considered as having lived in the household half of the recall period. For deceased members the exact time under observation (14 May 2014 to date of death) was used as a denominator. Statistical analysis was conducted in STATA version 13 (Stata corporation, Texas, USA). The procedures conducted were in accordance with the ethical standards of the Helsinki Declaration. Additionally, the National Research Ethics Board of the Ministry of Health of Liberia granted ethical approval and authorization to conduct this survey. An information sheet was read and explained to each respondent at the beginning of the interview. Participation was voluntary. For each respondent, oral informed consent was requested and this response was documented. No personal identifiable information was collected. Telephone numbers were kept confidential and stored securely. 768 (22%) persons of 3,500 that were contacted by the network provider from 19 December to 2 January 2015 responded to the text message and agreed to be contacted for the survey, 446 were reached by the surveyors and fulfilled the required eligibility criteria (overall response rate 13%; 446/3,500) (Table 1). They provided information for 3,363 household members, indicating an average household size of 7. 5 persons per household. As a result of the response outcome through early January 2015, the sample size was recalculated, adjusting the household size estimate (5 → 7. 5) and the expected non-response rate (50% → 90%). A second round of sampling was launched in order to reach the required numbers of respondents, with an additional 7,000 phone numbers contacted by text (total contacted, 10,500). Of the 7,000 texts messages sent to a new random sample of mobile phone owners between 29 January and 7 March 2015,781 customers replied with agreement to participate (11%; 781/7,000) (Table 1). 459 households were eligible for inclusion (Table 1); they represented 3,450 household members (average household size 7. 5). After both rounds of sampling, the survey included a total of 905 households. The total number of days in the recall period ranged from 215 days (first interview) to 293 days (last interview). At the beginning of the survey period, 6,813 household members were included in the sample (Table 2). The crude birth rate was 29. 1 births per 1,000 persons per year. The median age of the 905 respondents was 29 years (interquartile range: 23–36). Respondents came from all parts of Monrovia; between 31 and 146 persons/10,000 population per neighbourhood participated in the survey (Table 3). The proportion of households included was lower for precarious neighbourhoods and higher in more affluent neighbourhoods. Overall 55 deaths occurred during the recall period, indicating a crude mortality rate of 0. 33/10,000 (95%-CI: 0. 25–0. 43) persons per day. Fifty-five deaths occurred in 47 households, indicating that over the entire recall period, 5. 2% of households experienced at least one death. Six (0. 7%) households experienced more than one death. Forty-five (82%) of 55 deaths did not meet the EVD case definition, indicating a non-EVD mortality rate of 0. 27/10,000 (95%-CI: 0. 20–0. 36) persons per day. Deaths due to a cause other than EVD were chronic diseases (n = 20), injuries (n = 6), birth- or pregnancy-associated deaths (n = 3), “African sign” (n = 3), ulcers (n = 2), “old age” (n = 1), stomach pain (n = 1), food poisoning (n = 1), “no good care” (n = 1) and jaundice (n = 1). For six deaths the reason was unknown. EVD attributable deaths were 10 of 55 (18%; 95%-CI: 9–31%), indicating an EVD specific mortality rate of 0. 06/10,000 (95%-CI: 0. 03–0. 11) persons per day. Individuals that died of EVD had a lower median age than those dying of non-EVD causes (median age 32 and 36 years, respectively; interquartile range 25–33 and 21–56, respectively) and were more frequently reported to have died in an ETU (Table 4). Six (11%) of 55 deaths were of children under 5 years of age; their causes were reported as EVD (n = 1), asphyxia at birth (n = 1), “African sign” (n = 1), “no good care” (n = 1) and ulcers (n = 2). 277 (4%) of 6,813 household members were reported to have been sick in the 30 days prior to the survey. 19 of those sick reported suffering from more than one disease, resulting in 277 sick persons with 295 disease episodes. The most frequently reported diseases were malaria (2. 2% of all household members), acute respiratory infections (ARI) (0. 4%) and chronic diseases (0. 3%) (Table 5). One EVD case was reported during the 30 days preceding the survey. 10 (4%) of the 277 household members that had been sick in the 30 days prior to the survey were children under five years of age, 70% (7/10) of these children were reported to have had experienced an episode of malaria. 19 EVD cases were reported to have occurred between 14 May 2014 and the date of interview. However, three of the reported EVD cases did not meet case definition. Of the remaining 16 cases, four met the case definition for a suspect case, one for a probable case and eleven for a confirmed case. One additional death reported as a non-EVD death met the case definition for a suspect case. The overall attack rate of reported cases meeting the EVD case definition (n = 17) among all household members was 0. 25% (95%-CI: 0. 15–0. 40). Of 17 EVD cases, 10 were reported to have died (case fatality rate: 59%). Median age of the reported EVD cases was 29 years, 53% of cases were male. 76% were reported to have been hospitalised in ETUs. Date of symptom onset was available for 16 cases, eight of which had symptom onset in August, after which the number of cases declined (Fig 1). Nine (3%) of the 276 individuals, who had been sick during the 30 days prior to the survey with non-EVD diseases, did not seek any health care. Forty-three (16%) of the 276 sick people accessed health care in a government-run health facility. 112 (41%) accessed health care in private health facilities. 54 (20%) found treatment either in pharmacies or drug stores and 50 (18%) asked a health care worker (HCW) to take care of them at home, 6 (2%) visited a traditional healer, one sought healthcare at the church and one elsewhere. Of the 150 reported cases of malaria, 35% (52/150) did not seek care at neither a public nor a private health facility; an additional 16% (24/150) were reported to have been treated at home by a health care worker. We have shown that a mobile phone survey can be implemented in a context where access to the population is limited–be it due to the risk of infection by Ebola, as in this case or, potentially to other contexts such as insecure conflict environments, difficulties in transportation during rainy season or after natural disasters, or difficult access caused by the risk of kidnapping. There are several limitations introduced by using the mobile telephone methodology: First, the dependence on a network provider for random sampling created some delay and contributed to a lack of full transparency in the process. Second, data on population coverage of the network provider in Monrovia was unknown as were the socio-demographic characteristics of customers, leading to uncertainties regarding the representativeness of the sample for Monrovia’s whole population. Low rates of inclusion of households in precarious neighbourhoods of Monrovia suggest that the sample frame might not have been as representative of the population of greater Monrovia as implied by the provider. Thus, poverty associated diseases and deaths, including EVD, may be underestimated in this survey. Despite these limitations, mean age of the respondents and household size was similar to previously published estimates for Monrovia [23] and estimates for birth and death rates [15], morbidity [22,23] and health seeking behaviour [24] were in accordance with previous research indicating acceptable representation of Monrovia’s population. In future mobile phone surveys underrepresentation could possibly be mitigated with an adapted quota sampling strategy, if sociodemographic and -economic characteristics of the customers of the partnering company are available. Third, the respondents’ environment can have an influence on the quality of data gathered from mobile phone surveys. Mobile phone respondents can be in situations where survey participation is not feasible or the connection not adequate. Despite these potential limitations, studies have shown that the interview mode does not influence significantly the quality of data measurement [25]. Fourth, the initial response was much lower than expected by the provider but in keeping with response in telephone surveys performed in industrialized countries [26]. The low response necessitated a new round of sampling and extended the recall period. Our experience suggests that for future mobile phone based surveys, the sample size calculations should account for a proportion of non-response as high as in industrialized countries. As with all retrospective mortality studies, there is the potential for recall and survivor bias that may have affected the results. Additionally, stigma, fear, an environment of mistrust and the trauma of the experience may have led to under-reporting of EVD; the format of the survey–dependent on the reporting of sensitive health information to an unknown surveyor on the phone–may have exacerbated this effect. For operational reasons–given the context and the “no-touch-policy”–we could not compare the telephone survey method with a gold standard community based survey to evaluate the specificity and sensitivity. The mobile phone based survey was found to be a feasible and acceptable alternative method when data collection in the community is impossible. CMR was estimated below the emergency threshold of 1/10,000 persons per day. Our results suggest excess mortality in the population directly resulting from Ebola. However, non-EVD mortality did not increase to the extent originally expected. Importantly, the small proportion of sick household members presenting to official health facilities when sick might pose a challenge for future outbreak detection and mitigation. Substantial reported health-seeking behaviour outside of health facilities may also suggest the need for adapted health messaging and improved access to health care.

During the Ebola outbreak in 2014/2015 more than 4,800 people died of Ebola in Liberia. Health care providers in the field have assumed that closure of health facilities and mistrust in existing structures resulted not only substantial additional deaths from Ebola but also impacted on death rate of other diseases and on the way people tried to seek health care. We conducted a mobile phone survey in Monrovia to identify deaths and diseases a household had faced since the beginning of the Ebola outbreak and the kind of health care they sought. We estimated that the non-Ebola-related death rate in Monrovia was not higher than previous national estimates for Liberia. However, additional deaths occurred in the population of Monrovia directly resulting from Ebola. Of the household members that were sick of any disease during the survey period, 43% did not visit any health care facility. The high proportion among the sick household members that sought health care in pharmacies or drug stores or by health care workers among their peers but outside health facilities emphasizes the importance of ensuring access to non-Ebola-related health care as outbreak control is challenged by sick community members staying undiagnosed and untreated.

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Neurosteroids are endogenous modulators of neuronal excitability and nervous system development and are being developed as anesthetic agents and treatments for psychiatric diseases. While gamma amino-butyric acid Type A (GABAA) receptors are the primary molecular targets of neurosteroid action, the structural details of neurosteroid binding to these proteins remain ill defined. We synthesized neurosteroid analogue photolabeling reagents in which the photolabeling groups were placed at three positions around the neurosteroid ring structure, enabling identification of binding sites and mapping of neurosteroid orientation within these sites. Using middle-down mass spectrometry (MS), we identified three clusters of photolabeled residues representing three distinct neurosteroid binding sites in the human α1β3 GABAA receptor. Novel intrasubunit binding sites were identified within the transmembrane helical bundles of both the α1 (labeled residues α1-N408, Y415) and β3 (labeled residue β3-Y442) subunits, adjacent to the extracellular domains (ECDs). An intersubunit site (labeled residues β3-L294 and G308) in the interface between the β3 (+) and α1 (−) subunits of the GABAA receptor pentamer was also identified. Computational docking studies of neurosteroid to the three sites predicted critical residues contributing to neurosteroid interaction with the GABAA receptors. Electrophysiological studies of receptors with mutations based on these predictions (α1-V227W, N408A/Y411F, and Q242L) indicate that both the α1 intrasubunit and β3-α1 intersubunit sites are critical for neurosteroid action. Neurosteroids are cholesterol metabolites produced by neurons [1] and glia [2] in the central nervous system (CNS) that are thought to play important roles in both nervous system development and behavioral modulation [3]. Neurosteroid analogues are also being developed as sedative hypnotics [4], antidepressants [5], and anticonvulsants [6]. Gamma amino-butyric acid Type A (GABAA) receptors, the principal ionotropic inhibitory neurotransmitter receptors in the CNS, have been identified as the primary functional target of neurosteroids. The major endogenous neurosteroids—allopregnanolone and tetrahydroxy-desoxycorticosterone (THDOC) —are positive allosteric modulators (PAMs) of GABAA receptors, potentiating the effects of GABA at nanomolar concentrations and directly activating currents at micromolar concentrations. GABAA receptors are members of the pentameric ligand-gated ion channel (pLGIC) superfamily and are typically composed of two α subunits, two β subunits, and one γ or δ subunit [7]. There are 19 homologous GABAA receptor subunits (including six α, three β and two γ isoforms), with each subunit composed of a large extracellular domain (ECD), a transmembrane domain (TMD) formed by four membrane-spanning helices (TMD1–4), a long intracellular loop between TMD3 and TMD4, and a short extracellular C-terminus. These distinctive structural domains form binding sites for a number of ligands: GABA and benzodiazepines bind to the ECD, picrotoxin to the channel pore [8], and general anesthetics—such as propofol [9,10], etomidate [11], barbiturates [12], and neurosteroids—to the TMDs [13–18]. Substantial evidence indicates that neurosteroids produce their effects on GABAA receptors by binding to sites within the TMDs [13–15,19,20]. Whereas the TMDs of β-subunits are critically important to the actions of propofol and etomidate [11,21–26], the α-subunit TMDs appear to be essential for neurosteroid action. Mutagenesis studies in α1β2γ2 GABAA receptors identified several residues in the α1 subunit, notably Q241 in TMD1, as critical to neurosteroid potentiation of GABA-elicited currents [14,27]. More recent crystallographic studies have shown that, in homo-pentameric chimeric receptors in which the TMDs are derived from either α1 [16] or α5 subunits [17], the neurosteroids THDOC and pregnanolone bind in a cleft between the α-subunits, with the C3-hydroxyl substituent of the steroids interacting directly with α1Q241. Neurosteroids are PAMs of these chimeric receptors, and α1Q241L and α1Q241W mutations eliminate this modulation. These studies posit a single critical binding site for neurosteroids that is conserved across the six α-subunit isoforms [14,27]. A significant body of evidence also suggests that neurosteroid modulation of GABAA receptors may be mediated by multiple sites. Site-directed mutagenesis identified multiple residues that affect neurosteroid action on GABAA receptors, suggestive of two neurosteroid binding sites, with one site mediating potentiation of GABA responses and the other mediating direct activation [14,27]. Single channel electrophysiological studies as well as studies examining neurosteroid modulation of [35S]t-butylbicyclophosphorothionate (TBPS) binding, have also identified multiple distinct effects of neurosteroids with various structural analogues producing some or all of these effects, consistent with multiple neurosteroid binding sites [28–30]. Finally, neurosteroid photolabeling studies in the bacterial pLGIC, Gloeobacter ligand-gated ion channel (GLIC), demonstrate two neurosteroid binding sites per monomer [31], one analogous to the canonical intersubunit site and one located in an intrasubunit pocket previously shown to bind propofol [32,33] and the inhalational anesthetics [33,34]. Both of these sites contribute to neurosteroid modulation of GLIC currents, suggesting the possibility of analogous sites in GABAA receptors. We have developed a suite of neurosteroid analogue photolabeling reagents with photolabeling groups positioned around the neurosteroid ring structure to identify all of the neurosteroid binding sites on GABAA receptors and determine the orientation of neurosteroid binding within each site. Photolabeling was performed in membranes from a mammalian cell line that stably expresses α1His-FLAGβ3 receptors (rather than in detergent-solubilized receptors) to optimize the likelihood that the receptors were in native conformations and environment. Finally, we deployed a middle-down mass spectrometry (MS) approach, coupled with a stable-heavy isotope encoded click chemistry tag for neurosteroid-peptide adduct identification to circumvent challenges associated with MS identification (predominantly neutral loss) and quantification of neurosteroid-peptide adducts [35]. Using these approaches, we have identified three clusters of neurosteroid-photolabeled residues on the human α1β3 GABAA receptor. Computational docking studies, guided by the photolabeling data, were used to describe three binding sites and the orientation of the neurosteroids within each site. The docking studies were also used to predict critical residues to test the contribution of each of these sites to neurosteroid modulation of GABAA currents. Site-directed mutagenesis of these sites and electrophysiological studies indicate that at least two of three structurally distinct sites contribute to allosteric modulation of GABA currents. Allopregnanolone (3α-hydroxy-5α-pregnan-20-one) is a potent, endogenous PAM of GABAA receptors (Fig 1A). We synthesized three photolabeling analogues of allopregnanolone in which photolabeling moieties were placed at various positions around the steroid backbone. KK123 has a 6-diazirine photolabeling group on the C5-C6-C7 edge of the sterol, which is a likely binding interface with α-helices [36] and minimally perturbs neurosteroid structure [37]. KK123 is, however, an aliphatic diazirine and, as such, may preferentially label nucleophilic amino acids [38]. The two other reagents, KK202 and KK200, incorporate a trifluoromethylphenyl-diazirine (TPD) group at either the 3- or 17-carbon. These were designed to sample the space in the plane of the steroid off either the A-ring (KK202) or the D-ring (KK200). Following UV irradiation, TPD groups generate a carbene which can insert into any bond [39,40]. Thus, while the TPD groups are bulky and removed several angstroms from the neurosteroid pharmacophore, they should form an adduct precisely at their binding site in the GABAA receptor. Where feasible (KK123, KK202), an alkyne was incorporated in the photolabeling reagents to allow attachment of a fluorophore, purification tag, or an MS reporter tag (FLI-tag) via click chemistry [35]. A useful photoaffinity labeling reagent must bind to the same site on a protein as the ligand it mimics and should produce the same effects on protein functions. To determine whether our photoaffinity labeling reagents mimic allopregnanolone as modulators of GABAA receptor function, we assessed modulation of α1β3 GABAA receptors currents in Xenopus laevis oocytes, and enhancement of [3H]muscimol binding in human embryonic kidney (HEK) cell membranes expressing α1β3 GABAA receptors. KK123 enhanced GABA-elicited (0. 3 μM) currents 4. 2 ± 3. 3-fold at 1 μM (n = 5 cells) and 8. 2 ± 6. 7-fold at 10 μM (n = 7). KK123 (10 μM) also directly activated α1β3 GABAA receptors, eliciting 6. 3% ± 3. 8% (n = 5) of the maximum current elicited by a saturating concentration of GABA. KK123 potentiation of GABA-elicited currents and direct activation were absent in α1Q242Lβ3 GABAA receptors, indicating that KK123 closely mimics the actions of allopregnanolone (the human α1Q242L mutation is equivalent to rat α1Q241L and is known to selectively prevent neurosteroid action (Fig 1B and Table 1) [14,27]). KK200 and KK202 also potentiated GABA-elicited currents at 1 and 10 μM and directly activated the channels at 10 μM (Table 1). Positive allosteric modulation by KK202 was somewhat surprising, given that an ether-linked TPD group replaces the 3α-OH group thought to be critical for neurosteroid action [41,42]. While the effects of KK200 were abolished in α1Q242Lβ3 receptors, the potentiation by KK202 was reduced by 50% in α1Q242Lβ3 receptors, suggesting that KK202 may have actions at both the canonical neurosteroid site and other binding sites. Because photolabeling experiments were performed in membranes prepared from cells expressing α1β3 GABAA receptors, we also examined the ability of the photolabeling reagents to enhance [3H]muscimol binding in these membranes. A stable HEK-293 cell line was established with tetracycline-inducible expression of human α1His-FLAG β3 GABAA receptors (See Materials and methods); receptor density in these membranes was 20–30 pmol [3H]muscimol binding/mg membrane protein. Consistent with previous determinations [43], the average stoichiometry of the receptors was estimated at two α1 subunits and three β3 subunits using MS label-free quantitation [44] (spectral count). Allopregnanolone enhanced [3H]muscimol binding to these recombinant receptors 4-fold with a half maximal effective concentration (EC50) of 3. 9 ± 5. 6 μM (S1 Fig). KK123, KK200, and KK202 all enhanced [3H]muscimol binding with EC50 values similar to or lower than allopregnanolone (S1 Fig). Collectively, the electrophysiology and radioligand binding data indicate that KK123, KK200, and KK202 are functional mimetics of allopregnanolone. To determine whether KK123—which contains an aliphatic diazirine—photolabels GABAA receptors, we utilized the butynyloxy (alkyne) moiety on KK123 to attach a biotin purification tag for selective enrichment of photolabeled GABAA receptor subunits. HEK-293 cell membranes containing α1β3 GABAA receptors were photolabeled with 15 μM KK123, solubilized in SDS, and coupled via Cu2+-catalyzed cycloaddition to MQ112 (S2A Fig), a trifunctional linker containing an azide group for cycloaddition, biotin for biotin-streptavidin affinity purification, and a cleavable azobenzene group for elution of photolabeled proteins. The photolabeled-MQ112-tagged receptors were bound to streptavidin beads and eluted by cleavage of the linker with sodium dithionite. The purified, photolabeled GABAA receptor subunits were assayed by western blot using anti-α1 and anti-β3. A band at 52 kDa was observed with both α1 and β3 subunit antibodies in the KK123 photolabeling group (S2B Fig), indicating that both α1 and β3 subunits are photolabeled by KK123. In control samples photolabeled with ZCM42—an allopregnanolone photolabeling analogue containing a diazirine at the 6-carbon but no alkyne (S2C Fig) —neither α1 nor β3 subunits were purified. These data indicated that KK123 can photolabel both α1 and β3 subunits and is thus an appropriate reagent to use for site identification. A 35 kDa band was intermittently observed in replicate anti-α1 western blots (S2B Fig); this is likely to be a proteolytic fragment of the α1-subunit that retains the antibody-recognition epitope but was not further analyzed. Identification of sterol adducts in hydrophobic peptides has been impeded by multiple challenges, including peptide insolubility during sample digestion, ineffective chromatographic separation of hydrophobic TMD peptides, and neutral loss of sterol adducts from small hydrophobic peptides during ionization and fragmentation. To circumvent these problems, we employed middle-down MS to analyze GABAA receptor TMD peptides and their sterol adducts. This approach identifies each TMD as a single, large peptide and attenuates neutral loss of adduct, facilitating identification of the sites of neurosteroid incorporation. In our studies, α1His-FLAGβ3 GABAA receptors were photolabeled in native HEK cell membranes. The photolabeled proteins were then solubilized in n-dodecyl-β-D-maltoside (DDM) -containing lysis buffer. The pentameric GABAA receptors were purified using anti-FLAG agarose beads, and eluted receptors were digested with trypsin in the presence of the MS-compatible detergent DDM. These conditions generated peptides containing each of the GABAA receptor TMDs in their entirety. The peptides were separated using PLRP-S nano-liquid chromatography and analyzed on a Thermo ELITE orbitrap mass spectrometer. This workflow (S3 Fig) minimized protein/peptide aggregation, simplified MS1-level identification of TMD-sterol adducts, and optimized fragmentation of TMD peptides and their adducts. All eight of the TMD peptides were reliably sequenced with 100% residue-level coverage. In addition, the covalent addition of neurosteroid to the TMD peptides increased the hydrophobicity of TMD peptides and shifted their chromatographic elution to later retention times (S3 Fig). The delayed retention time was used as a critical criterion for identification of photolabeled peptides. Two photolabeled peptides were found in the mass spectra of tryptic digests of α1β3 GABAA receptors photolabeled with KK123 (Fig 2A and S4 Fig). A KK123 adduct of the α1-TM4 peptide, 398IAFPLLFGIFNLVYWATYKK123LNREPQLK423 (m/z = 875. 503, z = 4), was identified (add weight of KK123 = 316. 27). Site-defining ions in the fragmentation spectra identified the site of KK123 insertion as Y415, at the C-terminus of α1-TM4 (underlined in the sequence; see Fig 2A). In a separate series of experiments, α1β3 receptors were photolabeled with KK123, which was then coupled to FLI-tag using click chemistry. FLI-tag, an azide-containing tag, adds both charge and a heavy/light stable isotope pair to a photolabeled peptide, enhancing identification by creating doublets in the MS1 spectra [35]. MS1 level search for pairs of ions differing by 10. 07 mass units found two peptide ion features (m/z = 1,073. 246 and m/z = 1,076. 580, z = 3) that had identical chromatographic retention times (Fig 2B). Fragmentation spectra revealed both of these peptides as β3-TM4 peptide (426IVFPFTFSLFNLVYWLYKK123YVN445) with a KK123-FLI-tag adduct (adduct mass = 672. 432 and mass = 682. 441) on Y442 (Fig 2C). In the fragmentation spectrum, ions containing KK123 plus light FLI-tag (Fig 2C, black) were different by 10. 07 mass units from the corresponding fragment ions from KK123 plus heavy FLI-tag (Fig 2C, red), confirming that KK123 photolabels Y442 of the β3 subunit. β3-Y442 is located on the C-terminus of β3-TM4 in a homologous position to α1-Y415, the KK123 photolabeling site in α1-TM4 (Fig 2D, upper right panel). Thus, KK123 labeling data identified two discrete sites, one in α1 and the other in β3. We employed additional photolabeling reagents containing TPD groups arrayed around the sterol backbone to confirm whether the KK123-labeled residues represent neurosteroid binding sites and to determine the orientation of the neurosteroids in these sites. KK200, which has a TPD photolabeling group attached at C17 on the steroid backbone, has been previously used to map neurosteroid binding sites on GLIC [31]. Analysis of α1β3 receptors photolabeled with 15 μM KK200 detected two photolabeled TMD peptides: an α1-TM4 peptide, 398IAFPLLFGIFNKK200LVYWATYLNREPQLK423, was photolabeled with KK200 (m/z = 898. 002; z = 4); site-defining ions in the fragmentation spectra identified N408 as the modified residue (Fig 3A). The N408 residue (N407 in rat) has previously been shown to be critical to neurosteroid potentiation of GABA-elicited currents [14,15]. A β3-TM3 peptide, 280AIDMYLMGCNEM+DTTFVFVFLALLEYAFVNYIFFGRKK200GPQR313 (m/z = 1,188. 352; z = 4; N-ethylmaleimide [NEM]; 1,4-dithiothreitol [DTT]; alkylation adduct), was also photolabeled with KK200. Fragmentation spectra narrowed the possible sites of adduction to G308 or R309, both at the junction of TM3 with the M3–M4 intracellular loop (Fig 3B). Analysis of GABAA receptors photolabeled with KK202 (Fig 3C and 3D), identified two photolabeled peptides eluting two minutes apart. Both peptides were identified as the β3-TM3 peptide, 278VKAIDMYLMGCNEMFVFVFLALLEYAFVNYIFFGRGPQR313 (m/z = 811. 453, z = 6). Fragmentation spectra of the earlier eluting peptide localized labeling to a three-residue sequence, 278VKA280, at the N-terminus of β3-TM3 (Fig 3C). The fragmentation spectrum of the later eluting peptide, identified L294 as the site of adduction (Fig 3D). (The different retention time of the two photolabeled peptides is likely due to differences in peptide conformation and surface hydrophobicity resulting from incorporation of the photolabeling reagent into different residues.) An important test of whether the photolabeled sites constitute specific allopregnanolone binding sites is the ability of excess allopregnanolone to competitively prevent photolabeling. Photolabeling studies for site identification were performed using 15 μM photolabeling reagent and achieved levels of labeling efficiency varying from 0. 06% to 3. 0% (S1 Table). Because allopregnanolone has limited aqueous solubility (about 30 μM) and a large competitor excess is needed to demonstrate competition (particularly with an irreversibly bound ligand), we were limited to studying competition at the photolabeled residues that could be detected following photolabeling at a concentration of 3 μM. Accordingly, we measured the photolabeling efficiency obtained following photolabeling of α1β3 GABAA receptors with 3 μM KK123, KK200, or KK202 in the presence or absence of 30 μM allopregnanolone. KK123 photolabeled both α1-Y415 (0. 77% efficiency) and β3 -Y442 (0. 37% efficiency). For both of these residues, photolabeling was reduced by >90% in the presence of excess allopregnanolone (Fig 4A). KK200 photolabeled β3 -G308/R309 (0. 19% efficiency), and labeling was reduced by 98% in the presence of allopregnanolone. KK202 labeled both β3-L294 (0. 29% efficiency) and β3-278VKA280 (0. 21% efficiency) in TM3; labeling of both of these sites was undetectable in the presence of 30 μM allopregnanolone. Studies were also performed to determine whether the orthosteric agonist GABA (1 mM) enhanced photolabeling by 3 uM KK123 or KK200. Labeling efficiency was not significantly enhanced in the presence of GABA. This suggests that there is a small difference in neurosteroid affinity for closed versus open/desensitized states, which is consistent with the fact that neurosteroids have very low efficacy as direct activators of GABAA receptors [45]. Modification of ligand analogues with labeling groups at different locations has been used to determine the orientation of the ligands within their binding pockets [46]. Here, the six residues photolabeled by KK123, KK200, and KK202 were examined in a model of the α1β3 receptor created by threading the aligned sequence of the α1 subunit on the structure of the β3 subunit (PDB 4COF) [47]. The photolabeling sites grouped into the following three clusters: cluster 1 (brown circle), β3-L294 (KK202) and β3-G308/R309 (KK200); cluster 2 (red circle), α1-Y415 (KK123) and α1-N408 (KK200); and cluster 3 (blue circle), β3-Y442 (KK123) and β3-278VKA280 (KK202) (Fig 5A). In cluster 1 (brown circle, Fig 5A and 5D), β3-L294 faces into the β (+) /α (−) intersubunit cleft, and G308/R309 is at the junction between the bottom of TM3 and the TM3–4 intracellular loop. G308/R309 is two α-helical turns below β3-F301 (i. e., toward the intracellular terminus of TM3), a residue previously photolabeled by 6-azi pregnanolone in β3 homomeric receptors [13]. These data support neurosteroid binding in the β (+) /α (−) interface, consistent with the canonical THDOC and pregnanolone binding sites identified in crystal structures of α1 (+) /α1 (−) interfaces in chimeric proteins [16] and in substituted cysteine modification protection studies of α1β2γ2 receptors [18]. The pattern of labeling also indicates that the A-ring of the steroid is oriented upwards in the intersubunit cleft toward the center of the membrane, the D-ring is pointing toward the intracellular termini of the TMDs, and the C5-C6-C7 edge of the steroid is pointing toward the β3 (+) side of the cleft. Cluster 1 corresponds to a β3 (+) /α1 (−) intersubunit site. In cluster 2 (red circle, Fig 5A and 5C), N408 and Y415 are both on the C-terminal end of α1-TM4, facing toward TM1 within the same α1 subunit, consistent with an α1 intrasubunit neurosteroid binding site. N408, the residue labeled by the C17-TPD of KK200, is two α-helical turns closer to the center of TM4 than is Y415, the residue labeled by the C6-diazirine of KK123. This labeling pattern suggests that neurosteroids orient in this site with the A-ring pointing toward the ECD and the D-ring facing to the center of the TMD. Cluster 2 corresponds to an α1 intrasubunit site. In cluster 3 (blue circle, Fig 5A and 5B), Y442 is located at the C-terminal end of β3-TM4, and 278VKA280 is located on the TM2–TM3 loop near the extracellular end of β3-TM3. The adjacency of these two photolabeling sites suggests an intrasubunit neurosteroid binding site at the extracellular end of β3, analogous to the α1 intrasubunit site. The labeling of 278VKA280 in the extracellular loop by the C3-TPD group of KK202 suggests that neurosteroids orient in this site with the A-ring facing the ECD. Cluster 3 corresponds to a β3 intrasubunit site. A homology model of the α1β3 GABAA receptor based on the structure of a β3 homomeric GABAA receptor (PDB 4COF) [47] was used to examine the preferred energetic poses of neurosteroid binding to the three binding sites. The homology model was embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer and the structure refined by molecular dynamics. We then docked each of the three photoaffinity labeling reagents as well as allopregnanolone to each of the proposed binding sites, using a time course series of snapshots from the simulation trajectory to account for receptor flexibility. All of the neurosteroid photolabeling reagents docked in the three sites; the identified sites are relatively shallow with respect to the protein–lipid interface. Moreover, the neurosteroid analogues were all found to adopt multiple poses in each of the sites with minimal energy differences between the poses (see Materials and methods). Photolabeling data combined with the docking scores (binding energy) and population of a given pose were used to guide selection of the preferred steroid orientation in each site. In the α1 intrasubunit site, the poses clustered between TM1 and TM4. The preferred pose (Fig 5C) for allopregnanolone (lowest energy cluster of poses) shows the A-ring oriented toward the ECD with the walls of the predicted binding site lined on one side by N408 and Y415 and on the other by V227. Docking of KK200 in this site has a similar orientation with the A-ring oriented toward the ECD and the TPD group on the D-ring proximal to N408 (Fig 6A). Docking of KK-123 shows a preferred pose in which the A-ring is oriented toward the ECD and the C6-diazirine proximal to Y415 (Fig 6B). These data elucidate a prior finding that mutations to N408 and Y411 eliminate potentiation by steroid analogues that lack a hydrogen bonding group on the D-ring [48]. In the β3 intrasubunit site, the poses are clustered between TM3 and TM4. Allopregnanolone preferred a pose with the A-ring oriented toward the ECD near Y442 and the D-ring proximal to V290 (Fig 5B). KK123 was found to dock at the top of the TM helices with the A-ring oriented toward the ECD, placing the 6-diazirine in proximity to Y442 (Fig 6C). KK202 was found to dock in a similar orientation but lower in the TM region, with the TPD group in proximity to A280 and Y442 (Fig 6D). In the intersubunit site, the preferred pose for allopregnanolone was one of the lowest energy clusters of poses with the A-ring proximal to α1-Q242 (equivalent to rat α1-Q241), the D-ring pointing toward the cytoplasmic termini of the TMDs, and the D-ring facing β3-F301 (Fig 5D). Docking of KK200 showed a similar orientation although shifted slightly upwards toward the ECD, placing the A-ring near α1-Q242, the benzene ring of the TPD near β3-F301, and the diazirine in proximity to G308 (Fig 6E). The preferred pose of KK202 was closer to TM3 of the β3 subunit with the A-ring near α1-Q242 and the D-ring near the β3-F301 placing the diazirine in proximity to β3-L294 (Fig 6F). The orientation of allopregnanolone docked in our α1β3 model is nearly identical to the orientation of THDOC in the crystal structure of α1-GLIC [16] (PDB 5OSB). As a confirmation of our docking, we also docked allopregnanolone to the apo-neurosteroid crystal structures of the α1-GLIC [16] and α5-β3 chimeric [17] proteins (PDB 5OSA and PDB 5OJM, respectively) (S5 Fig). The preferred poses for allopregnanolone in the β3-α1 intersubunit site are nearly identical between the three models. The preferred poses are also very similar in the α1 intrasubunit site between our α1β3 homology model and the structures of the α-homomeric TMDs. The A-ring/D-ring orientation of allopregnanolone in the three neurosteroid sites was consistent with the orientations identified by the photolabeling data in all of the GABAA receptor structures. The calculated binding energies from the docking studies (S4 Table) indicate that the rank order of allopregnanolone affinity for the three sites is β3/α1 intersubunit site > α1 intrasubunit site > β3 intrasubunit site. The β3-α1 intersubunit binding site identified in our photolabeling studies has been extensively validated by site-directed mutagenesis as a functionally important site. Mutations on the α (−) side of the interface, including Q241 (rat) L/W and W246 (rat) L, have been shown to eliminate neurosteroid potentiation and gating of α1β2γ2 GABAA receptors [14,27,49]; mutations on the β (+) side of the interface, including F301A and L297A, have also been shown to partially reduce neurosteroid effect [17]. In the current study, we showed that α1Q242Lβ3 prevented the action of allopregnanolone, KK123, and KK200 while reducing the effect of KK202 in α1β3 receptors, confirming the β–α interface as a functionally significant neurosteroid binding site and validating the relevance of our photolabeling reagents (Fig 1B). Based on computational simulation and docking results, we also identified residues in the proposed α1- and β3-intrasubunit binding sites that we predicted could be involved in allopregnanolone binding or action (S2 Table and S3 Table for all mutated subunits tested). N408 and Y411 in α1-TM4 line one side of the putative α1 intrasubunit site, and V227 in α1-TM1 lines the other (Fig 5C). α1N407 (rat) A and α1Y410 (rat) W mutations have previously been shown to prevent neurosteroid potentiation of GABA-elicited currents in α1β2γ2 GABAA receptors [15]. Our data confirm that the double mutant α1N408A/Y411Fβ3 substantially reduces allopregnanolone potentiation of GABA-elicited currents (Fig 4B and 4C1, ***p < 0. 001 versus α1β3 wild-type). Allopregnanolone (1 μM) potentiation of GABA-elicited currents and direct activation (10 μM) of α1V227Wβ3 receptors was also significantly reduced in comparison to α1β3 wild-type (*p < 0. 05 and **p < 0. 01; Fig 4B, 4C1 and 4C2). To test whether these mutations selectively affected neurosteroid actions, we also compared the effect of propofol in α1V227Wβ3 and α1N408A/Y411Fβ3 to its effect on wild-type α1β3 receptors. Propofol action was not different between the mutant and wild-type receptors, indicating a selective effect on neurosteroid action (Fig 4B, 4C3 and 4C4). The finding that multiple mutations lining the α1-intrasubunit binding pocket selectively reduce allopregnanolone action buttresses the evidence that the photolabeled residues identify a specific, functionally important neurosteroid binding site. Multiple mutations within the putative β3-intrasubunit binding site were also tested. However, none of the mutations significantly altered potentiation or activation by allopregnanolone (S2 Table and S3 Table for all of the mutations that were tested). These data suggest that allopregnanolone occupancy of the β3 intrasubunit site does not contribute to channel gating. Direct activation of α1β2Y284Fγ2 receptors by THDOC has previously been shown to be markedly reduced in comparison to wild-type receptors [15], although we found no significant effect of the β3-Y284 mutation in α1β3 receptors (S2 Table and S3 Table). The difference in results between experiments in α1β3 and α1β2γ2 GABAA receptors suggests possible receptor subtype specificity in the functional effects of neurosteroid binding at a β-intrasubunit site. Collectively, the photolabeling, modeling, and functional data indicate that heteropentameric α1β3 GABAA receptors contain at least seven binding sites for neurosteroids, of three different types. The use of multiple photolabeling reagents also enabled determination of the orientation of neurosteroids in each proposed class of sites. At least two of these classes are involved in producing the allosteric effect of steroids, the β3-α1 intersubunit site (two copies per receptor) and the α1 intrasubunit site (two copies). Mutations of residues in the proposed β3 intrasubunit site (three copies) had no effect on modulation by allopregnanolone although residues were labeled by two photolabeling reagents and labeling was prevented by excess allopregnanolone. Accordingly, the functional significance of this proposed site is not known. Previous, site-directed mutagenesis studies using electrophysiology readout identified multiple residues, including α1-Q241, N407, Y410, T236, and β3-Y284, that selectively contribute to the positive allosteric effects of neurosteroids [14,27]. Based on homology to the structure of the muscle nicotinic acetylcholine receptor [50], it was hypothesized that there are two neurosteroid binding sites on GABAA receptors: an α1-intrasubunit site spanning Q241 and N407 and an intersubunit site between β3-Y284 and α1-T236. Subsequent data [16–18] have clearly established the existence of a β–α intersubunit site. Our photolabeling experiments and homology modeling now show that the previously identified residues contribute to multiple distinct neurosteroid binding sites, albeit differently than originally proposed. It is noteworthy that the α1-intrasubunit site was not identified in the X-ray crystallographic structures of α1-GLIC chimeras bound with THDOC or the α5-β3 chimera bound with pregnanolone. This is likely because the proteins with steroid bound in the intrasubunit site did not form stable crystals. Mutations in either the β3-α1 intersubunit site or the α1-intrasubunit site can ablate both potentiation and direct activation by allopregnanolone, indicating that these are not distinct sites mediating potentiation and direct activation. The data also do not conform to simple energetic additivity for the two sites. The observation that mutations in either binding site can largely eliminate neurosteroid effect indicates that these two sites do not function completely independently and suggests allosteric interaction between the two sites. Development of site-selective neurosteroid analogues (PAMs and antagonists) should facilitate clarification of the mechanisms of allosteric interaction between these two sites. In light of the demonstration of multiple neurosteroid binding sites in α1β3 GABAA receptors, the possibility of additional isoform-specific sites must be considered. The strong sequence homology between the TMDs of the six α-subunits and three β-subunits suggests that there will not be large isoform differences in the intersubunit site [27]. In contrast, the contribution of ECD residues to the α- and β-intrasubunit sites suggests possible isoform-specific differences. The sequence homology between the γ and δ subunits and α and β subunits suggests that there may also be intrasubunit neurosteroid binding sites in these isoforms. Identification of a neurosteroid binding site on a δ-subunit would be of particular relevance because GABAA receptors containing δ-subunits are particularly sensitive to neurosteroids [51–53]. High-resolution, cryo-electron microscopy structures of α1β3γ2 GABAA receptors [54–56] have been published since initial submission of this work. The structural homology between γ2 subunits and α and β subunits suggests that there may also be intrasubunit neurosteroid binding sites in the γ2 subunit. The existence of multiple sites in which neurosteroids bind with different orientation may also offer some explanation for the difficulty in identifying neurosteroid antagonists [57] and for the differences in single-channel electrophysiological effects of various neurosteroid analogues [28,30]. The possibility of multiple isoform-specific sites with distinct patterns of neurosteroid affinity, binding orientation, and effect offers the exciting potential for the development of isoform-specific agonists, partial agonists, and antagonists with targeted therapeutic effects. The human α1 and β3 subunits were subcloned into pcDNA3 for molecular manipulations and cRNA synthesis. Using QuikChange mutagenesis (Agilent), a FLAG tag was first added to the α1 subunit then an 8xHis tag was added to generate the following His-FLAG tag tandem (QPSLHHHHHHHHDYKDDDDKDEL), inserted between the fourth and fifth residues of the mature peptide. The α1 and β3 subunits were then transferred into the pcDNA4/TO and pcDNA5/TO vectors (ThermoFisher Scientific, Waltham, MA), respectively, for tetracycline-inducible expression. For X. laevis oocytes, point mutations were generated using the QuikChange site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA) and the coding region fully sequenced prior to use. The cDNAs were linearized with Xba I (NEB Labs, Ipswich, MA), and the cRNAs were generated using T7 mMessage mMachine (Ambion, Austin, TX). The tetracycline-inducible cell line HEK T-RexTM-293 (ThermoFisher) was cultured under the following conditions: cells were maintained in DMEM/F-12 50/50 medium containing 10% fetal bovine serum (tetracycline-free, Takara, Mountain View, CA), penicillin (100 units/ml), streptomycin (100 g/ml), and blastcidine (2 μg/ml) in a humidified atmosphere containing 5% CO2. Cells were passaged twice each week, maintaining subconfluent cultures. Stably transfected cells were cultured as above with the addition of hygromycin (50 μg/ml) and Zeocin (20 μg/ml). A stable cell line was generated by transfecting HEK T-RexTM-293 cells with human α1-8x His-FLAG pcDNA4/TO and human β3 pcDNA5/TO in a 150 mm culture dish, using the Effectene transfection reagent (Qiagen). Two days after transfection, selection of stably transfected cells was performed with hygromycin and zeocin until distinct colonies appeared (usually after two weeks). Medium was exchanged several times each week to maintain antibiotic selection. Individual clones (about 65) were selected from the dish and transferred to 24-well plates for expansion of each clone selected. When the cells grew to a sufficient number, about 50% confluency, they were split into two other plates, one for a surface ELISA against the FLAG epitope and a second for protein assay, to normalize surface expression to cell number [58]. The best eight clones were selected for expansion into 150 mm dishes, followed by [3H]muscimol binding. Once the best expressing clone was determined, the highest-expressing cells of that clone were selected through fluorescence-activated cell sorting (FACS). FACS was done against the FLAG epitope, using a phycoerythrin (PE) -conjugated anti-FLAG antibody. Fluorescent-activated cells (1 ml containing about 10 million cells) were sorted on the AriaII cell sorter (Washington University Pathology Core), collecting 0. 5% of the highest-fluorescing cells in a culture tube containing complete medium. The cells were plated in a 35 mm dish and expanded until a near confluent 150 mm dish was obtained. Cells were enriched for expression by FACS three times. A final FACS was performed to select individual cells into a 96-well plate, which resulted in only 10 colonies of cells. These colonies were expanded and assayed for [3H]muscimol binding; the highest-expressing clone was used for experiments. Stably transfected cells were plated into fifty 150 mm dishes. After reaching 50% confluency, GABA receptors were expressed by inducing cells with 1 μg/ml of doxycycline with the addition of 5 mM sodium butyrate. Cells were harvested after 48 to 72 hours after induction. HEK cells, after tetracycline induction, grown to 70%–80% confluency, were washed with 10 mM sodium phosphate/proteinase inhibitors (Sigma-Aldrich, St. Louis, MO) two times and harvested with cell scrapers. The cells were washed with 10 mM sodium phosphate/proteinase inhibitors and collected by centrifugation at 1,000 g at 4°C for 5 minutes. The cells were homogenized with a glass mortar Teflon pestle for 10 strokes on ice. The pellet containing the membrane proteins was collected after centrifugation at 34,000 g at 4°C for 30 minutes and resuspended in a buffer containing 10 mM potassium phosphate and 100 mM KCl. The protein concentration was determined with micro-BCA protein assay and stored at −80°C. [3H]muscimol binding assays were performed using a previously described method with minor modification [59]. Briefly, HEK cell membranes proteins (50 μg/ml final concentration) were incubated with 1–2 nM [3H]muscimol (30 Ci/mmol; PerkinElmer Life Sciences), neurosteroid in different concentrations (1 nM-10 μM), binding buffer (10 mM potassium phosphate, 100 mM KCl [pH 7. 5]), in a total volume of 1 ml. Assay tubes were incubated for 1 hour at 4°C in the dark. Nonspecific binding was determined by binding in the presence of 1 mM GABA. Membranes were collected on Whatman/GF-C glass filter paper using a Brandel cell harvester (Gaithersburg, MD). To determine the Bmax of [3H]muscimol binding, 100 μg/ml of proteins were incubated with 250 nM [3H]muscimol, with specific activity reduced to 2 Ci/mmol, for 1 hour at 4°C in the dark. The membranes were collected on Whatman/GF-B glass filter papers using manifold. Radioactivity bound to the filters was measured by liquid scintillation spectrometry using Bio-Safe II (Research Products International Corporation). Each data point was determined in triplicate. For all the photolabeling experiments, 10–20 mg of HEK cell membrane proteins (about 300 pmol [3H]muscimol binding) were thawed and resuspended in buffer containing 10 mM potassium phosphate, 100 mM KCl (pH 7. 5) at a final concentration of 1. 25 mg/ml. For photolabeling site identification experiments, 15 μM neurosteroid photolabeling reagent was added to the membrane proteins and incubated on ice for 1 hour. For the photolabeling competition experiments, 3 μM neurosteroid photolabeling reagent in the presence of 30 μM allopregnanolone or the same volume of ethanol was added for incubation. The samples were then irradiated in a quartz cuvette for 5 minutes, by using a photoreactor emitting light at >320 nm [59]. The membrane proteins were then collected by centrifugation at 20,000 g for 45 minutes. All of the photolabeling experiments to identify sites of neurosteroid photolabeling were performed at least three times. The photolabeled peptides and residues described in the text were all observed in replicate experiments. The amount of 10 mg of KK123 or ZCM42 photolabeled HEK membrane proteins were solubilized in 1 ml 2% SDS/PBS and incubated at room temperature for 2 hours. The protein lysate was collected by centrifugation at 21,000 g for 30 minutes. FLI-tag was clicked to the KK123- or ZCM-photolabeled proteins at room temperature overnight in PBS buffer containing 2% SDS, 100 μM FLI-tag [35], 2. 5 mM sodium ascorbate, 250 μM Tris [ (1-benzyl-1H-1,2, 3triazol-4-yl) methyl]amine, and 2. 5 mM CuSO4. The amount of 1% Triton/PBS was added to the protein lysate to an SDS final concentration of 0. 05%. The protein lysate was loaded onto a streptavidin agarose column. The flow through was reloaded to the column two times or till the flow through was colorless and the streptavidin column was dark orange yellow. The column was washed with 10 ml 0. 05% Triton/PBS and eluted by 10 ml 100 mM sodium dithionite/0. 05%Triton/PBS. The column was turned into colorless after elutions. The eluted proteins were concentrated into 100 μl with 30 kDa cutoff Centricon apparatus. The supernatant of the Centricon tube was added into SDS-sample loading buffer, loaded to a 10% SDS-PAGE, and transferred to a PVDF membrane, followed by western blot with polyclonal rabbit anti-α1 raised against a peptide mapping within a cytoplasmic domain of human GABAR α1 subunit [60] (Santa Cruz Biotechnology) or monoclonal anti-β3 antibody against 370–433 of mouse GABAR β3 subunit [61] (NeuroMab). The photolabeled membrane proteins were resuspended in lysis buffer containing 1% DDM, 0. 25% cholesteryl hemisuccinate (CHS), 50 mM Tris (pH 7. 5), 150 mM NaCl, 2 mM CaCl2,5 mM KCl, 5 mM MgCl2,1 mM EDTA, and 10% glycerol at a final concentration of 1 mg/ml. The membrane protein suspension was homogenized using a Teflon pestle in a motor-driven homogenizer and incubated at 4°C overnight. The protein lysate was centrifuged at 20,000 g for 45 minutes, and supernatant was incubated with 0. 5 ml anti-FLAG agarose (Sigma) at 4°C for 2 hours. The anti-FLAG agarose was then transferred to an empty column, followed by washing with 20 ml washing buffer (50 mM triethylammonium bicarbonate and 0. 05% DDM). The GABAA receptors were eluted with ten 1-ml 200 μg/ml FLAG peptide and 100 μg/ml 3X FLAG (ApexBio) in the washing buffer. The 10 ml effective elutions containing GABAA receptors (tested by western blot with anti-α1 or anti-β3 antibody) were concentrated by 100 kDa cutoff Centricon filters into 0. 1 ml. The purified GABAA receptors (100 ul) were reduced by 5 mM tris (2-carboxyethyl) phosphine (TCEP) at for 30 minutes followed by alkylation with 7. 5 mM NEM for 1 hour in the dark. The NEM was quenched by 7. 5 mM DTT for 15 minutes. These three steps were done at room temperature. Eight μg of trypsin was added to the protein samples and incubated at 4°C for 7–10 days. The digest was terminated by adding formic acid (FA) in a final concentration of 1%. The samples were then analyzed by an OrbiTrap ELITE mass spectrometer (ThermoFisher) as in previous work [13,31] with some modifications. Briefly, a 20 μl aliquot was injected by an autosampler (Eksigent) at a flow rate of 800 nl/min onto a home-packed polymeric reverse phase PLRP-S column (Agilent, 12 cm × 75 μm, 300 Å). An acetonitrile (ACN) 10%–90% concentration gradient was applied in the flow rate of 800 nl/min for 145 minutes to separate peptides. Solvent A was 0. 1% FA/water, and solvent B was 0. 1%FA/ACN. The ACN gradient was as follows: isocratic elution at 10% solvent B, 1–60 minutes; 10%–90% solvent B, 60–125 minutes; 90% solvent B, 125–135 minutes; 90%–10% solvent B, 135–140 minutes; isocratic solvent B, 140–145 minutes. For the first 60 minutes, a built-in divert valve on the mass spectrometer was used to remove the hydrophilic contaminants from the mass spectrometer. The survey MS1 scans were acquired at acquired at high resolution (60,000 resolution) in the range of m/z = 100–2,000, and the fragmentation spectra were acquired at 15,000 resolution. Data-dependent acquisition of the top 20 MS1 precursors with exclusion of singly charged precursors was set for MS2 scans. Fragmentation was performed using collision-induced dissociation or high-energy dissociation with normalized energy of 35%. The data were acquired and reviewed with Xcalibur 2. 2 (ThermoFisher). The MS experiments of identification of the photolabeling sites and competition of photolabeling were replicated at least three times. The LC-MS data were searched against a customized database containing the sequence of the GABAA receptor 8X His-FLAG-α1 and β3 subunit and filtered with 1% false discovery rate using PEAKS 8. 5 (Bioinformatics Solutions Inc.). Search parameters were set for a precursor mass accuracy of 30 ppm, fragmentation ion accuracy of 0. 1 Da, up to three missed cleavage on either side of peptide with trypsin digestion. Methionine oxidation, cysteine alkylation with NEM and DTT, any amino acids with adduct of KK123 (mass = 372. 16), KK200 (mass = 462. 27), KK202 (mass = 500. 31), KK123 with light FLI-tag (mass = 672. 4322), and KK123 with heavy FLI-tag (mass = 682. 44) were included as variable modification. The GABAA receptors were expressed in oocytes from the African clawed frog (X. laevis). Frogs were purchased from Xenopus 1 (Dexter, MI) and housed and cared for in a Washington University Animal Care Facility under the supervision of the Washington University Division of Comparative Medicine. Harvesting of oocytes was conducted under the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. The animal protocol was approved by the Animal Studies Committee of Washington University in St. Louis (approval No. 20170071). The oocytes were injected with a total of 12 ng cRNA in 5: 1 ratio (α1: β3) to minimize the expression of β3 homomeric receptors. Following injection, the oocytes were incubated in ND96 with supplements (96 mM NaCl, 2 mM KCl, 1. 8 mM CaCl2,1 mM MgCl2,2. 5 mM Na pyruvate, 5 mM HEPES, and 100 U/ml + 100 μg/ml penicillin + streptomycin and 50 μg/ml gentamycin [pH 7. 4]) at 16°C for 1–2 days prior to conducting electrophysiological recordings. The electrophysiological recordings were conducted using standard two-electrode voltage clamp. Borosilicate capillary glass tubing (G120F-4, OD = 1. 20 mm, ID = 0. 69 mm; Warner Instruments, Hamden, CT) were used for voltage and current electrodes. The oocytes were clamped at −60 mV. The chamber (RC-1Z; Warner Instruments, Hamden, CT) was perfused with ND96 at 5–8 ml min−1. Solutions were gravity-applied from 30-ml glass syringes with glass luer slips via Teflon tubing. The current responses were amplified with an OC-725C amplifier (Warner Instruments), digitized with a Digidata 1200 series digitizer (Molecular Devices) and were stored using pClamp (Molecular Devices). The peak amplitude was determined using Clampfit (Molecular Devices). The stock solution of GABA was made in ND96 bath solution at 500 mM, stored in aliquots at −20°C, and diluted as needed on the day of experiment. Stock solution of propofol (200 mM in DMSO) was stored at room temperature. The steroids were dissolved in DMSO at 10 mM and stored at room temperature. The α1β3 wild-type and mutant receptors were tested (see Table 1 and S2 and S3 Tables) for potentiation by steroids (3α5α-allopregnanolone, 3α5β-pregnanolone, KK123, KK200, and KK-202) and direct activation by steroids (allopregnanolone KK123, KK200, KK-202, and pregnanolone). As control, several receptor isoforms were tested for potentiation by propofol. For each receptor type, we also determined constitutive open probability (Po, const). To estimate Po, const, the effect of 100 μM picrotoxin (estimated Po = 0) on the holding current was compared to the peak response to saturating GABA + 100 μM propofol (estimated Po = 1). Po, const was then calculated as Ipicrotoxin ÷ (Ipicrotoxin − IGABA+propofol) [62]. Potentiation is expressed as the potentiation response ratio, calculated as the ratio of the peak response to GABA + modulator (steroid or propofol) to the peak response to GABA alone. The concentration of GABA was selected to produce a response of 5%–15% of the response to saturating GABA + 100 μM propofol. Direct activation by steroids was evaluated by comparing the peak response to 10 μM neurosteroid to the peak response to saturating GABA + 100 μM propofol. Direct activation by steroids is expressed in units of open probability that includes constitutive open probability. All data are given as mean ± SD and analyzed by one-way ANOVA followed by Dunnet’s multiple comparison to the control wild-type group. A homology model of the α1β3 GABAA receptor was developed using the crystal structure of the human β3 homopentamer published in 2014 (PDB ID: 4COF) [47]. In this structure, the large cytoplasmic loops were replaced with the sequence SQPARAA used by Jansen and colleagues [63] The pentamer subunits were organized as A α1, B β3, C α1, D β3, E β3. The α1 sequence was aligned to the β3 sequence using the program MUSCLE [64]. The pentameric alignment was then used as input for the program Modeller [65], using 4COF as the template; a total of 25 models were generated. The best model as evaluated by the DOPE score [66] was then oriented into a POPC membrane, and the system was fully solvated with 40715 TIP3 water molecules and ionic strength set to 0. 15 M KCl. A 100 ns molecular dynamics trajectory was then obtained using the CHARMM36 force field and NAMD. The resulting trajectory was then processed using the utility mdtraj [67], to extract a snapshot of the receptor at each nanosecond of time frame. These structures were then mutually aligned by fitting the alpha carbons, providing a set of 100 mutually aligned structures used for docking studies. The docking was performed using AutoDock Vina [68] on each of the 100 snapshots in order to capture the receptor flexibility. Docking boxes were built for the β3 intrasubunit site (cluster 3), the α1 intrasubunit site (cluster 2), and the β3-α1 intersubunit site (cluster 1). The boxes were centered around the residues photolabeled by KK123, KK200, and KK202 and had dimensions of 25 × 25 × 25 Ångströms, large enough to easily fit the linear dimensions of all of the steroids. For docking studies of allopregnanolone, the docking boxes were placed in the same locations but had smaller dimensions of 20 × 20 × 20 Ångströms. Docking was limited to an energy range of 3 kcal from the best docking pose and was limited to a total of 20 unique poses. The docking results for a given site could result in a maximum of 2,000 unique poses (20 poses × 100 receptor structures); these were then clustered geometrically using the program DIVCF [69]. The resulting clusters were then ranked by Vina score and cluster size and visually analyzed for compatibility with the photolabeling results, which is the photolabeling group oriented in the correct direction to produce the observed photo adducts. The inorganic salts used in the buffers, GABA, picrotoxin, and the steroids 3α, 5α-allopregnanolone, and 3α, 5β-pregnanolone were purchased from Sigma-Aldrich. Propofol was purchased from MP Biomedicals (Solon).

Neurosteroids are cholesterol metabolites produced by neurons and glial cells that participate in central nervous system (CNS) development, regulate neuronal excitability, and modulate complex behaviors such as mood. Exogenously administered neurosteroid analogues are effective sedative hypnotics and are being developed as antidepressants and anticonvulsants. Gamma amino-butyric acid Type A (GABAA) receptors, the principal ionotropic inhibitory neurotransmitter receptors in the brain, are the primary functional target of neurosteroids. Understanding the molecular details of neurosteroid interactions with GABAA receptors is critical to understanding their mechanism of action and developing specific and effective therapeutic agents. In the current study, we developed a suite of neurosteroid analogue affinity labeling reagents, which we used to identify three distinct binding sites on GABAA receptors and to determine the orientation of neurosteroid binding in each site. Electrophysiological studies performed on receptors with mutations designed to disrupt the identified binding sites showed that two of the three sites contribute to neurosteroid modulation of GABAA currents. The distinct patterns of neurosteroid affinity, binding orientation, and effect provide the potential for the development of isoform-specific agonists, partial agonists, and antagonists with targeted therapeutic effects.

lay_plos

Produced by: Wendy Knoller Final check by Kim [Scene: Central Perk. Everybody's sitting on the couch and Monica is eating a chunk of cake.] Monica: (really excited) Mmh... this cake is amazing! Rachel: My God, get a room! Monica: I would get a room with this cake. I think I could show this cake a good time! Phoebe: If you had to, what would you give up, food or s*x? Monica: (with no hesitation) s*x! Chandler: (looking at her) Seriously, answer faster! Monica: Oh, I'm sorry honey, you know, but when she said "s*x" I wasn't thinking about "s*x with you"! Chandler: (to Phoebe) It's like a giant hug. Phoebe: Ross, how about you. What would you give up, s*x or food? Ross: Food. Phoebe: Ok, how about... uhm... s*x or dinosaurs? Ross: Oh my God. It's like Sophie's Choice. Rachel: Oh God. What about you, Joe? What would you give up, s*x or food? Joey: Uhm... oh... I don't know, it's too hard. Rachel: No, you gotta pick one! Joey: Oh... food. No, s*x. Food! s*x! Food! Se-I don't know! Good God, I don't know, I want girls on bread! OPENING CREDITS [Scene: Central Perk. Rachel and Phoebe are looking at some photos and they're sitting next to the window.] Rachel: You gotta see these latest pictures of Emma. Phoebe: Oh, how cute! Rachel: Yeah. Phoebe: Oh, she looks just like a little doll! Rachel: Oh, no, no. That is a doll. Phoebe: Oh, thank God, 'cause that thing's really creepy! (looking outside the window) Look, there's Chandler. (he's on the street, talking to a woman) Rachel: Oh. Who is the blonde, she's pretty. Phoebe: OH! He's having an affair. Rachel: He's not having an affair! Phoebe: You know, I'm always right about these things. Rachel: No, you're not! Last week you thought Ross was trying to kill you! Phoebe: Well, I'm sorry but it's hard to believe that anyone would tell a story that dull just to tell it! (looking outside) See, there's something going on with them. Look, he's getting into the car with her! Rachel: Oh, that doesn't mean anything. Phoebe: Oh yeah? Well, let's see. (she takes her mobile phone) Ok, duck down. (they both get down to hide themselves. Phoebe calls Chandler) Chandler: (picking up the phone) Hello. Phoebe: Oh, hi Chandler. It's Phoebe. Uhm... I know that Monica is working today so...(back to Central Perk) ...I was wondering if you want to come to the movies with me and Rachel. Chandler: Oh, uhm... I have to work too. Yeah, I'm stuck at the office all day. Phoebe: (shocked) Oh, well, it's a shame that you-that you miss the movie 'cause we were gonna see, you know, either "Liar, Liar" or "Betrayal", or... "An Affair To Remember". Chandler: Those are all really old! Phoebe: Ok, then maybe it'll be, uhm... Rachel: (whispering) "Dude, Where's My Car?" Phoebe: (glancing at her) What? Rachel: They're in a caaar... Phoebe: (to Chandler at the phone) Okay, we-we'll talk to you later. Okay, bye. Rachel: Geez! Phoebe: Ok. Quick. We gotta find a cab and follow them. Rachel: Oh, yeah, ok. Let me just grab my night vision goggles and my stun gun. Phoebe: (patting her bag) I got them! [Scene: Monica's apartment. Chandler enters the door.] Chandler: Hi! Monica: Hey! You smell like perfume and cigarettes. Chandler: I was in the car with Nancy all day. Monica: Nancy doesn't smoke! Chandler: Well, at least the perfume is not mine, be thankful for that! Monica: So? What do you think of the house? Chandler: It's perfect. It's everything we've been looking for. Monica: Isn't it? Then what about the amazing wainscotting and the crown molding and the dormer windows in the attic? Chandler: And the wiggle wharms and the zip zorps? (pause) What were the things you said? Monica: Don't you love the huge yard? Chandler: And the fireplace in the bedroom. Monica: And Nancy said that it's really under price, because the guy lost his job and has to move in with his parents! Chandler: This is bringing out a lovely color in you! Monica: So? Do you think we should get it? Chandler: I don't know. What do you think? Monica: I think we should. Chandler: I do too. Monica: This is huge! Chandler: I know. Monica: How bad you wanna smoke, right now. Chandler: I don't know what you mean, giant talking cigarette! Oh, by the way, Phoebe called just as I was getting into Nancy's car, so if she asks you, I was at work all day. Monica: Gotcha. When do we tell them about this? Chandler: We don't. Not until it's a hundred percent. I mean, why upset everybody over nothing. Monica: Okay. Right. Oh my God that is gonna be so hard. Chandler: I know. Gooooood luck with it. [Scene: Joey's apartment. Everybody except Monica and Chandler is there.] Ross: I just can't see Chandler cheating! Rachel: I'm telling you guys, we followed them out to a house in Westchester, the went in for like forty-five minutes and then they came out looking pretty happy! Joey: Chandler? Forty-five minutes? Well, something is not right. I just can't believe he would do this to Monica! Ross: I know, and with the baby coming? Phoebe: So, should we tell her? Ross: I don't know. Phoebe, if one of us saw Mike with another woman would you want us to tell you? Phoebe: Why? Who'd you seen him with? Ross: No one, I'm just saying if... (Phoebe starts pinching him in his neck) Phoebe: TELL ME WHAT YOU KNOW! Ross: (yelling in pain) I know nothing! Mike's a great guy, it was hypothetical! Phoebe: All right. (she releases him). He is a good guy. You're right, he wouldn't cheat. Ross: Believe me, if I did see with someone, there's no way I... (Phoebe starts pinching him again) Phoebe : WHO DID YOU SEE HIM WITH? [Scene: Monica's apartment. Monica is cleaning with a vacuum and then she cleans it with a dust buster. The guys enter the room.] Rachel: Oh, look at her, so happy! Monica: If only there were a smaller one to clean this one! Joey: Hey, is uhm... is Chandler here? Monica: No, he's picking up dinner, why, what's up? Phoebe: Well, look, whatever happens, we're here for you and we love you. Monica (puzzled): All right... Ross: We think Chandler might be having an affair. Monica: What? Rachel: Phoebe and I saw Chandler with a blonde woman today outside on the street and then we followed them to a house in Westchester. Phoebe: They went in together. So sorry. Monica: Oh my God! Oh my God that's awful! What did you think of the house? (they all look confused and sorry for her) Phoebe: What? Joey: (walking towards her to hold her and support her) Monica, you understand what we are saying, right? Monica: Yeah, sure... uhm, I'm devastated, obviously... (to the rest) Did you think the neighborhood was homey? (Chandler enters) Chandler: Hey! Joey: (to Chandler) You son of a bitch! Chandler: Is it me, or have the greetings gone downhill around here? Monica: (goes to Chandler) Phoebe and Rachel saw you with Nancy today and... em... they think you're having an affair. Rachel: Who's Nancy? Ross: What's going on? Monica: (turns to them) Ok, alright, you guys, you'd better sit down, this is pretty big. Chandler: Yeah (motions them to sit and they do) I'm not having an affair. Nancy is our realtor. Joey: I knew he couldn't be with a woman for 45 minutes!! Phoebe: Why do you have a realtor? Monica: Uhm, she has been showing us houses outside of the city. Joey: (clearly shocked) What? Rachel: Are you serious? Monica: When we found out that we're gonna get this baby, Chandler and I started talking and we decided that we didn't want to raise a kid in the city. Phoebe: So you're gonna move? Ross: Oh my God. Joey: Shouldn't we all vote on stuff like this?! Rachel: What is wrong with raising a kid in the city? I'm doing it, Ross is doing it, Sarah Jessica Parker is doing it! Monica: And that's great for you guys, but we want a lawn and a swingset... Chandler: ...and a street where our kids can ride their bikes and maybe an ice-cream truck can go by. Ross: (sarcastic) So you wanna buy a house in the 50's? Phoebe: Have you thought about what you would be giving up? You can't move out of the city, what if you want Chinese food at 5am? Or a fake Rolex that breaks as soon as it rains or an Asian hooker sent right to your door? Ross: You know what, if you wanna look for a house, that's okay. Joey: No, no, it's not, don't listen to him! (to Ross) I'm gonna thump you! (points his fist at him) Ross: (to Joey) It's ok, because they have to get it out of their system, okay (back to Mon and Chan), but you're going to realize, this is the only place, you wanna be. (pause before Monica and Chandler speak, they look like they are looking for the right words) Chandler: Actually, we already found a house we love. Ross: What? Monica: And about an hour ago, we made an offer. (All the friends looked shocked and confused. There is a long silence.) Chandler: Bet you wish I was having an affair now, huh? TIME LAPSE Ross: You put an offer on a house? Monica: (smiling) It's so sweet. It really is. It has this big yard that leads down to this stream and then there's these old maple trees... (gets cut off) Phoebe: Wha..? Again with the nature, what are you? Beavers? Chandler: I know this is really hard and we're really sorry. Joey: Is this because I come over here without knocking and eat your food? (Walks towards the fridge) Because I can stop doing that, (looks at the fridge) I really, really think I can! Chandler: (goes towards Joey) You know that's not the reason Joe. (Joey hugs him and after, he takes something from the fridge and puts it in his mouth. He goes back to where he was standing before) Monica: We think if you saw it, you'd understand. I mean you guys were there. (Points to Rachel and Phoebe) It is beautiful, isn't it? Rachel: Yeah it is. Joey: What the hell are you doin'? Rachel: Well, it is, all right? When we were out there today, all I kept thinking was: I can't believe Chandler is screwing this woman, but MAN this would be a nice place to live! Phoebe: Yeah, but so is this. Ross: Yeah, I mean, if you moved there, you have to leave here. I mean, how can you leave this place? [We fade to some flashback scenes.] (from 1.01 - "The One Where Monica Gets a New Roommate - The Pilot") Rachel: (talking on the phone) C'mon Daddy, listen to me! All of my life, everyone has always told me, 'You're a shoe! You're a shoe, you're a shoe, you're a shoe!'. And today I just stopped and I said, 'What if I don't wanna be a shoe? What if I wanna be a- a purse, y'know? Or a- or a hat! No, I don't want you to buy me a hat, I'm saying that I am a ha- It's a metaphor, Daddy! Ross: You can see where he'd have trouble. Rachel: Well maybe I'll just stay here with Monica. Monica: Well, I guess we've established who's staying here with Monica... (from 1.18 - "The One With All the Poker") Ross: That money is mine, Green! Rachel: You're fly is open, Geller! Phoebe: You guys, you know what I just realized? 'Joker' is 'poker' with a 'J.' Coincidence? Chandler: Hey, that's...'joincidence' with a 'C'! (from 1.07 - "The One With The Blackout") Phoebe: [looking outside the window] Eww, look. Ugly Naked Guy lit a bunch of candles. [They all look at the window, grossed out, then flinch in pain.] Rachel: Ow, that had to hurt! (from 3.09 - "The One With All the Football") Phoebe: Hey, it's your Thanksgiving too, y'know, instead of watching football, you could help. The Guys: We will. (they don't move) Monica: Okay, Rachel, you wanna put the marshmallows in concentric circles. Rachel: No Mon, you want to put them in concentric circles. I want to do this. (Rachel sticks a marshmallow into Monica's nose. Monica takes it out of her nose by closing one nostril, and blowing.) Monica: Every year. (from 5.08 - "The One With the Thanksgiving Flashbacks") Joey: (he has a turkey on his head) It's stuck!!! Phoebe: (walks him to the kitchen) Easy. Step. How did it get on? Joey: I put it on to scare Chandler! Phoebe: Oh my God! Monica's gonna totally freak out! Joey: It smells really bad in here. Phoebe: Well, of course it smells really bad. You have your head inside a turkey's ass! (They hear Monica trying to unlock the door. So Phoebe quickly pushes his head down onto the table to make it look like the turkey is just sitting on a platter and not stuck on Joey's head.) Monica: Hey, did you get the turkey bast-Oh my God! Oh my God! (She sees someone is stuck in the turkey.) Who is that? Joey: It's Joey. (from 4.12 - "The one With the Embryos") Monica: I got it! How about, if we win, they have to get rid of the rooster? Rachel: Oooohh that's interesting. Chandler: If you win, we give up the birds. Joey: (shocked) Dah!! (Chandler motions for him to calm down.) Chandler: But if we win, we get your apartment. Joey: Oooooh! Monica: Deal! TIME LAPSE Ross: What was Monica's nickname when she was a field hockey goalie? Joey: Big fat goalie. Ross: Correct. Rachel claims this is her favorite movie... Chandler: Dangerous Liaisons. Ross: Correct. Her actual favorite movie is... Joey: Weekend at Bernie's. Ross: Monica categorizes her towels. How many categories are there? (They both confer) Joey: Everyday use. Chandler: Fancy. Joey: Guest. Chandler: Fancy guest. Ross: Two seconds... Joey: Uhh, 11! Ross: 11, unbelievable, 11 is correct. (The guys celebrate.) Ross: (to the girls) Chandler was how old when he first touched a girl's breast? Rachel: 14? Ross: No, 19. Chandler: Thanks man. Ross: Joey had an imaginary childhood friend. His name was? Monica: Maurice. Ross: Correct, his profession was? Rachel: Space cowboy! Ross: Correct! What is Chandler Bing's job? (The girls are stumped) Rachel: Ow...Oh Gosh! Ross: 10 seconds, you need this or you lose the game. Monica: It's umm, it has something to do with transponding. Rachel: Oh-oh-oh, he's a transponce-transpondster! Monica: That's not even a word! (Ross stops the clock, signifying the end of the lightning round.) Monica: NOOOOOOOOO!!!!! TIME LAPSE (The door opens and Joey and Chandler ride in on the big, fake dog in triumph) Rachel: Y'know what, you are mean boys, who are just being mean! Joey: Hey, don't get mad at us! No one forced you to raise the stakes! Rachel: That is not true. She did! She forced me! Monica: Hey, we would still be living here if hadn't gotten the question wrong! Rachel: Well it stupid, unfair question! Ross: Don't blame the questions! Chandler: Would you all stop yelling in our apartment! You are ruining moving day for us! [SCENE_BREAK] (from 5.15 - "The One With The Girl Who Hits Joey") Ross: Chandler!!! Chandler!!! (He opens the door to the apartment but is stopped by the chain; Chandler and Monica quickly stop making out and try to get dressed.) Chandler, I saw what you were doing through the window! Chandler, I saw what you were doing to my sister! Now get out here! Chandler: (To Monica) Wow! Listen, we had a good run. You know, what was it? Four? Five months? I mean, that's more than most people have in a lifetime! So, good-bye, take care, bye-bye then! (He kisses her and starts to climb out the balcony window) Monica: (She opens the door.) Hey Ross. What's up bro? (Ross spots Chandler and starts chasing him around the kitchen table. Chandler runs and hides behind Monica.) Ross: What the hell are doing?!! Rachel: (running from the guy's apartment with Joey in tow) Hey, what's-what's going on?! Chandler: Well, I think, I think Ross knows about me and Monica. Joey: (panicking) Dude! He's right there! Ross: (To Chandler) I thought you were my best friend, this is my sister! My best friend and my sister! I-I cannot believe this! Chandler: Look, we're not just messing around! I love her. Okay, I'm in love with her. Monica: I'm so sorry that you had to find out this way. I'm sorry, but iit-t's true, I love him too. (There's a brief pause.) Ross: (happily) My best friend and my sister! I cannot believe this. (He hugs them both.) (from 6.06 - "The One On The Last Night") Monica: Well, this is the last box of your clothes. I'm just gonna label it, "What were you thinking?" Rachel: Funny, because I was just gonna go across the hall and write that on Chandler. Phoebe: Ok, you guys, I don't mean to make things worse, but umm, I don't want to live with Rachel anymore. Monica and Rachel: What?! Phoebe: You're just so mean to each other! And I don't want to end up like that with Rachel. I still like you! Rachel: Well, Phoebe that's fine because I'm not moving. Monica: Whoa-whoa-whoa, Phoebe you gotta take her! Y'know, I-I-I said some really bad stuff about her, but y'know Rachel has some good qualities that make her a good roommate. She gets tons of catalogs and umm, she'll fold down the pages of the things she thinks that I'd like. Phoebe: What else? Monica: When I take a shower, she leaves me little notes on the mirror. Rachel: Yeah, I do. I-I do, do that. Phoebe: That's nice. I like having things to read in the bathroom. Monica: When I fall asleep on the couch after reading, she covers me over with a blanket. Rachel: Well y'know, I don't want you to be cold. Monica: And when I told her that I was gonna be moving in with Chandler, she was really supportive. (To Rachel) (Starts to cry) You were so great. You made it so easy. And now you have to leave. And I have to live with a boy!! (They both break down in tears.) TIME LAPSE (Monica closes the door and slowly walks into Rachel's old and now empty room.) Chandler: (entering) Hey. Monica: She really left. Chandler: I know. (He kisses her.) Monica: Thank you. Chandler: No problem roomie. (She turns around and hugs him.) Monica: Can I ask you a question? Chandler: Sure! Monica: What the hell is that dog doing here?! (She notices the dog sitting in the living room.) (from 1.09 - "The One Where Underdog Gets Away") Chandler: Little toast here. I know this isn't exactly the kind of Thanksgiving that all of you all planned, but for me, this has been really great, you know, I think because it didn't involve divorce or projectile vomiting. Anyway, I was just thinking, I mean, if you'd gone to Vail, and if you guys'd been with your family, if you didn't have syphilis and stuff, we wouldn't be all together, you know? So I guess what I'm trying to say is that I'm very thankful that all of your Thanksgivings sucked. All: That's so sweet. Ross: And hey, here's to a lousy Christmas. Rachel: And a crappy New Year. Chandler: Here, here! [Scene: We're back to the present. Chandler and Monica's. They're all still at the kitchen table.] Rachel: You can't move. You just... you just can't. Joey: Rachel's right. This is where you guys belong. Phoebe: Yeah, you don't wanna live in Westchester. That's like the worst of the Chesters. Ross: You know, sometimes when I'm alone in my apartment, I look over here and you guys... are just having dinner or... watching TV or something, but... it makes me feel better. And now when I look over, who am I gonna see? The Gottliebs, the Yangs? They don't make me feel so good. (Joey pats Ross on his back) Rachel: Yeah. So don't move, okay? Just stay here and... (nods towards Ross) maybe close your blinds at night. (The phone rings and Chandler goes to get it) Chandler: Hello? It's Nancy, they responded to our offer. Monica: And? (Chandler listens to what Nancy says) Chandler: (to Nancy) Okay, thanks... (to Monica) They passed. They said they wouldn't go a penny under the asking price. Monica: We can't afford that. Chandler: I know. Monica: Well, there you go. (Chandler and Monica hug) Joey: I'm really sorry you guys. Ross: Yeah. I'm sorry too. I'm even more sorry that that phone call didn't come before I told you about looking through the window. Rachel: Yeah, we're gonna let you be alone. Phoebe: (to Monica) You're gonna be okay? Monica: Yeah, we'll be okay. Ross: Love you guys. (he kisses Monica, he, Rachel and Phoebe leave.) Joey: You know, I'm really sorry I wasn't more supportive before. Chandler: That's okay, we understand. Joey: And about this Nancy thing... If you're not sleeping with her, should I? (Chandler gives Joey her business card, which he eagerly grabs and he leaves.) Monica: I know there'll be other houses, but it's just so... I love that one so much. Chandler: Yeah... Well, it's a good thing we got it then. Monica: What? Chandler: We got the house. Monica: Oh my God! Chandler: I just didn't want to tell you in front of them. Monica: Oh my God! My God! We've got the house !? Chandler: We're getting the house. (they hug) We're getting the house. Monica: And a baby... Chandler: We're growing up. Monica: We sure are. Chandler: So who's gonna tell them? Monica: (quickly) Not it! Chandler: Not it! Damn it! [SCENE_BREAK] [Scene: Central Perk. The entire gang is there, and Chandler and Monica are handing out presents.] Monica: Rachel, this is yours. Rachel: Aah! Why? What are these for? Chandler: You'll see. Monica: All right, everybody open them! (they all tear off the wrapping paper) Rachel: Ooh! Oh wow this is so beautiful. (she got a scarf) Phoebe: Oh! These are the ones I was looking at in the store. (she got earrings) Monica: I know. Ross: I love this. (he got a sweater) Joey: A meatball Sub? Thanks! (he got a meatball sandwich) Ross: Seriously you guys, what's going on? What are these for? Chandler: Well, I didn't know how to tell you before, but... We got the house. Monica: Enjoy! (they both run off, leaving Ross, Phoebe and Rachel stunned.) Joey: (speaking with his mouth full, enjoying his sandwich) What did they say?

Rachel and Phoebe see Chandler getting into a car with another woman. He dodges their questions by pretending to be at work but they follow him to the suburbs and find them entering a house. They believe he is cheating on Monica and tell Ross and Joey. The four of them tell Monica but Monica doesn't react the way they thought she would. It soon comes out they are looking to buy a house in the suburbs to raise their family (the woman having been their realtor). The group tries to convince them to stay in the city while reminiscing about the past few years. In the end Monica and Chandler get the house and tell everyone they are moving right after giving them gifts.

summ_screen_fd

[EXT. LAS VEGAS (STOCK) -- NIGHT] [SCENE_BREAK] [INT. DEVON HOTEL -- LOBBY -- NIGHT] (A MAN and A WOMAN walk through the lobby, their arms around each other.) Man: You're going to like my room. You can see the entire strip from my balcony ... and the bed. (He turns her around, her back against the wall. Her hands come up against his chest.) Woman: (nervously) I usually don't do this. I-I mean, especially with somebody that I just met. Man: Yeah? Well, I could tell that just by looking at you. (The MAN presses the elevator door button. The elevator bell dings.) Woman: Uh ... Man: Come on, don't worry, all right? You can trust me. Okay? Woman: Promise? Man: I never lie. (The MAN starts to kiss the WOMAN. He backs her up into the elevator.) Woman: You are so bad. Man: Yeah, I am. (He lifts her up and carries her into the elevator. He presses her against the elevator wall and starts kissing her. After a moment, the woman looks down and sees the dead body on the floor ... ) [SCENE_BREAK] [INT. DEVON HOTEL -- NIGHT] (SARA and GRISSOM arrive at the scene. They duck under the crime scene tape and walk toward BRASS who is already there.) Sara: Hey. Dispatch said suspicious circs. Brass: Yeah. We got an anonymous call to 9-1-1 at the same time the couple found him. A male voice said: "A man has collapsed at the Devon. Hurry." Grissom: Where's the man? (GRISSOM looks at the empty elevator. There are two cones on the floor ... and no body.) Brass: Oh, he's unconscious, but breathing. So paramedics took him to Desert Palm Hospital. There was no bullet wounds, no knife ... nothing. Sara: You got a name? Brass: Bob Fairmont. Upscale home developer. Sara: "A Fairmont Home, You'll Never Roam?" Those billboards are everywhere. Brass: Well, they took these pictures before they moved him. (BRASS hands GRISSOM a stack of photos. GRISSOM puts his kit down and looks through the photos.) Grissom: Well, this is as phony as a chappaquiddick neck brace. See how the clothes are all bunched. Sara: Collar's off to the side, leg fabric's gathered. Grissom: It's impossible to redress an unconscious person to make it look like they dressed themselves. You notice anything about the suit coat? (GRISSOM shows the photo to BRASS. BRASS takes it and looks at it.) Brass: Well, unless he's going to court or to church, there's no way he buttons all three buttons. (BRASS gives the photo back to GRISSOM.) Grissom: Very good, Jim. (to SARA) Why do they think they can fool us? HARD CUT TO BLACK. END OF TEASER. ROLL TITLE CREDITS. [SCENE_BREAK] [INT. DEVON HOTEL - LOBBY -- NIGHT] (SARA takes pictures of the elevator. GRISSOM studies the photos.) Brass: I'm going to go to the hospital to talk to Fairmont's wife. (SARA continues to take pictures.) Grissom: Call me if you learn anything. (BRASS leaves. NICK walks up to them.) Nick: Hey, guys. I just pulled the manager out of a restaurant. Bob Fairmont was staying in room 2927. Catherine's parking the car. Grissom: Let's go up. Sara: We'll meet you there. Nick: Okay. [SCENE_BREAK] [INT. DEVON HOTEL - HALLWAY -- NIGHT] (GRISSOM walks toward room 2927. In front of them is the door to the stairs. NICK, SARA and CATHERINE follow. They all stop in front of the closed room door.) Grissom: Mr. Fairmont was staying in Murder Central. Catherine: (agrees) Mmm. Nick: "Murder Central"? Sara: You never heard that phrase? Nick: (exasperated) Well, if I did, would I have asked the question? (SARA looks smug as she got a reaction from NICK. Grissom: Sara, you're with me in here. (to CATHERINE and NICK) You guys get the elevator. Catherine: All yours, girl. Sara: Thanks. (GRISSOM unlocks the hotel room door. He and SARA enter. CATHERINE and NICK walk back down the hallway.) Nick: (disgruntled) Sara doesn't know what Murder Central means any more than I do. Catherine: (puts a hand on NICK'S shoulder) Oh, Nick, give it up. We got a death imminent to worry about. [SCENE_BREAK] [EXT. HOSPITAL EMERGENCY ROOM -- NIGHT] (BRASS questions JULIA FAIRMONT) Julia Fairmont: My husband's in surgery. Can't this wait? Brass: I'm sorry, no. He was found under suspicious circumstances. Do you have any idea who may have redressed him or moved him? Julia Fairmont: (shakes her head) He usually goes with women who look a lot like me ... only younger. And I'm embarrassed to say that I've been flattered by that, at times. Brass: Do you have a name? Julia Fairmont: I was very much on the outside of that part of his life. Brass: I see. (BRASS puts his notebook back in his jacket pocket.) [SCENE_BREAK] [INT. DEVON HOTEL - ROOM 2927 -- NIGHT] (Standing side-by-side, GRISSOM and SARA look at the room in front of them. They're silent as they look around the room.) Sara: I know. The room's talking to us. They had champagne. (SARA walks into the room, GRISSOM remains by the door.) Sara: They were celebrating something. (She looks at the glasses.) Sara: No lipstick on either glass. 9-1-1 did say it was a man's voice that placed the call. Grissom: Smell the musk? Hint of bleach? (GRISSOM looks up at the mirror over the bed. He notices that the bed is mussed.) Grissom: Sexual intercourse. (SARA turns around to look at GRISSOM.) Sara: They kept the drapes open. A married man who's not worried about ... photographs, long lenses ... Grissom: Well, he's either careless or arrogant, maybe. Sara: Or he has a death wish. (SARA looks down and picks up a bra.) 34 C? If he was with a woman, who was the guy on the 911 call? [SCENE_BREAK] [INT. DEVON HOTEL - ELEVATOR -- NIGHT] (CATHERINE and NICK work on the elevator.) Catherine: Hand me that magnifier. (NICK hands her the magnifier.) Thank you. Nick: What do you got? Catherine: See those white specks? (Camera zooms for a close up of the white specks. Resume to present.) Nick: What do you think, cocaine? Catherine: No, I don't think so. Nick: How can you tell just by looking? Catherine: Never you mind. (CATHERINE takes a tape lift of the white specks.) Catherine: Let's just get this to trace. [SCENE_BREAK] [INT. DEVON HOTEL - ROOM 2927] (NICK opens the hotel room door and walks inside. GRISSOM and SARA are just putting on their goggles and taking out the ALS.) Nick: Okay, we're done in the elevator. You guys need a hand? Grissom: Yeah, you want to dust the champagne bottle? Sara: The average American hotel room is covered with stains invisible to the naked eye. Grissom: Yeah, but they're not all biological. Some are soda stains, food stains, whiskey stains, you know. Sara: No matter how clean or expensive the room seems that why always travel with nonoxinol nine. (They stand up.) Grissom: You sound like you're making a commercial. Nick, hit the lights, will you? (NICK turns the lights off.) Grissom: Okay, we're looking for the freshest stains. Sara: Soda, maybe ... maybe champagne ... oh! Someone's little soldiers ... more champagne. (They look up at the walls.) Grissom: Hmm ... "starlight, star bright, first star I see tonight." (GRISSOM takes off his goggles and heads for the door.) Grissom: Okay, I'll let you guys do the collection. Nick: Thanks a lot. (GRISSOM turns around.) Grissom: Oh, and Sara? Sara: Hmm. Grissom: Last hotel room nearest the stairwell -- easy entry and egress for an intruder and if the victim fights back, fifty percent less chance of being heard. Sara: Rooms only on one side. Murder Central. (GRISSOM nods his head and leaves the room. The door closes behind him.) Nick: (smiling) You are so busted. (SARA turns around to look at NICK. She smiles.) [SCENE_BREAK] [EXT. LAS VEGAS CITY (STOCK) - NIGHT] [SCENE_BREAK] [INT. CSI - DNA LAB -- NIGHT] (Camera close up of a scope view of the "white specks" found on the elevator floor. The magnification view is increased. Camera turns around and goes back up the viewer to the eye looking through the scope. End of scope view.) Catherine: I gave this to trace. Why do you have Greg: Well, because I deal in DNA. The smallest sliver of epidermal tissue. Catherine: This is skin. Greg: Scalp skin. Itchy. Can be embarrassing in social situations especially if one is wearing a dark shirt. Catherine: Dandruff. (GREG reaches out and brushes non-existent dandruff from CATHERINE'S shoulder.) Greg: Good chance whoever moved the guy into the elevator ... Catherine: Had a bad case of seborrhea. (CATHERINE deliberately leans forward and brushes the non-existent dandruff from GREG'S shoulder.) Catherine: Thanks. (CATHERINE turns to leave the lab. GREG stops her.) Greg: Hey, Catherine? Do you think Sara would ever go out with me? Catherine: (thinks about it) Sure. (deadpans) As long as you don't tell her it's a date. (CATHERINE leaves the lab.) [SCENE_BREAK] [INT. CSI - HALLWAY - NIGHT -- CONTINUOUS] (CATHERINE exits the DNA lab and walks down the hallway. WARRICK catches up with her.) Warrick: Hey, Cath... are you on this Fairmont case? Catherine: House mogul caught with his pants on? Yeah, I'm just rolling to the hospital to get his clothes. Why? Warrick: You know the Fairmont house was one of my first calls three years ago? Catherine: You serious? For what? Warrick: Shots fired. He shot himself while he was cleaning his gun. Catherine: Wow! Warrick: Yeah. And after hearing about tonight I'm wondering if that's what really happened. I'm going to pull his file and check it out. Catherine: Let me know. Warrick: I will. [SCENE_BREAK] [INT. DEVON HOTEL - ROOM 2927] (NICK picks up the used condom from the fixture on the wall.) Nick: Nice shot. (looks inside) Reservoir's still wet. (NICK bags it.) Sara: Why did he throw it? Nick: Mmm. I'm only impressed if he aimed. Sara: I wonder if the woman has any idea she left her DNA behind? Nick: Not rocket science. Man's inside, woman's out. [SCENE_BREAK] [INT. CSI -- HALLWAY] (BRASS fills GRISSOM in about the 911 tape.) Brass: Wife says the guy's a player. She doesn't know the women. Grissom: You tell her about the 911 tape? Brass: The male voice? Oh, I just got back from central dispatch. The tape's lost. New computer system. Grissom: Any ear-witnesses? Brass: The dispatcher says it could've been a woman whispering. Or a very old person, gender indeterminate. Grissom: That's about as good as an eyewitness. (GRISSOM'S phone rings. He answers it.) Grissom: Grissom. (pause) What? (pause) I'll be right there. (He hangs up and looks at BRASS.) Grissom: No lunch. (GRISSOM turns around and heads back down the hallway.) [SCENE_BREAK] SCENE #13 [INT. CSI - FORENSIC AUTOPSY] (ROBBINS goes over the findings with GRISSOM.) Grissom: We got a time? Robbins: Well, all indications are he was brain-dead from the time he collapsed at the hotel Grissom: From? (Quick CGI POV to: A camera close up of the deceased's forehead. White flash to a view of the brain where blood starts to leak out.) Robbins: (V.O.) An artery out-pouches blood floods the subarachnoid space. (End of CGI POV. Resume to present.) Robbins: Heart still beats, but the brain's dead. And it doesn't regenerate. Grissom: Aneurysm from trauma ... infection...? Robbins: Genetic predisposition. Both parents went that way. It increases a person's chance of stroke ten-fold. He looks like an athlete. Grissom: Brain dead at 38, otherwise healthy and strong. Is this a Frankenstein? Robbins: I was wondering when you'd ask. Prime donor candidate. Next of kin signed off. Grissom: How many organs they take? Robbins: Eight in under two hours. Grissom: Man, those harvest doctors move, don't they? (Quick flashback to: The heart monitor beeps. The doctor puts the first organ in the pan.) Doctor: Kidneys, stat! DOCTOR: Go, go, go! (Various flashes of the organs being removed and of various machinery monitoring the donor.) Doctor: Here comes the liver. Ready with a basin, please. Nurse: For ... DOCTOR: Right now. DOCTOR: And here we go! (The second organ goes into the pan marked: "Liver".) Doctor: Clamp it. DOCTOR: Get it back, get it back. DOCTOR: Ready and got it. DOCTOR: Here we go. (The final organ is placed in the pan. End of flashback. Resume to present.) Grissom: (nods) All right, I'll notify the PD about the organs. (GRISSOM leaves the room.) [SCENE_BREAK] [INT. CSI - DNA LAB] (GREG takes a swab from the condom. SARA reads through some papers as she waits.) Greg: So I was thinking. Maybe we could take our break at the same time. (beat) Later this shift. (beat) Together. Sara: Sure. Greg: (surprised) Really. (GREG tests the swab. SARA leans in to look.) Sara: Semen. No surprise there. Greg: Well, without the DNA sample from the hotel guy this test is pretty useless. (SARA goes back to looking at the photos.) Sara: I'm more interested ... in who the woman is. Greg: Well, just like the bra, I'm going to need a reference of her DNA in order to do anything, and that's not going to happen until ... (SARA rifles through the autopsy photos. She sees something.) Sara: What the heck? Greg: What? Sara: (looking up) Stripes. (Without further explanation, SARA walks out of the lab.) Greg: Stripes. [SCENE_BREAK] [INT. CSI - HALLWAY] (SARA and GRISSOM walk through the hallway. SARA shows GRISSOM the photos.) Sara: Transverse white bands that appear six weeks after the onset of symptoms of toxicity. Grissom: You got a closer view? (SARA hands GRISSOM the photo she was looking at. Camera zooms in to the fingernails to show the white stripes.) Grissom: Uh ... Sara: They look like mees lines to me. Grissom: Could be livor mortis. Sara: Or white striae, indicative of heavy metal poisoning. Grissom: We can't count on a photo to draw that kind of conclusion. We need the body. [SCENE_BREAK] [INT. CSI - FORENSIC AUTOPSY] (SARA, GRISSOM and ROBBINS look at the empty table.) Sara: Don't take this wrong, Dr. Robbins, but ... how do you release a body that's been redressed and dumped in an elevator? (ROBBINS pushes the table back into the cabinet.) Robbins: I deal with cause of death, which was an aneurysm and therefore, a natural, and based on that his body is released to a mortuary. (ROBBINS sits down at the computer monitor.) Sara: The whole transplant thing didn't raise a flag? Grissom: I thought I'd raised a flag with the good doctor when I told him I was going to notify the police department but evidently, I mis-communicated again. Sara: Great. (ROBBINS enters the search in the computer database.) (The results are as follows: [Name: FAIRMONT, ROBERT Cause of Death: Natural Date: 12/13/01 Time of Death: 4:00 a.m. Case #: 29574 Destination: Desert Haven Mortuary ] Robbins: All may not be lost. Here... body of Robert Fairmont released at 0400 hours to the Desert Haven Mortuary. Sara: I know that place. Grissom: Well, if they haven't embalmed him we can still get a blood sample test for heavy metal poisoning. Sara: Good. I'll drive. (SARA heads for the door. She turns around when she realizes that GRISSOM isn't moving.) Sara: You're not coming? Grissom: (looks up) You found it, you run with it. (SARA seems surprised.) Grissom: You can do it. Take Nick. Sara: Okay. (SARA leaves.) [SCENE_BREAK] [INT. DESERT HAVEN MORTUARY] (FURNACE POV: SARA looks in through the window. Her face shows her shock and surprise.) Sara: How long's he been in here? Randy Gesek: 92 minutes at 1,600 degrees. At, exactly, was it that you wanted to see? (SARA turns around to look at RANDY GESEK.) Nick: His fingernails. Randy Gesek: I'm very sorry. Nick: Who approved this cremation? (RANDY looks at his clipboard and sighs.) Randy Gesek: His wife, Julia Fairmont. Sara: Same person who approved the organ transplants. (RANDY nods. SARA turns to look at the furnace.) [SCENE_BREAK] [INT. CSI -- LAB] (Camera opens on the evidence bag of ROBERT "BOB" FAIRMONT'S ashes. The bag is dated 12/6. Case # 81585171. Labeled: "Ashes of Bob Fairmont" and "DOB 2-25- (SARA picks up a piece of ash and places it in a mortar. She grounds it with a pestle.) [SCENE_BREAK] [INT. POLICE DEPARTMENT - WAITING ROOM] (BRASS and SARA JULIA FAIRMONT.) Brass: You told me your husband was in surgery. Julia Fairmont: He was. Brass: Surgery to take his organs not to save his life. Julia Fairmont: He was technically ... dead. He wanted to donate his organs. I was following his wishes. Why are you being so hostile? Brass: Someone was poisoning your husband. Julia Fairmont: What? Sara: We don't know the dose or the duration but we do know the type of poison. We processed your husband's remains. (Quick series of flashes to match SARA'S narration. SARA in the lab taking the piece of ash. SARA testing a sample of the ash in the analyzer. SARA pressing the button to start the machine.) (Machine test results read as follows for "Alignment Mode Lamp 1": [Name: Se-02-196.1-lib3 Pulse: Wide Lamp: Se Buck sci Bkgnd: Off Wavelength: 196.0- Slit: 0.2 Pnt: 304.9V Energy: 2.652 ] Sara: (V.O.) Most poisons would be completely burned off by cremation but heavy metals are very resistant to heat. This heavy metal -- selenium. (White flash to the fire in the furnace. Printer prints results. End of flashback. Resume to present.) Sara: Did you notice that your husband's breath might have been garlicky? Julia Fairmont: Garlicky? Sara: The body will excrete demethyl selenide -- smells just like garlic. Julia Fairmont: Uh... no. Sara: The white stripes on his fingernails--did you notice those? Julia Fairmont: Um ... we didn't see each other a lot. Bob was busy building his company. Sara: That bother you, him never being home? (JULIA FAIRMONT looks at BRASS.) Julia Fairmont: I think it would bother any woman. Sara: Did you know that the most common choice of premeditated murder among women is poison? They cite its passivity. Julia Fairmont: I did not poison my husband. Sara: You cremated his body. Julia Fairmont: He wanted to be cremated. I cremated him. He wanted to donate his organs. I did that, too. Now, if there's nothing else, I will be at home preparing for my husband's memorial service. Excuse me. (Angry, JULIA FAIRMONT grabs her jacket and stands up to leave. SARA stops her.) Sara: Do you want us to notify you? (SARA and JULIA turn around to look at each other.) Julia Fairmont: Of what? (SARA stands up and walks toward JULIA FAIRMONT.) Sara: When we find out the exact amount of selenium given to your husband and over what period of time? Julia Fairmont: I thought you couldn't tell. Sara: Your husband's gone, but his organs are still out there. Julia Fairmont: (nods) Well, good. Yes, I would like to be notified. (SARA turns to look at BRASS.) [SCENE_BREAK] [INT. HOSPITAL] (NICK stands behind the physician as they explain the situation to the PATIENT, CARL MERCER, in the bed.) Carl Mercer: Straight up, am I going to die? Physician: You have only a small fraction of the poison that was in your donor's body--one organ. With nothing new coming in it should work its way out. Carl Mercer: I must've had 100 tests. How'd you doctors miss this? Nick: A heavy metal like selenium presents so rarely that it's Carl Mercer: You don't look like a doctor. Nick: (smiles) No, sir, no, I'm not. I'm Nick Stokes. I'm with the Las Vegas Crime Lab. Physician: I'll be right outside. Nick: Thank you, doctor. (The doctor leaves. NICK steps up toward the bed.) Nick: I was hoping I might be able to get a sample of that new kidney of yours. Carl Mercer: A sample? Nick: Biopsy. A sliver. Carl Mercer: You mean open me up again? Nick: Maybe an invasive scope. The only way I can get an accurate barometer of the poison in Mr. Fairmont's system is through one of his organs. Carl Mercer: I'm sorry, young man but this is my kidney now. (He shakes his head.) I waited four years. I can't have anyone cut into me for it. I just can't. Nick: Hey, I understand. Thank you for your time. (NICK glances down at the watch on the railing.) Nick: I like your watch, man, that's cool. Carl Mercer: It broke while I was down in surgery--stuck on 11:00. Think the Lord's trying to tell me something? (NICK chuckles.) Nick: Yeah, get it to a watch repair shop. (pause) I hope you get to feeling better, Mr. Mercer. (NICK leaves the room.) [SCENE_BREAK] [EXT. LAS VEGAS CITY (STOCK) - NIGHT] [SCENE_BREAK] [INT. CSI -- HALLWAY] (SARA updates GRISSOM.) Sara: Nick struck out on the kidney. I got four "no's" over the phone. One yes from a recipient in Illinois-- heart. Grissom: Useless-- heart has no memory for poison. Sara: Why can't we cut the middle man and just check the wife's house for selenium? (GRISSOM looks at SARA.) All right, I know, something about constitutional law. Get probable cause, then get a search warrant ... (GREG walks on into the hallway, directly in front of SARA and GRISSOM.) Greg: Sara! I was just looking for you. Still up for break? Sara: Sorry, Greggo-- hot case. I'm going to go look at Nick's champagne bottle. How you doing with our DNA? Greg: Uh ... inside-outside we're still looking for a reference for comparison but the epidermals are looking promising. Sara: Nice. (SARA leaves.) Grissom: You want to clue me in? Greg: Sara and I were just going out for dinner. Grissom: On the case, "Greggo". Greg: Oh. Right. (GREG turns back in to his lab. GRISSOM follows.) [SCENE_BREAK] [INT. HOSPITAL] (CLAUDIA GIDEON walks in and heads for the Nurse's station. CATHERINE is standing next to the station, waiting.) Claudia Gideon: Excuse me ... I'm Claudia Gideon, Bob Fairmont's secretary. I came to pick up his property. Nurse: Oh, I'm sorry, but Ms. Willows already asked for them. Catherine: His property is part of our investigation. I'm afraid I have to take it. Claudia Gideon: Oh, fine. I was just doing my job. Catherine: Yeah, me, too. Did you work for the Fairmonts three years ago, when Mr. Fairmont was shot? Claudia Gideon: I was out sick that day. Catherine: Interesting. (CATHERINE sees something on CLAUDIA'S sweater.) Catherine: You have dandruff. (CATHERINE turns to get tape from her kit.) Claudia Gideon: Is that really proper etiquette? Catherine: It is when I'm on a case. Do you mind? (CATHERINE takes a tape lift.) Claudia Gideon: Where did you say you worked? [SCENE_BREAK] [INT. POLICE DEPARTMENT - INTERVIEW ROOM] (BRASS questions CLAUDIA GIDEON.) Brass: I'm going to ask you again -- were you in that hotel room with Bob Fairmont? (CATHERINE enters the room and closes the door.) Claudia Gideon: I was not, no. Catherine: But you did redress him and place him in the elevator. Claudia Gideon: You don't know what you're talking about. Catherine: Well, that's dangerous to say to a scientist. (CATHERINE puts her file on the table.) Catherine: Dandruff is nothing more than sloughed-off skin. It has a nucleus just like a cell on your arm or your big toe. (CATHERINE puts the test results and photo in front of CLAUDIA GIDEON.) Catherine: These 13 DNA markers from this dandruff cell are a match to the dandruff that I recovered from your sweater. You were there. At that hotel. Claudia Gideon: Okay, Mr. Fairmont told me earlier that day to meet him in his hotel room at 9:00 to pick up some papers, okay? When I got there, he was in bed -- naked, (Quick flashback to: CLAUDIA GIDEON finds BOB FAIRMONT in bed naked. She looks shocked.) Claudia Gideon: (V.O.) ... unconscious. (CLAUDIA moves and picks up his clothes.) Claudia Gideon: (V.O.) He always told me reputation before health -- no scandals. I dragged him to the elevator. (CLAUDIA drags BOB FAIRMONT into the elevator. She leaves him there. The elevator doors close. End of flashback. Resume to present.) Catherine: Let me guess -- you snuck out down the stairwell. (She nods.) Brass: Who was Fairmont partying with in that room? Claudia Gideon: I don't know. Catherine: Well, we won't mind if we compare your DNA to the vaginal contribution of a condom that we recovered from Mr. Fairmont's room? Claudia Gideon: (nods) Sure, go ahead. Catherine: Good. We'd like to show you something else -- about that accidental shooting three years ago at your boss' house. [SCENE_BREAK] [INT. CSI - OFFICE] (On the monitor is a computer reinactment of the first shooting years ago. The green figure is sitting down at a table when the gun falls down and shoots the victim in the groin.) Warrick: In this reinactment, Fairmont shot himself while cleaning his gun? In this reenactment the story just doesn't add up. I think I fell for it because I was new and I wasn't too eager to talk to another guy about him almost shooting off his manhood. (BRASS sighs. WARRICK straightens the monitor.) Warrick: This is the room as it was then based off of my crime scene photos. This is crime scene reconstruction, new school. Works backward, reverse time. (On the monitor it shows a different scenario with the green figure standing in the foyer.) Warrick: The bullet hit here. (The screen backs up showing a red figure (the shooter) sitting in a chair and shooting the green figure (the victim) in the groin.) Warrick: The only logical place for that bullet to have been shot from is four meters away, one meter high. He didn't actually shoot himself. Someone shot him. Catherine: We're thinking it was you. Brass: Admission form from the ER, 9/6/98. The person who brought Fairmont in on the accidental shooting... was you. (BRASS puts a copy of the Record of Admissions Form from the Desert Palms Hospital in front of her. The application Information reads as follows: [Name: FAIRMONT, BOB Local Residence - Street: 41733 Calle Matria City - Town: Las Vegas State: NV County: Clark Date Admitted: 9/6/98 Time Admitted: 10:32 Patient Accompanied by: CLAUDIA GIDEON Address: 311 Sephill Rd, Las Vegas, NV 89109 Attending Physician: Dr. Tommy Liu Address: 826 Herrick Ln, Las Vegas, NV 89109 Alternate Physician: Dr. Sheryl Aragon Address: 311 Sephill Rd, Las Vegas, NV 89108 Next of kin or representative/Relationship: Tyson Green Brother-in-law Address: 493 Fairmark, Las Vegas, NV 89108 Person to notify in Emergency/Relationship: Tyson Green Brother-in-law Address: Same as Above ] Claudia Gideon: I didn't shoot him. Brass: You didn't poison him, either. All these bad things just happen to you. Claudia Gideon: I'm on call 24 hours a day, okay? Mr. Fairmont beeped me. I drove out to the house, and I took him in. Catherine: Where was his wife? (CLAUDIA shakes her head.) Claudia Gideon: You'd have to ask her. [SCENE_BREAK] [INT. CSI - INTERVIEW ROOM] (CATHERINE, WARRICK and BRASS requestion JULIA FAIRMONT.) Julia Fairmont: Yes, I shot him. He was supposed to go riding with me and he didn't show up till two in the morning. No calls, nothing. I was ... hurt. Catherine: So, you just sat there in the dark and aimed south of his belt. Julia Fairmont: I just wanted to scare him. (Quick flashback to: BOB FAIRMONT returns home and finds his wife angry and holding a gun on him.) Bob Fairmont: It was a business meeting. BOB FAIRMONT: I swear. BOB FAIRMONT: I-it wasn't like before. BOB FAIRMONT: I was thinking about you the whole time. (She shoots. He grunts from the impact. End of flashback. Resume to present.) Brass: So, why didn't you tell the truth then? Mrs. Fairmont: I wanted to, but Bob wouldn't let me. He said that it would ruin his career -- Fairmont Family Estates. Warrick: I'm presenting this case to the D.A., even if it is three years old. Claudia: My husband's dead. We had our problems but I loved him I don't ... I don't much care what you do now. Brass: Well, there's only two reasons a woman shoots a man. She either loves him or hates him. Catherine: Or both. [SCENE_BREAK] [INT. CSI - DNA LAB] (GRISSOM looks through the scope at the dandruff sample.) Greg: That's the sample Catherine pulled off of the secretary's blouse and the hotel man's clothes -- same source. Grissom: Secretary moved the body. Catherine: We just interviewed her. She copped to moving him. We're looking hard at the wife on the poisoning. Grissom: The secretary has dandruff. Catherine: We know. Dermatomycoses seborrhea -- we got it. Grissom: Pical eczemas like that require prescription medication. The primary ingredient of those medications -- selenium sulfide. (CATHERINE sighs at missing the obvious. She heads out the lab.) [SCENE_BREAK] [INT. FAIRMONT RESIDENCE -- NIGHT] (BRASS and CATHERINE are on the porch talking with JULIA FAIRMONT.) Julia Fairmont: I don't really feel right about doing this. Brass: Oh, this is your residence. Everything in Claudia's office is your property. (CATHERINE and BRASS step inside.) Catherine: She could be back from dinner at any moment. This shouldn't take long. (The door closes behind them. CATHERINE and BRASS walk into the office and start looking around. JULIA FAIRMONT waits by the door. CATHERINE opens the cabinet and finds a large brown bottle in the back. She picks it up and looks at the label.) (The label reads: [Pharmacy / 493 Fairmark Ave., Las Vegas,NV 89107 CLAUDIA GIDEON Fill Date: 7/21/01 Phone: (702) 555-0150 Rx No: 4529975 Dr: Doty "USE AS NEEDED FOR SEBORRHEA" NIZORAL A-D 24 OZ DATE: 11/07/01 DISCARD AFTER: REFILL: 1 BY: 11/7/02 ] (The front door slams shut. CLAUDIA GIDEON walks in carrying a storage box.) Claudia Gideon: That's mine. Brass: Could you tell us why you keep your shampoo in your office? Catherine: Shampoo full of the same poison found in Bob Fairmont's remains? Claudia Gideon: I don't know. Brass: Aw, you have to do better than that. Claudia Gideon: Maybe I pied it up at the pharmacy on lunch and put it there. I don't know. (CATHERINE opens the refrigerator and looks inside. She finds a small tub of garlic cream cheese.) Catherine: Cream cheese. (CLAUDIA scoffs.) Catherine: Garlic cream cheese ... Brass: Hides the bitter aftertaste. (Quick flashback to: BOB FAIRMONT eating a bagel loaded with cream cheese. End of flashback. Resume to present.) Catherine: It explains away his bad breath. (Quick flashback to: BOB FAIRMONT finishing the bagel with cream cheese and laughing. End of flashback. Resume to present.) Claudia Gideon: I did not poison him. (The cabinet door closes.) Catherine: We'll see. Julia Fairmont: Aren't you going to arrest her? (CLAUDIA scoffs.) Claudia Gideon: I haven't done anything. Julia Fairmont: Other than poison Bob. Claudia Gideon: Poison? I'm the one who looked out for him. Julia Fairmont: You? I ran his home. I entertained his clients. Claudia Gideon: Who made all of his dental appointments, huh? His haircuts? Who got his taxes in on time? Julia Fairmont: You covered for his mistresses ... Claudia Gideon: Why did he need mistresses? Brass: Ladies ... don't go there. Catherine: This is yours ... and these are yours. (CATHERINE hands each woman a key.) There's the door. We build a case before we make any arrests. [SCENE_BREAK] [INT. HOSPITAL] (NICK walks into CARL MERCER'S hospital room.) Nick: Hey, Mr. Mercer. I got your message. You wanted to talk to me? Carl Mercer: Did you find out who poisoned Mr. Fairmont? Nick: No. No, sir. ,I have found selenium in his office but... without actually knowing how much was in his system ... Carl Mercer: What if I could tell you? My body's rejecting the organ. Doctor says it's got nothing to do with poison. It's antibodies. Nick: I'm sorry. Carl Mercer: I told you this watch stopped while I was down in surgery? It's funny ... it's like it knew all along the kidney wasn't going to take. Anyway... there's one last good thing I can do before I leave this earth. So ... how do we do this? (NICK nods. Camera holds on CARL MERCER.) [SCENE_BREAK] [INT. CSI - GRISSOM'S OFFICE] (NICK talks to GRISSOM about CARL MERCER.) Nick: I can't even talk Warrick into splitting a sandwich with me and this guy's willing to give us his kidney? Grissom: You asked for it. Nick: Yeah, that's my point. Carl Mercer risks dying sooner to help our investigation but who protects his rights? Grissom: He has free will. Nick: Well, so do I. I want to retract our request. I don't think any investigation for the dead is worth hurting the living. (GRISSOM turns around. He doesn't say anything.) Nick: What? (SARA calls out to them from the doorway.) Sara: Guys? You're not going to believe this. [SCENE_BREAK] [INT. CSI - PRINT LAB] (SARA runs the print through the database.) Sara: Funny sometimes it is the simplest things that seems so difficult. I've been trying to match the partial from the champagne bottle to these known prints. It was upside down. (The computer beeps.) Grissom: Must've grabbed the bottle ... (Quick flashback to: BOB FAIRMONT in bed with someone. She reaches over and grabs the champagne bottle.) Grissom: ... upside-down to pour. (End of flashback. Resume to present.) (On the monitor, it shows that a match was found. The prints match up. GRISSOM looks at SARA.) Grissom: So ... who was he with? Sara: Mrs. Fairmont. Nick: (surprised) What? Player was in the hotel cheating with his own wife? [SCENE_BREAK] [INT. POLICE DEPARTMENT - INTERVIEW ROOM] (BRASS re-interviews JULIA FAIRMONT.) Julia Fairmont: I was trying to ... rekindle our marriage. I ... got the room ... brought the champagne. (Quick flashback to: BOB FAIRMONT in bed with his wife.) Bob Fairmont: (moaning) Oh, yes. Yes. (End of flashback. Resume to present.) Julia Fairmont: He squeezed me in between business meetings at the hotel. Brass: And you didn't think to tell me that when we first talked? (In the observation room, GRISSOM, SARA and NICK listen in on the questioning.) Julia Fairmont: No, because you asked me about him being redressed in an elevator ... and, besides, when I left him, he was ... alive watching the news. (Quick flashback to: JULIA kisses BOB FAIRMONT and leaves. The news can be heard on the television. End of flashback. Resume to present.) Brass: Oh, so, he had a stroke alone, after you left. (Quick flashback to: BOB FAIRMONT puts a hand to his forehead and collapses back on the bed. End of flashback. Resume to present.) Julia Fairmont: I'm not a doctor. I don't know when he had it. Brass: Well, CSI is testing that champagne bottle for selenium. Julia Fairmont: I didn't think poison caused strokes. Brass: You seem to know a lot about poison for not being a doctor. Julia Fairmont: I know a lot of things about a lot of things, but that doesn't mean that I killed my husband. [SCENE_BREAK] [OBSERVATION ROOM] (Upon hearing this, GRISSOM looks at SARA.) Grissom: Yeah ... but what never lies? [SCENE_BREAK] [INT CSI - EVIDENCE ROOM] (GRISSOM sifts through BOB FAIRMONT'S ashes. SARA sits on the table next to him and looks at the file.) Sara: Fairmont had three gold crowns. They should've survived the cremation. Grissom: At least partially. Heavy metal, after all. Sara: His wife ... had them removed at the mortuary. That adds new meaning to the phrase "gold digger." Grissom: Actually, that's how the phrase got started. People used to dig up the bodies to extract the gold. Sara: Now they just marry them. In some countries, this would be enough to have her arrested for murdering her husband. Grissom: What's the most important component in a poisoning. (Hmmm ... trick question. SARA thinks about it.) Sara: Poison. (GRISSOM looks up at SARA.) Grissom: Patience. (SARA smiles and shakes her head.) [SCENE_BREAK] [EXT. POLICE DEPARTMENT - PARKING LOT -- NIGHT] (JULIA FAIRMONT makes her way to her parked car. She staggers a little and appears to have difficulty walking. She makes it to her car and tries to unlock the door. NICK sees her duress and calls out to her.) Nick: Ms. Fairmont ... Ms. Fairmont, you feeling okay? (She waves him away.) Julia Fairmont: Yeah, I'm, I'm ... (JULIA doubles over in pain. NICK rushes over to help her.) Nick: Whoa, whoa, whoa, whoa, whoa, whoa. Julia Fairmont: Hey ... maybe I just need to sit down... for a minute. Nick: Yeah, yeah, let's sit down here in the car for a sec. (NICK opens the door and helps JULIA FAIRMONT sit inside.) Nick: There you go. Relax, relax, relax. (NICK takes out his cell phone and makes a call.) Nick: yeah, I need an ambulance. Uh, Vegas PD, west parking lot. [SCENE_BREAK] [INT. CSI -- OFFICE] (GREG is on the computer. NICK walks in through the hallway. He stops when he sees GREG at the computer, pauses for a moment, then walks by.) (GREG does a web search for "Julia Fairmont". He finds 900 matches.) (He narrows the search to "Julia Fairmont" in "Nevada". He finds 4 matches.) [SCENE_BREAK] [INT. CSI -- HALLWAY] (NICK continues through the hallway. He meets up with CATHERINE and GRISSOM.) Catherine: Hey, what's going on? We called the hospital. She's been released. Nick: ER doc gave her a shot of hydropazeen ... sent her on her way. Grissom: Hydropazeen? That's an antiemetic used to counteract heavy metal poisoning. Nick: Which means ... somebody's been poisoning our suspect. [SCENE_BREAK] [INT. POLICE DEPARTMENT] (BRASS questions CLAUDIA GIDEON.) (CLAUDIA GIDEON shakes her head: No.) Claudia Gideon: I don't know how it happened. Maybe she poisoned herself. Brass: Why would she do that? Claudia Gideon: (scoffs) Why would she hide my shampoo in my office? Brass: What, you know she did that? Claudia Gideon: Well, it wasn't her husband. He was already dead. She's trying to frame me for killing him. Brass: I mean, do you have anything beyond your suspicions that she did kill him? Claudia Gideon: She had his gold fillings removed. Doesn't that tell you something about her? [SCENE_BREAK] [INT. POLICE DEPARTMENT - INTERVIEW ROOM] (BRASS questions JULIA FAIRMONT.) Julia Fairmont: I had his watch and ring removed, too. Do you want to see them? They're in a box at home. Brass: You realize that doesn't look good? Julia Fairmont: It can look however it wants. I have nothing to hide. Brass: You mean since you yourself have been poisoned? Julia Fairmont: Yeah, why aren't you asking Claudia Richards about that? First my husband, then me? Brass: Why would she want to kill either one of you? Julia Fairmont: I don't know. Maybe because he refused ... to leave me for her. [SCENE_BREAK] [INT. POLICE DEPARMTENT - INTERVIEW ROOM] (BRASS questions CLAUDIA GIDEON.) Claudia Gideon: I had to wash his grungy coffee mug every night. I had to clean his toenail clippings off of his desktop. Why would I ever ask Bob Fairmont to leave anyone for me? Brass: So, you think it was all in Mrs. Fairmont's head? Claudia Gideon: He always said she was crazy. Brass: (scoffs) Okay, that's it. All right ... look, we're not leaving here till we get to the bottom of this. Claudia Gideon: Then I hope she talks soon. [SCENE_BREAK] [EXT. LAS VEGAS CITY (STOCK) - NIGHT] [SCENE_BREAK] [INT. CSI -- OFFICE] (SARA walks by the offices. She looks and sees GREG will working on the computer. She walks into the office.) Sara: Greg? Greg: (smiles) Hey. I was just printing something out for you ... on your hot case. (SARA steps inside and looks at the monitor.) Sara: From the internet? Greg: Yeah, I ... I was... on break, had some time ... thought ... hey ... (GREG gives SARA the print out of his finds. SARA looks at it.) Greg: Sorry, I drew a blank on the wife. (SARA leans in toward GREG.) Sara: (thrilled) I could really, really just kiss you right now. (Smiling, GREG bashfully turns his head away from SARA. SARA leaves the office. He sighs. When he turns back, SARA'S gone.) [SCENE_BREAK] [INT. CSI -- HALLWAY] (SARA shows GRISSOM the information. GRISSOM looks at a photo of "Claudia and John Gideon".) Grissom: So, this is Claudia. Sara: The secretary, ten years ago her husband, John Gideon. (Across the hallway, CLAUDIA GIDEON takes a drink from the water fountain. GRISSOM and SARA turn around to look at her. CLAUDIA GIDEON smiles back at them. She leaves.) Sara: Claudia's rich husband also died young-- 35. (GRISSOM looks down at the Obituary for "John Gideon, dies at 35".) Grissom: She donated his organs and cremated his body. Sara: We don't have to chase down these organs. Ralph Parks rejected the organ that he got from Claudia's husband. Grissom: An organ with a memory, maybe? Sara: The most. Liver. (SARA show GRISSOM the report. It reads with the following information: BLOOD TYPE: A-POS Rh: NEG DATE: 4-13-93 RECIPIENT INFORMATION: NAME: RALPH PARKS ORGAN(S): LIVER STATE: ARIZONA TRANSPLANT DATE: 5-3-93 DOB: 5-30-62 / GENDER: M / BLOOD TYPE: A-POS Rh: Neg NAME: JASON JUNG ORGAN(S): AORTA TRANSPLANT DATE: 4-15-93 ] Sara: He died two months after receiving it back in '93. Lived one state over in Arizona. I have the cemetery address. (NICK rushes up toward them holding a piece of paper.) Nick: Hey, Sara, got the warrant. Sara: Awesome. We'll keep you posted. (GRISSOM nods as SARA and NICK leave. He looks up at CLAUDIA GIDEON sitting in the waiting room.) [SCENE_BREAK] [EXT. CEMETARY] (A large crane lifts the coffin out of the ground as SARA and NICK watch.) [SCENE_BREAK] [INT. CSI - FORENSIC AUTOPSY] (ROBBINS holds the sheet over the body up. NICK looks up at the ceiling.) Robbins: Embalming certainly retards the decomposition process. Nick: Yeah, But it doesn't stop the smell. Robbins: The liver's degraded but any metal should still be there. (ROBBINS lifts the cloth over the metal pan to show NICK the liver inside.) Nick: We'll find out. (NICK takes the liver and leaves.) [SCENE_BREAK] [INT. CSI -- LAB] (NICK cuts pieces of the liver off. He puts them into a blender and crushes them. He takes the liver mixture and puts it in a small container. He takes a small sample of the mixture and puts it in the machine to analyze. The results read on the monitor.) (The printer prints the results.) [SCENE_BREAK] [INT. CSI -- LAB] (SARA walks into the lab. NICK reads the results off of the printout.) Sara: Nick, did you get anything Nick: Hey, Sara. Yeah. Sodium selenite-- selenium. 280 milligrams for a 180-pound man... (NICK gives the printout to SARA. She looks at it.) Sara: That's murder. Nick: Yeah, well, it would have been murder for a 300-pound man. Sara: This is different than the shampoo compound. Nick: Yeah, it contains polysorbate 80 and vitamin E. That combination's found in selenium supplements injected in animals. Sara: Animals? Like cats, dogs? Nick: Mostly livestock, grazers. Sara: To make up for the lack of selenium nutrient in the soil. How did Claudia get access to this stuff? Catherine: (o.s.) A dairy farm. (SARA and NICK look up to see CATHERINE standing in the doorway reading a file.) Catherine: "Gideon Dairy and Cheese." Largest supplier of milk in Connecticut, in fact. Brass talked to a local chamber-of-commerce type about Claudia's husband. You want to know who his secretary was? Sara: Yeah. (CATHERINE puts the open file on the table for NICK and SARA to look at. The photograph in the picture is of John Gideon and Julia Fairmont.) Sara: Our Julia Fairmont. (SARA looks up at CATHERINE and smiles.) [SCENE_BREAK] [INT. POLICE DEPARTMENT - INTERVIEW ROOM] (CATHERINE and SARA interview CLAUDIA GIDEON and JULIA FAIRMONT, together. SARA puts the photographs of each woman with JOHN GIDEON in front of each of them.) Sara: Ten years ago, you played secretary and you played wife. Claudia: We have never represented we didn't know each other previously. Julia: And I wasn't playing. I was a hard-working secretary. Catherine: And you loved your husband Right up to the day he died ... of 240 milligrams of selenium. Sara: Animal selenium, the kind used for cattle. Claudia: Well, we did live in grazing country although, I have to say, Julia knew more about livestock than I did. She rode horses. (CLAUDIA smiles.) Julia: I took the job because Mr. G let me board my horse at the family stables. Claudia: Oh, I hated the stables. The smell. Never stepped foot in them. Sara: Implicating you, the secretary. (SARA sits down.) Sara: I don't believe this. Catherine: Bravo. Ladies. You have got this down to a science. (CLAUDIA looks at JULIA. JULIA continues to look at CATHERINE.) Why did it happen the first time? Hubby ignored you or abused you or you got sick of him fooling around so you started slipping the ... (Quick flashback to: CLAUDIA stands in front of the table. In one hand she holds the bottle of selenium. In the other, she grabs the glass of drink. JULIA walks in on her.) Catherine: (V.O.) ... cow stuff into his chocolate milk? Julia must have caught you. (End of flashback. Resume to present.) Catherine: So the two of you struck a deal. (Quick flashback to: CLAUDIA looks at JULIA. JULIA puts the scenario in front of her together.) Claudia: I'm, uh ... I'm coming into a lot of money. (JULIA puts the things she's carrying down. She sits down and looks at CLAUDIA.) Julia: No, we are. (CLAUDIA smiles. End of flashback. Resume to present.) Sara: And then what? The two of you realized how quickly you could run through a couple million? So you went looking for another mark here in Nevada? Catherine: But the difference is that you actually loved Bob Fairmont. (Quick flashback to: JULIA in bed with her husband. End of flashback. Resume to present.) Catherine: You shot him because he was cheating but that didn't get in the way of your long-term goals, did it? (GRISSOM peers through the door and taps on the glass. CATHERINE looks up. She and SARA go to see what GRISSOM wants.) [SCENE_BREAK] [INT. POLICE DEPARTMENT - HALLWAY -- CONTINUOUS] (SARA and CATHERINE walk out of the interview room.) Grissom: I just came from the D.A. Their attorneys were there as well. Based on everything we have the D.A. Is not going to move forward. He says it's a reject. Sara: Brass doesn't have anything? Grissom: Brass is still in the field. (GRISSOM looks into the interview room and tells the two women: ) Grissom: You're free to go. (They stand up to leave. SARA looks at GRISSOM. She's not happy with the outcome of the case. CATHERINE is taking it in stride.) Julia: (to CATHERINE) Where's the ladies' room? Catherine: It's down the hall to your right. Julia: Thanks. Claudia: Pardon me. (The two women leave.) Catherine: Well, no big surprise. We have proof of poison but no way to prove which one of them did it. Sara: I like either one of them for the murder-- so will a jury. Catherine: She-said/she-said defense? It's never going to fly. They'll raise each other as a viable suspect just like they did with us. Grissom: That's what the D.A. said verbatim. Catherine: See you back at CSI. (CATHERINE leaves. SARA is completely unhappy with the results.) Sara: Wait. What? So, what, and just let them move on to another state, another husband? Grissom: It's not always up to us. (Without another word, SARA walks out into the parking lot. GRISSOM turns and watches her leave. He takes off after her.) [SCENE_BREAK] [EXT. POLICE DEPARTMENT -- PARKING LOT - NIGHT -- CONTINUOUS] (SARA walks across the sidewalk and heads for her car. GRISSOM rushes out to catch up with her.) Grissom: Sara ... look ... (SARA stops and finally turns around to look at GRISSOM. She's disillusioned.) Grissom: I know this isn't news to you but sometimes science isn't enough. Sara: What are we doing? Digging up graves, chasing prints -- if it's no good in court ... if the killers win ... Grissom: It isn't a competition. We don't win. (SARA turns her head as GRISSOM continues.) The courts are like dice. They have no memory. What works one week doesn't work the next. Sara: (nods and looks at GRISSOM.) I know that. I do. I know that. That's why I'm mad. Grissom: But, see, if you get mad, then they do win. Sara: You just said ... This is one of your riddles isn't it? Grissom: (gently) One of life's riddles ... (GRISSOM turns around to head back to the building. He turns back to SARA.) ... But, hey ... the good news? There's no statute of limitation on murder. (GRISSOM makes his way back to the building. SARA turns and walks away.)

The CSI team is called in when a famous real estate entrepreneur is found dead in a hotel elevator. When the body of Bob Fairmont, the real estate developer, is found, the team discovers that the crime scene has been tampered with and that the victim has been re-dressed. After tracing the cause of death to a lethal poison, the team is shocked to discover that Fairmont's organs have already been removed and donated, making the case a bit more difficult to solve.

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How do adapting populations navigate the tensions between the costs of gene expression and the benefits of gene products to optimize the levels of many genes at once? Here we combined independently-arising beneficial mutations that altered enzyme levels in the central metabolism of Methylobacterium extorquens to uncover the fitness landscape defined by gene expression levels. We found strong antagonism and sign epistasis between these beneficial mutations. Mutations with the largest individual benefit interacted the most antagonistically with other mutations, a trend we also uncovered through analyses of datasets from other model systems. However, these beneficial mutations interacted multiplicatively (i. e., no epistasis) at the level of enzyme expression. By generating a model that predicts fitness from enzyme levels we could explain the observed sign epistasis as a result of overshooting the optimum defined by a balance between enzyme catalysis benefits and fitness costs. Knowledge of the phenotypic landscape also illuminated that, although the fitness peak was phenotypically far from the ancestral state, it was not genetically distant. Single beneficial mutations jumped straight toward the global optimum rather than being constrained to change the expression phenotypes in the correlated fashion expected by the genetic architecture. Given that adaptation in nature often results from optimizing gene expression, these conclusions can be widely applicable to other organisms and selective conditions. Poor interactions between individually beneficial alleles affecting gene expression may thus compromise the benefit of sex during adaptation and promote genetic differentiation. The concept of a fitness landscape unites the three levels of evolutionary change – genotype, phenotype, and fitness – into a mathematical picture of the potential for, and constraints upon, adaptive evolution. By mapping genotypes to a measure of fitness, fitness landscapes guide our understanding of how epistasis – nonlinear interactions between the fitness effects of mutations – shapes evolution. Strong epistasis implies that landscapes are rugged, with many peaks, or locally optimally genotypes [1], [2]. The magnitude and form of epistasis is predicted to determine the number of evolutionary trajectories [3], [4], the rate and repeatability of adaptation [5]–[7], and the benefit of sex [8]. Recent experimental work with a wide variety of model organisms has revealed diminishing returns as a general trend of adaptation [9]–[13], with relatively few cases of synergy [11], [14] or sign epistasis [15] (i. e., the same mutation being beneficial or deleterious in different contexts [16]). Antagonism between adaptive mutations might imply that these populations are summiting peaks in their fitness landscapes with just a handful of genetic changes. This explanation might lead to further trends, such as a negative relationship between the initial selective coefficient of a mutation and its epistatic interactions that could prove to be a useful predictor of a saturating process of adaptation [17]. In order to definitely link diminishing returns to the ascent of local peaks, as well as to understand the existence of the peaks themselves, we must understand the phenotypes that link genotype and fitness in the adaptive landscape. Mathematically convenient formulations such as Fisher' s geometric model for adaptation near a single peak [18] have been used to interpret the trend toward antagonism [19]. This approach assumes stabilizing selection a priori. What remains unclear is what types of physiological interactions give rise to fitness landscapes of varying shape and form, as well the constraints upon mutational changes to underlying phenotypes. Models of metabolic pathways have been amongst the most successful in translating underlying biochemical phenotypes to fitness. The contribution of enzyme activities upon metabolic flux has been formalized via Metabolic Control Analysis (MCA) [20], [21]. The ability of this approach to predict the fitness consequences of changes in enzyme properties has been verified in experimental systems that vary from Escherichia coli in lactose-limited chemostats to the flight properties of butterflies (reviewed in [22]). Turning to multiple enzymes, MCA theory has suggested a general trend toward synergistic interactions between activity-increasing mutations in a metabolic pathway [23], [24]. A major limitation, however, has been that the costs of enzyme expression [25] have not been included in classical MCA. Whereas the dependence of flux through a metabolic pathway saturates with increasing levels of a given enzyme, the costs will continue to accumulate. The balance of these two selective factors will generate an intermediate optimum, and thus stabilizing selection. Inclusion of expression costs to MCA has enabled predictions of the optimum levels of a single enzyme [26], and was used to compare the differential utility of alternate, degenerate pathways [27]. An open question, however, is how the balance between catalytic benefits and expression costs plays out to optimize enzyme expression across many enzymes simultaneously. In order to study how evolution would simultaneously optimize expression of multiple genes, we have developed a model system of an engineered Methylobacterium extorquens AM1 (EM) in which we altered its central metabolism to be dependent upon a foreign pathway (Figure 1A for details). M. extorquens grows on methanol by oxidizing it first to formaldehyde, and then through a series of steps to formate, which is either fully oxidized to CO2 or incorporated into biomass [28]–[32]. In the EM strain we removed the endogenous pathway for formaldehyde oxidation in wild-type (WT) [28] and replaced it with two genes encoding a foreign pathway that oxidizes formaldehyde via glutathione (GSH) derivatives [29]. Eight populations dependent upon this introduced metabolic pathway evolved in methanol-containing medium via serial transfers for 900 generations [10], [33]. Adaptation of the unfit EM strain to grow on methanol consistently involved beneficial mutations that altered expression of the foreign GSH pathway (Figure 1B). When the GSH pathway was introduced, the two enzymes were cloned together on a single mRNA transcript behind a strong native promoter present on a medium copy plasmid (∼9 cell−1) [10], [29], [34], [35]. As such, the costs of expression outweighed the catalytic benefits, and among the targets of adaptation we identified by resequencing strains evolved in separate populations, we universally obtained beneficial mutations that decreased expression of these enzymes [10], [34]. These mutations reduced expression of the GSH pathway through three classes of underlying mechanisms: Class A decreased expression per gene copy, Class B reduced gene dosage by lowering plasmid copy number, and Class C integrated the introduced pathway into the host genome, which also reduced plasmid copy number [33], [34] (Figure 1B). In terms of epistasis, mutations in multiple genes along a single adaptive trajectory – including one mutation (here ‘A1’) reducing expression of the GSH pathway – have been shown to exhibit a general trend of diminishing returns that was devoid of sign epistasis [10]. However, here we are interested in uncovering the trends and mechanisms underlying epistatic interactions between mutations that arose in separate adapting lineages and affect expression of the same metabolic pathway. We combined independently-arising beneficial mutations affecting gene expression of this two-enzyme metabolic pathway and report strong antagonism and sign epistasis for fitness. These interactions were increasingly antagonistic for larger benefit mutations. Such strong antagonism did not stem from the effects of mutational combinations upon enzyme levels, but rather from the nonlinear mapping between enzyme expression and organismal fitness. By developing a quantitative model that relates expression cost and catalytic benefit to fitness, we characterized the overall shape of this fitness landscape and revealed that some of these single mutations can optimize multiple phenotypes simultaneously, leading to a big jump toward the single, global optimum. To explore the pattern of epistatic interactions between beneficial mutations affecting expression of the GSH-dependent pathway, we combined beneficial plasmid mutations that emerged during experimental evolution and affected distinct traits [34]. We focused upon Class A (decreased expression per copy) and B (reduced gene dosage) mutations because of their genetic tractability, and the prediction that these represent orthogonal mechanisms to achieve lower expression. We hypothesized that mutational combinations between these classes would result in enzyme levels that would be the product of the individual perturbations (Figure 1C). Three class A mutations, A1–A3, and one class B mutation, B5, occurred independently, whereas B2 and B3 were isolated together from the same plasmid. We generated 12 plasmids that paired each Class A mutation with each one from Class B, as well as with the B2–B3 pair, and measured their relative fitness via competitions with a fluorescently labeled ancestor [34] (Tables S1, S2). The observed fitness values for the mutational combinations were substantially less than expected based upon a simple multiplicative null model incorporating the single mutant effects (i. e., Wij = Wi×Wj; R2 = 0. 53, adj-R2 = 0. 32; Figure 2A). The increasingly strong antagonism for higher expected fitness values suggested a potential negative relationship between the selective coefficients observed for each mutation and the average epistasis that mutation exhibited with other mutations. We observed that the individually most beneficial mutations (large s) engendered the greatest antagonism (ε<0) when combined with other mutations, including several examples of sign epistasis (Figure 2B). Several theoretical arguments suggest that the geometry of fitness landscapes might induce correlations between the size of a mutation and the strength and direction of epistasis. Epistasis has been observed to be coupled to the mean fitness effect of mutations [36], [37]. A single beneficial mutation of large effect may appreciably change both the mean fitness of subsequent mutations and the remaining distance to the optimum, potentially skewing its own epistatic coefficients. Given the emerging empirical consensus and theoretical arguments for antagonistic epistatic interactions among beneficial mutations, we analyzed several other datasets to ask whether the strength and form of the relationship between selective effect and average epistatic effect held for intragenic and intergenic datasets. For this comparison we analyzed the relationship between s and ε for previous datasets from M. extorquens and E. coli where the beneficial mutations occurred consecutively in a variety of genes across the genome of a single adapting lineage [10], [11], combinations of mutations from two genes of the bacteriophage ID11 [12], and two datasets of within-protein interactions for β-lactamase [17], [38]. These datasets also displayed signs of a correlation between s and increasingly negative ε (as noted in [37]), with the exception of the intragenic data for β-lactamase (Figure S1). Negative trends in the relationship between initial selective coefficient and epistasis may seem like obvious evidence for diminishing returns. However, recent theoretical work has shown that in models where mutations have random effects and no tendency to be either synergistic or antagonistic, a pattern of diminishing returns occurs between mutations if they are selected conditioned on being beneficial in the ancestral background [39]. In Supplementary Text S1 we show this behavior in a simple model of evolution on fitness landscapes with no mean epistatic tendency and show how it leads to a pattern of diminishing returns between beneficial mutations as a form of regression to the mean. This analysis suggests that genotype-fitness data alone, without knowledge of the phenotypic effects of mutations or the physiological causes for trade-offs, might be insufficient to infer the mechanism underlying a pattern of epistasis. What physiological factors underlie the strong antagonism observed between mutations affecting expression of the foreign GSH pathway? A first possibility is that mutational combinations lead to smaller changes in protein expression than expected from the single mutants and that such antagonistic behavior at the level of expression phenotypes merely propagated through as observed antagonism at the level of fitness. Because we used combinations that largely derived from pairing mutations that reduced expression per copy (Class A) with those that decreased plasmid copy number (Class B), our null hypothesis was that these mechanisms should act independently to alter expression, such that the expression level of an A+B mutant pair would simply be the product of these two values. Consistent with this prediction, enzyme levels were well described by the null model of multiplicative independence between paired perturbations (Figure 3A, Table S1). A simple linear model of log-transformed changes in enzyme levels as a function of the presence of the single mutations with no interaction terms explains much of the variation for both FlhA and FghA (adjusted-R2 = 0. 85 (FlhA) and 0. 86 (FghA) ). This predictability can be seen in the high correlation between observed expression phenotypes for paired perturbations and those expected based upon the single changes. Since mutational combinations did not introduce epistatic interactions at the level of gene expression, we built a model of the fitness landscape based upon enzyme levels in order to ask how its shape would contribute to antagonism. Building upon earlier work on single enzymes [26] (Supplementary Text S2), we generated a model of the fitness landscape that calculates fitness as flux through the pathway above a threshold, minus the sum of two costs: The hyperbolic expression for catalysis has been used before [40] to effectively describe the dependence of steady-state flux to the levels of a single enzyme and incorporates a “Vmax” term for the pathway, and an Eh half-maximal enzyme level term. We only model FlhA concentration as beneficial to fitness even though FghA is absolutely required for growth on methanol [34]. This is because, over the parameter range of our perturbations, fitness appeared to rise monotonically with decreasing levels of FghA. This suggests FghA is a typical enzyme that has a low metabolic “control coefficient” [20], [21] and that it only limits catalysis at exceptionally low levels. None of our perturbations pushed FghA levels below 2%, and for comparison β-galactosidase levels in lactose-limited chemostats only impacted fitness significantly if they decreased activity to ≤1% [41]. The threshold flux term was added to the model to capture an unusual right-shift of the typical relationship between enzyme concentration and fitness observed with FlhA in these data, such that fitness approached zero even in the presence of measurable concentrations of functioning enzyme. We have observed similar behavior when manipulating levels of the analogous enzyme in the endogenous, tetrahydromethanopterin-dependent pathway for formaldehyde oxidation in WT (SM Carroll, CJM, unpublished). As both of these enzymes occur directly downstream of formaldehyde production, this threshold phenomenon may be explained by toxic effects of elevated steady-state formaldehyde concentrations at low enzyme levels. Finally, there are two cost terms for FlhA and FghA. The cost per molecule for each enzyme was treated as a linear function, consistent with prior work [26], [42]. The six parameters of this benefit - costs model were fit using the data from the EM ancestor, single mutants, as well as strains with inducible promoter plasmids (27 data points; Table S3). The inducible promoter plasmids contained a cumate-responsive repressor to modulate the levels of flhA-fghA from ancestral levels to lower values (Table S1). These data were critical for capturing the steep decline of the fitness landscape at low values of FlhA. The resulting benefit - costs model captured the curvature of the fitness landscape (Figure 4) and, unlike the simple multiplicative model, it was able to predict the 17 combinations of mutations that were not used for model fitting with high precision (R2 = 0. 98) (Figure 3B). From the perspective of the ancestral genotype, in the model fitness rises gently with decreased expression of either enzyme, but then declines rapidly upon reaching catalytically-limiting levels of FlhA. A similar cliff exists for low values of FghA [34], but at enzyme levels beyond the range of our dataset and below the detection threshold of our enzyme assay method (see Methods). Our fitness landscape model that precisely maps phenotypes to fitness allowed us to explore how much local topography may have influenced the direction of phenotypic change during evolution by de novo mutations (Figure 5). We compared the changes in enzyme expression caused by single beneficial mutations relative to three factors: 1) the local gradient in the fitness landscape for the ancestor (greater decreases of FlhA versus FghA because the former is more costly), 2) the direct vector pointing to the global fitness optimum and 3) equal proportional changes between the enzymes which might be expected due to the physical constraint of their being expression from a single transcript. All mutations moved toward the global optimum rather than ascend in the phenotypically steepest direction on the local fitness landscape. Mutations B2 and B5 affected copy number but, through mechanisms we do not currently understand, led to greater decreases in FghA than FlhA. In contrast, B3 was directly along the line of equivalent change in both enzymes. This mutation was identified along with B2 as a plasmid haplotype, and this B2–B3 combination allowed this lineage to accomplish a similar phenotypic (and fitness) change as the other mutants. We found that combining adaptive mutations that optimize expression of a two-enzyme pathway exhibited strong antagonistic interactions and sign epistasis. Fitness values of mutational combinations were generally less than expected relative to a null model of independent, multiplicative effects upon fitness. We further observed a negative relationship between s and ε for individual mutations. Other datasets of intragenic epistasis revealed similar trends between s and ε; however, this overall trend of epistasis (e. g., antagonism) does not imply a specific connection with properties of the individual mutations, such as s. For example, this trend will also arise as a consequence of regression to the mean when the beneficial mutations assayed are conditioned to be beneficial in the ancestral background and the effect of a mutation has a component that is independently distributed on each possible genetic background. Therefore, to extract biological insight from the quantitative relationship between s and ε, we must interrogate the mechanisms that lead to antagonistic epistasis. The first possible explanation for antagonism in our data would be non-linearities in the way mutations combined to affect enzyme expression. However, as expected from having chosen combinations that combined class A mutations with those from class B, these orthogonal mechanistic effects resulted in independent effects on enzyme expression that were jointly well predicted with a simple, multiplicative model. As in this system, many ecologically relevant genes are encoded on plasmids whose regulation and gene dosage may both be effected by separate sets of mutations. More broadly, mutations that influence different traits that make joint contribution to a higher phenotype such as fitness are common. At the level of individual genes, for example, catalytic improvement of an enzyme often results from the joint contribution of mutations that improve protein stability and those that enhance kinetic parameters [43]–[45]. The second factor that could generate antagonism is the curvature of the underlying fitness landscape for gene expression. Recent theory has shown that almost any formulation of fitness based upon multiple underlying phenotypes will generate epistasis at the level of fitness, even when the mutations – as we observed here – do not interact epistatically on the underlying trait phenotypes [46]. Previous models have formulated fitness as a function of gene expression correctly predicted the evolution of optimal levels of gene expression [26]. Here we extended this model framework to multiple enzymes and used it for the first time to interpret beneficial mutational effects from phenotype to fitness, which we characterized both individually and in combination. Our fitness landscape model was able to predict the fitness values of the mutation combinations with high precision (R2 = 0. 98). The asymmetry in the curvature of this fitness landscape results from the relatively gentle effects of expression costs relative to the sharp transition in fitness effects due to rate limitation upon catalysis [41]. This observed selection to maintain an intermediate optimum of enzyme levels is distinct from the selective neutrality on a catalytic plateau that was predicted by classical MCA analyses that did not incorporate expression costs [40]. Knowledge of the underlying fitness landscape allows us to understand aspects of the epistatic interactions not evident from fitness values alone. For example, we observed that the A3 and B5 mutations had fairly comparable individual fitness values (1. 420±0. 032 vs. 1. 457±0. 033; mean and 95% CI), but the former had three-fold more antagonistic epistasis than the latter (average = −0. 46 vs. −1. 34; t-test, p = 0. 025; Figure 6). The modeled fitness landscape illuminates the underlying reason for this difference. Both mutations rest near the peak value of enzyme expression, but on opposite sides (B5 has 70% the level of FghA as A3). This poises B5 such that it is much more sensitive to further reductions in expression than A3. Thus, although these two mutations are essentially equivalent if one only considers their fitness values, their locations in phenotypic space change their likelihood for antagonism and sign epistasis. One consequence of sign epistasis between mutations affecting the phenotypes like gene expression is a reduction of the benefit caused by recombination bringing beneficial mutations together into the same genome (i. e., Fisher-Muller model). This tradeoff between benefits and costs is inherent to gene expression, and thus results in stabilizing selection. These patterns of epistasis are likely very common, given the apparent ubiquity of stabilizing selection upon gene expression from microbes to primates [47]–[50]. With a complete fitness landscape defined by biochemical phenotypes, we can now interpret the genetic landscape in terms of what was accessible to individual mutations. In contrast to how selection acts upon standing genetic variation, the de novo mutations fixed in experimental populations ignored the phenotypically-local best direction of change and “jumped” towards the global optimum. This highlights that the classic quantitative genetics intuition of climbing in the direction of the steepest selection gradient [51], which is appropriate for small populations containing standing genetic variation, fails to capture the phenotypic potential of a sizeable pool of de novo mutations arising from large populations. In our case, it was not the large magnitude of expression change that was surprising, per se, but change in the ratio of their expression. Given that flhA and fghA are encoded on the same transcript, it was notable that all but one of the single mutations down-regulated FghA to a large extent while only cutting FlhA levels in half. Whereas this system allowed individual mutations to reach near-optimality, a multi-step trajectory was required for the directed evolution of the LacI repressor to reverse its regulatory logic [52]. In that system, the first round mutation simply broke the old logic to become constitutive, which in combination with two latter mutations allowed the “anti-LacI” phenotype to emerge and locate the fitness peak they predicted from a computational model. Our results suggest that relatively large moves in multi-phenotype space can emerge as winners, provided the genetic architecture at least allows rare mutations to achieve this possibility. Finally, the near optimal expression levels of these beneficial mutations becomes even more remarkable when considering that this optimization did not happen in isolation, but in adapting populations that contained many beneficial mutations simultaneously [33], [34], [53]. In varying environments, such diversity in large microbial populations may lead to genetically complex adaptation such as stable polymorphisms [54]. In a stable environment, this diversity leads to ‘clonal interference’ [55], a type of serial fixation that effectively sorts for mutations of the greatest effect amongst what was possible. This would have impacted the mutations that affected GSH pathway expression in two ways. Firstly, there were many different genetic solutions to reducing expression of these enzymes [34]. One type in particular - the Class C mutations that resulted from integration of the introduced plasmid into the host chromosome – occur at very high rates and emerged to detectable levels repeatedly, up to 17 times per population [33]. These mutations confer ∼⅔ the benefit of the Class A and B mutations [34], however, and were only found to rise to fixation in three of eight populations despite more than 100 observed occurrences [33]. In this regard, clonal interference aids finding optimal solutions by allowing only the best individual mutations to fix. Recently, however, it has been shown that fixation probability of contending mutations is only partly dependent upon their individual effect because they commonly hitchhike with other beneficial mutations present [56]–[58]. This leads to a second effect of clonal interference, which is competition between lineages carrying beneficial mutations affecting distinct phenotypic processes. Indeed, the ancestral genotype faced a variety of phenotypic challenges besides just optimizing expression of the GSH pathway [14], [59], [60]. Some of these mutations in other loci had beneficial effects up to 3× larger than those described here [10] and were segregating at the same time as mutations affecting expression of the GSH pathway [33], [53]. Even with so much turmoil in the populations, the eventual winners discovered nearly optimal solutions to this local, two-enzyme expression optimization in order to win the battle for fixation. Population size thus contributed to the fixation of optimal solutions by both increasing the number of mutations occurring and escaping drift in the first place, and by facilitating competition between multiple potential solutions. These factors conspired to allow selection to reward – when mutationally possible – lineages that made long-range, lucky jumps to distant peaks on the phenotypic landscape. The EM strain was generated previously by deleting the mptG gene of M. extorquens AM1 in the white strain WT CM502 [61] lacking carotenoid pigments due to an unmarked mutation in crtI (encoding phytoene desaturase) [62], followed by introduction of pCM410 [10]. Eight replicate populations seeded by the EM strain were grown in 9. 6 ml methanol (15 mM) minimal media incubated in a 30°C shaking incubator at 225 rpm. Populations were transferred to fresh media at a 1/64 dilution rate (thus six generations per growth cycle, Nfinal≈109) and propagated for 600 generations. One liter of minimal media consists of 100 ml of phosphate buffer (25. 3 g of K2HPO4 and 22. 5 g of NaH2PO4 in 1 liter of deionized water), 100 ml of sulfate solution (5 g of (NH4) 2SO4 and 0. 98 g of MgSO4 in 1 liter of deionized water), 799 ml of deionized water, and 1 ml of trace metal solution. One liter of the trace metal solution consists of 100 ml of 179. 5 mM FeSO4,800 ml of premixed metal mix (12. 738 g of EDTA disodium salt dihydrate, 4. 4 g of ZnSO4·7H2O, 1. 466 g of CaCl2·2H2O, 1. 012 g of MnCl2·4H2O, 0. 22 g of (NH4) 6Mo7O24·4H2O, 0. 314 g of CuSO4·5H2O, and 0. 322 g of CoCl2·6H2O in 1 liter of deionized water, pH 5), and 100 ml of deionized water [14]. All strains and plasmids used are indicated in Table S2. All plasmids constructed in this study were maintained in E. coli 10-beta strain (New England Biolabs) and were transferred to M. extorquens via electroporation [63] or tri-parental mating with the helper strain pRK2073 [64]. Plasmid DNA in E. coli was extracted using the QIAprep Spin MiniPrep Kit (Qiagen). The PmxaF expression vector pCM160 [65], its variant pCM410 in the EM strain expressing the flhA-fghA cassette [10], and the cumate-inducible vector pHC112 expressing the flhA-fghA cassette [34] have been described previously. In order to combine Class A and B mutations which accumulated on separate pCM410 derivatives during experimental evolution of the EM strain (Figure 1B, Table S1), Class B mutations (from pCM410B2B3, pCM410B2, pCM410B3, and pCM410B5) were moved to pCM410 derivatives bearing Class A mutations (pCM410A1, pCM410A2, and pCM410A3) through the procedures delineated below. Fragments containing B2–B3, B2, and B3 mutations were first obtained by digesting pCM410B2B3, pCM410B2, or pCM410B3 with SfiI and NheI. These were then ligated into the plasmid backbone of pCM410A1, pCM410A2, or pCM410A3 cut with the same enzymes. A fragment containing the B5 mutation was obtained by digesting of pCM410B5 with SfiI and SexAI and then ligated into the plasmid backbone pCM410A1, pCM410A2, or pCM410A3 cut by the same enzymes. The above procedures were also applied to introduce B2–B3, B2, B3, and B5 mutations into pHC112 in order to generate pHC112 derivatives that vary in their plasmid copy number. Fitness assays were performed by a previously described procedure [34]. Strains were first physiologically acclimated through one 4-day growth cycle in 9. 6 ml of minimal media supplemented with 15 mM methanol. In addition, for strains bearing cumate-inducible promoter plasmids (pHC112 derivatives), different concentrations of cumate (Table S1) were added to growth media to modulate the expression of FlhA and FghA enzymes. After this acclimation phase, each of these strains was mixed with a fluorescent variant (CM1232) of the EM ancestor [10] by a 1∶1 volume ratio, diluted 1/64 into 9. 6 ml of fresh growth media, and incubated in a 30°C shaking incubator at 225 rpm. The ratios of the two populations before (R0) and after (R1) competitive growth were quantified by a LSR II flow cytometer (BD Biosciences) for at least 50000 cell counts per sample. The forward scatter threshold of LSRII was adjusted to 300 to ensure unbiased detection of the test and reference strains despite their potential differences in cell size. Fitness values (W) relative to the reference strain were calculated by a previously described equation assuming an average of 64-fold size expansion of mixed populations during competitive growth [35]: In order to convert to absolute differences in growth rate, the EM ancestor grows under these conditions with a growth rate of 0. 0654+/−0. 0016 h−1 [10]. The activities of FlhA [66] and FghA [67] were assayed in three replicates as described using cells harvested from mid-exponential phase cultures. Cells were collected through centrifugation at 10,000× g for 10 min, frozen at −80°C, and used for enzyme assays within a week. Right before assays frozen cell pellets were suspended in 50 mM Tris-HCl buffer (pH 7. 5) and physically disrupted in tubes containing Lysing Matrix B and shaken at speed 6. 0 m/s on a FastPrep®-24 bead beater (MP Biomedicals) for 40 seconds. Insoluble debris in the cell lysate was removed by centrifugation at 13,000× g, 4°C for 15 min. The total protein concentration of the cell lysate was quantified using the Bradford method [68]. Kinetic analysis of FlhA and FghA activities over 10 min at 30°C was performed in 200 µl reaction mixtures using a SpectraMax M5 Plate Reader (Molecular Devices). The copy number of pCM410 derivatives in M. extorquens was quantified by a real-time PCR approach described previously [34]. Briefly genomic DNA of M. extorquens from mid-exponential phase cultures was extracted by an alkaline lysis method [69]. Detection of plasmid DNA was targeted at the kan gene using primers HC410p18 (5′-GAAAACTCACCGAGGCAGTTCCATAG-3′) and HC410p19 (5′-TCAGTCGTCACTCATGGTGATTTCTCA-3′). Detection of chromosomal DNA was targeted at the rpsB gene (encoding the 30S ribosomal protein S2) in the chromosome META1 using primers HCAM111 (5′-TGACCAACTGGAAGACCATCTCC-3′) and HCAM113 (5′-TTGGTGTCGATCACGAACAGCAG-3′). Real-time PCR experiments were performed in three replicates with the PerfeCTa SYBR Green SuperMix (Quanta Biosciences) on a DNA Engine Opticon2 (MJ Research), and the average threshold cycle (Ct) of each PCR reaction was determined using the Opticon Monitor v. 2. 02 software (MJ Research). Each real-time PCR reaction contained 25 ng of genomic DNA extracted from various strains and kan- or rpsB-specific primers. To establish a standard curve (SC) of plasmid copy numbers, 1,0. 1,0. 01, and 0. 001 ng of pCM410 (equivalent to 9. 09×107,9. 09×106,9. 09×105, and 9. 09×104 plasmid molecules, respectively) were mixed with 25 ng of genomic DNA (equivalent to 3. 03×106 genome copies) of the plasmid-less, white WT M. extorquens (CM502) [61]. The standard curve is a plot of ΔCt (i. e. Ctkan–CtrpsB) versus plasmid molecules on a log2 scale. For each strain, by interpolating its ΔCt value against the SC the absolute quantity of plasmid DNA can be estimated using the following equation: Multiplicative models predicting fitness or gene expression were fit and assessed as standard linear models following a log transformation of the response variable. The model for the fitness landscape was fit using a non-linear routine in Matlab. The raw data as well as commented code in Matlab and R that completely recreates the analysis and figures has been deposited at www. datadryad. org (doi: 10. 5061/dryad. 8hb23).

The pace and outcome of a series of adaptive steps in an evolving lineage depends upon how well different beneficial mutations stack on top of each other. We found that independent beneficial mutations that affected gene expression for a metabolic pathway did not work well together, and were often jointly deleterious. The most beneficial mutations interacted the most poorly with others, which was a trend we found common in other biological systems. Through generating a model that accounted for enzymatic benefits and expression costs, we uncovered that this antagonism was caused by a phenotype to fitness mapping that had an intermediate peak. This allowed us to predict the fitness effect of double mutants and to uncover that the single winning mutations tended to move straight to the peak in a single step. These findings demonstrate the importance of considering the phenotypic changes that cause nonlinear interactions between mutations upon fitness, and thus influence how populations evolve.

lay_plos

Rothmund-Thomson syndrome (RTS) is a rare autosomal recessive disorder characterized by skin rash (poikiloderma), skeletal dysplasia, small stature, juvenile cataracts, sparse or absent hair, and predisposition to specific malignancies such as osteosarcoma and hematological neoplasms. RTS is caused by germ-line mutations in RECQL4, a RecQ helicase family member. In vitro studies have identified functions for the ATP-dependent helicase of RECQL4. However, its specific role in vivo remains unclear. To determine the physiological requirement and the biological functions of Recql4 helicase activity, we generated mice with an ATP-binding-deficient knock-in mutation (Recql4K525A). Recql4K525A/K525A mice were strikingly normal in terms of embryonic development, body weight, hematopoiesis, B and T cell development, and physiological DNA damage repair. However, mice bearing two distinct truncating mutations Recql4G522Efs and Recql4R347*, that abolished not only the helicase but also the C-terminal domain, developed a profound bone marrow failure and decrease in survival similar to a Recql4 null allele. These results demonstrate that the ATP-dependent helicase activity of Recql4 is not essential for its physiological functions and that other domains might contribute to this phenotype. Future studies need to be performed to elucidate the complex interactions of RECQL4 domains and its contribution to the development of RTS. Rothmund-Thomson syndrome (RTS) (OMIM #268400) is a rare autosomal recessive disorder characterized by skin rash (poikiloderma), skeletal dysplasia, small stature, sparse or absent hair, gastrointestinal complications, and high predisposition to specific malignancies such as osteosarcoma (OS) and hematological neoplasms [1,2]. RTS and the related RAPADILINO and Baller-Gerold syndromes are associated with damaging germ-line mutations in RECQL4 [3–5]. The majority of RECQL4 mutations are located in its helicase domain, yet the physiological role of this domain remains unclear [6]. Helicases are enzymes that unwind double-stranded or more complex DNA and RNA structures using energy from ATP hydrolysis. The unwinding of double-stranded DNA (dsDNA) is necessary to allow access to the DNA during replication, repair, recombination and transcription [7]. In humans, five members of the RecQ helicase family have been identified: RECQL1, RECQL4, RECQL5, BLM and WRN. Mutations in three are associated with syndromes that present with premature aging and cancer-predisposition: WRN in Werner’s syndrome, BLM in Bloom’s syndrome, and RECQL4 in RTS [8,9]. RecQ-family helicases contain three highly conserved protein domains: an archetypal helicase domain, which contains seven conserved motifs that couple ATP hydrolysis to dsDNA strand separation [8,9]; the RecQ C-terminal (RQC) domain, which features a beta-hairpin motif, a winged-helix domain and a zinc-binding motif, for intervention in the binding of G quadruplex DNA and stabilization of DNA structures [10]; and the Helicase-and-ribonuclease-D-like C-terminal (HRDC) domain, which promotes stable DNA binding [11]. RECQL4 differs from the other family members as it has no HRDC domain and lacks a structurally conserved RQC domain. Instead, it contains the structurally unique domain called RecQ4-Zn2+-binding domain (R4ZBD) and, importantly, an N-terminal region of homology with the S. cerevisiae DNA replication initiation factor Sld2 [12,13]. Sld2 is an essential protein required for activation of replication origins in yeast [14], and RECQL4 is the putative mammalian homologue. The human RECQL4 gene is located at the long arm of chromosome eight (8q24. 3) and consists of 21 exons and 13 relatively short introns (<100 bp in length), yielding a full-length transcript of 3,627bp [3]. Mutations in the RECQL4 gene have been found in the majority of RTS patients, and also in the related RAPADILINO and Baller-Gerold Syndromes [1,6]. Most mutations are either nonsense or frameshift mutations and are predicted to create truncated proteins [6]. Over half of these impact the reading frame at exons 8–14 causing abnormal translation and or truncation of the RECQL4 protein with the presumed loss of DNA helicase function [6]. Very few mutations are located in the N-terminal Sld2 region, and those that are reported are primarily silent or missense [6]. This finding has led to the hypothesis that the N-terminal domain is critical for organismal viability, and that inactivation of the helicase function is a critical effect of the mutation spectrum that is found in RTS. RECQL4 has been well characterized biochemically in terms of its single-stranded DNA (ssDNA) binding, ATP hydrolysis and DNA unwinding ability [15–17]. In vitro, RECQL4 ATPase and helicase activity is completely abolished by point mutations in the canonical Walker A and B motifs [18]. However, helicase-dead mutants can rescue cellular lethality in RECQL4-deficient DT40 chicken cells and murine hematopoietic cells in vitro [19,20]. In addition, mutations that affect the helicase domain not only affect its activity but can also lead to protein truncation or unstable proteins, which does not allow the specific assessment of the physiological requirement of the ATP-dependent helicase RECQL4 in isolation. To understand the biological functions of RECQL4 helicase activity in a whole animal context, we generated a mouse model with a knock-in point mutation that specifically abolishes its ATP-dependent helicase activity (Recql4K525A) and compared this to two different truncating mutations Recql4G522Efs and Recql4R347*. Here we show that mice with a specific deficiency in Recql4 helicase activity are strikingly normal, in contrast to pathogenic effects of truncating mutations that remove the entire helicase domain and downstream part of the protein. We generated full-length wild-type mouse Recql4 protein, along with a K525A variant. The alanine substitution replaced a critical lysine, present in all Walker A motif-containing ATPases, that is necessary for ATP hydrolysis and corresponds to a previously analyzed human RECQL4 K508A mutation that lacks ATP-dependent helicase activity [15,21] (Fig 1A). To biochemically characterize an ATPase deficient mutation in murine Recql4, we established in vitro assays for Recql4 function. Both WT and K525A proteins had equivalent affinity for ssDNA binding, using an electrophoretic-mobility shift assay (EMSA) (Fig 1B). In ATPase assays, the WT protein hydrolyzed ATP in a DNA-dependent manner whereas Recql4K525A showed no activity above background levels (Fig 1C). Finally, we tested the ability of these recombinant proteins to unwind DNA, using a dsDNA substrate with a ssDNA loading site. If ATP-dependent helicase activity is present, the fluorophore-labeled strand is released from the quencher-labeled complementary strand providing a real-time fluorescent readout of unwinding activity (Fig 1D). Robust ATP-dependent unwinding of the substrate was observed when using WT-Recql4 protein, whereas no change in fluorescence was observed using the Recql4K525A mutant consistent with an inability to unwind DNA (Fig 1E). Together, our results demonstrate that the Recql4K525A protein is helicase and ATPase dead, despite equivalent protein stability and DNA binding properties. To understand the contribution of Recql4 helicase activity in the phenotypes of RTS, we generated an in vivo knock-in model of the K525A mutation. Sequencing of the Recql4 locus in targeted mice confirmed the change in nucleotides encoding lysine (AAG) to alanine (GCA), and the resultant introduction of a unique MslI restriction enzyme site in the mutant allele (Fig 2A). PCR amplification over the mutation site produces a 416 bp fragment in the wildtype that can cleaved to 361 bp (+55 bp) by MsiI when the mutation is present (Fig 2B). Finally, to determine the expression and stability of the mutant protein in vivo we generated a rat monoclonal antibody against the first 200aa from the N-terminal of murine Recql4 (clone 3B10). The K525A mutant protein has the same predicted molecular mass (~133kDa) as wild-type Recql4 and neither size nor abundance of the protein were affected by the mutation when assessed in thymocyte derived protein samples (Fig 2C). Taken together, these results demonstrate that the loss of helicase activity does not affect the expression or stability of the Recql4 protein in vivo. We observed a slightly sub-Mendelian ratio of homozygous Recql4K525A/K525A animals at weaning from heterozygous breeding pairs, although this was not statistically significant by chi-squared test (p = 0. 6255) (Fig 2D). Heterozygous and homozygous mice for the K525A mutation were viable and outwardly normal (Fig 2E). Further, we observed no difference across genotypes or sexes in 10-week old animals assessed for body weight and body composition (Fig 2F and 2G). Both male and female Recql4K525A/K525A animals were fertile and able to breed, and there was no difference in the survival when comparing the Recql4K525A/K525A and control genotypes using Kaplan Meier survival analysis (Fig 2H). Collectively these results show that, unlike the embryonic lethality of Recql4 null alleles [20,22], the Recql4K525A protein supports normal development and adult homeostasis. We previously reported that somatic deletion of Recql4 resulted in a fully penetrant bone marrow (BM) failure [20]. To determine the role of helicase activity in hematopoiesis, we assessed cohorts of adult wild-type, Recql4K525A/+ and Recql4K525A/K525A mice. Analysis of the peripheral blood (PB) revealed no changes in leukocyte or platelet numbers (Fig 3A and 3B). The absolute red blood cell counts were subtly increased in both the K525A/+ and K525A/K525A mutants, however, the hemoglobin levels were not changed compared to the WT (Fig 3C and 3D). Further analysis of the individual leukocyte subsets in the PB including granulocytes, macrophages, B lymphocytes, and CD4+ and CD8+ T lymphocytes revealed no significant differences in the proportions or absolute numbers of these populations (Fig 3E). Within the BM, the total numbers of leukocytes were comparable across genotypes (Fig 3F). Similarly, granulocyte and macrophage numbers were similar (Fig 3G). Within the B-lymphoid populations (B220+IgM-), the Pre-B, Pro-B, and Pre-Pro-B subpopulations were all unaltered in Recql4K525A/+ and Recql4K52A/K525A compared to WT littermates (Fig 3H), as were the erythroid subpopulations (Fig 3I). The frequencies and absolute numbers of the primitive hematopoietic stem cells (HSCs) and progenitors were also assessed. There were no major differences in the numbers of phenotypic long-term HSCs (LT-HSC), short-term HSCs (ST-HSC) or multipotent progenitor (MPP (Lin- c-kit+ Sca-1+ CD34/Flt3) ) fractions, nor in their myeloid committed subpopulations (Fig 3J–3L). There was an elevation of the phenotypic myelo-erythroid progenitor (MEP, Lin-c-kit+Sca-1-CD34/FcγRII) and colony forming unit-erythroid (CFU-E, Lin-LKS-CD41-FcγRII-CD150-CD105+) in the Recql4K525A/+ animals, however the absolute change was very small and not statistically significant in the Recql4K525A/K525A mice. The basis for this mild elevation in RBC counts and committed erythroid progenitors is currently undefined. In summary, unlike for complete deletion of Recql4, the abrogation of Recql4 helicase activity does not substantively perturb hematopoiesis in vivo. Our previous work demonstrated that the human helicase dead RECQL4 (K508A) was able to rescue in vitro B and T cell development from murine Recql4Δ/Δ hematopoietic cells [20]. To determine if this was also the case in vivo, T and B cell development was assessed from thymocytes and splenocytes respectively in 10-week-old mutant and WT mice. There was no difference in thymus cellularity or in the numbers of double positive CD4+/CD8+ nor the mature single positive CD4+ and CD8+ cells (Fig 4A and 4B). Analysis of early intra-thymic progenitor cells (double negative DN1-4) found no difference in the Recql4K525A/K525A compared to the WT littermates (Fig 4C). In the spleen there was no significant difference in the cellularity or number of mature B cells (Fig 4D and 4E). To determine the proportion of B cells in the follicular (FO) and marginal zone (MZ) of the white pulp of the spleen, we divided splenic cells with high expression of B220 and CD19 followed by analysis of CD21/CD23 expression (FO B cells: CD21lowCD23high; MZ B cells: CD21highCD23low). No shift of B cells in the follicular or marginal zone compartments was apparent (Fig 4F). Therefore, consistent with the prior retroviral rescue data in vitro, the ATP-dependent helicase function of Recql4 is not required for B or T cell development and homeostasis in adult mice. Given the role of RecQ helicases in repair of DNA damage [11], we sought to determine if there was a function for the ATP-dependent helicase activity of Recql4 during physiologically induced DNA damage occurring following B cell stimulation. We isolated mature B cells from WT, Recql4+/K525A and Recql4K525A/K525A spleens and stimulated them in vitro using bacterial lipopolysaccharide (LPS) and rmIL-4 to induce proliferation and immunoglobulin gene (Ig) class switch recombination. The proliferation of the cells in response to LPS was the same irrespective of Recql4 helicase status as measured by cell-trace violet dilution kinetics (Fig 4G and 4H). Additionally, the Recql4K525A/K525A cells underwent normal class switching to IgG1 (Fig 4I). Therefore maturation, physiological activation induced proliferation, DNA damage and immunoglobulin rearrangement of B cells do not require the helicase activity of Recql4. To further test the requirement for RecQ helicase activity we compared the response to DNA damaging agents in vitro. For these studies, we established GM-CSF dependent myeloid progenitor cell lines from R26-CreER Recql4fl/K525A and R26-CreER Recql4fl/+ (control genotype) by immortalization with HoxB8 [23]. To allow analysis of the requirement for RecQ helicase activity, the cells were treated with tamoxifen for 4 days to activate Cre-mediated deletion of the loxP flanked wild-type Recql4 (Recql4fl) allele. This resulted in the cells either becoming Recql4 heterozygous (Recql4Δ/+) or expressing only the K525A mutant allele (Recql4Δ/K525A). Isogenic cells from each genotype (pre-treated with tamoxifen for 4 days or untreated) were seeded in 96-well plates and the response to four different genotoxic agents was assessed: doxorubicin (topoisomerase II inhibitor), hydroxyurea (ribonucleotide reductase inhibitor), 4-nitroquinoline (oxidative DNA damage) and topotecan (topoisomerase I inhibitor). Cell viability was measured after 48 hours using the CellTiter-Glo Luminescent assay. As shown in Fig 5A–5D, R26-CreER Recql4Δ/K525A had a comparable IC50 to Doxorubicin, Hydroxyurea, 4-Nitroquinoline, and Topotecan as the non-tamoxifen treated isogenic controls (Figs 5A–5D and S3). Additionally, R26-CreER Recql4Δ/+ showed a similar IC50 to Doxorubicin, Hydroxyurea, and Topotecan, except for 4-Nitroquinoline, which exhibited a mildly increased resistance in the tamoxifen-treated cells in comparison to the non-tamoxifen treated controls. These results indicate that the role of Recql4 in non-physiological, pharmacologically induced DNA repair does not depend on its helicase activity. The results so far establish that the ATP-dependent helicase activity of Recql4 is not essential in vivo. To directly compare the effects of having a protein truncating/hypomorphic allele compared to a helicase-dead full-length Recql4 protein, we established three additional point mutant alleles (Fig 6A). The G522EfsX43 truncated mutation was generated as a co-incidental mutation during the Crispr/Cas9 mediate generation of the K525A allele and maps closely to the relatively common RTS associated mutations in human RECQL4, S523TfsX35 and C525AfsX33 [6]. We also identified an N-ethyl-N-nitrosourea (ENU) mutagenesis induced truncated mutation R347* (R355 in humans). Three RTS patients have been reported with p. Arg350GlyfsX21 mutations, mapping closely to this allele [6] (S2 Fig). A second ENU induced mutation, M789K mutation (V767 in human), was identified and used as a control for mutations within the Recql4 locus as this mutation was predicted to be benign. We crossed all the individual point mutant alleles (M789K, K525A, G522Efs and R347*) to the R26-CreER Recql4fl/fl line that we previously described and assessed the R26-CreER Recql4fl/PM in parallel with the R26-CreER Recql4fl/+ and R26-CreER Recql4fl/fl allele. [20]. At 8–10 weeks of age, mice were fed tamoxifen containing chow for 30 days to activate the Cre mediated deletion of the wild-type Recql4 floxed allele broadly throughout the body. Using this experimental design, the tamoxifen treated R26-CreER Recql4fl/fl animals (completely Recql4 deficient) developed fully penetrant BM failure [20]. The efficiency of Recql4 deletion was confirmed by PCR for genomic recombination (S1 Fig). Analysis of PB (Fig 6B) showed an approximately 50% reduction in leukocytes and erythrocytes in mice expressing the truncating mutations G522Efs and R347*, very similar to mice rendered null for Recql4 protein expression (Recql4Δ/Δ). The K525A and M789K mutation, as well as the heterozygous (Recql4fl/+) control, did not show any significant change in leukocyte or erythroid indices. In addition, analysis of individual lineages within the PB showed a similar pattern across granulocytes, B lymphocytes, and CD4+ and CD8+ T lymphocytes in the truncating and null mutant (Fig 6B–6D). When the BM was analyzed, leukocytes and erythroid precursors (CD71+Ter119+) from R26-CreERki/+Recql4Δ/G522Efs and Recql4Δ/R347* mice showed a dramatic decrease, consistent with the BM failure phenotype we had previously described in Recql4Δ/Δ (Fig 6E and 6F). The Recql4Δ/+ and K525A and M789K only expressing animals did not develop any phenotype after 30 days of treatment with tamoxifen and, whilst developing a pan-cytopenia in PB and BM, seven of eight G522Efs mice were still alive at the end of the treatment. In contrast, mice expressing the most severely truncating R347* allele developed a profound BM failure and three of five required euthanasia prior to 30 days of treatment, a phenotype similar to complete loss of Recql4 protein (Fig 6G). Collectively, these analyses establish that the ATP-dependent helicase is not required for the physiological functions of Recql4 in vivo, however mutations resulting in truncated protein products are deleterious. The N-terminal Sld2-domain of RECQL4 protein is unique among RecQ family members and has been shown to be critical for the initiation of DNA replication in chicken, Drosophila, Xenopus and human cells [12,17,19,24,25], mostly likely through Sld2-domain dependent recruitment of the MCM10 and CTF4 factors to origins of replication [26]. The importance of the Sld2-domain to cell viability is reflected in the mutation spectrum detected in RTS patients–it is rarely mutated and always intact in at least one allele [6]. These findings implied that RTS and related disorders were caused by the loss of activity of the canonical ATP-dependent helicase domain, whose role in the replication initiation function is less clear. While initial in vitro studies using purified full-length human RECQL4 protein did not detect any unwinding potential on long DNA substrates [27], it was later shown that RECQL4 could unwind shorter duplex regions if a single strand-loading region was provided [16,18]. This activity, together with the single-stranded DNA dependent ATP hydrolyzing activity, was lost in human Walker motif mutants RECQL4K508A and D605A. We now demonstrate that the murine Recql4K525A mutant protein is able to bind DNA but cannot hydrolyze ATP nor unwind DNA substrates, confirming that the mutant is helicase-dead and the homologue of human RECQL4K508A. Unexpectedly, however, we found that this protein behaved comparably to wild-type Recql4 in supporting viability, fertility and normal physiological development of mice. No phenotypes or symptoms consistent with RTS were observed. Perhaps more surprising, was the normal response of Recql4K525A/K525A cells in replication, and in their response to both physiologically or exogenously induced DNA damage. The most highly conserved domain of the RecQ helicases is the ATPase core. By analogy to multiple other DNA helicases (reviewed in ref. [28]), it was assumed that the ATP-dependent helicase activity of RECQL4 is essential for normal cellular DNA metabolism. Several studies have reported findings consistent with this interpretation. Complementation experiments in Drosophila showed that the helicase-dead K898N mutant could not rescue the viability of RecQ4 null mutants [29]. Similarly, a helicase-inactive human D605A mutant could not restore replication of xRecql4 depleted cells in Xenopus egg extracts [12]. Murine studies to date have only assessed complete nulls or severe hypomorphic alleles. The deletion of the exons that precede the helicase domain (exons 5–8) resulted in embryonic lethality by day 3–6 and a significantly truncated protein with no helicase or C-terminal domain [22]. When the entire helicase domain was deleted (exons 9–13; in-frame deletion), mice were viable but showed high rates of perinatal lethality [30] and a truncated protein product of 480aa was predicted. With the aim to maintain an intact protein, a study replaced the last helicase coding exon (13) with a neomycin cassette. 95% of animals died within 2 weeks after birth and a substantial number of short transcripts covering exon 1 to 12 were reported, encoding potentially truncated products [31]. Since all these models effectively create proteins that lack the helicase and C-terminal domain, it is unclear if the observed phenotype could be attributed to the absence of the helicase domain only. Our study demonstrates that mice carrying the K525A helicase-dead mutation, with a stable full-length Recql4 protein, were viable and fertile with no apparent phenotype. The in vivo studies reported herein support a conclusion that the ATP-dependent helicase activity of Recql4 is not essential for replication or viability and that other domains account for these functions. A range of prior in vitro studies have pointed to the importance of the N-terminal Sld2-like region. Lethality of RECQL4-depleted chicken cells was rescued by expression of the N-terminal region only [19]. In addition, the N-terminal domain of RECQL4 was shown to physically interact with several proteins involved in DNA replication in Xenopus laevis and human cell extracts [32,33]. In our in vivo experiments however, we have shown that one copy of the N-terminal region alone (R347* or G522Efs) is insufficient for viability, indicating that a certain level of expression or localization of full-length Recql4 protein is required even if it has no ATP-ase or unwinding capacity. The high frequency of chromosome abnormalities found in cells from RTS patients and the increased cancer incidence rates, suggest that RECQL4 may have a role in maintaining genome stability through DNA repair [2]. Prior studies have attributed several DNA repair functions of RECQL4 to its ATP-dependent helicase region, but they also have noted participation of the N-terminal region. Lu et al. found that a helicase-dead K508M could not rescue the loss of DNA end resection and homologous recombination (HR) repair after RECQL4 siRNA knock down, suggesting that the ATP-dependent helicase function of RECQL4 is involved in HR [34]. However, they also showed that it is the N-terminus of RECQL4 that physically interacts with MRN and CtIP [35]. In a similar fashion, a role for RECQL4 in non-homologous end joining (NHEJ) was linked to its interaction with the Ku70-80 by the N-terminal domain [36]. RECQL4 deficiency has been associated with modulation of core proteins involved in base excision repair (BER) such as POLB, FEN1, and APE1. The latter has shown to specifically interact with the N-terminal region of RECQL4 [37]. Herein, we observed no differences in the sensitivity of helicase-dead mutant cells compared to WT cells in response to various kinds of DNA damage (NHEJ, MMEJ, and HR for doxorubicin, BER for hydroxyurea, NER and BER for 4-nitroquinoline, and BER/SSBR for topotecan). Taken together there was no evidence for a role for the ATP-dependent function of Recql4 in the repair of pathological environmentally induced DNA damage. A recent study showed that RECQL4-depleted U2OS cells were also deficient in ATM dependent checkpoint activation in response to drug induced DNA DSBs. Complementation assays using helicase-inactive point mutants in Walker A (K508G) or Walker B motif (D605A and E606A) further indicated that this was the result of a lack of helicase activity [38]. The ATM pathway plays an equally important role in the physiological processes of DSB repair and recombination, such as V (D) J recombination in T cell development and class switch recombination in B cell activation [39–41]. In our in vivo analysis we did not detect any defect in T cell maturation at the CD4+/CD8+ double positive to CD4+ and CD8+ single positive transition or any earlier stage, nor did the mice develop any T cell lymphomas as a result of chromosomal anomalies. In addition, in vitro B cell activation and class switch recombination in helicase-dead splenic B lymphocytes was indistinguishable from that in WT cells, arguing that the helicase activity is not required for either physiological checkpoint activation or DNA damage repair. RTS patients, however, usually present with compound heterozygous mutations. It was reported that in 46% of RTS patients compound heterozygous mutations were present in the RECQL4 gene [6]. The majority of these mutations affect the helicase and C-terminal region and are predicted to create truncated proteins caused by an early stop codon, frameshift, or mis-splicing [6]. The phenotypes in RTS patients, although grossly similar, can vary widely in severity. The relatively common C525AfsX33 (12 alleles) for example, has been found in all three syndromes (RTS, RAPADILINO and Baller-Gerold) and no single mutation has been assigned to a specific set of clinical features. In our study we demonstrate that mice carrying a sole truncating mutation (G522Efs and R347*) presented with a BM failure reminiscent of the Recql4 null. This was not seen in the helicase-dead K525A, the M789K or the WT heterozygous null mice. Furthermore, when we assessed BM, PB, thymus, and spleen from heterozygous and homozygous K525A helicase-dead mutants, we did not find any significant change. A similar observation was made for the human WRN helicase. A naturally occurring single nucleotide polymorphism (R834C) was shown to have less than 10% of the WT helicase activity, but normal exonuclease activity. None of the heterozygous or homozygous carriers of this mutation developed Werner Syndrome (as defined by the clinical phenotype), clearly separating the WRN helicase function from other WRN functions [42]. Our findings demonstrate that helicase activity of Recql4 is also not required in vivo in mammals. Collectively, this study has demonstrated the ATP-dependent helicase activity of Recql4 is not physiologically essential for murine embryonic development or adult homeostasis, cellular replication and physiological DNA damage repair. However, mutations that create truncating proteins are not tolerated. Further studies will have to be performed to elucidate the complex interactions of Recql4 mutations, their role in RTS and the contribution of the individual Recql4 domains to its normal physiological function. All animal experiments were approved by the Animal Ethics Committee, St. Vincent’s Hospital, Melbourne, Australia (#007/14 and 011/15). Animals were euthanized by CO2 asphyxiation or cervical dislocation. The full-length WT and K525A mutant codon optimized cDNA sequence of the mouse Recql4 containing 3xFLAG tag at the C-terminus were cloned into vector pFL-EGFP and transferred to the Multibac expression system to generate baculovirus [43]. Baculovirus infected High 5 cells were resuspended in TNG buffer (20mM TEA pH7. 5,150mM NaCl, 10% glycerol, 1mM EDTA, 1x mammalian protease inhibitors (Sigma-Aldrich) and 1mM DTT. Mixtures were sonicated three times for 30 seconds on ice. Lysates were clarified by centrifugation at 50K x g 30 minutes. Anti-Flag M2–Affinity Gel (Sigma-Aldrich) resin was added and incubated for 60 minutes. Resin was extensively washed with TNG (without PI or EDTA), then washed overnight in TNG containing Benzonase nuclease (Sigma-Aldrich). Further washes were performed to remove the nuclease. Subsequently Recql4 was eluted with 100ug/ml Flag peptide in TNG. Helicase assays were performed according to Kaiser et al [18]. 80μl reactions containing 0. 5μM protein and 50nM of a 15nt 3’-overhang (OH) DNA substrate in assay buffer (20mM HEPES pH 8. 0,10mM NaCl, 5% Glycerol, 1mM MgCl2,0. 5mM TCEP) were assayed in an EnSpire 2300 microplate reader (Perkin Elmer) at 25°C. The DNA substrate (T3-Cy3 annealed to B1-Dab) contained a 3′-15nt polyT ssDNA loading site and a 15nt dsDNA part with a generic sequence. After recording baseline fluorescence for 60s (Excitation 530nm / Emission 580nm), the helicase reaction was initiated by adding ATP to a final concentration of 1. 25mM and the increasing Cy3-fluorescence as the quencher-labelled bottom-DNA strand is separated from the Cy3-labelled top-DNA strand, was recorded for 5 min. Measurements using H2O in place of ATP as well as reactions with buffer instead of protein served as blank and were subtracted from the ATP-data. PiColorLock phosphate detection reagent (Expedeon) was used to measure the presence of inorganic phosphate (Pi) release from ATP as a measure Recql4 ATPase activity. The proteins were assayed at 115nM in the presence of 1mM ATP and DNA in assay buffer (20mM HEPES pH 8. 0,10mM NaCl, 5% Glycerol, 1mM MgCl2,0. 5mM TCEP). Color change was measured at Abs650nm in an EnSpire 2300 microplate reader (Perkin Elmer) at 25°C. Electro-mobility shift assay (EMSA) was used to measure the relative binding of WT versus K525A mutant Recql4 to DNA binding. Protein was serially diluted from 300nM to 19nM and bound to 25nM single stranded DNA oligo XOm1 conjugated to IRDye680. Bound Protein-DNA complex was separated on a 6% TBE/Acrylamide gel. The gel was directly imaged on a Li-Cor Odyssey CLx near-infrared fluorescence imaging system (Millennium Science). Recql4K525A and Recql4G522Efs mice were generated using Cripsr/Cas9 methods by the Mouse Engineering at Garvan/ABR (MEGA) services (Garvan Institute, Darlinghurst, Australia). Lysine 525 was mutated to Alanine (AAG>GCA) in single cell C57Bl/6 embryos via sgRNA-directed gene targeting and homologous recombination with a single stranded DNA oligonucleotide substrate. Viable pups were screened by DNA sequencing and one C57Bl/6 male carrying the K525A mutation on one allele and a 2bp insertion (GA) after the T521 codon (G522Efs) on the other allele was identified as a founder. The chemically (ENU) induced Recql4M789K and Recql4R347X mutations were obtained from the Australian Phenomics Facility (APF, Canberra, Australia: IGL01381 and IGL01809). Recql4fl/fl mice (C57BL/6-Recql4tm2272Arte) have been previously described [20,44]. Rosa26-CreERT2 mice on a C57Bl/6 background were purchased from The Jackson Laboratory (B6. 129-Gt (ROSA) 26Sortm1 (cre/ERT2) Tyj/J, Stock Number: 008463) and have been previously described [20]. All lines were on a backcrossed C57Bl/6 background. ENU mutants were outcrossed at least 6 times and assessed across multiple generations to eliminate effects of any additional mutations. Tamoxifen containing food was prepared by Specialty Feeds (Perth, Australia) at 400mg/kg tamoxifen citrate (Sigma Aldrich) in a base of standard mouse chow. Genotyping of the K525A mutants was performed by PCR using the following primers: mRecql4 K525A MO36-F9: 5’-TAGACCTTATGAAACCTCAAAGCC-3’ and mRecql4 K525A MO36-R3: 5’- AGAACATTGGGCATTCGGC-3’ to yield a 591bp product, which was then digested with MslI (NEB) restriction enzyme to generate two fragments of 416 and 175bp for the WT or three fragments of 361,175 and 55bp for the K525A mutant. Primers for the M789K mutants are: mRecql4 M789K 1F: 5’- AATAGGTGGCAATGGGCAGG-3’ and: mRecql4 M789K 1R: 5’-GCACTCGGCGAAAGGATACA-3’ yielding a 420bp PCR product, uncut by MslI when M789K mutant, but cut in two (277 and 143bp) when WT. The presence of the G522Efs and R347X mutations was determined by KASP (competitive allele specific PCR) technology (LGC) with custom designed (G522Efs) or facility provided (R347X primer: 5’- GAAGGTGACCAAGTTCATGCTAAAGCGTTTGTTTTTCATGTTGAGTCG-3’, 5’- GAAGGTCGGAGTCAACGGATTCAAAGCGTTTGTTTTTCATGTTGAGTCA-3’, reverse primer 5’-GCTTCCCTAGACAGAGGGAACTATA-3’) sequences according to manufacturer instructions. Thymocyte lysates were prepared in RIPA buffer (50mM Tris, 150mM NaCl, 1% NP-40,0. 5% sodium deoxycholate, 0. 1% SDS, pH8. 0) plus Complete protease inhibitor (Roche, 04693116001) and PhosStop (Roche, 4906837001) tablets. 25μg whole protein extracts were fractionated on pre-cast NuPAGE BOLT 8% Bis-Tris polyacrylamide gels (Invitrogen) and transferred onto Immobilon-P PVDF membranes (Merck Millipore). Membranes were blocked with 5% milk in TBST and incubated overnight with rat monoclonal anti-mouse Recql4 antibody (clone 3B10, WEHI Antibody Services, Walter and Eliza Hall Institute Biotechnology Centre) or mouse anti-β-Actin (Sigma Aldrich, A1978). Membranes were then probed with HRP-conjugated goat anti-rat (Thermo Fisher Scientific, 31470) or anti-mouse (Thermo Fisher Scientific, 31444) secondary antibodies and visualized using ECL Prime Substrate (Amersham). Peripheral blood was analyzed on a hematological analyzer (Sysmex KX-21N, Roche Diagnostics). For flow cytometric analysis, red blood cells were lysed using a red blood cell lysis buffer (150mM NH4Cl, 10mM KHCO3,0. 1mM Na2EDTA, pH7. 3). Bones were flushed, spleens and thymus crushed, and single cell suspensions were prepared in PBS containing 2% FBS. Antibodies against murine Ter119, CD71, B220, IgM, CD43, CD19, CD21, CD23, Mac-1, Gr1, F4/80, CD4, CD8, TCRβ, CD25, CD44, Sca-1, c-Kit, CD34, FLT3, FcγRII/III (CD16/32), CD41, CD105, CD150, either biotinylated or conjugated with phycoerythrin, phycoerythrin-Cy7, peridinin chlorophyll protein-Cy5. 5, allophycocyanin, allophycocyanin eFluor780, eFluor660 or eFluor450 were all obtained from eBioscience, BioLegend or BD Pharmingen (S1 Table) [20,45,46]. Biotinylated antibodies were detected with streptavidin conjugated with Brilliant Violet-605. 30,000–500,000 cells were acquired on a BD LSRIIFortessa and analyzed with FlowJo software Version 9 or 10. 0 (Treestar). B cells were purified from single cell spleen suspensions using a B Cell Isolation kit (Miltenyi, 130-090-862); 106 cells per well were cultured in 6-well plates for 4 days in RPMI supplemented with 10% FCS, 100U/ml penicillin, 100ng/ml streptomycin, 2mM L-glutamine, 1 x MEM nonessential amino acids, 1mM sodium pyruvate, 50μM ß-mercaptoethanol, 15μg/ml LPS (Invivogen, tlrl-3pelps) and 10ng/ml recombinant murine IL-4 (Peprotech, 214–14), and stained with CellTrace Violet (Thermo Fisher Scientific, C34557) and rat anti-mouse IgG1-APC (BD Pharmingen, 550874) [47]. Stained cells were assessed using a LSRIIFortessa (BD) and data analysed using FACSDiva (BD) or FlowJo (Tree Star) software. Hoxb8 immortalized [23] R26-CreERT2 Recql4fl/+ (control) and R26-CreERT2 Recql4fl/K525A cells were maintained in IMDM, 10% FBS (non-heat inactivated) and 1% GM-CSF containing media (BHK-HM5 cell conditioned media). The cells were treated for 4 days with 400nM 4-hydroxy tamoxifen (Merck Millipore) then genotyped to confirm complete recombination. Cells were then plated at 10,000 cells/well in 96 well plates (Corning, CLS3610) and incubated for 48 hours with the indicated concentration of drugs in triplicates per dose (dose range Doxorubicin: 0–0. 5μM, Hydroxyurea: 0–0. 5mM, 4-Nitroquinoline: 0–2μM and Topotecan: 0–0. 5mM). Doxorubicin was obtained from St. Vincent’s Hospital Pharmacy. Hydroxyurea was purchased from Selleck. 4-Nitroquinoline and Topotecan were purchased from Sigma-Aldrich. Cell viability was measured using ATP-Lite (Perkin Elmer) as directed by the manufacturer and measured on an EnSpire plate reader (Perkin Elmer). Data were plotted and the IC50 value calculated using Prism 7 software. The dose-response curve was plotted as mean±SEM.

DNA helicases unwind double-stranded nucleic acids using energy from ATP to access genetic information during cell replication. In humans, several families of helicases have been described and one of particular importance is the RecQ family, where mutations in three of five members cause human disease. RECQL4 is a member of this family and its mutation results in Rothmund-Thomson syndrome (RTS). Prior studies have shown that defects in the helicase region of RECQL4 may contribute to the disease, but no studies have specifically assessed the biological effects of its absence in a whole animal model. In this study, we generated a mouse model with a specific point mutation resulting in a helicase-inactive Recql4 protein. We found that an absence of ATP-dependent helicase activity does not perturb the physiological functions of Recql4 with the homozygous mutants being normal. In contrast, when we assessed point mutations that generate protein truncations these were pathogenic. Our results suggest that the helicase function of Recql4 is not essential for its physiological functions and that other domains of this protein might account for its functions in diseases such as RTS.

lay_plos

Madame Merle, who had come to Florence on Mrs. Touchett's arrival at the invitation of this lady--Mrs. Touchett offering her for a month the hospitality of Palazzo Crescentini--the judicious Madame Merle spoke to Isabel afresh about Gilbert Osmond and expressed the hope she might know him; making, however, no such point of the matter as we have seen her do in recommending the girl herself to Mr. Osmond's attention. The reason of this was perhaps that Isabel offered no resistance whatever to Madame Merle's proposal. In Italy, as in England, the lady had a multitude of friends, both among the natives of the country and its heterogeneous visitors. She had mentioned to Isabel most of the people the girl would find it well to "meet"--of course, she said, Isabel could know whomever in the wide world she would--and had placed Mr. Osmond near the top of the list. He was an old friend of her own; she had known him these dozen years; he was one of the cleverest and most agreeable men--well, in Europe simply. He was altogether above the respectable average; quite another affair. He wasn't a professional charmer--far from it, and the effect he produced depended a good deal on the state of his nerves and his spirits. When not in the right mood he could fall as low as any one, saved only by his looking at such hours rather like a demoralised prince in exile. But if he cared or was interested or rightly challenged--just exactly rightly it had to be--then one felt his cleverness and his distinction. Those qualities didn't depend, in him, as in so many people, on his not committing or exposing himself. He had his perversities--which indeed Isabel would find to be the case with all the men really worth knowing--and didn't cause his light to shine equally for all persons. Madame Merle, however, thought she could undertake that for Isabel he would be brilliant. He was easily bored, too easily, and dull people always put him out; but a quick and cultivated girl like Isabel would give him a stimulus which was too absent from his life. At any rate he was a person not to miss. One shouldn't attempt to live in Italy without making a friend of Gilbert Osmond, who knew more about the country than any one except two or three German professors. And if they had more knowledge than he it was he who had most perception and taste--being artistic through and through. Isabel remembered that her friend had spoken of him during their plunge, at Gardencourt, into the deeps of talk, and wondered a little what was the nature of the tie binding these superior spirits. She felt that Madame Merle's ties always somehow had histories, and such an impression was part of the interest created by this inordinate woman. As regards her relations with Mr. Osmond, however, she hinted at nothing but a long-established calm friendship. Isabel said she should be happy to know a person who had enjoyed so high a confidence for so many years. "You ought to see a great many men," Madame Merle remarked; "you ought to see as many as possible, so as to get used to them." "Used to them?" Isabel repeated with that solemn stare which sometimes seemed to proclaim her deficient in the sense of comedy. "Why, I'm not afraid of them--I'm as used to them as the cook to the butcher-boys." "Used to them, I mean, so as to despise them. That's what one comes to with most of them. You'll pick out, for your society, the few whom you don't despise." This was a note of cynicism that Madame Merle didn't often allow herself to sound; but Isabel was not alarmed, for she had never supposed that as one saw more of the world the sentiment of respect became the most active of one's emotions. It was excited, none the less, by the beautiful city of Florence, which pleased her not less than Madame Merle had promised; and if her unassisted perception had not been able to gauge its charms she had clever companions as priests to the mystery. She was--in no want indeed of esthetic illumination, for Ralph found it a joy that renewed his own early passion to act as cicerone to his eager young kinswoman. Madame Merle remained at home; she had seen the treasures of Florence again and again and had always something else to do. But she talked of all things with remarkable vividness of memory--she recalled the right-hand corner of the large Perugino and the position of the hands of the Saint Elizabeth in the picture next to it. She had her opinions as to the character of many famous works of art, differing often from Ralph with great sharpness and defending her interpretations with as much ingenuity as good-humour. Isabel listened to the discussions taking place between the two with a sense that she might derive much benefit from them and that they were among the advantages she couldn't have enjoyed for instance in Albany. In the clear May mornings before the formal breakfast--this repast at Mrs. Touchett's was served at twelve o'clock--she wandered with her cousin through the narrow and sombre Florentine streets, resting a while in the thicker dusk of some historic church or the vaulted chambers of some dispeopled convent. She went to the galleries and palaces; she looked at the pictures and statues that had hitherto been great names to her, and exchanged for a knowledge which was sometimes a limitation a presentiment which proved usually to have been a blank. She performed all those acts of mental prostration in which, on a first visit to Italy, youth and enthusiasm so freely indulge; she felt her heart beat in the presence of immortal genius and knew the sweetness of rising tears in eyes to which faded fresco and darkened marble grew dim. But the return, every day, was even pleasanter than the going forth; the return into the wide, monumental court of the great house in which Mrs. Touchett, many years before, had established herself, and into the high, cool rooms where the carven rafters and pompous frescoes of the sixteenth century looked down on the familiar commodities of the age of advertisement. Mrs. Touchett inhabited an historic building in a narrow street whose very name recalled the strife of medieval factions; and found compensation for the darkness of her frontage in the modicity of her rent and the brightness of a garden where nature itself looked as archaic as the rugged architecture of the palace and which cleared and scented the rooms in regular use. To live in such a place was, for Isabel, to hold to her ear all day a shell of the sea of the past. This vague eternal rumour kept her imagination awake. Gilbert Osmond came to see Madame Merle, who presented him to the young lady lurking at the other side of the room. Isabel took on this occasion little part in the talk; she scarcely even smiled when the others turned to her invitingly; she sat there as if she had been at the play and had paid even a large sum for her place. Mrs. Touchett was not present, and these two had it, for the effect of brilliancy, all their own way. They talked of the Florentine, the Roman, the cosmopolite world, and might have been distinguished performers figuring for a charity. It all had the rich readiness that would have come from rehearsal. Madame Merle appealed to her as if she had been on the stage, but she could ignore any learnt cue without spoiling the scene--though of course she thus put dreadfully in the wrong the friend who had told Mr. Osmond she could be depended on. This was no matter for once; even if more had been involved she could have made no attempt to shine. There was something in the visitor that checked her and held her in suspense--made it more important she should get an impression of him than that she should produce one herself. Besides, she had little skill in producing an impression which she knew to be expected: nothing could be happier, in general, than to seem dazzling, but she had a perverse unwillingness to glitter by arrangement. Mr. Osmond, to do him justice, had a well-bred air of expecting nothing, a quiet ease that covered everything, even the first show of his own wit. This was the more grateful as his face, his head, was sensitive; he was not handsome, but he was fine, as fine as one of the drawings in the long gallery above the bridge of the Uffizi. And his very voice was fine--the more strangely that, with its clearness, it yet somehow wasn't sweet. This had had really to do with making her abstain from interference. His utterance was the vibration of glass, and if she had put out her finger she might have changed the pitch and spoiled the concert. Yet before he went she had to speak. "Madame Merle," he said, "consents to come up to my hill-top some day next week and drink tea in my garden. It would give me much pleasure if you would come with her. It's thought rather pretty--there's what they call a general view. My daughter too would be so glad--or rather, for she's too young to have strong emotions, I should be so glad--so very glad." And Mr. Osmond paused with a slight air of embarrassment, leaving his sentence unfinished. "I should be so happy if you could know my daughter," he went on a moment afterwards. Isabel replied that she should be delighted to see Miss Osmond and that if Madame Merle would show her the way to the hill-top she should be very grateful. Upon this assurance the visitor took his leave; after which Isabel fully expected her friend would scold her for having been so stupid. But to her surprise that lady, who indeed never fell into the mere matter-of-course, said to her in a few moments, "You were charming, my dear; you were just as one would have wished you. You're never disappointing." A rebuke might possibly have been irritating, though it is much more probable that Isabel would have taken it in good part; but, strange to say, the words that Madame Merle actually used caused her the first feeling of displeasure she had known this ally to excite. "That's more than I intended," she answered coldly. "I'm under no obligation that I know of to charm Mr. Osmond." Madame Merle perceptibly flushed, but we know it was not her habit to retract. "My dear child, I didn't speak for him, poor man; I spoke for yourself. It's not of course a question as to his liking you; it matters little whether he likes you or not! But I thought you liked HIM." "I did," said Isabel honestly. "But I don't see what that matters either." "Everything that concerns you matters to me," Madame Merle returned with her weary nobleness; "especially when at the same time another old friend's concerned." Whatever Isabel's obligations may have been to Mr. Osmond, it must be admitted that she found them sufficient to lead her to put to Ralph sundry questions about him. She thought Ralph's judgements distorted by his trials, but she flattered herself she had learned to make allowance for that. "Do I know him?" said her cousin. "Oh, yes, I 'know' him; not well, but on the whole enough. I've never cultivated his society, and he apparently has never found mine indispensable to his happiness. Who is he, what is he? He's a vague, unexplained American who has been living these thirty years, or less, in Italy. Why do I call him unexplained? Only as a cover for my ignorance; I don't know his antecedents, his family, his origin. For all I do know he may be a prince in disguise; he rather looks like one, by the way--like a prince who has abdicated in a fit of fastidiousness and has been in a state of disgust ever since. He used to live in Rome; but of late years he has taken up his abode here; I remember hearing him say that Rome has grown vulgar. He has a great dread of vulgarity; that's his special line; he hasn't any other that I know of. He lives on his income, which I suspect of not being vulgarly large. He's a poor but honest gentleman that's what he calls himself. He married young and lost his wife, and I believe he has a daughter. He also has a sister, who's married to some small Count or other, of these parts; I remember meeting her of old. She's nicer than he, I should think, but rather impossible. I remember there used to be some stories about her. I don't think I recommend you to know her. But why don't you ask Madame Merle about these people? She knows them all much better than I." "I ask you because I want your opinion as well as hers," said Isabel. "A fig for my opinion! If you fall in love with Mr. Osmond what will you care for that?" "Not much, probably. But meanwhile it has a certain importance. The more information one has about one's dangers the better." "I don't agree to that--it may make them dangers. We know too much about people in these days; we hear too much. Our ears, our minds, our mouths, are stuffed with personalities. Don't mind anything any one tells you about any one else. Judge everyone and everything for yourself." "That's what I try to do," said Isabel "but when you do that people call you conceited." "You're not to mind them--that's precisely my argument; not to mind what they say about yourself any more than what they say about your friend or your enemy." Isabel considered. "I think you're right; but there are some things I can't help minding: for instance when my friend's attacked or when I myself am praised." "Of course you're always at liberty to judge the critic. Judge people as critics, however," Ralph added, "and you'll condemn them all!" "I shall see Mr. Osmond for myself," said Isabel. "I've promised to pay him a visit." "To pay him a visit?" "To go and see his view, his pictures, his daughter--I don't know exactly what. Madame Merle's to take me; she tells me a great many ladies call on him." "Ah, with Madame Merle you may go anywhere, de confiance," said Ralph. "She knows none but the best people." Isabel said no more about Mr. Osmond, but she presently remarked to her cousin that she was not satisfied with his tone about Madame Merle. "It seems to me you insinuate things about her. I don't know what you mean, but if you've any grounds for disliking her I think you should either mention them frankly or else say nothing at all." Ralph, however, resented this charge with more apparent earnestness than he commonly used. "I speak of Madame Merle exactly as I speak to her: with an even exaggerated respect." "Exaggerated, precisely. That's what I complain of." "I do so because Madame Merle's merits are exaggerated." "By whom, pray? By me? If so I do her a poor service." "No, no; by herself." "Ah, I protest!" Isabel earnestly cried. "If ever there was a woman who made small claims--!" "You put your finger on it," Ralph interrupted. "Her modesty's exaggerated. She has no business with small claims--she has a perfect right to make large ones." "Her merits are large then. You contradict yourself." "Her merits are immense," said Ralph. "She's indescribably blameless; a pathless desert of virtue; the only woman I know who never gives one a chance." "A chance for what?" "Well, say to call her a fool! She's the only woman I know who has but that one little fault." Isabel turned away with impatience. "I don't understand you; you're too paradoxical for my plain mind." "Let me explain. When I say she exaggerates I don't mean it in the vulgar sense--that she boasts, overstates, gives too fine an account of herself. I mean literally that she pushes the search for perfection too far--that her merits are in themselves overstrained. She's too good, too kind, too clever, too learned, too accomplished, too everything. She's too complete, in a word. I confess to you that she acts on my nerves and that I feel about her a good deal as that intensely human Athenian felt about Aristides the Just." Isabel looked hard at her cousin; but the mocking spirit, if it lurked in his words, failed on this occasion to peep from his face. "Do you wish Madame Merle to be banished?" "By no means. She's much too good company. I delight in Madame Merle," said Ralph Touchett simply. "You're very odious, sir!" Isabel exclaimed. And then she asked him if he knew anything that was not to the honour of her brilliant friend. "Nothing whatever. Don't you see that's just what I mean? On the character of every one else you may find some little black speck; if I were to take half an hour to it, some day, I've no doubt I should be able to find one on yours. For my own, of course, I'm spotted like a leopard. But on Madame Merle's nothing, nothing, nothing!" "That's just what I think!" said Isabel with a toss of her head. "That is why I like her so much." "She's a capital person for you to know. Since you wish to see the world you couldn't have a better guide." "I suppose you mean by that that she's worldly?" "Worldly? No," said Ralph, "she's the great round world itself!" It had certainly not, as Isabel for the moment took it into her head to believe, been a refinement of malice in him to say that he delighted in Madame Merle. Ralph Touchett took his refreshment wherever he could find it, and he would not have forgiven himself if he had been left wholly unbeguiled by such a mistress of the social art. There are deep-lying sympathies and antipathies, and it may have been that, in spite of the administered justice she enjoyed at his hands, her absence from his mother's house would not have made life barren to him. But Ralph Touchett had learned more or less inscrutably to attend, and there could have been nothing so "sustained" to attend to as the general performance of Madame Merle. He tasted her in sips, he let her stand, with an opportuneness she herself could not have surpassed. There were moments when he felt almost sorry for her; and these, oddly enough, were the moments when his kindness was least demonstrative. He was sure she had been yearningly ambitious and that what she had visibly accomplished was far below her secret measure. She had got herself into perfect training, but had won none of the prizes. She was always plain Madame Merle, the widow of a Swiss negociant, with a small income and a large acquaintance, who stayed with people a great deal and was almost as universally "liked" as some new volume of smooth twaddle. The contrast between this position and any one of some half-dozen others that he supposed to have at various moments engaged her hope had an element of the tragical. His mother thought he got on beautifully with their genial guest; to Mrs. Touchett's sense two persons who dealt so largely in too-ingenious theories of conduct--that is of their own--would have much in common. He had given due consideration to Isabel's intimacy with her eminent friend, having long since made up his mind that he could not, without opposition, keep his cousin to himself; and he made the best of it, as he had done of worse things. He believed it would take care of itself; it wouldn't last forever. Neither of these two superior persons knew the other as well as she supposed, and when each had made an important discovery or two there would be, if not a rupture, at least a relaxation. Meanwhile he was quite willing to admit that the conversation of the elder lady was an advantage to the younger, who had a great deal to learn and would doubtless learn it better from Madame Merle than from some other instructors of the young. It was not probable that Isabel would be injured.

Madame Merle returns to Palazzo Crescentini, Mrs. Touchett's home in Florence. Madame Merle recommends Osmond to Isabel once more, telling her of his impressive traits. She ominously tells Isabel that she should interact with more men, so that she gets "used" to them - that is to say, so she learns which ones to despise. Uh... huh. Okay, Madame Merle. Ralph takes Isabel out on the town; she's delighted with Florence. Madame Merle has been there many times before, and can converse with them about the city, although she does not go out with them. Isabel loves Mrs. Touchett's house, and thinks that it is so rich with history and tradition that it's like living in the past. Gilbert Osmond pays a visit to Madame Merle and Isabel. Madame Merle and Osmond talk for most of the time and Isabel, for once, is mostly silent. Osmond invites Madame Merle and Isabel to his house next week. He suggests that he would like Isabel to meet Pansy. Isabel accepts the invitation. After Osmond leaves, Madame Merle commends Isabel for behaving just as she should have. Condescend much? Isabel is duly annoyed by this comment, and Madame Merle sweet-talks her back into a good mood. Isabel asks Ralph about Osmond, and Ralph confesses that he doesn't know much. He suspects that there's something shady about Osmond's sister. Ralph recommends that Isabel follows her own instincts, and doesn't listen to people's opinions about others. Ralph says that, as long as Isabel is with Madame Merle, she's in good hands. Isabel detects the usual trace of sarcasm when Ralph talks about Madame Merle, and asks him about it. Ralph says that Madame Merle is too perfect and too modest for how perfect she is. Right on the dot, Ralph. However, despite his misgivings about Madame Merle's creepy so-called perfection, he figures that Isabel can benefit from being friends with the older woman. We'll see about that....

booksum

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. application Ser. No. 10/141,494, filed on May 7, 2002, which is a continuation of U.S. application Ser. No. 09/608,940, filed on Jun. 30, 2000, now U.S. Pat. No. 6,383,156, which claims the benefit of U.S. provisional application Ser. No. 60/156,342, filed on Sep. 27, 1999. Each of the priority applications is hereby incorporated by reference herein. BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present application relates to orthopaedic braces adapted with an adjustable-length strut for use in stabilizing ajoint after invasive surgery. [0004] 2. Description of the Related Art [0005] In order to ensure the proper healing of a human joint after an injury or invasive surgery, it is often desirable to limit the pivotal motion of the human joint to a predetermined angular range between full extension and full flexion. The pivotal motion may be limited by a range of motion hinge disposed between an upper strut and a lower strut. In order for the orthopaedic brace to function properly, the struts must be adaptable to the body proportions of the patient. [0006] The following U.S. patents, which describe orthopaedic braces of this general type, are herein incorporated by reference to establish the nature of such range of motion braces, and how and why such equipment is used. U.S. Pat. No. 552,143 issued on Dec. 31, 1895; U.S. Pat. No. 649,237 issued on May 8, 1900; U.S. Pat. No. 4,776,326 issued to Young et al., on Oct. 11, 1988 entitled “Modular Lower Limb Bracing System”; U.S. Pat. No. 4,817,588 issued to Bledsoe on Apr. 4, 1989 entitled “Motion Restraining Knee Brace”; U.S. Pat. No. 4,982,732 issued to Morris on Jan. 8, 1991 entitled “Orthopedic Rehabilitation Knee Brace”; U.S. Pat. No. 5,052,379 issued to Airy et al., on Oct. 1, 1991 entitled “Combination Brace and Wearable Exercise Apparatus for Body Joints”; and U.S. Pat. No. 5,018,514 issued to Grood et al., on May 28, 1991 entitled “Knee Brace”. [0007] It is well known that the orthopaedic braces described in the aforementioned incorporated patents suffer various problems, shortcomings and disadvantages. In some cases such braces cannot be adjusted to fit the patient, rather, the braces come in various fixed sizes. Alternatively, the braces are not easily adjustable, requiring, for example, tools to change the size of the struts. Some braces require actual cutting or breaking off pieces of the struts to permanently change the length of the struts. Others rely upon friction, as from a tightening screw, to less than positively lock the strut at the desire length. [0008] It is thus an object of the present invention to provide an orthopaedic brace that is easy to adjust. [0009] It is thus another object of the present invention to provide an orthopaedic brace that is adjustable without a need for tools. [0010] It is thus further an object of the present invention to provide an orthopaedic brace that is adjustable without cutting or breaking a strut. SUMMARY OF THE INVENTION [0011] The present invention is an orthopaedic brace that has adjustable length struts. [0012] In one form, the present invention is an orthopaedic brace including a first strut, a second strut, a hinge disposed between the first and second struts, and an adjustment assembly disposed on one of the first and second struts. The hinge is configured to allow movement of one of the first and second struts about an axis defined by the hinge. The adjustment assembly is configured to cooperate with the one of the first and second struts to adjustably set an operative length of the one of the first and second struts. [0013] In another form, the present invention is an orthopaedic brace including an upper strut, a lower strut, a hinge disposed between the upper strut and the lower strut, and an adjustment assembly disposed on one of the first and second struts. The hinge is configured to allow movement of one of the upper and lower struts about an axis defined by the hinge. One of the upper and lower struts has a plurality of notches defining a plurality of strut length settings. The adjustment assembly is configured to cooperate with any one of the plurality of notches of the one of the first and second struts to selectively set a length of the one of the first and second struts. [0014] In yet another form, the present invention is an orthopaedic brace including an upper strut, a lower strut, a hinge disposed between the upper strut and the lower strut, an upper adjustment assembly disposed on the upper strut, and a lower adjustment assembly disposed on the lower strut. The hinge is configured to allow movement of one of the upper and lower struts about an axis defined by the hinge. The upper adjustment assembly is configured to cooperate with the upper strut to adjustably set a length of the upper strut. The lower adjustment assembly is configured to cooperate with the lower strut to adjustably set a length of the lower strut. [0015] Accordingly, the present invention improves upon the prior art by providing an orthopaedic brace strut that may be changed in length without the use of tools and with the ability to return to the original length, or some other length as desired. [0016] The present invention also provides for a single-action positive lock for a strut length adjustment assembly rather than relying on friction. The ability to size and resize the struts provides a cost-effective and comfortable means to apply an orthopaedic brace to virtually any joint on the human body and eliminates the need to carry large inventories of braces that cannot be sized. By providing a positive lock, the improved brace also better protects the patient and speeds recovery. [0017] The present invention also allows contoured wings, with cushioning material and/or non-slip material attached, to be used to limit movement of the brace after it has been attached and to provide increased comfort to the patient. BRIEF DESCRIPTION OF THE DRAWINGS [0018] The above-mentioned and other features and advantages of this invention, and the manner of attaining them, will become more apparent and the invention will be better understood by reference to the following description of embodiments of the invention taken in conjunction with the accompanying drawings, wherein: [0019] FIG. 1 is a side perspective view of an adjustable orthopaedic brace assembly having adjustable-length strut assemblies that embodies principles of the present invention showing the brace operatively connected to a human leg; [0020] FIGS. 2A and 2B are, respectively, top and underside perspective views of an adjustable-length strut assembly for the orthopaedic brace of FIG. 1 ; [0021] FIG. 3 is an exploded, perspective view of the adjustable-length strut assembly of FIGS. 2A and 2B ; [0022] FIGS. 4A and 4B are cross-sectional views through the adjustable-length strut assembly taken along line 4 - 4 of FIG. 3 ; [0023] FIG. 5 is a perspective view of a second embodiment of an adjustable-length strut assembly; [0024] FIG. 6 is a cross-sectional view through the second embodiment of the adjustable-length strut assembly taken along line 6 - 6 of FIG. 5 ; [0025] FIG. 7 is a cross-sectional view through the second embodiment of the adjustable-length strut assembly taken along line 7 - 7 of FIG. 6 ; and [0026] FIG. 8 is a perspective view of a third embodiment of an adjustable-length strut assembly. [0027] Corresponding reference characters indicate corresponding parts throughout the several views. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0028] An orthopaedic brace 10 is shown in FIG. 1 operatively attached to a leg 64 using a plurality of straps 54 mounted on an upper strut 12 and a lower strut 14 with a hinge assembly 16 disposed between the upper strut 12 and the lower strut 14. While only one side of the orthopaedic brace 10 is shown (i.e. the hinge assembly 16, the upper strut 12, and the lower strut 14 or “assembly”) it should be understood that an identical, but mirror image, assembly is provided on the opposite side of the leg 64. [0029] Each strut 12 and 14 is provided with a preferably identically configured wing assembly 18 although variations in either are contemplated, which is slidably mounted for adjustable movement on the elongated struts 12 and 14. Stated in another manner, each strut 12 and 14 is adjustable in length relative to the length of the strut between the hinge 16 and the straps 54 through adjustable strut assemblies 18. Such will be considered hereafter as the length adjustment of a strut. It should be appreciated that such assemblies 18 may be provided on both struts 12 and 14, or only on one of the two struts 12 and 14. As well, it should also be appreciated that adjustability of the length of a strut may be considered as either or both the adjustment of the assembly 18 relative to a strut ( 12 and/or 14 ), or as the adjustment of a strut ( 12 and/or 14 ) relative to the assembly 18. [0030] The adjustable mounting of the wing assembly 18 on elongated struts 12 and 14 allows the struts to telescope or move in and out, one in opposition to the other, of the respective wing assembly 18, as will be described subsequently, to accommodate long or short legs, as one example, or long or short arms, as another example. Because the structure and function of the wing assembly is similar regardless of whether mounted to the upper strut 12 or the lower strut 14, reference will be made to only the upper strut 12 in the following description and its wing assembly 18. As well, because the structure and function of the struts 12 and 14 are identical (assuming each strut terminates in a wing assembly 18 ), reference to strut 12 in the following description will be construed to pertain to strut 14. [0031] Referring to FIG. 2A, the wing assembly 18 has a wing body 20, which is preferably formed of a relatively rigid material, as for example plastic. The wing body 20 has an arcuate profile and is provided with one or more strap-retaining loops 22 for receiving the one or more adjustable straps 54 that are threaded through the loops 22 to encircle both the wing assembly 18 and a human limb, such as the leg 64 (as depicted in FIG. 1 ), thereby immovably securing the brace 10 to the leg 64, for example. FIG. 2B shows that the underside of the arcuate-shaped wing body 20 is provided with a generous layer of non-slip cushioning 50, both to pad the wearer&#39;s limb and to assure that the brace 10 remains in place. [0032] FIGS. 2B, 3, 4 A and 4 B reveal that the underside of the wing body 20 defines a unitary channel 46 that runs longitudinally down the entire length the wing body 20. While the channel 46 is generally open, splitting the cushioning 50 into two halves, a lip 48 portion of the wing body 20 overhangs the channel 46 at each of the side edges of the channel 46 down the entire longitudinal length of each side of the channel 46. The channel 46 with opposing lips 48 receives the elongated strut 12 and retains and guides the strut 12 as it telescopes in and out of the channel 46. The open nature of the channel 46 also helps to reduce the overall weight of the orthopaedic brace 10. [0033] Referring to FIGS. 2B and 3, the strut 12 has formed through its body a longitudinal slot 60. The length of the slot 60 may be varied depending upon the desired maximum and minimum lengths of the orthopaedic brace 10. Longitudinally spaced down each side of the slot 60 are a plurality of arcuate-shaped, stop notches generally designated 62. The notches 62 are equally divided into a plurality of notches 62 a that are mirror images of, and directly across the slot 60 from, a plurality of opposing notches 62 b, such that the opposing, arcuate-shaped pairs of notches 62 a and 62 b would define a circle if their ends were connected by an arc of constant radius equal to the distance from the center of the slot 60 to the center of the opposing notches. One end of the slot 60 contains an arcuate-shaped notch 62 c and the other end of the slot 60 contains a mirror image arcuate-shaped notch 62 d. Notches 62 c and 62 d are connected on each end to the outer ends of notches 62 a and 62 b. It should be appreciated that the notches may be shaped other than that shown. [0034] Referring to FIG. 3, it can be seen that the wing body 20 also defines a depression or chamber 28 on the top of the body 20 which is shown as circular but can be any shape. The wing body 20 also defines an aperture 26 of smaller diameter than the chamber 28 that extends through the center of the chamber 28 all the way to the slot 60 on the underside of the wing body 20. The chamber 28 and aperture 26 are adapted to house a positive-lock, adjustment or button assembly 30. [0035] The adjustment assembly 30 ( FIG. 3 ) has a generally flat pushbutton top 32 that has a cylindrical extension 34 extending downward away from and perpendicular to the top. The cylindrical extension 34 has a radius that allows it to freely travel through the aperture 26 and the slot 60 without engaging any of the notches 62 a and 62 b. With additional reference to FIGS. 4A and 4B, a threaded aperture 36 extends down through the center of the top 32 and the extension 34 and is adapted to receive a screw 42 from the underside of wing body 20. Fitting over the extension 34 is a biasing spring 38 of smaller diameter than the chamber 26. A retaining bushing 40, with a radius approximating that of the notches 62 a, 62 b, 62 c and 62 d, is secured to the adjustment assembly 30 (extension 34 ) from the underside of the wing body 20 by the screw 42, which runs through the aperture 28 into the threaded aperture 36 in the extension 34 and thus the button 32. The spring 38 is thereby secured and sandwiched between the underside 33 of the top of the button 32 and a bottom 27 of the chamber 28. [0036] FIGS. 2B and 4A show the positively locked position of the adjustment assembly 30. The spring 38 normally urges (biases) the push-button top 32 up and away from the bottom of the chamber 27 and thereby captively urges the attached bushing 40 up into the selected pair of opposing notches 62 a and 62 b to retain the strut 12. The bushing 40 prevents the strut 12 from longitudinally moving relative to the wing assembly 18 while the bushing 40 is within a notch. [0037] When a finger 66 applies downward pressure on the push-button top 32, the spring 38 is compressed and pushes the connected bushing 40 down out of the opposing notches 62 a and 62 b. With pressure still applied, the entire wing assembly 18 can be translated up or down the slot 46 (or vice-versa) until the pressure on the button 32 is removed and the bushing (stop member) 40 re-engages one of the pair of opposing notches 62 a and 62 b. [0038] FIGS. 5, 6 and 7 depict a second embodiment of a wing assembly, generally designated 118 that telescopes in the exact manner just described with respect to the wing assembly 18. The second embodiment functions the same as the wing assembly 18 with respect to the adjustment of the length of the strut 12. The wing assembly 18 is provided with at least one strap-retaining channel 72 that runs transversely across the wing member 20. A strap-retaining loop 74 extends longitudinally outward from an adjustment assembly housing 131 that retains the adjustment assembly 30 across the entire width of the channel 72 and is flush with the top of the adjustment housing 131. The loops 74 may be formed of plastic, metal, or other suitable material that is resilient enough to be repeatedly bent and still spring downward to retain the strap 54. The adjustment assembly 30 is structured and functions in like manner to the adjustment assembly 30. Features and/or functions not discussed below with respect to the wing assembly 118 should be considered to be the same as those features and/or functions with respect to the wing assembly 18 unless noted to the contrary. [0039] This configuration gives the wing assembly 118 a lower and sleeker profile that is less likely to get caught on obstructions during use. In addition, one end 78 of the retaining loop 74 is not connected to the wing body 20. The end 78 has a nub 80 to keep the strap 54 in place ( FIGS. 6 and 7 ). The end 78 may also have a snap or other positive locking mechanism that is releasably engageable with the wing assembly 118. Referring to FIG. 6, the retaining loop 74 can be pivoted or bent up at the unconnected end 78 in order easily to slip in the strap 54. When the end 78 is released, the nub 80 ensures that the strap 54 will not slip out of the retaining channel 72. The arrow in FIG. 5 depicts where and how another strap may be placed. [0040] FIG. 8 depicts a third embodiment of a wing assembly, generally designated 218. This third embodiment telescopes in the exact manner described with respect to the wing assemblies 18 and 118. Other features and/or functions not discussed below with respect to the wing assembly 218 should be considered to be the same as those features and/or functions with respect to the wing assemblies 18 and 118. [0041] The wing assembly 218 is similar in design/appearance to the wing assembly 118. The wing assembly 218 includes a body or housing 20 having a unitary retaining loop 74 that defines two channels 72 for receipt of straps ( 54 ). The adjustment assembly 230 is oval rather than round to provide easier manipulation, and is situated at an end of the body 20, proximate the strut 12. The adjustment assembly 230 is surrounded by an adjustment housing 231. [0042] Although the invention has been described in detail with reference to a preferred embodiment and an alternative embodiment, variations and modifications exist within the scope and spirit of the invention. Additional features of the invention will become apparent to those skilled in the art upon consideration of the detailed description of preferred embodiments exemplifying the best mode of carrying out the invention as presently perceived.

An orthopaedic brace includes a strut length adjustment assembly to change the operable length of the strut for sizing the brace on a patient without the need for special tools or cutting of the strut. The adjustment assembly includes a biased adjustment mechanism that coacts with a plurality of notches in the strut to variably set/position the strut relative to the adjustment assembly to set the struts length. Each upper and lower strut preferably includes a strut length adjustment assembly to independently set the length of each strut. The strut length adjustment assembly retains a strut and includes a strap retention mechanism that is configured to releasably engage the strap.

big_patent

THE MONSTER OF PELADON BY: BRIAN HAYLES PART FIVE 5:30pm - 5:55pm [SCENE_BREAK] 1: INT. COMMUNICATIONS ROOM AZAXYR: The sonic lance has a self-destruct circuit...which I have already pre-set by remote control. If the rebel should try to fire it, it will destroy itself. It will, of course, kill all those in the area. (SARAH up from the monitor in shock at the Ice Lord.) [SCENE_BREAK] 2: INT. CAVE OVERLOOKING CITADEL (The DOCTOR and ETTIS start to grapple with their swords in the middle of the cave. ETTIS kicks the back of the DOCTOR'S leg, knocking him to the ground. He stands on his sword before he can retrieve it and the DOCTOR rolls away to avoid the sudden thrust of ETTIS' free sword downwards. He gets to his feet but unarmed now, he has to avoid each deadly jab of ETTIS'S sword. He manages to grab his arm and throw the man down, dislodging the sword from his grasp and throwing at across the cave.) DOCTOR: Come on, Ettis, this is pointless. (ETTIS starts to clamber up.) ETTIS: Yes, Doctor, you're right. I am a fool! (He head butts the DOCTOR in the stomach. He falls to the ground. ETTIS picks him up and throws him into the rock face of the cave and then throws him over and onto the floor. The DOCTOR staggers to his feet and ETTIS knocks him back with an almighty punch to the face. He then starts to power up the lance. The DOCTOR manages to get back to his feet again...) DOCTOR: No, Ettis, no! (...only for ETTIS to land two more punches on him, once more knocking him to the ground. ETTIS returns to the lance and presses the firing switch. The cave is filled with a massive explosion.) [SCENE_BREAK] 3: INT. COMMUNICATIONS ROOM (SARAH watches events on the monitor in horror.) SARAH: You killed him! You killed the Doctor! AZAXYR: I was defending the safety of the citadel. The death of the Doctor was an unfortunate necessity. (He switches the monitor off.) AZAXYR: You would do well to accept the situation. [SCENE_BREAK] 4: INT. CAVE OVERLOOKING CITADEL (Aside from the drifting smoke, all is still in the cave. The DOCTOR lies to one side. He is not moving.) [SCENE_BREAK] 5: INT. COMMUNICATIONS ROOM SARAH: (To AZAXYR.) You still haven't won, you know? The rebels control the mines and you can't go down there because of the heat. AZAXYR: But we have switched off the heating controls. We merely have to wait for the temperature to return to normal. SARAH: Gebek and his miners know every inch of those tunnels. They won't surrender. AZAXYR: Again you underestimate me. The mines... (ECKERSLEY walks into the communications room.) AZAXYR: Ah, Eckersley. ECKERSLEY: Yes? AZAXYR: Just the man I wanted to see. The mines have a ventilation system, have they not, controlled from the refinery? ECKERSLEY: That's right, yes. (AZAXYR crosses to SARAH.) AZAXYR: Your miners can withstand heat, but they cannot live without air. Eckersley, you will go to the refinery at once and you will turn the ventilation system off. (SARAH runs across the room to plead with ECKERSLEY.) SARAH: No! No, tell him you won't do it! If they're forced out of the mines, they'll be massacred! ECKERSLEY: (Laughs.) Of course they won't! SARAH: They will! He's already killed the Doctor! (The smile disappears off ECKERSLEY'S face. He looks at AZAXYR.) AZAXYR: The Doctor was too dangerous to live, Eckersley. ECKERSLEY: (To SARAH.) Look, I'm sorry about the Doctor, believe me, but...he should never have got involved in local politics, and I'm not making the same mistake. (He leaves the room.) AZAXYR: Sskel, take her to the throne room and keep her with the others. (SSKEL takes SARAH'S arm roughly in his clamp-like hand and drags her from the room.) AZAXYR: (To ALPHA CENTAURI.) Now, Ambassador, we have work to do. There are several matters I wish to discuss with you concerning the future administration of this planet on more efficient lines. I shall require your help. [SCENE_BREAK] 6: INT. CAVE OVERLOOKING CITADEL (Within the cave, there is movement. The DOCTOR comes round and slowly gets to his feet, groaning. He looks at the still-smoking remains of the sonic lance and crosses over to it. He looks round further and suddenly a look of concern appears on his face. He runs from the cave.) [SCENE_BREAK] 7: INT. TUNNEL (He makes his way with some speed down one tunnel...) [SCENE_BREAK] 8: INT. ANOTHER TUNNEL (...but finds that he is still weak from his battle as he goes further along. He sits down on a rock to rest. No longer has he done so than he hears GEBEK calling from down the tunnel...) GEBEK: (OOV.) Doctor? DOCTOR: Gebek? (GEBEK runs up to him.) GEBEK: Oh, I was just on my way to find you. Well, did you manage to stop him? DOCTOR: Well, something did. The whole gun blew up. (GEBEK sighs with relief and smiles.) GEBEK: Thank goodness for that. (Puzzled.) But, er, what happened to Ettis? DOCTOR: I'm afraid he's dead. (GEBEK looks grim.) DOCTOR: How are thing's going? GEBEK: Oh, not too well. (He sits next to the DOCTOR.) GEBEK: We drove the Ice Warriors out of the mines but we couldn't break through to the citadel. Now the temperature dropping, soon they'll be able to come in after us. And worse than that... (He sniffs.) GEBEK: The air's getting stale. DOCTOR: Mmm, yes, I've noticed. Well they've probably switched the ventilation system off. GEBEK: We'll be gradually forced up to the upper levels. There are Ice wai...Warriors waiting at every exit. DOCTOR: Now where's the ventilation controlled from - do you know? GEBEK: Yes, the, er, refinery. DOCTOR: Well, you'd better take me there straight away. (They get up.) DOCTOR: And by the way, where's Sarah? (GEBEK hesitates.) DOCTOR: Well, answer me, man! Where is she? GEBEK: I left her looking after Rima, Doctor. But when I went back for her, he was dead. She was gone. DOCTOR: What? GEBEK: I'm sorry. DOCTOR: Well, it's not your fault, is it? Now don't worry, old chap, we'll find her. [SCENE_BREAK] 9: INT. THRONE ROOM (SARAH has been taken to the throne room. There, THALIRA and ORTRON attempt to comfort her...) THALIRA: We will never forget him, Sarah. He was a true friend to Peladon. His name will always be honoured. SARAH: (Stunned.) I still can't believe it. I...I can't believe that he's dead. You see, he was the most alive person I ever met. ORTRON: (Softly.) Well, perhaps he escaped somehow? There's always a chance. SARAH: That's what the Doctor used to say - "There's always a chance, that while there's life..." (She breaks off, unable to complete the sentence. The awkward silence is broken when a slightly hysterical ALPHA CENTAURI bursts into the room past SSKEL who is stood on guard outside in the passage...) ALPHA CENTAURI: Your Majesty! I have just had an exhausting meeting with Commander Azaxyr, discussing his plans for his future rule of this planet. ORTRON: (Outraged.) His rule? Queen Thalira rules Peladon! ALPHA CENTAURI: In name only from now on. Azaxyr plans an extension of martial law over the entire planet. All able-bodied citizens will be conscripted to work in the trisilicate mines. THALIRA: (Fiercely.) My people will never submit to this! ALPHA CENTAURI: I fear your people will have little choice, your Majesty. ORTRON: But surely the Federation won't approve? SARAH: Then it's up to us to get a message to the Federation and tell them what Azaxyr is up to. ALPHA CENTAURI: I've already tried that. Azaxyr has blocked all the communications circuits. I could activate the spatial distress beacon but it only sends a signal on a pre-set frequency - a general call for help. SARAH: A kind of SOS? Well, let's try it. Was the communications room guarded when you left? ALPHA CENTAURI: No, it was empty. SARAH: Then all we've got to do is get there - but how? ORTRON: (To THALIRA.) There is the secret passage behind the throne. THALIRA: In full view of the guard? SARAH: We've got to get past him. (SARAH jabs at thumb at SSKEL.) THALIRA: If we could entice him from the doorway, he's far too slow to prevent our escaping. ORTRON: Our escaping, your Majesty? THALIRA: Yes, Ortron. We intend to join our people in the mines. ORTRON: Your Majesty, I forbid it! THALIRA: (Fiercely.) What would you have me do, Orton? Stay here to become a puppet queen for Commander Azaxyr? See my people made slaves? (ORTRON drops his eyes.) ORTRON: Your Majesty, you put me to shame. (He turns to a smiling SARAH.) ORTRON: We shall assist you in your escape. SARAH: Well the first thing we need to do is create a diversion. (She looks over towards SSKEL outside the doorway and an idea comes to her.) SARAH: Your Majesty, do you think you could faint convincingly? (THALIRA smiles.) [SCENE_BREAK] 10: INT. PASSAGE OUTSIDE THRONE ROOM (LATER) (A short time later, ALPHA CENTAURI bustles out of the throne room and up to SSKEL.) ALPHA CENTAURI: The Queen is ill. (SSKEL swings round and sees THALIRA lying on the steps of the throne with SARAH and ORTRON attending to her.) ALPHA CENTAURI: You must summon medical assistance, immediately. (SARAH gestures to SSKEL who lumbers into the room. CENTAURI remains in the doorway and watches as he gets near to the Queen. SARAH suddenly pushes him from behind.) SARAH: Run everybody! [SCENE_BREAK] 11: INT. THRONE ROOM (But SSKEL has stepped onto the edge of the Queen's cloak and she is unable to move. She screams. As SARAH and ALPHA CENTAURI run off, ORTRON runs back to help the Queen.) ORTRON: Your Majesty! (He grabs SSKEL'S arm and pulls him off the Queen.) ORTRON: Run, your Majesty! (THALIRA runs with speed for the door but SSKEL has swung round to face ORTRON and blasts the High Priest with his sonic gun. ORTRON is caught full by the shot and falls to the floor. The Queen looks back and sees what has occurred. She runs to the fallen man.) THALIRA: Ortron! (SSKEL raises his gun at her as she tends to ORTRON, but at that moment, AZAXYR stalks into the room.) AZAXYR: No! (SSKEL lowers his gun while AZAXYR looks round and sees who is missing.) AZAXYR: (To THALIRA.) The Ambassador...and the Doctor's companion - where are they? (THALIRA thinks desperately.) THALIRA: They went to tunnels. They wanted to join Gebek and...the rebels. AZAXYR: How very convenient. Let there be no more of this foolishness, your Majesty. You can see for yourself the results of defying the Ice Warriors. Guard her. (He leaves. SSKEL stands over her as she takes off her cloak and covers the dead man with it.) [SCENE_BREAK] 12: INT. MINE TUNNEL (GEBEK leads the DOCTOR through a deserted mine tunnel. He suddenly sees something ahead and gestures to the DOCTOR to hide. They run back a short distance and hide behind a pit-prop and a pillar of rock. An Ice Warrior walks along the tunnel from the way that they would have taken and into a side tunnel. When it has gone, the DOCTOR and GEBEK set off again.) [SCENE_BREAK] 13: INT. TUNNEL OUTSIDE REFINERY (They come to the outside of the refinery. The junction box with the alarm equipment has been re-locked but someone is already inside. The DOCTOR gingerly approaches the door but the ever-vigilant GEBEK hears another noise and hurriedly signals to the DOCTOR that someone is approaching them from the same direction that they have just come from. The two hide behind a ledge of rock and watch as AZAXYR and an Ice Warrior come out of the tunnel. They both go inside the refinery and shut the heavy metal door behind them.) [SCENE_BREAK] 14: INT. REFINERY (ECKERSLEY is inside the well-lit refinery, sat at a large bank of controls on the right-hand side of the room. On the left-hand side of the room, next to a curtained alcove, is a smaller control on the wall marked "VENTILATION CONTROL". AZAXYR walks up to this to examine it.) AZAXYR: Good - you have switched off the ventilation system. ECKERSLEY: Yes, it won't be long now. AZAXYR: Excellent. The Ambassador and the Doctor's companion have foolishly taken refuge...in the mines. (ECKERSLEY smiles. Behind them, the door has swung open a few inches...) [SCENE_BREAK] 15: INT. TUNNEL OUTSIDE REFINERY (The DOCTOR and GEBEK come out of hiding and silently approach the open door. They stop to listen.) [SCENE_BREAK] 16: INT. COMMUNICATIONS ROOM (SARAH and ALPHA CENTAURI have made it back to the communications room. CENTAURI twitches over the communications console, much to the impatient SARAH'S irritation...) SARAH: Come on, we haven't much time! ALPHA CENTAURI: I am trying to remember the correct circuit. SARAH: Oh, hurry up! ALPHA CENTAURI: Please? You are making me nervous. SARAH: Oh, erm, sorry, I'll...I'll let you concentrate. (She tries to calm down. Smiling, she steps back from the console.) SARAH: I'll check what's going on. (She crosses to the two monitors and switches one of them on, flicking through pictures of empty passages in the citadel and equally empty tunnels. The fourth image shows the inside of the refinery with AZAXYR, ECKERSLEY and the Ice Warrior. SARAH clicks to another image of a tunnel but, interested more in what she has seen in the refinery, flicks back to this picture.) ALPHA CENTAURI: It is done. (CENTAURI crosses to SARAH at the monitor.) ALPHA CENTAURI: Though of course there is no guarantee that anyone will hear it, or that they will act on it if they do. SARAH: Look at this. ALPHA CENTAURI: It's Eckersley and Commander Azaxyr. SARAH: Oh, I know that. (Suspiciously.) Bit chummy though, aren't they? As if they were... (She points at the console.) SARAH: Can you get me sound on this? (CENTAURI flicks a switch and the sound comes through.) AZAXYR: (On monitor.) Yes, but I see no further need for this masquerade, Eckersley. ECKERSLEY: (On monitor.) Well I do. We agreed - till we succeeded, I stay undercover. AZAXYR: (On monitor.) But this planet is almost ours. ECKERSLEY: (On monitor.) Almost isn't quite good enough. Things could still go wrong. If they do - well, I'm just an innocent bystander. AZAXYR: (On monitor.) Very well, just so long as we can soon begin shipping trisilicate to Galaxy Five. (ALPHA CENTAURI almost screams out...) ALPHA CENTAURI: They are both traitors! [SCENE_BREAK] 17: INT. REFINERY ECKERSLEY: They've agreed our terms? AZAXYR: Subject to a time limit, so we must conclude matters quickly. [SCENE_BREAK] 18: INT. TUNNEL OUTSIDE REFINERY (Outside, the DOCTOR and GEBEK are also listening to the conversation...) ECKERSLEY: (OOV: Inside refinery.) Well, I'm ready to get things moving. How soon can you regain control of the mines? [SCENE_BREAK] 19: INT. REFINERY AZAXYR: Air will run out eventually, and then they must come up. ECKERSLEY: Yes, but that will take time. AZAXYR: And time is short! (ECKERSLEY crosses to the curtained alcove.) ECKERSLEY: Then we'll smoke them out...with Aggedor! (He pulls the curtain aside. Within it is a solid statue manifestation of the spirit that has been wreaking havoc in the mines.) [SCENE_BREAK] 20: INT. TUNNEL OUTSIDE REFINERY (The DOCTOR and GEBEK look at each other in surprise and shock.) [SCENE_BREAK] 21: INT. REFINERY (ECKERSLEY crosses to the main control bank.) ECKERSLEY: That'll get them on the move. (He presses settings on the console and then moves two adjacent levers in opposite directions. He then presses a large red button which is surrounded by six red lights in a hexagonal pattern. The lights come on and, with its rising electronic burbling sound, the statue fades away.) [SCENE_BREAK] 22: INT. MINE TUNNEL (Red and glowing, it materialises in a mine tunnel. A group of four miners jump back in shock but one of them is too late in running away and he is incinerated into nothing as the "ghost" fires at him. The image fades away...) [SCENE_BREAK] 23: INT. REFINERY (...and the statue materialises back in the alcove.) [SCENE_BREAK] 24: INT. TUNNEL OUTSIDE REFINERY (The DOCTOR mouths to GEBEK "stay there". GEBEK nods and the DOCTOR crosses to the other side of the doorway, ready to sneak a look through.) [SCENE_BREAK] 25: INT. COMMUNICATIONS ROOM (SARAH and ALPHA CENTAURI have continued to watch events in the refinery on the monitor.) ALPHA CENTAURI: It seems the Doctor was right - the appearances of Aggedor were a technological trick...controlled by Eckersley. But, er, why has he been doing this? SARAH: Well, it's obvious, isn't it? He was deliberately making things worse so he could persuade you to call in his Ice Warrior friends. (Suddenly, SARAH looks at the image in shock.) SARAH: Look! (On the monitor, the DOCTOR can be clearly seen looking through the partially opened doorway.) SARAH: Ambassador, look. Look, it's the Doctor! ALPHA CENTAURI: What? SARAH: (Exultantly.) He's alive! He's alive after all! (She thinks rapidly.) SARAH: Erm, you stay here and keep sending that SOS call. I'm going to join him. (Grinning with delight, she runs from the room.) [SCENE_BREAK] 26: INT. REFINERY (AZAXYR and ECKERSLEY receive a message from SSKEL over the intercom in the refinery as the DOCTOR continues to watch through the gap in the door.) SSKEL: (OOV: Over intercom.) The miners are fleeing from the lower levels. We are destroying them as they emerge. AZAXYR: (Into intercom.) See that all exits are blocked. They must not escape. (The DOCTOR carefully and quietly opens the door a little further in order that he can look round it at the statue of Aggedor. He peeks in.) AZAXYR: It seems that our scheme is working, Eckersley. ECKERSLEY: I'd better keep up the good work then. (Having had his suspicions confirmed, the DOCTOR retreats back outside. He continues to watch through the gap as ECKERSLEY re-sets the coordinates, adjusts the two levers and presses the main red switch.) [SCENE_BREAK] 27: INT. TUNNEL (Two guards and two miners turn a corner in the tunnel and cry out in fear at the apparition of Aggedor before them. It spits out its deadly fire and all four are vapourised.) [SCENE_BREAK] 28: INT. TUNNEL OUTSIDE REFINERY (The DOCTOR closes the refinery door and re-joins GEBEK.) GEBEK: (Whispers.) What's happening, Doctor? DOCTOR: (Whispers.) I'm afraid that it's Eckersley that has been summoning up Aggedor. And now he's using him to attack your miners. GEBEK: Well we must stop him! (GEBEK makes for the door but the DOCTOR holds him back.) DOCTOR: (Whispers.) No. If we show our faces in there, Azaxyr will wipe us out without a qualm. I'm sorry, Gebek. We've got to wait our chance. [SCENE_BREAK] 29: INT. REFINERY (ECKERSLEY switches the console off.) ECKERSLEY: Well, that should do for the moment. Give them another dose later. (He crosses to the alcove and closes the curtain.) AZAXYR: Excellent. Let us return to the citadel. [SCENE_BREAK] 30: INT. TUNNEL OUTSIDE REFINERY (The DOCTOR nods at GEBEK and the two rush to their hiding place behind the ledge of rock. The three within the refinery come out and ECKERSLEY attaches a small device to the top of the alarm junction box.) AZAXYR: You have re-set the alarm? ECKERSLEY: I can't, not from here. The late, lamented Doctor jiggered the control circuits. I'll have to re-set the alarm from the communications room. (He sets off down the tunnels.) AZAXYR: (To the Ice Warrior.) Guard the refinery. (The Ice Warrior nods and stands sentinel and AZAXYR follows ECKERSLEY into the tunnels. The DOCTOR and GEBEK see and hear this.) DOCTOR: (Whispers.) How long will it take them to get back to the communications room? GEBEK: (Whispers.) Five minutes, maybe ten - why? DOCTOR: (Whispers.) Well, that's how long we've got to get rid of our friend here, fix that alarm system and get back into the refinery. GEBEK: (Whispers.) But how? He'll blast us as soon as we break cover. DOCTOR: (Whispers.) Yes, I'm well aware of that. [SCENE_BREAK] 31: INT. TUNNEL (SARAH quietly makes her way through one of the tunnels.) [SCENE_BREAK] 32: INT. TUNNEL OUTSIDE REFINERY (She comes out at the refinery and the Ice Warrior guard swings round to aim at her with its sonic gun. She gasps and then sees that behind the Ice Warrior, the DOCTOR and GEBEK have come out of hiding and that the Peladonian is grasping a large rock in his hands. SARAH raises her hands in surrender and as a distraction.) SARAH: Don't shoot! No, I'm...! (GEBEK crashes the rock down onto the back of the Ice Warrior's helmet. The Martian crashes to the floor.) SARAH: Gebek! (She rushes up to the DOCTOR.) SARAH: Oh, Doctor, you...! (She hugs him.) SARAH: Oh, you're alive! I...I thought you'd been blown up in the cave?! DOCTOR: Luckily for me, that was Ettis. SARAH: I don't know, can't you ever stay out of trouble? DOCTOR: My dear Sarah, there's nothing I like more than a quiet life. SARAH: Oh! DOCTOR: Now, if you'll excuse me, I've got something very important to do in a very short space of time. (He crosses to the junction box, having to step over the Ice Warrior to do so.) DOCTOR: Look, can you get him out of the light? SARAH: Yes! GEBEK: Yes, Doctor, we'll take him... (SARAH and GEBEK grab a leg each of the Ice Warrior and start to drag him across the passage.) [SCENE_BREAK] 33: INT. TUNNEL (ECKERSLEY and AZAXYR reach the secret entrance to the citadel. The traitorous engineer pulls down the torch bracket and the door swings open. They go through it.) [SCENE_BREAK] 34: INT. PASSAGE (Within the citadel, AZAXYR stalks off towards the communications room while ECKERSLEY re-closes the door.) [SCENE_BREAK] 35: INT. TUNNEL OUTSIDE REFINERY (Watched by GEBEK and SARAH, the DOCTOR uses his sonic screwdriver to re-open the junction box.) [SCENE_BREAK] 36: INT. COMMUNICATIONS ROOM (ALPHA CENTAURI watches this on the monitor. Suddenly, AZAXYR and ECKERSLEY walk in and see him in surprise.) AZAXYR: Ambassador! (CENTAURI hurriedly switches off the image before the two new arrivals see it.) AZAXYR: What are you doing here? I was told you had taken refuge in the mines. ALPHA CENTAURI: Nonsense. I was simply trying to keep out of the way. AZAXYR: (Suspiciously.) Yes, but why here? (He looks round and crosses to the communications console.) AZAXYR: Could it be that you are trying to send a message to the Federation? (He spots the setting and hisses in anger.) AZAXYR: The spatial distress beacon! (His clamp-like hand slams down and switches the beacon off.) AZAXYR: You have been very foolish, Ambassador. ALPHA CENTAURI: I merely thought that things were getting out of control, it would be wise... (Coughs.) ...to summon more help for you. AZAXYR: (Angrily.) But things are not getting out of control, Ambassador. This planet is firmly under my command. If you disobey me again, Ambassador, you will take the consequences. ECKERSLEY: (Placatory.) Commander, I'm sure the Ambassador meant it for the best. (AZAXYR calms down.) AZAXYR: Very well, Eckersley. I will give you one last chance, Ambassador. You will come with me to the throne room - now! (AZAXYR storms out with ALPHA CENTAURI behind him. ECKERSLEY is following...) [SCENE_BREAK] 37: INT. TUNNEL OUTSIDE REFINERY (The DOCTOR continues his re-sabotage of the junction box as SARAH keeps a wary eye on the tunnel.) SARAH: (Whispers.) How's it going? DOCTOR: (Quietly.) Nearly finished. [SCENE_BREAK] 38: INT. COMMUNICATIONS ROOM (In the doorway of the communications room, ECKERSLEY calls down the outside passage.) ECKERSLEY: I'll catch you up. I forgot to switch the alarm on. (He comes back in and heads for the console.) [SCENE_BREAK] 39: INT. TUNNEL OUTSIDE REFINERY DOCTOR: Well, that's the alarm fixed - now for the door. [SCENE_BREAK] 40: INT. COMMUNICATIONS ROOM (ECKERSLEY switches the alarm on - just too late - and leaves the room.) [SCENE_BREAK] 41: INT. THRONE ROOM (With SSKEL in attendance, a scowling THALIRA sits on her throne and faces AZAXYR as ALPHA CENTAURI listens.) AZAXYR: Trisilicate production will be resumed immediately, your Majesty, using all available modern tools. I shall expect your Majesty's full cooperation. (THALIRA can barely keep her temper...) THALIRA: When my father signed treaties with the Federation, he could not have known it would lead to nothing but bloodshed! However...we must accept the consequences. AZAXYR: A wise attitude, your Majesty. (ALPHA CENTAURI cannot keep silent any longer and bursts out...) ALPHA CENTAURI: Do not believe him, your Majesty! The Federation has no part in this! Commander Azaxyr is a traitor - Eckersley too! They plan to ship the trisilicate to our enemies of Galaxy Five! AZAXYR: You were warned, Ambassador! (He steps back to enable SSKEL to have a clear shot at CENTAURI.) AZAXYR: Sskel! (SSKEL raises his sonic gun but THALIRA jumps to her feet.) THALIRA: No! (ECKERSLEY, as cool as ever, also steps forward.) ECKERSLEY: No. No, no. Whatever he knows, we still need him. ALPHA CENTAURI: Thank you, Eckersley, but you are still a traitor. THALIRA: (Shocked.) Then this is true, Eckersley? ECKERSLEY: Yes, your Majesty, it's true. But it doesn't make any difference to you, does it? THALIRA: Peladon has never dishonoured a treaty. Our loyalty is to the Federation. (AZAXYR steps back forward, hissing angrily.) AZAXYR: There is your loyalty. (He points to SSKEL'S sonic gun which the Ice Warrior raises and points at the Queen. She looks down at it coolly.) AZAXYR: You will obey...or perish! ECKERSLEY: (To ALPHA CENTAURI.) Now, Ambassador, you must be a lot brighter than you look. How come you're so well informed, hmm? ALPHA CENTAURI: You were betrayed by your own security system, Eckersley. ECKERSLEY: What? ALPHA CENTAURI: When you were conspiring with the Commander, Sarah and I overheard you on the monitor. ECKERSLEY: Sarah? AZAXYR: The girl! (He turns to THALIRA.) AZAXYR: You said she had joined the rebels in the mines. Where is she? (THALIRA, without taking her sardonic gaze off AZAXYR, resumes her seat on the throne. The Ice Lord turns to ALPHA CENTAURI.) AZAXYR: Where is she, Ambassador? (CENTAURI looks away and blinks. AZAXYR approaches menacingly.) AZAXYR: You will answer...if you value your life. (CENTAURI looks at ECKERSLEY for support but gets none. He turns back simpering to AZAXYR.) ALPHA CENTAURI: She...left before you arrived. When she saw the Doctor on the monitor, she went to the refinery. (AZAXYR hisses in shock at this revelation.) AZAXYR: The Doctor! ECKERSLEY: The refinery! AZAXYR: Sskel! Go to the refinery at once! If the Doctor is there, destroy him! (SSKEL lumbers out on his mission.) [SCENE_BREAK] 42: INT. TUNNEL OUTSIDE REFINERY (The DOCTOR re-wires the two connections and once more the lights within the box start to flash.) DOCTOR: Right, that's it. In you go. (GEBEK opens the door and SARAH walks in, followed by the miner. The DOCTOR re-closes the box.) [SCENE_BREAK] 43: INT. TUNNEL (SSKEL makes his way towards the refinery.) [SCENE_BREAK] 44: INT. TUNNEL OUTSIDE REFINERY (The DOCTOR uses his sonic screwdriver to re-lock the junction box.) [SCENE_BREAK] 45: INT. REFINERY (He then enters the refinery and GEBEK closes the door after him.) DOCTOR: Right, the first thing we've gotta do is ventilation back on. Give those miners of yours some air. (He crosses to the ventilation control, presses a switch and pulls a lever down.) DOCTOR: There, that should do it. (SARAH pulls the curtain back from the alcove, revealing the statue within.) SARAH: How about this, Doctor. DOCTOR: Yes, I've seen it. (He crosses to the console and takes the seat. SARAH follows him and points at the controls.) SARAH: Well, what's that thing then? DOCTOR: Mmm? SARAH: That thing - what is it? DOCTOR: Oh, that's just a simple matter projector, linked to a directional heat ray. Er, the projector sends an image of that... (He points at the statue.) DOCTOR: ...and the heat ray does the damage. GEBEK: The appearances of Aggedor were all done from here? DOCTOR: Yes, that's right - by Eckersley or one of his Ice Warrior friends. Very clever piece of work this. Quite an engineer, our friend Eckersley. [SCENE_BREAK] 46: INT. TUNNEL OUTSIDE REFINERY (SSKEL arrives at the refinery. He looks round and sees the fallen Ice Warrior on the floor. He then goes over to the door, aims his sonic gun and fires...) [SCENE_BREAK] 47: INT. REFINERY (The DOCTOR is still looking over the controls.) DOCTOR: Directional coordinates are here... (SARAH sniffs the air.) SARAH: Something's burning. (She looks round and sees a tiny pin-point of smoking red on the metal door.) SARAH: Doctor, look! [SCENE_BREAK] 48: INT. THRONE ROOM (THALIRA and ALPHA CENTAURI are still in the throne room with ECKERSLEY and guarded by two Ice Warriors.) ALPHA CENTAURI: What puzzles me, Eckersley, is your reason for this betrayal. ECKERSLEY: It's simply a matter of business, Ambassador. I'll get a percentage of all the trisilicate mined on Peladon, just to make me the richest and the most powerful man in the galaxy. THALIRA: Most powerful? ECKERSLEY: Yes, your Majesty. When the Ice Warriors have won, I shall be ruler of Earth. ALPHA CENTAURI: And what of Commander Azaxyr? ECKERSLEY: No, it's only military glory he's after. ALPHA CENTAURI: (Puzzled.) But the Ice Warriors have been loyal members of the Federation for many years. ECKERSLEY: Azaxyr's head of some kind of breakaway group. He wants to return to the good old days of death or glory. (AZAXYR walks back in.) ECKERSLEY: Ah, Commander. AZAXYR: It appears that the Doctor is indeed alive. But Sskel has him trapped...in the refinery. (THALIRA lowers her eyes at the inevitability.) [SCENE_BREAK] 49: INT. TUNNEL OUTSIDE REFINERY (Two more Ice Warriors join SSKEL as he continues to blast at the metal door.) SSKEL: Help me. (They line up opposite the door.) [SCENE_BREAK] 50: INT. REFINERY (Within the refinery, the DOCTOR frantically re-sets the controls.) [SCENE_BREAK] 51: INT. TUNNEL OUTSIDE REFINERY (As the three Ice Warriors blast at the door, more and more of the metal melts away. A larger and larger hole is being created...) [SCENE_BREAK] 52: INT. REFINERY (SARAH and GEBEK run back from the door as smoke pours through the gaping hole and over to the DOCTOR.) SARAH: (Shouts.) Hurry, Doctor! There's a whole crowd of them out there now! GEBEK: (Shouts.) They'll be through any minute, Doctor! (Through the hole, the green form of the attacking Ice Warriors can be seen...)

Sarah and Alpha Centauri decide to try and alert the Federation to what the Ice Warriors are up to but first they have to escape from the throne room.

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Breaking News Emails Get breaking news alerts and special reports. The news and stories that matter, delivered weekday mornings. SUBSCRIBE For decades, Hollywood producer Bob Weinstein appeared to live in the outsize shadow of his older brother, Harvey. Harvey Weinstein was the public face of the studios they co-founded, a fixture on the Oscar circuit and a major player in Democratic political circles. Bob Weinstein, by contrast, seemed to shy away from the glare of fame. "My style is more low-key. Outside of the movie business I tend to be known as the 'quiet brother,' which is fine by me," Bob Weinstein wrote in an essay for Vanity Fair in 2003. "It actually works out for the best that only one of us likes the spotlight. There wouldn't physically be enough room in it for both." But this month, following bombshell reports alleging that Harvey Weinstein sexually harassed and assaulted dozens of women over decades, Bob Weinstein has been forced into the center of a roiling scandal, emerging as a vehement critic of his embattled brother even as he also faces an allegation of sexual misconduct. Harvey and Bob Weinstein after the red carpet opening of the movie "Inglourious Basterds" in Los Angeles in November 2011. J.Emilio Flores / Corbis via Getty Images file Bob Weinstein, 63, blasted Harvey Weinstein, 65, last Thursday as a "very sick man" and a "world class liar" whose remorse rings "hollow." Just five days later, Variety reported that Amanda Segel, a former executive producer of the sci-fi series "The Mist," claimed that Bob Weinstein made repeated romantic overtures to her and would not take no for an answer. "Bob kept referring to me that he wanted to have a friendship," Segel told Variety. "He didn't want a friendship. He wanted more than that. My hope is that 'no' is enough from now on." Bert Fields, a lawyer for Bob Weinstein, denied the allegations. "Variety's story about Bob Weinstein is riddled with false and misleading assertions by Ms. Segel and we have the emails to prove it," he said in part, adding: "There is no way in the world that Bob Weinstein is guilty of sexual harassment." A representative for Harvey Weinstein has said "any allegations of non-consensual sex are unequivocally denied by Mr. Weinstein." Both brothers have also come under fresh scrutiny for their brash tactics, which some have called abusive, at their firms, Miramax and The Weinstein Company. The brothers’ behavior is recounted in "Down and Dirty Pictures," a 2004 book by veteran entertainment journalist Peter Biskind. Movie mogul Jeffrey Katzenberg, who condemned Harvey Weinstein following explosive reports in The New York Times and The New Yorker, leveled harsh words this week at Bob Weinstein, saying he had significant "issues" with him when they worked together in the 1990s. "Bob Weinstein was genuinely abusive with people in my company," Katzenberg, the former chairman of Walt Disney Studios, said in an interview with The Wall Street Journal on Tuesday. (Disney acquired Miramax in 1993.) "There's the fascinating thing about this," Katzenberg said, apparently referring to the overall scandal. "The one person who did reveal himself in an unacceptable way to me was, in fact, Bob." Now, the alleged misconduct threatens to take down everything they've built. Although Bob Weinstein said he took steps in recent years to distance himself from his disgraced brother, their fates may be intertwined. Some people close to the company say it will be difficult for Bob Weinstein to retain a leadership role in the Weinstein Co. — if it survives at all. And the Weinstein name may be forever tainted, making it difficult for him to start fresh in Hollywood. The Weinstein Co. exec insists he had no idea about "the type of predator that he was" and is sickened by Harvey’s seeming lack of remorse. "I want him to get the justice that he deserves." For nearly 30 years, Bob Weinstein has lived in the shadow of his older brother Harvey. While Harvey, 65, was the very public face of Miramax and then The Weinstein Co. from Sundance to Cannes to Hollywood, palling around with stars and schmoozing Oscar voters, Bob, 62, has served on TWC's board and tended to Dimension, their genre label, turning out movies like the Scream and Scary Movie franchises that routinely made more money than all but Harvey's biggest hits. Now, in the wake of the dozens of allegations charging Harvey with three decades of sexual harassment, abuse and even rape, he is out, fired from the company he co-founded with Bob in 2005. And Bob, thrust into an unaccustomed limelight, is forced to try to pick up the pieces amid the growing chaos. He insists that the company — which is expected to undergo a name change — can survive. But four of his fellow board members have resigned, and his COO David Glasser and other key members of his 150-employee staff have yet to commit to stay with the company. Amid widespread predictions that The Weinstein Co. will be forced to shut down or sell all or parts, Bob maintains, "There is a plan to come out on the other side." But right now, even as he struggles to right the company (of which he and Harvey each own about 20 percent), Bob is also coping with his own sense of shame and betrayal, expressing sympathy for Harvey's victims while also questioning whether he should have done more in the face of Harvey's alleged abusive behavior. Bob, who worked mostly in Los Angeles while Harvey presided over TWC's New York offices, says he's barely spoken to his brother over the past five years. "I could not take his cheating, his lying and also his attitude toward everyone," he says. While Bob says he knew his brother was unfaithful to wife Georgina Chapman, he insists he had no idea about "the type of predator that he was" and is sickened by Harvey's seeming lack of remorse. "I have a brother that's indefensible and crazy," says Bob, adding, "I want him to get the justice that he deserves." Amid the chaos and uncertain future at TWC, Bob agreed to a 45-minute phone interview with The Hollywood Reporter. During the talk, he often became emotional when discussing his brother and the company they co-founded. He refused to discuss certain specifics, such as the claim in The New York Times that he and the board were aware of Harvey's settlements with women during Harvey's most recent contract negotiation, or the fate of certain TWC movies, such as the year-end title The Current War. But he opened up on the personal aspect of the scandal and said he believed the Film Academy should expel his "sick and depraved" brother. How do you assess what has happened? I find myself in a waking nightmare. My brother has caused unconscionable suffering. As a father of three girls, I say this with every bone in my body — I am heartbroken for the women that he has harmed. I'm a fighter. For my entire adult life, I fought for the films I want to see the light of day. I have fought for my employees, who have dedicated their lives to achieving the vision of this company that me and my brother founded. But I cannot fight what is indefensible. The members of the board, including myself, did not know the extent of my brother's actions. I know him on a personal level better than anyone. It's hard to describe how I feel that he took out the emptiness inside of him in so many sick and depraved ways. It's a sickness but not a sickness that is excusable. It's a sickness that's inexcusable. And I, as a brother, understood and was aware as a family member that my brother needed help and that something was wrong. I was also the object of a lot of his verbal abuse — at one time physical abuse. And I am not looking for one bit of sympathy from anyone. I do not put myself in the category at all of those women that he hurt. But it's a complicated situation when it's your brother doing the abusing to you as well. I saw it and I asked him to get help for many years. And that's the truth. He avoided getting the help. We begged him. This hurts, but I don't feel an ounce of remorse coming from him, and that kills me, too. When I heard his written, lame excuse … Not an excuse. When I heard his admission of feeling remorse for the victims and then him cavalierly, almost crazily saying he was going to go out and take on the NRA, it was so disturbing to me. It was utter insanity. My daughters all felt sick hearing this because we understood he felt nothing. I don't feel he feels anything to this day. I don't. One question that is on everybody's mind: He is your brother — how in the world did you not know this was going on? First of all, let me tell you something that people don't know. For the last five years, I've probably talked to my brother 10 times on any personal level. That's the fracture that's gone on. Since Dimension started, we ran two separate companies. So many of the people that he does business with — actors, actresses — I've never even met and they know it. I wanted to lead a separate existence. So we were leading two separate divisions. I actually was quite aware that Harvey was philandering with every woman he could meet. I was sick and disgusted by his actions. But that's the extent of what [I knew]. I said, "Harvey, you're just cheating. Why do you constantly cheat?" I could see it. But I wasn't in the room with him. For me, I thought he was literally just going out there cheating in a pervasive way. It wasn't like he even had a mistress. It was one after another and that I was aware of. But as far as being in a room and hearing the description in The New York Times? No way. No F-in' way was I aware that that was the type of predator that he was. And the way he convinced people to do things? I thought they were all consensual situations. I'll tell you what I did know. Harvey was a bully, Harvey was arrogant, he treated people like shit all the time. That I knew. And I had to clean up for so many of his employee messes. People that came in crying to my office: "Your brother said this, that and the other." And I'd feel sick about it. Why did you tolerate his behavior? Because it didn't rise to a certain level. I would often counsel people and say, "You know what, you have a choice here. Leave. Leave, please leave." I don't know why some of them stayed. So I would just try to mend a broken fence. There is no mending this. This is not a broken fence. [But] I will not quit and leave the business that I built, rightfully so, and leave the films and filmmakers that I was involved in. When you guys were negotiating Harvey's 2015 employment contract … That I'm not gonna [discuss]. I'm sorry, I'm not gonna be litigated in this article. There is an investigation going on, let that investigation take its course. Do you think Harvey should be kicked out of the Academy? Yes, I do. I was gonna actually write [to the Academy]. And I will do it. I am gonna write a note to them saying he definitely should be kicked out of the Academy. You issued a statement earlier today saying that the company is not for sale despite reports that it could be shut down or its parts sold off. If it's not for sale, what is the plan to salvage this company? We are thinking about how to do that. We had an employee meeting the other day, all 180 of the employees. And me and David Glasser addressed them all. Talk about emotions cascading. These innocent people are hurt, so many of them not knowing anything about my brother's behavior. But I was touched because — and I did not know this was coming — they said, "We don't want Harvey to have the last word on this company. We want to stay. We believe in the films, we believe in you, Bob, David Glasser, we believe in ourselves. And we definitely don't want to let him win." And that's a part of the human story that nobody is hearing. I know they're saying, "Shut this company down." Well, they didn't shut Fox News down, they didn't shut NBC down. My brother is the one that should pay with everything. And I mean literally — whether it's criminal or otherwise — I will be supportive of all of that. But I don't think the people that are the employees of this company or the company itself should pay. Harvey was the face of the company. That's what he loves. It's actually part of his whole thing, being famous. This brother is not that brother. This brother made just as much money, ran a successful division, more successful financially than Harvey's. But I'm a different guy and I run it differently and people know it and they know I can be successful and we don't need to do any of the Harvey stuff. And there is a plan. All I'm trying to do right now is go forward, figure out a plan, me and David Glasser and the board members have an idea of what we'd like to do, that we think would be the responsible thing to do for all the critics, rightfully so, with regards to the TWC side and yet for people to keep their jobs. And the pieces of the business that still can be resurrected and continue, we think that they should. Do you have a new name for the company? We're coming up with one. And it won't be familial, I promise you that. Any effort to keep the company going will depend on financial backing. Aren't the finances of this company in trouble? We have good enough finances, the banks gave us their support today. What does that mean? With scandal, sometimes people can just say, "We don't want to be associated." They're supporting us. And in terms of surviving, we have enough good product and things on our slate. We are moving quickly, and there are people interested in participating financially on the condition, rightfully so, that Harvey Weinstein is out for good. But you have people like Lin-Manuel Miranda trying to get In the Heights extricated from the company. You have Disney dropping you guys from producing Artemis Fowl. Apple and Amazon have scrapped big TV projects. Collaborators are turning their backs on you. How do you survive that? We will, is my answer, and there are a lot of things in process with regards to a lot of those kinds of things that will get resolved in an amicable way and a good financial way. I really don't want to get into the particulars of any one situation. All I can say is that there is a plan to come out on the other side. And also the other side that makes the public rightfully feel happy that what Harvey stood for exists no longer. The public deserves that. The victims deserve that. Everybody deserves that. Those bankers, they look at my slate and they know that I've made them money for many, many years. And lately I have had a cold hand, there is no two ways about it, on the Dimension side, but I have a fuckin' great slate coming up and they're aware of it. And this is a business where all the sudden you walk into Stephen King's It one day. And things change. It is that kind of business. I have Six Billion Dollar Man, I'm closing a deal on it. I have Paddington 2, Paddington 3 is coming two years later. I'm back in the wheelhouse of what I've done. And when I'm back on, there is money to be made, there's jobs to be had. Harvey knew how to take credit, win his awards. But for me, there are no Academy Awards. Bob's not going to the Golden Globes or the Oscars unless somebody invites me. There have been reports that Jay Z might buy Harvey's stake in the company … I'd love nothing more than that, but as far as I know, that is not a fact. There are predictions for a raft of civil litigation against the company and against Harvey personally. How can the company withstand that? That's for lawyers and people like that to go deal with. That's what they do. And things end up getting resolved, they do. My hope is that the community will allow us the time to get it right for those that deserve to continue on. That's all I can say and that's what I'm trying to do. But it's going to take time. I cannot do it in a week. Has anyone in Hollywood reached out to you with support? Yes, they have. David Glasser had a meeting with David Hutkin, who is the CFO, and the banks have been supportive. They want to see us succeed and they see that there is a product line of many movies on my side, TV shows that have been done. That they say this company can still be viable. And there have been other people that have been supportive, other companies that are doing business with us, that are sticking by us. What about the talent agencies? They are going to be key to any company going forward. Have they expressed support? There have been men and women, actresses, actors, directors, but especially women I would say, who are so properly disgusted with my brother's actions. Their attitude is when there is finally the entire divorce, when there's the plan in place, when there is the separation in place … I have a reputation that's different than Harvey, obviously, and I work differently than him. And David Glasser is his own man. We have our own status in the industry. And they're saying we will give the two of you a chance. You have to make the separation. [With] my brother having contractual financial rights in this company, the divorce is going to be as speedy as we can make it. It's in process. But Harvey's going to fight his firing. Anybody can do what they want to do. I cannot control other people's actions. But he was fired by the board, okay? I was on that board. I fired him. He can fight. It will be a losing fight. He may be fired but he still maintains his ownership interest in the company, correct? That is correct and we are going to seek to sever that. It can't be done that quickly. But I am on it 24/7 and so is David Glasser and so is the board of directors that remain and so are the shareholders. This is being dealt with. Given your relationship with Harvey, there is a contingent out there who believe that you were responsible for the leak of this information, especially the internal HR memo that made it to The New York Times. Is that true? That's totally untrue. I could take a lie detector test on that. I didn't and, you know, Harvey is suspicious of everybody. People that are liars — lying to his wife, to his children, to everyone — well, they have to turn around and say, "Who stabbed me?" It's unbelievable that even to this moment he is more concerned with who sold him out. I don't hear concern or contrition for the victims. And I want them to hear that. Harvey has no remorse whatsoever. I have spoken to him two times [since news broke], hoping to hear, "Oh, my God, what have I done?" I didn't hear that. What did you hear? I heard a guy who still was fighting to get back and I was disgusted by it. Do you know how disgusted I am? I divorced my brother five years ago. Literally. And those that know me personally in this company understood how I could not take being around him on any level. And certainly my daughters and my family knew it. I could not take his cheating, his lying and also his attitude toward everyone. I had to divorce myself to survive. Nobody is perfect. I'm not perfect. If I made mistakes, I apologize to everyone for not standing up stronger and sooner. But if you want to take my head and the company's and everybody else's. … If I lose at the end of the day, then I lose it. But I'll fight for what I believe is right. And I'll apologize for my own lack of strength at times. Do you believe that the 2015 incident with model Ambra Battilana and the NYPD investigation derailed the deal for ITV to acquire the company's TV division? Again, this is where I draw the line in terms of getting into a specific. I don't want to comment on that kind of specific. I'll ask a general question then: Do you believe that Harvey's antics hurt the business in the past? I'm being honest. I don't know what hurt or didn't. I haven't had a relationship with him as a brother for many, many years. But I'm ashamed that he is my brother, to be honest, and I am ashamed that these are his actions. I'm not thinking at this moment about dissecting what may or may not have affected any past situation. I'm so in the here and now and feel sick. Mira Sorvino came out and told her story [in The New Yorker]. I have been a personal friend of Mira for the last several years. We reconnected when I rented a house in Malibu. Mira, her husband and her kids have come over and spent many hours together with us. [But] she never told me. And now I literally was texting with her, and she said, "Are you mad at me?" And I said, "Mad at you? I'm so proud of you, but I feel sick that you had to hold that kind of thing in." [Harvey] should never be allowed back, ever. Ever. He lost his rights. He didn't lose his rights to be rehabilitated as a human being. But as far as being in this town again? I mean, give me a break. What do you think Harvey will do with the rest of his life? He lived for this business and he lived for the outside [persona]. There were no insides to this, as far as I can see. So unless there becomes an inner person inside there, I have no idea what he'll do. Will you cooperate with any police investigation? A hundred percent, if it came to that. If I had anything to offer, I would. A hundred percent. There have been stories that once, when Harvey got physical with you, he broke your nose? He didn't break my nose, but he got physical and there were several people there, and he assaulted me. And I should've done something then. Why did you not? All I can tell you is it's like asking any other victim why they didn't stand up. I regret that beyond all measure. I live with that. That was a defining moment for me of cowardice on my own part. Not easy to live with. It doesn't absolve me of my own cowardice, but this is the thing that happens, this is the nature of that whole syndrome. And it's disgusting on every level. But hopefully, the more it's talked about — I was happy when people started to come out. It made other people braver. Well, you've had some anger issues in the past as well … Yes, but I've done my work. Without getting into details, I've done enough work, and I've faced my own self. There are those that do the work and those that don't. I did it. I'm not that guy and that's not the way I operate. Any last thing you'd like the entertainment community to hear from you with regard to this situation? I'm mortified and disgusted by my brother's actions. And I am sick for the victims. And I feel for them. I feel for them.

Bob Weinstein's prior knowledge of the sexual harassment and assault allegations now piling up against his brother, Harvey Weinstein, isn't clear. What is clear is that the brothers were in the midst of a rift that the scandal would only expose. According to the Los Angeles Times, the brothers with a reputation for noisy spats had for years pursued their own interests at the Weinstein Company-Bob favoring commercial flicks, Harvey preferring high-brow films-when Harvey reportedly punched Bob during a meeting six years ago. "I divorced my brother five years ago" and since then, "I've probably talked to my brother 10 times on any personal level," Bob Weinstein tells the Hollywood Reporter. "I could not take being around him." In 2015, it was Bob Weinstein who alerted the Weinstein Company's board of directors to a sexual harassment allegation made against his brother by a female employee, the Times reports. Publicly, however, the brothers played nice to appease their mother, sources say. Upon her death last year at age 90, outright war was no longer out of the question. But if Bob Weinstein intended to take full control over the company he built with his brother, he might've been overconfident. Now facing his own allegations of harassment and bullying, he may only hold onto his offshoot label Dimension Films. There's also the possibility he could start a new label, but that may be a challenge without his brazen brother leading the charge, reports NBC News.

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Spinal muscular atrophy is a severe motor neuron disease caused by inactivating mutations in the SMN1 gene leading to reduced levels of full-length functional SMN protein. SMN is a critical mediator of spliceosomal protein assembly, and complete loss or drastic reduction in protein leads to loss of cell viability. However, the reason for selective motor neuron degeneration when SMN is reduced to levels which are tolerated by all other cell types is not currently understood. Widespread splicing abnormalities have recently been reported at end-stage in a mouse model of SMA, leading to the proposition that disruption of efficient splicing is the primary mechanism of motor neuron death. However, it remains unclear whether splicing abnormalities are present during early stages of the disease, which would be a requirement for a direct role in disease pathogenesis. We performed exon-array analysis of RNA from SMN deficient mouse spinal cord at 3 time points, pre-symptomatic (P1), early symptomatic (P7), and late-symptomatic (P13). Compared to littermate control mice, SMA mice showed a time-dependent increase in the number of exons showing differential expression, with minimal differences between genotypes at P1 and P7, but substantial variation in late-symptomatic (P13) mice. Gene ontology analysis revealed differences in pathways associated with neuronal development as well as cellular injury. Validation of selected targets by RT–PCR confirmed the array findings and was in keeping with a shift between physiologically occurring mRNA isoforms. We conclude that the majority of splicing changes occur late in SMA and may represent a secondary effect of cell injury, though we cannot rule out significant early changes in a small number of transcripts crucial to motor neuron survival. Autosomal recessive Spinal Muscular Atrophy (SMA) is a leading genetic cause of infant mortality, with a carrier frequency of 1∶50 and an annual incidence of 1 in 10,000 live births [1]. Affected individuals develop symmetrical, proximal weakness resulting from neurogenic muscle atrophy, and ultimately leading, in the most severely affected individuals, to death from respiratory failure. The pathological correlate of these symptoms is selective loss of large alpha motor neurons in the ventral horn of the spinal cord. The vast majority of cases are caused by homozygous deletion of the survival motor neuron 1 (SMN1) gene [2] with subsequent reduction in levels of the SMN protein [3]. SMN is highly conserved in evolution and ubiquitously expressed. Complete loss of SMN, which is incompatible with life [4], is prevented by production of SMN from the SMN2 gene, a near identical paralogue of SMN1 which has arisen from an inverted duplication event in recent evolution. The presence of a translationally silent C-T transition in SMN2 exon 7, however, leads to disruption of an exonic splice-enhancer (ESE) element and exon 7 skipping in the majority of SMN2 derived transcripts [5], [6]. The functional consequence is that SMN2 produces only very little full length SMN (FL-SMN), while the SMND7 isoform is translated into an unstable protein that is rapidly degraded [7]. Disease severity is broadly proportional to residual SMN levels, which is a function of SMN2 copy number, although other modifying factors are involved in some cases [8], [9]. The mechanism by which SMN deficiency leads to selective lower motor neuron loss in SMA is poorly understood and difficult to reconcile with its ubiquitous expression unless either SMN has a motor neuron-specific function or motor neurons are selectively vulnerable to a deficiency in the general function of SMN common to all cells. Currently, the best characterised function of SMN is as part of a multi-protein complex which is critical for the core steps in the assembly of small nuclear ribonuclear proteins (snRNPs), components of the spliceosome, the cellular machinery that controls splicing of pre-mRNAs [10], [11]. In particular, SMN acts in the cytoplasmic assembly of Sm core proteins on snRNAs, which is a prerequisite for import of snRNPs into the nucleus [12]. SMN levels and the activity of snRNP assembly vary during development and according to tissue type. In the mouse spinal cord, snRNP assembly is highest during embryogenesis and early postnatal development and then falls to a baseline level when myelination occurs [13]. Mouse models of SMA, which have reduced SMN levels, show a drop in snRNP assembly activity as measured by in vitro assays, while steady state snRNP levels measured in tissues are only mildly reduced [14]. Interestingly, a subset of snRNPs belonging to the minor spliceosome seems to be differentially affected [14], [15]. Several strands of evidence support the notion that reduced snRNP assembly is associated with motor neuron degeneration. The subtle motor neuron loss that has been reported by some investigators at late stages in mice heterozygous null for Smn can be accelerated by crossing with mice heterozygous null for Gemin2, another core component of the SMN complex. This is associated with a reduction in snRNP assembly [16]. In zebrafish, a failure of embryonic motor axon growth can be induced by silencing not only SMN, but also gemin2. The observed defects can be rescued by direct injection of U snRNPs [17]. However, other studies in zebrafish indicate that the axonal degeneration phenotype is not coupled to U snRNP assembly [18]. Reducing SMN levels in HeLa cells by RNAi leads to an increased error rate in splice-site pairing, which has also been observed in fibroblasts from SMA patients [19]. Finally, a recent study in SMA mice found that at end-stage, widespread splicing abnormalities can be found in several tissues including the spinal cord. Importantly, different transcripts were found to be altered in a tissue-specific manner [15]. While both snRNP assembly dysfunction and splicing abnormalities have been documented in models of SMA, several questions remain. It is still unclear whether splicing abnormalities cause motor neuron loss in SMA, or whether they are a late occurrence in disease, either as a consequence of spliceosome dysfunction or the severe physiological alterations secondary to respiratory distress, hypoxia and malnutrition. If spliceosome dysfunction is critical for disease pathogenesis, the mechanism of splicing alterations needs to be further delineated. In addition, the role of SMN in spliceosome assembly is only one of many functions that are potentially altered in SMA, including roles in transcription regulation [20]–[22], axonal transport of mRNA and RNPs [23], [24] as well as regulation of local translation at the neuromuscular junction [25]. This study had two aims. First, we wanted to address the question of whether abnormal splicing events are a cause or consequence of SMA. We utilised an exon-specific microarray to examine the transcriptome of spinal cord samples harvested from SMA and control mice at pre-symptomatic, early symptomatic and late-symptomatic stages, to test the hypothesis that widespread alteration of splicing precedes disease onset. Secondly, we used the difference in temporal mRNA expression pattern between SMA and control mice to identify neuronal pathways disturbed by SMN deficiency. The mouse model used in this study (Smn−/−; SMN2; SMNΔ7) is the most commonly used model of severe SMA and has been described previously [26]. The maximum lifespan of SMA mice was 14 days. Subtle differences in weight compared to the control littermates (Smn+/+; SMN2; SMNΔ7) were discernible before P7, but were not reliably predictive of genotype in individual mice (Figure 1A and 1B). SMN protein levels were markedly reduced in SMA mice at all time points as measured by Western blot (Figure 1C) and immunohistochemistry (Figure 1D). At P7 a failure in the righting reflex became apparent. Importantly, this coincided with a drop in numbers of large motor neurons in the spinal cord (Figure 2A and 2B). No discernible differences in phenotype or pathology were present at P1, indicating that, in the SMNΔ7 mouse model of SMA, the disease develops in a motor system in which embryonic development has been relatively normal. By P13, mice appeared emaciated, were unable to right themselves, and showed signs of respiratory distress (Figure 1B). There was a corresponding loss of >30% of lower motor neurons from the ventral horn in SMA mice. At late-symptomatic stage (P13), a similar relative loss of motor neurons was evident across the entire length of the spinal cord, (Figure 2C), justifying the use of whole spinal cord for RNA analysis. However, absolute numbers differed depending on the region examined, reflecting the differential innervation of limb and trunk musculature by motor neurons originating in the cervical, thoracic or lumbar cord. To understand the scale of change in mRNA expression in the spinal cord, we first performed gene-level comparisons between genotypes at each time point, using the core probe sets on the array and GeneSpring software (Agilent Technologies, Santa Clara, CA, USA). When using an arbitrary p-value threshold of 0. 05 and a fold-change threshold of 1. 5 as cut-off for biological significance, the expression of 142 genes was increased or decreased in the spinal cord of SMA mice compared with their control littermates at late-symptomatic stage (P13), with a maximal fold-change of 3. 9 (Table S1). Importantly, the degree of change between SMA and control mice was much smaller at the pre-symptomatic (P1) and early-symptomatic (P7) stages, with only 12 and 23 genes changed, respectively (Table 1 and Table 2; Figure 3A). This finding argues against a critical function of SMN in transcription regulation, but also shows that if widespread splicing changes occur in SMA, this does not lead to a pre-symptomatic systemic change in whole transcript level mRNA expression, which might be expected if mis-spliced transcripts are subject to nonsense-mediated decay. An additional data analysis, which we refer to as the ENSG analsysis, was performed in which probes were grouped into sets, each corresponding to a gene in the Ensembl (version 49) annotation database [27]. Time point-specific differential expression between cases and controls was quantified by fitting a linear model on a gene-by-gene basis [28], [29]. Of 21,911 genes examined, 693 genes exhibited case/control differences at P13, as opposed to 92 at P7, and 83 at P1 (Figure S1, Tables S2, S3, S4, see Text S1 for details of the linear model). This is in accordance with the parallel analysis described above. We next examined changes between time points for each genotype. Overall, the number of genes differentially expressed between time points in control mice was higher by a factor of ten compared to the number of genes differentially expressed between genotypes at individual time points (Figure 3B), suggesting that the immediate post-natal period is associated with major changes in spinal cord gene expression in normally maturing mice. A key aim of this study was to assess the amount of splicing variation in the spinal cord of an SMA mouse model compared to control mouse spinal cord at several time points during disease progression. Because exon-specific microarrays are relatively novel, and analysis methods have not been fully developed and validated, we used two complementary statistical approaches to investigate the number of differentially expressed exons. First we used an ANOVA test in GeneSpring (Agilent) to select exons which show a significant difference between exon-level and transcript-level signal and then calculated the splicing index (SI) [30], for these exons. The SI is the logarithm of the ratio of array probe set intensities (corresponding to expression levels of individual exons) to overall gene-level expression. So an SI value of 0 indicates equal expression of a particular exon in relation to the gene as a whole between genotypes i. e. no differential alternative splicing. Using a splicing index of |SI|≥0. 5 as an arbitrary threshold we identified 252 potential alternative splicing events at the late-symptomatic stage (P13), but only 5 at the early symptomatic (P7) and 16 at the pre-symptomatic (P1) stage (Figure 4A). This initial analysis suggests that alternative splicing events are a consequence of disease progression in SMA, rather than the primary cause. Since the splicing index method is known to lead to inaccuracies if complex splicing patterns are present, such as splicing of multiple exons in one gene, and might thus underestimate the level of differential exon use present, we next performed an analysis comparing expression levels of individual exons between genotypes at each time point. The rationale for this is that each instance of differential splicing between genotypes will lead to at least one exon being differentially expressed. So the number of differentially expressed exons provides an upper bound for the number of differential splicing events. In this analysis, which we refer to as the ENSE analysis, probes were grouped into sets, each corresponding to an Ensembl exon [27]. Time point-specific differential expression between cases and controls was quantified by fitting a linear model on an exon-by-exon basis [28], [29]. The p-value cut-off for significant differences between genotypes at the exon-level was chosen to balance sensitivity with a reasonable false discovery rate (FDR), as estimated by a permutation-based analysis (Text S1 and Table S5). At a p-value threshold of 1e-4, there were 812 significantly differentially expressed exons at P13, compared to 66 at P7, and 72 at P1 (Figure 4B, Tables S6, S7, S8); a total of 211,567 exons were examined. See Materials and Methods and Text S1 for further details of this analysis. This provided additional evidence that the vast majority of alternative splicing events occur at late-stage disease in the SMNΔ7 mouse model of SMA. We further observed that for genes with at least one differentially expressed probe set under the ENSE annotation, but no significant gene-level change under the ENSG annotation, i. e. genes for which there is some evidence of differential splicing events, it is mostly one, and never more than two, exons that are significantly altered. To assess whether the differentially expressed exons were associated with a particular intron type, we cross-referenced our data with a publicly available database of U12 introns, i. e. sequences recognised by the U12 snRNA containing minor spliceosome, but not the U2 snRNA containing major spliceosome [31]. Overall, the frequency of U12 introns in genes containing exons differentially expressed between genotypes was 0. 19%, as opposed to the expected frequency of approximately 0. 35% (0. 13% at P1,0% at P7,0. 22% at P13). Moreover, only one gene (Vash1, ENSG000000712460, Vasohibin-1) contained a differentially expressed exon directly adjacent to an U12 type intron, while all other exons were remote from U12 type introns. This analysis suggests that genes spliced by the minor spliceosome are not preferentially affected by SMA, even though components of the minor spliceosome were shown to be disproportionately affected by SMN deficiency [14]. While the results obtained from the SMNΔ7mouse model afforded important insights into the dynamics of gene- and exon-level expression changes over time, to ensure that our findings were not restricted to this particular transgenic model of SMA, but applicable to SMA mouse models in general, we performed a similar analysis on the more severe but genetically less complicated Smn−/−; SMN2 mouse model of SMA [32]. The Smn−/−; SMN2 animals, in which complete absence of mouse Smn is rescued by the introduction of the human SMN2 transgene, have a maximum lifespan of 6 days. Previous studies showed that at P1, there are normal motor neuron numbers, whereas at P5, there is about a 35% loss compared to litter mates with normal mouse Smn (Smn+/+; SMN2) [32]. Early synaptic abnormalities are seen from P2 in this model [33], although neuromuscular junctions appear normal at P1, indicating normal pre-symptomatic development [34]. Exon-array analysis of spinal cord harvested from pre-symptomatic (P1) and late-symptomatic (P5) animals mirrored the principal findings in the SMNΔ7 mouse at both gene and exon level. When examining gene expression in Genespring using core probe sets, more changes were present at late-symptomatic stage than at the pre-symptomatic stage (3 genes up- or down-regulated at P1,160 genes up- or down-regulated at P5 with p≤0. 05 and fold change >1. 5). Even more striking was the result of the splicing index analysis, which showed 27 potential alternative splicing events at P5, but none at P1 when choosing a splicing index cut-off of 0. 5. These results showed that our findings in the SMNΔ7 mouse model were not caused by a specific effect of the SMNΔ7 transgene but are likely to be a generalised phenomenon in SMA. To validate the findings of our microarray experiments, we performed semi-quantitative and/or quantitative RT-PCR focussing on genes that showed at least one exon with differential expression at all time points (Cdkn1, Snrp1a, Chodl, Mccc2, Uspl1, ChAT, Figure S2). In addition, we performed qRT-PCR on spinal cord samples obtained from E16 embryos for some of the targets to examine whether changes were already present pre-natally. All qRT-PCR results matched the changes at gene-level seen in the array experiments, underlining the robustness of the array findings. Chodl, the gene encoding chondrolectin is a C-type lectin with unknown in vivo function. Interestingly, in situ hybridisation shows that this gene is highly expressed in anterior horn cells (Allen Brain Atlas http: //mousespinal. brain-map. org) and might have important, if currently unknown, motor neuronal functions. We observed a progressive reduction in Chodl expression over time. Of note, the exon array data were indicative of differential expression of the 3′ end of Chodl, and validation by qRT-PCR using primers spanning both constitutive exons as well as two alternative 3′-terminal exons was in keeping with a preferential loss of the Chodl -001 isoform (ENSMUST23568) and relative sparing of Chodl-002 (ENSMUST69148) (Figure 5A and 5B). The relative reduction of Chodl expression appeared to be spinal cord specific and could not be demonstrated in either skeletal muscle or kidney. However, even these tissues showed a trend towards increased expression of the Chodl-002 isoform in SMA compared to control mice (Figure 5C). Immunohistochemistry for Chodl showed strong immunoreactivity of anterior horn cells, in keeping with the published in situ data. There was reduced Chodl immunoreactivity in SMA mice, but remaining anterior horn cells retained substantial Chodl staining, which indicated that the reduced Chodl expression is at least partially due to loss of motor neurons (Figure S4). Uspl1 (ubiquitin specific peptidase like 1), a gene encoding part of the ubiquitin-dependent protein degradation pathway was found to be up-regulated in SMA at all time points. In addition, Uspl1 showed a consistent change in both splicing index and Ensembl exon-level analysis of differential splicing. Uspl1 exon 2 (ENSMUSE00000351955) is a cassette exon absent in transcripts Uspl1-006 (ENSMUST00000121416) and -007 (ENSMUST00000117878). The increased use of Uspl1 exon 2 in the SMA mice led to an isoform shift with relatively higher levels of exon 2 containing transcripts Uspl1 -001-005 (Figure 6). Of note, this pattern was much more pronounced in muscle than in spinal cord, and less evident in kidney (Figure 6B). In spinal cord samples, the degree of isoform shift was more pronounced at symptomatic than at pre-symptomatic stages (Figure 6C). Immunohistochemistry for Uspl1 showed ubiquitous cytoplasmic staining in all spinal cord cells with grey matter preference. In keeping with the only mild overall increase in Uspl1 expression at mRNA level (Figure S2F), no difference in immunoreactivity was evident between genotypes Figure S4) and Western blotting showed no significant difference in the main protein isoform identified between genotypes. Further interesting changes at all time points were detected including the whole-gene up-regulation of Snrpa1 (average 1. 8 fold). Snrpa1 encodes one of the many protein components of the spliceosomal A complex [34], which is associated with the U2 snRNA. Western blotting showed a consistent small expression increase (Figure S4). While no other spliceosomal components were differentially expressed, the Snrpa1 change might reflect a compensatory response of the cell to Smn deficiency. Another change included down-regulation of isoforms ENSMUST00000091326 and ENSMUST00000022148 of Mccc2, the gene encoding the methylcrotonoyl-CoA carboxylase beta chain, a mitochondrial enzyme involved in amino acid metabolism (Figure S2D). Interestingly, the array data suggest that, while two Mccc2 isoforms were expressed at lower levels in SMA compared to controls, a third isoform (ENSMUST00000109383) was in fact up-regulated. This was confirmed by qRT-PCR and semi-quantitative PCR (Figure S3). In the SMNΔ7 mouse model, the early postnatal days appeared particularly relevant to disease development. Our earlier finding of massive gene expression changes between time points in post-natal wild-type mice (Figure 3B) indicates that events relevant to disease in the SMN Δ7 mouse model coincide with transcriptome changes associated with normal post-natal development or maturation. To analyse which pathways were physiologically activated during this time, we first compared gene expression in control mice (Smn+/+; SMN2; SMNΔ7) between P1 and P7, and subsequently between P7 and P13. Using GO-Elite software, we identified pathways enriched with genes involved in spinal cord cell proliferation, axon development, oligodendrocyte development and myelination as significantly altered, reflecting physiological events during the rapid growth of the spinal cord. When the same analysis was performed for the SMA mice, a strikingly different pattern emerged. With the exception of two GO IDs pertaining to nervous system development, all physiologically activated pathways were absent in both the P1 v P7 and P7 v P13 analyses Table 3). To investigate whether this dramatic change in gene expression pathways was mirrored by a difference in proliferating spinal cord cells, we performed Western blotting and immunohistochemistry for the proliferation marker PCNA. Our preliminary results indicate that there is indeed a reduction in the number of proliferating cells in the SMA spinal cord (Figure S5). At P13, genes relating to cellular responses to DNA damage became prominent in the SMA mice, which could not be explained by a significant amount of spinal cord gliosis (Figure S5). Of note, the gene with the highest-fold change between genotypes at P13 was Cdkn1a, a cyclin dependent kinase inhibitor activated by p53 in response to DNA damage. This analysis suggested that in the SMNΔ7 mouse model there is an inhibition or a failure of activation of the normal physiological pathways of post-natal spinal cord maturation. In this study we undertook a detailed assessment of transcriptional changes over time in the spinal cord of a commonly used severe mouse model of SMA, using time points correlated with key phenotypic and pathological changes. We identified alterations in a subset of genes involved in post-natal neurodevelopmental pathways in SMA, and showed that splicing alterations are only a late occurrence in disease. Survival, weight development and motor phenotype of our SMNΔ7 mouse colonies were similar to that described by others [26], [35], with a change in outward appearance and development of motor deficits apparent at P7. Recent studies have identified morphological changes at the neuromuscular junction as early events in SMA [33], [35] with neurofilament accumulation occurring as early as P5 in the SMNΔ7 model. This structural change at the distal end of the motor neuron is closely followed in our study by a significant loss of large motor neurons at P7, indicating that although synaptic changes are the earliest identified feature of SMA it ultimately is a disease of the entire lower motor neuron. Of note, motor neuron loss was present to a similar degree across the entire spinal cord, in contrast to the previous finding of a rostral-caudal gradient with relative sparing of the lumbar region [35]. Motor neuron loss at P7 was reflected in the reduction at transcript level of choline acetyl transferase (ChAT), the key enzyme in motor neuronal synthesis of acetylcholine (Figure S2E). This finding, as well as that of reduced levels of Chodl mRNA, which seems to be highly expressed in anterior horn cells, shows that even though whole spinal cord was used for the array, important cell-type specific changes were detectable. The key finding of this study is that alternative splicing events are a late occurrence in SMA, and are therefore unlikely to contribute to early disease pathogenesis. Zhang et al. [15] described widespread splicing abnormalities in several tissues including the spinal cord at end-stage disease in the same mouse model employed in this study and attributed this finding to dysfunction of the spliceosome secondary to SMN deficiency. Importantly, even though the time point of analysis is not absolutely identical to ours, there is considerable overlap between Zhang et al.' s data set obtained at P11 and our P13 data set, when raw data are analysed in the same way (Figures S6, S7; Tables S1, S9, S10, S11). However, it remains unclear from these data whether splicing abnormalities had preceded the onset of symptoms, as would be predicted from the crucial role of SMN in spliceosome assembly, particularly in embryonic and early post-natal development [13]. In addition, the presence of widespread splicing defects in organs other than the spinal cord is difficult to interpret in the light of apparent tissue specificity of the disease if splicing abnormalities are indeed thought to be relevant to the mechanism of motor neuron degeneration. To determine the degree of variation in splicing between SMA and control mice, we utilised the splicing index, but also examined changes at individual exons as a measure of the maximum number of alternative splicing events present. The absolute number of changes found in late-symptomatic mice was not large given the large number of exons examined (>200,000). When the same analysis was performed, comparing between genotypes at pre-symptomatic and early symptomatic time points, only very few exon-level changes were present. Importantly, we were able to confirm a similar pattern of exon expression changes in the more severe Smn−/−; SMN2 mouse model, corroborating our main finding in the SMNΔ7 mice. Overall, our data indicate that the majority of splicing changes are not a direct consequence of SMN deficiency, but rather a consequence of disease progression, probably representing physiological isoform-shifts in response to cell stress. There is evidence that oxidative stress can induce shifts in alternative splicing, and that neurons may be more vulnerable to this process than other cells [36]. We would therefore argue that SMN deficiency to the degree observed in either the SMNΔ7 or Smn−/−; SMN2 mouse, although associated with severe reduction in snRNP assembly capacity in vitro [14], does not lead to a systemic breakdown of splicing fidelity in vivo until the disease is well established. While our results do not support the hypothesis that widespread, systematic splicing abnormalities cause SMA, we cannot rule out the possibility that splicing of one or several transcripts is critically affected by SMN deficiency, and that the few splicing changes observed early in our mice contribute to SMA pathogenesis, followed by a cascade of loss of splicing fidelity or secondary effects. In fact, at least one of the genes identified by our array (Uspl1) is differentially spliced between SMA and control mice even at the embryonic phase, albeit to a lesser degree than at the symptomatic stages. Of note, the isoform shift observed in this particular gene is more pronounced in muscle than in spinal cord, and less marked in kidney, an organ not affected by SMA pathology. Independent of whether or not splicing changes are ultimately responsible for SMA disease initiation, our study identified several pathways that might shed light on SMA pathogenesis and disease progression. Analysis of transcriptional changes between genotypes in this study took place on a background of large scale physiological changes between time points, reflecting rapid neuronal development in the early post-natal days. Analysis of the changes between time points identified several pathways related to normal neuronal development. Surprisingly, in the SMA mice the majority of physiological transcriptional changes seen in control mice were absent even between the early time points P1 and P7. This finding not only indicates that abnormal post-natal neuronal development might underlie early events in SMA but might explain general delayed growth and failure to thrive in the SMA mice. In this study, we examined gene expression in the entire spinal cord and are unable to distinguish which cell types contribute most to expression changes, even though changes that plausibly originate from motor neurons, such as ChAT and Chodl, are clearly present. The neurodevelopmental pathways identified as altered in our study can be associated with other cell types, including oligodendrocytes and cells in the posterior spinal cord. While this is a preliminary finding, it clearly warrants further studies examining the role of non-motor neuronal cells in SMA. Human autopsy cases indicate involvement of sensory neurons in the dorsal root ganglia, Clarke' s column and the thalamus in SMA [37]–[40], although the majority of studies were undertaken before the molecular diagnosis of SMA was available. More recent clinical studies showed that severe SMN-related SMA is associated with widespread neuronal degeneration, including sensory pathways [41]. Subtle sensory neuron abnormalities have also been detected in a severe mouse model of SMA [42]. To our knowledge, however, there is no study systematically investigating the role of non-motoneuronal cells in SMA spinal cord. In conclusion, our data show that alternative splicing events predominantly occur late in SMA, while alterations of post-natal neurodevelopmental pathways precede overt symptom onset. Further studies should continue to focus on the role of SMN in the post-natal maturation and development of the neuromuscular system including spinal cord motor neurons. Transgenic Smn+/−; SMN2; SMNΔ7 [26] mice were maintained as heterozygous breeding pairs in standard animal facilities in Oxford. Homozygous Smn−/−; SMN2; SMNΔ7 mice reached the disease end-point by post-natal day 13 (P13). Five mice of each genotype were sacrificed at age P1, P3, P5, P7, P9, P11 and P13 for motor neuron counts, and 4 mice of each genotype at P1, P7 and P13 for RNA and protein extraction. Mice were genotyped using DNA extracted from tail-tips and standard PCR procedures. For motor neuron counts, mice were terminally anaesthetised with i. p. pentobarbitone and transcardially perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS. For RNA and protein extraction, mice were killed by i. p. injection of pentobarbitone. Smn+/−; SMN2 mice (Jackson labs strain no. 005024) were maintained as heterozygote breeding pairs under standard SPF conditions in animal care facilities in Edinburgh [43]. Litters produced from SMA colonies were retrospectively genotyped using standard PCR protocols (JAX® Mice Resources). All animal breeding and procedures were performed in accordance with Home Office and University guidelines. Spinal cords were dissected, post-fixed in 4% PFA for 2 hours, cryoprotected in 30% sucrose overnight, embedded in OCT medium and rapidly frozen in liquid nitrogen-cooled isopentane. 20 µm thick horizontal sections were cut on a cryostat and stained with 0. 5% Cresyl violet with 0. 04% acetic acid. A minimum of 30 non-adjacent sections covering the entire spinal cord segment of interest were scrutinised for large, polygonal, Nissl positive cells in the ventral horn of the spinal cord anterior to the central canal. Only cells with a clearly present nucleolus were counted to avoid double counting of neurons. Motor neuron counts were performed blinded to genotype. At P13, cords were macroscopically divided into cervical, thoracic and lumbar segments using the cervical and lumbar enlargements as landmarks. For the time-course, only lumbar spinal cord was utilised. 6 µm thick paraffin section were cut and stained with standard Haematoxylin and Eosin. For immunohistochemistry, sections were incubated with the primary antibody (mouse anti-SMN antibody (1∶320, BD Transduction lab), rabbit anti-Uspl1 (1∶600, Santa Cruz), mouse anti-Chodl (1∶200, abcam), rabbit anti-PCNA (1∶2500, abcam), goat anti-Chat (1∶400, Chemicon) for 40 minutes at room temperature or at 4°C overnight. Antibody binding was visualised using a Dako REAL EnVision kit according to manufacturer' s instructions. Immunohistochemistry was carried out on several sections taken from two different paraffin blocks for two animals per genotype. Representative images are shown. Whole spinal cords were rapidly dissected and snap-frozen on dry ice. RNA was extracted using the Qiagen RNeasy Mini RNA extraction kit according to the manufacturer' s instructions. The quality and RNA integrity was assessed on a BioAnalyzer; all samples had a RNA Integrity Number (RIN) ≥9 (Agilent Laboratories, US). 1 ug starting RNA was ribosome depleted using the Ribominus Human/MouseTranscriptome Isolation kit (Invitrogen). Labelled sense ssDNA for hybridization was generated with the Affymetrix GeneChip WT sense target labelling and control reagents kit (Affymetrix, UK) according to the manufacturer' s instructions. Sense ssDNA was fragmented and the distribution of fragment lengths was measured on the BioAnalyzer. The fragmented ssDNA was labelled and hybridized to the Affymetrix GeneChip Mouse Exon 1. 0 ST Array (Affymetrix). Chips were processed on an Affymetrix GeneChip Fluidics Station 450 and Scanner 3000. For the gene-level analysis, core probe sets which map to the same transcript cluster were grouped together and RMA (Robust multi-array analysis) [44] normalised in GeneSpring GX10. 1. 02. Differentially expressed genes were identified using an unpaired t-test; selecting genes with 1) a p-value less than or equal to an arbitrary threshold of 0. 05 and 2) a fold change difference between genotypes ≥1. 5. The selected genes were sorted according to gene ontology using GenMAPP' s GO-Elite (http: //www. genmapp. org/go_elite/go_elite. html). Only MAPPFinder ontologies with ≥3 genes changing and a permuted p-value of ≤0. 05 were reported. At the exon level, core probe sets were PLIER (Probe Logarithmic Intensity Error) normalised in GeneSpring GX 10. 1. 02 (Agilent). Transcript probe sets that had detection above background (DABG) p-value≤0. 05 in both SMA and control groups were retained. An ANOVA test was used to identify significant differences between exon-level signal and transcript level signal. As recommended in the Affymetrix White Paper [44], [45], exon level probe sets exhibiting exon-to-transcript intensity ratios >5 were excluded from the ANOVA (where log2[exon-to-transcript ratio] = log2[exon expression]−log2[transcript expression]). This filter removed probes with high background and cross-hybridisation potential. The p-value threshold for the ANOVA was selected to control for a false discovery rate of 0. 05 using the Benjamini-Hochberg multiple testing procedure [46]. For exons selected on the basis of the ANOVA, the spicing index, SI (log2[exon-to-transcript expression ratio]), was calculated and used as a measure of differential splicing between genotypes. See [45], [47] for further details. A significantly differentially spliced exon was defined to be one having both an FDR-controlled ANOVA p-value≤0. 05 and |SI|≥0. 5 (on the log scale, corresponding to a fold change up or down of approximately 1. 4 in the absolute exon/transcript ratio between genotypes). CEL files were preprocessed using RMA without background correction (see Text S1). Publicly available custom chip-definition files (CDFs) (http: //brainarray. mbni. med. umich. edu/Brainarray/Database/CustomCDF/CDF_download. asp) were used to group probes into sets. Parallel analyses, based on two different CDFs were performed. The first CDF, referred to as ENSE, defines a probe set for each Ensembl exon. The second, ENSG, defines a probe set for each Ensembl gene [27]. There were 211,567 and 21,911 probe sets for the ENSE and ENSG analyses respectively. At each probe set, a linear model was fitted using the limma package (version 2. 16. 5), to quantify evidence of genotype differences within each time point (see Text S1 for details). A permutation-based analysis was conducted to estimate the FDR at a variety of p-value thresholds (see Text S1 and Table S5). The p-value thresholds selected were 1e-4 for ENSE, and 1e-3 for ENSG, as these thresholds controlled the FDR at a reasonably low level. Statistical analyses were performed using R [28], version 2. 8. 1. Snap frozen whole spinal cords were homogenised in RIPA lysis buffer (50 mM Tris-Cl, pH 7. 5,150 mM NaCl, 0. 1% (w/v) SDS, 1% (v/v) sodium deoxycholate, 1% (v/v) TX-100) and 1% (v/v) protease inhibitors by sonication at 50% output for 15 sec. Homogenates were chilled on ice for 20 min and clarified by centrifugation at 15,800 g for 20 min at 4°C. Protein samples (50 µg/well) were electrophoresed through 12% SDS polyacrylamide gels and transferred to 0. 2 µm nitrocellulose membranes (Millipore). Membranes were blocked with 5% (w/v) milk powder in TBS-T, pH 8. 0, for 1 hr and incubated with the primary antibody (mouse anti-SMN (1∶1,000, BD Transduction labs), mouse anti-actin (1∶1,000, Abcam), rabbit anti-SNRPA1 (1∶1000, abcam), goat anti-CHAT (1∶500, Chemicon), rabbit anti-USPL1 (1∶500, Santa Cruz), rabbit anti-PCNA (1∶200, abcam) in 3% (w/v) BSA in TBST overnight at 4°C. Blots were probed with HRP-conjugated antibodies (1∶10,000, Amersham) and developed using ECL reagents (Amersham). Western blots were repeated three times on biological replicates, and representative blots are shown. RNA was reverse transcribed into cDNA using random hexamer primers (Invitrogen) and Expand Reverse Transcriptase (Roche) under standard conditions. RT-PCR was performed using Taq DNA Polymerase (Sigma) or Expand High Fidelity PCR system (Roche). Real-time PCR was performed using Fast SYBR Green chemistry (Applied Biosystems) and a StepOnePlus Real-time PCR machine (Applied Biosystems). Real-time PCR Primers were designed with Primer Express software (Applied Biosystems). Primer concentrations were optimised to yield low Ct values and minimal primer dimer formation (commonly 300 nM for both forward and reverse primers). GAPDH was used as the endogenous control, as there was no differential expression between genotype in the array and in qRT-PCR experiments. All primer pairs were tested to have similar amplification efficiency to GAPDH when tested on serial cDNA dilutions over 4 log. Fold change was calculated using standard ΔΔCt calculations. The average fold change per time point was calculated from four biological replicates at each time point, and an unpaired two-tailed t-test was used to test for significantly different gene expression at each time-point. Error bars represent the standard deviation of the mean.

The identification of mutations in the Survival Motor Neuron (SMN) gene as the cause of the severe motor neuron disorder spinal muscular atrophy is one of a number of discoveries implicating selective motor neuron vulnerability to defects in processing of RNA and its associated ribonucleoprotein complexes. An unresolved issue is whether loss of the general cellular function of SMN in spliceosomal assembly, which is predicted to result in widespread defects in mRNA splicing, is directly responsible for motor neuron death. We have used exon-specific microarrays to assess the degree of altered splicing in the spinal cord in a mouse model of SMA. Our finding that the vast majority of splicing changes are a late feature of the disease and may represent a shift to alternative isoform expression, rather than loss of splicing fidelity, provides evidence that widespread splicing disturbance is not a primary feature of the disease pathogenesis but a secondary effect of cell injury in a late phase of the disease. However, our study cannot rule out a role for subtle early changes in one or a few transcripts crucial to motor neuron survival expressed at low levels or in only in a sub-population of spinal cord cells.

lay_plos

Gene expression of DNA viruses requires nuclear import of the viral genome. Human Adenoviruses (Ads), like most DNA viruses, encode factors within early transcription units promoting their own gene expression and counteracting cellular antiviral defense mechanisms. The cellular transcriptional repressor Daxx prevents viral gene expression through the assembly of repressive chromatin remodeling complexes targeting incoming viral genomes. However, it has remained unclear how initial transcriptional activation of the adenoviral genome is achieved. Here we show that Daxx mediated repression of the immediate early Ad E1A promoter is efficiently counteracted by the capsid protein VI. This requires a conserved PPxY motif in protein VI. Capsid proteins from other DNA viruses were also shown to activate the Ad E1A promoter independent of Ad gene expression and support virus replication. Our results show how Ad entry is connected to transcriptional activation of their genome in the nucleus. Our data further suggest a common principle for genome activation of DNA viruses by counteracting Daxx related repressive mechanisms through virion proteins. DNA viruses require the transport of their genome into the nucleus to initiate replication. Cells perceive the introduction of foreign nucleic acids or unscheduled replication as danger signals and activate a DNA damage response that leads to cell cycle arrest and/or apoptosis. To ensure proper replication, DNA viruses express ‘early’ viral genes to degrade or displace key regulators of cellular antiviral machinery. In return, cells repress incoming viral genomes through a network of transcriptional repressors and activators that normally control cellular homeostasis [reviewed in 1], [2]. The nuclear domains thought to be responsible for repressing viral genomes are ND10 or promyelocytic nuclear bodies [PML]-[NBs; reviewed in 3,4] named after the scaffolding PML protein. PML-NBs are interferon inducible, dot-like nuclear structures associated with proteins with transcriptional repressive functions. These include HP-1, Sp100, ATRX and Daxx [summarized in 4], [5]. Daxx (death domain associated protein) was first described as a modulator of Fas-induced apoptotic signaling [6]. When chromatin-bound, Daxx inhibits basal gene expression from various promoters by binding to transcription factors (e. g. p53/p73, NF-kappaB, E2F1, Pax3, Smad4 or ETS1), ATRX, histone deacetylases and core histones to form a repressive chromatin environment [7]–[13]. In contrast, Daxx localization to PML-NBs reduces its repressive capacity and facilitates apoptosis through p53 family members [5], [7], [14]. PML-NBs are found in close proximity to replication centers of DNA viruses (e. g. adenoviruses (Ads), herpes simplex virus (HSV-1), human cytomegalovirus (HCMV) and human papillomavirus [HPV]; [ 15], [16]–[18]. Gene expression from these viruses is repressed via the PML-NBs, suggesting a role in antiviral defense [19]–[22]. To counteract genome repression, viral genome activation involves PML-NB disruption or degradation of Daxx, Sp100 and/or PML via different mechanisms. HCMV gene expression is initiated by proteasomal degradation of Daxx via tegument protein pp71 of the incoming particle [23]. Early HSV-1 gene expression requires PML degradation, mediated by the virus encoded ubiquitin ligase ICP0. Furthermore, in order to activate viral gene expression, transcriptional repression by Daxx and ATRX needs to be relieved [3], [24], [25]. HPV early gene expression is supported by reorganization of PML-NBs through the minor capsid protein L2 [26]. At the beginning of infection, Ads express the immediate early protein E1A from the E1A promoter. E1A binds and displaces the transcriptional repressor Rb from E2F transcription factors. This results in the auto-stimulation of E1A expression and the activation of the downstream viral expression units E1B, E2, E3 and E4 as well as promoting cellular gene expression. The early E1B-55K protein forms a SCF-like E3-ubiquitin ligase complex with the viral E4orf6 and several cellular factors. This complex degrades factors (for example, factors of the DNA damage response) to ensure progression of the replication cycle [summarized in 1], [2], [27]. E1B-55K protein complex also targets Daxx for proteasomal degradation counteracting its repressive effect [21]. In contrast to HSV-1, PML is not degraded by Ads but relocalized into track-like structures through the E4orf3 protein [28], [29]. Despite the well-characterized mechanism of E1A dependent transactivation of early Ad genes, it is unclear how the E1A transcription is efficiently initiated before other viral genes are expressed. The genome enters the cell as a transcriptionally inactive nucleoprotein complex, which is highly condensed by the histone-like viral protein VII inside the capsid shell. Partial disassembly of the endocytosed capsid releases the endosomolytic internal capsid protein VI, permitting endosomal membrane penetration [30], [31] and transport towards the nucleus. After import through the nuclear pore complex, Ad genomes associate with PML-NBs and replication centers are established [30], [31], [reviewed in 32], [33]–[35]. Endosomal escape and subsequent transport are facilitated by Nedd4 ubiquitin ligases, which are recruited through a conserved PPxY motif in protein VI. Ads with mutated PPxY motif do not bind Nedd4 ligases and have reduced infectivity, showing the importance of this interaction for the onset of gene expression from the viral genome [36]. Here we report that Ad capsid proteins and cytoplasmic entry steps are linked to initiation of the adenoviral E1A expression by counteracting Daxx mediated transcriptional repression. Using the Ad system, we further show that capsid proteins from several other DNA viruses share and complement this function. This suggests a conserved mechanism among DNA viruses and provides insights into the very early virus-host interactions required to establish an optimal cellular environment for productive infection. The capsid protein VI participates in two crucial steps in the nuclear delivery of the Ad genome. Firstly, protein VI is required for lysis of endosomal membranes. Secondly, it is needed for efficient post-endosomolytic transport, mediated by the cellular ubiquitin ligase Nedd4 that binds to a conserved PPxY motif in protein VI. Mutating the PPxY motif interferes with capsid transport toward the nucleus and efficient viral gene expression [30], [36]. To investigate the role of protein VI during post-endosomolytic steps required for the onset of viral replication, we constructed replication competent Ads containing the E1 region with either wildtype (wt) protein VI (HH-Ad5-VI-wt, depicted in the Figure S1) or mutant “M1” protein VI in which the PPSY motif was mutated to PGAA that abolished Nedd4 interaction [HH-Ad5-M1; Fig. S1; [36]]. Following infection of U2OS cells, we observed that M1 virus replication was attenuated compared to wt (Figure 1A and S1B). This is in agreement with our previous observations showing reduced infectivity of an E1-deleted M1 Ad vector compared to the corresponding E1-deleted wt Ad vector [36]. To distinguish between capsid transport and possible more downstream effects, we infected cells with different amounts of replication competent wt and M1 viruses. Then, we determined the genome copy numbers in nuclear and cytoplasmic fractions by qPCR and the efficiency of the initiation of virus replication by quantification of E2A stained replication centers (detailed in Figure S2). Compared to wt, fewer M1 virus genomes accumulated in the nucleus associated fraction, independent of the amount of input virus. In contrast, initiation of virus replication for M1 genomes was reduced for low, but not at high physical particle per cell ratios (Figure S2) suggesting defects downstream of virus nuclear transport. Therefore, the expression of the early viral proteins E1A, E1B-55K and E2A in wt and M1 infected cells was analyzed by western blot, starting 8 h post infection (p. i.) and throughout the whole replication cycle (Figure 1B, left panel). We observed that expression of E1A in M1 virus infected cells was reduced compared to wt (Figure 1B, right panel) and accordingly, all other gene products were expressed with a delayed kinetic. This observation can be explained by the initial lower levels of E1A expression, because E1A is required for full activity of Ad downstream promoters [37]. Thus, we next investigated if the reduced E1A protein expression in M1-infected cells was due to reduced transcriptional activation of the E1A promoter following infection. We isolated and quantified newly synthesized E1A mRNA from cells infected with wt and M1 virus starting as early as 1–2 h p. i. (Figure 1C). The results confirmed that, at 1–4 h p. i., M1-infected cells showed reduced levels of newly synthesized E1A mRNA compared to wt-infected cells. Interestingly this reduction was gradually compensated throughout the first hours of infection (Figure 1C, compare 1–2 h, 3–4 h and 5–6 h) suggesting that low levels of initially made E1A were sufficient to compensate for the M1-defect in E1A transcription. The high particle per cell ratio requirement for transcriptional activation and the reduced levels of E1A mRNA and E1A protein expression for the M1 virus indicated that the PPxY motif in protein VI not only affects transport towards the nucleus, but also early viral gene expression, presumably through separate mechanisms. We previously showed that protein VI contains nucleo-cytoplasmic transport signals [38]. To test if protein VI could play a direct role in the initial activation of the viral genome, we first analyzed whether protein VI from incoming Ad capsids is imported into the nucleus. Using nucleo-cytoplasmic fractionation, we observed rapid protein VI accumulation in the nuclear fraction after infection (Figure 2A). Fractionation does not discriminate between nuclear (inside) or nucleus-associated (outside) accumulation of protein VI (e. g. capsid-associated at the microtubule organizing center). Thus, we investigated the subcellular localization of protein VI derived from entering viral particles by confocal microscopy in synchronous infected cells. Within one hour, we observed protein VI specific signals in dot-like structures inside the nucleus for wt- and the M1-virus. Using antibodies (Ab) against PML, we showed some protein VI associated with PML-NBs (Figure 2B). We confirmed the association of some protein VI with PML-NBs in a virus free system by transfecting protein VI-mRFP alone or together with EGFP-PML expressing plasmids into U2OS cells. Transfected proteins were detected via the mRFP and EGFP signal or with specific Ab for endogenous PML (“endogenous” highlighted throughout the text and in figures by the suffix “e”, e. g. ePML). The results show that protein VI was able to independently associate with PML-NBs (Figure 2C). Using a serie of protein VI mutants, we mapped the region of protein VI required for PML-NB association (Figure S3). This analysis revealed that the N-terminal amphipathic helix was required for efficient PML-NB targeting, because a mutant (VI-delta54) deleted of the amphipathic helix showed a diffuse nuclear distribution (Figure S3). We repeatedly observed the clustering of PML in transfected cells, suggesting PML-NB structure modulation resulting from protein VI expression. In summary, these data showed that some protein VI from incoming Ad particles is targeted into the nucleus, where some of it consistently localizes adjacent to PML-NBs, suggesting an involvement in additional intranuclear steps. It was recently reported by some of the co-authors of this work that the transient PML-NBs resident factor Daxx suppressed Ad replication and was degraded late in the infection cycle [21]. The observation that some protein VI was associated with PML-NBs prompted us to investigate whether PML itself, or PML-NB-associated factors such as Daxx, interact with protein VI. These interactions could provide an explanation for the reduced transcription of the E1A promoter observed for the M1 virus. Cells were infected with HH-Ad5-VI-wt or -VI-M1 and harvested after 24 h. Lysates were subjected to immunoprecipitation (IP) using PML or Daxx specific Ab and analyzed by western blot (Figure 3A). The data showed that protein VI could be precipitated from both wt and M1 infected cells using either PML or Daxx specific Ab. In contrast to virus infected cells, we did not detect co-precipitated protein VI following cotransfection and IP with different PML isoforms, suggesting an indirect association of PML and protein VI, presumably bridged by other viral or infection induced factors (Figure 3B). In contrast, co-IP of protein VI with Daxx also occurred after isolated transfection of protein VI-wt as well as protein VI-M1 suggesting that the interaction is independent of other viral factors (Figure 3C). We next asked whether Daxx interaction with protein VI could explain the reduced replication of HH-Ad5-VI-M1. For these assays, we used the hepatoma derived cell line HepaRG, because of its close resemblance to primary cells [39], and HepaRG cells depleted of Daxx (HAD, Daxx was depleted with shRNA expressing lentiviral vectors [20]). We infected Daxx-depleted HAD and HepaRG parental cells with HH-Ad5-VI-wt and HH-Ad5-VI-M1 and determined virus yields and gene expression at 12,24 and 72 h p. i. (Figure 3). The M1 virus was more strongly attenuated in HepaRG cells than in U2OS cells (compare to Figure 1), while Daxx depletion strongly enhanced virus production for both viruses and nearly restored the M1 virus yields to wt levels (Figure 3D). This improvement of Ad permissivity was confirmed by an increase of expression of all analyzed viral genes, including gene products from the E1A and E1B promoters (Figure 3E). The data showed that Daxx depletion was sufficient to increase Ad gene expression for both viruses, emphasizing the role of Daxx in viral genome repression. In addition, wt but not M1 mutant protein VI could counteract Daxx mediated inhibition indicating that the PPxY motif of protein VI plays a significant role in initiating viral gene expression. Next, we asked whether the Ad immediate early E1A and early E1B promoters are targeted by Daxx mediated repression and if this is the case whether it can be reversed by protein VI. To this end, we constructed luciferase expression vectors controlled by the Ad E1A and E1B promoters and measured luciferase expression in protein VI-wt or protein VI-M1 transfected H1299 cells (Figure 4A). Unlike VI-M1, VI-wt was able to stimulate expression from the E1A promoter ∼2. 5-fold and ∼1. 5-fold from the E1B promoter (Figure 4A). To show direct association of protein VI with E1 promoters, we performed chromatin immunoprecipitation assays (ChIP) at 48 h p. i from M1- or wt virus infected cells, using protein VI specific serum and Ad promoter-specific primers (Figure 4B). The results show that the VI-wt protein was much more strongly associated with the E1A and E1B promoter in infected cells than the VI-M1 protein, which is also reflected in their relative activation ability (Figure 4B, compare with 4A). To analyze whether protein VI associated activation of Ad early promoters is involved in Daxx de-repression, we cotransfected the E1B promoter driven luciferase expression vector in absence or presence of Daxx with protein VI-wt or VI-M1 expression vectors. Protein VI-wt, but not VI-M1, alleviated Daxx repression implying a role for the PPxY motif (Figure 4C). Although there was less binding to protein VI compared to the E1A promoter, we observed a strong effect on the activation of luciferase expression in that experiment. We also tested if protein VI (wt or M1) stimulates other Ad promoters using luciferase expression vectors for all viral promoters. The data showed that protein VI-wt was able to stimulate most of the Ad promoters in absence of other viral factors to various degrees (Figure S4). The strongest induction was observed for the immediate early E1A and E2A early promoter, which is in agreement with the weak E2A expression observed in HepaRG cells in M1-virus infected cells and the restoration of E2A expression following Daxx depletion (see Figure 3E). In contrast, E3 and E4 promoter activation was weak with no clear difference between wt and M1. In the context of an ongoing virus infection, the transcriptional activation of both promoter groups (E1/E2 vs. E3/E4) was shown to be regulated by E1A but via different mechanisms [40], [41]. Thus, our data showed that protein VI might also play a minor role in the transcriptional activation of the E1/E2 promoter group. Altogether, the promoter analysis suggests that protein VI plays a so far not recognized role in the Ad gene expression program. We next asked how the PPxY motif of protein VI contributes to Daxx de-repression. In previous work, we showed that this motif mediates protein VI interaction with cytoplasmic Nedd4 ubiquitin ligases [36]. Overexpression of protein VI and/or Nedd4 did not result in a change of steady-state Daxx levels (data not shown) suggesting that de-repression was not achieved through Daxx degradation as e. g. as shown for HCMV. However, when we tested if protein VI targets Nedd4 ligases to PML-NBs our analysis showed that protein VI-wt, but not VI-M1 targets Nedd4 ligases towards PML-NBs. This targeting required the PPxY motif and the amphipathic helix, but was independent of catalytical Nedd4 activity suggesting that Nedd4 ligases could be involved in other steps of counteracting Daxx repression by protein VI (Figure S5). As a next step, we therefore analyzed whether the subcellular distribution of Daxx was altered in response to protein VI and Nedd4 expression. In non-transfected cells, endogenous Daxx (eDaxx) is nuclear in steady state with some Daxx localizing to dot-like intranuclear structures resembling PML-NBs (Figure 5a). When we transfected expression vectors for protein VI-wt or VI-M1 into U2OS cells, nuclear localization of eDaxx was lost and eDaxx colocalized with transfected protein VI in the cytoplasm (Figure 5b and e). In contrast, following transfection of expression vectors for protein VI-wt and Nedd4 ligases, eDaxx remained nuclear and instead protein VI-wt colocalized with Nedd4 ligases in the cytoplasm (Figure 5c). When we transfected expression vectors for Nedd4 ligases and protein VI-M1, protein VI retained the capacity of translocating eDaxx to the cytoplasm (Figure 5f). These data suggested that binding of Nedd4 to the PPxY motif of protein VI efficiently competed with protein VI-dependent cytoplasmic translocation and/or cytoplasmic retention of Daxx. This effect did not require Nedd4 ubiquitin ligase activity (Figure 5d). Thus, our results suggested that the PPxY motif present in wt protein VI could influence the dynamic nucleo-cytoplasmic distribution of Daxx. To continue our analysis in a more physiological setting, we analyzed the subcellular localization of Daxx during Ad entry (Figure 6). In uninfected control cells, Daxx localized to the nucleoplasm and into PML-NBs. Within the first hour of infection, Daxx remained largely nuclear in wt- as well as M1-virus infected cells. Occasional cytoplasmic Daxx was never virus particle-associated. In contrast to non-infected cells, we observed a trend towards intranuclear displacement of Daxx from PML-NBs and PML clustering following infection (Figure 6A, red arrows), which could be clearly distinguished from Daxx spots in uninfected cells. This suggests that incoming viruses displace Daxx from PML-NBs by a mechanism independent of the PPxY motif of protein VI and prior to initial viral gene expression. Because we noticed occasionally large PML-NBs in infected cells, we next quantified the number of PML-NBs in wt- and M1-infected cells compared to non-infected cells. The results showed that on average, infected cells had less PML-NBs than non-infected cells, supporting our observation that PML-NBs were clustering (Figure 6B) and that the effects where PPxY motif independent. To show that the Daxx displacement from PML-NBs in the very early infection phase was caused by protein VI, we analyzed Daxx dissociation from PML-NB also in VI-wt and VI-M1 transfected cells (Figure S6). Compared to non-transfected cells, expression of protein VI-wt or VI-M1 led to translocation and cytoplasmic colocalization of Daxx (as seen in Figure 5). In addition, in several cells, Daxx was partially or completely displaced from PML-NBs and PML formed large nuclear clusters similar to those observed in infected cells (Figure S6, red arrows). We also transfected cells with expression vectors for HCMV pp71 tegument protein, known to interact with Daxx [42]. Unlike for protein VI, in pp71 transfected cells, Daxx remained nuclear and localized to some degree with PML into pp71 induced, ring-like structures also partially displacing Daxx from PML-NBs (Figure S6). To directly follow Daxx displacement from PML-NBs and from the nucleus, we used microinjection of recombinant protein VI (Figure 7 and Videos S1, S2, S3). We transfected U2OS cells with Daxx-mCherry and PML-GFP expression constructs, and injected the cytoplasm with either control buffer, recombinant VI-wt or with recombinant VI-M1 (Figure 7B) and followed the distribution of Daxx-mCherry using live-cell imaging (Figure 7A). Daxx-mCherry was exclusively localized to the nucleoplasm and PML-NBs, while PML-GFP showed an intranuclear dot-like distribution with some cytoplasmic aggregates at higher levels of expression. Cytoplasmic injection of protein VI-wt or VI-M1 led to displacement of Daxx from PML-NBs and cytoplasmic accumulation of Daxx within minutes of injection (Figure 7A, first and second row compared to buffer controls in the last row). We quantified the cytoplasmic accumulation of Daxx by measuring nuclear Daxx fluorescence loss following microinjection. This quantification revealed that Daxx nuclear export occurred more rapidly post injection of protein VI-wt than VI-M1, suggesting that the PPxY motif accelerated the process of Daxx displacement (Figure 7C). Notably, Daxx displacement was paralleled by a strong increase in intranuclear mobility of PML-GFP and by fusion events between individual bodies (Videos S1 and S2), thus providing evidence that the large clustered PML-NBs, observed in fixed cells, result from the mobilization of Daxx out of the bodies. We also microinjected recombinant protein VI (VI-delta54), lacking the amphipathic helix required for PML-NB targeting of protein VI, to see whether PML-NBs association is required for Daxx displacement. In contrast to protein VI-wt and VI-M1, injection of VI-delta54 only transiently displaced Daxx from PML-NBs and did not result in Daxx cytoplasmic translocation (Figure 7A third row and Video S3). The Daxx residence time in PML-NBs is ∼2 seconds [43]. Therefore our observation could be explained by competitive binding of VI-delta54 to Daxx, which could transiently prevent Daxx from association with PML-NBs. In summary, these data strongly suggested that protein VI from incoming adenoviral capsids can displace Daxx from PML-NBs, which in turn affects the PML-NB architecture leading to the accumulation of PML in large intranuclear clusters. Our analysis further indicate that association of protein VI with PML-NBs through the amphipathic helix is not strictly required for Daxx displacement from PML-NBs and that the PML-NB rearrangements take place prior to or are concomitant with the initiation of adenoviral transcription. Our data showed that protein VI activates the Ad E1 promoters by reversing Daxx repression, presumably until newly synthesized E1A can secure the Ad gene expression program. In this case, virion proteins derived from other DNA viruses known to abrogate Daxx repression should be able to substitute this function. To test this possibility, we tested whether the expression from the E1A promoter can be activated by the HCMV pp71 tegument protein or by the HPV L2 minor capsid protein, which both target Daxx [26], [44]. Similar to protein VI-wt, pp71 and L2 were able to stimulate the Ad E1A promoter (Figure 8A). Furthermore, we observed that like protein VI-wt, pp71 and L2 could also drive efficient E1A and E1B expression from a subviral construct, preserving the virus context encoding the E1A and E1B transcription units (Figure 8B, lane 3,6 and 7). These results show that non-adenoviral virion proteins are also capable of inducing immediate early adenoviral gene expression in the absence of any further Ad protein. This induction of gene expression was through mediating transcriptional activation, as shown by elevated E1A and E1B mRNA levels (Figure 8C). Similarly, this result confirmed that elevated E1A mRNA and protein expression levels driven by protein VI require the PPxY motif, thus directly linking entry and early viral gene expression (Figure 8B, lanes 1–4). To extend the analysis for other regions of protein VI, we used the expression construct encoding protein VI-delta54, lacking the amphipathic helix, which is required to target protein VI to PML-NBs (Figure S3d). The results showed that like protein VI-M1, the construct expressing VI-delta54 only marginally stimulated the E1A promoter (compare wt-, M1 and delta54 in Figure 8A and C). In contrast, the expression of protein VI-delta54 resulted in somewhat elevated protein expression levels compared to VI-M1 suggesting that it might promote E1A expression on a post-transcriptional level. This could result from the diffuse localization of VI-delta54 in the nucleoplasm of transfected cells (compare with Figure S3). In summary, this analysis showed that efficient transcriptional activation of the E1A promoter requires the amphipathic helix in addition to the PPxY motif. If the HCMV tegument protein pp71, that is known to remove Daxx repression from the immediate early HCMV promoter [45], activates the Ad E1A promoter, it was conceivable to speculate that protein VI would also be able to stimulate the immediate early HCMV promoter. To test this hypothesis, we constructed viral vectors encoding wt- or M1-mutated protein VI where the E1 region was replaced by a HCMV promoter controlled GFP (wt) or mCherry (M1) expression unit. We transduced U2OS cells with M1-vectors and increasing amounts of wt virus and quantified gene expression using fluorescent activated cell sorting. The results showed partial restoration of the (HCMV promoter controlled) marker gene expression from VI-M1 vector transduced cells only in cells that were co-transduced with the M1-vector and the wt-vector (Figure S7). This analysis suggested that protein VI stimulated the HCMV promoter in trans, like pp71 could stimulate the Ad E1A promoter in trans (Figure S7). Taken together the effects that protein VI has on the E1A promoter are comparable, and moreover compatible and interchangeable, with the HCMV or papillomavirus virion derived immediate early enhancing activities. Because protein VI, pp71 and L2 can stimulate Ad E1A expression independently, we next asked if they could compensate for the lack of functional PPxY motif in the replication competent HH-Ad5-VI-M1 virus. We transfected cells with expression vectors for protein VI-wt, VI-M1 and VI-delta54 (Figure 9A) and HCMV tegument protein pp71 and HPV small capsid protein L2 (Figure 9B) followed by infection with HH-Ad5-VI-wt or HH-Ad5-VI-M1 virus. The analysis showed that protein VI-wt was able to fully compensate for the M1 mutation in the virus and restored progeny virus production to wt levels, while protein VI-M1 was not able to rescue virus production and VI-delta54 resulted only in partial rescue (Figure 9A). Amazingly, HCMV pp71 and HPV L2 were also fully capable of complementing the M1 mutant virus and restored progeny virus production to wt levels (Figure 9B). Lastly, we wanted to know if the adenoviral protein VI capsid protein was also able to stimulate an immediate early promoter in the context of a non-related virus infection. We transfected U2OS cells with protein VI-wt and VI-M1 or a control vector and infected the transfected cells with a murine cytomegalovirus (MCMV) expressing luciferase under the control of the HCMV immediate early promoter (MCMV-Luc). Luciferase expression was measured 2 h after a synchronized infection to quantify the activation of the immediate early promoter. The results showed that only protein VI-wt was able to stimulate immediate early promoter in the context of MCMV infection (Figure 9C). Taken together these results showed that protein VI promotes immediate early gene expression from the adenoviral E1A promoter, but it was also able to act on the immediate early gene expression of a non-related virus. In summary, our analysis provides an intriguing mechanistic basis for cross genome activation of at least three unrelated DNA viruses. Our data suggest that initiation of viral gene expression can be achieved in cases where the respective virion proteins of one virus are capable of removing Daxx dependent transcriptional repression from the genome of the other virus. Here, we show that the capsid protein VI is necessary for efficient initiation of Ad gene expression by activating the E1A promoter and promoting initial expression of the E1A transactivator, a function that had not been previously identified. E1A is a crucial global transcriptional activator promoting early adenoviral gene expression [37]. We show that E1A transcription and E1A protein expression at the onset of viral gene expression are reduced when cells are infected with an Ad mutant in which the PPxY motif in the capsid protein VI is inactivated. E1A mRNA production in this mutant increases with time and reaches wildtype levels, suggesting that newly expressed E1A compensates for the mutation in protein VI and drives adenoviral gene expression as soon as critical concentrations have been reached [37]. In addition, protein VI also stimulates other E1A dependent Ad promoters in the absence of any viral protein suggesting that it may act as a capsid derived E1A surrogate prior to the onset of E1A expression. Thus, protein VI is an important regulator of viral gene expression and links virus entry to the onset of gene expression. This is at least in part mediated by counteracting transcriptional repression imposed by the cellular Daxx protein and can be substituted by functionally homologous capsid proteins from unrelated DNA viruses. In the nucleus, Daxx associates with chromatin and PML-NBs. PML-NB association with Daxx is thought to alleviate gene repression and activate apoptosis, while chromatin bound Daxx is thought to act in a transcriptionally repressive manner [7], [46], [47]. A dynamic equilibrium of Daxx between PML-NBs and chromatin association may thus govern the response status of the host cell upon infection. Moreover, an antiviral interferon response increases expression of PML and sensitizes cells for apoptosis. Artificial knock down of PML increases replication of Ad and other viruses, an observation that supports antiviral functions of PML [reviewed in 4], [21]. However, PML knock down also decreases Daxx steady state levels by an unknown mechanism, showing that antiviral activity might be mediated by Daxx rather than PML [21]. This would be in line with our observation that Daxx knock down has much stronger pro-replicative effects on Ads. Here we demonstrate that Daxx directly represses Ad E1 promoters. So far, it has been shown that Daxx inactivates the major immediate early promoter of HCMV [45], is recruited to HSV genomes via SUMO dependent pathways [48] and is likely to associate with incoming avian sarcoma virus (ASV) and human immunodeficiency virus (HIV) genomes [49], [50]. Therefore, Daxx could act as a cytoplasmic and/or nuclear DNA sensor and may be part of a cellular innate defence mechanism against DNA virus infection (or other pathogens) by simply assembling repressive complexes on incoming DNA [51]. This is supported by two recent studies showing that Daxx selectively represses procaryotic DNA expression [52] and that frequent epigenetic silencing of integrated retroviral genomes could be reversed by Daxx depletion, showing epigenetic control of pathogen DNA by Daxx associated mechanisms [53]. Daxx mutants that fail to associate with the HSV genome also fail to induce repression on the HSV genome, underlining the important role of Daxx as part of the cellular innate antiviral defence mechanism [48]. If Daxx serves in antiviral intrinsic immunity to repress viral genomes, virion proteins are viral countermeasures. Several structural proteins from viral particles have been reported to interact with Daxx, including tegument protein pp71 [HCMV]; [ 42,54], minor capsid protein L2 [HPV; 26], DENVC [Dengue virus; 55], p6 [HIV GAG; 56], nucleocapsid protein PUUV-N [Hantavirus; 57], Integrase [ASV], [ HIV; 49,53] and protein VI (Ad, this study). The best studied is the tegument protein pp71 of HCMV, which enhances infectivity and replication through activation of the immediate early promoter. This requires colocalization of the viral genome with PML-NBs and Daxx degradation via pp71 [23], [42], [44], [58], [59]. In addition, pp71 was also shown to activate gene expression from HSV-1, a different herpesvirus, showing that its function is not restricted to HCMV [60]. Unlike for HCMV, degradation of Daxx [through E1B-55K; 21] during Ad infection requires early gene expression. Here we observe quantitative removal of Daxx from PML-NBs upon infection without degradation before gene expression is established. We propose that this is caused by protein VI derived from the entering capsid, which partially associates with PML-NBs during entry. Similar to what we observe early in infection, transfected protein VI also displaces Daxx from PML-NBs and translocates it into the cytoplasm. Similarly, microinjected protein VI leads to rapid exclusion of Daxx from PML-NBs and cytoplasmic accumulation suggesting active removal following protein VI nuclear import. Deletion of the N-terminal amphipathic helix from protein VI, which serves as PML-NB targeting domain, still mediated the transient dissociation of Daxx from PML-NBs suggesting that competitive binding and a short residence time of Daxx in PML-NBs can also cause Daxx removal from PML-NBs [43]. Daxx depletion from PML-NBs also provokes intranuclear mobility and clustering of PML, reminiscent of infected cells and showing that Daxx contributes to the integrity of PML-NBs, which confirms previous observations [10]. Ad-wt, but not a virus with the mutated PPxY-motif in protein VI, counteracts Daxx repression for efficient viral gene expression. Protein VI wt also induces a more rapid Daxx displacement from PML-NBs and subsequent nuclear export than its mutated counterpart. In contrast, binding of Nedd4-family ubiquitin ligases to the PPxY of protein VI abolished cytoplasmic translocation of Daxx at steady state, suggesting that Nedd4 binding to protein VI competes with the interaction between Daxx and protein VI. Increasing the efficiency of Daxx mobilization in the nucleus, and simultaneously preventing Daxx nuclear export or limiting the time Daxx resides in the cytoplasm through competitive binding to Nedd4, could lead to efficient derepression and prevent Daxx from activating apoptosis (via JNK pathways), which could explain why Nedd4 binding is beneficial for the virus [10], [61]. Displacing Daxx from PML-NBs immediately after virus entry prevents antiviral apoptotic processes, possibly increasing Daxx mediated repression by epigenetic silencing [reviewed in 5]. We observe Daxx removal from PML-NBs for wt- as well as M1 mutated protein VI. In contrast, only wt-VI shows a strong stimulation and direct association with viral E1 promoters as determined by ChIP. In addition, proper transcriptional activation of the E1A promoter required the presence of the amphipathic helix. Thus, reversal of Daxx repression by protein VI from viral promoters might provide an additional explanation for Nedd4 function and the role of the PPxY motif. Targeting Nedd4 to viral promoters via the PPxY could result in ubiquitylation of histone or the histone-like DNA bound viral protein VII or other Daxx interactors, to open the chromatin structure for transcription. In this scenario, protein VI would prevent formation or disassemble already bound repressive complexes from viral promoters via the PPxY motif and Nedd4. This would explain why the M1 mutant still displaces Daxx from PML-NBs, but retains only a minor capacity of stimulating viral gene expression presumably through interfering with the assembly of new Daxx repressive complexes. This model would also support the observation that, like protein VI-M1, protein VI without amphipathic helix (but intact PPxY and still capable of Daxx binding) hardly stimulates the E1A promoter. This mutant is diffusely distributed in the nucleus showing that the helix contributes to proper intranuclear targeting of protein VI. Mislocalization therefore could reduce the capacity to remove or prevent assembly of Daxx repressive complexes on the E1A promoter. How this mutant still retains some capacity of stimulating E1A protein expression (and as a consequence partially rescues the M1-virus) without activating the E1A promoter is currently unclear. Removal of Daxx by components of incoming virions to initiate gene expression is a common viral strategy. Our experiments are the first to show that the consequences are not virus-family specific, but provoke global changes in transcriptional activity that allow transcriptional activation of one viral genome (here the Ad or MCMV genome) by the virion protein of unrelated viruses (here pp71 from HCMV and L2 from HPV; or the CMV promoter by protein VI). All three virion proteins (VI, pp71 and L2) target Daxx repressive complexes. The details of these interactions are not fully understood but they share similarities as highlighted in the model in Figure 10. We suggest that activation of viral gene expression for the three viral systems (Ad, HCMV and HPV) involves prevention and removal of Daxx repressive complexes. This is achieved by preventing Daxx-PML interaction or association of Daxx repressive complexes with the viral genome and (in some cases) involves the degradation of components of the complex (Figure 10). Neither pp71 nor L2 contain a PPxY motif suggesting different modes of action on Daxx or components of the Daxx repressive complex. Protein VI is also not restricted to Ads in its de-repressive activity and is able to stimulate the immediate early HCMV promoter. Several other viral capsid proteins have been reported to encode PPxY motifs [reviewed in 62]. The research focus for those motifs has been on their role in virus budding despite the presence of these proteins in the capsids of many viruses during virus entry. If virion derived PPxY (and related motifs) are part of a more general activation mechanism for several viruses then this could also mean that co-infections with different viruses, frequently observed in vivo, could promote each other. Similarly it is an interesting question, whether superinfections of a latently infected cell by another de-repressive virus would support reactivation of the latent genome. Epidemiological data from a recent study show that Ad/HCMV co-infections in vivo happen as often as mono-infections and the authors suggest that this could reflect co-viral reactivation [63]. Our data would provide a mechanistic basis for this observation, which is potentially applicable to several types of viral co-infections. Lastly, we believe that gene regulatory functions of viral structural proteins should be considered when addressing safety issues for the application of viral vectors (e. g. adenoviral vectors) in therapeutic settings where (re) activation of unrelated (latent) viruses is unwanted. U2OS, H1299 and HepaRG cells were grown in Dulbecco' s modified Eagle' s medium supplemented with 10% fetal calf serum (FCS), 100 U of penicillin, 100 µg of streptomycin per ml in a 5% CO2 atmosphere at 37°C. For HepaRG and HAD (Daxx knock down) cells media was supplemented with 5 µg/ml of bovine insulin and 0. 5 µM of hydrocortisone [20], [21]. Tagged protein VI, PML, Daxx and Nedd4 expression vectors have been described previously [36], [64]. E1A was expressed from constructs encompassing the left part of the viral genome including left inverted terminal repeat (ITR) and the E1 genes (pPG-S3). N-terminal flag-tagged human PML-isoforms I-VI were expressed from pLKO. 1-puro vector (kindly provided by R. Everett). Codon optimized HPV (type 16) L2 expression vector was kindly provided by M. Mueller, DKFZ Heidelberg. Expression vector pCGN71 [65] encodes an XbaI-BamHI PCR fragment 0corresponding to the HCMV strain AD169 UL82. Dual luciferase assays were performed according to manufacturers instructions and have been described previously [66]. Promoter constructs are based on the pGL3-basic vector (Invitrogen, cloning details will be provided upon request). E1-deficient viral vectors BxAd5-VI-wt-GFP and BxAd5-VI-M1-mCherry are based on human Ad serotype 5 and have been cloned using homologous and site-specific recombination using bacterial artificial chromosomes (BACs) as described in detail recently [36]. Replication competent wt virus HH-Ad5-VI-wt is identical to the previously described H5pg4100 [67]. The virus mutant HH-Ad5-VI-M1 carries an altered PPxY motif in the protein VI open reading frame [PPSY = >PGAA; Fig. S1; [36]]. Viruses were constructed, propagated and titrated on HEK293 cells as detailed in Figure S1. For immunofluorescence analysis cells were washed in PBS and fixed for 20 min using 4% paraformaldehyde. Detection of endogenous antigens using primary and secondary Ab was done in IF-buffer (PBS with 10% FCS and 0. 2% Saponin) followed by washing and embedding in Prolong Gold (Invitrogen). A list of primary and secondary Ab used in this study is given in Protocol S1 in Text S1. Images are presented as maximum image projections if not indicated otherwise. For protein analysis total-cell lysates were prepared and analyzed by western blot using standard protocols. The list of the antibodies used in this study and details for immunoprecipitation (IP) procedures are given in Protocol S1 and Protocol S2 in Text S1. H1299 cells were infected with HH-Ad5-VI-wt or HH-Ad5-VI-M1 at 50 fluorescence forming units/cell (FFU/cell) and harvested 24 h p. i. ChIP analysis was performed as described previously with some modifications [68], [69]. For ChIP, proteins from 2×106 cells were cross-linked to DNA with 1% formaldehyde in PBS for 10 min at room temperature. The reaction was quenched and cells were washed with PBS and harvested by scraping off the dish. Nuclei were isolated by incubation of cross-linked cells with 500 µl buffer I (50 mM Hepes-KOH, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0. 5% NP-40,0. 25% Triton X-100) for 10 min on ice and pelleted by centrifugation. The nuclei were subsequently washed with 500 µl buffer II (10 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 0. 5 mM EGTA), pelleted again and resuspended in 500 µl buffer III (1% SDS, 10 mM EDTA, 50 mM Tris-HCl). Chromatin was fragmented by sonication using a Bioruptor (Diagenode) to an average length of 100–300 bp. After addition of 10% Triton X-100, cell debris were pelleted by centrifugation (20,000× g, 4°C) and supernatants were collected. Chromatin was diluted with dilution buffer (0. 01% SDS, 1. 1% Triton X-100,1. 2 mM EDTA, 16. 7 mM Tris-HCl, 167 mM NaCl). To reduce non-specific background, chromatin was pre-incubated with salmon-sperm DNA protein-A agarose beads (Upstate). Antibodies were added and incubated for 16 h at 4°C. Fifty µl agarose beads were added to precipitate the chromatin-immunocomplexes for 4 h at 4°C. Beads were washed once with low-salt buffer (0. 1% SDS, 1% Triton X-100,2 mM EDTA, 20 mM Tris-HCl, 150 mM NaCl), once with high-salt buffer (0. 1% SDS, 1% Triton X-100,2 mM EDTA, 20 mM Tris-HCl, 500 mM NaCl), once with LiCl-wash buffer (0. 25 M LiCl, 1% Nonidet P-40,1% Na-deoxycholate, 1 mM EDTA, 10 mM Tris-HCl) and twice with TE buffer. Chromatin was eluted from the beads in elution-buffer (50 mM Tris-HCl pH 8. 0,10 mM EDTA, 1% SDS) for 10 min at 95°C. Proteinase K was added for protein degradation and samples were incubated for 1 h at 55°C. For preparation of input controls, samples were treated identical to IP samples except that non-specific Ab were used. qPCR analysis was performed using a Rotor Gene 6000 (Corbett Life Sciences, Australia) in 0. 5 ml reaction tubes containing 1/100 dilution of the precipitated chromatin, 10 pmol/µl of each synthetic oligonucleotide primer (E1A fwd 5′TCCGCGTTCCGGGTCAAAGT3′; E1A rev5′GTCGGAGCGGCTCGGAG3′; E1B fwd 5′GGTGAGATAATGTTTAACTTGC3′ ¸ E1B rev 5′TAACCAAGATTAGCC CACGG3′), 5 µl/sample SYBR Green PCR Master Mix (Applied Biosystems). The PCR conditions used: 7 min at 95°C, 45 cycles of 12 s at 95°C, 40 s at 60°C and 15 s at 72°C. The average Ct-value was determined from triplicate reactions and normalized against non-specififc IgG controls with standard curves for each primer pair. The identities of the products obtained were confirmed by melting curve analysis. For qPCR analysis, U2OS cells were infected with 1,10 and 200 physical particles/cell and genome copy numbers were determined in nuclear and cytoplasmic fractions using hexon specific primers [70]. 4sU (Sigma) was added to the cell culture media for 1 h, made up to a final concentration of 200 µM, during indicated time points throughout infection. Cells were harvested using Trizol reagent (Invitrogen) and total RNA isolated by phenol-chloroform extraction. Biotinylation and purification of 4sU-tagged RNA (newly transcribed RNA), was performed as described previously [71]. Five hundred ng of each newly transcribed RNA per reaction was reverse transcribed in 25 µl reactions using Superscript III (Invitrogen) and oligo-dT primers (Invitrogen) following the manufacturer' s instructions. PCR was performed on a Light Cycler (Roche Molecular Biochemicals). Each reaction, every sample in duplicates, was carried out using 5 µl of cDNA (1∶10 dilution) and 15 µl reaction mixtures of Quantitect SYBR Green PCR master mix and 0. 5 µM of the primers. PCRs were subjected to 10 min of 95°C hot-start, and SYBR Green incorporation was monitored for 45 cycles of 95°C denaturation for 10 s, 58°C annealing for 3 s, and 72°C elongation for 10 s. The data were analyzed using the ΔΔCt method using GAPDH as an endogenous reference, and the mock-infected sample as a calibrator. Values were normalized to 100% for wt-infected cells. The E1A 13S mRNA specific and the GAPDH specific primers were described in [72]. Primers used are listed below: E1A13S-fwd (5′-GGC TCA GGT TCA GAC ACA GGA CTG TAG), E1A13S-rev (5′-TCC GGA GCC GCC TCA CCT TTC), GAPDH-fwd (5′-TGG TAT CGT GGA AGG ACT CA), GAPDH-rev (5′-CCA GTA GAG GCA GGG ATG AT). Details for microinjection are given in the Figure 8 and video legends (Video S1). Briefly, U2OS cells were cotransfected with PML-GFP and Daxx-mCherry expression plasmids and cultivated on a heated stage (37°C) in CO2 stabilized medium attached to a SP5 confocal microscope (Leica) equipped with a microinjection device (Eppendorf). Microinjected cells were imaged within a single confocal plane at the nuclear midsection at 20 s intervals for 10 frames prior to injection and 40 frames post injection. Injected proteins were purified as His-tagged proteins using standard procedures and dialyzed into transport buffer as detailed previously [30], [36]. Data are presented as mean, error bars as standard deviation (STD). Statistical analysis was done using paired students t-test except for Figure 6B where a two-tailed two sample t-test was used. The p-values are indicated. Human Daxx CAG33366. 1, Protein VI AAA96411. 1, Human Adenovirus Type 5 HY339865, PML-I AAG50180, PML-II AF230410, PML-III S50913, PML-IV AAG50185, PML-V AAG50181, PML-VI AAG50184, HCMV pp71 ACZ79993. 1, humanized HPV L2 (HPV16).

To initiate infection, DNA viruses deliver their genome to the nucleus and express viral genes required for genome replication. Efficient transport is achieved by packing the viral genome as a condensed, transcriptionally inactive nucleo-protein complex. However, for most DNA viruses, including Adenoviruses (Ads), it remains unclear how the viral genome is decondensed and how transcription is initiated inside the nucleus. Cells control unwanted gene expression by chromatin modification mediated through transcriptionally repressive complexes. A key factor in repressive complex assemblies is the transcriptional repressor Daxx. The Ad structural capsid protein VI is required for endosomal escape and nuclear transport. Here we show that protein VI also activates the Ad E1A promoter to initiate Ad gene expression. This is achieved through the removal of Daxx repression from the E1A promoter, which requires a conserved ubiquitin ligase interacting motif (PPxY-motif) in protein VI. We further show that capsid proteins from other unrelated DNA viruses also activate the Ad E1A promoter and support Ad replication by counteracting Daxx repression, functionally replacing protein VI. Our data suggest that reversal of Daxx repression by virion proteins is a widespread mechanism among DNA viruses that is not restricted to a single virus family.

lay_plos

Kinesin-12 motors are a little studied branch of the kinesin superfamily with the human protein (Kif15) implicated in spindle mechanics and chromosome movement. In this study, we reconstitute full-length hKif15 and its microtubule-targeting factor hTpx2 in vitro to gain insight into the motors mode of operation. We reveal that hKif15 is a plus-end-directed processive homotetramer that can step against loads of up to 3. 5 pN. We further show that hKif15 is the first kinesin that effectively switches microtubule tracks at intersections, enabling it to navigate microtubule networks, such as the spindle. hKif15 tetramers are also capable of cross-linking microtubules, but unexpectedly, this does not depend on hTpx2. Instead, we find that hTpx2 inhibits hKif15 stepping when microtubule-bound. Our data reveal that hKif15 is a second tetrameric spindle motor in addition to the kinesin-5 Eg5 and provides insight into the mechanisms by which hKif15 and its inhibitor hTpx2 modulate spindle microtubule architecture. The human mitotic spindle is a microtubule-based protein machine with bipolar geometry that mediates chromosome segregation (Dumont and Mitchison, 2009). Initial assembly of the spindle requires the separation of centrosomes by the homotetrameric plus-end-directed molecular motor Kif11 (Eg5) —a member of the kinesin-5 family (Tanenbaum and Medema, 2010). In vitro reconstitution experiments reveal that Kif11 can drive the outward sliding of anti-parallel microtubules thus providing a mechanistic basis for centrosome separation (Kapitein et al., 2005). Although essential for centrosome separation, Kif11 is not required to maintain subsequent spindle bipolarity due to the compensatory activity of the Kinesin-12 Kif15 (hKlp2) (Tanenbaum et al., 2009; Vanneste et al., 2009). Furthermore, overexpression of hKif15 can overcome the absolute requirement for hKif11 in centrosome separation (Tanenbaum et al., 2009; Sturgill and Ohi, 2013). This functional redundancy has led to a model in which hKif15, like Kif11, can slide apart anti-parallel microtubules. Kinesin-12 proteins from Xenopus (xKlp2) (Wittmann et al., 1998), sea urchin (Rogers et al., 2000), and rat (Liu et al., 2010) are dimeric plus-end-directed motors that are targeted to the spindle by the microtubule-associated protein (MAP) Tpx2 (targeting protein for xKlp2) (Wittmann et al., 1998). hKif15 is also recruited to the spindle microtubules by hTpx2 in human cells (Tanenbaum et al., 2009; Vanneste et al., 2009) with the interaction between dimeric hKif15 and hTpx2 hypothesised to enable the motor to cross-link and slide anti-parallel microtubules (Tanenbaum et al., 2009). However, this model has been challenged by recent cell-biological studies showing that hKif15 localizes to kinetochore (k) -fibres (parallel microtubule bundles) and contributes to the generation of forces that counter those generated by hKif11 (Sturgill and Ohi, 2013; Vladimirou et al., 2013). Understanding how hKif11 and hKif15 cooperate during mitosis has important implications for cancer therapy because the clinical efficacy of hKif11 inhibitors, currently used as monotherapy, has proven largely disappointing (Rath and Kozielski, 2012). hKif15 is therefore emerging as a potentially important therapeutic target (Groen, 2013). To understand how hKif15/hKif11 operate within the spindle, we will require a detailed mechanistic understanding of how each motor interacts with microtubules and generates force. Such information is already available for Kif11. In this study, we provide the first insight into the properties of the Kinesin-12 family motor hKif15. Full-length human His6-Kif15 (hKif15), His6-Kif15-eGFP, and His6-Tpx2 (hTpx2) were expressed in insect SF9-cells and the recombinant proteins purified to near homogeneity by sequential affinity chromatography using a cation-exchange and a Co-NTA matrix (Figure 1A). Analysis of His6-hKif15 on native PAGE revealed that the protein migrates at ∼730 KDa (Figure 1B). Given the predicted molecular weight of His6-hKif15 (164. 8 kDa) our data suggest the presence of hKif15 tetramers, although hydrodynamic analysis of the frog (xKlp2) and Sea urchin (KRP180) Kif15 orthologues indicated that the motor is dimeric with a sedimentation coefficient of 8. 1S and 8. 3S respectively (Wittmann et al., 1998; Rogers et al., 2000). To further characterise human Kif15, we subjected His6-hKif15 to glycerol gradient ultracentrifugation on 5–40% glycerol gradients. At physiological ionic strengths (35 mM sodium phosphate buffer, 0-150 mM NaCl) both His6-hKif15 and His6-hKif15-eGFP appear to be monodispersed and have apparent sedimentation coefficients of ∼12S (Figure 1C). However, increasing the salt concentration to 300 mM NaCl converts hKif15 into a species that runs at ∼8S (Figure 1C). Taken together, our data show that hKif15 exists as a tetramer at physiological ionic strength that can be forced to dissociate into a dimer at high ionic strength. To further confirm that hKif15 can form tetramers we performed a size-exclusion chromatography combined with multi angle light scattering (SEC-MALS), which allows determination of the absolute molecular weight. This analysis confirmed the presence of tetrameric hKif15 motors (molecular weight of 744. 4 ± 14. 1 kDa) (Figure 1D, 1st peak, blue line). We could also detect a dimeric population of motors (molecular weight of 360. 1 ± 6. 5 kDa) indicating that there is some degree of complex dissociation in solution under these conditions (Figure 1D, 2nd peak, orange line). 10. 7554/eLife. 01724. 003Figure 1. Kif15 is a tetramer. (A) Coomassie stained SDS-PAGE gel of purified His6-hTpx2, His6-hKif15, and His6-hKif15-GFP. (B) Tetrameric His6-hKif15 on a 4–16% Native-PAGE gel stained with coomassie. Calculated molecular weight: His6-hKif15: 165 kDa. (C) Coomassie stained SDS-PAGE gels of fractions 1–23 out of 25 from 5–40% glycerol gradients loaded with either ∼5 µg His6-hKif15 or His6-hKif15-eGFP at different salt concentration (see table ‘below’ that summarises the apparent S values of hKif15 (eGFP) at different salt concentrations). (D) Elution profile (grey line, A280, left y-axis) from a size-exclusion chromatography (SEC) run with subsequent multi angle light scattering (MALS) analysis. Outcome of the MALS analysis for the peaks is presented in coloured lines (blue-hKif15 tetramer, orange hKif15 dimer, MW, right y-axis). Molecular weight ± uncertainty is given above each peak. Please note that the presence of dimeric hKif15 species is specific to the protein preparation used in this experiment only (‘Materials and methods’). Standard preparations do not contain any significant proportion of dimeric hKif15 (see panels B and C this Figure). DOI: http: //dx. doi. org/10. 7554/eLife. 01724. 003 Next, we used total internal reflection fluorescence (TIRF) -microscopy, to observe the movement of single hKif15-eGFP motors on polarity-marked microtubules that were stabilised with the non-hydrolysable GTP-analogue GMP-CPP. These experiments were done in buffer conditions (BRB20,50 mM KCl) that would be expected to preserve hKif15 in a tetrameric state (from here on hKif15-eGFP refers to single tetrameric motors). The majority hKif15 motors move either by directional movement towards the plus-end of individual microtubules (Figure 2A, ‘P’) or by bidirectional 1D-diffusion (Figure 2A, ‘D’, Figure 2B, right, Figure 2—figure supplement 1D, right) along the microtubule lattice. These types of movement are equally likely at all salt conditions tested (Figure 2—figure supplement 1A) and single motors can switch between both modes (Figure 2—figure supplement 1B). Processively moving, plus-end-directed motors move at a speed of 137. 8 ± 4. 1 nm•s−1 median ± SEM, though we observed velocities up to 550 nm•s−1 (Figure 2B, left). The run length of hKif15 motors is 1. 9 ± 0. 09 µm median ± SEM (max. 9. 6 µm) (Figure 2B, middle) and their residency time 26. 3 ± 2. 8 s median ± SEM (max. >180 s) (Figure 2B, right). Processive runs can include pauses up to 20. 5 s (median ± SEM: 5. 0 ± 0. 4 s) at a frequency of 0. 30 ± 0. 04 pauses•µm−1 mean ± SD (n = 162, three different experiments) without motor-dissociation from the microtubule lattice (Figure 2—figure supplement 1B, D, middle). Additionally, when motors reach the microtubule end, more than 90% remain attached (Figure 2—figure supplement 1D, left note orange column). Motors dwell at the tip for 18. 8 ± 3. 5 s median ± SEM (max. >152 s) (Figure 2A, (asterix), Figure 2—figure supplement 1D, left) and may switch to diffusional movement on the end-proximal microtubule lattice (Figure 2—figure supplement 1C). While the majority of hKif15 motors either moves directionally to the plus-end or diffuses along the lattice, we also observed a small proportion of motors that move processively towards the minus-end of polarity-marked microtubules (87. 5% plus-end-directed motors vs 12. 5% minus-end-directed motors, n = 504) (Figure 2A, right kymograph, ‘MP’). Minus-end-directed motors move at a speed similar to plus-end-directed motors, but have a significantly reduced run length/residency time and do not undergo directional reversals (Figure 2C). 10. 7554/eLife. 01724. 004Figure 2. hKif15 is a processive tetramer. (A) Kymographs showing typical behaviour of eGFP-labelled hKif15 motors on GMP-CPP stabilised microtubules (processive movement–P, diffusion–D, plus-end dwelling–asterix, processive minus-end directed movement–MP). Pictures on the left of each kymograph show orientation of the polarity labelled microtubule. (B) Frequency distributions showing kinetic properties of processively moving plus-end-directed eGFP-labelled hKif15 motors. Coloured lines within the column plots are Gaussian or exponential decay fits. Insets show the respective median ± standard error of mean and maximum value of the distribution as well as its sample size. All values are derived from kymographs as shown in (A). (C) Frequency distributions showing kinetic properties of eGFP-labelled hKif15 motors that move processively to the minus-end of a microtubule. Coloured lines within the column plots are Gaussian or exponential decay fits. Insets show the respective median ± standard error of mean and maximum value of the distribution as well as its sample size. All values are derived from kymographs as shown in (A). (D) Kymographs showing a processively moving hKif15-eGFP motor (left) and a diffusive hKif15-eGFP motor (right) that photo-bleach in three and four steps respectively, indicating presence of four eGFP molecules in each motor. Plot below the kymograph shows the intensity along motor traces in arbitrary units. Intensity had been locally corrected for background intensity. Horizontal green lines within the graphs indicate the fitted average intensity (arbitrary units ± standard deviation) in the respective section of the trace. Photo-bleaching steps were manually defined. Please note that for the last bleaching step in the right kymograph we cannot formally exclude a dissociation event. (E) Example traces showing the movement of a single molecule (hKif15 tetramer) as a function of time (1 ms boxcar filtered). The motor steps out of the trap centre (black dotted line) until it detaches at variable loads between 1. 5 and 3. 5 pN and reattaches (in the trap centre) for subsequent movements. Movements can also be bidirectional (green arrowhead, compare with Figure 2A). DOI: http: //dx. doi. org/10. 7554/eLife. 01724. 00410. 7554/eLife. 01724. 005Figure 2—figure supplement 1. TIRF-based analysis of hKif15 motility. (A) Kymographs showing the behaviour of hKif15-eGFP motors on GMP-CPP stabilised microtubules in dependency indicated salt concentrations apart from the standard condition used throughout the experiments (BRB20,50 mM KCl, Figure 2A) (processive movement—P, diffusion—D). (B) Kymographs showing moving hKif15-eGFP motors that convert from diffusive (‘D’) into processive movement (‘P’) or vice versa and show extensive pauses during processive movement (horizontal sections of the trace). (C) Kymographs showing end dwelling hKif15 motors that switch to diffusive movements and roam the end proximal region of the microtubule lattice. (D) Additional frequency distributions showing kinetic properties of processively and diffusively moving eGFP-labelled hKif15 motors. Coloured lines within the column plots are Gaussian or exponential decay fits. Insets show the respective median ± standard error of mean and maximum value of the distribution as well as its sample size. (E) Kymograph showing additional processive moving hKif15-eGFP motors at different velocities that photo-bleach in two steps, indicating the presence of (at least) three eGFP molecules per motor. The speed for each slope-section is given (please compare with velocity distribution in Figure 2B, left panel). Graph below the kymographs show the intensity along motor trace in arbitrary units. Intensity had been locally corrected for background intensity. Horizontal green lines within the graphs indicate the fitted average intensity (arbitrary units ± standard deviation) in the respective section of the trace. Photo-bleaching steps were manually defined. Compare with Figure 2D. DOI: http: //dx. doi. org/10. 7554/eLife. 01724. 00510. 7554/eLife. 01724. 006Figure 2—figure supplement 2. Analysis of bead motility and microtubule-binding in laser trap experiments. (A) Poisson statistics for the bead-binding probability of n ≥ 1 (blue) and n ≥ 2 (orange) motors fitted to data from a hKif15 dilution series (brown). Number of analysed beads per concentration >50 except for the highest where n = 30 (B) Velocity frequency distributions of hKif15-linked polystyrene beads at zero load followed by DIC microscopy. Orange line within the column plot is a Gaussian fit. Inset shows the respective median ± standard error of mean. Dotted blue line is the TIRF-derived velocity distribution fit from Figure 2B copied for the purpose of comparison. (C) Poisson statistics for the bead-binding probability of n ≥ 1 (blue) and n ≥ 2 (orange) motors fitted to data from a kinesin-1 dilution series (black). Number of analysed beads per concentration n = 25 except for the three lowest where n = 50. DOI: http: //dx. doi. org/10. 7554/eLife. 01724. 006 To confirm that motile hKif15 motors are tetramers, we imaged hKif15-eGFP motors under conditions that caused photobleaching and measured the change in fluorescence intensity with time (‘Materials and methods’). This analysis showed that both processive and diffusive motors bleach in up to four steps, indicating the presence of four eGFP molecules per motor (Figure 2D). Furthermore, processive tetramers are evident throughout the velocity distribution (Figure 2—figure supplement 1E). These data are in line with our biochemical analysis (Figure 1) and confirm that the observed hKif15-eGFP motors are indeed tetramers. Within the mitotic spindle, motors will experience varying loads and thus it is essential to understand how a motor responds to force. We therefore investigated the mechanical properties of hKif15 motors using our single-bead laser trap set-up (Carter and Cross, 2005). Single hKif15 motors were adsorbed onto 550-nm diameter polystyrene beads and steered to the microtubule surface with the laser trap at which point the motor can bind to the lattice (see ‘Materials and methods’ for details and Figure 2—figure supplement 2A). With the trap turned off (zero-load) the mean velocity of beads moving on the microtubule was consistent with our measurements of hKif15-eGFP by TIRF microscopy (Figure 2—figure supplement 2B). Next, we held the bead in the trap and allowed the motor to walk along the microtubule, away from the centre of the trap. The motion of the bead reports the stepping actions of the motor. Figure 2E shows two position traces in which hKif15 motors make a number of processive steps along the microtubule before detaching at relatively low loads between 1. 5 and 3. 5 pN. We also observed that beads move out the trap before moving back in the opposite direction without detaching (green arrowhead). These events may reflect the minus- and plus-end-directed diffusive motion observed in our TIRF experiments (Figure 2A). Together with our TIRF microscopy, we conclude that hKif15 is a homotetramer that undergoes either diffusive movement along microtubules, processive plus-end-directed stepping over long-distances or short duration movements to the minus-end. Our single molecule TIRF experiments also revealed a remarkable property of hKif15 motors as they approach microtubule intersections. The well-studied kinesin-1 will normally pass intersections and only in rare cases the motor will pause or switch microtubule tracks (Ross et al., 2008). In contrast, hKif15 motors show significantly elevated pause and switch events at intersections. Every fifth motor that approaches an intersection switches its microtubule track (Figure 3A, B; Videos 1 and 2). In general all events—pass, switch, pause, and dissociation at intersections—are equally likely. In this way, hKif15 is able to roam microtubule networks and Figure 3C demonstrates that one tetramer (see bleaching steps, lower panel) can indeed navigate along several different microtubules: the motor starts processive movement on microtubule #1 (trace A), eventually dissociates and rebinds to microtubule #2. On this, the motor diffuses a short distance (trace B) and dissociates again, just to rebind to microtubule #1. It processively follows microtubule #1 (trace C) and sequentially switches to microtubules #3 (trace D) and #4 (trace E), ending at the plus-end of microtubule #4 without detaching from the microtubule lattice (s) once again (see also Video 3). Thus, Kif15 is a processive motor that is able to navigate microtubule networks by effectively switching microtubule tracks. 10. 7554/eLife. 01724. 007Figure 3. hKif15 effectively switches microtubules at intersections. (A) Kymographs showing the processive switch of an eGFP-labelled hKif15 motor at a microtubule–microtubule intersection. The image in the upper left gives an overview of the microtubule positons, bar = 5 µm. The schematic below depicts the microtubules whose line scans are depicted as kymographs on the right. The numbering of the microtubules corresponds to that of the respective kymograph and the assigned letters to the traces within the kymograph. White arrows depict the direction of the line-scan; the dotted black arrow depicts the path of the hKif15 motor. Velocities of single trace-sections are given. Please note that the motor never leaves the microtubule lattice during the switch event (yellow dotted line between the kymographs). (B) Quantification of motor behaviour at microtubule intersections. Definitions as following: Pass–motor passes on the same microtubule and continues movement (may include a pause at the intersection); Switch–motor switches the microtubule and continues movement (may include a pause at the intersection); Pause–motor pauses for more than 5 s and does not continue movement after intersection (may dissociate or bleach at some point); Dissociate–motor immediately dissociates at intersection. (C) Essentially as in (A), now following a motor along four microtubules. Movement involves two dissociation/re-association (Kymographs: trace A/trace B, trace B/trace C) and two microtubule switch events (trace C/trace D, trace D/trace E) during switch events the motor never leaves the microtubule lattice (yellow dotted line between the kymographs). Plot below the kymograph shows the intensity along motor traces in arbitrary units. Intensity had been locally corrected for background intensity. Horizontal green lines within the graphs indicate the fitted average intensity (arbitrary units ± standard deviation) in the respective section of the trace. Photo-bleaching steps were manually defined and show that the observed motor bleaches in three steps indicating presence of four eGFP molecules. DOI: http: //dx. doi. org/10. 7554/eLife. 01724. 00710. 7554/eLife. 01724. 008Video 1. hKif15 effectively switches microtubules at intersections. Video of the events summarised in Figure 3A (hKif15-eGFP—cyan, microtubules—yellow). The switching hKif15-eGFP motor is marked by the cyan arrowhead. DOI: http: //dx. doi. org/10. 7554/eLife. 01724. 00810. 7554/eLife. 01724. 009Video 2. hKif15 effectively switches microtubules at intersections. Video of another hKif15-eGFP motor switching polarity-marked microtubules (hKif15-eGFP—cyan, seed—red, microtubule extension—yellow). The switching motor is marked by the cyan arrowhead. Please note that the motor moves for some time towards the minus-end of the microtubule it switched onto and subsequently changes direction towards the plus-end. DOI: http: //dx. doi. org/10. 7554/eLife. 01724. 00910. 7554/eLife. 01724. 010Video 3. hKif15 effectively switches microtubules at intersections. Video of the events summarised in Figure 3C (hKif15-eGFP—cyan, seed—red, microtubule extension—yellow). The switching hKif15-eGFP motor is marked by the cyan arrowhead. DOI: http: //dx. doi. org/10. 7554/eLife. 01724. 010 The tetrameric organisation of hKif15 would support the hypothesis that the motor drives extensile sliding of anti-parallel microtubule overlaps like the tetrameric Kif11 motor (Tanenbaum et al., 2009). To establish, whether tetrameric hKif15 indeed is able to cross-link microtubules on its own, we carried out experiments, in which biotinylated HiLyte-647-labelled GMP-CPP-microtubules were attached to a cover slip followed by addition of short XRhodamin-labelled GMP-CPP-stabilized- ‘transport’ microtubules and 1. 4 nM tetrameric hKif15-eGFP. By drawing kymographs, we found that hKif15 could mediate the cross-linking of microtubules (Figure 4) on its own, but that there was very little microtubule–microtubule sliding activity. Besides pivoting microtubules, we did observe episodes of bidirectional short duration sliding indicating a tug-of-war (Figure 4, right) and rarely the transport of short microtubules (Figure 4, left): behaviours that were recently reported for the yeast Kinesin-8 Kip3 (Su et al., 2013). However, we have no clear evidence to support the idea that hKif15 can drive continuous extensile sliding of anti-parallel microtubule bundles like hKif11. This is presumably because the motors frequently pause during processive excursions on the lattice and dwell at the ends for a long period (Figure 2—figure supplement 1B–D). Consistent with this, we only observed discontinuous short distance movement and pivoting of microtubules (around their ends) on surface-bound hKif15 motors, but not continuous microtubule sliding in microtubule gliding assays (Videos 4 and 5). 10. 7554/eLife. 01724. 011Figure 4. hKif15-eGFP can transport short microtubules as a cargo. Kymographs showing examples of processive (left) and tug-of-war-type transport (right), with an overview image of the crosslinked microtubules on top. Left: hKif15-eGFP motors drive slow (26 nm•s−1) processive movement of a cargo microtubule. The moving microtubule (magenta) is attached via Kif15 motors (cyan) to a substrate microtubule (yellow) for which the plus-end is orientated left in the overview image on top and at the bottom of the kymograph. hKif15 motors can be seen stably associated with one end of the cargo microtubule, which must be the plus-end because the motors move in a plus-end direction and pause at the end (see Figure 2A, Figure 2—figure supplement 1C, see also schematics to the left). Thus, the cargo microtubule moves plus-end leading towards the plus-end of the substrate microtubule. That is, the microtubules are parallel. Right: hKif15-eGFP motors drive tug-of-war-type movement, characterised by frequent and rapid direction changes during movement. DOI: http: //dx. doi. org/10. 7554/eLife. 01724. 01110. 7554/eLife. 01724. 012Video 4. Surface-bound hKif15 does not support continuous sliding of GMP-CPP stabilised microtubules (white) over long distances. DOI: http: //dx. doi. org/10. 7554/eLife. 01724. 01210. 7554/eLife. 01724. 013Video 5. Surface bound hKif15 does not support continuous sliding of GMP-CPP stabilised microtubules (white) over long distances. DOI: http: //dx. doi. org/10. 7554/eLife. 01724. 013 So far our experiments show that hTpx2 is not required for hKif15 to bind microtubules or for the motors processivity and ability to crosslink microtubules (as a tetramer). However, cell-biological experiments showed that hTpx2 is essential for hKif15 to be recruited to the mitotic spindle (Tanenbaum et al., 2009; Vanneste et al., 2009). To resolve this contradiction, we first analysed possible complex formation of recombinant hKif15 and hTpx2 in solution by ultracentrifugation in glycerol gradients. With nanomolar concentrations of each protein, we could not observe any complex formation over a range of different salt conditions (50 mM HEPES pH7. 5,0/150/300/450 mM NaCl [data not shown] and 35 mM sodium phosphate buffer [Figure 5A]). However, a 30-fold excess of hTpx2 dimers (2. 5 µM hTpx2 [dimer] + 80 nM hKif15 [tetramer]) allowed the formation of a hKif15-hTpx2 complex of ∼14. 0S, which included approximately one third of the provided hKif15 (Figure 5A, lower gels). The dissociation constant of the hKif15–hTpx2 complex is likely to be in the µM range arguing that its formation in vivo is less likely. In contrast, using a microtubule sedimentation assay, we observed that at low nM concentrations, hTpx2 (30 nM, dimer) does enhance the affinity of hKif15 (15 nM, tetramer) binding to GMP-CPP stabilised microtubules when in the presence of the non-hydrolysable ATP analogue AMP-PNP (the fitted Kd increasing ∼twofold from 993 nM to 490 nM; Figure 5B). This observation could explain why cell-biological experiments show that association of hKif15 with the mitotic spindle depends on hTpx2 (Tanenbaum et al., 2009; Vanneste et al., 2009). It is, however, unclear why hTpx2 further increases the microtubule binding affinity of a motor that is already highly processive. 10. 7554/eLife. 01724. 014Figure 5. hTpx2 inhibits hKif15 motility by increasing its microtubule affinity. (A) Formation of a stable hKif15/hTpx2 complex occurs only at low µM concentration of Tpx2. Coomassie stained SDS-PAGE gels showing the first 23 of 25 fractions of a 5–40% glycerol gradient in 35 mM sodium phosphate buffer loaded with the indicated purified proteins alone and in mixture at the indicated concentrations. While hKif15 and hTpx2 do not form a stable complex at nanomolar concentrations, a vast excess of 2. 5 µM dimeric hTpx2 drives formation of a hKif15/hTpx2 complex of 14. 0S. (B) Above: coomassie stained SDS-PAGE gel of a typical microtubule co-sedimentation experiment of 15 nM tetrameric hKif15 in the presence or absence of 30 nM dimeric hTpx2 at different concentrations of taxol-stabilised microtubules (SN–supernatant, P–pellet). Below: Quantification of pelleted tubulin and bound hKif15 shown above. Crosses indicate the SD of the average from three independent experiments. Deviation in tubulin concentration is due to partial microtubule instability in phosphate buffer at low tubulin concentration and the microtubule stabilising effects of hTpx2 (compare pelleted tubulin at 0. 25 µM with and without hTxp2). (C) Kymographs show the effect of 5 nM dimeric hTpx2 on the motility of 5 nM tetrameric hKif15 on GMP-CPP stabilised microtubules. Graph below left shows the fraction of motile and static motors normalised to overall microtubule length and subtracted by the fraction of static motors in the control, which sets motile motors in control to 1. Error bars show SD of three independent experiments. Graph below right shows the velocity distribution of motile motors in the presence of hTpx2, coloured lines are Gaussian fits revealing a bimodal distribution, compare with Figure 2B. (D) Side-by-side comparison of hKif15 (blue) and Drosophila kinesin-1 (green) stepping in the absence and presence of hTpx2 in the laser trap. Stepping traces for each kinesin are from the same bead before flow-in of 36 nM hTpx2 (left trace), after flow-in of hTpx2 (right trace). The asterix in the above right trace marks the deliberate movement of the stage, showing motor maintained attachment to the microtubule. DOI: http: //dx. doi. org/10. 7554/eLife. 01724. 014 To address the impact of hTpx2 on moving hKif15 tetramers, we added hTpx2 in equimolar (i. e., 5 nM) concentration to our hKif15-eGFP TIRF setup. Addition of hTpx2 decreased the proportion of motile motors on microtubules by 70%. The remaining motors still showed both directional and diffusive movement, indicating that inhibition by hTpx2 is not selective for either type of movement (Figure 5C, kymographs and left graph below). Further, the velocity distribution of remaining processive motors (compared to Figure 2A) now shows a bimodal distribution with a high proportion of slow motors (42. 1 ± 1. 7 nm•s−1 median ± SEM) and fewer motors at the median speed of hKif15 motors in the absence of hTpx2 (114. 9 ± 3. 2 nm•s−1 median ± SEM) (Figure 5C, right graph below). This may reflect the higher probability of a fast moving motor from engaging hTpx2 molecules that are bound on the microtubule lattice. To further confirm the inhibition of hKif15 by hTpx2 and to estimate the functional impact of hTpx2 inhibition on hKif15 functions beside movement, we included hTpx2 in our optical trap experiment. To do this, we captured a hKif15-linked bead and confirmed that it underwent stepping on the microtubule (Figures 5D, 1. blue trace). We then exchanged the buffer to allow 18 nM dimeric hTpx2 access to the hKif15-microtubule complex and again recorded the bead position. The hKif15 motor could no-longer step processively, although it remained bound to the microtubule lattice (4/5 cases; Figures 5D, 2. blue trace). To rule out that the bead had not simply detached, we moved the microscope stage and observed an increase in force indicating an intact microtubule-motor connection (see silver asterisk in Figure 5D). During this period, the bead can maintain microtubule attachment at forces that—in the absence of hTpx2—would have forced dissociation of the motor (compare also with Figure 2E). Thus, hTpx2 increases the force-holding capability of microtubule-bound hKif15. We can rule out a generic ‘roadblock’ inhibition-mechanism: Firstly, kinesin-1 motility is unaffected by the presence of hTpx2 (n = 5; Figure 5D, green traces). Secondly, hTpx2 already inhibits hKif15 motility at low nanomolar concentrations when only single molecules are present on the lattice. Kinesin-1 motility in the presence of hTpx2 also shows that inhibition of bead motility in case of hKif15 is not simply mediated by hTpx2-bead-microtubule interactions. Thus, hTpx2 is a selective inhibitor of hKif15 motor stepping presumably by forming stable trimeric hKif15-hTpx2-microtubule complexes. In this study, we reveal that hKif15 is a second tetrameric spindle motor in addition to the tetrameric Kinesin-5 hKif11 (Eg5). This finding is in line with initial cell-biological experiments that concluded that these two motors are redundant with Kif15 becoming essential for spindle assembly when hKif11 is inhibited (Tanenbaum et al., 2009; Vanneste et al., 2009). Indeed, hKif15, like Kif11, is only able to step under low loads in the 1–3 pN range (Korneev et al., 2007). However, our data show that Kif15 motors have a number of biophysical properties that distinguish it from Kif11: while both motors are able to undergo plus-end-directed movement or 1-D diffusion (Kwok et al., 2006; Kapitein et al., 2008; Figure 6, ‘A’), the median velocity of hKif15 is 10-fold higher than that of Kif11 and processive runs have a fourfold longer duration (Korneev et al., 2007). In the case of hKif15, both these movement types occur at the same ionic strength. In contrast, processive and diffusive movement by Kif11 is exclusive and depends on the ionic strength (Kapitein et al., 2008). Additionally, to our knowledge, pauses during processive runs have not been reported for Kif11. 10. 7554/eLife. 01724. 015Figure 6. Schematic model summarising the biophysical and biochemical properties of hKif15 and illustrating how the motor may operate within a k-fibre (parallel microtubule bundle). A–hKif15 can move uni-directionally towards the plus-end (minus-end-directed motion can also occur albeit with lower frequency) by processive stepping or by bi-directionally diffusion along the microtubule lattice. B–hKif15 can switch between microtubule tracks. (box) Model for the sequence of events during switch or pass movements at intersections via a bridge structure of the hKif15 tetramer that resolves into a pass (1) or switch event (2) depending on which motor domain pair detaches first from the microtubule lattice. C–hKif15 has a significant plus-end dwell time and therefore might modulate plus-end dynamics like other k-fibre motors (Stumpff et al., 2012). D–Being a tetramer, hKif15 can crosslink two microtubules and potentially resist sliding of microtubules within the fibre. Note that this link is dynamic as the motor still can step or diffuse within a bundle. (box) Once hKif15 and hTpx2 have formed a complex on the microtubule lattice, this crosslink could become static, thereby forming a fixed structural link in the fibre, which can sustain higher loads than hKif15-only crosslinks. E–hKif15 powers transport of (small) microtubule fragments. DOI: http: //dx. doi. org/10. 7554/eLife. 01724. 015 The uniqueness of hKif15 is also reflected in the motors ability to track-switch during processive movement—a property shared with Dynein (Ross et al., 2008). It is easy to comprehend how dynein switches between microtubule tracks, as this motor is capable of making variable step sizes, significantly larger than 8 nm (Reck-Peterson et al., 2006). However, kinesins step with a step size of ∼8 nm (Svoboda et al., 1993; Valentine et al., 2006; Yardimci et al., 2008; Huckaba et al., 2011; Jannasch et al., 2013) suggesting that it is unlikely for a single hKif15 motor domain-pair to mediate switching without leaving the lattice. In this regard, we show that within our 500 ms time resolution single motors do not leave the lattice during switching (Figure 3). Furthermore, switching events often include a pause at the intersection (Figure 3C, traces ‘D’ and ‘E’). As hKif15 is a motile tetramer, we propose that a switch starts with attachment of the second, free head-pair to the lattice of the intersecting microtubule, while the first, engaged head-pair of the same molecule stays attached to the old track. This hKif15 bridge can now pause at the intersection or resolve by detachment of the second domain-pair into a pass or by detachment of the firstdomain-pair into a switch event (Figure 6, ‘B’+box). Microtubule switching is a feature that is very helpful to navigate through the complex microtubule arrangement within the k-fibre. Thereby each switch event is equivalent to a new independent run on a new microtubule, so that a single molecule can travel a distance of ‘median run-length × (nswitches+1) ’ as we have shown in Figure 3C: traces C-E sum up to 7. 4 µm run length in total, with single traces at 1. 6,3. 7 and 2. 1 µm. Thus, even with a moderate median run length of 2 microns, a single molecule would have a high probability of reaching the plus-end (kinetochores) of the k-fibre, regardless of any discontinuity within the fibre or roadblocks owing to structural microtubule-associated elements (Figure 6, ‘B’+box). In contrast, short running, non-switching hKif11, that additionally is constantly gathered at the spindle poles by a dynein-dependent minus-end-directed flux (Ma et al., 2010), cannot travel far enough into the k-fibre network to reach the kinetochore/plus-end. In vivo data backs up this hypothesis, as hKif11 localises mainly to the spindle pole and fades towards the spindle midzone, while hKif15 is localised uniformly along the k-fibres (Vanneste et al., 2009; Sturgill and Ohi, 2013). Moreover, Kif15 motors also modulate the capacity of k-fibre microtubule bundles to generate an inward-directed force within the spindle (in opposition to hKif11) (Sturgill and Ohi, 2013). One possibility is that, due to their prolonged end dwell time, hKif15 motors may influence plus-end dynamics at kinetochores (Figure 6, ‘C’). Such is the proposed role of the Kinesin-8 Kif18a, which is reported to dampen microtubule dynamics in vitro (Du et al., 2010; Stumpff et al., 2012). Tetrameric hKif15 motors, like Kif11, can form cross-links between microtubules, we show that hKif15 crosslinks are dynamic as we observed limited tug-of-war movements of crosslinked cargo microtubules. Such cross-links may be reinforced by inhibitory hTpx2 (see below) turning dynamic hKif15 crosslinks into static structural hKif15/hTpx2 crosslinks that can resist higher forces than hKif15 only crosslinks (Figure 6, ‘D’+box). Our data does not, however, provide any evidence that hKif15 drives a Kif11-like continuous extensile sliding of anti-parallel microtubules in vitro, though we cannot rule out that in vivo other additional factors are involved in such an activity. This factor is unlikely to be hTpx2 since our data shows this protein to inhibit the stepping of hKif15. How this mechanism is regulated within the spindle will be an important future work, but the fact that hTpx2 and hKif11 are constantly transported to the spindle pole (Ma et al., 2010) might hint to a partition of the spindle into a hKif11 active zone around the poles and a hKif15 active zone towards the spindle equator. Overall, our data provide the first insight into the biochemical and biophysical properties of the full-length human Kinesin-12 hKif15. We reveal that hKif15 is a distinct class of kinesin that assembles into stable tetramers, which are highly processive, can navigate microtubule networks by switching track and form high load-bearing microtubule–microtubule crosslinks when bound to the regulatory factor hTpx2. These data shed new light on the mechanism by which hKif15 motors control spindle and chromosome mechanics. The hKIF15 ORF was amplified in five 800- to 1000-bp sized fragments from a cDNA library (derived from hTERT immortalised retinal pigment epithelial (hTERT-RPE1 cells) ) and subsequently joined by overlap extension PCRs using Pfu Ultra AD polymerase (Agilent, Stockport, UK). The complete hKIF15 ORF was cloned via AseI/NotI into a modified (bases 2391–2426 of the pIEx/Bac1-vector coding the Strep-Tag II were replaced by the His6/TEV-cleavage site element of p11 [DNASU Plasmid ID: EvNO00085126, DNASU Plasmid Repository at Arizona State University], flanked by a 5′ NcoI and a 3′ NdeI site. [gift form Miho Katsuki, Riken, Japan]) pIEx/Bac1-vector (MERCK, Darmstadt, Germany) opened with NdeI/NotI. For hKIF15-eGFP an AscI-site was introduced in front of the NotI-site and the eGFP inserted via AscI/NotI before the hKIF15 ORF was inserted as above. We observed that the hKIF15 ORF is toxic to Escherichia coli cells when antibiotics other than ampicillin (e. g., kanamycin, gentamycin) are used, so viral genome generation by the Invitrogen Bac-to-Bac system (Life Technologies, Paisley, UK) using DH10BAC cells is not possible. The hTPX2 ORF was amplified from I. M. A. G. E. 3509275 (ATCC clone MG1537) and cloned into the pFastBacM13 vector (MPI-CBG, Dresden, Germany) via SpeI/SalI. Assembly of viral genomes was carried out according to manufacturer protocols and transfection competent baculovirus particles were generated and used for transfection of 500 ml–1 L SF9-cells expression cultures according to (Wasilko et al., 2009). Cells were harvested at 250-g in a SLA-3000 rotor (Thermo Scientific, Waltham, MA, USA) and resuspended in lysis buffer (50 mM HEPES pH 7. 5,150 mM NaCl, 1. 5 mM MgCl2,3 mM EGTA, 5% glycerol, 0. 1% Tween-20,0. 1 mM ATP, complete protease inhibitor [Roche, Burgess Hill, UK]). For hKif15 purification, lysates were cleared at 48k-g in a SS-34 rotor (Thermo Scientific), diluted 1: 3 (to 50 mM NaCl final) with 50 mM sodium phosphate buffer and adjusted to pH 7. 0. Protein was allowed to bind to SP-sepharose (GE Healthcare, Little Chalfont, UK) in batch for 2 hr at 4°C. hKif15 was directly eluted by applying a threefold bed volume of 50 mM sodium phosphate buffer pH 7. 5,50 mM NaCl, 1 mM MgCl2,5% glycerol, 0. 05% Tween-20,0. 1 mM ATP onto Talon-beads (Takara Biotech, Saint-Germain-en-Laye, France). Again, protein was allowed to bind 2 hr at 4°C in batch. Talon-beads where washed with 10-fold bed volume of 50 mM sodium phosphate buffer pH 7. 5,300 mM NaCl, 1 mM MgCl2,5% glycerol, 0. 05% Tween-20,0. 1 mM ATP and a 15-fold bed volume of 50 mM sodium phosphate buffer pH 7. 5,100 mM NaCl, 1 mM MgCl2,5% glycerol, 0. 05% Tween-20,10 mM imidazole, 0. 1 mM ATP. Protein was eluted with 50 mM sodium phosphate buffer pH 7. 5,150 mM NaCl, 1 mM MgCl2,10% glycerol, 50 mM imidazole, 0. 1 mM ATP and purity as well as concentration determined by SDS-PAGE against a BSA standard using ImageJ. For each protein preparation, the oligomerisation state and its dispersity have been checked by glycerol gradients or native PAGE. Note that for the SEC-MALS experiment, protein was eluted with 35 mM sodium phosphate buffer pH 7. 0,1 mM MgCl2,50 mM imidazole, 0. 05 mM ATP and snap frozen in the absence of glycerol in 500 µl aliquots and stored in liquid nitrogen. For purification of hTpx2 lysates were cleared and diluted 1: 3 (to 50 mM NaCl) with 50 mM sodium phosphate buffer pH 7. 5 as done for hKif15. Protein was allowed to bind to SP-sepharose in batch for 2 hr at 4°C. Beads were washed with a fivefold bed volume of 50 mM sodium phosphate buffer pH 7. 5,100 mM NaCl, 1 mM MgCl2,5% glycerol, 0. 05% Tween-20 and protein was eluted with a threefold bed volume of 50 mM sodium phosphate buffer pH 7. 5,300 mM NaCl, 1 mM MgCl2,5% glycerol, 0. 05% Tween-20,10 mM imidazole onto Talon-beads. Protein was allowed to bind 2 hr at 4°C in batch and beads were washed with a 25-fold bed volume of 50 mM sodium phosphate buffer pH 7. 5,150 mM NaCl, 10 mM imidazole, 1 mM MgCl2,5% glycerol, 0. 05% Tween-20. Protein was eluted with 50 mM sodium phosphate buffer pH 7. 5,75 mM NaCl, 150 mM imidazole, 10% glycerol. Purity and concentration was determined as above. All protein was snap frozen in 5–20 µl aliquots and stored in liquid nitrogen. Glycerol gradients were essentially carried out as described in McClelland and McAinsh (2009) in the presence of 1 mM DTT and complete protease protein inhibitor at 4°C for 15 hr over night. We checked for hKif15/hTpx2 interactions in 50 mM HEPES pH 7. 5,1 mM MgCl2,1 mM EGTA, 0. 1 mM ATP, 0/150/300/450 mM NaCl (150 mM NaCl also ± free tubulin in ATP or AMP-PNP) and 35 mM sodium phosphate buffer pH7. 0,1 mM MgCl2,1 mM EGTA, 0/150/300/500 mM NaCl, 0. 1 mM ATP or AMP-PNP. The 5-mlgradient was fractionated by hand in fractions of 200 µl each. The protein was TCA-precipitated and fractions 1–23, together with 1/2 input, analysed on the same SDS-PAGE gel stained with coomassie (SimplyBlue, Invitrogen). Protein bands were quantified with ImageJ and peak values were determined by fitting a Gaussian distribution to the data using Origin 8. 5. Selected proteins from the High/low molecular weight gel filtration calibration kit (GE Healthcare) were used in standard calibration runs. Protein samples were concentrated to ∼2 mg/ml in 35 mM sodium phosphate buffer pH 7. 0,1 mM MgCl2 and loaded onto a size exclusion column (Wyatt, Santa Barbara, CA, USA) connected to a Dawn Heleos II MALS detector and Optilab T-rEX refractometer (Wyatt). A dn/dc value of 0. 185 was used for all calculations. Please note that for the SEC-MALS experiment only, protein-purification was modified in that hKi15 was finally eluted in 35 mM sodium phosphate buffer pH 7. 0,1 mM MgCl2,50 mM imidazole, 0. 05 mM ATP and snap frozen in the absence of glycerol, which is likely to be the reason for the appearance of dimeric hKif15 in the SEC-MALS analysis that otherwise are absent in the preparation used in all the other experiments (as shown by native PAGE and glycerol gradients, see Figure 1B, C). Microtubules for the sedimentation assay were grown in presence of 1 mM GTP in BRB80 (80 mM PIPES pH 6. 8,1 mM MgCl2,1 mM EGTA) by stepwise increasing the taxol concentration in solution. Polymerised microtubules were washed once with BRB80+Taxol using a Beckman Airfuge and resuspended in 35 mM sodium phosphate buffer pH 7. 0,1 mM MgCl2,1 mM EGTA and 1 mM DTT. Samples were mixed in the same buffer at the indicated concentrations (16. 5 nM tetrameric hKif15,33 nM dimeric hTpx2) and incubated for 20 min at room temperature in the presence of 2 mM ATP or AMP-PNP. Samples were spun for 20 min at 45k rpm in a TLA100 rotor (Beckman Coulter, High Wycombe, UK) using thick-wall polyallomer tubes (Beckman Coulter). The supernatants were TCA precipitated and analysed together with the resuspended pellets by SDS-PAGE. Protein bands were quantified using ImageJ. Since taxol stabilised microtubules are less stable in 35 mM sodium phosphate over long time periods and that hTpx2 has a stabilising effect on microtubules, we also quantified the actual amount of precipitated tubulin in order to compare both setups with and without hTpx2. This gives rise to the horizontal error bars in Figure 5B. Preparation of sialylated coverslips and flow chamber setup (with double sided tape) was essentially carried out as described in Bechstedt et al. (2011), except for the initial cleaning step with piranha solution, which had been substituted by incubation with 7. 4% hydrochloric acid at 60°C over night. For single molecule assays, GMP-CPP (Jena Bioscience, Jena, Germany) stabilised, polarity-marked microtubules labelled with XRhodamin- and HiLyte-647-linked tubulin (Cytoskeleton, Denver, CO, USA) were adsorbed to the glass surface via anti beta tubulin antibodies (TUB 2. 1, Sigma-Aldrich, St. Louis, MO, USA) after the flow chamber has been blocked with 1% Pluronic F-127 (Sigma-Aldrich) and 1 mg/ml kappa casein. The flow chamber was loaded with 1–10 nM His6-hKIF15-GFP (tetrameric) in BRB20 (20 mM PIPES pH6. 8,1 mM MgCl2,1 mM EGTA), 50 mM KCl, 2 mM ATP, 0. 1 mg/ml kappa casein, 80 µg/ml glucose, 40 µg/ml glucose-oxidase, 16 µg/ml catalase, 1 mM DTT, sealed with VALAP (vaseline, lanolin, paraffin mixed 1: 1: 1) and imaged at 25°C on a Olympus CELLR/TIRF microscope (Olympus, Southend-on-Sea, UK) equipped with a ImagEM emCCD camera (Hamamatsu Photonics, Welwyn Garden City, UK), an environmental chamber and a stage-top-incubator (Okolab, Ottaviano, Italy) using a 100x NA 1. 49 objective with 1. 6x auxiliary magnification. To visualise hKif15–eGFP movement, 3-min time-lapse videos were recorded at 2 frames per second (fps) with a 100 ms exposure using a 488 nm laser line. For the bleaching experiments, videos were recorded at 4 fps. The position of polarised microtubules before and after the time-lapse video was determined by capturing a single image with the 561 nm (100 ms exposure) and 640 nm (100 ms exposure) laser lines. In General, kymographs were generated and analysed with ImageJ and the MultipleKymograph plugin (http: //www. embl. de/eamnet/html/body_kymograph. html). For the hTpx2 inhibition experiments, equimolar dimeric hTpx2 was directly loaded together with hKif15 into the flow chamber. Imaging was started after a short incubation period of 5 min at room temperature. To prevent a strong dilution of both proteins on the microtubule lattice, a microtubule density of 150 µm tubulin per field of view (51. 2 × 51. 2 µm) was not exceeded. For microtubule sliding experiments, biotinylated (Cytoskeleton) and HiLyte-647-labelled GMP-CPP-microtubules were adsorbed via streptavidin to a PEG-biotin-coated (Surface Solutions Switzerland, Dübendorf, Switzerland) cover slip that has been blocked with 1 mg/ml kappa casein. Short XRhodamin-labelled GMP-CPP-microtubules were added as cargo to the imaging-mix (as above). Again, 3 min time-lapse videos at 2 frames per second (fps) were recorded to track hKif15-GFP and cargo microtubule movement. Exposure times were 100 ms with the 488 nm laser line and 150/200 ms with the 561 nm laser. Positions of the HiLyte-647-labelled substrate microtubules were determined by capturing a single image (100 ms exposure) with a 640 nm laser line both before and after the time-lapse video. For the most part, instrumentation and sample preparation was the same as described in Carter and Cross (2005) with the following changes: motors were non-specifically bound to polystyrene beads (550 nm, NIST calibration size standards, Polysciences, Warrington, PA, USA) by incubation in a solution of 80 mM Pipes, 2 mM MgSO4,1 mM EGTA, 1 mM DTT, 3 mg/ml D-Glucose, 0. 2 mg/ml casein and 1 μM ATP at pH 7. 0. Before addition to the flow cell, the incubated bead–motor solutions were diluted in an assay buffer: 20 mM Pipes, 2 mM MgSO4,1 mM ATP, 1 mM EGTA, 1 mM DTT, 3 mg/ml D-Glucose, 0. 4 mg/ml casein, 4 μM taxol, and a glucose oxidase/catalase-based oxygen scavenging system. For improved stability, recordings were made in flow-cells sealed with Dow Corning high vacuum grease (Dow Corning, Barry, UK). Motor concentration was reduced until no more than one third of beads showed any binding. A bead-binding event was defined as a bead that remained attached to the microtubule for at least 2 s after the laser trap has turned off. Each bead was tested for binding on two or more different microtubules. Bead-binding data was fitted with two poisson probability curves using a linear least-squares method (Svoboda and Block, 1994). This analysis showed that our experimental hKif15 and Kinesin-1 data fit a ≥1 poisson distribution (the probability that a bead binds by one or more motors), rather than the ≥2 distribution (the probability that a bead binds by two or more motors). These data confirm that we are observing single motor (i. e., tetramer) motility at the low concentrations we used in our trapping experiments (3. 3 and 6. 5 nM hKif15, see Figure 2—figure supplement 2A). Some hKif15-linked beads bound in a diffusive non force-producing state to the microtubule, a behaviour that was rarely observed with kinesin-1-linked beads and fits to our observation of diffusive motion in our TIRF assays. In the case of the hTpx2 flow-through recordings, the samples were not sealed at the sides to allow wash-through. A bead with active motor was recorded and a further 3–4 cell volumes of assay buffer with 18 nM dimeric hTpx2 was carefully passed through the cell whilst retaining the same bead in the trap. The trapped bead was focused close to the lower cover-glass surface during these solution changes, this offered some boundary layer protection during the solution flow and also greatly reduced the chances of a second bead entering the trap. Following recordings in the presence of hTpx2, a further 3–4 cell volumes of assay buffer were passed through the flow cell to make the final recordings following hTpx2 wash out. Bead position data were recorded at 88 kHz and averaged down to 22 kHz for analysis and storage.

Before a cell can divide, it produces an extra copy of all its chromosomes, and it must then ensure that each daughter cell ends up with one copy of each chromosome. During the division process, a structure called the spindle forms in the cell. This spindle is made of thread-like extensions called microtubules that grow from two poles at opposite ends of the cell. These microtubules are responsible for getting the chromosomes to line up in the middle of the cell, and then pulling half of the chromosomes to one end of the cell, and half to the other end. The cell then divides into two daughter cells. Two motor proteins-so-called because they consume chemical energy to 'walk' along the microtubules-have important roles in this process: Kif11 motor proteins mainly drive the formation of the spindle and thus division of the chromosomes. A cell that does not contain Kif11 can only divide if it contains extra copies of a second motor protein called Kif15: this suggests that Kif15 can serve as some sort of back up for Kif11. Normal cells only divide when new cells are needed for growth or to replace old cells that have died. Cancer cells, on the other hand, divide in a way that is not controlled. Drugs that interfere with Kif11 have been developed in the hope that they will stop cancer cells dividing, but these drugs have not been very effective in clinical tests, possibly due to the Kif15 back up. Scientists hope, therefore, that a better understanding of the role of Kif15 may lead to improved cancer treatments. Drechsler et al. have isolated individual Kif15 motor proteins and used advanced microscopy techniques to study them in action. These experiments showed that Kif15 motor proteins can travel long distances along a single microtubule, and can also switch to a different microtubule at intersections. This movement of Kif15 is stopped when they bump into Tpx2 proteins, which are sitting on the microtubules. Together, these proteins can also form links between microtubules that can withstand high forces. These properties provide a starting point to understand how Kif15 can act as a back up for Kif11 in cells. In the future, it will be important to work out how Kif11 and Kif15 motor proteins work together in teams to build the spindle.

lay_elife

Glycolytic potential (GP) in skeletal muscle is economically important in the pig industry because of its effect on pork processing yield. We have previously mapped a major quantitative trait loci (QTL) for GP on chromosome 3 in a White Duroc × Erhualian F2 intercross. We herein performed a systems genetic analysis to identify the causal variant underlying the phenotype QTL (pQTL). We first conducted genome-wide association analyses in the F2 intercross and an F19 Sutai pig population. The QTL was then refined to an 180-kb interval based on the 2-LOD drop method. We then performed expression QTL (eQTL) mapping using muscle transcriptome data from 497 F2 animals. Within the QTL interval, only one gene (PHKG1) has a cis-eQTL that was colocolizated with pQTL peaked at the same SNP. The PHKG1 gene encodes a catalytic subunit of the phosphorylase kinase (PhK), which functions in the cascade activation of glycogen breakdown. Deep sequencing of PHKG1 revealed a point mutation (C>A) in a splice acceptor site of intron 9, resulting in a 32-bp deletion in the open reading frame and generating a premature stop codon. The aberrant transcript induces nonsense-mediated decay, leading to lower protein level and weaker enzymatic activity in affected animals. The mutation causes an increase of 43% in GP and a decrease of>20% in water-holding capacity of pork. These effects were consistent across the F2 and Sutai populations, as well as Duroc × (Landrace × Yorkshire) hybrid pigs. The unfavorable allele exists predominantly in Duroc-derived pigs. The findings provide new insights into understanding risk factors affecting glucose metabolism, and would greatly contribute to the genetic improvement of meat quality in Duroc related pigs. In past decades, thousands of quantitative trait loci (QTLs) have been detected for economically important traits in livestock through genetic linkage studies [1]. The recent availability of livestock genome sequences and high-density SNP chips has allowed detection of significant association of nucleotide polymorphisms with complex traits, and effective identification of causal mutations for some monogenic traits [2]–[4]. Despite these progresses, uncovering the quantitative trait genes (QTGs) or nucleotides (QTNs) for complex traits remains a challenging task. Only a handful of QTNs have been convincingly identified in livestock [5]–[7]. More recently, several studies in human, mouse and Drosophila have shown that the integration of phenotypic traits, genetic and gene transcript data enables researchers to identify expression QTL (eQTL), untangle gene-based regulatory networks, infer relationship between gene expression levels and phenotypic traits, and thereby detect novel trait-causing genes [8]–[10]. Therefore, the integrative analysis would play a key role in acquiring the knowledge of mechanisms responsible for livestock complex traits. Indeed, some researchers have identified a number of promising candidate genes for muscle traits and blood lipid traits of pig by integrative analyses of GWAS (or linkage mapping), eQTL and trait-correlated expression [11]–[14]. Glycogen storage diseases (GSD) characterized by defects in glycogen metabolism and excess glycogen stored in liver and muscle are multifactorial disorders. Both human and animals suffer from GSD. Agricultural researchers often measure the glycogen content or glycolytic potential [GP = 2× (glucose + glycogen + glucose-6-phosphate) + lactate] in skeletal muscle of farm animals at slaughter. However, instead of the GSD diagnostic indicator, the GP value is mainly used for the prediction of meat quality development during the conversion of muscle to meat, because GP is a determinant of multiple meat quality characteristics, such as pH, color, water holding capacity (drip loss), tenderness and processing yields [15], [16]. GSD or extreme GP phenotype can be caused by genetic variation of various enzymes or transporters, which are involved either directly in the synthesis or breakdown of glycogen or in the utilization of its catabolite, glucose-1-phosphate [17]. To date, only one responsible gene, PRKAG3, has been elaboratively evidenced to impact glycogen and GP levels in Hampshire and its related synthetic lines [18], [19]. Although more than 10 different QTLs for the trait have been reported (http: //cn. animalgenome. org/cgi-bin/QTLdb/index), QTGs or QTNs underlying these QTLs remain unexplored. We have previously identified a major QTL for GP and pH values at 42 cM on chromosome 3 in a large scale White Duroc × Erhualian F2 intercross [20]. To decipher the molecular basis of the GP QTL, we herein performed an integrative genetic analysis by utilizing extensive data sets of GP-related traits, high-density genotypes and gene expression profiling from three experimental populations. We show compelling evidence that a splice mutation in the PHKG1 gene is the QTN underlying the major QTL effect on GP and its related traits. We first performed a genome-wide association study (GWAS) for GP and its components including residual glycogen & glucose (RG), glucose-6-phosphate (G-6-P) and lactate on the White Duroc × Erhualian F2 intercross, in which 877 phenotyped F2 individuals and its parents/grandparents were genotyped by using Illumina Porcine SNP 60K Beadchips [21]. The quality control filtering and data processing of the GWAS data are described in the Methods. Quantile-quantile plots with genome control λGC values are shown in Figure S1. We found no evidence of systematic inflation of association test results. Consistent with our previous QTL results [20], the GWAS results demonstrated that Sus Scrofa chromosome 3 (SSC3) contained the most genome-wide significant locus (P = 5. 85×10−22) for both GP and RG with the top SNP ss131031160 (pSNP) at 17. 09 Mb (Figure 1). However, this lead SNP was not associated with the glycolysis intermediate product G-6-P and end-product lactate (Figure S2). These results suggest that the underlying QTG likely influences the conversion between glycogen and glucose (or G-6-P) rather than the conversion between G-6-P to lactate. The pSNP ss131031160 explains 19. 6% of the phenotypic variance in RG of the F2 population. To validate and fine map the SSC3 QTL for RG, we conducted a second GWAS on 433 Sutai pigs, a Chinese synthetic line derived from a cross between Duroc and Taihu including Erhualian and Meishan after more than twenty-year selection. Besides 62163 SNPs on the Illumina Porcine SNP 60K Beadchip, 53 SNPs derived from DNA sequence comparison between Erhualian and Duroc (see Materials and Methods) within a 1-Mb region surrounding the top pSNP were genotyped for these Sutai pigs. As expected, we confirmed the strong association signal on SSC3 in the Sutai population. The pSNP ss131565361 (P = 9. 06×10−19) at 16. 92 Mb (Figure 1) explains 53. 6% of the RG variance. This locus also significantly influenced ultimate pH (measured 24 hours after slaughter) and drip loss (Figure S2). Based on the LOD drop off 2, the empirical confidence intervals of the QTL in the F2 and Sutai populations was 920 kb (16. 92–17. 84 Mb) and 630 kb (16. 47–17. 10 Mb) respectively (Figure 2A). Therefore, the most likely QTL interval was their overlapping region of 180 kb (16. 92–17. 10 Mb). This interval contains 7 annotated genes: GUSB, VKORC1L1, NUPR1L, CHCHD2, PHKG1, SUMF2 and CCT6A (Figure 2A). Using the data of digital gene expression profiles (DGE) tested in longissimus muscle samples from 497 F2 animals, we identified genome-wide eQTL for the 7 annotated genes in the critical region. We operationally defined cis-eQTL as any association between a gene expression and SNPs within 2 Mb of the gene location. We found 3 cis-eQTL each for PHKG1, SUMF2 and GUSB (Figure 2B). Intriguingly, the lead SNP for the cis-eQTL affecting PHKG1 expression was identical to the top pSNP (ss131031160) for GP in the GWAS on the F2 population. The major allele (G) at this SNP was associated with lower residual glycogen and higher expression of PHKG1 (Table 1 and Figure 2C). PHKG1 encodes a catalytic subunit of the phosphorylase kinase (PhK), which can mediate glycogen breakdown. Therefore, the co-localization of the top SNP in both GWAS and eQTL mapping coupled with the biological function highlights PHKG1 as the most likely QTG underlying the major locus on SSC3. To search for coding variants in the PHKG1 gene, we isolated and sequenced PHKG1 cDNA from muscle samples of 6 F2 individuals representing three genotypes at the pSNP ss131031160 (2 GG, 2 GA and 2 AA animals). In total, we detected 14 polymorphisms (Table S1). Of note, we found a 32-bp deletion/insertion (c. del/ins32; Figure 3A&B) polymorphism at exon10 that alters the open-reading frame and causes a premature stop codon, leading to a truncated and nonfunctional protein product. To test if the 32-bp deletion was also present in the genomic DNA (gDNA), we sequenced the 140 bp genomic region encompassing the deletion using gDNA samples of the above-mentioned 6 F2 animals. We found only one single nucleotide substitution (C>A) in the region. This SNP (g. 8283C>A) lies 5 bp upstream of the c. del/ins32 (Figure 3C&D), where the pyrimidine (C or T) is highly conserved among vertebrates, including chicken, mouse, dog, cow, sheep and human. The C→A nucleotide transvertion may weaken the strength of the pyrimidine-rich signal near the 3′ end of the PHKG1 intron 9, which is known to correlate with splicing efficiency [22]. Using a web-available tool Alternative Splice Site Predictor (ASSP, http: //wangcomputing. com/assp/index. html), we found that the splice site score (1. 652) of the mutant sequence was much lower than the wild-type counterpart (3. 659), and even lower than the default cutoff value (2. 2) for acceptor sites set in the predictor. Therefore, the mutation appears to attenuate or inhibit the role of the constitutive acceptor splice site and this consequently evokes another cryptic acceptor at 32 bp downstream of the mutation, which results in a loss of 32 bp in mature mRNA during PHKG1 transcription (Figure 3E). To test if the 32-bp deletion in exon 10 was directly caused by the SNP g. 8283C>A, the effect of the variant on splicing was assessed using a minigene splicing assay (Figure S3). Two minigene expression vectors carrying either the wild-type or the mutant PHKG1 g. 8283C>A segment were constructed and transiently transfected into HeLa cells and 293T cells (Figure S3A). The minigene transcripts in these transfected cells were analyzed by RT-PCR, using specific primers complementary to exon 9 and exon 10 of the minigene. We found two amplicons of 78 bp and 62 bp from the mutant construct and only one amplicon of 94 bp from the wild-type construct (Figure S3B). Sanger sequencing analysis revealed that the two truncated amplicons corresponded to two aberrant splicings of the first 16 and 32 nucleotides (nt) at 5′ end of exon 10 (Figure S3C-E). The presence of novel aberrant splicing of 16-nt may be due to that the minigene constructs only contain partial PHKG1 sequence or there is a special splicing factor in the transfected cells. Anyway, the result clearly demonstrates that the variant PHKG1 g. 8283C>A is responsible for the aberrant splicing of 32-nt observed in vivo. To obtain further evidence for the causality of the candidate QTN (SNP g. 8283C>A), we conducted the concordance test between the SNP genotypes and the QTL genotypes on 9 parental boars (6 F1 boars and 3 Sutai boars). The QTL genotypes of these boars were determined by the marker-assisted segregation analysis (see Materials and Methods). We found that the SNP genotypes were completely concordant with the QTL genotypes in these boars (Table S2). In contrast, the two top pSNPs ss131031160 and ss131565361 in the GWAS exhibited disconcordance with the QTL genotypes in the 9 parental sires. Concordantly, the association of the g. 8283C>A SNP with RG phenotype was strongest among all SNPs genotyped in Sutai pigs, and was at the same significance level as that of the top GWAS SNP ss131031160 in the F2 population due to their complete linkage disequilibrium (Table S3). In addition, we re-sequenced a 10-kb segment covering 1 kb upstream of the PHKG1 gene to its 3′ end on all 9 sires (6 F1 and 3 Sutai sires) with deduced QTL status. A total of 142 variants were identified (Table S4). We found that 7 Q chromosomes of these boars shared the same haplotype while 11 q chromosomes corresponded to multiple divergent haplotypes (Figure S4). Surprisingly, a q haplotype from Sutai boars was nearly identical to the Q haplotypes except for three variants, of which only one (g. 8283C>A) showed co-segregation with QTL genotypes across all the 9 sires (Figure S5). This strongly supports the causality of the g. 8283C>A mutation. Haplotype analysis showed that no other variants within the PHKG1 region were in complete LD with the SNP g. 8283C>A across all experimental populations. Based on that, we tried to determine whether other variants can also result in partial QTL effect. When the SNP g. 8283C>A was included as a fixed effect in the model, the QTL effects on PHGK1 expression and RG trait vanished in these populations (Figure S6A). Furthermore, we divided the Sutai pigs into three genotype groups (AA, CA, CC). No eQTL effect was detected within each group (Figure S6B). It thus suggests that even there is another regulatory SNPs, their effects are likely negligible. Aberrant mRNA, like the truncated PHKG1 transcript caused by the g. 8283C>A SNP, tend to be degraded by a known mechanism of nonsense-mediated mRNA decay (NMD) in the cell. To analyze whether the mutation interferes with PHKG1 mRNA expression levels, qRT-PCR experiments were performed on total RNA isolated from 293T cells transiently transfected by the wild-type and mutant PHKG1 minigenes. The mutant minigene produced nonsense mRNAs at 56% level of normal expression (Figure S7). The finding indicates that the PHKG1-32del mRNA bearing the premature termination codon was most likely degraded by NMD. In fact, electropherogram of RT-PCR products amplified from PHKG1 mRNA in homozygous animals showed that the intensity of the mutant DNA band of 114 bp was much lower than the normal band of 146 bp (Figure 3A), implying NMD of the mutant PHKG1 mRNA. Accordingly, the PHKG1 cDNA sequence chromatograms illustrated that the fluorescence intensity of wild-type allele (Wt or q) were 2–3 fold stronger than that of the mutant allele (Mt or Q) in animals heterozygous for the g. 8283C>A SNP (Figure 3F). To more accurately estimate the difference in abundance between Wt and Mt transcripts, we designed two pairs of primers: one (Common-5′-FP/RP) for amplification of both transcripts, and the other (Wt-3′-FP/RP) for the specific amplification of Wt transcript (Figure 4A). After performing qRT-PCR with the two primer sets, we quantified the levels of total mRNA transcripts relative to wild-type transcripts. In heterozygotes, the ratio of (Mt+Wt) to Wt transcripts was 1. 4∶1 (Figure 4B), suggesting that the 60% of Mt transcripts were degraded by NMD. Interestingly, we found that mutant homozygotes (AA or QQ) had PHKG1 Wt, as RT-PCRs using the primers specific for the Wt generated amplicons corresponding to Wt in these mutant homozygotes (Figure 4C). We further showed that, only about one-eighth of PHKG1 transcripts was Wt (Figure 4B). The result suggests that the g. 8283C>A mutation could strongly decrease but not completely abrogate the original splicing form. Consistently, the PHKG1 Wt transcript level is highest in muscle samples from CC animals, followed by AC and AA individuals (Figure 4C). Similar tendency was observed for the PHKG1 Wt protein level in the three genotypes by Western-blot analysis (Figure 4D). Thus, it is evident that the g. 8283C>A mutation affects the expression of PHKG1 at both transcription and translation levels. PHKG1 is a critical enzyme in the glycogen metabolism. We expected that the PHKG1 mRNA expression level would be significantly associated with the RG phenotype. However, we did not observe such strong correlation (r = −0. 08, P = 0. 106; Figure 5B) using the DGE data in the F2 population. We then made a close examination on the DGE data. We found that the DGE tag for PHKG1 was located in a region upstream of the 32-bp deletion, which therefore can not distinguish Wt from Mt transcripts. To correct for the biased effect of the abnormal Mt transcripts, we further used qRT-PCR to measure the Wt level on 117 F2 individuals and 104 Sutai pigs, of which the number of CC, CA and AA animals at the g. 8283C>A locus are nearly identical (Figure 5A). We then examined the correlation between the qPCR-data and the RG phenotypes. As a result, the PHKG1 Wt (functional) transcript level was significantly correlated with RG phenotype (r≤−0. 4, P<10−5) in the two populations (Figure 5B). Such strong correlation were not detected for any other cis-regulated genes within the critical region of the SSC3 QTL (Figure S8), strengthening the causal relationship between PHKG1 and RG. PHKG1 is a catalytic subunit of the phosphorylase kinase (PhK). The g. 8283C>A splice mutation in the PHKG1 gene thus likely influence the enzymatic activity of PhK and glycogen phosphorylase. To verify the assumption, we tested the enzymatic activity of PhK in 18 muscle samples―6 per genotype group (CC, AC, and AA). As expected, the AA group showed>6-fold reduction in enzyme activity as compared to the CC and AC groups (Figure 6). It is reasonable to speculate that reduced PhK enzyme' s activity would lead to slow breakdown of glycogen in muscle, which in turn cause excess accumulation of glycogen in the tissue. This is consistent with the SSC3 QTL effect on GP or RG trait. Considering the inter-relationship of GP with other meat quality attributes, we evaluated the effects of the PHKG1 g. 8283C>A mutation on all meat quality traits measured in the 864 F2 individuals from the White Duroc × Erhualian intercross, 431 Sutai pigs and 140 three-way hybrid DLY [Duroc × (Landrace × Yorkshire) ] pigs. In the F2 population, there was a tendency towards lower ultimate pH and higher drip loss in AA pigs with higher GP compared to CA and CC individuals (the mean values of pH 24h in AA, CA and CC groups were 5. 70,5. 72 and 5. 77, respectively; for drip loss: 1. 01%, 0. 85% and 0. 92%, respectively), although the mean differences did not reach statistical significance, partially due to a low frequency (5%) of the AA genotype in the population. In Sutai pigs, each genotype group (AA, AC or CC) at the PHKG1 g. 8283C>A site has more than 100 individuals. Significant effects of the causal mutation were observed on almost all meat quality traits in this population (Tables 2 and S5). Especially, the RG level in the longissimus muscle (LM) of AA animals was 4 times higher (P = 9. 03×10−54) than those in CC animals. Coincidently, drip loss, rate of pH decline and Minolta a* and b* were higher (P<0. 01) in AA pigs than CC pigs. AA pigs also had lower (P<0. 001) intramuscular fat content (IMF) and marbling score compared to CC pigs. In the DLY hybrid commercial population, all 140 individuals were genotyped for the PHKG1 g. 8283C>A mutation and 53 surrounding SNPs using the OpenArray platform (see Materials and Methods). Again, we observed the significant effects of the g. 8283C>A mutation on RG, pH and drip loss (Table 2). Moreover, this mutation showed stronger associations with these traits than any other surrounding SNPs (Figure S9). These results indicate that the A allele has unfavorable effects on these meat quality traits. To reveal the allele frequency of the PHKG1 g. 8283C>A mutation in diverse pig breeds, we genotyped this mutation in a broad panel of 629 animals representing 5 European commercial breeds, 2 Chinese domestic breeds, and wild boars from China and Europe. The A allele causing excess glycogen content occurred at high frequency (0. 70) in White Duroc and at medium frequency (0. 32) in Red Duroc, but it was nearly absent in other domestic and wild breeds (<0. 03; Table 3). The PHKG1 mutant allele was absent in wild boars, suggesting that the mutation likely happened after domestication. However, due to limit number of wild boars examined in this study, we cannot rule out the possibility that it is a standing variation. To date, only a handful of QTG and QTN have been identified for complex traits in farm animals. To the best of our knowledge, this is the first study that has used a system genetics approach including GWAS, eQTL mapping and causality modeling to identify QTG and QTN for complex traits in pigs. Our study supports the PHKG1 gene as a QTG for GP-related traits based on the following findings: (1) GWAS on the F2 and Sutai populations enable us to define the major QTL for GP within an interval of 180-kb on SSC3, which contains only 7 genes including PHKG1. (2) Of three positional candidate genes with cis-eQTLs signals, only PHKG1 has the cis-eQTL peak SNP that is identical to the pQTL peak SNP. (3) PHKG1 is a catalytic subunit of the phosphorylase kinase (PhK), which is critical to glycogen degradation. (4) The wild-type transcript level of PHKG1 was significantly correlated to glycogen content in muscle. Furthermore, our study shows the following evidences for the PHKG1 g. 8283C>A mutation as QTN underlying the SSC3 QTL effect: (1) The mutation caused abnormal splicing of PHKG1 mRNA, resulting in a shift in the reading frame with a premature stop codon. (2) The aberrant mRNA transcripts were diminished by NMD, which reduced the PHKG1 protein level and led to PhK enzyme deficiency. (3) The mutation shows the complete concordance between their genotypes and the deduced QTN genotypes across all parental boars. No other variants in the PHKG1 gene exhibited such concordance. (4) This mutation has a consistent and significant effect on GP-related traits across all tested populations. Here, we illustrate that the QTN (g. 8283C>A) is a splice site mutation affecting both transcription and translation levels of the PHKG1 gene, which has at least three implications. One is that cis-eQTL mapping for annotated genes residing the QTL region can not only contribute to identification of regulatory QTNs, but also benefit characterization of protein-altering QTNs including splice and nonsense mutations that affect transcript levels by NMD, just like our identified QTN. Another implication is that if a gene is subjected to alternative splicing, it is necessary to determine the levels of its different transcripts (instead of only the total amount of these transcripts) by deep RNA sequencing and then examine their individual relationship with phenotypic traits. The third one is that we could identify a cis QTL for PHKG1 protein level that overlaps with the mapped cis-eQTL and pQTL on SSC3 if a big data set on protein abundance was available, and find a strong relationship between protein level and GP-related traits. This information could add a new dimension for the search of QTG. Several studies have recently demonstrated the feasibility of high-throughput proteome quantification and revealed the variation and genetic control of protein abundance in humans and mice [23], [24]. Hopefully, whole proteome analysis will be more efficient and routinely added to integrative genomic analysis to accelerate the identification of QTG and QTN for complex traits in livestock. Phosphorylase kinase (PhK) is comprised of four different subunits with a stoichiometry of (αβγδ) 4; α, β, and δ are regulatory, while γ is catalytic. Each of these subunits has isoforms or splice variants differentially expressed in different tissues. Mutations in 4 PhK subunit genes (PHKA1, PHKA2, PHKB and PHKG2) have been implicated in low PhK activity in liver and/or muscle [25]–[29]. No variant in the muscle isoform of the PhKγsubunit (PHKG1) has been reported for muscle PhK deficiency [17]. To our knowledge, this study is the first one to confirm the association of the PHKG1 mutation with PhK deficiency, muscle glycogenosis and meat quality traits in pigs. Pork with high GP (>180 µmol/g) often has low ultimate pH and water holding capacity, and is therefore called “acid meat”. The R225Q mutation in PRKAG3 gene was the first identified causal mutation for acid meat [19]. This gain-of-function mutation causes an increased glucose uptake and glycogen synthesis in skeletal muscle [30], [31]. Its unfavorable allele (225Q) is dominant and specifically present in Hampshire and related breeds [19]. In contrast, the PHKG1 g. 8283C>A mutation that we identified is a loss-of-function mutation causing the defect in glycogen degradation. This variant occurs predominantly in Duroc or Duroc-crossed pigs. The mutant allele A appears to be partially recessive, since the average RG value of AC heterozygotes approximates to that of CC homozygotes rather than AA homozygotes (Table S3). Costa et al. [32] have reported that 9. 8% of DLY hybrid pigs free of the PRKAG3 225Q allele still show the acid meat phenotype with GP higher than 180 µmol/g. In this study, we found that the PHKG1 QTNs are segregating in DLY hybrid pigs with a frequency of 1. 7% (9/540) for the homozygous mutant with GP beyond 180 µmol/g. Therefore, we speculate that a substantial proportion of acid meat is likely caused by the PHKG1 g. 8283C>A mutation. It was observed that the mutation had negative effects on almost all meat quality traits but did not impact any growth traits (e. g. carcass weight; see Table S6) in the F2, Sutai and DLY populations. However, Duroc and two Duroc-derived lines carry the mutation at relatively high frequencies (Table 3). The result may suggests that meat quality has not been the primary breeding goals in these pig populations. In conclusion, we identified a causal mutation in the PHKG1 gene associated with excess glycogen content in Duroc and its related pigs. The finding highlights the important role of genes encoding PhK subunits in the formation of the GP-related traits. More intriguingly, our finding would be of considerably importance for the pig industry as we can develop a diagnostic DNA test to effectively eliminate the PHKG1 undesirable allele from nucleus herds and consequently improve meat quality. All procedures involving animals followed the guideline for the care and use of experimental animals established by the Ministry of Agriculture of China. The ethics committee of Jiangxi Agricultural University specifically approved this study. Three experimental populations were involved in this study: a White Duroc × Erhualian F2 intercross, a Chinese Sutai half-sib population and a commercial DLY hybrid population. The F2 population was established as described previously [33]. Briefly, two White Duroc boars were mated to 17 Erhualian sows. Nine F1 boars and 59 F1 sows were then intercrossed to produce a total of 1912 F2 animals in 6 batches. Sutai is a Chinese synthetic line that is derived from Chinese Taihu (50%) and Western Duroc (50%) after over 18 generations of artificial selection. The Sutai population comprised offspring of 4 sires and 55 dams. A total of 930 F2 individuals, 434 Sutai pigs and 540 DLY pigs were used in this study. The F2 and Sutai pigs were weaned at day 46 and 28 respectively, and males were castrated at day 90 and 18 respectively. The two populations were raised at an experimental farm of Jiangxi Agricultural University in Nanchang city during their fattening period and were slaughtered for phenotype recording at the age of 240±3 days. The DLY pigs grew up at a farm of Xiushui city (about 110 miles away from Nanchang) until the slaughter weight of 90–100kg. All pigs were transported and slaughtered at the same commercial abattoir in Nanchang where the pigs were fasted for 15–20 hours with water available ad libitum. After slaughter, muscle samples were removed within 30 minutes postmortem from the longissimus (LM) and semimembranosus (SM) muscles for RNA isolation and the measurement of meat quality traits. The meat characteristics including the ultimate pH (or pH 24h, measured at 24h postmortem), drip loss, Minolta color parameters (L*, a*, b*), subjective scores of color and marbling, intramuscular fat content (IMF), glycolytic potential (GP), residual glycogen (RG), glucose-6-phosphate (G-6-P) and lactate were measured as described previously [20], [34]. Genomic DNA was isolated from ear, blood or spleen tissues with a standard phenol/chloroform method. A total of 1020 animals from the F2 population were genotyped for 62163 SNPs on the Illumina PorcineSNP60 BeadChip [21] according to the standard manufacture' s protocol. The 60K Beadchip has 62,163 SNP, of which 54,920 can be mapped to the current pig genome assembly (Sus Scrofa Build 10. 2) [35]. The quality control (QC) procedures were carried out by Plink v 1. 07 [36]. Briefly, animals with call rate>0. 9 and Mendelian error rate <0. 05, and SNP with call rate>0. 9, minor allele frequency>0. 05, P value>10−5 for the Hardy-Weinberg equilibrium test were included. A final set of 39414 informative SNPs on 930 F2 pigs were used for subsequent analyses. A total of 434 Sutai pigs were also genotyped using the Illumina PorcineSNP60 BeadChip. To fine map the SSC3 QTL, we increased the marker density in this QTL region with additional 53 SNPs (Table S7), which were identified through comparison of our own whole-genome sequence data from 4 F0 Erhualian sows and the Duroc reference genome sequence (Sus scrofa Build 10. 2). All Sutai pigs were further genotyped for the 53 SNPs by using the TaqMan OpenArray Genotyping System (Life Technologies). After QC filtering as described above, 44560 SNPs were included for further analysis. We also genotyped 140 DLY pigs using our developed OpenArray 53-SNP panel. Out of the 53 SNPs, 28 passing QC were included in association analysis. These genotype data are deposited in the Dryad repository (http: //dx. doi. org/10. 5061/dryad. 7kn7r). The allelic effect of each SNP on phenotypic traits was tested using a general linear mixed model [37]. The model included a random polygenic effect, and the variance-covariance matrix was proportionate to genome-wide identity-by-state [38]. The formula of the model is given in a mathematic expression:, where Y is the vector of phenotypes; µ is the overall mean; b is the vector of fixed effects including sex and batch effects; w is the vector of slaughter weight of individuals considered as covariate; c is the vector of SNP effects with Erhualian allele substitute to White Duroc allele; a is the vector of random additive genetic effects with α∼N (0, Gσα2), where G is the genomic relationship matrix calculated from the corrected pedigree and σα2 is the polygenetic additive variance; k is the regression coefficient of slaughter weight and e is the vector of residual errors with e∼N (0, Iσe2), where I is the identity matrix and σe2 is the residual variance. X, S and Z are incidence matrices for b, w and c respectively. All single-marker GWAS were conducted by GenABEL packages [39]. The genome-wide significance threshold was determined by the Bonferroni method, in which the conventional P-value was divided by the number of tests performed [40]. A SNP was considered to have genome-wide significance at P<0. 05/N and chromosome-wide significance at P<1/N, where N is the number of SNPs tested in the analyses. The genome-wide and chromosome-wide significant thresholds were respectively 1. 27e-6 (0. 05/39414) and 2. 54e-5 (1/39414) for the F2 population, and 1. 12e-6 (0. 05/44560) and 2. 24e-5 (1/44560) for the Sutai population. The phenotypic variance explained by the top SNPs was estimated by (Vreduce– Vfull) /Vreduce, where Vfull and Vreduce are residual variances of models for association analysis with and without SNP term, respectively. The influence of population stratification was assessed by examining the distribution of test statistics generated from thousands of association tests and assessing their deviation from the null distribution (that expected under the null hypothesis of no SNP associated with the trait) in a quantile-quantile (Q-Q) plot [41]. The Q-Q plot was constructed using R software. Linkage disequilibrium (r2) was estimated for SNPs around the QTL region in the two populations by using PLINK v1. 07 [36]. Total RNA was extracted from longissimus dorsi muscle samples of 497 F2 animals using Trizol (Invitrogen). RNA quantity and integrity were assessed using a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific) and a 2100 Bioanalyser (Agilent). Genome-wide transcripts were assayed by digital gene expression (DGE) system and data processing was conducted as described previously [11], [42]. In brief, the raw tags were first filtered to produce the clean tag data. For mapping clean tags to reference transcript sets or to the pig reference genome (Sus Scrofa Build 10. 2) [35], we created virtual libraries containing all the possible 17-base length sequences of these resources located next to an NlaIII restriction site. The reference transcript sets were downloaded from the database of PEDE (Pig Expression Data Explorer; http: //pede. dna. affrc. go. jp/) and pig unigene in NCBI (ftp: //ftp. ncbi. nih. gov/repository/UniGene/Sus_scrofa/). The redundant transcripts overlapped between the two databases were removed from the reference transcript set. For monitoring the mapping events on both strands, virtual sense and antisense tag sequence databases were generated for both full gene and cDNA sequences using in-house Perl scripts. The clean tag sequences were then mapped using SOAP2 [43] allowing up to one mismatches in 21-bp tag sequences. Sense and antisense tag sequences that unsuccessfully mapped to reference transcripts or mapped to multiple genes were filtered. The number of clean tags that uniquely mapped to the reference transcript sequence of each gene was calculated and then normalized to TPM (number of tags mapped to each gene per million clean tags) as expression level of transcript. The DGE data of 497 F2 animals were adjusted for gender, batch and kinship using a robust linear regression model [44]. Associations between gene expression levels and the RG phenotype were evaluated with Pearson correlation coefficient by R software. eQTL mapping was performed for the DGE profiles in 497 F2 animals using mixed linear model implemented by mmscore function of GenABEL in R package. Sex and batch were considered as fixed effects, and the genetic co-variances among samples were also taken into account by fitting kinship matrix derived from whole genome SNP genotypes. Bonferroni correction was applied to adjust the multiple tests. All the above mentioned analyses were carried out with R software. The entire cDNA sequence of PHKG1 were determined by RT-PCR (reverse transcriptase-polymerase chain reaction) and RACE (rapid amplication of cDNA ends) analysis. First, RNA was extracted from skeletal muscle of 6 F2 pigs (2 of each QTL genotype) using Trizol (Invitrogen), then cDNA was synthesized using the PrimerScript RT reagent Kit With gDNA Eraser (Takara) for RT-PCR or the 5′- & 3′- Full Race Kits (Takara) for RACE. The corresponding primer pairs are listed in Table S8. To get full-length gene sequence, we amplified 14 overlapping segments from genomic DNA, using standard PCR condition and primers reported in Table S9. All PCR products were purified using the QIAquick PCR Purification Kit (Qiagen) and sequenced on both strands using the same primers and BigDye Terminator v3. 1 Cycle Sequencing kits (Applied Biosystems) and a 3130 DNA Analyzer (Applied Biosystems). The sequence traces were assembled and analyzed for polymorphisms using the SeqMan program (DNASTAR). A 140-bp fragment (Figure 3C) encompassing the g. 8283C>A mutation was amplified by PCR from genomic DNA using primers (PHKG1-F1: 5′-ATC CCT GTG CTT GCT GGT G-3′; PHKG1-R1: 5′-CCC GGC GGT ACT GGT AAT-3′), digested using enzyme TaqαI and size fractionated by 2% agarose gel electrophoresis. The A allele was represented by two fragments of 78 and 62 bp, and the C allele by uncut amplicons. Association between the PHKG1 g. 8283C>A polymorphism and meat quality traits was calculated using the least square means method of GLM (General Linear Model) procedure in R software. For the F2 and Sutai populations (n = 930 and 434 respectively), sex and batch was included in the model as fix effects, carcass weight as a covariate. A total of 540 DLY pigs were genotyped for this mutation. The residual glycogen content and other meat quality traits were determined for muscle samples from 140 DLY pigs. The model for DLY included sex and harvest batch as fix effects, and carcass weight as a covariate. The associations between SNP genotypes (i. e. ss131031160 SNP and PHKG1 g. 8283C>A) and expression levels of three genes (PHKG1, GUSB and SUMF2) were established by One-Way ANOVA analysis in R software. The relationship between the transcript level of PHKG1 and the RG content was calculated using Pearson correlation in R software. QTL genotypes of 6 F1 boars in the F2 population and 3 Sutai F0 boars were determined by marker-assisted segregation analysis as described previously [45]. Briefly, a Z-score was calculated for each sire; the score is the log10 of the H1/H0 likelihood ratio where H1 assumes that the boar is heterozygous at the QTL (Qq), while H0 postulates that the boar is homozygous QQ or qq. Boars were considered to be Qq when Z>2, QQ or qq when Z <−2, and of undetermined genotype if −2<Z<2. Quantitative RT-PCR (qRT-PCR) reactions were performed in a final volume of 10 µl containing 1 µl of 2. 5-fold diluted cDNA (corresponding to 20 ng of starting total RNA), 5 µl Power SYBR Green PCR Master Mix (Applied Biosystems), forward and reverse primers (2 pM each) and 3. 6 µl free water. PCRs were conducted on an ABI7900HT instrument (Applied Biosystems) under the following cycling conditions: 10 min at 95°C followed by 40 cycles at 95°C for 15 sec and 60°C for 50 sec. Two primer sets Common-5' -FP/RP and Wt-3' -FP/RP (Figure 4A; Table S10) were used to respectively test total and wild-type transcript (Wt) levels of PHKG1. Beta Actin (ACTB) was included as endogenous controls. Expression of all assays was measured in triplicates and average values of the triplicates were used for the analysis. The quantification of transcripts was performed by the comparative Ct (2-ΔΔCt) method. The presence of PHKG1 Wt transcript in animals with three QTL genotypes was assayed by RT-PCR using the primers 5′-CAC CCC AAC ATC ATA CAG CT-3′ and 5′-ACA GAA GCC AGC ACC GTC-3′ (RT-3' -RP). PCR products of 698-bp were electrophoresed on 1% agarose gel and visualized by UV illumination (Figure 4C). Total protein from pig muscle tissue was extracted using a Protein Extraction Kit (Applygen). Protein samples separated on SDS-PAGE were transferred onto polyvinylidene difluoride membranes and incubated with rabbit anti-PHKG1 (Proteintech) and rabbit anti-mouse β-Actin (as loading control; Beijing Zhong Shan-Golden Bridge Biological Technology) antibodies. Anti-mouse or anti-rabbit secondary antibodies conjugated with horseradish peroxidase were used and visualized using chemiluminescent substrate (Tiangen Biotech). Phk activity of 18 F2 animals were determined on their frozen muscle samples using Phk Colorimetric Assay kit (Genmed Scientifics) according to the manufacturer' s instructions.

Glycogen storage diseases (GSD) are a group of inherited disorders characterized by storage of excess glycogen, which are mainly caused by the abnormality of a particular enzyme essential for releasing glucose from glycogen. GSD-like conditions have been described in a wide variety of species. Pigs are a valuable model for the study of human GSD. Moreover, pigs affected by GSD usually produce inferior pork with a lower ultimate pH (so-called "acid meat") and less processing yield due to post-mortem degradation of the excess glycogen. So far, only one causal variant, PRKAG3 R225Q, has been identified for GSD in pigs. Here we reported a loss-of-function mutation in the PHKG1 gene that causes the deficiency of the glycogen breakdown, consequently leading to GSD and acid meat in Duroc-sired pigs. Eliminating the undesirable mutation from the breeding stock by a diagnostic DNA test will greatly reduce the incidence of GSD and significantly improve pork quality and productivity in the pig.

lay_plos

Mitochondrial complex III (CIII2) and complex IV (CIV), which can associate into a higher-order supercomplex (SC III2+IV), play key roles in respiration. However, structures of these plant complexes remain unknown. We present atomic models of CIII2, CIV, and SC III2+IV from Vigna radiata determined by single-particle cryoEM. The structures reveal plant-specific differences in the MPP domain of CIII2 and define the subunit composition of CIV. Conformational heterogeneity analysis of CIII2 revealed long-range, coordinated movements across the complex, as well as the motion of CIII2’s iron-sulfur head domain. The CIV structure suggests that, in plants, proton translocation does not occur via the H channel. The supercomplex interface differs significantly from that in yeast and bacteria in its interacting subunits, angle of approach and limited interactions in the mitochondrial matrix. These structures challenge long-standing assumptions about the plant complexes and generate new mechanistic hypotheses. The canonical mitochondrial electron transport chain (mETC), composed of four integral membrane protein complexes (complexes I–IV; CI–CIV) located in the inner mitochondrial membrane (IMM), transfers electrons from NADH and succinate to molecular oxygen. The concomitant pumping of protons (H+) across the IMM establishes an electrochemical proton gradient that is used by ATP synthase to produce ATP (Nicholls, 2013). Whereas the atomic details of the respiratory complexes and several supercomplexes (higher-order complex assemblies) are known for yeast, mammals and bacteria (Vinothkumar et al., 2014; Fiedorczuk et al., 2016; Gu et al., 2016; Letts et al., 2016; Wu et al., 2016; Zhu et al., 2016; Zickermann et al., 2015; Blaza et al., 2018; Guo et al., 2017; Letts et al., 2019; Agip et al., 2018; Parey et al., 2018), the high-resolution structural details of the respiratory complexes and supercomplexes of plants have remained mostly unknown. Complex III (CIII2), also called the cytochrome bc1 complex or ubiquinol-cytochrome c oxidoreductase, is an obligate dimer that transfers electrons from ubiquinol in the IMM (reduced by CI, CII, or alternative NADH dehydrogenases) to soluble cytochrome c in the intermembrane space (IMS) (Nicholls, 2013). This redox reaction is coupled to the pumping four H+ to the IMS. CIII2 is composed of three conserved subunits present in all organisms (cytochrome b, COB; cytochrome c1, CYC1; and the iron-sulfur ‘Rieske’ subunit, UCR1), as well as a varying number of accessory subunits present in eukaryotes (Iwata et al., 1998; Xia et al., 2013; Xia et al., 1997). Each CIII monomer contains one low-potential heme b (bL) and one high-potential heme b (bH) in COB, a heme c in CYC1, a 2Fe-2S iron-sulfur cluster in UCR1, as well as two quinone-binding sites (QP and QN close to the positive/IMS and negative/matrix sides respectively) in COB. Given that CIII2 is a dimer, these sites in each CIII monomer are symmetrical within the dimer in isolation. However, the symmetry may be broken when CIII assembles into asymmetrical supercomplexes (Letts et al., 2016; Letts et al., 2019; Letts and Sazanov, 2017). CIII2’s redox and proton pumping occur via the ‘Q-cycle’ mechanism (Cramer et al., 2011), which allows for efficient electron transfer between ubiquinol (a two-electron donor) and cyt c (a one-electron acceptor). To this end, one electron is transferred from ubiquinol in the Qp site to the 2Fe-2S in the UCR1 head domain. The head domain then undergoes a large conformational swinging motion from its ‘proximal’ position close to the QP site to a ‘distal’ CYC1 binding site adjacent to heme c1 (Zhang et al., 1998). The electron is then transferred via heme c1 to cyt c bound to CIII2 in the IMS. Of note, the UCR1 head domain belongs to the opposite CIII protomer relative to COB and CYC1. The second electron donated by ubiquinol is transferred via hemes bL and bH to a quinone in the QN site, reducing it to ubisemiquinone. The cycle is repeated to regenerate ubiquinol in the QN site, ultimately reducing two molecules of cyt c and pumping four protons. In eukaryotes, the large CIII2 accessory subunits exposed to the mitochondrial matrix have homology to mitochondrial processing peptidases (MPP) of the pitrilysin family (Gakh et al., 2002). These metalloendopeptidases—composed of an active β subunit and an essential but catalytically inactive α subunit—cleave mitochondrial signal sequences of proteins that are imported into the mitochondria (Gakh et al., 2002). Whereas in yeast the CIII2 accessory subunits with MPP homology (ScCor1/Cor2) have completely lost MPP enzymatic activity, the mammalian CIII2 homolog (UQCR1/UQCR2 heterodimer) retains basal activity to only one known substrate (the Rieske subunit) (Gakh et al., 2002; Taylor et al., 2001). Hence, in yeast and mammals this enzymatic activity is carried out by soluble MPP heterodimers in the mitochondrial matrix. In contrast, in vascular plants, there is no additional soluble MPP enzyme, and all MPP activity is provided by the CIII2 MPP heterodimer (MPP-α/β). Thus, in plants CIII2 serves a dual role as a respiratory enzyme and a peptidase (Braun et al., 1992; Emmermann et al., 1993; Eriksson et al., 1994; Braun et al., 1995; Eriksson et al., 1996; Braun and Schmitz, 1995a; Glaser and Dessi, 1999). This integration of respiratory and peptidase activities may have occurred early in eukaryogenesis (Braun and Schmitz, 1995b). The bioenergetic implications of this dual function of plant CIII2 remain unknown. Complex IV (CIV), also called cytochrome c oxidase, transfers electrons from cyt c to molecular oxygen, reducing it to water (Nicholls, 2013). The redox reaction is coupled to the pumping of four protons into the IMS. Like CIII2, CIV is composed of three conserved subunits (COX1, COX2, COX3) and a variable number of accessory subunits, depending on the organism. Electrons are transferred from cyt c to oxygen via COX1’s dinuclear copper (CuA), heme a and copper-associated heme a3 (CuB, binuclear center). The passage of protons from the matrix to the IMS occurs through distinct ‘channels’ formed by protonatable amino-acid residues (Rich, 2017; Rich and Maréchal, 2013; Yoshikawa and Shimada, 2015; Wikström et al., 2018). It is currently believed that, whereas yeast CIV pumps protons through the K and D transfer pathways (named after key amino-acid residues in each pathway), mammalian CIV uses an H channel in addition to the K and D channels (Rich, 2017; Rich and Maréchal, 2013; Yoshikawa and Shimada, 2015; Wikström et al., 2018; Maréchal et al., 2020). The contribution of the K, D, H pathways in plant CIV has not been characterized. In the IMM, respiratory complexes can be found as separate entities or as higher-order assemblies known as supercomplexes (Schägger and Pfeiffer, 2000). Although it was initially hypothesized that supercomplexes would allow for direct substrate channeling between complexes, evidence has mounted against this view (Gu et al., 2016; Letts et al., 2016; Letts et al., 2019; Yu et al., 2018; Sousa et al., 2016; Blaza et al., 2014; Fedor and Hirst, 2018). Instead, supercomplexes may have roles in improving the stability of the complexes, providing kinetic advantages to the electron transfer or reducing the production of reactive oxygen species or of aggregates in the IMM (Letts and Sazanov, 2017; Milenkovic et al., 2017). Supercomplexes of various stoichiometries between CIII2 and CIV (e. g. SC CIII2+CIV, SC CIII2+CIV2) have been seen (Schägger and Pfeiffer, 2000; Eubel et al., 2004; Eubel et al., 2003). In plants, the CIII2-CIV supercomplex of highest abundance is a single CIII dimer with a single CIV monomer (SC III2+IV) (Eubel et al., 2004). High-resolution structures of the model yeast Saccharomyces cerevisiae and Mycobacterium smegmatis CIII2-CIV supercomplex (SC III2+IV2) have recently been determined (Hartley et al., 2019; Rathore et al., 2019; Gong et al., 2018; Wiseman et al., 2018). Although there is currently no high-resolution structure for a mammalian CIII2-CIV supercomplex, the supercomplex between CI, CIII2 and CIV (SC I+III2+IV, the respirasome) shows a distinct interaction interface between CIII2 and CIV relative to the SC III2+IV2 from yeast and bacteria (Gu et al., 2016; Letts et al., 2016). Similar to that seen in comparative tomographic studies of plant SC I+III2 and bovine and yeast SC I+III2+IV (Davies et al., 2018), the above SC III2+IV2 studies revealed that, while the general configuration of the individual CIII2 and CIV are conserved, the location of the binding interface between CIII2 and CIV in the supercomplex is divergent, with different subunits involved in the different organisms. For plant CIII2 and CIV, the only currently available structural information is from low-resolution, 2D-averages of negative-stain EM samples from A. thaliana (Dudkina et al., 2005) and potato (Bultema et al., 2009). High-resolution structures or atomic models for CIII2, CIV or their supercomplexes are not currently available for the plant kingdom. Here we present the cryoEM structures of CIII2 and SC III2+IV from the vascular plant Vigna radiata (mung bean) at nominal resolutions of 3. 2 Å and 3. 8 Å, respectively. Using focused refinements around CIV, we achieved a nominal resolution for CIV of 3. 8 Å. The structures reveal plant CIII2 and CIV’s active sites, as well as the plant-specific configuration of several CIII2 and CIV subunits. The structures also show the SC III2+IV binding interface and orientation, which is unique to plants. Additionally, using cryoEM 3D-conformation variability analysis (Punjani and Fleet, 2020), we were able to visualize the swinging motion of CIII2’s UCR1 head domain at 5 Å resolution in the absence of substrate or inhibitors. We also observed complex-wide coupled conformational changes in the rest of CIII2. These results question long-standing assumptions, generate new mechanistic hypotheses and provide the initial structural basis for the development of more selective agricultural inhibitors of plant CIII2 and CIV. By docking the individually refined CIII2 and CIV models into the SC III2+IV composite map (Figure 1, Figure 1—figure supplement 2, Video 3), we were able to define the binding interface of the plant supercomplex. Direct contacts between the complexes occur in one site in the matrix side and one site in the IMS (Figure 8). Site 1 (matrix side), shows a single hydrophobic contact between one residue of VrQCR8 (Pro31) and one residue of VrCOX2 (Trp59) (Figure 8B). Our cryoEM reconstruction also contains a short stretch of weak, unassigned density near the first modelled residue of VrCOX5c (BtCOX6c, ScCox9), which could maximally represent an additional four amino acids. If so, the N-terminus of VrCOX5c could potentially provide additional contacts with CIII2. However, this density may also correspond to a bridging lipid bound between the two complexes. The limited matrix-side contacts in V. radiata are in stark contrast to the supercomplex interface in S. cerevisiae, where binding is dominated by interactions between ScCor1 (VrMPP-β) and ScCOX5a (VrCox4) on the matrix side. Instead, V. radiata’s site 2 (IMS side) provides the bulk of the protein-protein interactions of the supercomplex, with a hydrophobic interaction between VrQCR6 and VrCOX5c (Leu26 and Leu51 respectively) as well as an interface between VrQCR6 (Pro19 and Lys20) and VrCOX4 (Arg114-Phe117) (Figure 8C). Despite the potential for lipid bridges at the matrix leaflet of the IMM, there are no direct contacts in the membrane and no protein contact at the IMS leaflet of the IMM. The overall limited binding interactions between V. radiata’s CIII2 and CIV, and, consequently, the lower expected stability of the plant supercomplex compared to yeast' s, are consistent with the fact that (CIII2+CIV) n supercomplexes have only been experimentally identified in a few of the plant species studied (Dudkina et al., 2006; Braun, 2020). Unsurprisingly given the large differences in contacts, there is a significant difference in the angle between CIII2 and CIV in V. radiata versus yeast, resulting in a more ‘open’ orientation in V. radiata (Figure 8—figure supplement 1). This orientation leads to a difference of 18° in the angle between the CIII2 and CIV as measured by the relative positions of the bh-hemes in CIII2 and the a-hemes in CIV. The difference in orientation also results in a larger estimated distance between the CIII2- and CIV-bound cyt c in V. radiata (~70 Å) than in yeast (~61 Å) (Figure 8—figure supplement 1E). Plant CIV subunit composition has been previously analyzed by mass spectrometry of proteins from 2-dimensional blue-native gels (BN-PAGE) (Millar et al., 2004; Klodmann et al., 2011; Senkler et al., 2017). Although these studies were mostly in agreement, several putative CIV subunits—including several putative plant-specific subunits—differed between datasets. Given the considerable technical challenges in obtaining plant CIV samples, experimental evidence for the stoichiometric presence of these putative subunits in complex IV remained limited, with strongest evidence for COX-X1, COX-X2 and COX-X4 (Senkler et al., 2017; Braun, 2020). The structure of V. radiata CIV presented here offers a complementary approach to determine the complex’s subunit composition. Our structure shows that CIV obtained from etiolated V. radiata sprouts is composed of 10 subunits (three mitochondrially encoded subunits and seven accessory subunits). Only three of the previous plant-specific candidates are seen in our structure (COX-X2, COX-X3 and COX-X4). Moreover, structural analysis shows that COX-X2, COX-X3 and COX-X4 are homologs of mammalian and yeast CIV subunits (BtCOX4/ScCOX5, BtCOX7c/ScCox8 and BtCOX7a/ScCox7, respectively) rather than being plant-specific subunits. Although mass spectrometry analysis of our mixed sample also shows some evidence for the occurrence of COX-X1, this protein is not present in our structure, and its function remains unknown. Our structure provides a new definition for plant CIV’s composition; however, we note that the arrangement may differ between free CIV and that in supercomplexes. Moreover, its composition may be dynamically regulated in different metabolic states (e. g. different light or oxygen levels), as is known to occur for certain subunit isoforms in other organisms (Burke et al., 1997). The 3DVA allowed us to observe the full swing of the UCR1 (Rieske subunit) head domain (Figure 4, Video 10) without the need for any CIII2 inhibitor to capture the proximal, distal or intermediate positions in the mobile state (Esser et al., 2019). As such, it provides direct confirmation for a multitude of previous crystallographic, mutational, kinetic and molecular dynamics studies, mostly done in the presence of inhibitors, that showed the flexibility and mobility of this domain (Xia et al., 2013; Cooley, 2013; Berry and Huang, 2011; Izrailev et al., 1999; Huang and Berry, 2016). Together, the findings definitively show that the swinging motion is an inherent property of the UCR1 in the absence of substrates. Moreover, the conformational heterogeneity analysis suggests that the movement of the UCR1 head domains is coordinated between the CIII2 protomers to a large degree (Figure 4C, Video 10). Determining the nature of and mechanism for this inter-protomer coordination will have implications on electron transfer in CIII2 and its supercomplexes. Unfortunately, however, this conformational flexibility precluded us from building an atomic model for the head domain. Thus, we were not able to evaluate the H-bonding pattern of the UCR1 head domain with COB, or the implications of such pattern to the Q-cycle electron bifurcation mechanism (Xia et al., 2013; Ho et al., 2020; Belt et al., 2017). Similarly, the resolution of the 3DVA was not sufficient to evaluate changes in the positions of COB’s cd1 and ef helices. These helices are critical components of the UCR1’s COB binding ‘crater’ (Xia et al., 2013), and their position changes in response to binding of different CIII2 inhibitors (Esser et al., 2006), with important implications for the Q-cycle mechanism. It is important to note that 3DVA only reveals conformational changes, with no information on kinetics or occupancy rates. Nonetheless, we demonstrate here that cryoEM conformational heterogeneity tools such as 3DVA (Punjani and Fleet, 2020) and others (Zhong et al., 2020) are a valuable complementary approach to study the conformational changes of CIII2’s Q-cycle in its native state, as well as in the presence of inhibitors and substrates. Moreover, the 3DVA revealed that CIII2 can undergo different types of complex-wide motions that are coordinated across sides of the membrane and between protomers (Videos 6–9). Changes at the ‘top’ of the complex on one side of the membrane co-vary (i. e. are coupled) with movement at the ‘bottom’ of the complex on the other side of the membrane. This long-range conformational coupling across the entire CIII2 could be the basis for symmetry-breaking and coordination of the UCR1 head domain motion between the CIII2 protomers. The long-range conformational coupling is particularly relevant in the context of plant CIII2’s dual roles in signal-peptide processing and respiration. The potential interdependence between these two functions was investigated using CIII2 inhibitors (Eriksson et al., 1994; Eriksson et al., 1996), ultimately leading to the interpretation that these functions are independent (Glaser and Dessi, 1999). In the presence of the CIII2 respiratory inhibitors antimycin A (QN-site inhibitor) and myxothiazol (QP-site inhibitor) at concentrations that inhibit ~90% of spinach CIII2’s respiratory activity, the complex’s peptidase activity is inhibited 30–40% (Eriksson et al., 1994; Eriksson et al., 1996). Given that higher concentrations of inhibitors are needed to elicit MPP inhibition than respiratory inhibition, the authors initially speculated that the effects on the peptidase activity could be due to the inhibitors preventing necessary conformational changes (Eriksson et al., 1994). However, when crystal structures of metazoan CIII2 in complex with these inhibitors became available and revealed the locations of the inhibitor binding sites (Iwata et al., 1998; Xia et al., 1997; Zhang et al., 1998), the large distances between these sites and the MPP domain were interpreted to reinforce the notion that the dual roles of plant CIII2are independent, as long-range coupled conformational changes were deemed unlikely (Glaser and Dessi, 1999). In contrast, our 3DVA results showed that long-range coupled motions are intrinsic to V. radiata CIII2. Moreover, our atomic model of plant CIII2 revealed additional contacts and secondary-structure elements not previously seen in other organisms that enhance the interaction between the MPP domain and the rest of the complex. For example, the extended N-termini of MPP-α and -β bridge across the dimer and provide plant-specific contacts with CIII2’s membrane subunits (Figure 3, Figure 3—figure supplements 1–2). Moreover, UCR1’s longer N-terminus in plants also provides plant-specific contacts with the MPP domain (Figure 2—figure supplements 1 and 3; Figure 3—figure supplement 1C). Given its span across the membrane and its domain-swapping across protomers, UCR1 may have roles as a ‘conformational coupler’ beyond its essential function in the Q-cycle. Together, our 3DVA results challenge long-standing assumptions on plant CIII2’s suitability for conformational coupling and call for a re-evaluation of the relationship between the respiratory and the processing activities of the plant complex. The orientation and binding interfaces of SC III2+IV vary significantly among organisms (Hartley et al., 2019; Rathore et al., 2019; Gong et al., 2018; Wiseman et al., 2018; Sousa and Vonck, 2019). For instance, the CIII surface used by yeast to bind to CIV is instead used by mammals to bind to CI (Hartley et al., 2019). Given these disparities, it is not surprising that the differences seen in the VrCIV subunits are concentrated in the subunits that form the supercomplex interfaces in the different organisms (Figure 6). Moreover, while some of the supercomplex interactions in V. radiata are reminiscent of the supercomplex interface in yeast, there are significant differences in the protein: protein sites, interacting subunits and angle of orientation within the SC (Figure 8 and Figure 8—figure supplement 1). In yeast, the main interface is on the matrix side, with the N-terminal helical domain of ScCox5a (VrCOX4) interacting with ScCor1 (homolog of VrMPP-β). In V. radiata, this interface is lacking, as plant COX4 does not possess the ~100 amino-acid helical N-terminal domain present in yeast and mammals (Figure 6 and Figure 6—figure supplement 1). In contrast, the main supercomplex interface in mung bean is in the IMS, driven by contacts between VrQCR6 and VrCOX4. In the yeast supercomplex, the homologs of VrQCR6 and VrCOX4 (ScQCR6 and ScCOX5a/b) also interact in the IMS side, but in a much more limited fashion. In light of VrCIII2’s conformational heterogeneity and the potential interdependence between respiratory and peptidase functions of plant CIII2, an intriguing possibility is that matrix-side interactions between CIII2 and CIV are minimized in the plant supercomplex to prevent steric constraints on the MPP domain, which is catalytically active and likely requires flexibility for its peptidase activity. Thus, the plant-specific supercomplex interface may have evolved to accommodate the particularities of plant CIII2’s dual respiratory and processing functions. Nevertheless, whereas the details differ, the overall location of the CIII2: CIV interface in V. radiata and yeast is similar. A related observation has been made for the supercomplexes between CIII2 and CI (SCI+III2) of plants, yeast and mammals as seen by sub-tomogram averaging (Davies et al., 2018). In this case, although the interfaces between CI and CIII2 in the different organisms were also similar, there was a ~10° difference in the angle between CI and CIII2. Additional functional/structural studies of supercomplexes from organisms of diverse phylogenetic origins could determine whether the location of the supercomplex interface has been achieved by convergent or divergent evolution. In turn, this would shed light on the evolution and potential functional significance of the interface sites. What can already be concluded is that—as seen in yeast (Hartley et al., 2019; Rathore et al., 2019) —the benefit of the SC III2+IV arrangement in plants does not involve direct electron transfer from CIII2 to CIV by simultaneously bound cyt c on each complex, as the calculated distance between the bound cyt c is too large (~70 Å, Figure 8—figure supplement 1). Recent quantitative-proteomics estimations of the stoichiometry of plant respiratory-chain components indicate that the average plant mitochondrion contains ~6500 copies of CIII monomers (i. e. ~3250 CIII2), ~2000 copies of CIV and ~2250 copies of cytochrome c (Fuchs et al., 2020). This implies a maximum of ~2000 copies of SC III2+IV and, thus, a roughly 1: 1 ratio between cyt c and SC III2+IV. (The ratio has been estimated to be 2–3 in S. cerevisiae [Stuchebrukhov et al., 2020]). Based on recent theoretical analyses of electron flow between CIII2 and CIV (Stuchebrukhov et al., 2020), at this low 1: 1 ratio, electron flow would be limited by the time constant of cyt c equilibrating with the bulk IMS phase. Under these conditions, the formation of SC III2+IV in the plant mitochondrion would provide a kinetic advantage to electron flow between CIII2 and CIV by reducing the distance between them relative to CIII2 and CIV freely diffusing in the plane of the membrane. It is important to note that this possible kinetic advantage does not imply substrate trapping or channeling between the complexes, and is thus consistent with a single cyt c pool (Stuchebrukhov et al., 2020). Our work provides the first high-resolution structure of SC III2+IV in plants, revealing plant-specific features of the complexes and supercomplex. Detailed comparisons of plant CIII2 and CIV sites with existing structures of inhibitor-bound complexes in other species will allow for the development of more selective inhibitors for plant CIII2 and CIV, frequently used as agricultural herbicides and pesticides (Esser et al., 2014). The structures also allow for the generation of new mechanistic hypotheses—for example, related to proton translocation in CIV—and a re-evaluation of long-standing assumptions in the field—for instance, related to CIII’s capacity for long-range coordinated motion and the relationship between its respiratory and processing functions. Together with biochemical, cellular and genetic studies, further comparative analyses of these atomic structures with the growing number of respiratory complexes and supercomplexes across the tree of life will allow for the derivation of the fundamental principles of the respiratory electron transport chain. The cryoEM dataset used in this paper is the same sample, grid and micrographs as those used in Maldonado et al., 2020. CryoEM data processing for the structures reported in this paper and those reported in Maldonado et al., 2020 diverged after 2D classification (see Figure 1—figure supplement 1). Further data processing for the structures shown here is described in detail below. V. radiata seeds were purchased from Todd’s Tactical Group (Las Vegas, Nevada, USA). Seeds were incubated in 1% (v: v) bleach for 20 min and rinsed until the water achieved neutral pH. Seeds were subsequently imbibed in a 6 mM CaCl2 solution for 20 hr in the dark. The following day, the imbibed seeds were sown in plastic trays on damp cheesecloth layers, at a density of 0. 1 g/cm2 and incubated in the dark at 20 °C for 6 days. The resulting etiolated mung beans were manually picked, and the hypocotyls were separated from the roots and cotyledons. The hypocotyls were further processed for mitochondria purification based on established protocols (Millar et al., 2007). Briefly, hypocotyls were homogenized in a Waring blender with homogenization buffer (0. 4 M sucrose, 1 mM EDTA, 25 mM MOPS-KOH, 10 mM tricine, 1% w: v PVP-40, freshly added 8 mM cysteine and 0. 1% w: v BSA, pH 7. 8) before a centrifugation of 10 min at 1000 x g (4 °C). The supernatant was collected and centrifuged for 30 min at 12,000 x g (4 °C). The resulting pellet was resuspended with wash buffer (0. 4 M sucrose, 1 mM EDTA, 25 mM MOPS-KOH, freshly added 0. 1% w: v BSA, pH 7. 2) and gently centrifuged at 1000 x g for 5 min (4 °C). This supernatant was then centrifuged for 45 min at 12,000 x g. The resulting pellet was resuspended in wash buffer, loaded on to sucrose step gradients (35% w: v, 55% w: v, 75% w: v) and centrifuged for 60 min at 52,900 x g. The sucrose gradients were fractionated with a BioComp Piston Gradient Fractionator connected to a Gilson F203B fraction collector, following absorbance at 280 nm. The fractions containing mitochondria were pooled, diluted 1: 5 in 10 mM MOPS-KOH, 1 mM EDTA, pH 7. 2 and centrifuged for 20 min at 12,000 x g (4 °C). The pellet was resuspended in final resuspension buffer (20 mM HEPES, 50 mM NaCl, 1 mM EDTA, 10% glycerol, pH 7. 5) and centrifuged for 20 min at 16,000 x g (4 °C). The supernatant was removed, and the pellets were frozen and stored in a −80 °C freezer. The yield of these mitochondrial pellets was 0. 8–1 mg per gram of hypocotyl. Frozen V. radiata mitochondrial pellets were thawed at 4°C, resuspended in 10 ml of chilled (4 °C) double-distilled water per gram of pellet and homogenized with a cold Dounce glass homogenizer on ice. Chilled KCl was added to the homogenate to a final concentration of 0. 15 M and further homogenized. The homogenate was centrifuged for 45 min at 32,000 x g (4 °C). The pellets were resuspended in cold Buffer M (20 mM Tris, 50 mM NaCl, 1 mM EDTA, 2 mM DTT, 0. 002% PMSF, 10% glycerol, pH 7. 4) and further homogenized before centrifugation at 32,000 x g for 45 min (4 °C). The pellets were resuspended in 3 ml of Buffer M per gram of starting material and further homogenized. The protein concentration of the homogenate was determined using a Pierce BCA assay kit (Thermo Fisher, Waltham, Massachusetts, USA), and the concentration was adjusted to a final concentration of 10 mg/ml and 30% glycerol. Washed membranes were thawed at 4°C. Digitonin (EMD Millipore, Burlington, Massachusetts, USA) was added to the membranes at a final concentration of 1% (w: v) and a digitonin: protein ratio of 4: 1 (w: w). Membrane complexes were extracted by tumbling this mixture for 60 min at 4 °C. The extract was centrifuged at 16,000 x g for 45 min (4 °C). Amphipol A8-35 (Anatrace, Maumee, Ohio, USA) was added to the supernatant at a final concentration of 0. 2% w: v and tumbled for 30 min at 4°C, after which gamma-cyclodextrin (EMD Millipore, Burlington, Massachusetts, USA) was added stepwise to a final amount of 1. 2x gamma-cyclodextrain: digitonin (mole: mole). The mixture was centrifuged at 137,000 x g for 60 min (4 °C). The supernatant was concentrated with centrifugal protein concentrators (Pall Corporation, NY, NY, USA) of 100,000 MW cut-off, loaded onto 10–45% (w: v) or 15–45% (w: v) linear sucrose gradients in 15 mM HEPES, 20 mM KCl, pH 7. 8 produced using factory settings of a BioComp Instruments (Fredericton, Canada) gradient maker and centrifuged for 16 hr at 37,000 x g (4 °C). The gradients were subsequently fractionated with a BioComp Piston Gradient Fractionator connected to a Gilson F203B fraction collector, following absorbance at 280 nm. For grid preparation, the relevant fractions were buffer-exchanged into 20 mM HEPES, 150 mM NaCl, 1 mM EDTA, pH 7. 8 (no sucrose) and concentrated to a final protein concentration of 6 mg/ml and mixed one-to-one with the same buffer containing 0. 2% digitonin (w: v), for a final concentration of 0. 1% digitonin (w: v). Mitochondrial membrane extractions were diluted in 2X BN-loading buffer (250 mM aminocaproic acid, 100 mM Tris-HCl, pH 7. 4,50% glycerol, 2. 5% (w: v) Coomassie G-250), loaded on pre-cast 3–12% NativePAGE Bis-Tris gels (Invitrogen, Carlsbad, CA) and run at 4 °C. The cathode buffer was 50 mM Tricine, 50 mM BisTris-HCl, pH 6. 8 plus 1X NativePAGE Cathode Buffer Additive (0. 02% Coomassie G-250) (Invitrogen, Carlsbad, CA) and the anode buffer was 50 mM Tricine, 50 mM BisTris-HCl, pH 6. 8. Gels were run at 150 V constant voltage for ∼30 min, after which the cathode buffer was switched for a ‘light blue’ cathode buffer containing 50 mM Tricine, 50 mM BisTris-HCl, pH 6. 8 plus 0. 1X NativePAGE Cathode Buffer Additive (0. 002% Coomassie G-250) (Invitrogen, Carlsbad, CA). The settings were changed to 200 V constant voltage and run for another ∼90 min. The CI in-gel NADH-dehydrogenase activity assay was performed based on Schertl and Braun, 2015. The BN-PAGE gel was incubated in 10 ml of freshly prepared reaction buffer (1. 5 mg/ml nitrotetrazoleum blue in 10 mM Tris-HCl pH 7. 4). Freshly thawed NADH stock (20 mM) was added to the container with the gel, to a final concentration of 150 μM. The gel with the complete reaction buffer was rocked at room temperature for ∼10 min. Once purple bands indicating NADH-dehydrogenase activity appeared, the reaction was quenched with a solution of 50% methanol (v: v) and 10% acetic acid (v: v). Spectroscopic activity assays were performed based on Letts et al., 2019; Huang et al., 2015; Barrientos et al., 2009, with modifications. Reduced-decylubiquinone (DQ): cyt c activity was measured by spectroscopic observation of cyt c reduction at 550 nm wavelength at room temperature using a Molecular Devices (San Jose, CA) Spectramax M2 spectrophotometer. Reactions were carried out in 96-well plates. DQ (Santa Cruz Biotechnology, Dallas, TX) was freshly reduced. The required amount of ethanol-diluted 100 mM DQ was aliquoted and further diluted to ~300 μl with 100% ethanol. A couple of lithium borohydride crystals were added to reduce the DQ, turning the solution transparent. Excess lithium borohydride was quenched by the dropwise addition of 1 N HCl until no further bubbles were observed. The ethanol was then evaporated with a stream of argon gas until a volume of ~50 μl was obtained. This reduced-DQ was added to a master mix reaction buffer (100 mM HEPES, pH 7. 8,50 mM NaCl, 10% glycerol, 0. 1% CHAPS, 1 mg/ml BSA, 0. 25 mg/ml 4: 1 asolectin: cardiolipin in 0. 1% CHAPS, 25 U/ml SOD, 4 μM KCN, 15 μM piericidin) at a final concentration of 100 μM and mixed by vortexing. The pH of the reaction buffer master mix was checked to be 7–8 with pH strips. The reaction master mix was aliquoted, and CIII2 inhibitors antimycin A and myxothiazol were added at 1 μM final concentration where relevant and mixed by vortexing. Protein samples (5 μg) were added to the respective aliquots of reaction buffer to a total volume of 200 μl and mixed by vortexing. The reaction was initiated by addition of equine cyt c (Sigma Aldrich, St Louis, MO) to a final concentration of 100 μM, briefly mixed by pipetting and plate stirring for 10 s before recording for 3 min every 4 s. Measurements were done in 3–5 replicates, averaged and background-corrected. An extinction co-efficient of 28 mM−1 cm−1 (Huang et al., 2015) was used in the calculations. Statistical significance was determined using two-tailed t-tests. Spectroscopic activity assays were performed based on Letts et al., 2019; Huang et al., 2015; Barrientos et al., 2009, with modifications. Cytochrome c oxidase activity was measured by spectroscopic observation of the oxidation of reduced cyt c at 550 nm wavelength at room temperature using a Molecular Devices (San Jose, CA) Spectramax M2 spectrophotometer. Reactions were carried out in 96-well plates. Equine cyt c (Sigma Aldrich, St Louis, MO), diluted in 20 mM HEPES, pH 7. 4,50 mM NaCl, 10% glycerol buffer, was freshly reduced based on manufacturer’s instructions with modifications. Dithiothreitol (DTT) was added at a 10 mM final concentration. After ~20 min and a visible change in color, cyt c reduction was confirmed spectroscopically. Given the spectrophotometer’s specifications, the simultaneous measurement of A550 and A565 was suboptimal (A550: A565 ratio of ~9). Therefore, the A550: A575 was measured instead, as per manufacturer’s recommendations. Cyt c reduction was confirmed at A550: A575 ratio ~22–24. For the spectroscopic activity assay, the reaction master mix consisted of 20 mM HEPES, pH 7. 4,50 mM NaCl, 10% glycerol, 0. 1% CHAPS (w: v) with additional 4 μM KCN wherever appropriate. Protein samples (5 μg) were added to the respective aliquots of reaction buffer to a total volume of 200 μl and mixed by vortexing. The reaction was initiated by addition of reduced cyt c to a final concentration of 100 μM, briefly mixed by pipetting and plate stirring for 10 s before recording for 3 min every 4 s. Measurements were done in 3–4 replicates, averaged and background-corrected. An extinction co-efficient of 28 mM−1 cm−1 (Huang et al., 2015) was used in the calculations. Statistical significance was determined using two-tailed t-tests. The sample used for mass spectrometry was the sample used to blot the cryoEM grid that was used for here and in Maldonado et al., 2020. This sample corresponds to concentrated, pooled peak two fractions from the sucrose gradient shown in Maldonado et al., 2020 (fractions 10–11, Figure 1—figure supplement 2H). This sample is roughly equivalent to fractions 11–13 from Figure 1—figure supplement 4 here. Thus, the mass spectrometry results of this mixed sample include complex I subunits in addition to CIII2 and CIV subunits. See below for full dataset availability and accession codes. Samples were digested with the S-Trap micro (PROTIFI) digestion. Digestion followed the S-trap protocol. The proteins were reduced and alkylated, the buffer concentrations were adjusted to a final concentration of 5% SDS, 50 mM TEAB, 12% phosphoric acid was added at a 1: 10 (v: v) ratio with a final concentration of 1. 2% and S-trap buffer (100 mM TEAB in 90% MEOH) was added at a 1: 7 ratio (v: v) ratio. The protein lysate S-trap buffer mixture was then spun through the S-trap column and washed three times with S-Trap buffer. Finally, 50 mM TEAB and 1 µg of trypsin (1: 25 ratio) was added and the sample was incubated overnight with one addition of 50 mM TEAB and trypsin after two hours. The following day the digested peptides were released from the S-trap solid support by spinning at 1 min for 3000 x g with a series of solutions starting with 50 mM TEAB which is placed on top of the digestion solution then 5% formic acid followed by 50% acetonitrile, 0. 1% formic acid. The solution was then vacuum centrifuged to almost dryness and resuspended in 2% acetonitrile, 0. 1% triflouroacetic acid (TFA) and subjected to Fluorescent Peptide Quantification (Pierce). Digested peptides were analyzed by LC-MS/MS on a Thermo Scientific Q Exactive plus Orbitrap Mass spectrometer in conjunction Proxeon Easy-nLC II HPLC (Thermo Scientific) and Proxeon nanospray source. The digested peptides were loaded on a 100 micron x 25 mm Dr. Masic reverse phase trap where they were desalted online before being separated using a 75 micron x 150 mm Magic C18 200 Å 3U reverse phase column. Peptides were eluted using a 70 min gradient with a flow rate of 300 nL/min. An MS survey scan was obtained for the m/z range 300–1600, MS/MS spectra were acquired using a top 15 method, where the top 15 ions in the MS spectra were subjected to HCD (High Energy Collisional Dissociation). An isolation mass window of 2. 0 m/z was used for the precursor ion selection, and normalized collision energy of 27% was used for fragmentation. A twenty second duration was used for the dynamic exclusion. Tandem mass spectra were extracted and charge state deconvoluted by Proteome Discoverer (Thermo Scientific). All MS/MS samples were analyzed using X! Tandem (The GPM, thegpm. org; version X! Tandem Alanine (2017. 2. 1. 4) ). X! Tandem was set up to search the Uniprot Vigna radiata database (October 2019,35065 entries) the cRAP database of common laboratory contaminants (http: //www. thegpm. org/crap; 117 entries) plus an equal number of reverse protein sequences assuming the digestion enzyme trypsin. X! Tandem was searched with a fragment ion mass tolerance of 20 PPM and a parent ion tolerance of 20 PPM. Carbamidomethyl of cysteine and selenocysteine was specified in X! Tandem as a fixed modification. Glu->pyro Glu of the n-terminus, ammonia-loss of the n-terminus, gln->pyro Glu of the n-terminus, deamidated of asparagine and glutamine, oxidation of methionine and tryptophan and dioxidation of methionine and tryptophan were specified in X! Tandem as variable modifications. Scaffold (version Scaffold_4. 8. 4, Proteome Software Inc, Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 98. 0% probability by the Scaffold Local FDR algorithm. X! Tandem identifications required score of at least 2. Protein identifications were accepted if they could be established at greater than 6. 0% probability to achieve an FDR less than 5. 0% and contained at least two identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii et al., 2003). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters. The sample (6 mg/ml protein in 20 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0. 1% digitonin, pH 7. 8) was applied onto glow-discharged holey carbon grids (Quantifoil, 1. 2/1. 3 300 mesh) followed by a 60 s incubation and blotting for 9 s at 15°C with 100% humidity and flash-freezing in liquid ethane using a FEI Vitrobot Mach III. CryoEM data acquisition was performed on a 300 kV Titan Krios electron microscope equipped with an energy filter and a K3 detector at the UCSF W. M. Keck Foundation Advanced Microscopy Laboratory, accessed through the Bay Area CryoEM Consortium. Automated data collection was performed with the SerialEM package (Schorb et al., 2019). Micrographs were recorded in super-resolution mode at a nominal magnification of 60,010 X, resulting in a pixel size of 0. 8332 Å2. Defocus values varied from 1. 5 to 3. 0 µm. The dose rate was 20 electrons per pixel per second. Exposures of 3 s were dose-fractionated into 118 frames, leading to a dose of 0. 72 electrons per Å2 per frame and a total accumulated dose of 51 electrons per Å2. A total of 9816 micrographs were collected. Software used in the project was installed and configured by SBGrid (Morin et al., 2013). All processing steps were done using cryoSPARC and RELION 3. 0 (Zivanov et al., 2018; Punjani et al., 2017) unless otherwise stated. Motioncor2 (Zheng et al., 2017) was used for whole-image drift correction of each micrograph. Contrast transfer function (CTF) parameters of the corrected micrographs were estimated using Ctffind4 (Rohou and Grigorieff, 2015). After motion correction and CTF correction, a set of 8541 micrographs was selected for further processing. Automated particle picking using crYOLO (Wagner et al., 2019; Wagner and Raunser, 2020) resulted in ~1. 5 million particles. The particles were extracted using 4002 pixel box binned two-fold and sorted by reference-free 2D classification in Relion using (--max_sig 5), followed by re-extraction at 5122 pixel box. Reference-free 2D classification in Relion resulted in the identification of 502,224 particles that were then imported into cryoSPARC for further reference-free 2D classification. A set of 121,702 particles were identified by 2D classification in cryoSPARC to contain CIII2 alone or SC III2+IV (Figure 1—figure supplement 1). These particles were subjected to ab initio model generation with four targets to remove contaminant particles resulting in a set of 99,937 particles across three classes. Each individual class was subjected to an additional round of ab initio model generation with three targets. This separated CIII2 alone particles from the SC III2+IV class and allowed the recovery of CIII2 alone particles from the poor particle class. CIII2 alone particles from across the ab initio model generation jobs were pooled, defining a final class of 48,111 particles. The multiple rounds of ab initio model generation resulted in only one good class of 28,020 SC III2+IV particles. Poses for these two particle sets (CIII2 alone and SC III2+IV) were refined using cryoSPARC’s Homogeneous Refinement (New) algorithm including Defocus Refinement and Global CTF refinement. This resulted in reconstructions at 3. 2 Å and 3. 8 Å resolution for CIII2 alone and SC III2+IV respectively, according to the gold standard FSC criteria (Figure 1—figure supplement 1; Scheres and Chen, 2012). In parallel, a set of 69,876 particles were identified by further 2D and 3D classification in Relion (Figure 1—figure supplement 2). These particles, which contained a mixture of SC III2+IV and CIII2 alone particles, were aligned using a SC III2+IV model and mask. They were then subjected to five rounds of masked classification using a CIV model and mask aligned with the position of CIV in the SC. Three parallel masked classifications (all using T = 8) varied in the degree of rotational searches, with either no searches, 0. 1° sampling interval over +/- 0. 2° search range and 3. 7° sampling interval over +/- 7. 5° search range. The masked 3D classification without searches was repeated successively for three rounds, inputting the best particles from the previous round into the subsequent round. The best CIV class from each 3D classification were selected and combined while removing overlaps (any particle within 200 pixels of another was considered as an overlap and discarded). This 3D classification strategy resulted in a set of 38,410 particles. The coordinates of these particles were used to extract two sets of re-centered SC particles, one centered on CIII2 and one centered on CIV. These two sets of particles were independently 3D-refined, CTF-refined and Bayesian-polished using a model and mask centered around CIII2 or CIV respectively. The CIV-centered shiny particles were subjected to a final round of 3D classification, defining a final set of 29,348 CIV particles. Although this final round of 3D classification did not improve the nominal resolution of the map, it increased map quality at the periphery of the complex. These final CIII2 and CIV classes resulted in reconstructions at 3. 7 Å and 3. 8 Å resolution for CIII2 and IV from the SC respectively, according to the gold standard FSC criteria (Figure 1—figure supplement 1; Scheres and Chen, 2012). These two maps were aligned with the full SC map and combined to make a composite map using Phenix. 3D variability analysis (3DVA) was performed on CIII2 alone in cryoSPARC using their built-in algorithm (Punjani and Fleet, 2020). Two separate instances of 3DVA were performed, each solving for the four largest principal components. The first instance used a mask around the entire CIII2 and data was low-pass filtered to 6 Å resolution to remove the influence of high-resolution noise from the amphipol detergent belt. The second instance used a mask focused around the IMS domain of CIII2 and was low-pass filtered to 5 Å resolution. All other parameters were kept as default. Starting template models for V. radiata CIII2 was ovine CIII2 (PDB: 6Q9E). Starting template models for V. radiata CIV were from S. cerevisiae (PDB: 6HU9) and bovine (PDB: 5B1A) CIV. Additionally, starting models for the V. radiata subunits were generated using the Phyre2 web portal (Kelley et al., 2015). Real-space refinement of the model was done in PHENIX (Liebschner et al., 2019; Goddard et al., 2018; Pettersen et al., 2004) and group atomic displacement parameters (ADPs) were refined in reciprocal space. The single cycle of group ADP refinement was followed by three cycles of global minimization, followed by an additional cycle of group ADP refinement and finally three cycles of global minimization (Letts et al., 2019). The refined CIII2 and CIV models were docked into the SC III2+CIV map without subsequent refinement. Molecular graphics and analyses were performed with UCSF Chimera (Pettersen et al., 2004) and ChimeraX (Goddard et al., 2018) developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311 and R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases. PyMOL Molecular Graphics System, Version 2. 0 Schrödinger, LLC was also used.

Most living things including plants and animals use respiration to release energy from food. Respiration requires the activity of five large protein complexes typically called complex I, II, III, IV and V. Sometimes these complexes combine to form supercomplexes. The complexes are similar across plants, animals and other living things, but there are also many differences. Detailed structures of the respiratory complexes have been determined for many species of animals, fungi and bacteria, highlighting similarities and differences between organisms, and providing clues as to how respiration works. Yet, there is still a lot to learn about these complexes in plants. To bridge this gap, Maldonado et al. used a technique called cryo electron microscopy to study the structure of complexes III and IV and the supercomplex they form in the mung bean. This is the first study of the detailed structure of these two complexes in plants. The results showed many similarities to other species, as well as several features that are specific to plants. The way the two complexes interact to form a supercomplex is different than in other species, as are several other, smaller, structural features. Further examination of complex III revealed that it is flexible and that movements are coordinated across the length of the complex. Maldonado et al. speculate that this may allow it to coordinate its role in respiration with its other cellular roles. Understanding how plant respiratory complexes work could lead to improvements in crop yields or, since respiration is required for survival, result in the development of herbicides that block respiration in plants more effectively and specifically. Further researching the structure of the plant respiratory complexes and supercomplexes could also shed light on how plants adapt to different environments, including how they change to survive global warming.

lay_elife

In recent years, the East African region has seen an increase in arboviral diseases transmitted by blood-feeding arthropods. Effective surveillance to monitor and reduce incidence of these infections requires the use of appropriate vector sampling tools. Here, trapped skin volatiles on fur from sheep, a known preferred host of mosquito vectors of Rift Valley fever virus (RVFV), were used with a standard CDC light trap to improve catches of mosquito vectors. We tested the standard CDC light trap alone (L), and baited with (a) CO2 (LC), (b) animal volatiles (LF), and (c) CO2 plus animal volatiles (LCF) in two highly endemic areas for RVF in Kenya (Marigat and Ijara districts) from March–June and September–December 2010. The incidence rate ratios (IRR) that mosquito species chose traps baited with treatments (LCF, LC and LF) instead of the control (L) were estimated. Marigat was dominated by secondary vectors and host-seeking mosquitoes were 3–4 times more likely to enter LC and LCF traps [IRR = 3. 1 and IRR = 3. 8 respectively] than the L only trap. The LCF trap captured a greater number of mosquitoes than the LC trap (IRR = 1. 23) although the difference was not significant. Analogous results were observed at Ijara, where species were dominated by key primary and primary RVFV vectors, with 1. 6-, 6. 5-, and 8. 5-fold increases in trap captures recorded in LF, LC and LCF baited traps respectively, relative to the control. These catches all differed significantly from those trapped in L only. Further, there was a significant increase in trap captures in LCF compared to LC (IRR = 1. 63). Mosquito species composition and trap counts differed between the RVF sites. However, within each site, catches differed in abundance only and no species preferences were noted in the different baited-traps. Identifying the attractive components present in these natural odors should lead to development of an effective odor-bait trapping system for population density-monitoring and result in improved RVF surveillance especially during the inter-epidemic period. Rift Valley fever virus (RVFV) is transmitted primarily by mosquitoes and there are periodic outbreaks of this disease in humans and domestic animals in Africa and the Arabian Peninsula [1], [2]. Key mosquito vectors involved in the enzootic transmission include flood water Aedes spp. as the primary vectors, and other epizootic culicine vectors such as Mansonia, Culex and Anopheles spp. as the secondary vectors [3]. In Kenya, the number of suspected vectors continues to rise with increasing isolation of the virus from additional species [3]. Since human vaccines and therapeutic treatments are not available for RVFV, surveillance is essential for early warning to ensure that devastating outbreaks and/or sporadic infections are prevented. Efficient surveillance is essential for early detection of increased vector abundance and detection of pathogens in trapped mosquitoes. This requires a systematic collection of mosquito samples and routine testing of mosquito pools for arboviruses in order to assess the status of transmission and to allow for informed decision-making [4]. However, fluctuations in mosquito abundance and arboviral infections pose a challenge for mosquito based surveillance programs, since different surveillance strategies are required to detect different arboviral vectors and infection rates and potential and transmission rates. This is particularly problematic in the case of early detection and during the inter-epidemic periods (IEP), when transmission foci are sporadic and mosquito infection rates are low. Therefore, detection of mosquito infections when there is low transmission requires the collection of large samples of mosquitoes. For West Nile virus, 700 mosquitoes are needed for a modest detection probability of 0. 5 when the natural infection rate is 0. 1% for mosquito surveillance programs in the early season or in areas of low transmission [5]. Trapping large numbers of mosquitoes for detection of RVFV can be accomplished by improving the efficiency of existing surveillance traps, such as the standard CO2-baited CDC light trap. One way to improve trapping efficiency is by exploiting the host-seeking behavior of female mosquito vectors. Adult female mosquitoes use host-emitted olfactory cues to locate hosts to obtain blood meals [6]. Domestic animals including cattle, sheep, camels and goats serve as hosts for these vectors of RVFV. However, sheep appear to be more susceptible to RVF infections than cattle or camels [7], [8]. Whether or not animal susceptibility is associated with increased attraction is unclear; however, it is clear that sheep are preferred hosts of these vectors. We hypothesized that body odors from sheep are important cues used by RVF mosquitoes. The present study was carried out to investigate the response of mosquito vectors of RVFV to the CO2-baited CDC light trap combined with sheep skin odors, in a field setting. All experiments were conducted at two ecologically distinct sites: Ijara and Marigat districts, which are highly endemic areas for epidemic Rift Valley fever (RVF) in Kenya [3], and are currently under active surveillance for arbovirus activities. Ijara District is located in the North Eastern Province of Kenya and is characterized by a semi-arid to arid climate. Mosquitoes were sampled at Kotile (1. 97°S, 40. 19°E) (near Masalani) and Sangailu (1. 31°S, 40. 71°E), which is around 60 m above sea level. The average annual rainfall is 540 mm with bimodal peaks recorded from March–June and September–December each year. However, the interannual rainfall variability is very high and reaches abnormal levels leading to floods during El Niño years. Minimum temperatures are always above 20°C, and maximum temperatures reach 30°C to 34°C with a high seasonal and interannual variability. The predominant vegetation is Acacia-Commiphora deciduous bushland and thicket (Savannah, Shrubland, open to very open shrubs), which is much degraded due to overgrazing around the settlement areas. The road leading from Masalani to Sangailu demarcates the boundary between these semi-arid landscapes and the more moist Tana River delta and Boni Forest towards the coast. Boni Forest is an indigenous open canopy forest that forms part of the Northern Zanzibar-Inhamdare Coastal Forest Mosaic. The second study site is Marigat district, located in the Kenyan Rift Valley 250 km northwest of Nairobi where traps were set in surrounding villages/communities of N' gambo (0. 50°N, 36. 06°E) and Salabani (0. 55°N, 36. 06°E). The study site covers the basin between Lake Baringo and Lake Bogoria with the town of Marigat as an economic center and lies about 1000 m above sea level. The climate is hot and dry with high rainfall variability, both annually and inter-annually. The average annual rainfall is 650 mm with weak bimodal peaks recorded from March–May and June–August. Temperatures vary from 30 to 35°C, but can rise to 37°C in some months. The low lying arid part of the Baringo basin consists of northern Acacia-Commiphora bushlands and thickets but it has experienced severe land degradation caused by uncontrolled grazing and deforestation. Prosopis juliflora (Sw.) DC, locally called mathenge, was introduced to Baringo in the early 1980s for fuelwood production and reforestation as a mitigation measure to stop desertification. The plant was introduced at two sites but now covers large areas, i. e. N' gambo village, one of the vector sampling sites. Three indigenous human communities live in this area, the Ilchamus, Pokot and Tugen. They earn their living through pastoralism and agro-pastoralism keeping large numbers of cattle and small livestock such as sheep and goats. The Perkerra irrigation scheme (growing of vegetables, maize seed production), fishing and tourism provide additional income to these communities. Sheep are the most susceptible among livestock hosts afflicted by RVFV [1], [8], [11], and the living animal has been exploited as a lure in trapping mosquito vectors [12]. Its role in the enzootic maintenance of the RVFV [13] is the reason why it is the preferred domestic animal currently being used as sentinels in an ongoing surveillance program for RVF at the two study sites. The study was conducted with the approval of the national ethics review committee based at the Kenya Medical Research Institute (KEMRI) and is renewed on an annual basis after a scientific audit. The Animal use component was also given approval by the KEMRI Animal Use and Care committee (KEMRI-AUCC). KEMRI-AUCC complies with the national guidelines for care and use of laboratory animals in Kenya developed by the Kenya Veterinary Association and the Kenya lab animal technicians association 1989. The KEMRI-AUCC which approved the study protocol has an assurance identification number A5879-01 from the Office of Laboratory Animal Welfare (OLAW) under the Kenyan department of health and human services. For purposes of livestock use, funds from the project were used to purchase animals to monitor RVFV seroprevalence and used for all experimental activities described in this study. These animals were owned and maintained for the study by the project. The project bought 492 animals comprising 5 sentinel herds; two in Marigat, three in Ijara district (one in Kotile and 2 in Sangailu). The animals were left with the owners as part of their flocks but they were not allowed to sell or slaughter them because the project was monitoring the animals. The animals were reverted back to the owner at the end of the project activity. Any newborns born out of the tagged animals belonged to the farmers. We worked in collaboration with the department of veterinary services and veterinary doctors mandated by the government to do livestock sampling and research. The above terms were stipulated well in an agreement between the farmers and the International Centre of Insect Physiology and Ecology (icipe), the hosting institution for the AVID Project Consortium. Experiments were conducted in October and December 2010 during the rains to ascertain the presence of mosquitoes. This comprised 10 replicates of 4 treatments per district. The treatment-trap combinations consisted of the standard CDC light trap alone (L) and baited with (a) animal volatiles (LF), (b) CO2 (LC), or (c) CO2 and animal skin volatiles (LCF) using fur obtained from living sheep. The animal volatiles consisted of fresh sheep (Ovis aries Linnæus) hair samples shaved from the belly and back areas of the animals (avoiding the head and anal regions) daily. The animal fur was wrapped in five layers of aluminum foil, kept in a cold box (10°C) and immediately transported to the trapping site (located between 2 to 5 km). Once at the trapping site, approximately 19 g of the animal fur were placed in each canister (cylindrical in shape with a diameter of 9. 5 cm and height 22. 5 cm) designed from Brass mesh wire (mesh size, 0. 15 mm, McNichols Co, Tampa FLA). With an inter-trap distance of 40±2 m, the traps were hung in trees 1. 5±0. 2 m off the ground and activated within 30 min of sunset (1800–1830) and trap contents collected within 30 min after sunrise (0600–0630 hours). Treatments and control were assigned to a predetermined similar area following a Latin square design with days as replicates. Traps were rotated on every trapping day to minimize variability due to trap placement. Dry ice (1 kg) was used as the CO2 source, which was delivered in Igloo thermos containers (∼2 L) (J. W. Hock, Gainesville, FL) with a 13-mm hole in the bottom center. Treatments with the canisters containing fur (which released skin volatiles) were hung at the base of the standard CDC trap (battery-powered model 512, John W Hock Co., Gainesville, FL) and when in the presence of CO2 directly in the air flow. All bait canisters were boiled in l0% bleach solution after each nightly trapping to eliminate any residual odor. Mosquitoes caught daily from each of the treatments were anesthetized using triethylamine and identified morphologically to species using taxonomic keys 14–16. When large numbers of mosquitoes were trapped, they were anesthetized, sorted from other insects and immediately stored in 15 or 50 mL centrifuge tubes, and transported in a liquid nitrogen shipper to the laboratory where they were later identified and the total number by species for each treatment-trap were recorded. Trap count data were analyzed per district and were also subdivided into four categories (i. e., key primary vectors, primary vectors, secondary vectors and non-vectors) based on the relative importance and involvement of member species in RVFV transmission 2,3. The four main categories of trapped mosquitoes recorded in the different treatments were further categorized as follows: flood water Aedes species (key primary vectors; Aedes mcintoshi and Aedes ochraceus); primary vectors (Aedes sudanensis/Aedes tricholabis); secondary vectors (Mansonia and Culex spp.) and non-vectors, which do not fall into any of these categories (Table 1). Analysis of key primary and primary RVFV vectors was limited to Ijara district where they were mainly encountered and secondary vectors limited to Marigat district where they occurred in substantial numbers (Table 1). Daily count of mosquitoes recorded in the various trap treatments were analyzed using a generalized linear model with negative binomial error structure and log link using R 2. 11. 0 software [17]. Using the treatment L only (control) as the reference category, the incidence rate ratios (IRR) that mosquito species chose other treatments (LCF, LC and LF), instead of the control, were estimated. The IRR for the control is 1 (unity) and values above this indicates better performance and values below under performance of the treatments relative to the control. The distribution of RVFV mosquitoes captured per treatment-trap combination for the two districts are contained in Table 1. Mosquito species composition and trap captures differed markedly between the two districts which might suggest varied habitat preferences for each mosquito species. Differences in abundance were observed between the treatments with no clear pattern of preference of any species for a particular trap treatment. Some species were not caught in all replicates, and it is unclear if such variability was due to overall low population densities or the mosquitoes failing to enter (or to respond to) the traps. In general, traps baited with CO2 (LCF and LC) captured more mosquitoes than those without (LF and L) (Table 1 and Figures 1 and 2). There was a significant effect of treatments compared to the unbaited CDC trap on overall mosquito captures from Marigat (χ2 = 20. 68, df = 3, p<0. 001) and from Ijara (χ2 = 37. 51, df = 3, p<0. 001). Trap catches from Marigat indicate that, compared to L only, LC and LCF traps caught 3–4 times more host-seeking mosquitoes [IRR = 3. 1 for LC and IRR = 3. 8 for LCF]. LCF traps recorded higher mosquito catches compared to LC traps (IRR = 1. 23) although the difference was not statistically significant (Figure 1). Similarly, the LF trap caught slightly more mosquitoes (IRR = 1. 03) than L only but was not significantly different (Figure 1). Similar findings were observed at Ijara where there was a significant treatment effect on mosquito catches (χ2 = 37. 51, df = 3, p<0. 001). Carbon dioxide (LC), CO2+fur (LCF) significantly increased trap captures by 6. 5 and 8. 5 times, respectively, compared to the control. The LF caught more than the control, L, but this was not statistically significant (Figure 1). Trap catches at Ijara were dominated by flood water aedine mosquitoes categorized as key and primary RVFV vectors; these species were sparse or absent at Marigat. There was a highly significant effect of treatments on key primary RVFV vectors (χ2 = 199. 99, df = 3, p<0. 001). For this group, relative to the control, there was a 4. 0- and 6. 5-fold significant increase in captures recorded in LC and LCF traps, respectively (Figure 2). Additionally, LCF capture rates were significantly higher than LC capture rates (IRR = 1. 63). A significant effect of treatments on RVFV primary vectors was also evident (χ2 = 74. 24, df = 3, p<0. 001). Compared to the control, the treatments LF, LC and LCF caught 2. 9,31 and 42 times as many primary vectors. Interestingly, for this group, there was a 34% significant increase in captures for traps baited with LCF compared to LC (IRR = 1. 34) (Figure 2). Marigat yielded very low catches for key primary vectors and there was a total absence of primary vectors. Therefore results are only presented for secondary vectors. For secondary vectors at Marigat, there was a highly significant effect of treatments on the mosquito catches (χ2 = 22. 94, df = 3, p<0. 001). Relative to the control, there were 3 to 4-fold increases in captures for LC and LCF traps, respectively (Figure 2). Comparable captures were recorded for LF and L traps with only a slight increase recorded in LF baited traps relative to the control (IRR = 1. 04). Captures rates, although not significant were higher for LCF traps than LC traps (IRR = 1. 24) (Figure 2). Mosquito collections within this category at Ijara were low and dominated by Cx. pipiens s. l. with an observed increase in captures in the other treatments compared to L. The non-vectors category included species of the genera Ficalbia, Coquilettidia, Anopheles and Aedes (Stegomyia). Members of these genera occurred in low numbers in both districts, especially at Ijara (Table 1). However, data for Marigat suggest a bias in trap captures in LCF and LC, compared to L although there were no significant differences in the captures between these treatments, while similar trap captures were observed for the LF and L-baited traps. Non-mosquito species notably beetles and moths were trapped in addition to mosquitoes but were not included in our data. The results demonstrate that more mosquitoes were caught in traps that contained a release of the combination of sheep odors+CO2 and were in most cases the most attractive bait compared to the conventional CO2-baited light trap. This confirms that odors emanating from sheep fur play a role in host-location by these mosquitoes. The attractive effect was highly evident in captures of flood water aedines comprising key and primary RVFV vectors as well as secondary vectors. The effectiveness of sheep is supported by a study on blood meal patterns during a RVF outbreak where widespread feeding on sheep was observed [18]. Moreover, most mosquitoes belonging to the Culex, Mansonia and Aedes genera have been reported to feed opportunistically and readily on mammals [19]–[21]. The entire animal body emanations comprising breath and skin volatiles influence the outcome of mosquito host-seeking process [22]. Research has indicated that animal skin emanations have a kairomonal (attractive) effect on mosquitoes while breath volatiles have an allomonal or repellent effect [23]. Skin body odor may be the primary factor for mosquito attraction and discrimination when mosquitoes are in close proximity of a host. It is therefore not surprising that addition of skin volatiles captured in sheep fur enhanced captures of mosquitoes attracted to sheep hosts when combined with the conventional CO2-baited light trap. The effect of sheep skin odors emanating from fur was not evaluated alone but in combination with CO2 and/or light which are known attractants for mosquitoes and other biting flies. Although animal odors enhanced trap captures when added to either CO2-baited light trap or light trap only, the captures were greater in the CO2-based blend than in the combination without CO2. However, the crude animal skin odor in traps is imperfect because of possible loss of volatile attractive components over time compared to the dynamic production from live animals. Therefore it would be beneficial to identify the attractive compounds and develop a synthetic blend. In some replicates there was a decrease in trap catches when host odor was added to light or to CO2. This could be attributed to variation in attractiveness of the batches of animal fur used in the daily trapping experiment as odors used were not from the same animal; low occurrence of targeted mosquitoes, as observed at the districts for certain vector categories; a difference in preferred host other than sheep e. g. Cx. poicilipes between LCF and LC baited traps (Table 1); and volatiles from fur are a static system and most volatile compounds evaporate first and therefore the odor profile changes. The effect of host odors did not markedly influence trap catches of the non-vector category. The low abundance of mosquitoes was insufficient to observe a significant preference in trap catches for the different treatments used; even though there was an increase in trap catches for those baited with host odors compared to light only. The effect of CO2 on trap catches was not evaluated independently; however, its effect was evidenced in the difference in trap catches between LC and L baited traps. The data support its role in enhancing trap captures [24], especially for RVFV vectors. Our experimental setup excluded landing response as a measurement, instead focussing solely on trap catch. The goal was to evaluate animal fur containing skin emanations that provided attractive stimuli. However, the large response of these mosquitoes to CO2, suggests that it can serve as a good positive control for evaluating candidate synthetic attractants of skin origin for this group of arbovirus vectors of medical and veterinary importance. Our preliminary trials (data not shown) and earlier studies highlighted the importance of these attractants in flight activation of mosquitoes towards host odors [25], [26]. This justifies the inclusion of these well-known long-range attractants in trap design. Our data suggest that host skin odors other than CO2 are important in enhancing mosquito trap captures in concurrence with studies reporting enhanced effect of mosquito attraction to animal skin volatiles in the presence of CO2 or light [22], [27], [28]. It is well-known that many nocturnally-active hematophagous insects are attracted to light [29], [30]. In conformity with earlier findings [31], our results show that light as a visual cue is enhanced by sheep skin odors and CO2. Besides being non-specific, previous studies have argued that CO2 activates mosquitoes to initiate host-finding, but may not necessarily attract it and at close range, can actually act as a deterrent [26] and be of limited use in host discrimination [32]. Although this was not the subject of our study, CO2 increased trap captures in the presence of host skin odors, in agreement with previous research [24], [25], [33]. The observed trap captures recorded in the LCF traps were generally higher compared to those caught in the LC traps. Nonetheless, among the mosquito species trapped, differences in capture rate were not observed between the LCF and LC traps. Therefore, CO2-baited light traps may be adequate for monitoring and surveillance of these species. However, for effective arbovirus disease surveillance, an improved sampling method is vital especially during the inter-epidemic period where transmission foci are sporadic and infected vectors are rare. Emphasis needs to be placed on increasing the collections with an additional advantage of depicting the dynamics of populations. Beyond the already described finding that animal odor inclusion increases trap catch with CO2 present, there were some cases where it suppressed trap catch. In some cases, lower catches of the LCF trap were noted on days with light showers; therefore, precipitation may have interfered and reduced mosquito attraction to skin odor baits as observed before by Olanga et al. [34]. However there was no record of variation in weather patterns during the study period. Another possibility is the variation from fur samples used in this study. Samples were obtained from various animals without prior assessments of their degree of attractiveness. Animals in a herd are known to vary greatly in their attractiveness to mosquitoes [35], [36]. Reduced attraction due to loss of important volatile compounds during the fur extraction process remains plausible. A higher number of Cx. poicilipes were collected in CO2-baited light traps than in similar traps baited only with host skin odor, although the difference in trap captures was not significant. This suggests that sheep are not preferred hosts for this species. However, the effect of CO2 in the presence of host-related odors may be variable and a strong attraction response may be observed with often different responses between species [37], [38]. This observation might emphasize the importance of trap placement in the sampling process, though it is not certain if this species would be attracted to the host that emanates the greatest amount of CO2 in nature. Differential catches of Cx. pipiens s. l. and An. gambiae s. l. to odors from sheep fur were recorded at the different sites (Table 1). Among the species complexes captured, there are known marked differences in olfactory responses between members of the complexes [39], [40]. Culex pipiens preferentially feed on birds [41] although they can adapt and readily feed on mammals in proportions possibly based on host abundance [42]. The observed differences in trap responses in the highest trap treatments (i. e., LC and LCF) at both districts may indicate different spatial feeding preferences in geographically separate populations. Related response patterns of discrete populations of mosquito species to host odors has been reported [42], [43], as such, it may be worthwhile to include a preference test involving odors from other livestock hosts in field bioassays. Only volatiles from skin emanations captured in the fur were tested in this study. Studies on other volatile sources involved in host attraction to hematophagous flies have been reported from feces [44] and urine [45], [46]. In this regard, other sources of attractive odors might contribute to the attraction of mosquitoes and combination using these odors may be worth investigating. Laboratory bioassays have commonly been used to evaluate the effect of semiochemicals on mosquito behavior whilst minimizing other environmental variables. However, such an approach is inadequate for predicting effects on natural populations and on ecosystem-level features [47]. Alternatively, insect behaviors have been assessed in the field by baiting traps with extracts of animal volatiles [42], [48]. Use of whole animals provides another approach but it becomes difficult to delineate individual contributions of attractants from breath or skin emanations or other exogenous compounds to the overall trap catches. Our design followed a field-based approach to evaluate the role of skin emanations on mosquito trap catches. The design can account and provide for an understanding of heterogeneities which dramatically influence dynamics of natural systems. This is similar to the trapping design employed by Njiru et al. [49] and Jawara et al. [50] to investigate mosquito captures in conventional traps baited with human foot odors trapped on nylon stockings. Although the contribution of geographical variability to the total variance was not estimated, possible experimental confounders such as time, location and environmental influence are unlikely to affect the overall observed results as the present experiments were performed at a variety of sites with different animals of the same species and treatment traps treated alike. As such, it is likely that many of the mosquitoes approaching the trap had the opportunity to sample more than one of the treatment-traps, and may have made a choice between them. Albeit the above mentioned challenges, the use of crude volatiles in the field approach presented in this paper can contribute to the evaluation of the effect of host volatiles in the standard CDC light trap. In conclusion, the addition of sheep skin odor to the CO2-baited light trap improved trap catches of RVFV vectors in line with similar findings reporting enhanced effect of animal skin odors and other cues such as CO2 [27], [28], [44]. Our results indicate host skin olfactory cues are important signals in mediating mosquito host location. The finding is also in accordance with the consensus that additional compounds other than CO2 from animal skin may be exploited by mosquitoes in host location [51]–[53]. Sheep skin odor contributes to the attraction of host-seeking RVFV mosquito vectors. Identification of chemicals emanated by sheep might provide the basis for the development of improved devices to sample these vectors. However, refinements into an effective monitoring tool requires identifying and understanding the specific behavioral effects of the attractive components present in these skin odors which is currently underway.

The East African region is a major epizootic center for endemic and emerging mosquito borne-arboviruses such as Rift Valley fever virus (RVFV), as evidenced by the increasing frequency and magnitude of this disease. The absence of vaccines or prophylactic drugs for most of these diseases emphasizes the need for accurate sampling of mosquito vector populations and testing for arboviruses. Accurate surveillance is crucial for early warning of potential or assessing mitigation of existing outbreaks. However, it is a challenge to sample mosquitoes in adequate numbers during the inter-epidemic periods (IEP) because this period is characterized by low mosquito population densities, sporadic transmission foci and low mosquito infection rates. Therefore more efficient tools are needed to increase capture rates so maximized virus detection probability in the mosquitoes can be achieved for assessing risk and outbreak predictions. This can be accomplished by exploiting the host-seeking behavior of adult female mosquitoes and the olfactory cues used to locate a potential host. Here, odors emanating from fur of sheep, a susceptible host for RVFV, is shown to improve trap capture rates of mosquito vectors of RVF in a standard surveillance trap. These data provide for future investigations to identify attractive components present in these natural odors, so that they can be incorporated into existing traps to serve as a population density-monitoring tool for improved arbovirus disease surveillance during IEP.

lay_plos

Two-component systems (TCSs) are important for the adaptation and survival of bacteria and fungi under stress conditions. A TCS is often composed of a membrane-bound sensor histidine kinase (SK) and a response regulator (RR), which are relayed through sequential phosphorylation steps. However, the mechanism for how an SK is switched on in response to environmental stimuli remains obscure. Here, we report the crystal structure of a complete cytoplasmic portion of an SK, VicK from Streptococcus mutans. The overall structure of VicK is a long-rod dimer that anchors four connected domains: HAMP, Per-ARNT-SIM (PAS), DHp, and catalytic and ATP binding domain (CA). The HAMP, a signal transducer, and the PAS domain, major sensor, adopt canonical folds with dyad symmetry. In contrast, the dimer of the DHp and CA domains is asymmetric because of different helical bends in the DHp domain and spatial positions of the CA domains. Moreover, a conserved proline, which is adjacent to the phosphoryl acceptor histidine, contributes to helical bending, which is essential for the autokinase and phosphatase activities. Together, the elegant architecture of VicK with a signal transducer and sensor domain suggests a model where DHp helical bending and a CA swing movement are likely coordinated for autokinase activation. Protein phosphorylation is an essential signal carrier. Bacteria respond to transient living environments through transmembrane-integrated sensor histidine kinases (SKs), which act in concert with their intracellular cognate response regulators (RRs) to elicit necessary adaptive responses that are critical for their survival and virulence. The SKs and RRs have evolved into a two-component signal transduction system (TCS), whereby stimulation of the SK autophosphorylates at a conserved histidine residue to initiate a signaling cascade [1]. The phosphoryl group is transferred from the SKs to their cognate RRs, some of which lead to quickly reprogram bacteria by altering the transcriptional level of specific downstream target genes [2]. Because of the wide prevalence in bacteria and fungi, TCSs have been considered attractive targets for the development of potential therapeutics to control bacterial infections [3], [4]. Sensor domains are key modulators for SKs [5]–[7]. PAS domains (acronym for Per, ARNT, and SIM from Drosophila) are sensors in a majority of SKs, which respond to alterations in the redox potential, oxygen content, light, and small molecules in their environments [8], [9]. Because of their broad involvement in biological processes, the structure and function of the PAS domains in interactions with a variety of ligands have been extensively studied [10], [11]. The oligomeric dynamics of PAS domains in cooperation with local conformational changes can affect the stability of the entire SK, which is thought to be part of the mechanism of signal sensing and transduction [10], [12]. The enzymatic activities of SKs are modulated by HAMP domains, which are commonly found in histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis, and phosphatase proteins [6]. Structural dynamics of the HAMP domain are believed to mediate transmembrane signal transductions [13]. The NMR structure of a HAMP domain from the putative transmembrane receptor Af1503 in Archaeoglobus fulgidus revealed an unusual knobs-to-knobs interhelical structure, which suggests a coordinated helical rotation model for the HAMP domain in signal transmission [14]. This model was further supported by a series of experimental structures of the HAMP mutants and detailed bioinformatics analyses [15], [16]. Several other laboratories reached a consensus conclusion using homologous HAMP domains from different TCSs that the dynamic properties of the HAMP domains are essential in mediating signal transduction [17]–[22]. The C-terminal catalytic and ATP-binding domain (CA) of the SKs, also called HATPase_c, in addition to the DHp domain (dimerization and histidine phosphorylation domain), phosphorylates a conserved histidine residue in the middle of DHp helices [2], [23]. The active site of an SK is assembled with the CA and DHp domains [23], [24]. The plasticity of the DHp domain and CA positioning is implicated in the on/off switch of the temperature sensor DesK kinase from Bacillus subtilis [25]. VicRK is a well-characterized TCS that is highly conserved and essential for survival and virulence in a wide range of firmicute bacteria, including Streptococci, Bacilli, and Staphylococci [26]. Because of its critical role in cell wall synthesis, VicRK is also referred to as WalRK [27]. The VicRK in S. mutans, which is an important pathogen in caries etiology, regulates acid production and tolerance conducive to dental caries, including proton expulsion (F1F0-ATPase) [28]–[31]. VicK belongs to the type IA family based on sequence conservation of the DHp and CA domains [32]. In addition, VicK has one HAMP domain and one PAS domain. However, the function of the PAS domain is not clear because no ligand has been identified [26]. Recently, a deletion experiment indicated that the HAMP and PAS domains are essential for VicK phosphatase activity [33]. Given the vital role of TCSs in bacterial adaptation and pathogenicity, decades of research have focused on the molecular mechanisms of TCS signaling cascades [34]–[36]. Although the structure and functions of individual domains are well known, the signal transduction mechanisms remain largely unknown. Toward this end, we determined a crystal structure for a streptococcal VicK that harbors HAMP transducer and PAS sensor domains. Our crystal structure of the nearly full-length VicK comprises an elegant construction of multiple domains and reveals novel insights into the molecular mechanisms of the VicK histidine kinase. The S. mutans VicK has one transmembrane domain (TM, aa 9–30) that anchors itself to the cytoplasmic membrane (Figure 1A). Following the TM domain, a HAMP signal transducer domain and PAS sensor domain are directly connected to the CA domain through a DHp domain. As several attempts to express full-length VicK (aa 1–450) resulted in insoluble protein, the entire intracellular region (TVicK, aa 31–450) was successfully purified and crystallized. TVicK had a Km of 44. 5 µM and a Kcat of 0. 413 min−1 (Table S1). Its kinetic parameters were similar to the TM-truncated VicK homologue of S. pneumoniae [33]. Static light scattering revealed a single species of VicK at a molecular weight of 103. 2 kDa, indicating that the holoenzyme exists as a stable dimer in solution (Figure 1B). VicK crystals diffracted slightly better than 3 Å; however, because of strong anisotropy, the final structure was refined up to only 3. 3 Å (Table S2). The overall structure of VicK comprises a dimer in the shape of a long slim rod (Figure 1C). The longest dimension of this molecule is nearly 150 Å (Cα distance). Each monomer contains a series of helices (α1–α11). One asymmetric unit contains two VicK dimers. Remarkably, the total buried surface area is 7590. 8 Å2 upon VicK dimerization. The dimer interface consists of three tight hydrophobic contact patches (Figure 1D, I–III). In addition, the HAMP, PAS, and DHp domains are organized as dimers, which are connected by long, straight coiled-coils of helices α2 and α6 (Figure 1C). The C-terminal ends of the VicK dimer harbor two monomeric CA domains (Figure 1C). The N-terminal end of monomer A (Na, aa 31–37) and both C-terminal tails (Ca or Cb, aa 433–450) are disordered. The HAMP domain (aa 36–86) is located at the uppermost position within the N-terminal region of the VicK structure (Figure 1C). Helices α1 and α2 of each VicK monomer form a parallel four-helical coiled-coil that is connected with loops L1 (Figure S1A). S. mutans HAMP shares approximately 45% identity to other Streptococci; however, it shows little (∼5%) identity to A. fulgidus Af1503, although the critical residues at positions a and d are mostly conserved (Figure 2A). The helical interactions within the HAMP domain can be grouped into three shells (Figure S1B). The outer shell is formed by hydrophilic and polar residues at positions b, e, and g of the coiled-coil in addition to two residues from loop L1 (Figure 2A). The residues at the b and g positions in helix α1 are rich in basic residues with long side chains, whereas the corresponding positions in α2 are rich in polar residues (Figure S1B, gold). The middle shell is formed with hydrophobic residues, including leucine, isoleucine, and valine (Figure S1B, green). These residues form the canonical knobs-into-holes packing of coiled-coils [37]. Central to the VicK HAMP bundle are the hydrophobic residues that are all in van der Waals contacts and display knobs-to-knobs or x-layer packing (Figures 2A and S1B, blue). The HAMP outer shell is distinct with three bound rings visible on the electrostatic surface (Figure S1C). Within this bundled structure, there are three pairs of hydrophobic residues in the knobs-to-knobs packing solely from two α2 helices. Two Leu71 residues compose the inside core of the first ring (Figure 2B) and two Leu78 residues are inside the core of the second ring (Figure 2C). Strikingly, the Phe82 pair forms π-π stacking inside the third ring, which is further stabilized by two Tyr56 residues from two L1 loops (Figure 2D). Indeed, Tyr56 is completely conserved in Streptococci, whereas Phe82 can be replaced by leucine in Af1503 (Figure 2A). In other HAMPs, position 82 can be Ile, Val, or Leu, whereas position 56 is typically Leu, Phe, or Tyr [15]. These residues are also capable of making van der Waals contacts if placed into the VicK HAMP domain (unpublished data). Notably, Gly54 is absolutely conserved in these HAMP homologs (Figure 2A). The PAS domain (aa 87–198) in VicK, which is located downstream of the HAMP domain, adopts a canonical fold (Figure 3A). Both PAS domains can be well aligned (root mean square deviation [rmsd] of 1. 2 Å) except for the large shift of loop L5 (Figure S2A). The five β-strands form a core of β-sheets, which are sandwiched by two α2 helices on one side and helices α3–5 on the other. The loop L3 and the connected helix α5 form a surface layer on the top of the β-sheet with two flexible loops, L4 and L5, on both edges. Three substantial binding pockets (S1–3) with a large cavity and inside tunnel are observed on this surface (Figure 3B). The tunnel is lined by mostly hydrophobic residues, including Leu108, Ile116, Leu127, Ile142, Phe178, and Leu195. The VicK PAS domains form a unique dimer, which is mediated by a leucine-zipper (Figure 3C). This leucine-zipper further forms hydrophobic networks with the canonical PAS domain, which suggests that the leucine-zipper is an integral part of the VicK PAS domains (Figure S2B). The residues Leu89, Leu96, Leu100, and Lys93 are in key positions to make van der Waals contacts (Figure 3C). The substitution of Leu100 with arginine was previously shown to disrupt VicK autokinase activity in S. pneumoniae [38]. It is likely that the L100R mutation destabilizes the dimerization of the VicK PAS domains, which sheds light on the functional importance of PAS dimerization. To look for clues of ligands for VicK PAS domains, we compared the VicK PAS domains with representative structures of all ligand-bound PAS domains according to a recent comprehensive review by Henry and Crosson [11]. Despite their limited sequence identities (6%–10%), all PAS domains can be structurally aligned to VicK with low rmsd of 2–2. 5 Å (Figures S3 and S4). The relatively large shifts were observed in loops L3 to L5 and helix α5 of the PAS domains of NifL, FixL, DcuS, and PhoQ, which form pockets for a variety of ligands to bind. Therefore, the VicK PAS domain may bind some ligands differently from these PAS domains because it has a unique cavity and tunnel properties. The C-terminal end of VicK (aa 199–450) contains a histidine-specific ATPase, which is often divided into one DHp domain and one CA domain with a short linker (aa 270–278) (Figure 1A). The dimeric architecture of these domains looks like a butterfly, wherein the DHp domain (aa 199–269) is a four-helix bundle of helices α6 and α7 with a phosphoryl receptor histidine located in the middle (Figure S5A). Surrounding this helical core are two CA domains with a layer of four helices (α8 to α11) on a layer of β-sheets (β7 to β12), wherein α10 is a short helix (Figure S5B). The two loop regions, L7 (aa 303–308) and L11 (aa 391–395) in the CA domain, called the lid, could not be well defined because of a weak electron density and are labeled with light dashed lines. One linker (aa 270–274), which connects the DHp and CA domains, was also disordered (Figure S5A, the monomer in magenta). Both CA domains (aa 278–450) of VicK adopt a classical histidine kinase fold and are well aligned with an rmsd of 1. 3 Å despite the missing loop L11 (Figure S5B). The CA domain of VicK is similar to HK853 (rmsd of 1. 7 Å) except for structural differences in loops L7 and L8 in addition to the loop L11 region (Figure S5C). We could not clearly define ATP binding because of the weak electron density at the current resolution. Unlike the HAMP domain, the DHp domain forms an anti-parallel four-helical coiled-coil. Interestingly, the two monomers of the DHp domain bear an asymmetric fold (Figure 4A). The two monomers are well aligned with an rmsd of 2. 2 Å for the bottom part of the coiled-coil (aa 219–255), and the relatively large rmsd of the alignment is mostly caused by flexibility of loop L6. However, the upper parts of helices α6 and α7 are remarkably different, wherein helix α6 bends ∼25° at Pro222 toward the central DHp axis and α7 moves ∼11° away in the opposite direction to avoid a possible clash. When aligning the VicK DHp domain with T. maritima HK853, which is a symmetric four-helix coiled-coil, we observed similar bending of monomer A (Figure 4B). The bending is coordinated with shifts of the upper helices, whereas the bottom helices remain stationary, and the upper part of helix α6 of monomer A moves toward the center axis, which possibly drives the other helices away. Helix α6 of the DHp domain has an intrinsic plasticity for bending (Figure 4C). The bending region is absolutely conserved among over 50 VicK homologs in Streptococci, Lactobacilli, Lactococcus, and Enterococci (Figures 4C, the bottom and S6). Consistently, the DHp domain region (aa 211–225) was predicted to have low helical probability (Figure 4C, the top). Therefore, we hypothesized that the low helical propensity of the DHp domain may play a role in His217 phosphorylation. To test this, we mutated Pro222 to glycine and measured its autokinase activity (Table S1). The P222G mutant retained full activity when compared to wild-type (wt) VicK, as did the T221A mutant. We continued to mutate residues in the bending region, including Val212, Val215, Ser213, and Ser216 to alanine (VS2AA), which is statistically favorable in an α-helix [39]. All mutants, including the combined mutants VSP2AAA, VST2AAA, and VSTP2AAAA, bound S. mutans VicR similarly to wt VicK, which suggested that they likely retain the correct conformation (Figure S7). The autokinase activity of the single and combined mutants described above was analyzed using γ32P-ATP (Figure 4D; Table S1). As the Thr221 and Pro222 mutants (T221A, P222A, P222G, and TP2AA) retained nearly full activity compared to wt VicK, the two combined mutations of VSP2AAA and VSTP2AAAA showed significantly reduced activity. Although we cannot rule out other effects from multiple sites of mutation, these data are consistent with the model that the low helical propensity of the DHp domain in addition to Pro222 are important for VicK autokinase activity. As VicK is a multifunctional enzyme, we tested whether the helical bending region of the DHp domain is important for phosphatase activity. Here, we used Phos-tag gel mobility shift assay (PMS) to detect the phosphorylated S. pneumoniae VicR. As a control, over 90% of VicR was phosphorylated by acetyl-phosphate (AcP), which resulted in a mobility retardation shift (Figure 5A, lane 2). Further incubation with wt VicK completely removed the phosphate group from VicR (lane 3, top gel). It is interesting to note that in the absence of 5 mM ATP, little dephosphorylation of VicR was observed (lane 3, middle gel), which suggested that ATP is required for VicK phosphatase activity. We tested the phosphatase activity of the DHp mutants described above. To our surprise, the single proline or threonine mutations (P222A and T221A) abolished VicK phosphatase activity (Figure 5A, lanes 5 and 7, top gel). In contrast, the P222G and VS2AA mutants, similar to wt VicK, dephosphorylated VicR within the time course of this assay (lanes 6 and 9). Consistently, the combined mutants VSP2AAA, VST2AAA, and VSTP2AAAA, which contain Pro222 and Thr221 substitutions, also showed little phosphatase activity (lanes 10–12). It is interesting to note that the VSP2AAA mutant had substantially more phosphatase activity than the single P222A mutant likely because VSP2AAA mutant lost partially kinase activity (Figure 4D). We also analyzed some of these mutants using high performance liquid chromatography (HPLC). Phosphorylated S. pneumoniae VicR eluted at ∼7. 6 min compared with the native VicR at ∼8. 8 min (Figure 5B, run 1). After incubation with VicK in the presence of ATP, phosphorylated VicR completely shifted back to 8. 8 min (Figure 5B, run 2). A clear conversion to unphosphorylated VicR was also observed when incubated with the VS2AA mutant (Figure 5B, run 3). All other VicK mutants, including T221A, P222A, TP2AA, and VST2AAA, failed to dephosphorylate VicR (Figure 5B, runs 4–7). Together, our data suggest that Pro222 and its neighbor, Thr221, are the key residues in the bending region required for the phosphatase activity of VicK. Although the overall conformation of each CA domain in VicK is the same, their positions relative to the DHp domain show dramatic differences, which generates an asymmetrical ATPase dimer (Figure 6A). The CA domain rotates ∼61° and further translates ∼20 Å down along an axis parallel to the DHp domain. When aligning the CA domain with the symmetric dimeric structure of T. maritima HK853, we found that monomer A takes completely different positions (Figure S8, magenta). The large shift of the CA domain of monomer A moves its active site to the in cis phosphoryl acceptor His217. Recently, a crystal structure of the B. subtilis YycG CA domain bound to ATP was solved, which is 47% identical to the CA domain of S. mutans VicK [40]. These two structures of the CA domains aligned well with an rmsd of 1. 47 Å of all backbone atoms, which is where the ATP from the structure of the YycG CA domain was modeled nicely in the active pocket of the VicK CA domain. To our surprise, we found that the γ-phosphate of ATP approached His217 to form two hydrogen bonds with εΝ or δN of His217 (Figure 6B, yellow dashed lines). In addition, Leu265 from monomer B is close to His217 to form van der Waals interactions (Figure 6B, grey dashed lines). Arg220 of monomer A and Arg269 of monomer B are also in close contact with His217. In addition to the ATP binding pocket, the CA domain positions itself toward the DHp domain to form a large interface (Figure S9). Many residues are involved in the direct interaction between the DHp and CA domains (Figure 6C). Arg294, Asp326, Gln330, Asn334/337, and Arg385 of the CA domain form hydrogen bonds with Glu218, Thr221, Glu253, Arg256, and Arg259 residues around the middle region of the DHp domain. In addition, Arg382 and Asp387 form hydrogen bonds with Arg206 and Glu207 at the upper part of the DHp domain. Phe295, Ile298, Phe383, and Ile403 of the CA domain also form van der Waals contacts with Asn214, Thr224, Ser225, and Tyr229 of the DHp domain. Ile403, which is located on the back of the ATP binding pocket of VicK, is one of the key residues that contribute to the hydrophobic interaction with Leu267 and the aliphatic side chain of Glu218. To test whether these interactions are important for autokinase activity, we generated a series of mutants and analyzed their autokinase activity (Figure 6D). The Arg294, Gln297, Ile298, Asn334, and Asn337 mutations (R294A, Q297A/I298A, and N334A/N337A) were nearly as active as wt VicK (Figure 6D, lanes 5,7, and 9). In contrast, mutations of Asp326, Gln330, Arg382, Arg385, and Phe383 (D326A/Q330A, R382A/R385A, and F383A/R385A) dramatically suppressed VicK autokinase activity (Figure 6D, lanes 8,10, and 12), whereas the dual mutations of D326A/N337A and R382A/R385A completely eliminated VicK autokinase activity (Figure 6D, lane 13). As references, two mutations in the ATP-binding pocket (K341A/Y342A) and a deletion of the first G loop (del392–395, RAQG) negatively affected autokinase activity (Figure 6D, lanes 11 and 14). Two Ile403 mutations I403W and I403S did not significantly affect VicK autokinase activity (Figure 6D, lanes 3 and 4). Interestingly, the I403W mutation was found to dramatically increase autokinase activity in HK853 (I448W) [23]. Together, the CA domain of monomer A can position itself toward the DHp domain to form a large interface, which is further centered by two groups of key residues. One group is D326/Q330, which forms hydrogen bonds with Arg259 of the DHp domain. Another group is R382 and R385, which form hydrogen bonds with Glu207 and Glu218 of the DHp domain. F383 inserts into a hydrophobic pocket in the DHp domain and further stabilizes the interactions between the CA and DHp domains. Therefore, a mutant (D326A/Q330A/R382A/R385A) that disrupts both groups of key residues completely eliminated the autokinase activity (Figure 6D, lane 13). The CA domain of VicK monomer B stays away from the DHp domain and represents an inactive state (Figures 1C and S5A). A relatively small interface is formed mainly by van der Waals interactions between Phe383, Ile403, and Leu399 from the CA domain and Phe211, Val215, and Leu264 from the DHp domain, which are further stabilized by several hydrogen bonds between Arg382, Arg385, Asp271, and Glu218 (Figure S10). The buried surface of this interface is 623 Å2, which suggests that it may not be sufficiently stable. Phe383 and Leu399 appear to be the most important residues because they insert into helices α6 and α7 for extensive van der Waals interactions with Leu264 and Val215. When compared with T. maritima HK853 alone, the VicK CA domain rotates ∼76° along an axis vertical to the DHp domain (Figure S8). Overall, the CA domain remains inactive by positioning itself away from the DHp domain and this interface may act as a hinge. Our crystal structure revealed a long rod-shaped VicK holoenzyme (Figure 1). The three modules of the HAMP, PAS, and DHp/CA domains are connected through two groups of straight helices. Such an extended VicK molecule may provide sufficient surface and accessibility for potential ligands or protein partners to interact with. The long shaped molecules often have the largest radius of gyration and tend to polymerize. VicK and its homologs have one or two integrated TMs rather than being free cytosolic proteins and are localized as clusters within the membrane [26]. In B. subtilis, YycG proteins center in the division ring possibly through interactions with FtsZ, a tubulin-like protein [41]. In contrast, approximately 420 dimeric VicK holoenzymes are randomly distributed as clusters throughout the periphery of one S. pneumoniae cell [42]. This clustering characteristic indicates that its function might require direct interaction between individual molecules, which is consistent with the physical properties of rod-shaped molecules. A long-standing question is how HAMP domains serve in signal transduction because they often directly connect TMs and extracellular sensors. The HAMP domains may undergo a 26° rotation, which is derived from the unusual knobs-to-knobs packing of the solution structure of Af1503 HAMP, when they receive transmembrane signals [14], [15]. The crystal structure of concatenated HAMP domains from Pseudomonas aeruginosa Aer-2 implicates that the conformational dynamics may also serve a role in signal transduction [20]. Consistently, a series of structure and functional experiments have shown that intrinsic thermodynamic instability is required for HAMP signaling [16], [17], [19], [21], [43]. The VicK HAMP domain appears to be a stable four-helix coiled-coil with classical knobs-into-holes interactions, wherein three pairs of hydrophobic residues from helix α2 form a central hydrophobic core with knobs-to-knobs packing, and it is unlikely that structural alternations of the HAMP domain play a central role in signal transmission (Figures 2 and S1B). The VicK HAMP domain is further stabilized by Phe82, which is conserved in the majority of streptococcal species (Figures 2A and S11). However, variable residues, including isoleucine, valine, and occasionally threonine, are present at position 82 in some VicK homologs, which suggests that the stability of HAMP domains may vary in different species. It is worth noting that the α2 helices of the HAMP domain connect with downstream PAS domains through continuous helices, part of which (aa 86–103) form a fairly rigid leucine-zipper (Figure 3C). This long helical structure most likely serves two purposes: (1) The HAMP domain might be further stabilized by additional interlock packing of the coiled-coil; and (2) The HAMP domain can easily transfer any conformational change down to the PAS domain. In contrast, the connection of the α6 helices between the PAS and DHp domains, particularly a short linker between α6 and β5, is rather flexible. Thus, the VicK molecule also appears to have a potential thermodynamic property for signal transduction from the HAMP or PAS domains to the catalytic CA domains. The VicK PAS domains form a stable dimer through the short leucine-zipper (aa 89–103) (Figure 3C). In general, canonical PAS domains are rather flexible and readily subjected to ligand-induced conformational changes [9]. The three loops, L3–L5, form a mobile surface that could be regulated by unknown ligands (Figure 3B). Winkler et al. recently demonstrated that deletion of the PAS domain but not a triple mutation (D133N, N136Y, and L140R) of S. pneumoniae VicK reduced the autokinase activity and, more dramatically, phosphatase activity [33]. A similar deletion experiment also showed that the PAS domain is essential for the phosphatase activity of T. maritima ThkA [44]. Our S. mutans VicK structure shows that Leu135 (equivalent to N136 of S. pneumoniae VicK) stabilizes helix α4 by interactions with Ile116 and Ile139 (equivalent to L140 of S. pneumoniae VicK), which contributes to the hydrophobic cavity of the PAS domain (unpublished data). Therefore, it is likely that an Ile139 to arginine mutation only disrupted potential ligand binding but not dimerization. In contrast, an L100R mutation disrupted dimerization of the PAS domain, and in turn, the kinase activity of S. pneumoniae VicK [38]. In ligand-free VicK, helix α5 adapts an open conformation when compared to flavin adenine dinucleotide (FAD) -bound NifL, heme-bound FixL, and malate-bound DcuS (Figure S4). Thus, loop L3 is able to provide a sufficient cavity and tunnel for potential ligands to bind. Interestingly, when aligned with PhoQ, helix α4 and loop L3 of the VicK PAS domain adapt significantly different conformations and the trajectory of helix α5 is similar. Unfortunately, despite our efforts, no ligand specific to the VicK PAS domain has been experimentally identified. Only when such ligands are identified will it be possible to analyze how induced conformational changes regulate VicK catalytic activities. Therefore, this remains an interesting area for future research. The local region around the phosphoryl receptor histidine of DHp domains is highly conserved [45]. We found that this region has a low helical propensity and is subject to significant helical bending, which is consistent with its low helical propensity. The helical bending most likely helps place His217 in close proximity to the CA domain to allow hydrogen bonds to form with the γ-phosphate of ATP (Figures 4 and 6B). Similarly, the DHp domain of B. subtilis DesK bends 50–54° when His188 is phosphorylated [25]. The HK853 DHp domain also bends 20° when bound to its cognate RR468 [46]. Therefore, the helical bending appears to be an intrinsic property of the DHp domain and is relevant to its function. Indeed, we showed that the combined mutations, through introducing alanines (VSP2AAA and VSTP2AAAA) into the DHp domain, abolished VicK autokinase activity (Figure 4D). Residues Pro222 and Thr221 are conserved among VicK homologs (Figure 4C). Interestingly, these residues are essential for phosphatase activity because mutations of either P222A or T221A abolished the phosphatase activity of VicK (Figure 5). Proline, which produces a kink and 18–35° bending that affects the thermodynamic stability of the α-helix [47], plays key roles in the transmembrane signaling of several proteins, including G-protein-coupled receptors and voltage-gated potassium channels [48]. In addition to proline, threonine and serine residues are able to bend a helix 3–4° larger than alanine, which is important for the channel gating of voltage-dependent connexin32 [49], [50]. Our data in this study demonstrate that Pro222 and Thr221 are essential for phosphatase activity in VicK (Figure 4D). Glycine may also serve a similar role as proline. The glycine in the middle of an α-helix attributes unique flexibility [51]. B. subtilis DesK has a glycine instead of proline present in this region [25]. Consistently, our P222G, as opposed to the P222A mutant, had phosphatase activity similar to wt VicK (Figure 5A, lane 6). It is worth noting that the DHp domain is not significantly bent in the structure of B. subtilis Spo0B and Spo0F complex compared with T. maritima HK853 and RR468 complex, although a glycine residue is localized adjacent to the phosphoryl receptor histidine [52]. However, it is possible that the crystal structure of the Spo0B and Spo0F complex captures only one state of the DHp domain of Spo0B kinase. Our structure has shown that monomer A positions toward to its own His217 to form an active state, which is consistent with the findings using heterodimeric kinase mutants of HK853 and PhoR [46]. A flexible linker between the CA and DHp domains has been postulated to play a role in CA domain swinging [23]. In addition, the small interface created by Arg382, Phe383, and Arg385 provides a docking site as well as sufficient freedom for the CA domain to rotate (Figure S10). In the HK853 and RR468 complex, the CA domain swings ∼37° along the axis of the DHp domain when compared with free HK853 [46]. Crystal structures of B. subtilis DesK have shown that the CA domains could position themselves differently relative to the DHp domain [25]. The buried surface of the active monomer A is 1140 Å2. However, this interface is mainly composed of hydrophilic and polar residues, which suggests that these contacts may only be transient (Figure 6C). Our mutagenesis experiments showed that these residues are important for VicK autokinase activity (Figure 6D). Interestingly, while Ile403 mediates van der Waals contacts with Asn214 and Glu218 in the active state, it also contributes to the interface in the inactive state (Figures 6C and S10). Thr221 mediates a hydrogen bond with Asn337 and van der Waals interactions with Phe295 (Figure 6C). The T221A mutation eliminated phosphatase activity but did not affect the autokinase activity (Figures 4D and 5). In contrast, Winkler et al. found that a T221R mutant of S. pneumoniae VicK completely abolished its autokinase activity and greatly reduced phosphatase activity [33]. It is possible that the large arginine side chain may block the CA domain from properly accessing the DHp domain for active site formation. It is important to note that the asymmetrical positioning of the CA domains and the different conformations of each monomer of the DHp domain are captured in a unique crystal-packing environment. The two VicK dimers form an anti-parallel tetrameric packing where the active CA position is stabilized by direct interactions with HAMP and PAS domains from another dimer in the same asymmetric unit (Figure S12). The N terminus of the HAMP domain makes contacts with the inactive CA domain of the VicK dimer from another asymmetric unit through the extended N terminus of monomer B (Nb) (Figure 1C). However, the overall structures of the HAMP and PAS domains remain symmetric. Together, coordinated helical shifts of DHp and movement of the CA domains can be combined into a model to illustrate the activation steps for VicK (Figure 7). When both CA domains are inactive, they stay relaxed and further away from the phosphoryl acceptor histidine (I). Upon stimulation, one helix bends ∼25° toward the DHp central axis in coordination with the global shifts of DHp to expose the phosphoryl acceptor histidine. Consequently, one CA domain in cis rotates ∼61° to reach this histidine for initial phosphorylation (II). It is likely that this activation does not happen simultaneously for both CA domains because the DHp domain allows only one helix to bend at a time (Figure 4B). To fully activate both histidines (IV), VicK may go through an intermediate state that is similar to the inactive state (III) before the second CA domain rotates. Finally, the CA domains swing back to the initial state (IV to I) through several steps that remain to be determined. S. mutans VicK (aa31–450) was cloned into vector pET15 (Novagen). S. mutans VicR (aa1–235) and S. pneumoniae VicR (aa1–234) were cloned into pETHis vector. All these constructs were expressed with N terminal 6× Histidine tag in Escherichia coli BL21/DE3 Rosetta (Novagen). VicK protein was purified through nickel affinity agarose (Qiagen), Q sepharose, phenyl sepharose, and Superdex S200 (GE Healthcare). The two VicR homologs were simply purified by nickel affinity agarose and gel filtration. Protein preps were concentrated down to 8 mg/ml in a buffer of 10 mM Tris (pH 8. 0), 100 mM NaCl, 300 mM ammonium acetate, 2 mM DTT, and 1 mM β-mecaptoethanol (β-ME). The preliminary crystals were obtained using the screen kits of JCSG Plus (Qiagen) and Index (Hampton Research). Most crystals from these initial screening could only diffract to ∼4. 5 Å. The later optimizations of those crystals defined the best condition of 2. 3–2. 8 M Sodium formate, 50 mM Tris (pH 7. 4–8. 6), and 4% PEG 4000 at room temperature. Selenium-methionine labeled protein was prepared following a standard protocol [53]. Bar-like crystals were grown for 2–4 d and immediately frozen in liquid nitrogen after quickly soaked with crystal growth buffer and additional 12% isopropanol as a cryo-protectant. Data were collected in BM17U, Shanghai Synchrotron Radiation facility (SSRF), China and processed by HKL2000 [54] and CCP4 suite [55]. Data collection statistics are summarized in Table S2. The selenium sites were located using SHELXD [56]. Heavy atom positions were refined and phases were calculated with PHASER [57]. The real-space constraints were applied to the electron density map in DM [58]. An initial model was then built manually using COOT [59]. The model was further refined using PHENIX with stereochemistry and secondary structure information as restraints [60]. The structure and refinement statistics are summarized in Table S2. Model analyses were performed using a variety of programs. The structural alignments were calculated in Coot [59]. Similar folds searches were carried out using Dali server [61]. The helical bending was calculated using a program HELANAL [62]. The buried surface areas were calculated in CNS [63]. The electrostatic potential surfaces was calculated and graphed by CCP4mg [64], while other graphics were made using Pymol (DeLano Scientific LLC). The VicK protein prep at ∼4 mg/ml was first resolved on a size exclusion column (Shodex KW-802. 5) in a buffer of 50 mM HEPES (pH 7. 0) and 200 mM Na2SO4 at 25°C. Data were then collected on a DAWN HELEOS II laser photometer with an emission at 658 nm (Wyatt, USA). Molecular mass was calculated using ASTRA V (Wyatt, USA). All VicK mutants were generated by our modified Quikchange mutagenesis protocol [65]. All these mutants were purified as wt VicK described above. β–ME was excluded from buffer in the final protein preps. The autokinase activity of VicK was measured by using isotope γ32P-ATP (Perkin-Elmer, NEG002Z001MC) according to a recent protocol published by Winkler and his colleague [33]. The working solution of the hot ATP was freshly made by mixing the hot ATP with an equal volume of 3 mM cold ATP. The VicK proteins at 0. 5–10 µM were incubated with a concentration gradient (0–80 µM) of the hot ATP working solution in 10 µl reaction buffer of 50 mM Tris (pH 7. 5), 200 mM KCl, and 10 mM MgCl2 for 0. 25 to 8 min at room temperature. The measurements were setup at four different time points. The protein concentration and time points for each VicK protein were pre-determined to have the signals within linear ranges. The reactions were stopped by adding 3 µl 4× SDS loading buffer. The resulted mixtures were then subjected to 12% SDS-PAGE before the gels were dried and scanned in Typhoon 9410 (GE Healthcare). Quantifications were carried out using a program Totallab Quant. The autokinase parameters were derived from non-linear regression of the Michaelis-Menten equation. For simple comparison of the various VicK mutants, the VicK protein preps of 2 µg were mixed with 0. 3 µl hot ATP working solution in 15 µl reaction buffer and further incubated for 30 min at room temperature. The reactions were stopped with 4× SDS loading buffer and separated in 15% SDS-PAGE. The gels were dried before being exposed to X-ray film. S. pneumoniae VicR at concentration of 4 µM was phosphorylated in 50 mM AcP, 50 mM Tris (pH 7. 4), 50 mM KCl, 2 mM MgCl2, and 20% glycerol for 1 h at 37°C [66], [67]. The phosphorylated VicR was then mixed with VicK wt and mutants at a final concentration of 4 µM for another hour at 37°C. AcP was diluted by at least 10-fold in the latter reaction. The resulted mixtures were first analyzed by PMS [68]. Briefly, the regular 8% SDS gels (29∶1) were prepared with additional 50 µM phos-tag acrylamide (Wako) and 100 µM MnCl2. The gels were run at 120 V for 120 min at 4°C for the best mobility shift and stained with coomassie blue. The reactions of phosphorylated VicR were also analyzed by HPLC [67]. Briefly, additional 20% glycerol was added into reactions of VicR after treated with AcP as described above, which were further mixed with HPLC running buffers (A: 0. 1% trifluoric acid; B: 0. 1% trifluoric acid and 100% acetonitrile) to reach 40% acetonitrile. HPLC was run using a reverse-phase C8 column (4. 6 mm×250 mm) (Agilent 1200). The phosphorylation state of VicR was confirmed by PMS as described above. Homologs of the full-length VicK with >50% sequence identities were initially pooled from Genebank using a program BLAST [69]. Redundant entries of 96%–100% identity were identified using a multiple alignment program CLUSTAL [70] and subsequently removed, resulting in >50 unique homologs with 50%–96% identities (Figure S11). The DHp regions corresponding to amino acids 197–269 of these VicK homologs were used to generate the conservation logo using Weblogo server (weblogo. berkeley. edu). Redundant sequences (with 100% identity) of the DHp domain were further removed, resulting in >40 unique sequences (Figure S6). The helicity of the DHp was analyzed by a comprehensive secondary structure prediction program Phyre, which scores each amino acid as 0–9 for a helical probability [71]. DHp engineering was carried out through reiterative process of mutations and secondary structure calculation. Those unfavorable amino acids to α-helices within the bending region of DHp domain were determined according to statistical analyses [39]. The coordinates of the structure and relevant information have been deposited into the Protein Data Bank (4I5S).

Two-component signal transduction systems (TCSs) are promising targets for new antimicrobial research because they help bacteria and fungi adapt and survive. One of the main components of TCSs is a sensor histidine kinase (SK), which relays extracellular signals to intracellular pathways. Despite intensive research, a full-length structure of an SK has yet to be solved. In this study, we report the first crystal structure of the complete cytoplasmic region of VicK, an important SK in the tooth decay pathogen S. mutans. VicK is composed of several domains (HAMP, PAS, DHp, and catalytic and ATP binding domain [CA]) in addition to a short transmembrane domain. We find that the dimeric VicK protein has an elegant rod-shaped structure with the domains linearly connected like beads on a string. The structure suggests that VicK kinase activates itself by helical bending of the DHp domain and coordinated swinging around of the catalytic CA domain to engage with the target histidine. Structure-based mutagenesis experiments also helped us to identify key residues that are required for VicK' s opposing phosphatase activity. Our studies of the multi-modular VicK protein suggest a sequential kinase activation model that may involve helical bending of the DHp domain and repositioning of the CA domains.

lay_plos

This study describes two longitudinal serological surveys of European Bat Lyssavirus type 1 (EBLV-1) antibodies in serotine bat (Eptesicus serotinus) maternity colonies located in the North-East of France. This species is currently considered as the main EBLV-1 reservoir. Multievent capture-recapture models were used to determine the factors influencing bat rabies transmission as this method accounts for imperfect detection and uncertainty in disease states. Considering the period of study, analyses revealed that survival and recapture probabilities were not affected by the serological status of individuals, confirming the capacity of bats to be exposed to lyssaviruses without dying. Five bats have been found with EBLV-1 RNA in the saliva at the start of the study, suggesting they were caught during virus excretion period. Among these bats, one was interestingly recaptured one year later and harbored a seropositive status. Along the survey, some others bats have been observed to both seroconvert (i. e. move from a negative to a positive serological status) and serorevert (i. e. move from a positive to a negative serological status). Peak of seroprevalence reached 34% and 70% in site A and B respectively. On one of the 2 sites, global decrease of seroprevalence was observed all along the study period nuanced by oscillation intervals of approximately 2–3 years supporting the oscillation infection dynamics hypothesized during a previous EBLV-1 study in a Myotis myotis colony. Seroprevalence were affected by significantly higher seroprevalence in summer than in spring. The maximum time observed between successive positive serological statuses of a bat demonstrated the potential persistence of neutralizing antibodies for at least 4 years. At last, EBLV-1 serological status transitions have been shown driven by age category with higher seroreversion frequencies in adults than in juvenile. Juveniles and female adults seemed indeed acting as distinct drivers of the rabies virus dynamics, hypothesis have been addressed but their exact role in the EBLV-1 transmission still need to be specified. Chiroptera is the second largest order of mammals after Rodentia. They have a worldwide geographical range, with the exception of Antarctica and are represented by more than 1 100 different species [1], of which 36 are found in Europe. Of these 36,34 are reported in France, and all of them are strictly protected by national [2,3] and international [4] legislation as they are sensitive to the destruction of their habitat. With their long lifespan regardless of their size, their unique flying ability as a mammal, and their overactive immune system, they are considered exceptional mammals and such fundamental innate abilities have recently attracted the interest of the scientific community [5–8]. More than 200 viruses have been associated with bats [9]. They were recently discovered to be potentially at the origin of the Zaire Ebola virus [10,11], and they have also been linked to other illnesses related to coronaviruses (Severe Acute Respiratory Syndrome, Middle Eastern Respiratory Syndrome), filoviruses (Ebola and Marburg), henipaviruses (Hendra and Nipah), and Lyssaviruses [12–14]. Rabies is a severe and lethal disease transmitted by the saliva of an infected animal through bite, dogs being the main source of human infection. The current lyssavirus taxonomy includes 14 lyssavirus species of the Rhabdoviridae family, order Mononegavirales [15], of which 12 species have been isolated in bats (reservoirs of the Mokola virus (MOKV) and Ikoma lyssavirus (IKOV) still remain to be identified [16,17]). Phylogenetic analyses suggest that all these lyssaviruses have bat origins [18–20]. The number of recorded species will certainly increase in the future as suggested by the latest isolations of Gannoruwa Bat Lyssavirus in a fruit bat (Pteropus medius) in Sri Lanka, a new candidate in the formal classification of lyssaviruses [21]. In Europe, bat lyssavirus was documented for the first time in 1954 in Hamburg, Germany [22]. From 1977 to 2016,1, 175 bat lyssavirus cases were recorded from the North to the South of the continent [23]. To date, 4 different lyssavirus species have been isolated in European bats. Initially, European bat lyssaviruses were genetically described into 2 different groups named European bat lyssavirus type 1 (EBLV-1) and European bat lyssavirus type 2 (EBLV-2) [24]. Recently, 2 new lyssavirus species represented by the Bokeloh Bat Lyssavirus (BBLV) located in Germany and in France [25,26] and the West Caucasian Bat Virus (WCBV) located in southern Russia [27] have been identified. A putative Lleida bat virus was detected in Spain in Miniopterus schreibersii but does not yet have a taxonomic status [28]. Most European bat cases have been recorded as belonging to EBLV-1 (>95%), which is associated with the serotine bat, Eptesicus serotinus [29], and with E. isabellinus in Spain, a sibling species of E. serotinus [30]. EBLV-1 molecular characterization has separated this species into 2 sublineages, EBLV-1a and EBLV-1b [31]. Lineage 1a shows a western-eastern European distribution from Russia to central France, while variant 1b exhibits a southern-northern European distribution from Spain to Denmark [32]. Except for 5 EBLV-2 cases in Pond bats (Myotis dasycneme) in the Netherlands [33], all other EBLV-2 cases were isolated from Daubenton’s bats (Myotis daubentonii) within a distribution area including the Netherlands, United Kingdom, Switzerland, Germany and Finland [34–36]. Among this viruses, only EBLV-1 and EBLV-2 have been associated with human cases with two identified case per virus species [37]. In France, bat lyssavirus was identified for the first time in 1989 in the Lorraine region (North-East France) (Briey and Bainville) and a bat rabies surveillance program was consequently initiated [38]. Epidemiosurveillance and research programs to estimate the public health risks associated with the infection of native bats by Lyssavirus were then strengthened following the report of the French Ministry of Agriculture [39], leading to the consolidation of the network involving both local veterinary services and the French National Bat Conservation Network (SFEPM). From 1989 to present, 78 bat lyssavirus cases—75 EBLV-1 cases in common serotine bats, 1 EBLV-1 case in common pipistrelle (Pipistrellus pipistrellus) and 2 cases of BBLV in Natterer' s bats (Myotis nattereri) —have been diagnosed in France (E. Picard-Meyer, under revision) and the issue of seasonality in the probability of detecting cases has been raised recently [40]. To gain a better understanding of virus transmission, active surveillance programs during population monitoring were set up in addition to the passive surveillance program. As shown in the synthesis made by Picard-Meyer for the 2004–2009 period, the sampling of such programs involved blood and saliva samples from more than 300 bats on 18 sites [41]. In such cross-sectional surveys, the bats were sampled on various sites throughout France and no data from marked individuals were available to allow a longitudinal study. This study proposes the first longitudinal survey of EBLV-1 in mono-specific serotine colonies, the main bat species found infected by this lyssavirus. Multi-state models, a category of capture-recapture analysis, have been used to attempt explaining EBLV-1 virus exposure. Originally developed for estimating the abundance of animal populations, capture-recapture methods have recently attracted attention in the field of veterinary epidemiology [42,43]. When appropriate data are available, capture-recapture models can also be used directly to estimate disease-associated mortality and epidemiological parameters, such as infection and recovery rates [44]. In this study, as the serological status of individuals could change, data were analyzed using a multistate capture-recapture approach [45]. Multistate models indeed allow individuals in a population to be distributed across multiple sites or among different disease states [46,47]. More precisely, we used multievent capture-recapture models [48], an extension of multi-state models, to determine the factors influencing bat rabies transmission while accounting for imperfect detection and uncertainty in disease states. Survival, capture, transition and judgment probabilities were assessed by hypothesizing, based on lyssaviruses literature, that EBLV-1 exposure in bat maternity colony was driven differently according to the age of individuals and the period of time. To our knowledge, this is the first attempt of describing EBLV-1 circulation in its reservoir, over time, and by such approach and methodology. Two maternity roost sites of serotine bat colonies located in the East of France (Fig 1) were monitored. The sites were located in Universal Transverse Mercator (UTM) 32U zone in villages bordered by Moselle River and surrounded by hardwood forest, cropland and grassland. The climate is semi-continental and the landscape relatively flat with altitude ranging from 167 to 374 meters. Site A was the roof of a house in Ancy-sur-Moselle in the Moselle department, while site B—8. 5 km from site A—was the garden shed of a house in Pagny-sur-Moselle in the Meurthe-et-Moselle department. Both sites were chosen following the detection of bat cadavers in 2009 and 2012 for site A and 2011 for site B (E. Picard-Meyer, under revision). On site A, 6 dead animals in 2009 and one dead animal in 2012 tested positive for lyssavirus with reference techniques [49–51]. On site B, 2 dead individuals tested positive in 2011. The infection was shown to be caused by the EBLV-1b variant, which is endemic in the region. They were the first detection of EBLV-1 infections in these municipalities. Capture-recapture sessions were completed in summer (July and August) and spring (May) between 2009 and 2015 (during 7 years) for site A, and between 2011 and 2015 (during 5 years) for site B. Capture sessions were organized by the French Agency for Food, Environmental and Occupational Health & Safety (ANSES) and the Commission for the Protection of Water, Heritage, Environment, Subsoil and Chiroptera (CPEPESC) of Lorraine Region, the naturalist association in charge of the study and protection of bats in the region. Residents provided full informed consent to have their residences used in the study. The trapping session dates were set up to avoid disturbing the bats during the parturition period. Captures were held at nightfall, when serotine bats are known to leave their roost to forage. Harp traps were used because they are the most suitable device when a large number of animals can be expected [52]. Moreover, these traps are considered the most effective for capturing bats without harming them [53]. Traps were placed at the exits of the roof from where the bats usually emerged. To avoid injury, they were handled carefully and firmly by trained people wearing gloves and adequately vaccinated against rabies. As soon as the bats were removed from the traps, they were placed temporarily in cotton bags and then held in the palm of the hand with fingers curled around the body [54]. The sex and age class were recorded for each animal and biological samples collected. Dry synthetic fiber swabs (classiqSwabs, COPAN, France) were soaked with saliva to assess EBLV-1 virus excretion as well as viral RNA detection. Blood was collected from the antebrachial vein along the propatagium, and more recently on the uropatagium, a method found more effective, to evaluate the serological status of each individual with respect to EBLV-1 exposure. Blood was collected using filter paper as described by Wasniewski et al. [55] and subsequently stored at -16°C till the analysis. Swabs were stored in 0. 3 mL of DMEM culture medium (Dulbecco’s minimum essential medium, Invitrogen, France) at -80°C for further testing in the laboratory. Lipped bat bands (split metal bat rings, PORZANAZTD, East Sussex, United Kingdom) positioned on the forearm were used to mark the animals [56]. Each bat captured was assigned a single record number, allowing for follow-up over the successive capture sessions. After sampling, all the bats were immediately released at the site of night-time capture. None of the bats appeared sick or were euthanized during the study period. All the animals were handled in strict accordance with good animal practices and according to the EUROBAT guideline [57]. Field work and animal sampling were performed in accordance with French legislation. Because bats are protected species in France, prior formal authorization by the French Ministry of the Environment was granted for their trapping, handling, and sampling [58] and colony monitoring was undertaken following local authorization by the Prefect of the Lorraine Region [59]. In France and within the European Union, the legal framework for using under experimentation purposes is governed by Regulation 2010/63/EU of the European parliament and of the council of 22 September 2010 (applicable and translated in French in 2013) and handling of wildlife animal in the field does not require any prior specific ethical approval. During each capture session, the captured animals were recorded and classified into different serological states concerning EBLV-1 neutralizing antibodies, “S” (“NEG”/ “POS”/ “INC”). The “NEG” state included EBLV-1 seronegative individuals, i. e. bats that had never been in contact with the virus, and consequently susceptible to future infection or previously exposed but with a non-detectable level of EBLV-1 antibodies. The “POS” state included EBLV-1 seropositive animals. Seropositive animals were defined as animals that had been in contact with the virus and seroconverted. This state included both bats that were potentially protected against infection by antibodies, and sick bats. Because the serological test was occasionally inconclusive (analysis not feasible), or no blood was sampled, an “INC” state was included. To address this particular issue and to allow the use of such a dataset, an extension of the multistate capture-recapture framework was used. This extension is known as the multievent model [47,67]. When an individual is observed in the field, its status can still remain unknown or uncertain, e. g. sex status [68], reproductive status [69] but also epidemiological status [42,47] and such a model accounts for uncertainties in the assessment of a state. The different probabilities assessed during the study were as follows: The models were fitted using the E-SURGE program [70]. Both sites were maternity colonies mainly composed of females with roost site fidelity and juvenile males leaving the colony at the end of their first summer [71], males were consequently discarded from the dataset to avoid bias in the survival analysis. Multiple capture sessions were conducted occasionally within the same season (between 2 and 5), and the detection/non-detection data were merged into a single capture session per year and season. Survival, recapture, transition and judgment probabilities were all computed by considering the serological status“S” (POS/NEG/INC) as explanatory variable. Age class “a” (juvenile/adult) was also considered as a potential explanatory variable for survival probabilities as juveniles could harbor higher mortality rate as demonstrated in serotine bat biology study [72]. Regarding serological transition probabilities, a previous EBLV-1 study suggested seasonal fluctuation in Myotis myotis colonies [73], we consequently hypothesized that serotine colony could by driveen by a comparable dynamic and included the season “s” (spring/summer) as explanatory variable. This study being the only known EBVL-1 longitudinal studies on serotine monospecific colonies, we also assumed based on classical bat rabies virus (RABV) studies that transmission rate could vary according the age [74] and included age class “a” (juvenile/adult) in candidates models. The year “y” and/or season “s” (spring/summer) effects and their interaction were considered with regard to recapture probabilities as weather variations are suspected to impact trapping efficiency. Possible interactions with the serological status were also assessed to determine whether there were any specific infection patterns. All model combinations to estimate survival, transition, capture and judgment probabilities fit accordingly. Akaike' s Information Criterion with a correction for small sample sizes (AICc) was used to assess the relative model fit. The model with the lowest AICc was selected as the model that fitted the data best [75]. When the ΔAICc was lower than 2 (Δi = difference between AICc and the lowest AICc value), the most parsimonious model was selected (i. e. the one with the fewest variables). To compute antibody prevalence and its standard error, we used the traditional abundance estimate and corrected the number of animals that tested positive or negative in each session by the corresponding recapture probability [46]. To account for “INC” observations, bats were assigned a “POS” or “NEG” status using the Viterbi algorithm [76]. For each site, a logistic regression was used to assess the effect of season and year on the estimated prevalence. The number of positive and negatives cases was used as the response variable, and the AICc was used to compare models either incorporating or excluding time variables. On site A, 15 capture sessions were undertaken between 2009 and 2015, corresponding to a total of 320 bat captures (including single captures and recaptures). The distribution of the number of captures and recaptures per year and season is presented in Table 1. Among the 214 marked animals, 81 individuals (38%) were recaptured once, 19 individuals (9%) were recaptured twice, 5 individuals (2%) were recaptured 3 times and 1 individual was captured 5 times within the study period. Within the studied 201 individuals were females (94%) and 13 were males (6%). All males but 2 were identified as juveniles. Both adults had a single capture history. By comparison, on site B, where 12 capture sessions were undertaken between 2009 and 2015, there was a total of 473 bat captures, single captures and recaptures combined. The distribution of the number of captures and recaptures per year and season is also presented in Tables 1 and 2. Among the 221 marked animals, 125 individuals (57%) were recaptured once, 60 individuals (27%) were recaptured twice, 36 individuals (16%) were recaptured 3 times, 21 individuals (10%) were recaptured 4 times, 7 individuals (3%) were recaptured 5 times and 1 individual were captured 7 times, 8 times and 9 times within the study period. 156 captured bats were females (71%) while 65 were males (30%). It should be noted that no other bat species have been identified within the study period, excepted one time where a Miniopterus schreibersii individual was trapped. Many different serological status histories were observed during the study (S1 Table). Thus, some animals evolved from a negative to positive status, some did the reverse, and occasionally some changed several times (See Supplementary material describing the frequencies of the different capture histories, inconclusive results were ignored for a better clarity). This process revealed that it was more frequent for a seropositive status to become seronegative than the opposite (Table 3). Indeed, 5 and 9 animals seroconverted (from NEG to POS) while 10 and 21 animals seroreverted (from POS to NEG) on sites A and B respectively. When analyzing oral swabs, all the tested animals were found negative for RNA detection in the saliva, apart from 5 individuals all captured in July 2009 on site A. Viral RNA was detected during a first capture for 4 animals and during a second capture for one animal. Among the animals captured for the first time, 3 animals (2 females—one adult, and one juvenile—and one juvenile male) were only captured once. Among the two recaptured bats, one adult female was sampled again during the next capture session but was surprisingly found to be seronegative, with no RNA detection. The history of the second recaptured bat, found RNA-positive in the second capture of the same year, was notable. The adult female bat was indeed negative for both RNA and serology in July 2009 then, 3 days later, positive for RNA but inconclusive as to its serological status (the blood sample could not be assessed) and negative for virus excretion yet seropositive for EBLV-1 antibody detection in August 2010, meaning one year later saliva has been detected RNA-positive. All the virus isolation tests failed to detect a live virus except for one seropositive juvenile female captured only once in July 2009 on site A, just after the positive testing of dead bats. All the samples from site B were negative for the presence of an infectious virus. Individual movements between the 2 colonies, 8 kilometers away, were possible but difficult to quantify as only one female marked on site B in July 2012 was found on site A in August 2013. The best models for both sites A and B included effects for the year and season on recapture probabilities (Tables 4 and 5). No specific pattern was detected for survival probability on either site, neither age category nor serological status affecting survival. We found an effect of the interaction of serological status and age on the transition probability for site B, while the judgment probability depended on the serological status for site A only. On site A, the best-ranked model indicated that the survival probability of female bats was 0. 86 [0. 76–0. 93]. The transition probability from seropositive to seronegative was 0. 99 [0. 02–0. 99] (a boundary estimate that was difficult to interpret) and 0. 21 [0. 09–0. 40] from seronegative to seropositive. The probability of judging a positive result as positive was 1 while the probability of judging a negative result as negative was 0. 81 [0. 75–0. 86]. On site B, the best-ranked model indicated that the survival probability of female bats was 0. 78 [0. 73–0. 83]. The transition probability from seropositive to seronegative was 0. 89 [0. 53–0. 98] and 0. 25 [0. 06–0. 61] from seronegative to seropositive for adult female bats and 0. 15 [0. 07–0. 27] and 0. 05 [0. 02–0. 12] for juveniles female bats respectively. The evolution of corrected bat EBLV-1 seroprevalence on both sites A and B are presented in Fig 2. On site A, seroprevalence varied from 34% in summer 2010 [28. 0–41. 0] and summer 2012 [27. 5–41. 9] to 0% in spring 2013 [0–1. 5] and spring 2015 [0–6. 1]. The trend appeared to indicate a constant decrease in seroprevalence over time during the study, with an approximate 2–3 year oscillation interval (2011–2013). The logistic regression detected a significantly lower frequency of seropositive cases from 2011 to 2013 than in 2010 (OR2011 = 0. 26 [0. 19–0. 37]; OR2012 = 0. 65 [0. 47–0. 90]; OR2013 = 0. 40 [0. 29–0. 55]) and a lower frequency of seropositive cases in spring than in summer (ORspring = 0. 46 [0. 35–0. 60]). On site B, seroprevalence peaked at 70% [59. 9–78. 9] in spring 2013 then progressively decreased to reach 21. 6% [11. 5–34. 9] in May 2015. The logistic regression did not detect any significant differences between seasons, but the frequency of seropositive cases in 2013 was higher than in 2012 (OR2013 = 2. 40 [1. 61–3. 60]). The analysis of the two roost site colonies using multievent models within the study period did not evidence any impact of the serological status on individuals’ survival or recapture probabilities. This supports previous observations that bats could harbor exposure events without any impact on their mortality rate. These results are indeed comparable to the survival analysis computed for big brown bats (Eptesicus fuscus) affected by RABV in the United States [77] and for a Myotis myotis colony affected by EBLV-1 in Spain [73]. The detection rates of bats were also demonstrated to be uncorrelated to the serological status, indicating that seropositivity does not induce a potential behavioral change in bats that could impact the recapture probability. This finding supports the hypothesis that, in our study, observed seroprevalence of a capture session can be regarded as an unbiased estimation of the percentage of animals wo have been exposed to EBLV-1 in the colony. Recapture probabilities on both sites were shown to be affected by seasonal and annual variations. This temporal dependency could be due to changes in climate and weather conditions over seasons and years, known to affect the emergence of bats and consequently the effectiveness of captures using a harp trap [78]. EBLV-1 virus-neutralizing antibodies have been found in various bat field studies, principally through single captures [41,79,80] or in successive captures of mono-specific colonies like Myotis myotis [73], Eptesicus isabellinus [81] and also in longitudinal studies of multi-species colonies [82–84]. In these previous studies, seroprevalence varied greatly according to the site location, species and time (month and year). This study, to our knowledge, is the first extensive longitudinal analysis of 2 mono-species serotine colonies, a species currently considered as the main EBLV-1 reservoir. Our study demonstrated that individual serological transition scenarios are highly variable. We found seroconversions (from seronegative to seropositive), seroreversions (from seropositive to seronegative) in addition to occasional multiple seroreversions and seroconversions in succession (about 10/393 individuals). It should be noted that such multiple reversions could be questionable as they may also reflect limitations of the serological tests [85] performed furthermore on small amounts of blood. Globally, on both sites, seroreversions were more frequent than seroconversions, suggesting that the 2 studies could have occurred at the end of the rabies epizootic wave. This hypothesis could be supported by the fact that prior to the first established EBLV-1 case in July 2009, approximately 30 to 40 individuals were found dead by the house owner, but the animals were unfortunately not collected and analyzed. Previous longitudinal studies in Spain have shown the seropositive status of Myotis Myotis over 3 years [84]. The maximum length of time observed between positive serological statuses in our own study was 4 years (2 individuals on site A), suggesting the possible persistence of seropositivity over several years. In this study, cut-off level used to discriminate positive from negative animals was determined to minimize the risk of false positive results. This caution was taken to provide reliable identification of positive individuals and to avoid false conclusion in the statistical analysis, but could have resulted in an underestimation of seroprevalence and in low statistical power. Lyssaviruses are excreted only at certain periods, and the chance of finding the virus or RNA in bat saliva during active surveillance field studies is relatively poor [41,79]. Interestingly, and for the first time in EBLV-1 longitudinal study, 5 individuals from site A were found with viral RNA in saliva in July 2009 (four were sampled in the same capture session, in the beginning of July and 1 was sampled three days later), during the same period in which bat mortality was observed. Of the 5 positive samples, one was demonstrated as effectively infectious, showing that RNA in the mouth cavity can be concomitant to virus excretion. One initially seronegative animal was captured several times in succession. Saliva was found RNA positive 3 days later and, when captured again 1 year later, the animal was again found seropositive, supporting the hypothesis that seropositivity persists for a long time after infection or that alternate subclinical infection occurred. The risk of cross-contamination regarding the four RNA positive samples collected during the same capture session can’t be completely ruled out with certainty although usual precautions to avoid false-positive PCR results were strictly followed during bat handling and in the laboratory. However, the raw-data of this capture shown that the RT-PCR positive swabs were collected and analyzed intercalated with RT-PCR negative swabs, suggesting that laboratory cross-contamination is unlikely. The first attempt to define the temporal dynamic infection of serotine bat colonies was undertaken through a one-year study [86]. In this latter study, seroprevalence declined from 74% to below 10% within a few months (from spring to fall). In contrast, Sera-Cobo at al. (2013) found a significantly higher antibody prevalence in summer when maternity colonies are present in most localities. This pattern was magnified by the presence of multi-species colonies compared to mono-specific colonies, with social contacts between bats. Colony formation, conferring thermodynamic and social advantages to reproductive females during pregnancy and lactation, could indeed increase the rate of rabies exposure due to hypothetically higher probabilities of inter-individual and inter-species interactions. In our study, site A data revealed higher prevalence in summer than in spring, supporting the conclusion that numerous inter-individual interactions of the colony during the post-partum season (care for the juveniles) could increase the probability of exposure. On site A, corrected seroprevalence decreased over time with significantly higher seropositive frequencies in 2010 (34% of seropositive bats) than in 2011–2013, while on site B, a peak of infection was observed in 2013 (70% of seropositive bats), midway through the 5-year-study. Our data on serotine colonies thus appear to confirm the cyclic temporal hypothesis of bat infections already proposed for Myotis Myotis, with an estimated 2–3 year cycle for site A at the time of the study [73]. The model suggests that after the initial introduction of EBLV-1 into the susceptible bat colony, the seroprevalence of the colony increases then, depending on the period, tends to oscillate, its amplitude decreasing year after year. Ecological studies performed in the bordering of Luxembourg and Germany, a hundred kilometers far from the study area, have shown that females were forming maternity colonies at the middle of April and had a philopatric behavior, meaning that each year the breeding colony invests the same maternity site [72]: This eight years study also shown that median period of birth was happening in the middle of June. The young bats usually make their first flights at around three weeks old, and at six weeks they can forage for themselves. Breeding colonies usually disperse by early September, although a few bats may use the colony site as a roost until early October [72]. Reproduction seems to take place in the autumn, but very little is known about the mating behavior. Hibernation of serotine bats occurs between October and end of March. However, very few information is indeed known about this period. Based on RABV rabies model transmission in the United States, the potential impact of hibernation on the virus’s capacity to remain in animals populations has been raised [74]. The hypothesis is that hibernation could allow infected individuals and their pathogens to survive, infected virus particles being potentially hosted and preserved in brown fat [87]. The relationship between the incubation period, hibernation season and annual birth pulse could indeed generate complex dynamics that should attract more attention in bat rabies studies. With a long incubation period, infected bats could survive long enough to enter hibernation and be responsible for infectious contacts in the main transmission season that follows, maintaining a reservoir until the birth pulse provides a new supply of immunologically naïve bats. Further model predictions fitted this assumption and showed that adult female bats were infectious earlier in the year, whereas infectious juveniles appeared later in the summer [74]. In a previous study, female serotine bats were shown to be more exposed to EBLV-1 than males, probably due to their gregarious social behavior, males being more solitary [88]. Similar findings were also highlighted in the framework of RABV transmission in Brazilian free-tailed bats [89] big brown bats [77] and in vampire bats [90]. In our study, occurring in breeding colonies, only females were assessed and we have shown evidence from site B that seroreversions were significantly more frequent than seroconversions. The seroreversion frequencies of adult females were higher than those of other transition states in juvenile females. Hence, adult female serotine bats appear to be a good indicator of EBLV-1 epizootic dynamics. This raises the question of whether adult female bats are more exposed to the virus due to the mating period in September and whether they could play a major role in virus maintenance, acting as a potential source of virus transmission. Because the reproductive status of adult females could potentially drive inter-individual exposure and transmission differently, it would be valuable to consider the reproductive status as ‘pregnant’, ‘lactating’ and ‘non reproductive’ for further studies. However, such age-related rabies dynamics we detected could also reflect the greater difficulties in characterizing EBLV-1 dynamics in juveniles due to their lower occurrence than adults in the population. The possibility of maternal antibodies transfer in juveniles via the placenta or during lactation also raises questions. Indeed, although this phenomenon has been known and measured for in experimental animals or domestic animals [91–93], the situation regarding bats and EBLV-1 is unknown. Its impact on the antibody level in juveniles, and therefore, on the evaluation of exposure, would need to be clarified. All the EBLV-1 cases detected on sites A and B were detected from end of June to start of August and all determined in dead bats identified morphologically as juveniles (E. Picard-Meyer, under revision), again raising the question of the key role of age in the virus’s transmission. The influx of susceptible young in summer could act as a crucial driver of EBLV-1 dynamics. The role of susceptible young in transmission dynamics has indeed already been raised in previous discussion on zoonotic diseases [94,95]. Such biological enigmas need to be clarified and additional studies are still needed, especially in the framework of age-related bat EBLV-1characterization. The means and rate of bat-to-bat transmission in serotine populations still need to be clarified. This is a difficult question to solve in field studies and the legal status of bats in Europe due to the decline of bat populations (all the 36 species are strongly protected by European regulation (Council Directive 92/43/EEC 1992) [96]) has made experimental studies difficult to implement. Only one experimental study of EBLV-1 infection in caged serotine bats was carried out in 2009 through different means of inoculation [97]. It appeared that the environmental contamination of bats is unlikely as none of the intranasally-inoculated bats seroconverted. Infection through bites was indicated as having the greatest potential for inter-bat transmission, as the subcutaneous route of inoculation was found to be relatively efficient [97]. Despite living in close proximity to humans, human contacts with serotine bats are rarely reported. It should be noted that during this study, despite the discovery of two infected bat colonies, no sanitary incidence has been reported, nor in human neither in domestic animals. To date, only 2 EBLV-1 induced human deaths have been reported in Voroshilovgrad, Ukraine (1977) and in Belgorod, Russia (1985) [98]. Experimental infections have also shown evidence of a very limited risk of an EBLV spillover from bat to fox [99]. A few natural EBLV-1 spillover cases have been so far reported in a sheep, a stone marten, 2 cats and a fruit bat [29,100]. The risk of transmission to other species thus appears very low. However, to avoid any risk of contamination, protective measures such as personal protective equipment, post-exposure rabies prophylaxis or a booster dose in the event of exposure have been established for bat biologists in Europe [96] and France [101,102]. EBLV-1 antibody carriage in serotine bats was not correlated with mortality probability. In both site A and B, peak-seroprevalences (34 and 70% respectively) were detected one or two years after the first detection of EBLV-1 positive carcasses. While we detected oscillation seroprevalences in time, at annual level, seroprevalences were found higher in summer compare to spring, suggesting that rearing period could increase virus circulation. We pointed out differences of serological statuses between adult female and juveniles and the need for further assessment. A better understanding of this mechanism, whether of ecological, biological and/or immunological origin, is indeed a real challenge and of great interest as elucidating zoonotic virus persistence in bats concomitant to unaffected survival could help to solve human health challenges.

A multi-annual survey of two serotine bat (Eptesicus serotinus) maternity colonies previously found exposed to European Bat Lyssavirus type 1 (EBLV-1) was assessed using capture-recapture methodology. The two roosting site were located in the North-East of France. Animals were trapped, banded, and blood samples were collected to study their status regarding EBLV-1 exposure. Using capture-recapture models, the authors found that seropositive status of bats did not affect the survival abilities of individuals. Seroprevalence of EBLV-1 antibodies within the study showed an oscillation interval of approximately 2-3 years and a higher evidence of contact with the virus in summer than in spring. The maximum duration observed between successive positive serological statuses in the bat population also demonstrated a survival for at least 4 years after the exposition. This study confirms the ability of bats to survive despite circulation of lyssaviruses within the colony. Bats could indeed provide a valuable key to improving human health, currently facing numerous zoonotic epidemic issues.

lay_plos

Neutrophils and dendritic cells (DCs) converge at localized sites of acute inflammation in the skin following pathogen deposition by the bites of arthropod vectors or by needle injection. Prior studies in mice have shown that neutrophils are the predominant recruited and infected cells during the earliest stage of Leishmania major infection in the skin, and that neutrophil depletion promotes host resistance to sand fly transmitted infection. How the massive influx of neutrophils aimed at wound repair and sterilization might modulate the function of DCs in the skin has not been previously addressed. The infected neutrophils recovered from the skin expressed elevated apoptotic markers compared to uninfected neutrophils, and were preferentially captured by dermal DCs when injected back into the mouse ear dermis. Following challenge with L. major directly, the majority of the infected DCs recovered from the skin at 24 hr stained positive for neutrophil markers, indicating that they acquired their parasites via uptake of infected neutrophils. When infected, dermal DCs were recovered from neutrophil depleted mice, their expression of activation markers was markedly enhanced, as was their capacity to present Leishmania antigens ex vivo. Neutrophil depletion also enhanced the priming of L. major specific CD4+ T cells in vivo. The findings suggest that following their rapid uptake by neutrophils in the skin, L. major exploits the immunosuppressive effects associated with the apoptotic cell clearance function of DCs to inhibit the development of acquired resistance until the acute neutrophilic response is resolved. Leishmaniasis is a vector-borne disease initiated by the bite of an infected sand fly. Based on exhaustive findings in the murine model of cutaneous leishmaniasis due to Leishmania major, the clinical course of disease is thought to depend on the balance of activating cytokines, produced largely by Th1 cells, and deactivating cytokines, produced largely by Th2 cells and subsets of regulatory T cells [1]. Even in genetically resistant C57BL/6 mice, however, that develop self-limiting lesions due to a strongly polarized Th1 response, the early growth of the parasite is unrestrained, suggesting that innate killing mechanisms and the development of acquired resistance are avoided or delayed [2]. There is evidence that the acute neutrophilic response is itself critical to the early establishment of infection in the skin [3], [4]. Inoculation of L. major by the bite of a sand fly, or by needle injection, induces an intense infiltration of neutrophils that phagocytose the majority of parasites but fails to kill them, and neutrophil depletion prior to sand fly challenge leads to more rapid parasite clearance [5]. The manner in which the acute neutrophilic response inhibits the development of immunity to L. major infection is not understood. Neutrophils and DCs are normally located in distinct anatomical compartments, but converge at sites of inflammation in response to infection or tissue injury. The essential function of neutrophils in phagocytosis and killing of bacteria and in tissue repair is well described [6], [7]. Their additional role in modulating the adaptive response is suggested by their ability to release chemokines, cytokines, and anti-microbial peptides, [8], [9], and by more recent findings suggesting that activated neutrophils can deliver both activation signals and microbial antigens to DCs [10], [11]. By contrast, engulfment of apoptotic cells, including neutrophils, by DCs under steady state conditions has been shown to suppress DC maturation and is thought critical to the maintenance of peripheral tolerance [12]–[14]. Thus, the immunologic outcome of neutrophil - DC interactions may vary depending on the activation state of the neutrophils, their type of cell death, and the presence or absence of additional danger signals in the microenvironment in which these encounters occur. Importantly, the cross-talk between neutrophils and DCs has not been investigated in the context of any vector borne pathogen for which the co-localization of these cells at the site of transmission by bite or injection by needle in the skin is apt to be especially pronounced. In the present studies, we have monitored the sequence of inflammatory events following infection with L. major in the mouse ear dermis. We provide clear evidence that dermal DCs are preferentially infected via their capture of parasitized neutrophils in the skin, and that the Leishmania specific CD4+ T cell response is compromised until the acute neutrophilic response is resolved. We investigated the sequence of local inflammatory responses and identified the cells harboring L. major following injection of Lm-RFP metacyclic promastigotes (2×105) in the ear dermis of C57BL/6 mice. Myeloid populations were identified as CD11b+ cells, and further classified based on their expression of additional markers (Figure 1A) as follows: neutrophils (Ly6CintLy6G+, region 1); inflammatory monocytes (Ly6ChiLy6G−CD11c−MHCII−, region 2); monocytes/macrophages (Ly6ChiLy6G−CD11c−MHCII+, region 3); monocyte-derived DCs (Ly6ChiLy6G−CD11c+MHCII+, region 4); dermal DCs (Ly6C−Ly6G−CD11c+MHCII+, region 6); and dermal macrophages (Ly6C−Ly6G−CD11c−MHCII+, region 5). The cells in region 5 were uniformly F4/80+ (data not shown). The CD11b+ cells recovered from naïve ears included few neutrophils and inflammatory monocytes, and relatively greater numbers of dermal DCs and macrophages. The total number of CD11b+ cells recovered from the infected ears increased slowly over the first week, and expanded dramatically over the second week (Figure 1B). A prominent and transient neutrophil infiltrate accounted for the earliest increase in myeloid cells in the site, beginning at 1 hr, peaking at 12 hr, and dropping markedly between 1- 4 days (Figure 1C). Interestingly, neutrophils were found infiltrating the site again at day 7, and by day 14 their numbers exceeded the peak numbers observed during the first wave of the neutrophilic response. Comparison of L. major infected and sham injected mice demonstrated that at 1 hr the initial neutrophil infiltrate was induced, at least in part, by the tissue injury associated with the needle injection. At subsequent time points, however, the recruitment was dependent on the infectious status of the inoculum (Figure 1I). The increase in the number of inflammatory monocytes (Figure 1D) lagged slightly behind the neutrophil response, beginning at 12 hr and peaking at 24 hr. Similarly to the neutrophils, their numbers dropped markedly by 4 days but began to increase again by day 7. Very few of the CD11b+Ly6ChiLy6G− cells recovered from the site during the first week of infection were MHCII+ or CD11c+ (Figure 1F), of note because of recent findings implicating monocyte-derived DCs formed at the infection site as crucial to the induction of protective immunity during the active stage of disease [15]. The number of macrophages and DCs remained relatively unchanged from steady state conditions until 7 days post-infection, marking the onset of their massive accumulation in the site (Figure 1G and 1H). By analyzing the total population of RFP+ gated cells, we could follow the subsets of infected cells in the injection site over time (Figure 2A–C). Regions 1–6 define to the same subsets of myeloid cells as the corresponding regions in figure 1A, and in each case their CD11b expression was confirmed (data not shown). By contrast, many of the infected cells in region 7 were CD11b−, and their identity was not established using additional markers. Considering the total population of RFP+ cells (Figure 2D), low numbers were recovered at 1 and 4 hr which significantly increased between 4–12 hr and dramatically increased between 7–14 days (Figure 2D). In Figures 2E–L, the infected subsets are expressed both as a percentage of the total infected cells and their absolute numbers recovered from the ear dermis at each time point. Neutrophils were the predominant infected cells during the first 1–12 hrs (Figure 2E). At 12 hrs, 72% of the infected cells were neutrophils, with the remainder inflammatory monocytes, macrophages, DCs and other populations of CD11b+ and CD11b− cells. At 24 hr, neutrophils still represented approximately 32% of the total RFP+ cells. By day 4, the percentage of neutrophils in the RFP+ gate had dropped to fewer than 1%. Interestingly, their numbers began to increase again by day 7, and by day 14, the absolute number of infected neutrophils in the site exceeded the peak numbers observed during the first wave, although they remained <5% of the total population of infected cells. The inflammatory monocytes in the RFP+ gate also demonstrated two phases of recruitment, the first peaking at day 1 when they represented 22% of the total RFP+ cells, and the second at day 7 (Figure 2F). Their absolute numbers were greatest at day 14, again reflecting the massive expansion in the total number of infected cells at this time point. Very few of the RFP+ inflammatory monocytes recovered during the first 4 days were MHCII+ or CDllc+, while at 7 and 14 days, the majority of the RFP+ Ly6ChiLy6G− cells were MHCII+ and CD11c− (Figure 2G), reflecting the early stage of their differentiation to macrophages in the site. By day 14, appreciable numbers of infected monocyte-derived DCs (Figure 2H) were recovered, though they still represented only around 4% of the total RFP+ cells. By contrast, the infected macrophages (Figure 2I), expressed both as a percentage and absolute number of infected cells, started to increase at 4 days, and accounted for up to 20% of the total RFP+ cells at 14 days. The RFP+ dermal DCs remained few in number and <5% of the RFP+ cells over the first 24 hr, and while they remained a low percentage of the RFP+ cells at later time points, their absolute numbers increased markedly at 7 days and especially at 14 days post-infection (Figure 2J). In summary, our detailed analysis of infected cells in the L. major loaded dermis confirmed that neutrophils rapidly infiltrating the site represent the vast majority of infected cells over the first 12 hr, with the infections transitioning to inflammatory monocytes, and finally to monocyte derived macrophages and DCs during the active stage of disease. Given their predominance both as the earliest infiltrating and parasitized cells in the injection site, we investigated the influence of neutrophils on the subsequent program of infection and immune response. In vitro studies have suggested that macrophages can acquire L. major by phagocytosing infected, apoptotic neutrophils [16], [17]. To investigate the fate of infected neutrophils and their internalized parasites, Lm-RFP metacyclic promastigotes were injected into the ears of LYS-eGFP mice [18], in which neutrophils (CD11bhiGr-1hiF4/80−MHCII−), including those recovered from the skin, are eGFPhi [5]. eGFPhiRFP+ infected neutrophils were purified by cell sorting (Figure 3A) and injected into the ears of C57BL/6 mice. Analysis of a stained, cytospin preparation of the sorted cells just prior to injection indicated that approximately 30% of the parasites had already been released from the neutrophils during the 4–5 hr collection. Four hours after injection, the vast majority of the RFP+ cells recovered from the ear (90%) were found in an eGFP− population (Figure 3B), suggesting that in addition to the free parasites present in the inoculum, most of the remaining parasites were released from the infected neutrophils and available to be taken up by host cells in the skin. These cells were CD11clo, and their F4/80 and CD11b expression indicated that they were endogenous macrophages/monocytes or neutrophils (Figure 3D). Of the RFP+ cells that retained their eGFP fluorescence, approximately half appeared to be the injected population of intact, infected neutrophils (Figure 3C). The remaining RFP+eGFP+ cells were CD11c+, suggesting that the capture of infected neutrophils in the skin was largely accomplished by DCs. Of the total number of CD11c+RFP+ cells, 68% were eGFP+ (Figure 3E), suggesting that most of the DCs acquired their parasites via uptake of infected neutrophils. Of note, the eGFP fluorescence in these cells was reduced relative to the starting population of infected neutrophils. To rule out the possibility that the eGFPhiRFP+ infected neutrophils could have differentiated into CD11c+ cells, or that a small contaminating population of CD11c+ cells in the purified eGFPhiRFP+ infected neutrophils was responsible for the RFP+eGFP+CD11c+ cells observed in figure 3B, we sorted eGFPhiRFP+ infected neutrophils (donor, CD45. 2), and injected them into the ears of B6SJL mice (host, CD45. 1). Analysis of CD45. 1 expression on the subpopulations of RFP+ cells indicated that virtually all of the RFP+eGFP+CD11c+ cells were CD45. 1+, ruling out their donor origin (Figure S1). To investigate whether DCs might favor engulfment of infected neutrophils over uninfected neutrophils in the skin, equal numbers of eGFPhiRFP− uninfected and eGFPhiRFP+ infected neutrophils (Figure 3A) were co-injected into the ears of C57BL/6 mice. The analysis of eGFP+ gated cells recovered four hours later confirmed that DCs are able to take up neutrophils in vivo, representing approximately 11% of the eGFP+ cells (Figure 3F). Importantly, and despite their exposure to equivalent numbers of infected and uninfected neutrophils, an average of 66% of the CD11c+eGFP+ cells were RFP+ (Figure 3F and G), indicating that the dermal DCs favored the uptake of the infected neutrophils. The capture of mammalian cells by DCs and others phagocytes is a common event during tissue remodeling and at infection sites, where cells dying by apoptosis expose signals that are recognized for engulfment. The best studied ‘eat me’ signal on apoptotic cells is phosphatidylserine (PtdSer) whose outer membrane exposure can be quantified by staining with Annexin V. When neutrophils were recovered from the ear dermis of LYS-eGFP mice 12 hr after infection, 53% of the infected neutrophils compared to 19% of the uninfected neutrophils were Annexin V+ (Figure 4A and B). TUNEL staining confirmed a higher degree of apoptosis in the infected population of neutrophils recovered from the injection site (Figure 4C and D). These findings suggest that the uptake of L. major leads to accelerated apoptosis and earlier exposure of PtdSer on neutrophils infiltrating the injection site, which may favor their recognition and capture by DCs in the skin. To investigate neutrophil - DCs interactions following injection of the parasite directly, we evaluated dermal DCs recovered from C57BL/6 mice 24 hr after infection with Lm-RFP parasites, and stained for neutrophil derived-myeloperoxidase (MPO) and elastase (NE). In addition, mice were treated with two neutrophil-depleting antibodies: the GR-1 specific antibody RB6-8C5, which recognizes an epitope shared by Ly6G and Ly6C, and the Ly6G specific antibody, 1A8. Administration of 1A8 one day before infection depleted 85% of the CD11b+GR1hiLy6Cint neutrophils present in the ear dermis 24 hr after infection (Figure 5A and C). The remaining neutrophils showed lower GR1 staining, likely due to competition with the surface bound 1A8 antibody. The CD11b+GR1intLy6Chi population was unaffected. By contrast, and consistent with the prior reports [19], [20], the RB6-8C5 antibody depleted both neutrophils and a population of inflammatory monocytes (Figure 5B and C). Furthermore, the neutrophil depletion achieved using RB6-8C5 was virtually complete (99%). Neither reagent affected the total number of DCs recovered from the ear at 24 hr, or the number RFP+ DCs as a percentage of the total population of RFP+ cells (Figure 5D and E). Gating on RFP+ or RFP− dermal DCs (Figure 5F), MPO staining on cells recovered from the control treated mice was observed in an average of the 58% of the RFP+ DCs, suggesting that the majority of the infected DCs acquired their parasites via uptake of infected neutrophils (Figure 5G and H). By contrast, only a low proportion (<5%) of the RFP−DCs were MPO+, although because far more RFP−DCs were recovered from the site compared to RFP+DCs (Figure 5F), the percentage of RFP−DCs staining for MPO was on average 60% of the total population of MPO+ DCs (data not shown). The intracellular MPO staining in the majority of the infected DCs was comparable to the MPO staining observed in the neutrophils themselves, and greater than the MPO staining observed in the inflammatory monocytes recovered from the site (Figure S2), reinforcing the conclusion that the acquisition of the MPO marker by infected DCs was due to their uptake of infected neutrophils. Importantly, the RFP+ DCs recovered from the RB6-8C5 treated mice were virtually all MPO−, confirming that the uptake of parasites in the absence of neutrophils or inflammatory monocytes does not upregulate the expression of MPO in the DCs. The number of RFP+ DCs recovered from the 1A8 treated mice that stained for MPO was also significantly reduced, though an average of 24% of the cells were still MPO+ cells, consistent with the incomplete neutrophil depletion using this antibody (Figure 5G and H). Staining for NE, while relatively weak compared with MPO, reinforced the MPO result in that the majority of the RFP+ DCs recovered from the non-depleted mice stained positive for NE (Figure S3). Finally, RFP+ DCs recovered from control treated mice 14 days after infection were mainly MPO− (Figure 5I), suggesting that following the resolution of the acute neutrophilic response, infected neutrophils were no longer the main source of parasite delivery for DCs in the skin. We further characterized the possible subsets of the RFP+ DCs recovered from the site based on their expression of Langerin and CD103. As reviewed [21], and confirmed in our analysis of the DCs recovered from the ear dermis 24 hr after infection, the DC subsets include Langerhans cells (LC) and migratory LC (CD11c+MHCII+Lang+CD103−), Langerin+ DC (CD11c+MHCII+Lang+CD103+), and Langerin− DC (CD11c+MHCII+Lang−CD103−) (Figure S4). The RFP signal was associated exclusively with the Langerin− DCs. To address whether neutrophils might modulate the antigen presentation functions of DCs during the early stages of infection, the expression of activation markers on infected DCs recovered from the ear dermis 3 days after infection in neutrophil-depleted (RB6-8C5) or control treated C57BL/6 mice was compared (Figure 6A–C). Expression of MHC class II, CD86 and CD40, but not CD80, was increased on RFP+ DCs recovered from the neutrophil depleted mice (Figure 6B and C). Functional studies involving these infected DCs required pooling of dermal cells from 10 mice (20 ears) for each treatment group in order to obtain a sufficient source of antigen and antigen presenting cells for the co-culture assays. Using CD11c+ RFP+ cells that were normalized for their RFP signals by cell sorting (Figure 6D), the infected DCs from neutrophil depleted mice were more efficient than the infected DCs from the control treated mice in activating Leishmania-primed T cells from healed mice to secrete IFN-γ, observed in two independent experiments (Figure 6E). To evaluate the influence of neutrophils on CD4+ T cell priming to L. major- derived antigen in vivo, B6. SJL congenic mice were depleted of neutrophils 24 hr prior to infection with L. major SP-OVA or control 3′NT transgenic parasites in the ear. CFSE-labeled, naïve OT-II CD4+ T cells specific for OVA were adoptively transferred into the same recipients. Draining lymph nodes were harvested on day 6 and dilution of CFSE fluorescence was determined on CD45. 2+ and CD4+ gated cells. Infection of control treated mice with Lm SP-OVA failed to induce OT-II proliferation above the background levels (6–7%) observed in control treated or neutrophil depleted mice infected with Lm 3′NT (Figure 6E). By contrast, mice treated with 1A8 and RB6-8C5 had an average of 20% and 34% of the gated cells in division, respectively (Figure 6F). We also assessed the ability of CD45. 2+ OT-II CD4+ cells to produce IFNγ, IL-10 and IL-17, following ex-vivo restimulation with PMA/ionomycin for 4 hours in the presence of brefeldin-A. A percentage of proliferating CD45. 2+ OT-II CD4+ cells from 1A8 and RB6-8C5 treated mice (29% and 25%, respectively) produced IFNγ (Figure 6G). Neither IL-10- nor IL-17A-producing T cells were detected (data not shown). The influence of early neutrophil depletion on CD4 priming was no longer apparent when OT-II cells were transferred 14 days post-infection with Lm SP-OVA (Figure 6H), at a time when infected DCs no longer harbored neutrophil markers (Figure 5I). Taken together, these findings suggest that the favored uptake of infected neutrophils by dermal DCs effectively prevents the activation of Leishmania-specific CD4+ T cells until the acute neutrophilic response is resolved. We have recently described the efficient capture of L. major metacyclic promastigotes by neutrophils at the site of needle inoculation or infected sand fly bite, and the powerful effects of early neutrophil depletion in promoting rather than compromising host resistance to sand fly transmitted infection [5], [22]. The current studies provide an underlying mechanism to explain the immunomodulatory role of neutrophils in the L. major loaded dermis. Under steady state conditions, DCs are strategically positioned in peripheral and lymphoid tissues to sense microorganisms and endogenous stress signals, including apoptotic cells. Neutrophils, by contrast, are present mainly within the blood, and circulate in a non-activated state with a half-life of 6–7 hrs. Following inoculation of L. major into the skin by needle or by the bite of an infected sand fly, the parasites are taken up by neutrophils that are rapidly recruited to and accumulate with DCs at the injured site. We observed that phagocytosis of L. major significantly accelerated the rate of neutrophil apoptosis, which was associated with the favored uptake of infected over uninfected neutrophils by DCs in the skin. More importantly, for the majority of infected DCs in the skin their initial encounter with the parasite occurred via capture of infected neutrophils, with a negative impact on CD4+ T cell priming. These studies confirm the previous findings in L. major [5], [23], recently extended to L. infantum [24], that neutrophils are rapidly recruited to and accumulate in the inoculation site, and represent the predominant parasitized cell during the first 1–12 hours of infection in the skin. The inflammatory and infectious process induced by L. major in the skin may be regulated in a tissue specific manner, since recent observation by Gonçalves et al. [25] and confirmed by our own studies (data not shown) have revealed that when L. major metacyclics are introduced into the peritoneal cavity, neutrophils are neither the first infiltrating nor predominant infected cells. Our kinetic analysis of the L. major loaded dermis revealed that the rapid neutrophilic response is initiated in part by signals generated by the tissue injury produced by the needle injection itself, since a transient recruitment was observed in sham injected mice, and amplified by more durable signals derived from the parasite and/or from infected cells [26]. The fate of the infected neutrophils was followed by transfer of eGFPhiRFP+ cells into the ear dermis of C57BL/6 mice. By 4 hr, the majority of RFP+ cells recovered from the site were endogenous neutrophils and monocytes/macrophages that were eGFP−, consistent with our prior in vivo imaging results that readily captured infected neutrophils undergoing apoptosis and releasing viable parasites for subsequent uptake by other cells in the skin [5]. Thus, the ‘Trojan Horse’ hypothesis as originally proposed [17], in which neutrophils serve as a vector for silent entry of Leishmania into macrophages, has not been directly substantiated in these studies. We cannot, however, dismiss the possibility that phagosomal degradation of the eGFP signal occurred rapidly following engulfment of the infected neutrophils by macrophages. It is also possible that clearance of neutrophil-derived, apoptotic bodies by infected macrophages would still contribute to their deactivation and promote the intracellular survival and growth of the parasite, as proposed. By contrast to macrophages, the evidence for the uptake of L. major infected neutrophils by DCs in the skin seems clear. Firstly, CD11c+ cells were the only endogenous cells associated with both the RFP and eGFP signals. Secondly, when the infections were initiated by RFP L. major metacyclics, the majority of the RFP+ DCs recovered from the injection site at 24 hr also stained positive for neutrophil-derived MPO and elastase. In studies by Ng et al. [27], two-photon imaging captured dermal DCs but not Langerhans cells taking up Leishmania promastigotes in the skin. We also found Langerin− dermal DCs as the major infected DC subset in the skin, but conclude based on their staining for neutrophil markers, and the absence of these markers in DCs that have taken up parasites in the absence of neutrophils, that the majority of the infected DCs acquired their parasites via engulfment of infected neutrophils. Favored uptake of infected over uninfected neutrophils was also observed, correlated with their accelerated expression of apoptotic markers that may have targeted them for more efficient recognition and clearance by DCs. Neutrophil ingestion of other microbial pathogens, notably E. coli [28], Str. pneumoniae [29], [30], C. albicans [31], Sta. aureus [32], and M. tuberculosis [33], has also been found to accelerate their apoptotic program. The findings involving Leishmania are inconsistent on this point, with delayed or enhanced expression of PtdSer observed on neutrophils obtained from human blood or the mouse peritoneal cavity and exposed to Leishmania in vitro [34]–[37]. The current studies are the first to compare the apoptotic profile of tissue infiltrated neutrophils that have taken up parasites, or not, in the inflamed dermis. Apoptosis is an active process to regulate cellular homeostasis. Efferocytosis refers to the capture of apoptotic cells by phagocytes, primarily macrophages and immature DCs (iDC), and is itself thought to be a homeostatic mechanism to resolve inflammation and to maintain peripheral tolerance [13]. Recognition and engulfment of apoptotic cells, including apoptotic neutrophils, by DC is known to inhibit their production of pro-inflammatory cytokines, expression of costimulatory molecules, and their ability to stimulate T-cell proliferation [14], [38], [39]. The exploitation of these inhibitory signals by microbial pathogens is suggested by in vitro studies showing that M. tuberculosis-induced activation of human iDC can be inhibited by their co-culture with apoptotic neutrophils [40], and that Plasmodium falciparum-infected erythrocytes can inhibit the maturation of mouse DCs by binding to CD36, a known recognition receptor for apoptotic cells [41]. The present studies are the first to demonstrate efferocytosis involving neutrophils and DCs in an infection driven inflammatory setting in vivo. The sequestration of Leishmania antigens within apoptotic neutrophils would seem an especially efficient process to exploit the immunosuppressive signals conferred by the clearance of dying cells by DCs. Removing host neutrophils as a source of apoptotic cells was sufficient to reconstitute the immune function of infected DCs. It should be noted that in contrast to recent studies [42], we did not observe a reduction in either the total number of DCs nor infected DCs recovered from the ear following neutrophil depletion (Figure 5D and E). We would offer that while the prior study was confined to cells migrating out of the ear dermis ex vivo, our analysis was based on the greater recovery of cells following enzymatic digestion of the tissue. By comparing the ex vivo APC function of infected DCs recovered from the skin of mice depleted or not of neutrophils, and normalized for their RFP signals, the inhibitory effects of neutrophil uptake on DC maturation and Leishmania specific T cell activation could be formally demonstrated. The consequence of this inhibition in effectively delaying the onset of Leishmania specific T cell priming in vivo was directly supported by the enhanced, early OT-II priming to Lm-derived OVA in the neutrophil depleted mice. The neutrophil - DC interactions that inhibit T cell priming following needle challenge with L. major might be relevant to more general vaccination protocols in which an acute neutrophilic infiltrate accumulates at the site of antigen deposition. A recent report by Yang et al. [43] described the negative influence of neutrophils on the T and B cell responses to protein antigens administered by needle in the footpad. It is clear, however, that apoptotic neutrophils can also provide a source of immunogenic molecules to DC, especially for cross-priming, and especially if accompanied by extrinsic maturation signals [44], [45]. The relative paucity of activation signals associated with the phagocytosis of Leishmania promastigotes by neutrophils is suggested by the fact that the parasite traffics to a non-lytic compartment, avoids activation of the NADPH oxidase, and survives capture by these cells [5], [37]. It should be noted that PtdSer exposure on the parasites themselves has been suggested to facilitate their silent entry into macrophages, [46], [47], and may be especially relevant to their initial survival in neutrophils. Following neutrophil depletion, or the resolution of the first wave of neutrophils in the site, the majority of the infected DCs recovered from the skin lacked neutrophil markers, and are presumed to have taken up the parasite directly. By contrast to the absence of activation signals associated with the direct uptake of L. major metacyclic promastigotes by macrophages, the activation of human and mouse DCs following their phagocytosis of these organisms in vitro is well described [48]. Direct uptake might allow for parasite antigens to be more accessible to the MHC class I and II processing machinery, for parasite encoded TLR agonists to more efficiently engage their respective receptors, and for activation pathways to proceed in the absence of the inhibitory signals induced by apoptotic cell clearance. By two weeks, the priming conditions had clearly improved, and neutrophil depletion did not further enhance the CD4+ T cell response, despite the reappearance of neutrophils in the site. In contrast to the initial wave, however, the infected neutrophils recovered at two weeks represented a small percentage of the total population of infected cells, and the majority of infected DCs no longer harbored neutrophil markers. It is likely that the conditions of neutrophil recruitment to and activation in the skin during the active stage of disease, possibly Th17 driven at this later time, are distinct from those associated with the acute infiltrate, and that the influence of these respective neutrophil populations on the anti-leishmanial response will also be distinct. In the current studies, there was a significant difference in the effects of the neutrophil depleting antibodies, 1A8 and RB6-8C5, in potentiating the early OT-II response to infection with Lm SP-OVA in the skin. The 1A8 treatment critically confines the enhanced priming observed to specific depletion of Ly6G+ neutrophils. The more powerful effects observed with the RB6-8C5 antibody is consistent with the more efficient depletion of neutrophils that was achieved, although the removal of an additional population of GR-1+ myeloid cells with suppressor activity [49], [50] cannot be discounted. While our studies have employed a relatively high dose, needle challenge in order to recover a sufficient number of infected cells from the ear dermis for analysis, it should be emphasized that the initial wave of neutrophil recruitment to the infected sand fly bite site is more massive, localized, and sustained compared to the needle injection site [22]. This may explain why the ablation of the early neutrophilic response had such a strong effect in promoting protection against sand fly transmitted infection as compared to needle challenge [5], [51]–[53]. Thus, the impact of the early neutrophil - DC interactions described in these studies may be especially relevant to the inflammatory conditions elicited by natural sand fly transmission, as well as to that of other vector borne pathogens, in promoting the early establishment of infection and the progression of disease. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Animal Care and Use Committee of the NIAID, NIH (protocol number LPD 68E). All mice were maintained at the NIAID animal care facility under specific pathogen-free conditions. Female C57BL/6 and B6. SJL congenic mice, and RAG1-deficient OT-II CD4+ TCR transgenic mice were purchased from Taconic Laboratories. C57BL/6 LYS-eGFP knock-in mice [18] were a gift from T. Graf (Albert Einstein University, NY) and were bred at Taconic Laboratories. Experiments were carried out using different lines of L. major: L. major Friedlin strain FV1 (MHOM/IL/80/FN); a stable transfected line of L. major FV1 promastigotes expressing a red fluorescent protein (Lm-RFP), L. major FV1 promastigotes expressing a portion of the ovalbumin gene encoding amino acids 139 to 386 containing the class II restricted epitope recognized by OT-II TCR transgenic CD4+ T cells (Lm- SP-OVA), and L. major V1- transfected with the control plasmid expressing Leishmania donovani 3′ nucleotidase-nuclease (Lm-NT). Transfected lines were generated as described previously [54]–[55]. Parasites were grown at 26°C in medium 199 supplemented with 20% heat-inactivated FCS (Gemini Bio-Products), 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 40 mM Hepes, 0. 1 mM adenine (in 50 mM Hepes), 5 mg/ml hemin (in 50% triethanolamine), 1 mg/ml 6-biotin (M199/S), and 50 µg/ml of Geneticin (Gibco). Infective-stage, metacyclic promastigotes of L. major were isolated from stationary cultures (4–5 days old) by negative selection using peanut agglutinin (PNA, Vector Laboratories Inc). For flow cytometric studies of dermal and draining lymph node cells, mice were infected with the specified number of metacyclic promastigotes in the ear dermis by i. d. injection in a volume of 10 µl. In parallel, sham mice received i. d. injection of DMEM in a volume of 10 µl. To obtain chronically infected mice, animals were infected 16–20 weeks previously with 104 L. major FV1 metacyclic promastigotes in the left hind footpad. Ear tissue was prepared as previously described [2]. Briefly, the two sheets of infected ear dermis were separated, deposited in DMEM containing 100 U/ml penicillin, 100 µg/ml streptomycin, and 0. 2 mg/ml Liberase CI purified enzyme blend (Roche Diagnostics Corp.), and incubated for 1 h and 30 min at 37°C. Digested tissue was placed in a grinder and processed in a tissue homogenizer (Medimachine; Becton Dickenson). Retromaxillary (ear) lymph nodes were removed, and mechanically dissociated using tweezers and a syringe plunger. Tissue homogenates were filtered through a 70 µm cell strainer (Falcon Products). Single-cell suspensions were incubated with an anti-Fc-γ III/II (CD16/32) receptor Ab (2. 4G2, BD Biosciences) in RPMI without phenol red (Gibco) containing 1% FCS and stained with fluorochrome-conjugated antibodies. The following antibodies were used: APC- anti-mouse CD11c (HL3, BD Biosciences), PE-Cy7- anti-mouse CD11c (N418, eBioscience), PerCP-Cy5. 5 or PE-Cy7- anti-mouse CD11b (M1/70, eBioscience); PerCP-Cy5. 5- anti-mouse Ly6C (HK1. 4, eBioscience); FITC- anti-mouse Ly6G (1A8, eBioscience); FITC- anti-mouse GR-1 (RB6-8C5, BD Biosciences); eFluor anti-mouse F4/80 (BM8, eBioscience), Alexafluor-700 anti-mouse MHC II (M5/114. 15. 2, eBioscience), APC- anti-mouse CD103 (M290, eBioscience), A488- anti-mouse Langerin (929F3. 01, Dendritics), APC- anti-mouse CD40 (1C10, eBioscience), FITC- anti-mouse CD80 (16-10A1, eBioscience), PerCP-Cy5. 5- anti-mouse CD86 (GL-1, BioLegend), APC- anti-mouse CD4 (RM4–5, eBioscience), PerCP-Cy5. 5- anti-mouse CD45. 2 (104, eBioscience); APC-eFluor 780 anti-mouse CD45. 1 (A20, eBioscience), FITC- anti-mouse myeloperoxidase (MPO) (8F4, Hycult), anti-human neutrophil elastase (NE) (H-57, Santa Cruz), FITC conjugated using and amine reactive probe (Sigma-Aldrich). The isotype controls used (all obtained from BD Biosciences) were rat IgG1 (R3–34) and rat IgG2b (A95-1). The staining of surface and intracytoplasmic markers was performed sequentially: the cells were stained first for their surface markers, followed by a permeabilization step with BD Cytofix/Cytoperm (BD Biosciences) and staining for Langerin, MPO or NE. For intracellular detection of cytokines, cells were first stimulated with Leukocyte Activation Cocktail, plus GolgiPlug (BD Biosciences) according the manufacturers' instructions for 4 h in vitro. Following surface staining and permeabilization, cells were then stained with a combination of anti-mouse antibodies: PerCP-Cy5. 5 anti-IL17A (eBio17B7, eBioscience) APC anti-IFN-g (XMG1. 2, eBioscience), PE anti-IL-10 (JES5-16E3, BD Bioscience) in Perm/Wash buffer (BD Bioscience). Intracellular staining was carried out for 30 minutes on ice. The data were collected and analyzed using CELLQuest software and a FACScalibur or FacsDIVA software and a FacsCANTO flow cytometer (BD Biosciences). Neutrophils, dendritic cells, macrophages and monocytes from the ear dermis were identified based on size (forward scatter) and granularity (side scatter) and by surface phenotype as indicated in the text and figure legends. Infected DCs (CD11c+RFP+) were purified using a FACSVantage or a FACsAria (BD Biosciences) cell sorter on cells recovered from the ear dermis 3 days after infection with 2×106 L. major-RFP. For the analysis of the capacity of infected, dermal DCs to induce the secretion of IFNγ by L. major specific T cells, 4×104 (Exp. 1) or 4. 5×104 (Exp. 2) infected dermal DCs pooled from 10 mice (20 ears) for each treatment group were co-cultured with 1×105 T cells purified by negative selection (Miltenyi Biotec) from draining lymph nodes (dLNs) of B6 mice with a healed, primary infection with L. major FV1. After 3 days, culture supernatants were analyzed for IFN-γ production by ELISA (eBioscience). For adoptive transfer experiments, CD4+ T cells were purified from spleens and lymph nodes of RAG1-deficient OT-II CD4+ TCR transgenic mice by negative selection (Miltenyi Biotec). Purified CD4+ T cells were incubated at 2. 5–5×107 cells/ml in PBS with 0. 5 µM CFSE (Invitrogen) for 10 min at 37°C. The reaction was stopped with 10% normal mouse serum, and the cells were washed twice with cold PBS/0. 1% BSA. B6. SJL congenic mice received intravenously (i. v.) 2–5×105 CFSE-labeled, purified CD4+ OT-II T cells either the same day or 14 days after challenge in the ear dermis with 105 metacyclic promastigotes. Six days after adoptive transfer, the dLNs were removed and analyzed by flow cytometry. To obtain neutrophils recruited to the site of infection in the skin, LYS-eGFP mice were inoculated in the ear dermis with 2×106 Lm-RFP. Twelve hours later the ear tissue was prepared as described above and infected (RFP+eGFPhi) and uninfected (RFP−eGFPhi) neutrophil populations were sorted from dermal tissue using a FACSVantage or a FACsAria (BD Biosciences) cell sorter. Sorted populations were washed once and immediately analyzed for apoptosis or injected into the ear dermis of C57BL/6 and B6SJL recipient mice in a volume of 10 ul. Sorted, infected (RFP+eGFPhi) and uninfected (RFP−eGFPhi) neutrophil populations were stained with Annexin-V-APC and 7-AAD (BD Biosciences) as recommended by the manufacturer. For TUNEL assays, neutrophil populations were fixed in 4% paraformaldehyde, and then labeled with the Beckman Coulter Mebstain Apoptosis kit using biotinylated dUTP. Cells were then incubated with streptavidin-conjugated APC (BD Pharmingen) for 30 min at room temperature. Cells were analyzed by flow cytometry. Neutrophils were depleted employing a single i. p. injection of 0. 5 mg RB6-8C5 (anti-Gr-1), or 1 mg of 1A8 (anti-Ly6G, BioXCell), or GL113 (control IgG, BioXCell), 1 d prior to parasite injection. The efficiency and specificity of the depletions were evaluated on dermal cell preparations, and on heparinized whole blood. Statistical significance between groups was determined by the unpaired, two-tailed student' s t test using Prism software (GraphPad).

Prior studies in mice have shown that the inoculation of Leishmania major into the skin by sand fly bite or by needle provokes a massive recruitment of neutrophils that take up the parasite, and that this response somehow suppresses immunity since neutrophil depletion results in better control of the infection. We investigated how neutrophils recruited to the injection site might interact with and suppress the function of dendritic cells (DCs) in the skin. Infected neutrophils recovered from the skin expressed increased levels of apoptotic markers compared to uninfected neutrophils, and were efficiently taken up by dermal DCs when injected back into the skin. When dermal DCs were permitted to take up parasites in the absence of neutrophils, their expression of activation markers and their ability to present Leishmania antigens were enhanced. Neutrophil depletion also enhanced the activation of Leishmania specific CD4+ T cells in vivo. The results suggest that for insect borne pathogens like Leishmania that provoke a strong inflammatory response at the site of infection, the immunosuppressive effects associated with the apoptotic cell clearance function of DCs will inhibit the early development of immunity.

lay_plos

Dobbin of Ours Cuff's fight with Dobbin, and the unexpected issue of that contest, will long be remembered by every man who was educated at Dr. Swishtail's famous school. The latter Youth (who used to be called Heigh-ho Dobbin, Gee-ho Dobbin, and by many other names indicative of puerile contempt) was the quietest, the clumsiest, and, as it seemed, the dullest of all Dr. Swishtail's young gentlemen. His parent was a grocer in the city: and it was bruited abroad that he was admitted into Dr. Swishtail's academy upon what are called "mutual principles"--that is to say, the expenses of his board and schooling were defrayed by his father in goods, not money; and he stood there--most at the bottom of the school--in his scraggy corduroys and jacket, through the seams of which his great big bones were bursting--as the representative of so many pounds of tea, candles, sugar, mottled-soap, plums (of which a very mild proportion was supplied for the puddings of the establishment), and other commodities. A dreadful day it was for young Dobbin when one of the youngsters of the school, having run into the town upon a poaching excursion for hardbake and polonies, espied the cart of Dobbin & Rudge, Grocers and Oilmen, Thames Street, London, at the Doctor's door, discharging a cargo of the wares in which the firm dealt. Young Dobbin had no peace after that. The jokes were frightful, and merciless against him. "Hullo, Dobbin," one wag would say, "here's good news in the paper. Sugars is ris', my boy." Another would set a sum--"If a pound of mutton-candles cost sevenpence-halfpenny, how much must Dobbin cost?" and a roar would follow from all the circle of young knaves, usher and all, who rightly considered that the selling of goods by retail is a shameful and infamous practice, meriting the contempt and scorn of all real gentlemen. "Your father's only a merchant, Osborne," Dobbin said in private to the little boy who had brought down the storm upon him. At which the latter replied haughtily, "My father's a gentleman, and keeps his carriage"; and Mr. William Dobbin retreated to a remote outhouse in the playground, where he passed a half-holiday in the bitterest sadness and woe. Who amongst us is there that does not recollect similar hours of bitter, bitter childish grief? Who feels injustice; who shrinks before a slight; who has a sense of wrong so acute, and so glowing a gratitude for kindness, as a generous boy? and how many of those gentle souls do you degrade, estrange, torture, for the sake of a little loose arithmetic, and miserable dog-latin? Now, William Dobbin, from an incapacity to acquire the rudiments of the above language, as they are propounded in that wonderful book the Eton Latin Grammar, was compelled to remain among the very last of Doctor Swishtail's scholars, and was "taken down" continually by little fellows with pink faces and pinafores when he marched up with the lower form, a giant amongst them, with his downcast, stupefied look, his dog's-eared primer, and his tight corduroys. High and low, all made fun of him. They sewed up those corduroys, tight as they were. They cut his bed-strings. They upset buckets and benches, so that he might break his shins over them, which he never failed to do. They sent him parcels, which, when opened, were found to contain the paternal soap and candles. There was no little fellow but had his jeer and joke at Dobbin; and he bore everything quite patiently, and was entirely dumb and miserable. Cuff, on the contrary, was the great chief and dandy of the Swishtail Seminary. He smuggled wine in. He fought the town-boys. Ponies used to come for him to ride home on Saturdays. He had his top-boots in his room, in which he used to hunt in the holidays. He had a gold repeater: and took snuff like the Doctor. He had been to the Opera, and knew the merits of the principal actors, preferring Mr. Kean to Mr. Kemble. He could knock you off forty Latin verses in an hour. He could make French poetry. What else didn't he know, or couldn't he do? They said even the Doctor himself was afraid of him. Cuff, the unquestioned king of the school, ruled over his subjects, and bullied them, with splendid superiority. This one blacked his shoes: that toasted his bread, others would fag out, and give him balls at cricket during whole summer afternoons. "Figs" was the fellow whom he despised most, and with whom, though always abusing him, and sneering at him, he scarcely ever condescended to hold personal communication. One day in private, the two young gentlemen had had a difference. Figs, alone in the schoolroom, was blundering over a home letter; when Cuff, entering, bade him go upon some message, of which tarts were probably the subject. "I can't," says Dobbin; "I want to finish my letter." "You CAN'T?" says Mr. Cuff, laying hold of that document (in which many words were scratched out, many were mis-spelt, on which had been spent I don't know how much thought, and labour, and tears; for the poor fellow was writing to his mother, who was fond of him, although she was a grocer's wife, and lived in a back parlour in Thames Street). "You CAN'T?" says Mr. Cuff: "I should like to know why, pray? Can't you write to old Mother Figs to-morrow?" "Don't call names," Dobbin said, getting off the bench very nervous. "Well, sir, will you go?" crowed the cock of the school. "Put down the letter," Dobbin replied; "no gentleman readth letterth." "Well, NOW will you go?" says the other. "No, I won't. Don't strike, or I'll THMASH you," roars out Dobbin, springing to a leaden inkstand, and looking so wicked, that Mr. Cuff paused, turned down his coat sleeves again, put his hands into his pockets, and walked away with a sneer. But he never meddled personally with the grocer's boy after that; though we must do him the justice to say he always spoke of Mr. Dobbin with contempt behind his back. Some time after this interview, it happened that Mr. Cuff, on a sunshiny afternoon, was in the neighbourhood of poor William Dobbin, who was lying under a tree in the playground, spelling over a favourite copy of the Arabian Nights which he had apart from the rest of the school, who were pursuing their various sports--quite lonely, and almost happy. If people would but leave children to themselves; if teachers would cease to bully them; if parents would not insist upon directing their thoughts, and dominating their feelings--those feelings and thoughts which are a mystery to all (for how much do you and I know of each other, of our children, of our fathers, of our neighbour, and how far more beautiful and sacred are the thoughts of the poor lad or girl whom you govern likely to be, than those of the dull and world-corrupted person who rules him?)--if, I say, parents and masters would leave their children alone a little more, small harm would accrue, although a less quantity of as in praesenti might be acquired. Well, William Dobbin had for once forgotten the world, and was away with Sindbad the Sailor in the Valley of Diamonds, or with Prince Ahmed and the Fairy Peribanou in that delightful cavern where the Prince found her, and whither we should all like to make a tour; when shrill cries, as of a little fellow weeping, woke up his pleasant reverie; and looking up, he saw Cuff before him, belabouring a little boy. It was the lad who had peached upon him about the grocer's cart; but he bore little malice, not at least towards the young and small. "How dare you, sir, break the bottle?" says Cuff to the little urchin, swinging a yellow cricket-stump over him. The boy had been instructed to get over the playground wall (at a selected spot where the broken glass had been removed from the top, and niches made convenient in the brick); to run a quarter of a mile; to purchase a pint of rum-shrub on credit; to brave all the Doctor's outlying spies, and to clamber back into the playground again; during the performance of which feat, his foot had slipt, and the bottle was broken, and the shrub had been spilt, and his pantaloons had been damaged, and he appeared before his employer a perfectly guilty and trembling, though harmless, wretch. "How dare you, sir, break it?" says Cuff; "you blundering little thief. You drank the shrub, and now you pretend to have broken the bottle. Hold out your hand, sir." Down came the stump with a great heavy thump on the child's hand. A moan followed. Dobbin looked up. The Fairy Peribanou had fled into the inmost cavern with Prince Ahmed: the Roc had whisked away Sindbad the Sailor out of the Valley of Diamonds out of sight, far into the clouds: and there was everyday life before honest William; and a big boy beating a little one without cause. "Hold out your other hand, sir," roars Cuff to his little schoolfellow, whose face was distorted with pain. Dobbin quivered, and gathered himself up in his narrow old clothes. "Take that, you little devil!" cried Mr. Cuff, and down came the wicket again on the child's hand.--Don't be horrified, ladies, every boy at a public school has done it. Your children will so do and be done by, in all probability. Down came the wicket again; and Dobbin started up. I can't tell what his motive was. Torture in a public school is as much licensed as the knout in Russia. It would be ungentlemanlike (in a manner) to resist it. Perhaps Dobbin's foolish soul revolted against that exercise of tyranny; or perhaps he had a hankering feeling of revenge in his mind, and longed to measure himself against that splendid bully and tyrant, who had all the glory, pride, pomp, circumstance, banners flying, drums beating, guards saluting, in the place. Whatever may have been his incentive, however, up he sprang, and screamed out, "Hold off, Cuff; don't bully that child any more; or I'll--" "Or you'll what?" Cuff asked in amazement at this interruption. "Hold out your hand, you little beast." "I'll give you the worst thrashing you ever had in your life," Dobbin said, in reply to the first part of Cuff's sentence; and little Osborne, gasping and in tears, looked up with wonder and incredulity at seeing this amazing champion put up suddenly to defend him: while Cuff's astonishment was scarcely less. Fancy our late monarch George III when he heard of the revolt of the North American colonies: fancy brazen Goliath when little David stepped forward and claimed a meeting; and you have the feelings of Mr. Reginald Cuff when this rencontre was proposed to him. "After school," says he, of course; after a pause and a look, as much as to say, "Make your will, and communicate your last wishes to your friends between this time and that." "As you please," Dobbin said. "You must be my bottle holder, Osborne." "Well, if you like," little Osborne replied; for you see his papa kept a carriage, and he was rather ashamed of his champion. Yes, when the hour of battle came, he was almost ashamed to say, "Go it, Figs"; and not a single other boy in the place uttered that cry for the first two or three rounds of this famous combat; at the commencement of which the scientific Cuff, with a contemptuous smile on his face, and as light and as gay as if he was at a ball, planted his blows upon his adversary, and floored that unlucky champion three times running. At each fall there was a cheer; and everybody was anxious to have the honour of offering the conqueror a knee. "What a licking I shall get when it's over," young Osborne thought, picking up his man. "You'd best give in," he said to Dobbin; "it's only a thrashing, Figs, and you know I'm used to it." But Figs, all whose limbs were in a quiver, and whose nostrils were breathing rage, put his little bottle-holder aside, and went in for a fourth time. As he did not in the least know how to parry the blows that were aimed at himself, and Cuff had begun the attack on the three preceding occasions, without ever allowing his enemy to strike, Figs now determined that he would commence the engagement by a charge on his own part; and accordingly, being a left-handed man, brought that arm into action, and hit out a couple of times with all his might--once at Mr. Cuff's left eye, and once on his beautiful Roman nose. Cuff went down this time, to the astonishment of the assembly. "Well hit, by Jove," says little Osborne, with the air of a connoisseur, clapping his man on the back. "Give it him with the left, Figs my boy." Figs's left made terrific play during all the rest of the combat. Cuff went down every time. At the sixth round, there were almost as many fellows shouting out, "Go it, Figs," as there were youths exclaiming, "Go it, Cuff." At the twelfth round the latter champion was all abroad, as the saying is, and had lost all presence of mind and power of attack or defence. Figs, on the contrary, was as calm as a quaker. His face being quite pale, his eyes shining open, and a great cut on his underlip bleeding profusely, gave this young fellow a fierce and ghastly air, which perhaps struck terror into many spectators. Nevertheless, his intrepid adversary prepared to close for the thirteenth time. If I had the pen of a Napier, or a Bell's Life, I should like to describe this combat properly. It was the last charge of the Guard--(that is, it would have been, only Waterloo had not yet taken place)--it was Ney's column breasting the hill of La Haye Sainte, bristling with ten thousand bayonets, and crowned with twenty eagles--it was the shout of the beef-eating British, as leaping down the hill they rushed to hug the enemy in the savage arms of battle--in other words, Cuff coming up full of pluck, but quite reeling and groggy, the Fig-merchant put in his left as usual on his adversary's nose, and sent him down for the last time. "I think that will do for him," Figs said, as his opponent dropped as neatly on the green as I have seen Jack Spot's ball plump into the pocket at billiards; and the fact is, when time was called, Mr. Reginald Cuff was not able, or did not choose, to stand up again. And now all the boys set up such a shout for Figs as would have made you think he had been their darling champion through the whole battle; and as absolutely brought Dr. Swishtail out of his study, curious to know the cause of the uproar. He threatened to flog Figs violently, of course; but Cuff, who had come to himself by this time, and was washing his wounds, stood up and said, "It's my fault, sir--not Figs'--not Dobbin's. I was bullying a little boy; and he served me right." By which magnanimous speech he not only saved his conqueror a whipping, but got back all his ascendancy over the boys which his defeat had nearly cost him. Young Osborne wrote home to his parents an account of the transaction. Sugarcane House, Richmond, March, 18-- DEAR MAMA,--I hope you are quite well. I should be much obliged to you to send me a cake and five shillings. There has been a fight here between Cuff & Dobbin. Cuff, you know, was the Cock of the School. They fought thirteen rounds, and Dobbin Licked. So Cuff is now Only Second Cock. The fight was about me. Cuff was licking me for breaking a bottle of milk, and Figs wouldn't stand it. We call him Figs because his father is a Grocer--Figs & Rudge, Thames St., City--I think as he fought for me you ought to buy your Tea & Sugar at his father's. Cuff goes home every Saturday, but can't this, because he has 2 Black Eyes. He has a white Pony to come and fetch him, and a groom in livery on a bay mare. I wish my Papa would let me have a Pony, and I am Your dutiful Son, GEORGE SEDLEY OSBORNE P.S.--Give my love to little Emmy. I am cutting her out a Coach in cardboard. Please not a seed-cake, but a plum-cake. In consequence of Dobbin's victory, his character rose prodigiously in the estimation of all his schoolfellows, and the name of Figs, which had been a byword of reproach, became as respectable and popular a nickname as any other in use in the school. "After all, it's not his fault that his father's a grocer," George Osborne said, who, though a little chap, had a very high popularity among the Swishtail youth; and his opinion was received with great applause. It was voted low to sneer at Dobbin about this accident of birth. "Old Figs" grew to be a name of kindness and endearment; and the sneak of an usher jeered at him no longer. And Dobbin's spirit rose with his altered circumstances. He made wonderful advances in scholastic learning. The superb Cuff himself, at whose condescension Dobbin could only blush and wonder, helped him on with his Latin verses; "coached" him in play-hours: carried him triumphantly out of the little-boy class into the middle-sized form; and even there got a fair place for him. It was discovered, that although dull at classical learning, at mathematics he was uncommonly quick. To the contentment of all he passed third in algebra, and got a French prize-book at the public Midsummer examination. You should have seen his mother's face when Telemaque (that delicious romance) was presented to him by the Doctor in the face of the whole school and the parents and company, with an inscription to Gulielmo Dobbin. All the boys clapped hands in token of applause and sympathy. His blushes, his stumbles, his awkwardness, and the number of feet which he crushed as he went back to his place, who shall describe or calculate? Old Dobbin, his father, who now respected him for the first time, gave him two guineas publicly; most of which he spent in a general tuck-out for the school: and he came back in a tail-coat after the holidays. Dobbin was much too modest a young fellow to suppose that this happy change in all his circumstances arose from his own generous and manly disposition: he chose, from some perverseness, to attribute his good fortune to the sole agency and benevolence of little George Osborne, to whom henceforth he vowed such a love and affection as is only felt by children--such an affection, as we read in the charming fairy-book, uncouth Orson had for splendid young Valentine his conqueror. He flung himself down at little Osborne's feet, and loved him. Even before they were acquainted, he had admired Osborne in secret. Now he was his valet, his dog, his man Friday. He believed Osborne to be the possessor of every perfection, to be the handsomest, the bravest, the most active, the cleverest, the most generous of created boys. He shared his money with him: bought him uncountable presents of knives, pencil-cases, gold seals, toffee, Little Warblers, and romantic books, with large coloured pictures of knights and robbers, in many of which latter you might read inscriptions to George Sedley Osborne, Esquire, from his attached friend William Dobbin--the which tokens of homage George received very graciously, as became his superior merit. So that Lieutenant Osborne, when coming to Russell Square on the day of the Vauxhall party, said to the ladies, "Mrs. Sedley, Ma'am, I hope you have room; I've asked Dobbin of ours to come and dine here, and go with us to Vauxhall. He's almost as modest as Jos." "Modesty! pooh," said the stout gentleman, casting a vainqueur look at Miss Sharp. "He is--but you are incomparably more graceful, Sedley," Osborne added, laughing. "I met him at the Bedford, when I went to look for you; and I told him that Miss Amelia was come home, and that we were all bent on going out for a night's pleasuring; and that Mrs. Sedley had forgiven his breaking the punch-bowl at the child's party. Don't you remember the catastrophe, Ma'am, seven years ago?" "Over Mrs. Flamingo's crimson silk gown," said good-natured Mrs. Sedley. "What a gawky it was! And his sisters are not much more graceful. Lady Dobbin was at Highbury last night with three of them. Such figures! my dears." "The Alderman's very rich, isn't he?" Osborne said archly. "Don't you think one of the daughters would be a good spec for me, Ma'am?" "You foolish creature! Who would take you, I should like to know, with your yellow face?" "Mine a yellow face? Stop till you see Dobbin. Why, he had the yellow fever three times; twice at Nassau, and once at St. Kitts." "Well, well; yours is quite yellow enough for us. Isn't it, Emmy?" Mrs. Sedley said: at which speech Miss Amelia only made a smile and a blush; and looking at Mr. George Osborne's pale interesting countenance, and those beautiful black, curling, shining whiskers, which the young gentleman himself regarded with no ordinary complacency, she thought in her little heart that in His Majesty's army, or in the wide world, there never was such a face or such a hero. "I don't care about Captain Dobbin's complexion," she said, "or about his awkwardness. I shall always like him, I know," her little reason being, that he was the friend and champion of George. "There's not a finer fellow in the service," Osborne said, "nor a better officer, though he is not an Adonis, certainly." And he looked towards the glass himself with much naivete; and in so doing, caught Miss Sharp's eye fixed keenly upon him, at which he blushed a little, and Rebecca thought in her heart, "Ah, mon beau Monsieur! I think I have YOUR gauge"--the little artful minx! That evening, when Amelia came tripping into the drawing-room in a white muslin frock, prepared for conquest at Vauxhall, singing like a lark, and as fresh as a rose--a very tall ungainly gentleman, with large hands and feet, and large ears, set off by a closely cropped head of black hair, and in the hideous military frogged coat and cocked hat of those times, advanced to meet her, and made her one of the clumsiest bows that was ever performed by a mortal. This was no other than Captain William Dobbin, of His Majesty's Regiment of Foot, returned from yellow fever, in the West Indies, to which the fortune of the service had ordered his regiment, whilst so many of his gallant comrades were reaping glory in the Peninsula. He had arrived with a knock so very timid and quiet that it was inaudible to the ladies upstairs: otherwise, you may be sure Miss Amelia would never have been so bold as to come singing into the room. As it was, the sweet fresh little voice went right into the Captain's heart, and nestled there. When she held out her hand for him to shake, before he enveloped it in his own, he paused, and thought--"Well, is it possible--are you the little maid I remember in the pink frock, such a short time ago--the night I upset the punch-bowl, just after I was gazetted? Are you the little girl that George Osborne said should marry him? What a blooming young creature you seem, and what a prize the rogue has got!" All this he thought, before he took Amelia's hand into his own, and as he let his cocked hat fall. His history since he left school, until the very moment when we have the pleasure of meeting him again, although not fully narrated, has yet, I think, been indicated sufficiently for an ingenious reader by the conversation in the last page. Dobbin, the despised grocer, was Alderman Dobbin--Alderman Dobbin was Colonel of the City Light Horse, then burning with military ardour to resist the French Invasion. Colonel Dobbin's corps, in which old Mr. Osborne himself was but an indifferent corporal, had been reviewed by the Sovereign and the Duke of York; and the colonel and alderman had been knighted. His son had entered the army: and young Osborne followed presently in the same regiment. They had served in the West Indies and in Canada. Their regiment had just come home, and the attachment of Dobbin to George Osborne was as warm and generous now as it had been when the two were schoolboys. So these worthy people sat down to dinner presently. They talked about war and glory, and Boney and Lord Wellington, and the last Gazette. In those famous days every gazette had a victory in it, and the two gallant young men longed to see their own names in the glorious list, and cursed their unlucky fate to belong to a regiment which had been away from the chances of honour. Miss Sharp kindled with this exciting talk, but Miss Sedley trembled and grew quite faint as she heard it. Mr. Jos told several of his tiger-hunting stories, finished the one about Miss Cutler and Lance the surgeon; helped Rebecca to everything on the table, and himself gobbled and drank a great deal. He sprang to open the door for the ladies, when they retired, with the most killing grace--and coming back to the table, filled himself bumper after bumper of claret, which he swallowed with nervous rapidity. "He's priming himself," Osborne whispered to Dobbin, and at length the hour and the carriage arrived for Vauxhall.

Another kid at the school was Cuff, who was the popular, bullying jock type. Everyone who was there will always remember the day Dobbin saw Cuff about to beat up George. It's not really clear how old everyone is at this point. We'll hazard a guess that George was 9ish, Dobbin 11ish, and Cuff 14ish. Point being that Cuff was way bigger than George. Dobbin intervened, and he and Cuff planned to fight after school. The fight was very much one-sided, with a half-crazed Dobbin "licking" Cuff. After this, Dobbin's reputation went way up, and the other kids started being so nice to him that he actually began doing really well in school. Turns out he wasn't stupid after all, but just too miserable to study or otherwise function. The other result of the fight? Dobbin and George became best friends. Or rather, George condescended to let Dobbin be really attached to him. Since all this happened, however, Dobbin's dad has become an alderman and has gotten knighted, meaning he is now Sir Dobbin, and is thus a much higher social class than when Dobbin was at school. Dobbin is now Captain Dobbin and is an officer in George's regiment. The reason for all this history? To explain why George invites Dobbin to Vauxhall as kind of a fifth wheel. Dobbin comes over to the Sedley house, sees Amelia, and instantly falls in love with her. The five young people hang out, having a good time, until it's time to go. Jos has a few strong drinks for the road, or maybe to give himself some liquid courage to ask Becky to marry him.

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A tweet from Twitter Inc. announcing its initial public offering is shown in this photo illustration in Toronto, September 12, 2013. LOS ANGELES/ SAN FRANCISCO When Twitter goes public in coming weeks, one of the biggest winners will be a 47-year-old financier who guards his secrecy so zealously that he employs a person to take down his Wikipedia entry and scrub his picture from the Internet. Over the past two years, Suhail Rizvi, founder of New York private equity firm Rizvi Traverse Management, has quietly amassed a stake of more than 15 percent in the microblogging phenomenon for himself and his investors at a cost of more than $1 billion, according to three people with knowledge of his investments. While Rizvi was known to be an investor in Twitter, the extent of his involvement had not previously been reported. Twitter made its IPO registration documents public late Thursday, setting the stage for the most closely watched initial public offering since Facebook's in 2012. Rizvi Traverse is listed as one of the institutional shareholders with at least a 5 percent ownership stake, but no further details were disclosed. The shares Rizvi purchased were distributed among investors via multiple vehicles, sources said, and the size of his personal stake is not known. People with direct knowledge of his investment activities say that Rizvi, backed by Chris Sacca, a former Google executive and Twitter investor, was instrumental in attracting large private investors to the microblogging site, serving as matchmaker between the company's founders and global financiers from Wall Street to Riyadh. Rizvi declined to comment for this article. Sacca, a longtime friend, gave him an entree into tech investing in 2011 - when Twitter was still struggling to make money. From there, Rizvi scored stakes in some of the most sought-after Internet startups, from Facebook Inc before it went public to Square and Flipboard. Rizvi's string of tech deals came amid intense competition among hedge funds and private equity investors to secure shares in startups, highlighted by Russian billionaire Yuri Milner's 2009 investment in Facebook. With tech companies waiting later than ever to go public, some investors believed they may miss out on the biggest gains if they wait to buy shares in public markets, when a company's value may no longer rise exponentially. DEEP-POCKETED INVESTORS The son of an Iowa psychology professor, Rizvi has networked with rich and powerful people including Queen Noor of Jordan and Google Inc's Larry Page and Eric Schmidt, devising financing schemes that leveraged his access to deep-pocketed investors, according to people who know Rizvi. Those who invest with Rizvi include British billionaire Richard Branson and Jeffrey Skoll, the former eBay executive and film producer, according to people with knowledge of the matter. It is not clear whether they are among the investors he brought into Twitter. Before turning his attention to the Internet, Rizvi's deal-making focused on Hollywood. He helped Hugh Hefner take Playboy Enterprises private; bought and then sold the Hollywood film studio behind the "Twilight" series; and led the buyout of a leading talent agency, International Creative Management (ICM). Rizvi is not alone among entertainment investors who have turned their focus to Silicon Valley. Former News Corp executive Peter Chernin's Chernin Group has invested in Tumblr, Pandora and Flipboard, while Michael Ovitz, the talent agent and former Disney CEO, has invested in Ron Conway's SV Angel funds, the tech incubator Y Combinator and venture capital firm Andreessen Horowitz. But few have operated on the scale of Rizvi. Twitter has a policy of restricting outside investors to only a handful, but Rizvi has had more freedom to bring in additional investors since he bought a large slice of the company. "He's not to be underestimated. His approach to traditional media as well as technology has put him in a great position," said Jeremy Zimmer, chief executive of United Talent Agency, a competitor of ICM. "His ICM investment was viable and gave him a seat at the table and a chance to make a sound investment in Twitter." TAG-TEAMING TWITTER In late 2010, Sacca approached Rizvi with an offer: Sacca's friend Evan Williams had stepped down as CEO of Twitter and was seeking to sell 10 percent of the company. Rizvi soon snapped up the shares for $340 million, according to people familiar with the matter. Following that first transaction, the two men formed a highly efficient tag team, the sources said. Sacca would seek shareholders who wanted to cash out, while Rizvi helped raise money to purchase the stock. The friends successfully pitched JPMorgan Chase & Co on a deal to buy more than $400 million worth of Twitter shares in 2011. Months later, Rizvi recruited Kingdom Holding Co, Saudi Prince Alwaleed's investment company, to buy an additional $300 million in stock in a separate vehicle. JPMorgan Chase and representatives for Alwaleed did not respond to requests for comment on the deal. By mid-2013, investment vehicles managed by Rizvi and Sacca had collectively bought more than $1 billion in shares, a stake that at one point amounted to nearly 20 percent of Twitter before it was diluted in recent months, sources said. Twitter declined to comment. Rizvi's Twitter connections opened the doors to other investing opportunities. Last year, Rizvi led a $200 million investment in Square, the mobile-payment processing company founded by Twitter co-founder Jack Dorsey, at a $3.25 billion valuation. On September 24, Rizvi led a $50 million financing round for the news reader app Flipboard, whose founder, Mike McCue, once sat on Twitter's board. But he has not always gotten his way in Silicon Valley. Although Rizvi indirectly owns some shares of Pinterest, Rizvi failed to purchase a significant stake when the online scrapboard site raised $100 million in 2012 and $200 million this year. It was unclear what stymied his attempts. "He fought tooth and nail for an allocation" but failed, an angel investor in Pinterest recalled. "He was willing to pay." It also remains unclear whether Rizvi's Internet investments other than Twitter will pan out. Some industry insiders note that he missed out on the huge gains that were made with very early investments in social media, and companies including Square and Flipboard remain unproven. Rizvi was able to buy only $100 million in Facebook shortly before its IPO, thus limiting his returns, according to people with knowledge of the matter. FROM IOWA FALLS TO SILICON VALLEY Rizvi, who owns a sprawling three-home compound in Greenwich, Connecticut, and a 1.65-acre Palm Beach, Florida, estate near Bill Gates and Michael Bloomberg, was born in India but moved to Iowa Falls, a town of 5,200 people, when he was five. Along with his older brother Ashraf, a hedge fund manager, Rizvi graduated from the University of Pennsylvania's Wharton business school. Both now serve on the undergraduate school's executive board. Rizvi worked as a real estate analyst while at Wharton, then he started and sold a telecom company. With the proceeds, he financed his first big buyout in 1995, when he bought the electronic manufacturing business of a Puerto Rico phone company. Refocusing it on making higher-cost equipment, he spent the next four years boosting the company's annual revenue from $10 million to $450 million. He did not break into the elite circles of media investing until 2004, after he founded Rizvi Traverse with John Giampetroni, a New York private equity investor. In 2005 the ICM talent agency, which represented stars like Mel Gibson and Robert Redford, was looking for financing as it struggled with the defection of key agents and stars. Rizvi took a controlling stake in ICM for $100 million, including $95 million in debt financing from Merrill Lynch & Co, according to a person with knowledge of the deal. Rizvi Traverse put up just $5 million. Later, he refinanced the agency for $300 million against future revenue from assets like "Friends," the popular show in which ICM holds a stake. That $300 million was enough to repay Merrill Lynch and provide a hefty return for Rizvi and his partners. ELUSIVE ON THE WEB Even as Rizvi's circles have broadened, he has continued to keep a low profile, both in his personal style - sometimes flying commercial, one Hollywood executive said - and in his hands-off approach to tech investing. Despite bringing capital, he has not taken a board seat at Twitter, Square or Flipboard. Rizvi's trusted lieutenant, Ben Kohn, has instead been the face of the firm on the West Coast, several entertainment industry executives said. Known for an aggressive negotiating style that contrasts with Rizvi's more reserved stance, Kohn manages a dozen employees out of a small office in Los Angeles. Rizvi's growing network includes the likes of Vivi Nevo, the secretive Time Warner Inc shareholder who also famously prided himself on being "UnGoogleable," mutual friends of the two men said. But while Nevo is now easy to find online, Rizvi maintains an elusive Web presence. The only readily found picture of Rizvi is a snapshot of him sitting with Twitter CEO Dick Costolo and Alwaleed in a New York hotel after the prince bought Twitter shares. The photo, on a Saudi news outlet, had irked Twitter executives and Rizvi, as they had preferred to keep the transaction quiet. (Reporting by Ronald Grover in Los Angeles and Gerry Shih in San Francisco; Additional reporting by Sue Zeidler in Los Angeles and Sarah McBride in San Francisco; Editing by Edwin Chan, Jonathan Weber, Tiffany Wu and Douglas Royalty) SAN FRANCISCO — The list of newly minted millionaires from Twitter ’s public offering will be longer than the average tweet. But the list of who stands to make into the hundreds of millions of dollars is tiny, and shows that sometimes when you roll the dice early in a company’s life the payoff can be enormous. A public filing Thursday listed some of Twitter’s biggest winners — five investment firms and a host of board members and current and former executives — that own sizable stakes in the company. But missing from the filing were two co-founders, several investors who took a chance on a scrappy young start-up with no business model and a few who were cautious in Twitter’s early years and threw the proverbial lottery ticket away. Among the more obvious winners listed were Evan Williams, who co-founded the company seven years ago. With a 12 percent stake worth about $1.2 billion based on Twitter’s most recent valuation of its shares, he is Twitter’s largest shareholder. Jack Dorsey, another co-founder and Twitter’s chairman, owns just under 5 percent of the company, worth about $483 million. Missing from the filing entirely was Biz Stone, who co-founded Twitter with Mr. Williams and Mr. Dorsey in 2006 and left the company in 2011. Mr. Stone, two people close to the company say, sold many of his early shares and could end up earning just 1 percent of Mr. Williams’s windfall. Noah Glass, the company’s oft-forgotten fourth co-founder, was also absent. Well-known investors like venture capitalist Marc Andreessen, who bet his own money on Twitter in its early days, also did not make the cut. That’s not to say that people not named in Thursday’s filing don’t stand to make, in many cases, millions of dollars when Twitter starts selling its shares. The Securities and Exchange Commission requires that companies list only investors that own more than 5 percent stakes, as well as compensation for top executives. The biggest winner among venture capital firms listed in the filing is Benchmark Capital, which owns more than 6 percent of the company and stands to make as much as a billion-dollar return on a check the firm wrote in 2009 when Twitter was still a 25-employee company. Union Square Ventures and Spark Capital, which invested in Twitter’s earliest rounds, have both hung on to more than a 5 percent stake in the company. Twitter eventually became Spark’s largest investment. According to the filing, DST Global, the investment firm founded by the Russian billionaire Yuri Milner, has managed to accumulate more than 5 percent of Twitter since it first invested two years ago. Perhaps the most surprising name among Twitter’s biggest shareholders was Suhail Rizvi, a little-known Hollywood investor who has quietly built up more than a 5 percent stake in Twitter through his private equity firm Rizvi Traverse Management. Mr. Rizvi, whose clients include Richard Branson, was first introduced to Twitter in 2010 through a long-time friend and early Twitter investor, Chris Sacca. Mr. Sacca, the founder of Lowercase Capital, approached him about buying an employee’s equity. That employee turned out to be Mr. Williams, who had left the company in October 2010 and wanted to sell around 10 percent of Twitter. Mr. Rizvi agreed to pay somewhere north of $300 million, according to someone familiar with Rizvi Traverse Management who spoke on the condition of anonymity. Mr. Rizvi continued to invest directly from employees on behalf of his clients, investing around $300 million on behalf of Prince Alwaleed bin Talal of Saudi Arabia and $400 million for JPMorgan Chase. Mr. Andreessen, a co-founder of Netscape, made a personal investment in Twitter in 2007, two years before he started his venture capital firm Andreessen Horowitz with Ben Horowitz. In 2011, the firm invested $80 million in Twitter after buying shares in secondary markets. The investment, which valued Twitter at around $4 billion even though it was still losing money, was criticized by other venture capitalists on Sand Hill Road, who believed Mr. Andreessen and his partners thought too highly of Twitter. Two years later, Andreessen Horowitz has already doubled the return on its investment based on Twitter’s own valuation of its stock last August, which put the company at $9.7 billion. The firm stands to quadruple its return if Twitter goes public at the $16 billion market valuation that some analysts are expecting. But one of Twitter’s luckiest investors may be Mike Maples, who put money into Twitter by happenstance, shortly after moving to Silicon Valley from Austin, Tex., in 2006. His first investment was a $25,000 check he wrote for a podcasting start-up called Odeo, which sputtered after Apple announced it would give away its podcasting services. Mr. Williams called up Odeo’s investors, which also included Josh Kopelman, a seed investor at First Round Capital; Mitch Kapor, a software pioneer; and Charles River Ventures, a venture capital firm, and offered to give their money back and pay what he had spent at Odeo out of his own pocket. But Mr. Maples told him to let it ride. “I told him, ‘Just as long as you let me invest in your next company, you can keep it,’ ” Mr. Maples recalled. “I don’t care if it’s a massage parlor.” That next company ended up being a microblogging service with no long-term design plans, and no revenue model, called Twttr. (The vowels were added later). On Thursday, Mr. Maples said he did not know how many shares in the company he owned. Like any good gambler, he said, he was too superstitious to count them before the offering. But, he said of his eventual return, “It will be good.” Odeo’s other investors did not get so lucky. They happily agreed to take back investments that today could be worth hundreds of millions. In those investors’ place will be a coterie of the co-founders’ friends like Greg Yaitanes, who was one of Twitter’s first nine investors, something Mr. Yaitanes recently described as a “nice badge of honor.” An Emmy-award winning television director, Mr. Yaitanes first began using Twitter when his childhood friend from Wellesley, Mass., Biz Stone, asked him to participate in Twitter’s testing phase. Later, when it came time to raise money, Mr. Stone called Mr. Yaitanes to see if he was interested in investing. Mr. Yaitanes recalled the conversation. He said yes. “My now ex-wife said if I invested in Twitter she would divorce me and now we’re divorced,” Mr. Yaitanes joked. “In hindsight, I wish I had put in more.”

As Twitter prepares to go public, there's plenty of people waiting in the wings to cash in; yesterday's filings reveal investment firms, board members, and execs will have the highest payouts, but one mysterious Hollywood financier also will add a good chunk of change to his bank account. Suhail Rizvi, whose clients include Richard Branson and Prince Alwaleed bin Talal of Saudi Arabia, has quietly set his hands on 15% of the company-split among himself and investors-at a cost of more than $1 billion, Reuters reports. Some 10% of that came from Twitter's co-founder Evan Williams, who sold the shares to Rizvi in 2010 for $340 million. It's still unclear how much is Rizvi's personally-thanks in part to his love of secrecy. Reuters has this great example: The 47-year-old pays someone to take down his Wikipedia page and ensure no photos of him appear on the Internet. It's doubtful Rizvi will get the largest single payout, however. The best bet there is Williams himself, whose 12% stake is worth $1.2 billion, reports the New York Times.

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Media playback is not supported on this device 'I would hope that I would win against Serena' Serena Williams disputes John McEnroe's claims that she would struggle to be in the world's top 700 if she was on the men's circuit. But the male player ranked 701 in the world - Dmitry Tursunov - believes he could beat her. Speaking to US radio station NPR, seven-time Grand Slam champion McEnroe said of former world number one Williams, who has won an Open-era record 23 Grand Slams: "If she played the men's circuit she'd be, like, 700 in the world." He qualified the comments by saying: "That doesn't mean I don't think Serena is an incredible player, and I suppose anything's possible; maybe at some point a women's tennis player can be better than anybody." But he added: "I just haven't seen that in any other sport, and I haven't seen it in tennis. If she had to just play the men's circuit, it would be an entirely different story." Williams later responded on Twitter: "Dear John, I adore and respect you but please please keep me out of your statements that are not factually based. "I've never played anyone ranked "there" nor do I have time. Respect me and my privacy as I'm trying to have a baby. Good day sir." Williams, 35, won her 23rd Grand Slam title at the Australian Open in January. It later transpired she was pregnant at the time 'She is pregnant, and I'm not' Tursunov, 34, was once ranked as high as 20th in the world. The Russian told BBC World Service Sport he did not think McEnroe was "trying to talk women's tennis down" but said "the reality" was that "men are stronger in general". "I would hope that I would win against Serena," he added. "It would be a similar argument to: who would run faster, the fastest woman or the fastest man? Tennis is becoming more and more a physical sport, so it's going to be hard for a woman to beat the men. "It's not black and white, there are lots of factors to take into account. Physically I might not be in the best shape of my life but as an overall package I'm much better than my ranking would suggest. She is pregnant, and I'm not. "I've never heard John say anything absolutely stupid - he knows his stuff. What he said about her being an incredible player is correct - explosive, powerful and she puts in a lot of work. But I would hope that I would win." Battle of the sexes A crowd of 30,000 watched Billie Jean King take on Bobby Riggs in Houston in 1973 Tennis matches between men and women have occurred before, mostly notably back in 1973 when Billie Jean King took on fellow American Bobby Riggs. Riggs, the world number one in the 1940s, retired in 1951 and at the age of 55 believed he could beat any of the top female players. King originally declined to play Riggs and Australian Margaret Court - at the time the top female player in the world - stepped in. Riggs won 6-2 6-1. But later that year, the top ranked women's player King - 26 years younger than Riggs at 29 - took him on in an exhibition match at the Houston Astrodome and won 6-4 6-3 6-3. A third 'Battle of the Sexes' match took in 1992 between American former world number one Jimmy Connors, aged 40 at the time, and Czech and American Martina Navratilova, who was 35. The match took place under special rules to make it more competitive - Connors was allowed only one serve per point, and Navratilova was allowed to hit into half the doubles court. Connors won 7-5 6-2. 'But Seriously,' Tennis Great John McEnroe Says He's Seeking 'Inner Peace' Enlarge this image toggle caption Maddie Meyer/Getty Images Maddie Meyer/Getty Images In the late 1970s and early 1980s, tennis great John McEnroe triumphed three times at Wimbledon and four times at the U.S. Open. But all his achievements on the court did not prepare him for life off of it. After his professional career ended, he dabbled as a talk show host and as an art collector and appeared in movies and TV shows. Above all, McEnroe wanted to be a rock guitarist in his wife's band, but, he admits: "That was not going to happen." His wife, singer Patty Smyth, told him, "I want to play mixed doubles with you at Wimbledon." To which he replied, "Well, you don't play tennis." And she said, "Exactly." During his tennis career, McEnroe became known for outbursts on the court when he thought umpires had missed a call. In one classic exchange, he yelled at an official, "You cannot be serious! That ball was on the line!" YouTube That line has followed him for decades. "If a day goes by where I don't hear that at least a couple times, it's a miracle," McEnroe says. So he has decided to embrace it: His first memoir was called You Cannot Be Serious, and his new memoir is called But Seriously. Interview Highlights On not taking himself too seriously Believe it or not, I didn't take myself too seriously back then.... Even though I'm extremely disappointed that the last seven years of my career I didn't play as well as I thought I was, or get better, or keep improving, I didn't want to quit tennis at 26 or 27. But Seriously by John McEnroe Hardcover, 277 pages purchase close overlay Buy Featured Book Your purchase helps support NPR programming. How? On reinventing himself after his pro career ended I was actually going through what turned out to be a separation and divorce from my first wife, [actress Tatum O'Neal], so I was unable to really think about anything else. We had three kids together and my head was all over the place and I couldn't even think about... the transition that I was anticipating I was going to be making.... I was sort of lost, but was open enough to experiment... so that I can find myself again, which isn't easy when you've peaked in your career at 26 years old. On why there aren't more great male American tennis players right now There's a lot of reasons, but the biggest one to me is the cost of it: the cost of play, the cost to train, the cost to get a court. All of this factors into the difficulty of getting a champion. The truth is... the game has become more athletic than ever, and quicker, you need to be more athletic, and our best athletes mainly are playing in basketball or football.... If you take a court the size of a tennis court and you decide you want to use it for a soccer field, say, you could fit a lot more kids.... When you talk about schools, they say: Well, it's better if we put a little soccer field in there and we get 20 kids running around kicking a ball.... Whereas tennis doesn't come as easily. Listen: John McEnroe on Serena Williams 5:03 On calling Serena Williams the best female tennis player in the world Garcia-Navarro: We're talking about male players but there is of course wonderful female players. Let's talk about Serena Williams. You say she is the best female player in the world in the book. McEnroe: Best female player ever — no question. Garcia-Navarro: Some wouldn't qualify it, some would say she's the best player in the world. Why qualify it? McEnroe: Oh! Uh, she's not, you mean, the best player in the world, period? Garcia-Navarro: Yeah, the best tennis player in the world. You know, why say female player? McEnroe: Well because if she was in, if she played the men's circuit she'd be like 700 in the world. Garcia-Navarro: You think so? McEnroe: Yeah. That doesn't mean I don't think Serena is an incredible player. I do, but the reality of what would happen would be I think something that perhaps it'd be a little higher, perhaps it'd be a little lower. And on a given day, Serena could beat some players. I believe because she's so incredibly strong mentally that she could overcome some situations where players would choke 'cause she's been in it so many times, so many situations at Wimbledon, The U.S. Open, etc. But if she had to just play the circuit — the men's circuit — that would be an entirely different story. Garcia-Navarro: Many people over the years, including, we should mention Donald Trump, the President, wanted you to play her, and you seemed to have at least thought about it. McEnroe: Well I've thought about it. I didn't really want to do it, personally. I don't know, people always seemed — I would say why don't they go ask Roger Federer? Or someone, you know they added the old fart that's you know 25 years over the hill. And I think I can still play and I think I could still — I mean my kids don't think I can beat her anymore. Maybe I should get her now because she's pregnant, but the truth is that I think that sometimes --I don't know why in tennis, I get it's that one battle of the sexes when Bobby Riggs played Billie Jean. Garcia-Navarro: Billie Jean one of the most famous, iconic and most watched, I think tennis matches at the time. McEnroe: Yeah, it was no question. I think there was the most, the biggest attendance at the Houston Astrodome, and it was great that Billie Jean did that but...OK, but that doesn't mean, talk about other sports. If you go look at the times, for example, of the world's fastest females — and you know maybe it will change! You know my daughter, one the things she says is 'You're a feminist, Dad.' OK. I started with two boys, I got four girls now and I'm all for it and I'm trying to just get with it and figure it out. Garcia-Navarro: So, you're a feminist. McEnroe: Maybe at some point a women's tennis player can be better than anybody. I just haven't seen it in any other sport, and I haven't seen it in tennis. I suppose anything's possible at some stage. Garcia-Navarro: You really think at 60, you could possibly beat Serena Williams? Maybe pregnant. McEnroe: The way you put that makes me think that you have your doubts. Garcia-Navarro: Far be it from me to question you Mr. McEnroe. McEnroe: Well, you know, my kids do, so feel free to. But there's people that because of course as you get older — I'm not sure how athletic you are and how often you get out in whatever sport it is, but I have kept at it regularly. I've done it sort of doing this playing some other guys close to my age even though they keep getting younger and younger. Obviously, if I was going to do something like that, I would train very seriously for that to make sure my body was at, like, the peak it could be. Absolutely — to try and be as ready as I possibly could, but I bring things to the table, certainly until recently. I may be way past it, but I can still bring a few things to the table and so that's why I guess people still find it interesting to even talk about. On where to go from here I need to make sure that I enjoy the upcoming 10 — hopefully 20 — years of my life and just appreciate the ride that it's been, and be able to continue to... find that inner peace, in a way, because that's difficult for me. I grew up a perfectionist getting pushed, pushed, pushed a lot.... Especially when my dad passed away a few months ago, I said, Wait a second, you've got to just take a step back here and smell the roses a little bit more. That would be my Number One goal moving ahead. Radio producer Peter Breslow, radio editor Stacey Samuel and Web producers Beth Novey and Wynne Davis contributed to this story. Is Serena Williams only as good as the male tennis player ranked 700th in the world? According to tennis veteran John McEnroe, quite possibly yes. That’s what he said in an interview that aired on Sunday — and his assessment sparked some debate online and a stern rebuke from Williams on Twitter where she said his statements are “not factually based” and that she has “never played anyone ranked ‘there.’” https://twitter.com/serenawilliams/status/879466071404810241 https://twitter.com/serenawilliams/status/879466221112094720 Update: McEnroe responded to Williams and critics who say he may have unfairly ranked her. In an interview on “Mad Dog Sports Radio,” show host Chris Russo called the reaction “innocuous” and told McEnroe, “you did nothing wrong with this Serena thing!” “We have a solution. Why don’t we just have them play together?” McEnroe referring to men’s tennis and women’s tennis. “I mean, the people jumping on me, ‘how dare you have an opinion on this.’ It’s not something that I haven’t not said in the past 20 years. I love Serena, by the way.” So, what exactly did McEnroe say to get this kind of response from the top player in women’s tennis? In an interview that aired on Sunday, NPR’s Lulu Garcia-Navarro asked McEnroe about his new book, “But Seriously,” and questioned the way he qualified hsi praise for Williams as “the best female player in the world.” https://twitter.com/yashar/status/879315322473525248 Garcia-Navarro: “Why say female player?” McEnroe: “Well because if she was in, if she played the men’s circuit she’d be like 700 in the world.” Garcia-Navarro: “You think so?” McEnroe: “Yeah. That doesn't mean I don't think Serena is an incredible player. I do, but the reality of what would happen would be I think something that perhaps it'd be a little higher, perhaps it'd be a little lower. And on a given day, Serena could beat some players. I believe because she's so incredibly strong mentally that she could overcome some situations where players would choke 'cause she's been in it so many times, so many situations at Wimbledon, The U.S. Open, etc. But if she had to just play the circuit — the men's circuit — that would be an entirely different story.” Williams is the world-record holder of 23 Grand Slam titles, which is far more than the seven Grand Slam titles McEnroe won in his career, and even more than the 18 Grand Slam titles won by the male tennis player with the most, Roger Federer, who has 18. But whatever measuring stick McEnroe was using to size up Williams, the internet did not take kind to the comparison and immediately clapped back with their reactions. https://twitter.com/reneeygraham/status/879285218745745409 https://twitter.com/RealMikeWelch/status/879360700283486210 https://twitter.com/HallTamyra/status/879056426827513856 Others didn’t quite see it the same way. In fact, the question of whether male professional tennis players are better than women is one that’s been discussed for decades. A conversation on the site Quora includes some similar conclusions from several people. “I don't think it's up for debate. The top male tennis players serve faster, hit harder, and cover more court than female professional tennis players. And that's okay!” one user wrote. “It has been demonstrated that the ~300 men's player could easily beat both Williams sisters in singles, in a challenge during one recent Aussie Open,” another wrote. Others on Twitter were not too far behind. https://twitter.com/tofucoffins/status/878974296227532800 https://twitter.com/AndrewJuge/status/879124504458539008 Williams has weighed in on the issue before. In one interview, she admitted that she would lose against tennis champ Andy Murray if they were to ever go toe to toe. “No, it's true. It's a completely different sport. The men are a lot faster and they serve harder, they hit harder, it's just a different game. I love to play women's tennis. I only want to play girls, because I don't want to be embarrassed," Williams told David Letterman in 2013. Three years later, Williams took on the gender debate in sports, saying that sexism plays a role in the way women are judged in sports. “If I were a man, I’d have been considered the greatest a long time ago,” she said in 2016. https://twitter.com/GMA/status/814090393549553664 Based on Williams’ reaction to McEnroe on Monday, it doesn’t seem like there is another “Battle of the Sexes” match in the works. (The first was actually in Ramona in 1973 between former Wimbledon champion Bobby Riggs and Margaret Court, then ranked No. 2 in the world). What do you think of McEnroe’s comments and Williams’ response? Have some thoughts to share? Join me in a conversation: Shoot me a private email with your thoughts or ideas on a different approach to this story. As always, you can also send us a tweet. Email: luis.gomez@sduniontribune.com Twitter: @RunGomez Read The Conversation on Flipboard. UPDATES: 11:55 a.m.: This article was updated with a response from John McEnroe This article was originally published Monday, June 26 at 6:25 p.m.

Serena Williams is an "incredible player" but she would be ranked "like 700" if she was on the men's circuit, John McEnroe said during an NPR interview Sunday, igniting a fierce debate and drawing a stern response from Williams. "I adore and respect you but please please keep me out of your statements that are not factually based," she tweeted Monday, adding: "I've never played anyone ranked 'there' nor do I have time. Respect me and my privacy as I'm trying to have a baby. Good day sir." During the interview, McEnroe said he hadn't seen it in any other sport, but "I suppose anything's possible; maybe at some point a women's tennis player can be better than anybody." McEnroe's remarks also drew an online backlash, with critics pointing out that Williams has won 23 Grand Slam titles to McEnroe's seven and managed to win the last one while pregnant, the San Diego Union-Tribune reports. The BBC spoke to the man ranked No. 701 in the world, Dmitry Tursunov. The Russian says he thinks he could beat Williams. "Tennis is becoming more and more a physical sport, so it's going to be hard for a woman to beat the men," he says. When NPR asked McEnroe if he had thought about playing Williams, he suggested she play Roger Federer instead. "My kids don't think I can beat her anymore," McEnroe said. "Maybe I should get her now because she's pregnant."

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Inter-individual variation in gene regulatory elements is hypothesized to play a causative role in adverse drug reactions and reduced drug activity. However, relatively little is known about the location and function of drug-dependent elements. To uncover drug-associated elements in a genome-wide manner, we performed RNA-seq and ChIP-seq using antibodies against the pregnane X receptor (PXR) and three active regulatory marks (p300, H3K4me1, H3K27ac) on primary human hepatocytes treated with rifampin or vehicle control. Rifampin and PXR were chosen since they are part of the CYP3A4 pathway, which is known to account for the metabolism of more than 50% of all prescribed drugs. We selected 227 proximal promoters for genes with rifampin-dependent expression or nearby PXR/p300 occupancy sites and assayed their ability to induce luciferase in rifampin-treated HepG2 cells, finding only 10 (4. 4%) that exhibited drug-dependent activity. As this result suggested a role for distal enhancer modules, we searched more broadly to identify 1,297 genomic regions bearing a conditional PXR occupancy as well as all three active regulatory marks. These regions are enriched near genes that function in the metabolism of xenobiotics, specifically members of the cytochrome P450 family. We performed enhancer assays in rifampin-treated HepG2 cells for 42 of these sequences as well as 7 sequences that overlap linkage-disequilibrium blocks defined by lead SNPs from pharmacogenomic GWAS studies, revealing 15/42 and 4/7 to be functional enhancers, respectively. A common African haplotype in one of these enhancers in the GSTA locus was found to exhibit potential rifampin hypersensitivity. Combined, our results further suggest that enhancers are the predominant targets of rifampin-induced PXR activation, provide a genome-wide catalog of PXR targets and serve as a model for the identification of drug-responsive regulatory elements. Adverse reactions to drug treatment constitute a substantial health problem that is a leading cause of morbidity and mortality in hospitalized patients [1]. Differential expression of drug metabolizing enzymes and drug transporters is a major determinant of inter-individual drug response variability [2]–[5]. By sequestering and metabolizing drug compounds in the liver and intestine, these enzymes and transporters effectively determine whether target organs and tissues are exposed to optimal drug dosages. Several coding mutations in these proteins have been detected which lead to adverse outcomes [6]–[10] and reduced drug activity [11], [12]. Regulatory elements, including promoters and enhancers, also likely play an important role that has so far been largely uncharacterized [13], [14]. The systematic identification of drug-responsive regulatory elements would thus provide a unique resource to discover novel genetic variants that lead to differences in drug response. The vast majority of pharmaceutical compounds are metabolized by the cytochrome P450 family (CYP) of enzymes. Of these, CYP3A4 is the most abundantly expressed in sites of drug disposition in the liver [15] and is also thought to be responsible for the metabolism of at least 50% of prescribed pharmaceuticals [16]. CYP3A4 activity can vary 5–20 fold between individuals [17] and its mRNA expression can vary as much as 120 fold [18]. Only a few single nucleotide polymorphisms (SNPs) in the immediate CYP3A4 locus have been found to be associated with CYP3A4 hepatic expression [19]–[21], suggesting that its variable expression could be caused by other genes and distant regulatory elements. CYP3A4 is one of many targets of the nuclear receptor PXR (coded by NR1I2), which is expressed predominantly in the liver and intestine [22] and is essential for activating Phase I and II enzymes in response to xenobiotics. PXR' s broad substrate specificity allows it to be activated by a wide variety of drugs including the antibiotic rifampin, the malaria resistance drug artemisinin, the hypolipidemic agent mevastatin, and the chemotherapeutic agent paclitaxel [2]. Relatively little is known about the mechanism by which PXR drives CYP3A4 transcription in vivo, although PXR response elements have been identified in the putative CYP3A4 promoter [23] and upstream cis-regulatory elements [24], [25] that drive its expression in vitro. Additional PXR responsive enhancers have been found for other CYPs [26], [27]. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) of PXR-bound DNA elements in livers from mice treated with PCN (a mouse PXR agonist) identified >3,000 drug-induced binding sites [28]. ChIP-seq for other drug-associated transcription factors such as LXR, RXR and PPARA has also been carried out in mouse liver [29]. However, the inherent drug metabolism differences between mouse and humans, in particular for PXR and the mouse homolog of CYP3A4 [22], [30], [31], hinder the ability to directly translate these results to humans. To identify PXR-associated regulatory elements in a genome-wide manner, we carried out RNA sequencing (RNA-seq) and ChIP-seq with antibodies for PXR and three different enhancer marks on primary human hepatocytes treated with rifampin or vehicle control. These included the E1A binding protein p300 (EP300/p300) which has been used to identify functional enhancers in vivo with high success rates [32], and two histone marks, H3K4me1 and H3K27ac. H3K4me1 marks both poised and active regulatory regions [33] while H3K27ac was shown to selectively mark active regions [34], [35]. We identified thousands of sequences that had rifampin induced ChIP-seq peaks. A reporter validation screen of proximal promoters associated with these peaks yielded only a few functional rifampin-dependent sequences. A similar assay for distal enhancers resulted in the identification of several novel drug-dependent enhancers. Analyses of nucleotide variants in selected sequences found a common African haplotype in the GSTA locus to possibly affect rifampin sensitivity. Our RNA-seq analyses found several differentially expressed genes, the majority of which are known to be involved in drug response. The number of differentially expressed genes using a p-value cutoff, after adjustment for multiple testing less than or equal to 0. 05, was 157 (Table S1). Amongst them, 11 were CYPs, with the top differentially expressed gene being CYP3A4, similar to our qPCR results. Of the eleven differentially expressed genes identified by qPCR, seven (64%) were also found to be differentially expressed by RNA-seq. It is worth noting that two (CYP2C9, CYP2C19) of the four genes that didn' t replicate in the RNA-seq data, showed a non-statistically significant induction by rifampin in our RNA-seq. We observed a massive recruitment of PXR binding across the genome following rifampin treatment. PXR-bound DNA fragments clustered into 1,158 discrete peaks with DMSO treated cells versus 6,302 after treatment with rifampin (Figure 1A, Table S2), with only 239 overlapping in both datasets (Figure 1B). Rifampin treatment led to a small increase in the percentage of promoters (25. 6% versus 24. 1%) bound by PXR and a larger increase for intronic (35. 18% versus 29. 9%) and exonic (5. 3% versus 1. 8%) regions (Table S2). In contrast, there was a reduction in the percentage of rifampin-induced PXR binding sites in intergenic regions (33. 9% versus 44. 2%). An analysis of the location of rifampin-induced PXR peaks found them to be enriched at transcription start sites (TSSs), but not at a particular location upstream or downstream to the TSS (Figure S1). The binding of p300 was also more extensive after rifampin treatment, with 13,811 peaks compared to 10,253 in DMSO-treated cells (Figure 1A, Table S2). There was a larger overlap between rifampin and DMSO treated ChIP-seq peaks compared to PXR, with 4,374 (31. 7%) in common between the two sets (Figure 1B, Table S2). We also observed a change in the functional distribution of binding sites, with rifampin increasing the percentage of intronic (42. 6% versus 36. 3%) and intergenic (38. 0% versus 31. 7%) p300 binding versus a small change in exons (3. 8% versus 2. 6%) and a reduction in promoter binding (15. 5% versus 29. 4%) (Table S2). This result was consistent with our observation that only 1,076 rifampin-induced p300 peaks overlapped rifampin-induced PXR peaks (Table S2). In contrast to PXR and p300, the distribution of histone marks was relatively stable, with about 49,000 enriched islands of H3K4me1 activity and about 40,000 H3K27ac islands in both treatments (Figure 1A). We also observed a large overlap between rifampin and DMSO treated H3K4me1 (82. 07%) and H3K27ac (87. 89%) enriched islands (Figure 1B, Table S2). Combined, these results suggest that histone marks are more stable in response to rifampin treatment compared to PXR and p300. We next looked at overlaps between the different ChIP-seq peaks. Amongst the 6,302 PXR rifampin treated peaks 1,037 (16%) overlapped p300 and around half overlapped histone marks (3,553 for H3K27ac and 2,942 for H3K4me1). This was similar for PXR peaks in the DMSO treated cells (Table S2). For p300 we observed a greater overlap with histone marks, with ∼70% of the peaks overlapping either H3K27ac (9,487/13,811) and H3K4me1 (9,840/13,811). In the DMSO treated cells, we observed a much higher overlap for p300 peaks with the active H3K27ac mark (9,487/10,253; 92%) versus H3K4me1 (7,789/10,253; 76%), suggesting that the p300 peaks in this condition tend to be in active regions. There are multiple examples of promoter nucleotide variants that are associated with inter-individual drug response [4], [5], [13], [38], [39]. We thus sought to identify drug-dependent promoters in our dataset which may harbor common variants with novel effects on drug response. We selected 227 promoters for 200 genes (some genes had more than one promoter; Table S3) from the LightSwitch Promoter Collection (SwitchGear Genomics) for genes whose expression was induced by rifampin or reside near rifampin-induced ChIP-seq peaks (Table S3). Of the 227 promoters, 154 overlap a PXR peak, 45 overlap p300,164 overlap H3K27ac and 84 overlap H3K4me1 (Table S3). This library consists of ∼1,000 bp proximal promoter fragments cloned into pLightSwich_Prom vector (see Methods, Table S3). We also included two positive controls: 1) The beta-actin promoter (ACTB), a strong constitutive promoter that should not be induced by rifampin and 2) The CYP3A4 proximal promoter, which is known to be induced by rifampin. We tested the 227 promoters in HepG2 cell lines co-transfected with human PXR and treated with rifampin or vehicle control (DMSO). Out of the 227 tested promoters, 179 were found to be functional promoters (>2 fold luciferase activity above empty vector) in the DMSO treated cells (Figure 2A, Table S3). Among those promoters, only 10 exhibited >2 fold increase in promoter activity upon rifampin treatment including our CYP3A4 proximal promoter control (Figure 2A). To confirm that the effects of rifampin were mediated through PXR, we also tested the 10 rifampin-induced promoters (including CYP3A4) in a similar assay, only this time without co-transfecting human PXR, and found only 2 of them to be induced by rifampin (>2 fold increase in promoter activity upon rifampin treatment) and at much lower levels (Figure 2B, Table S3). In addition, the CYP3A4 promoter was also not induced by rifampin in this assay. In both experiments, our ACTB control was a strong promoter, but not induced by rifampin. The overall lack of rifampin-sensitive promoters and previous results finding a role for enhancers in driving this drug response [24]–[27] suggests that other regulatory sequences, such as enhancers, may be involved in driving the effects of rifampin treatment on gene expression. Since most of the promoters tested in our assay did not demonstrate increased activity in the presence of rifampin, we broadened our search for inducible regulatory elements to include enhancers. To be more stringent in our analyses, we selected regions across the genome which showed PXR rifampin-induced binding in addition to all three enhancer marks. For both the DMSO and rifampin treatments, we generated a merged track of all four marks, with each region in the track overlapping one to four peaks/island. If, for example, a p300 peak is near a PXR peak, but they don' t overlap, while both overlap a H3K4me1 and/or a H3K27ac island, they were considered all as one region. Only 225 such regions were present in the DMSO treatment, while 1,387 were identified in the cells treated with rifampin (Figure 3A). Of the latter group, 1,297 regions were exclusive to the rifampin treatment and termed Rifampin-Induced Regions (RIRs) for downstream analyses. CYP3A4 is by far the most well studied target of PXR, with well characterized regulatory sequences: the proximal promoter, a −7. 5 kb upstream xenobiotic responsive enhancer module (XREM) [24], and a −11 kb constitutive liver enhancer module of CYP3A4 (CLEM4) [25]. A second potential XREM, putatively regulating CYP3A7, was additionally identified intergenically between CYP3A7 and CYP3A4 [26]. Our ChIP-seq data completely recapitulates this picture of regulation in primary human hepatocytes, with two large RIRs encompassing multiple rifampin-induced peaks (Figure 3B). It is also worth noting that the CYP3A4 locus is one of the few in which we observed a substantial difference in rifampin-induced enrichment of the H3K4me1 and H3K27ac marks. To identify enriched biological pathways and functions within the set of 1,297 RIRs, we carried out a genomic analysis using the Genomic Regions Enrichment of Annotations Tool (GREAT [40]). Our top enriched term (p-value 1. 85×10−9; binominal fold enrichment), originating from Pathway Commons (http: //www. pathwaycommons. org), was ‘xenobiotics’ (Figure 3C). This was attributed to RIRs residing near the following genes: ABCB4, ACSL1, ADH1A, ADH6, AKR1C2, AKR1C3, ALDH1A1, CNDP2, CYP26A1, CYP2A6, CYP2B6, CYP2C19, CYP2C8, CYP2C9, CYP2W1, CYP3A4, CYP3A7, CYP4F12, CYP4F3, CYP7A1, GCLC, GCLM, GSTA2, GSTO1, GSTO2, HNF4A, MAT1A, MAT2A, MGST2, MGST3, NCEH1, NNMT, PAPSS2, PTGIS, SLC35D1, SULT1B1, SULT2A1, UGDH, UGT1A1. In addition, we observed significant (FDR adjusted p-value≤0. 05) gene ontology enrichment terms for drug catabolic processes and other terms fitting with drug response (Figure 3C, Table S4). We performed a similar analysis for RIR neighboring genes using QIAGEN' s Ingenuity Pathway Analysis (IPA, QIAGEN Redwood City, www. qiagen. com/ingenuity) and found enrichment for the PXR/RXR Activation Canonical pathway (Figure S2, Table S5). Combined, these results suggest that our RIRs are enriched near drug-associated genes. Although ChIP-seq is a valuable tool for the identification of putative regulatory elements, functional studies are essential for the validation of such sequences. We selected forty-nine putative enhancer sequences for validation using two different selection criteria: 1) Forty-two RIRs residing near drug-associated genes as manually determined by the literature. 2) Seven rifampin-induced PXR ChIP-seq peaks harboring SNPs that are in linkage disequilibrium (LD) with pharmacogenomics or drug related genome-wide association study (GWAS) lead SNPs, termed GWAS linked peaks (GLPs) (Table S6). Previous studies have shown that several GWAS SNPs reside near potential regulatory elements that encompass SNPs that are in LD with the lead GWAS SNP [41]–[43]. To increase our chances to identify these regulatory elements, we used our rifampin treated PXR-ChIP-seq dataset, instead of the RIRs, since it had a larger number of peaks. SNPs in LD with pharmacogenomics GWAS hits were significantly enriched near rifampin-dependent PXR peaks compared to SNPs in LD with non-pharmacogenomic GWAS hits (p<0. 0001, Chi-squared test). None of the sequences chosen overlapped promoter regions (i. e. were within −2500/+500 bp of a TSS). Candidate enhancer sequences were cloned into the pGL4. 23 (Promega) enhancer assay vector, which contains a minimal promoter followed by the luciferase reporter gene. Since the peaks (or islands) enriched for the two histone marks are relatively long (average length ∼5 kb, Table S2), we selected shorter sequences within each RIR that encompass only the PXR/p300 peaks along with additional flanking sequence, up to 500 bp on each side of the peak. We also used different positive controls: 1) The ApoE liver enhancer [44] whose activity should not be enhanced by rifampin. 2) A CYP3A4 promoter-enhancer combination (p3A4-362 (7836/7208ins) [24] (Table S7) whose activity should be increased by rifampin treatment. All constructs were tested for their enhancer activity in HepG2 cells transfected with human PXR and treated with either rifampin or DMSO, as previously done for the promoter screen. Out of the 49 sequences tested, 19 (38. 7%) showed significant reporter expression levels versus the empty vector (≥2 two fold) in either condition: 15 RIRs and 4 GLPs (Figure 4A, Table S7). Among these 19 positive enhancers, we observed three types of enhancers: 1) Seven enhancers that were active at similar levels with DMSO and rifampin, termed ‘rifampin independent’. 2) Five enhancers that were active without rifampin, but whose expression levels significantly increased upon rifampin treatment, termed ‘rifampin increased’. 3) Seven enhancers that were active only when treated with rifampin and were called ‘rifampin dependent’. Two of these (GLP1 and GLP2) are located in the CYP2C locus (Figure S3) and contain SNPs in LD with pharmacogenomics GWAS SNPs for warfarin maintenance dose [45], [46], acenocoumarol maintenance dose [47] and response to clopidogrel therapy [48]. Combined, these results show that enhancer activity can be modulated by rifampin. We next determined whether common nucleotide variation within functional, drug-dependent enhancers could alter their activity. For these experiments we selected five enhancers that were either rifampin increased or dependent and near important drug-response genes. These included RIR7, which overlaps the putative CYP3A7 XREM (Figure 3B; chr7: 99339411–99341549; hg19) [26] and was rifampin dependent in our assays. We also selected RIR46, which is located in the glutathione S-transferase alpha (GSTA) locus near GSTA2 (chr6: 52609942–52611507; hg19) (Figure S4) and was rifampin-increased in our assays. The GSTA family of enzymes are known to be involved in the metabolism of various xenobiotics [49]. We also selected three different GLP sequences: GLP1,2, and 5. Both GLP1 (chr10: 96507473–96508107; hg19) and GLP2 (chr10: 96696182–96696970; hg19) are located in the CYP2C locus (Figure S3), which harbors several CYP metabolizing enzymes and has been analyzed extensively in various pharmacogenomic studies. GLP5 (chr2: 234672744–234673398; hg19) harbors a single SNP, rs3771341, that is in LD with several GWAS lead SNPs correlated with altered bilirubin levels [50]–[53] and was rifampin-increased in our study. This element is located in the UDP glucuronosyltransferase 1 family, polypeptide A cluster (UGT1A), ∼4 kb upstream of the UGT1A1 transcription start site (Figure S5). UGT1A enzymes have important roles in the metabolism of xenobiotics and both coding and promoter variants within them have been associated with adverse drug reactions [54]. We determined common haplotypes in all five sequences using the phased 1000 Genomes data (Table S8). Common haplotypes for all five sequences (Table S8) were then cloned into our enhancer assay vector (pGL4. 23) either by amplifying DNA from individuals from various ethnic backgrounds from the studies of pharmacogenomics in ethnically diverse populations (SOPHIE) cohort [55] or by site-directed mutagenesis and sequence verified. The sequences were then tested for enhancer activity in HepG2 cells transfected with human PXR and treated with either rifampin or DMSO, and compared to the ancestral haplotype. Out of the five tested enhancers, one haplotype in RIR46 showed a substantial difference in enhancer activity. For RIR46, we observed 1. 85-fold (4. 01/2. 16) increase in response to rifampin for haplotype 3, however after adjusting for multiple testing the variance in the response was too high to be significant (adjusted p-value = 0. 095 by FDR; ANOVA; Figure 4B, Table S8). This haplotype is present at a frequency of 6. 7% in the 1000 Genomes AFR population. It is worth noting that our success rate in finding haplotypes that significantly alter enhancer activity was low. Nonetheless, the observation that a haplotype in RIR46 could possibly affect enhancer activity suggests that nucleotide variants in these enhancers could lead to differential enhancer activity. By carrying out RNA-seq and ChIP-seq on rifampin and DMSO treated human hepatocytes, we have uncovered numerous drug-dependent regulatory elements. We observed that promoters bearing a rifampin-dependent signature were largely unable to independently induce the expression of a reporter upon rifampin treatment. This result suggested that other gene regulatory elements, such as enhancers, could constitute the predominant group of target sequences that are activated by this drug. An analysis of nucleotide variation in these enhancers showed that specific variants could affect enhancer activity, raising the possibility that nucleotide variants in enhancers could contribute to pharmacogenomic phenotypes more broadly. Our RNA-seq analyses that combined eight different individuals identified 157 differentially expressed genes, using a p-value cutoff that adjusted for multiple testing less than or equal to 0. 05 (Table S1). Many of these genes are known to be involved in drug response and the top differentially expressed gene was CYP3A4, fitting with its role as the most abundantly expressed gene in sites of drug disposition in the liver [15]. The number of differentially expressed genes (157), using our 0. 05 cutoff, was much lower than the number of RIRs (1,297) and PXR and p300 rifampin treated ChIP-seq peaks. This difference could be attributed to having multiple regulatory elements regulating the same gene. It could also be due to our conservative RNA-seq differential expression cutoff, which if relaxed would increase our gene list. Another cause for this can be that several of our identified peaks are not functional regulatory elements. Our functional characterization for example, found only 19 of the 49 tested sequences (38. 7%) to have significant reporter expression levels versus the empty vector (≥2 two fold). Of note though, that this could also be due to the different cell types and conditions that were used in this assay, as later described. Similar studies have been conducted to detect environmentally induced regulatory elements [28], [56]–[59]. For example, testing for changes in estrogen receptor α and RNA polymerase II occupancy due to estradiol, tamoxifen or fulvestrant treatment in MCF-7 cells, found differences in ligand regulation with tamoxifen leading to downregulation while fulvestrant increased RNAPII occupancy [58]. In our study, we observed a large rifampin-induced recruitment of PXR and p300 binding across the genome. This was particularly notable in the CYP3A locus, which was radically altered by rifampin treatment. For the most part, the regulatory hotspots that we identified in this region are consistent with established literature. It is worth noting, however, that we saw almost equal recruitment of PXR and p300 to the XREMs that putatively regulate CYP3A4 and CYP3A7, despite the fact that we observed substantially less CYP3A7 mRNA induction by qPCR and RNA-seq. We did not observe large changes in the assayed histone marks, H3K4me1 and H3K27ac. This could be attributed to these sites being poised to be activated by various xenobiotic responses, though H3K27ac has been previously shown to mark active enhancers [34], [35]. To get at these differences more systematically, we performed a second analysis in which H3K4me1 and H3K27ac peaks were called for the rifampin treatment using the DMSO treatment as the control (Table S9). We observed 110 differential islands for H3K27ac, and only 2 for H3K4me1 (one of which was in the CYP3A4 locus). The fact that we observed more rifampin-induced peaks for H3K27ac is consistent with its hypothesized role in marking active enhancers. Combined, these results suggest that while these two marks are incredibly stable in the face of massive changes in PXR/p300 binding, there are still measurable drug-induced changes in chromatin structure. Previous reports have found enhancers to be responders of drug treatment [24]–[27]. Our functional validation results broadly suggest that promoters on their own are largely incapable of driving the effects of rifampin induction. Instead, our results imply that enhancers appear to be induced by rifampin, and through their interaction with promoters, drug response genes are activated. We tested over 200 promoters of genes based on relatively loose selection criteria: that their respective genes either showed increased expression following rifampin treatment, were tagged by our rifampin ChIP-seq peaks, or were near them. On the other hand, for enhancers we required that candidates had all four marks. While it is possible that our promoter selection criteria missed out on important drug response promoters, we would still expect to achieve a higher rifampin induction success rate than what we observed in our assays (10/227; 4. 4%). While our assays may have not been ideal for these purposes, i. e. testing promoters in vitro in PXR-transfected HepG2 cells, both our negative and positive controls showed the expected results (Figure 2A). Furthermore, rifampin-induced promoters tested in non-PXR transfected conditions showed much lower induction by the drug (Figure 2B). Further systematic assays including ones carried out in vivo will be needed to better address this hypothesis. While 12 out of the 19 functional sequences identified by our screen exhibited basal enhancer activity, 7 sequences exhibited enhancer activity only upon rifampin treatment (Figure 4A). These sequences would not have been identified by conventional ChIP studies conducted in physiological conditions, nor would they be validated in functional assays without drug treatment. Together our results suggest that ChIP-seq datasets are dependent on the environmental conditions in which they were performed, and that there are likely many hidden enhancers which only become active following a specific stimulus. We identified several functional enhancers that were rifampin-increased or rifampin-induced whose location was near pharmacogenomic-associated variants. One of these elements is RIR46, which resides near GSTA2, a Phase II enzyme involved in the detoxification of numerous drugs [49], and is thus a likely target. Coding variants in GSTA2 have been shown to affect its detoxification efficiency [60], [61] and promoter variants in GSTA2 have been suggested to affect its expression levels [62], [63]. We identified a haplotype present in the 1000 Genomes AFR population that confers increased rifampin sensitivity (Figure 4B). Both GSTA2 and GSTA1 (which is 28 kb downstream to GSTA2), have been shown to have important roles in catalyzing carcinogenic substrates and nucleotide variation in them has been shown to be associated with cancer [60], [64]. Previous work has shown a significant difference in the distribution of coding SNPs in both these genes between African Americans and Caucasians [60]. However, these coding SNPs are not associated with prostate cancer disease status [60], suggesting that other factors could be playing a role. Future studies could examine whether variants in RIR46 or other RIRs are associated with these phenotypes. It is worth noting that there are several caveats to our study. Our ChIP-seq experiments only analyzed hepatocytes from a single donor at a single time point (24 hours post treatment), selected for being commonplace in rifampin induction studies. It is possible that there are enhancers that play a role much earlier than 24 hours post-treatment. To test regulatory sequences in reporter assays, it was also necessary to clone smaller fragments from each RIR bearing the PXR and p300 peaks. It is possible that we missed flanking sequences that were essential for enhancer function. Furthermore, because we had a limited amount of primary hepatocytes from the same donor, our reporter assays were carried out in PXR-transfected HepG2 cells, which could result in false negatives. Finally, we employed the pGL4. 23 vector, which is a commonly used enhancer assay vector, but possesses a very short TATA-box containing minimal promoter that may not be compatible with all of the enhancer sequences tested. Promoter regions upstream of drug metabolizing enzymes and transporters are presumed to be the major targets of xenobiotic activators of PXR, such as rifampin. Our work challenges this conception and strongly supports the idea that the direct targets could be enhancer elements, which may subsequently interact with promoters to enhance gene transcription. Combined, our results show that ChIP-seq in combination with drug treatment has a large potential in identifying novel regions in the genome associated with drug response. These regions can provide exceptional candidates for the detection of nucleotide variants associated with inter-individual differences in drug response. Our methodology could easily be adapted to other drugs/target/tissue combinations. With whole genome datasets becoming more widely used as a clinical toolbox, the ability to highlight these important drug response regions in the genome is of extreme importance. Cryopreserved human hepatocytes from a 19 year old Caucasian male donor with no history of medications (Lot Hu8080, Life Technologies) were thawed in CHRM recovery media (Cat# CM7000, Life Technologies) and cultured in CHPM plating media (Cat#CM9000, Life Technologies) on 6-well collagen-coated plates (Life Technologies). After 6 hours, the media was swapped with maintenance media, consisting of phenol-free Williams E media containing culture incubation supplements (Cat#CM4000, Life Technologies) and 0. 01% DMSO or 10 µM rifampin (Sigma) for 24 hours. The rifampin dose (10 µM for 24 hours) was used based on previously reported assays achieving high induction rates in human hepatocytes [65], [66]. Dexamethasone, which is included separately with the culture incubation supplements, was not added to the media as it can activate PXR. Cultured hepatocytes were treated with DMSO or rifampin for 24 hours in triplicate. The cells were then washed with PBS, and lysed directly with Buffer RLT from the RNAeasy mini kit (Qiagen) with the on-column DNase digestion step. One µg of total RNA was used to generate cDNA using the RT2 First Strand Kit (Qiagen). Gene expression levels for 84 genes of interest was determined using the “Drug Metabolism: Phase I Enzymes” RT2 Profiler PCR Array (Qiagen). Five housekeeping genes (B2M, HPRT1, RPL13A, GAPDH, ACTB) were used to control for loading. Fold induction was calculated using the ΔΔCt method [67]. Total RNA was acquired as described for qPCR for two replicates each of DMSO and rifampin treated hepatocytes. Libraries were made with ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicenter). Briefly, 3–5 ug of total RNA were subjected to rRNA removal using Ribo-Zero Magnetic Kit (Epicenter) prior to library construction. 5 ng of the rRNA-depleted sample was fragmented enzymatically and annealed with random hexamer to create the first strand of cDNA. Upon removal of the RNA template transcript by RNase, Terminal-Tagging Oligo (TTO), a known 5′-sequence tag, a 3′-random sequence, and a terminally blocked 3′ end to prevent priming of DNA synthesis, was added to create cDNA with known sequence tags at their 5′ and 3′ ends for directionality. Upon purification, adaptors with barcodes were added to cDNA fragments and enriched by 15 cycles of PCR. Sequencing was carried out on an Illumina HiSeq. The resulting reads were demultiplexed and aligned to the human genome (hg19) using TopHat v2. 0. 10 [68]. Read counts for each gene in the RefSeq annotation were obtained using NGSUtils [69] so as to allow comparison to the RNA-seq data from the other 7 primary hepatocytes treated with and without rifampin and sequenced with SOLiD as described in [37]. Analysis for differential expression across the nine replicates was performed using DESeq2 [70]. DESeq2 was chosen due to its capability in handling multifactorial experimental designs, in this case treated with rifampin versus control and SOLiD sequences versus Illumina. DESeq2 was then used to perform a likelihood ratio test between the model of treatment plus sequencing type versus the simplified model of just sequencing type in order to identify genes differentially expressed upon treatment with rifampin. Twelve million cells (an entire 6 well plate) per immunoprecipitation were fixed with 1% formaldehyde for 15 min and quenched with 0. 125 M glycine. The remainder of the ChIP-seq protocol was carried out by Active Motif Inc., as follows. Chromatin was isolated by adding lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA sheared to an average length of 300–500 bp. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer. Extrapolation to the original chromatin volume allowed quantitation of the total chromatin yield. An aliquot of chromatin (30 ug) was pre-cleared with protein G agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using 4 ug of antibody against PXR (sc-25381, Santa Cruz), 4 ug of antibody against p300 (Cat#sc-595, Santa Cruz), 20 ug of antibody against H3K27ac (Cat#ab4729, Abcam) and 5 ug antibody against H3K4me1 (Cat#ab8895, Abcam). Due to the limited amount of cryopreserved human hepatocytes from this donor, we elected to do various enhancer-associated ChIP-seq marks instead of additional replicates. Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65 C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. ChIP and input DNAs were prepared for amplification by converting overhangs into phosphorylated blunt ends and adding an adenine to the 3′ ends. Illumina adaptors were added and the library was size-selected (175–225 bp) on an agarose gel. The adaptor-ligated libraries were amplified for 18 cycles. The resulting amplified DNAs were purified, quantified, and tested by qPCR at the same specific genomic regions as the original ChIP DNA to assess quality of the amplification reactions. Amplified DNA libraries were sequenced on the Illumina Genome Analyzer II. The 36-nt sequence reads identified by the Sequencing Service were mapped to the genome using the ELAND (Illumina). Default settings were used: only tags that map uniquely, have no more than 2 mismatches, and that pass quality control filtering were considered. PXR and p300 peaks were called against input using MACS version 1. 4 [71] and for H3K27ac and H3K4me1 using SICER version 1. 1 [72]. A p-value cutoff of 1×10−7 was used for MACS and an FDR of 0. 01% was used as a cutoff for SICER, and the remaining parameters set to the default. For the alternate analysis presented in Table S9, we used a more stringent threshold in SICER of 1×10−10. For the analysis of the distribution of PXR ChIP-seq peaks following rifampin treatment, we used knownGene from the UCSC Genome Browser to define the transcription start site (TSS) as the center and pybedtools [73] for the calculations followed by matplotlib (http: //matplotlib. org) to plot. Selected promoters were obtained from the LightSwitch Promoter Collection (SwitchGear Genomics) and sequence verified in the pLightSwitch_Prom vector. Candidate enhancers were amplified from human genomic DNA (Roche) using oligonucleotides designed in Primer3 with 16 bp overhangs (5′-GCTCGCTAGCCTCGAG-3′ and 5′CGCCGAGGCCAGATCT-3′) complementary to the sequence flanking the BglII and XhoI sites in the pGL4. 23 vector (Promega). Primers were designed to encompass the PXR/p300 ChIP-seq peak plus up to 500 bp of sequence on either side of the peak. PCR products were cleaned using the QIAquick PCR Purification Kit (Qiagen) and cloned into BglII and XhoI digested pGL4. 23 using the Infusion HD cloning system (Clontech). Haplotypes were generated either by PCR amplification of DNA from various ethnic individuals or by site-directed mutagenesis using mutant primers amplified by PfuUltra High Fidelity DNA polymerase (Agilent) followed by DpnI digestion of the parental DNA (New England Biolabs) and transformation into competent cells. Sequences were verified by Sanger sequencing using RVPrimer3 (5′-CTAGCAAAATAGfGCTGTCCC-3′) and a custom reverse primer (5′-TCTTCCATGGTGGCTTTACC-3′). We manually curated the NHGRI GWAS catalog [74], dated December 15th, 2011, to identify 397 SNPs from 92 pharmacogenomic GWAS studies. Using data from the 1000 Genomes March 2012 Interim release (http: //1000genomes. org), we identified a larger set of SNPs in linkage disequilibrium with those SNPs (r2>0. 8) in the race/ethnicity corresponding to the pharmacogenetics study. Ten rifampin-induced PXR peaks overlapped at least one of these GWAS-LD SNPs (Table S6) and seven were selected for functional enhancer assays. To determine enrichment, we compared overlap of the LD blocks defined by the 397 lead SNPs from pharmacogenomic GWAS to 5,117 lead SNPs from non-pharmacogenomic GWAS. These assignments were curated manually. Each list of SNPs was intersected with the 6,302 rifampin-induced PXR peaks, resulting in 27 and 162 overlapping SNPs respectively. Using a two-tailed Chi-squared test with Yates' correction, we found the enrichment to be highly significant (p<0. 0001). HepG2 cells (ATCC) were maintained in D-MEM (Life Technologies) supplemented with FBS (JRS Scientific), Penicillin-Streptomycin and Glutamine (UCSF Cell Culture Facility). On the day of transfection, the cells were trypsinized, washed, and diluted to a density of 1 million cells/ml in Opti-MEM I Reduced Serum Media (Life Technologies). Ninety thousand cells and 125,000 cells were added to each well of a 96-well clear bottom black tissue culture plate (Greiner Bio-One) containing the transfection mixture for the promoter and enhancer assay respectively. The transfection mixture consisted of 50 ng of human PXR (PXR cDNA cloned into the pcDNA3. 1 (+) vector), 10 ng of pGL4. 74,21. 5 ul Opti-MEM, 100 ng of reporter construct, and 0. 48 ul of Lipofectamine LTX reagent (Life Technologies). After 18 hours of incubation at 37 degrees Celsius, the cells were washed with 150 ul Opti-MEM and the media was replaced with 100 ul of Opti-MEM supplemented with 10 uM Rifampin or 0. 1% DMSO. After 24 hours of incubation, the cells were washed with PBS and the promoter assay cells were placed at −80 degrees Celsius for 30 minutes to lyse and the enhancer assayed cells were lysed with 25 ul of Passive Lysis Buffer (Promega). All LightSwitch promoter vectors were measured with the LightSwitch Luciferase Assay reagents (SwitchGear Genomics) following the manufacturers protocol. For enhancers, reporter activity was measured using the Dual-Luciferase Reporter Assay System (Promega). Both assays were measured on a microplate luminometer (Promega). We used the 1000 Genomes March 2012 Interim release (), which contained genotypes for 379 White, 286 Asian, 181 Latino, and 246 African race/ethnicity individuals. For each gene RIR/GLP region, we identified haplotypes determined by computationally phasing the SNPs in the program PLINK v1. 07 [75], again using the corresponding race/ethnicity. ChIP-seq and RNA-seq data has been made publically available through NCBI (ChIP-seq BioProject ID: PRJNA239635; RNA-seq BioProject ID: PRJNA239637).

Drug response varies between individuals and can be caused by genetic factors. Nucleotide variation in gene regulatory elements can have a significant effect on drug response, but due to the difficulty in identifying these elements, they remain understudied. Here, we used various genomic assays to analyze human liver cells treated with or without the antibiotic rifampin and identified drug-induced regulatory elements genome-wide. The testing of numerous active promoters in human liver cells showed only a few to be induced by rifampin treatment. A similar analysis of enhancers found several of them to be induced by the drug. Nucleotide variants in one of these enhancers were found to alter its activity. Combined, this work identifies numerous novel gene regulatory elements that can be activated due to drug response and thus provides candidate sequences in the human genome where nucleotide variation can lead to differences in drug response. It also provides a universally applicable method to detect these elements for other drugs.

lay_plos

Rhadinoviruses establish chronic infections of clinical and economic importance. Several show respiratory transmission and cause lung pathologies. We used Murid Herpesvirus-4 (MuHV-4) to understand how rhadinovirus lung infection might work. A primary epithelial or B cell infection often is assumed. MuHV-4 targeted instead alveolar macrophages, and their depletion reduced markedly host entry. While host entry was efficient, alveolar macrophages lacked heparan - an important rhadinovirus binding target - and were infected poorly ex vivo. In situ analysis revealed that virions bound initially not to macrophages but to heparan+ type 1 alveolar epithelial cells (AECs). Although epithelial cell lines endocytose MuHV-4 readily in vitro, AECs did not. Rather bound virions were acquired by macrophages; epithelial infection occurred only later. Thus, host entry was co-operative - virion binding to epithelial cells licensed macrophage infection, and this in turn licensed AEC infection. An antibody block of epithelial cell binding failed to block host entry: opsonization provided merely another route to macrophages. By contrast an antibody block of membrane fusion was effective. Therefore co-operative infection extended viral tropism beyond the normal paradigm of a target cell infected readily in vitro; and macrophage involvement in host entry required neutralization to act down-stream of cell binding. The γ-herpesviruses chronically infect most mammals and cause disease in humans [1] and economically important ungulates [2]. Vaccines have failed to prevent infection. In part this reflects that host entry is ill-understood. Oral entry is often assumed, because the archetypal Epstein-Barr virus (EBV) causes acute tonsillitis. However clinical presentation occurs at least 1 month after EBV acquisition [3] and correlates better with peak salivary shedding. Thus, it may reflect host exit rather than entry, and because virus shedding involves reactivation from circulating B cells [4] entry and exit routes need not coincide. Lymphocryptoviruses such as EBV are known only in primates; Rhadinoviruses such as the Kaposi’s Sarcoma-associated Herpesvirus (KSHV) are more widespread [5]. KSHV in nasal secretions [6], and pulmonary Kaposi’s Sarcoma complicating AIDS [7], suggest respiratory infection. Consistent with this idea, Ovine herpesvirus-2 and Alcelaphine herpesvirus-1 show salivary and nasal shedding, and infect in nebulized form [8]; Equid γHV-5 causes respiratory disease [9] and can be isolated from the respiratory tract [10]; and Bovine herpesvirus-4 colonizes the respiratory tract [11], and persists in calves after intranasal (i. n.) inoculation [12]. MuHV-4 (strain MHV-68) [13,14] allows rhadinovirus infection to be analysed in mice. Like EBV and KSHV, it is B cell tropic. MuHV-4 infects readily mice i. n., and infects orally only if it spills into the respiratory tract [15]. When given i. n. under anesthesia it infects the lungs [16]; without anesthesia it infects the upper respiratory tract [17]; and it transmits sexually from female mice to co-caged males [18]. How MuHV-4 transmits in its natural host of yellow-necked mice [19] is unknown. Analysis of wood mice identified DNA from MuHV-4 or a related virus in lungs [20], and lung inoculation is used widely for experimental infections. Therefore MuHV-4 is suited to analyzing this mode of host entry. A key unknown is the first infected cell type. Lungs contain type 1 AECs, which comprise 95% of the alveolar surface area; type 2 AECs, which secrete surfactant; macrophages, which phagocytose inhaled debris [21]; and some lymphocytes. By 5 days after i. n. inoculation, MuHV-4 antigens are detectable in type 1 AECs and large mononuclear cells [16]. DNA of i. n. -inoculated, replication-deficient MuHV-4 has been detected by PCR of B cells from lung homogenates [22,23], suggesting that they are a primary infection target. However MuHV-4 normally binds to heparan, which B cells express poorly [24,25], and shows a post-binding block to B cell infection that is not overcome until virions pass through myeloid cells [26]. PCR detects replication-deficient MuHV-4 DNA also in association with B cells recovered from splenic homogenates after intraperitoneal (i. p.) inoculation [22,23], when in situ analysis shows no evidence of B cell infection [27]. Therefore the PCR signals associated with lung B cells might reflect adsorbed inoculum debris rather than infection. Any understanding of MuHV-4 infection must encompass its dependence on heparan for cell binding [24], a characteristic common to many rhadinoviruses [28–30]. The MuHV-4 gp70 and gH/gL bind to heparan [31,32], and gp150 further inhibits heparan-independent binding until heparan is engaged [24]. Thus, gL-gp70- MuHV-4 infects poorly both in vitro and in vivo, unless gp150 is disrupted also [33]. The Bovine Herpesvirus-4 gp150 homolog, gp180, is functionally similar [34]. While most transformed epithelial cells express abundant heparan, most in vivo epithelia do so only basolaterally [35]. Thus, heparan-dependent host entry by incoming, apical virions is not necessarily straightforward. The olfactory neuroepithelium, which we have analysed previously [17], unusually expresses both basolateral and apical heparan. The genital epithelium lacks apical heparan [36]; entry here may depend on epithelial trauma, as MuHV-4-infected cells are seen underneath rather than in the stratified squamous epithelium [18]. We asked which cell type MuHV-4 targets in the lungs and how this relates to heparan binding. Our aim was to understand better rhadinovirus entry into new hosts and so establish a rational basis for infection control. Four days after i. n. MuHV-4 inoculation, viral antigens were expressed in morphologically typical type 1 AECs, whose extensive cytoplasmic projections line the lung air spaces (Fig. 1A). This tropism was confirmed by co-staining lungs for viral antigens and the type 1 AEC marker podoplanin (PDP) [37] (Fig. 1B) —at 4 days post-inoculation >50% of viral antigen+ cells were PDP+ (Fig. 1C). The morphologically distinct remainder expressed the macrophage / granulocyte marker CD68 [38–40]. However at 1 day post-inoculation viral antigens were confined to CD68+ cells. Therefore while infection spread to type 1 AECs, it appeared to start in macrophages or granulocytes. MuHV-4-immune sera recognize mainly lytic antigens. As much early lung infection is latent [41], we tracked host entry more comprehensively via lytic cycle-independent [42] eGFP expression from a viral HCMV IE1 promoter (Fig. 2A). At 1 day post-inoculation, 238/238 eGFP+ cells on lung sections of 3 mice were CD68+. None was PDP+. MuHV-4 rendered incapable of uncomplemented lytic spread by disruption of its ORF50 transactivator (ORF50-) showed a similar eGFP distribution: 78/78 eGFP+ cells on lung sections from 5 mice expressed CD68, and none expressed PDP (Fig. 2B). Therefore CD68+ cells were direct infection targets. More than 80% of eGFP+ cells expressed also the tissue macrophage marker F4/80 [39] and the alveolar macrophage / dendritic cell marker CD11c [38,40] (Fig. 2C). Macrophage populations are heterogeneous and often not clearly demarcated [38], but combined CD11c, CD68 and F4/80 expression argued that most infected cells were alveolar macrophages. The lack of HCMV IE1-driven eGFP expression in type 1 AECs at day 1 post-inoculation established that they were uninfected rather than just slow to initiate lytic infection. We saw also no eGFP expression in B cells at day 1 by either eGFP+ wild-type (WT, >100 eGFP+ cells counted from 3 mice) or eGFP+ORF50- MuHV-4 (>50 eGFP+ cells counted from 3 mice) (S1 Fig). Nor did MuHV-4 with EF1α promoter-driven eGFP expression show a day 1 infection of B220+ lung B cells or type 1 AECs (S1 Fig, >100 eGFP+ cells counted from 3 mice). Therefore incoming virions targeted selectively alveolar macrophages. Viral lytic gene expression suggests but does not prove lytic replication. To determine how alveolar macrophage infection contributed to virus production we infected lysM-cre mice with MuHV-4 in which cre-mediated recombination switches fluorochrome expression from mCherry+ (red) to eGFP+ (green) (MHV-RG) [26]. LysM-cre mice express cre from the myeloid lysozyme locus [43]. To identify cre+ lung cells we crossed them with a reporter strain in which cre activates Zsgreen expression from a widely expressed promoter [44]. PDP+ cells lacked Zsgreen (S2 Fig). CD68+ cells expressed Zsgreen in an endosomal distribution. Type 2 AECs (surfactant protein C precursor+, CD68-, MAC-2-) also expressed Zsgreen—in a uniform rather than endosomal distribution. However in C57BL/6 mice given HCMV-IE1-eGFP+ MuHV-4 they remained eGFP- (S3 Fig) and so were not infected. Thioglycollate-induced Gr-1+ peritoneal exudate neutrophils of lysM-cre x Zsgreen mice were Zsgreen+, consistent with published data [43], but lung cells expressing neutrophil markers (Gr-1 or myeloperoxidase) were not (S2 Fig). They also lacked eGFP expression by HCMV-IE1-eGFP+ MuHV-4. Therefore we interpreted MHV-RG switching in lysM-cre mice as an infection of alveolar macrophages. Some switching in lung dendritic cells was possible, but they express little lysM [43] or CD68 [39], so there was no other indication that dendritic cells were a significant target for incoming virions. The MHV-RG recovered from lysM-cre lungs at 1 day post-inoculation was >96% eGFP+, indicating that essentially all of it had passed through an alveolar macrophage (Fig. 3A). Surprisingly the MHV-RG recovered after 4 days showed only 85% switching. This reduction in % switched virus with time was consistent and statistically significant (Fig. 3B), suggesting that virions could also enter the lungs without infecting a macrophage, but that virus production was then delayed. We confirmed myeloid infection as the predominant route by infecting CD11c-cre mice (Fig. 3C), as alveolar macrophages (and dendritic cells) are CD11c+ [38]. Again the virus recovered from lungs after 1 day was >96% eGFP+, and that recovered after 4 days was >85% eGFP+. The prominent viral lytic antigen expression in type 1 AECs at day 4 post-inoculation (Fig. 1) but not day 1 implied that they receive and amplify virus from alveolar macrophages. Their lack of HCMV IE1-driven or EF1α-driven eGFP expression at day 1 (Figs. 2, S1) excluded acute infection. Day 1 MHV-RG-infected lung sections (Fig. 3D) confirmed this: >95% of fluorescent cells were morphologically typical alveolar macrophages and PDP-. Of these 92. 6 ± 4. 3% were eGFP+ (mean ± SD of counts from 8 mice), confirming efficient viral fluorochrome switching in infected myeloid cells. However at day 4, when 63. 7% of fluorescent cells were PDP+, 48. 5 ± 10. 6% of these were eGFP-mCherry+ (mean ± SD of counts from 8 mice). Thus, not all type 1 AEC infection was down-stream of myeloid cell infection. And while no eGFP+ cells were PDP+ 1 day after i. n. eGFP+ORF50- MuHV-4 inoculation of C57BL/6 mice (Fig. 2), 35. 5 ± 29. 7% of eGFP+ cells were PDP+ after 5 days (mean ± SD, 6 sections from 3 mice) (S4 Fig). Therefore a delayed type 1 AEC infection without prior macrophage infection seemed to account for the reduction in % virus switching between days 1 and 4 in Fig. 3A-3C. Either virions eventually penetrated AECs, or macrophages licenced AEC infection without becoming infected themselves, for example by recycling virions from early endosomes to exosomes and releasing them in a licensed form [45]. Most lung macrophages express CD169 [46], so we used CD169-DTR mice [47] to test their importance for host entry (Fig. 4). Mice were given diphtheria toxin i. p. for 2 days, then given eGFP+ ORF50- MuHV-4 i. n. for 1 day. Staining lungs for CD169, CD68, F4/80 and CD206 (Fig. 4A) showed that CD169 expression was ablated completely. CD206 and F4/80 expression were ablated >90%, consistent with most lung macrophages expressing CD169. EGFP+ cell numbers in toxin-treated mice were <10% those of untreated controls (Fig. 4B). CD68+ cells were still seen, but they were larger than in undepleted mice and lacked other alveolar macrophage markers. Such cells were not evident in undepleted infected mice, and so seemed likely to be bone marrow-derived monocytes or granulocytes, recruited in response to alveolar macrophage ablation. They were clearly poor infection targets. Therefore alveolar macrophages played a crucial role in virus acquisition, for which lineage-distinct bone marrow-derived myeloid cells [48] did not substitute. Host entry via lung macrophages was unexpected, as MuHV-4 infects the lungs efficiently [49] and myeloid cells poorly [26]. The cell-free virions used for in vivo infection poorly infected ex vivo lung macrophages, peritoneal macrophages or the monocyte line RAW-264 (Fig. 5A). Virus stocks containing cell debris infected macrophages with reasonable efficiency. However cell-associated virus infections would occur only after host entry. Efficient alveolar macrophage infection by cell-free virions evidently depended on their normal tissue context. MuHV-4 that lacks heparan binding due to gp70 and gL disruption infects mice i. n. 100-fold less well than the WT, unless its gp150, which inhibits heparan-independent binding, is disrupted too [33]. This result implies that host entry requires heparan engagement. We confirmed it by pre-incubating WT virions with soluble heparin (Fig. 5B). Seroconversion by virus-specific serum IgG ELISA—the the most sensitive measure of low dose infection in immunocompetent mice [33]—was reduced 10–100-fold by the heparin treatment. Pre-incubating virions with heparin also reduced NMuMG epithelial cell infection 100-fold (Fig. 5C). However, while low dose heparin inhibited RAW-264 monocyte infection better than NMuMG cell infection—presumably because RAW-264 cells have little heparan for virion binding to start with [26]—there was also a heparin-resistant component of infection, such that the maximal inhibition was only 4-fold. Therefore the efficiency of lung infection and its susceptibility to inhibition by heparin both suggested an initial interaction with epithelial cells rather than macrophages. To identify heparan+ cells in the lungs we stained sections with mAb 10E4, which recognizes sulfated heparan [50] (Fig. 6A), and with mAb NAH46, which recognizes non-sulfated heparan [51] (Fig. 6B). Staining by both mAbs co-localized with PDP (type 1 AECs) and not with CD68 (alveolar macrophages). To distinguish apical from basolateral heparan, we co-stained sections for the alveolar basement membrane component collagen IV (Fig. 6C). Sulfated heparan (mAb 10E4) co-localized completely with collagen IV and so was just basolateral. Unsulfated heparan (mAb NAH46) also co-localized largely with collagen IV. However there were also NAH46+ surfaces abutting the air spaces that lacked collagen IV. Therefore specifically unsulfated heparan was accessible to incoming virions on the apical AEC surface. One day after WT MuHV-4 inoculation, viral antigens were evident as isolated dots—presumably virions. Of these 94. 8 ± 3. 9% associated with PDP+ type 1 AECs (>300 dots counted on 6 sections from 3 mice). By contrast strong cellular staining—presumably lytic infection—was confined (78/78 cells) to CD68+ macrophages (Fig. 7A). Therefore incoming virions bound to type 1 AECs but infected macrophages. Similarly 94. 0 ± 5. 1% of inhaled replication-deficient MuHV-4 virions associated with PDP+ cells (ORF50-, Fig. 7B, >300 dots on 6 sections from 6 mice). In mice given WT eGFP+ MuHV-4,109/113 lytically infected cells (96. 5%) were eGFP+ and 109/140 eGFP+ cells (77. 9%) were strongly lytic antigen+ (5 sections from 3 mice) (Fig. 7C). Therefore eGFP expression identified most lytically infected cells, plus eGFP+lytic antigen- cells that were presumably latently infected. Only 8. 7 ± 5. 9% of antigen+ dots (virions) were associated with eGFP+ cells. Therefore most of the cells binding input virions remained uninfected. In mice given eGFP+ORF50- MuHV-4, the lack of new lytic infection allowed us to identify diffusely antigen+ cells as those accumulating input virions (Fig. 7D). 32/35 antigen+ cells (91. 4%) were eGFP+, and 32/46 eGFP+ cells (69. 6%) were antigen+ (8 sections from 4 mice). The greater number of eGFP+ cells reflected presumably that detectable eGFP expression could come from a single infecting virion, but only cells accumulating multiple virions would be detectably antigen+. Nonetheless antigen accumulation and eGFP expression showed a significant correlation (p<0. 0001 by Fisher' s exact test). By contrast only 7. 1 ± 3. 8% of extracellular virions (isolated antigen+ dots) were associated with eGFP+ cells. Therefore type 1 AECs bound most input virions, but alveolar macrophages then accumulated the virions and became infected, consistent with their function of collecting particulate alveolar antigens [52]. MuHV-4 infection is endocytic [53] and virion antigens did not accumulate inside PDP+ cells. Therefore poor virion endocytosis seemed a likely explanation for type 1 AEC non-infection. Consistent with this idea, i. n. Herpes simplex virus type 1, which binds to heparan but penetrates most cells at the plasma membrane [54], showed abundant early type 1 AEC infection without marked macrophage involvement (S5 Fig). The γ-herpesviruses transmit from immune carriers and elicit salivary antibody responses [55]. Therefore shed virions may carry glycoprotein-specific antibodies. Also virions entering immune hosts or vaccinees could acquire antibodies before reaching mucosal surfaces. Therefore how bound antibodies affect host entry is important to understand. In vitro, immune sera inhibit MuHV-4 epithelial and fibroblast infections, but fail to inhibit and even enhance myeloid infection due to productive IgG Fc receptor (FcR) -mediated uptake [56]. This reflects that sera neutralize in vitro mainly by blocking cell binding, for which FcR binding can substitute. We tested 2 MuHV-4 glycoprotein-specific mAbs for their effect on lung infection: 8F10, which binds to gH/gL and blocks its interaction with heparan [57], and SC-9E8, which binds near the gB fusion loops and blocks membrane fusion [58] (Fig. 8). In vitro both mAbs inhibited fibroblast infection, but while SC-9E8 inhibited RAW-264 monocyte infection 8F10 enhanced it (Fig. 8A). Enhancement reflects that FcR binding improves significantly cell-free MuHV-4 attachment to RAW-264 cells [56]. In vivo virion capture via type 1 AECs was efficient already, so here mAb 8F10 simply failed to neutralize (Fig. 8B, 8C). By contrast mAb SC-9E8, which neutralizes independent of cell binding [58], was effective at blocking host entry. Most transformed epithelial cell lines display both sulfated and unsulfated heparan and are bound by both gp70 and gH/gL [33]. However only gH/gL binds well to unsulfated heparan (10E4-NAH46+) [17], as displayed by the apical alveolar epithelium (Fig. 6). Accordingly, an additional block of gp70 heparan binding by mAb LT-6E8 [33] did not affect host entry (Fig. 8C). To understand better the in vivo fate of 8F10-opsonized virions, we exposed WT and ORF50- eGFP+ MuHV-4 to mAb 8F10 or not, gave them i. n., and 1 day later stained lung sections for eGFP expression (Fig. 8D). Pre-incubation with 8F10 increased significantly the number of eGFP+ lung macrophages. Epithelial infection was unaffected, consistent with this occurring via macrophages rather than directly. Pre-incubating WT MuHV-4 with mAb 8F10 or not and staining lungs for virion antigens 5h after i. n. inoculation (Fig. 9) showed that 8F10 reduced significantly the virion binding to PDP+ cells without compromising their accumulation in CD68+ cells. Therefore blocking cell binding with antibody was insufficient to block host entry, because it merely diverted virions to where they were already going: macrophages. How rhadinoviruses first infect new hosts has been little explored. Even for lymphocryptoviruses there is considerable uncertainty. In vitro analyses have focussed on epithelial cell and lymphocyte infections. However MuHV-4, which enters the upper respiratory tract via olfactory neurons, entered the lungs via alveolar macrophages. Most entry models envisage a target cell with a specific binding receptor that is infected readily ex vivo. Alveolar macrophages lacked the key initiator of MuHV-4 binding—heparan—and were infected poorly ex vivo. These data suggest a new paradigm, in which virions with a sophisticated fusion machinery can exploit normal host pathways for entry. One such pathway appeared to be particle scavenging by alveolar macrophages; another was opsonization-dependent endocytosis. Thus, in vivo neutralization had to act post-binding. As immune sera neutralize MuHV-4 mainly by blocking cell binding [56], this explains their limited capacity to block host entry [59]. In vivo cell diversity and interaction provide a dimension lacking from most cell lines. Co-operative infections are one reflection of this and are a recurring theme in γ-herpesvirus host colonization: EBV can infect epithelial cells by transfer from B cells [60], and MuHV-4 infects olfactory sustentacular cells via binding to adjacent neurons [17]. The homeostatic interactions of macrophage with other cell types [61] make them prime candidates to participate in co-operative infections. Here, epithelial heparan binding licensed virions for macrophage infection, and interaction with macrophages in turn licensed epithelial infection. How might epithelial cell binding license macrophage infection? The MuHV-4 gp150 promotes virion release from heparan-deficient surfaces by inhibiting heparan-independent binding [24]. (Inhibition of EBV epithelial cell infection by gp350 [60] may analogously promote virion release.) Thus, initiating MuHV-4 infection requires heparan for binding and to engage gp150. A lack of heparan normally limits macrophage infection by non-opsonized, cell-free virions. If heparan is provided, or if the need for heparan is bypassed by gp150 disruption or by virion opsonization, then infection works well [26]. Therefore type 1 AECs licensed macrophage infection by their apical heparan capturing virions and engaging gp150. How might virion interaction with myeloid cells license epithelial infection? The next step in MuHV-4 entry after binding is endocytosis. Virions lacking gL show an endocytic defect that can be reproduced by gH/gL-directed neutralization, implying that gH/gL engages a pro-endocytic ligand [57]. (This ligand is not heparan as distinct mAbs block gH/gL heparan binding and virion endocytosis.) Thus, a paucity of pro-endocytic ligands may strand heparan-engaged virions on type 1 AECs. (By contrast the olfactory epithelium shows no barrier to virion uptake after binding [17,26].) The MuHV-4 gB and gH change conformation between endocytosis and fusion; fusion itself involves further changes [53,62]. Myeloid-derived virions display constitutively the intermediate, post-endocytic forms of gB and gH and overcome a post-binding block to B cell infection [26]. We envisage that they overcome the post-binding block to type 1 AEC infection in the same way. The tropism-associated gB and gH conformation changes occur in early endosomes, whereas fusion occurs in late endosomes [63]. Therefore virions sorted from myeloid early endosomes to exosomes could be released in a tropism-switched form without a need for myeloid infection. This would explain how follicular dendritic cells transfer MuHV-4 from marginal zone to follicular B cells without becoming infected [27]. A similar cycling through alveolar macrophage endosomes would explain delayed type 1 AEC infection without prior macrophage infection. Myeloid cells have roles in host colonization by several lymphotropic viruses [64,65] including the human MuHV-4 ortholog, KSHV [66,67]. Although MuHV-4 myeloid infection is sparse at steady state [68], inefficient ex vivo and not prominent in disease states, it is important for host entry, lymph node colonization [69] and systemic spread [27]. Myeloid cells engulf environmental antigens at many mucosal surfaces [70–72] and this can be extensive [73]. Therefore myeloid contributions to human γ-herpesvirus infections may be under-estimated. The amount of antibody attached to shed virions is unknown, but salivary antibody has clear potential to affect tropism [74,75]. Membrane fusion blocks or strong IgA responses might block host entry, because all infection requires fusion and mucosal IgA has a strong outward flux. However MuHV-4 elicits little post-binding neutralization [76] and little IgA [77]; antibody protects by effector recruitment [78] and poorly blocks host entry [59]. MuHV-4 is adapted to B cell physiology like EBV [79] but also to myeloid physiology, which encompasses the capture, transport and transfer of environmental antigens to B cells. Such adaptation is unlikely to be unique. Therefore γ-herpesvirus infection control strategies must consider not just the defensive functions of myeloid cells, but also their potential vulnerabilities. C57BL/6J, BALB/c (Harlan UK or Animal Resources Centre, WA), LysM-cre [43] (Jackson Laboratories), Ai6-ZSgreen1 [44], CD11c-cre [80] and CD169-DTR mice [47] were bred in the Department of Pathology, Cambridge or the Herston Medical Research Centre, Queensland. For infection, 6–12 week old mice were anesthetized with isoflurane and virus (30μl) pipetted onto the nares, from where it was inhaled. For luciferase imaging, mice were injected i. p. with 2mg D-luciferin, anesthetised with isoflurane and monitored for light emission with a charge-coupled device camera (IVIS lumina, Caliper Life Sciences). CD169-DTR mice (CD169+/DTR) were depleted of CD169+ cells by i. p. diphtheria toxin (2μg / mouse) (Sigma-Aldrich). Animal experiments were approved by the Cambridge University ethical review board, the UK Home Office (Project Licence 80/2538), and the University of Queensland animal ethics committee. BHK-21 cells (ATCC CCL-10), RAW-264 (ATCC TIB-71), 3T3–50 [15], NMuMG (ATCC CRL-1636) and 3T3-cre cells [24] were grown in Dulbecco’s Modified Eagle’s Medium with 2mM glutamine, 100IU/ml penicillin, 100μg/ml streptomycin, and 10% fetal calf serum (PAA laboratories). Macrophages were recovered from mice by post-mortem bronchial or peritoneal lavage, then adhered to tissue culture plastic (1h, 37°C) and washed in medium to remove contaminating B cells. The remaining cells were >95% CD68+. All viruses were derived from a BAC-cloned MuHV-4 genome [81] with an HCMV IE1 promoter-driven eGFP expression cassette. In some experiments this loxP-flanked cassette was retained to identify infected cells by eGFP expression; for fluorochrome-switching [26], luciferase-expressing [15] and EF1α promoter driven eGFP viruses [82] it was removed by passage in 3T3-cre cells. ORF50- MuHV-4 was grown and titered in 3T3-ORF50 cells, with ORF50 expression induced by doxycycline (1μg/ml). Herpes simplex virus type-1 (HSV-1) expressing eGFP from an HCMV IE1 promoter was grown and titered on BHK-21 cells [83]. MuHV-4 was recovered from infected cell supernatants by ultracentrifugation (13,000 x g, 2h). Any cell debris was removed by low speed centrifugation (500 x g, 5min) and filtration (0. 45μm). HSV-1 was recovered from infected cells by low speed centrifugation (500 x g, 10min) then sonicated (3 x 1min). Cell-associated MuHV-4 was prepared as for HSV-1 by centrifugation and sonication of infected BHK-21 cells. All virus stocks were titered by plaque assay and stored at-70°C. Infectious virus was measured by plaque assay [24]: dilutions of virus stocks or organ homogenates were incubated with BHK-21 cells (2h, 37°C), overlaid with 0. 3% carboxymethylcellulose, cultured for 2 (HSV-1) or 4 days (MuHV-4), then fixed (4% formaldehyde) and stained (0. 1% toluidine blue) for plaque counting. To assay fluorochrome switching by MHV-RG, plaque assays were performed at limiting dilution in 96 well plates (12–24 wells per dilution), and plaques scored as red or green fluorescent under ultraviolet illumination after 4 days. To assay infection by eGFP expression, cells were infected with eGFP+ virus (2h, 37°C), cultured overnight (18h, 37°C) in the presence of phosphonoacetic acid (100μg/ml) to prevent further spread, trypsinized, washed x2 in PBS, and analysed for eGFP expression with a FACS Calibur (BD Biosciences). To assay neutralization, viruses were incubated (1h, 37°C) with dilutions of mAbs 8F10 (anti-gH/gL, IgG2a), SC-9E8 (anti-gB, IgG2a), or as a negative control 28. 14. 8 (anti-H2Db, IgG2a, American Type Culture Collection HB-27), then assayed for infectivity as above. Organs were fixed in 1% formaldehyde / 10mM sodium periodate / 75mM L-lysine (24h, 4°C), equilibrated in 30% sucrose (18h, 4°C), then frozen in OCT. 9μm sections were air dried (1h, 23°C), blocked with 0. 3% Triton X-100 / 5% normal goat serum (1h, 23°C), then incubated (18h, 4°C) with combinations of primary antibodies to eGFP (rabbit pAb, Abcam), mCherry (rabbit pAb, Badrilla), B220 (rat mAb RA3–6B2, Abcam), CD11c (hamster mAb N418, Abcam), CD169 (rat mAb 3D6. 112, Abcam), CD206 (rat mAb MR5D3, Santa Cruz), unsulfated heparan (mouse mAb NAH46, Seikagaku Corporation), sulfated heparan (mouse mAb 10E4, Seikagaku Corporation), CD68 (rat mAb FA-11, Biolegend), PDP (goat pAb, R&D Systems), surfactant protein C precursor (goat pAb, Santa Cruz Biotechnology), MAC-2 (rat mAb M3/38, eBioscience), GR-1 (rat mAb RB6–8C5, R&D Systems), myeloperoxidase (goat pAb, R&D Systems), F4/80 (rat mAb CI: A3–1, Serotec), collagen IV (rabbit pAb, Abcam), HSV-1 (rabbit pAb, Abcam), and MuHV-4 (rabbit pAb). After incubation with primary antibodies (16h, 23°C), sections were washed x3 in PBS, incubated (1h, 23°C) with Alexa633-conjugated goat anti-rat IgG pAb, streptavidin-conjugated Alexa568 and Alexa488- or 568-conjugated goat anti-rabbit IgG pAb (Invitrogen), washed x3 in PBS, and mounted in Prolong Gold + DAPI. Fluorescence was visualised with a Leica TCS SP5 or Zeiss LSM 510 confocal microscope or a Nikon epifluorescence microscope, and analysed with ImageJ. MuHV-4 virions were disrupted with 0. 05% Triton-X100 in 50mM sodium carbonate pH = 8. 5, and coated (18h, 4°C) onto Maxisorp ELISA plates (Nalge Nunc). The plates were washed x3 in PBS / 0. 1% Tween-20, blocked with 2% bovine serum albumin in PBS / 0. 1% Tween-20, incubated with 3-fold dilutions of serum from MuHV-4-exposed mice (1h, 23°C), washed x4 in PBS / 0. 1% Tween-20, incubated (1h, 23°C) with alkaline phosphatase-conjugated goat anti-mouse IgG-Fc pAb (Sigma Chemical Co.), washed x5, and developed with nitrophenylphosphate substrate (Sigma). Absorbance was read at 405nm (Biorad), and the presence of a MuHV-4-specific response determined by comparison with sera from age-matched, uninfected controls.

All viral infections start with host entry. Entry into cells is studied widely in isolated cultures; entry into live hosts is more complicated and less well understood: our tissues have specific anatomical structures and our cells differ markedly from most cultured cells in size, shape and behaviour. The respiratory tract is a common site of virus infection. Size dictates where inhaled particles come to rest, and virus-sized particles can reach the lungs. Rhadinoviruses chronically infect both humans and economically important animals, and cause lung disease. We used a well-characterized murine example to determine how a rhadinovirus enters the lungs. At its peak, infection was prominent in epithelial cells lining the lung air spaces. However it started in macrophages, which normally clear the lungs of inhaled debris. Only epithelial cells expressed the molecules required for virus binding, but only macrophages internalized virus particles after binding; infection involved interaction between these different cell types. Blocking epithelial infection with an antibody did not stop host entry because attached antibodies increase virus uptake by lung macrophages; but an antibody that blocks macrophage infection was effective. Thus, understanding how rhadinovirus infections work in normal tissues provided important information for their control.

lay_plos

Background Plasma is the liquid portion of blood, containing nutrients, electrolytes (dissolved salts), gases, albumin, clotting factors, hormones, and wastes. Many different parts of plasma are used in treating the trauma of burns and surgery and for replacing blood elements that are lacking due to diseases such as hemophilia. According to estimates, each year about one million people in the United States receive products manufactured from human plasma. Plasma-derived products are purified from plasma pools by a process known as fractionation. This procedure involves a series of steps so that a single plasma pool yields several different protein products such as albumin and immune globulins. Plasma used for plasma-derived products manufactured and distributed in the United States may only be collected at facilities licensed and registered with the FDA. Centers require donors to provide proof that they are in the United States legally and have a local permanent residence. About 85 percent of plasma comes from paid donors in a commercial setting and is known as source plasma. The remaining 15 percent of plasma comes from volunteer donors and is known as recovered plasma. Units of plasma collected as source plasma contain approximately 825 milliliters; recovered plasma from whole blood donations contains approximately 250 milliliters. Thus, more than three times as many donated units of recovered plasma are required to make up a plasma pool equal in volume to one comprising only source plasma. Corporation, Baxter Healthcare Corporation, Bayer Corporation, and Centeon LLC. An additional 1.8 million liters of plasma are collected from approximately 8 million volunteer (not paid) donors, who contribute 12 to 13 million whole blood donations each year. These donors give blood at American Red Cross blood centers and independent blood centers represented by the trade group, America’s Blood Centers, and the plasma is recovered for further manufacturing. Plasma collected by the American Red Cross is fractionated under contract by Baxter Healthcare and the Swiss Red Cross and returned to the American Red Cross for distribution. Plasma collected at centers represented by America’s Blood Centers is sold only to the Swiss Red Cross, which manufactures the various plasma products and sells them through U.S. distributors. Paid donors typically receive between $15 and $20 for the 2 hours of time required to remove whole blood, separate the plasma from the cells and serum, and reinfuse the latter back into the donor. Source plasma donors may donate once every 48 hours but no more than twice a week. Whole blood donors may only donate once every 56 days because their red cells are not reinfused as they are with paid donors. All donors are tested for certain viruses known to be transmitted through blood, including HBV, HCV, and HIV. The specific screening tests check for the presence of hepatitis B surface antigen (HBsAg), antibodies to hepatitis C (anti-HCV), HIV-1 antigen, and antibodies to HIV types 1 and 2 (anti-HIV). Donors with positive test results are rejected from making further donations. The positive unit and all previously donated plasma units not pooled for manufacture in the preceding 6 months are retrieved, and those professional services that receive the plasma products are notified according to federal regulations (21 CFR 610.46). Risk of Infectious Units Entering Plasma Pools Is Somewhat Higher for Donations From Paid Plasma Donors Than for Donations From Volunteers The risk of incorporating a potentially infectious plasma unit into a plasma pool for HIV, HBV, or HCV is somewhat higher for donations from paid donors than for donations from volunteer donors. Information we obtained on viral marker rates for volunteer donors from the American Red Cross and for paid donors from the American Blood Resources Association (which represents paid plasma collection centers) showed viral marker rates among individuals who offer donations to paid plasma centers to be one and a half times higher than rates among those who come to volunteer blood centers. This is due to higher HCV rates among paid donors. In addition, incidence rates of HIV, HBV, or HCV are higher among paid donors than they are for volunteer donors, according to our review. These rates include donors who pass the initial screening tests and donate but who subsequently seroconvert and whom a screening test later detects during another donation as being positive. Thus, potentially infectious units from these donors could be incorporated into a plasma pool for manufacturing. HIV incidence rates are 19 times higher for paid donors than for volunteer donors; HBV and HCV rates are 31 times and 4 times higher, respectively. included in that pool if it were made exclusively from donations from paid donors. Manufacturer Reductions in Plasma Pool Sizes Tend Not to Benefit Frequent Users Concerns have been raised about the size of plasma pools because larger pools expose recipients of plasma products to more donors, raising the risk of infection. Manufacturers have recently taken steps to reduce the size of the plasma pools they use for producing plasma derivatives. Modeling techniques indicate that this effort can affect infrequent users of these products by minimizing their exposure to a certain number of donors. Frequent users of plasma products, such as hemophiliacs, however, tend not to benefit from these techniques because of the large number of different pools to which they are exposed during their lives. As recently as a year ago, FDA believed that initial fractionation pools contained 1,000 to 10,000 source plasma units or as many as 60,000 recovered plasma units. In response to inquiries from your Subcommittee, however, FDA obtained information from plasma manufacturers showing that after adjusting for the combination of intermediates, pooling of material from several hundred thousand donors for single lots of some products sometimes took place. For example, albumin can be added during intermediate processing steps or to a final product, such as factor VIII, for use as an excipient or stabilizer. This albumin often comes from another plasma pool containing donations that are not in the original pool. Because of concerns about pool size, the four major plasma fractionators voluntarily committed to reducing the size of plasma pools, measured by total number of donors, to 60,000 for all currently licensed U.S. plasma products, including factor VIII, factor IX, albumin, and immune globulin intravenous. This measurement takes into account the composition of starting pools, combining of intermediates from multiple pools, and use of plasma derivatives as additives or stabilizers in the manufacturing process. Prior production streams are still being processed and distributed, however, so that products distributed through the end of 1998 may still be produced from pools that exceeded the 60,000-donor limit. further manufactured by Baxter Healthcare. Seventy-five percent of all American Red Cross plasma manufactured by the Swiss Red Cross is now at the 60,000-donor limit, with plans for all production to adhere to the limit in the near future. In a study employing the modeling technique noted above, researchers found that limiting the number of donors in a pool may only be marginally beneficial for infrequent recipients, who might be exposed to an emergent unknown infectious agent with a low prevalence in the donor population, which current manufacturing processes did not inactivate or remove. As an example, the researchers calculated that for an agent with a prevalence of 1 in 500,000 (for example, a rare or emerging virus), a pool comprising 10,000 donations would yield a 2 in 100 chance of exposure to that agent for a one-time recipient. For frequent users of plasma products (that is, 100 infusions during a lifetime), however, this same pool size of 10,000 would yield an 86 in 100 chance of exposure to that agent, assuming that the products would come from different pools. Reducing the number of donors in a pool does not significantly decrease this effect. Thus, these modeling data suggest that smaller plasma pool sizes will reduce the likelihood of transmission of viral agents to infrequent users of plasma products but will have only a minor impact on frequent recipients of such products. In addition, risk of exposure does not always equate with risk of infection. In fact, risk of exposure is always greater than or equal to risk of infection. For example, the recent transmission of HCV by a plasma derivative that had not undergone viral inactivation procedures showed that the risk of seroconversion for recipients of this product increased with the number of positive HCV lots infused and the quantity of HCV viral material infused. Not all recipients were infected, however, because the highest percentage of seroconversions seen with the highest levels of HCV virus infused did not exceed 30 percent. Not all recipients experience seroconversions because of two factors: (1) each recipient’s dose and (2) the reduction of infectiousness due to steps in the manufacturing process in addition to viral removal and inactivation. Risk of Infection Reduced Through Viral Inactivation and Removal Techniques As mentioned, certain infectious units could make it through the donor screening, deferral, and testing process. Manufacturers have, therefore, introduced additional steps in the fractionation process to inactivate or remove viruses and bacteria that may have gotten into plasma pools. These techniques virtually eliminate enveloped viruses such as HIV, HBV, and HCV. They are only partly effective, however, against nonenveloped viruses such as HAV and human parvovirus. All types of plasma derivatives undergo viral inactivation or removal. The two main methods of inactivation are heat treatment and solvent-detergent treatment. To be effective, inactivation techniques must disrupt the virus, rendering it noninfectious. Heat treatment is accomplished either by exposing the freeze-dried product to dry heat or suspending it in a solution. Another technique heats the completely soluble liquid product with the addition of various stabilizers such as sucrose and glycine. The second technique, solvent-detergent washing, exposes the product to an organic solvent to dissolve the lipid coat of viruses, rendering them inactive without destroying the plasma-derived products. The lipid membrane contains critical viral proteins needed for infection of host cells. Disrupting the viral lipid envelope renders the virus noninfectious. Solvent-detergent inactivation is only partly effective, however, in eliminating nonlipid-coated viruses such as HAV or human parvovirus. Assessing the amount of viral clearance obtained through a particular inactivation or removal process determines the effectiveness of these different procedures. This assessment is based on the amount of virus that is killed or removed and therefore the extent to which these processes eliminate viruses through manufacturing. Individual manufacturing steps can be specifically designed for viral clearance, or they may be intended primarily as a purification process that will also help in killing or removing viral agents. To meet FDA approval of their particular inactivation or removal technique, manufacturers must separately validate each clearance step. cause infection, research has shown that infection is much more likely to occur with higher concentrations of virus. Proper viral inactivation and removal steps have resulted in no documented cases of HIV, HCV, or HBV transmission from plasma products since 1988. Recent Noncompliance With Current Good Manufacturing Practices Could Jeopardize Plasma Products’ Safety Although viral inactivation and removal techniques have proven to be highly effective, they are only useful if the steps in the manufacturing process are carried out properly. Recent FDA inspections of plasma fractionation facilities have found many violations of current good manufacturing practices. The lack of strict adherence to these practices could compromise the safety of plasma products. The objective of good manufacturing practices is to ensure that plasma products are safe, effective, adequately labeled, and possess the quality purported. Plasma manufacturers should operate in compliance with applicable regulations, which require adherence to current good manufacturing practices and quality assurance principles. In addition, each manufacturer must adhere to the standard operating procedures it has established for its facilities. To ensure that manufacturing processes, including inactivation procedures, follow current good manufacturing procedures, FDA is authorized to inspect plasma fractionation establishments. If the inspectors identify problems, FDA has a range of actions it may take. For violations deemed serious, these actions can include issuing warning letters, seeking a consent decree, or suspending a facility’s license. When an inspection reveals deficiencies, FDA may issue a warning letter to the facility, which does not suspend operations but gives the facility an opportunity to correct deficiencies. A warning letter notifies a firm that FDA considers its activities to be violating statutory or regulatory requirements and that failure to take appropriate and prompt corrective action may result in further FDA action. For some serious violations, FDA may seek a consent decree against a firm or individual—a court-ordered action that either mandates corrective actions that must be taken or prohibits the firm’s operation unless and until such actions are taken. FDA may pursue an action to suspend a facility’s license if the agency has documented deficiencies that constitute a danger to health, necessitating immediate corrective action. In such instances, the manufacturer would not be conforming to the standards in its license or the regulations. Recent FDA inspections conducted at the four major fractionation companies found many potential deviations in each company’s adherence to current good manufacturing practices. A recent inspection by FDA of Alpha Therapeutic’s facility observed 139 potential deviations from current good manufacturing practices or standard operating procedures; this has recently resulted in a consent decree with FDA. An FDA inspection of Baxter Healthcare’s fractionation facility observed 96 potential deviations. Bayer Corporation’s Berkeley, California, facility was cited for 30 potential deviations, and an inspection of Bayer’s Clayton, North Carolina, facility observed 77 potential deviations. Finally, an inspection of Centeon’s facility observed 87 potential deviations, which resulted in a consent decree filed in January 1997. The consent decree required Centeon to cease distribution of all but two of its products, while it brought its manufacturing standards into compliance with FDA statutes and regulations. In May 1997, FDA authorized the distribution of Centeon’s products from the facility, but, in a subsequent inspection completed in July 1998, FDA found that Centeon had failed to fully comply with the consent decree, and the company was notified to immediately cease manufacturing, processing, packing, holding, and distributing all biological and drug products manufactured at that facility. The company may, however, manufacture products deemed medically necessary. 54 percent of albumin lots for one company failed final container inspection because of visible evidence of protein material. To overcome these problems, the major fractionation companies have taken certain steps, such as increasing quality assurance and quality control and production staff and training, implementing capital investments at the fractionation facilities, and validating equipment processes. Many of the facilities slowed production as the firms reallocated resources to work on their corrective actions. In addition, FDA has taken several actions within the last year to better ensure manufacturer compliance with current good manufacturing practices. In a previous study examining the safety of the blood supply, we found inconsistencies in FDA’s inspection practices. As a result of this and an Office of Inspector General study examining FDA’s regulatory role in the field of biologics, FDA adopted a new inspection program. Under this program, FDA has designated two groups of investigators: one to focus on blood banks and source plasma collection centers and another to focus on plasma fractionation and manufacturers of allergenic products, therapeutics, licensed in vitro diagnostics, and vaccines. This approach is intended to ensure that all FDA current good manufacturing practice inspections are conducted by a single agency unit using a similar approach. If properly implemented, these actions by plasma manufacturers and FDA should help alleviate the problems related to adherence to current good manufacturing practices and quality assurance. This concludes my prepared statement, Mr. Chairman. I will be happy to respond to any questions that you or Members of the Subcommittee may have. The first copy of each GAO report and testimony is free. Additional copies are $2 each. Orders should be sent to the following address, accompanied by a check or money order made out to the Superintendent of Documents, when necessary. VISA and MasterCard credit cards are accepted, also. Orders for 100 or more copies to be mailed to a single address are discounted 25 percent. U.S. General Accounting Office P.O. Box 37050 Washington, DC 20013 Room 1100 700 4th St. NW (corner of 4th and G Sts. NW) U.S. General Accounting Office Washington, DC Orders may also be placed by calling (202) 512-6000 or by using fax number (202) 512-6061, or TDD (202) 512-2537. 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GAO provided information on blood plasma safety, focusing on: (1) comparing the risks of incorporating infected plasma from donations from volunteer donors with those from paid donors into the manufacturing process; (2) the impact on frequent and infrequent plasma users when pooling large numbers of plasma donations into manufactured plasma products; (3) the safety of end products from plasma after they have undergone further manufacturing and inactivation steps to kill or remove viruses; and (4) the recent regulatory compliance history of plasma manufacturers. GAO noted that: (1) viral clearance techniques have made the risks of receiving an infected plasma product extremely low when manufacturers follow all the procedures in place to ensure safety; (2) although paid plasma donors are over one and a half times more likely to donate potentially infectious units, several initiatives by the paid plasma industry have greatly reduced the chances of these units being included in the plasma production pool; (3) these initiatives include using only repeat donors and a 60-day inventory hold on all units to allow manufacturers to retrieve units from donors who subsequently test positive for disease or are otherwise disqualified; (4) even with those initiatives in place, plasma donated by paid donors poses a higher risk of infection; (5) limiting the number of donors whose plasma is pooled for production into plasma products helps to reduce the risks of viral transmission for recipients of these products; (6) a more significant step in reducing risk of infection takes place in manufacturing, during which all plasma products undergo viral removal or inactivation procedures, which virtually eliminate enveloped viruses; (7) epidemiological and laboratory data on the transmission of viruses through plasma products since the introduction of viral removal and inactivation procedures support the value of these procedures; (8) the effectiveness of these processes is limited, however, in reducing transmission of nonlipid enveloped viruses; (9) voluntary initiatives by the commercial plasma industry, technological advances from increasingly sophisticated screening tests, and viral removal and inactivation procedures are only effective if manufacturers of finished plasma products adhere to current good manufacturing practices; (10) not all of the major manufacturing companies producing plasma products adhere to these practices; (11) recent Food and Drug Administration (FDA) inspection reports highlight many instances of noncompliance with current good manufacturing practices; (12) this has led to consent decrees between FDA and two companies, temporary suspensions of production at one company's facility, and shortages of some plasma products; (13) a lack of strict adherence to these practices related to viral removal and inactivation procedures could compromise the safety of plasma products; and (14) actions being taken by FDA and the plasma manufacturers since these problems were identified should help to alleviate some of these problems.

gov_report

Natural killer T (NKT) cell development depends on recognition of self-glycolipids via their semi-invariant Vα14i-TCR. However, to what extent TCR-mediated signals determine identity and function of mature NKT cells remains incompletely understood. To address this issue, we developed a mouse strain allowing conditional Vα14i-TCR expression from within the endogenous Tcrα locus. We demonstrate that naïve T cells are activated upon replacement of their endogenous TCR repertoire with Vα14i-restricted TCRs, but they do not differentiate into NKT cells. On the other hand, induced TCR ablation on mature NKT cells did not affect their lineage identity, homeostasis, or innate rapid cytokine secretion abilities. We therefore propose that peripheral NKT cells become unresponsive to and thus are independent of their autoreactive TCR. Natural Killer T (NKT) cells represent a subset of T cells in mice and humans that express NK cell markers and recognize a small class of glycolipid (auto-) antigens [1], [2]. Most mouse NKT cells express an invariant Vα14-Jα18 (Vα14i) TCRα rearrangement (Vα24-Jα18 in humans). In principle, all TCRβ-chains are able to pair with this Vα14i-TCR chain [3]. However, the selection of NKT cells by endogenous glycolipids presented by the monomorphic MHC class I-like CD1d induces a strong bias towards TCRs containing Vβ8, Vβ7, or Vβ2 [1], [3], which is abrogated in the absence of selection [3], [4]. Recently, crystallographic analysis demonstrated a conserved binding mode of the NKT cell TCR to various glycolipids, where only germline-encoded residues were in direct antigen contact, reminiscent of innate pattern-recognition receptors [5]. Moreover, several observations suggest that this receptor is inherently auto-reactive [1], [2] and thereby determines NKT cell identity and influences their function. The expression of several inhibitory NK cell receptors on NKT cells was suggested to control their self-reactivity and avoid autoimmune activation [6], [7]. During development in the thymus, the few T cells expressing a Vα14i-TCR are selected upon recognition of self-lipids on double-positive thymocytes. Although several good candidates have been put forward [8]–[10], the exact nature of the selecting glycolipids remains controversial. Homotypic interactions involving the SLAM family (SLAMf) receptors 1 and 6 are additionally required for NKT cell differentiation [11]. Auto-reactive activation during thymic selection is thought to induce a substantially stronger TCR stimulus in comparison to that during the development of conventional T cells [12], [13]. As a consequence, expression of the transcription factors Egr1 and Egr2 is strongly increased [13], which in turn directly induce PLZF, the key transcription factor controlling NKT cell differentiation, migration, and functions [13]. Interestingly, the homeostatic proliferation of NKT cells after adoptive transfer was similar in CD1d-deficient and wild-type mice, indicating that this process is mostly cytokine-driven and does not depend on continued TCR-mediated self-lipid-recognition [14], [15]. However, as the transferred cells contained CD1d, a role for antigen could not be completely excluded. In addition, tonic antigen-independent TCR signals might contribute to NKT cell maintenance and phenotype. During immune responses, NKT cell activation depends mostly on two parameters: engagement of the TCR and the presence of proinflammatory cytokines released from antigen-presenting cells activated by innate immune pathways such as toll-like receptor (TLR) signals. Lipids derived from different bacteria [16]–[19] were shown to directly activate mouse and human NKT cells in a TLR- and IL-12-independent manner, and NKT cells are required for productive immune responses against these pathogens. NKT cells can also be activated indirectly through cytokines such as IL-12, IL-18, or type I interferons (IFNs) [20]. However, it remains controversial whether, depending on the strength of the cytokine signal, weak responses to self-antigens presented by CD1d are an additional obligate requirement. In one study, CD1d-dependent signals were found to be necessary for full NKT cell activation in response to all tested pathogens [20]. In contrast, others reported that IL-12-dependent NKT cell activation after LPS injection [21] or MCMV infection [22] is independent of either foreign or self-glycolipid antigen presentation by CD1d. Upon activation, the most distinguishing feature of NKT cells is their ability to rapidly produce and secrete large amounts of cytokines (Th1 and Th2 cytokines, among others). Their fast, effector-like response could be based on steady-state expression of cytokine mRNA in mice [23], [24] that was suggested to be a consequence of tonic self-reactive activation [2]. Recently, it was reported that human NKT cells do not constitutively express cytokine mRNAs. Instead, rapid cytokine-induced innate IFNγ production by NKT cells was suggested to rely on obligate continuous recognition of self-lipids, which retains histone acetylation patterns at the IFNG locus that favor transcription [25]. Another characteristic feature of NKT cells, their surface marker expression reminiscent of memory or recently activated T cells, was also connected to their inherent autoreactivity [2]. To thoroughly address the open questions regarding the nature and importance of TCR signaling for NKT cells, we generated a novel mouse model that allowed us to study the extent of Vα14i-TCR-mediated auto-antigen recognition in the periphery and its relevance for NKT cell identity. Furthermore, we monitored the fate of NKT cells after TCR ablation. Our results prove the inherent self-reactivity of the NKT cell TCR and demonstrate that although essential for positive selection, tonic TCR signaling is not required for NKT cell homeostasis, lineage identity, and rapid cytokine secretion. In order to produce large numbers of NKT cells in a physiological manner and to manipulate the expression of the semi-invariant Vα14i-TCR in a conditional fashion, we generated Vα14iStopF knock-in mice. To this end we cloned a productive Vα14-Jα18 rearrangement, including the Vα14 leader exon, intron and 1. 8 kb of upstream regulatory sequence, and 0. 2 kb intronic sequence downstream of Jα18. These elements were inserted by homologous recombination 3′ of Jα1 upstream of the Cα constant region of the Tcrα locus (Figure 1A). Expression of putative upstream rearrangements is aborted by four SV40 polyA sites at the 5′ end of the construct, and expression of Vα14i is rendered conditional through a loxP-flanked STOP cassette. We obtained over 80% (271 of 325) homologous recombinant ES cell clones during gene targeting, indicating an unusually high targeting efficiency of our construct (Figure S1A). The development of conventional T and NKT cells, identified by staining with mouse CD1d-PBS57-tetramers (tetramer+), occurs unperturbed in Vα14iStopF/wt heterozygous mice. In homozygous Vα14iStopF/F mice, T cell development is abolished due to transcriptional termination of TCRα expression before the Cα exons (Figure 1B). We bred Vα14iStopF to CD4-Cre mice, in order to express the inserted Vα14i-chain in double-positive thymocytes, mimicking the physiological timing of TCRα-chain rearrangement and expression [26], [27]. On average 23 times more thymic and 43 times more splenic NKT cells were generated in these, compared to wild-type mice (Figures 1B and 2A–E). Around 9% of the tetramer+ T cells in CD4-Cre Vα14iStopF/wt mice expressed the CD8 co-receptor (over 80% as CD8αβ heterodimer; Figures 1C and S1B, C), which is also expressed by some human NKT cells, but normally not in mice [28]. The proportions of CD4− CD8− double negative (DN) and CD4+ cells were comparable between transgenic (tg) and wild-type NKT cells (Figure 1C). Furthermore, the tgNKT cells were largely comparable to wild-type NKT cells with respect to Vβ-chain bias (Figure 1D) and surface phenotype (Figure 1E). Finally, we found that NKT cells from CD4-Cre Vα14iStopF/wt animals expressed the critical transcription factors promyelocytic leukemia zinc finger (PLZF), GATA binding protein 3 (GATA-3), and T-helper-inducing POZ/Krüppel-like factor (Th-POK) (Figure 1F) [28], [29]. Interestingly, we also detected a substantial proportion of the recently described NKT17 subset in the transgenic animals. These DN NK1. 1− NKT cells express the transcription factor ROR-γt and were shown to produce the cytokine IL-17 upon activation (Figure 1F) [29], [30]. Premature TCRα expression leads to aberrant T cell development in transgenic mouse models [26], [27]. To directly compare the consequence of premature to CD4-Cre-mediated timely Vα14i-TCRα-chain expression in our knock-in approach, we bred our mice to a germline Cre-deleter strain (Nestin-Cre) [31]. Compared to CD4-Cre-induced Vα14i-TCRα-chain expression, premature expression in Cre-deleter Vα14iStopF/wt led to significantly reduced numbers of NKT cells in thymus and spleen, especially of CD4+ NKT cells (Figure 2A–C). In addition, we found reduced thymocyte counts and a significant increase of most likely lineage-“confused” DN (CD4− CD8−) tetramer-negative T cells (Figure 2D, E). In fact Cre-deleter Vα14iStopF/wt mice strongly resemble the “first generation” Vα11 promoter-driven (Vα11p) Vα14i transgenic mice in these respects (Table S1) [32]. Moreover, in Cre-deleter Vα14iStopF/wt mice, we observed increased proportions of Vβ9-, Vβ10-, and Vβ14-containing Vα14i-TCRs, which can recognize α-GalCer-loaded tetramers, but most likely not endogenous self-glycolipids [3], [4], pointing to perturbed positive selection (Figure 2F). CD4-Cre Vα14iStopF/wt mice produce more NKT cells than any of the previously reported models, including mice with a Vα14i allele derived from a NKT cell nuclear transplantation experiment [11], [32]–[35]. A comparison of different Vα14i-transgenic models demonstrates that both the correct timing and endogenous control of TCR expression control favor NKT cell development (Table S1). Our analyses therefore showed that physiological timing of Vα14i-TCRα-expression at endogenous levels in CD4-Cre Vα14iStopF/wt mice contributes to the production of large numbers of correctly selected, bona fide NKT cells. To test the functionality of our transgenic NKT cells, we injected CD4-Cre Vα14iStopF/wt mice with the NKT cell ligand α-Galactosylceramide (α-GalCer) and determined their cytokine production directly ex vivo. The transgenic NKT cells were able to mount a rapid and robust cytokine response. Although a reduced proportion of transgenic NKT cells responded, in absolute cell numbers there was a 6–10-fold increase compared to wild-type NKT cells (Figure 3A). We did not observe significant steady-state cytokine production by transgenic or control NKT cells, and we detected only minor increases in cytokine levels in the serum of some of these mice (Figure S1D). Since cytokine production also varies with NKT cell maturation, we analyzed NKT cell development in CD4-Cre Vα14iStopF/wt mice in more detail. This revealed a strong bias toward immature fractions in the thymus, due to the dramatic increase in NKT cell progenitors. In the periphery, 20% of NKT cells fully matured, as judged by the expression of NK1. 1 and other NK cell markers (Figure 3B, C). This view is further supported by the reduced proportion of CD69 and T-bet-expressing NKT cells in CD4-Cre Vα14iStopF/wt compared to wild-type mice (Figure 3D). The expression of both CD69 and T-bet strongly correlated with NK1. 1 surface levels (Figure S1E, F). This also explains the higher intracellular PLZF expression in CD4+ and DN NKT cells of CD4-Cre Vα14iStopF/wt animals in comparison to control animals (Figure 1F), as it was shown that PLZF expression is downregulated during NKT cell development [36]. Reduced maturation seems to be a common feature in mice with overabundance of NKT cells (Figure S1G and Table S1) [33]. Indeed, a comparison of different Vα14i-tg mice suggests that independently of the total number of NKT cells generated, the size of the homeostatic niche for mature NKT cells appears to be around two million cells (Table S1). IL-15 is critical for the final maturation of NKT cells [37] and together with IL-7 required for their peripheral maintenance [14], [38]. NKT cells compete with NK cells for these resources [38]. The halved number of NK cells in CD4-Cre Vα14iStopF/wt mice (Figure 3E) suggests that the availability of these and maybe other cytokines might be insufficient due to the dramatically increased NKT cell numbers. The fact that a similar effect was observed in Vα11p-Vα14itg mice (Figure 3E) underscores this notion. These results let us conclude that while large amounts of NKT cells can be produced in mice, depending on the mode of Vα14i expression, the number of fully mature NKT cells is restricted by homeostatic constraints, some of which are shared with NK cells. The strong self-lipid-induced TCR stimulus that early NKT cell progenitors receive in the thymus can be visualized through high GFP expression under the control of the Nur77 gene locus, reporting TCR signal strength [12]. However, the subsequent loss of GFP in mature NKT cells suggests that these cells are either not exposed to or not responsive to self-antigens. In order to answer this question and to study NKT cell TCR-autoreactivity in the periphery, we investigated the consequences of Vα14i-TCR signals for conventional naïve T cells. We wondered whether Vα14i-TCR expression on naïve T cells, lacking inhibitory receptors and generally a NKT cell “identity”, would lead to activation upon (self-) lipid recognition and what cellular fate (s) are elicited by such activation. To this end, we generated mice enabling us to exchange the endogenous TCR-repertoire present on naïve peripheral T cells for a Vα14i-restricted TCR repertoire. The induction of Cre expression in Mx-Cre CαF/Vα14iStopF mice inactivates the CαF allele and simultaneously turns on the Vα14iStopF allele, leading to substitution of endogenous TCRα-chains with the Vα14i TCRα-chain (Figure 4A). As mentioned above, the Vα14i-chain can pair with all TCRβ-chains [3], although only Vβ2-, Vβ7-, and Vβ8-containing Vα14i-TCRs can recognize endogenous lipids such as iGb3 [3], [4]. Since TCRs containing one of these Vβ-chains constitute approximately 30% of the CD4+ and CD8+ peripheral T cell pool (Figure 1D and unpublished data), we predicted that our genetic switch experiment should generate sufficient numbers of T cells able to recognize self-lipids. In Mx-Cre transgenic mice, Cre expression can be induced through injection of dsRNA, such as poly (I: C) [39]. However, low-level “leaky” recombination occurs also in absence of an inducer [39], [40], leading to increased numbers of tetramer+ T cells in naive Mx-Cre CαF/Vα14iStopF mice (Figure S2A). Therefore, splenocytes were depleted of tetramer+ T cells by magnetic cell separation (MACS, Figure S2A), and 20×106 purified cells were injected intravenously (i. v.) into recipient animals lacking conventional αβ T cells and NKT cells (Cα−/− or Vα14iStopF/F). After cells were allowed to engraft for 2 wk, the TCR switch was induced by poly (I: C) injection. Importantly, except for a short-term activation of the immune system, poly (I: C) injection in Mx-Cre mice per se has no significant long-lasting effect on peripheral conventional T cells [40], [41] or on the number and phenotype of NKT cells (unpublished data). To definitely exclude any effect of poly (I: C) injection on our results, we waited 2–4 mo before analyzing the animals after the induced TCR switch. We found significant numbers of tetramer+ CD4+ and CD8+ T cells as a result of this switch experiment (Figure 4B–E). “Unloaded” tetramers did not stain these cells, demonstrating that they were not reactive against CD1d itself (Figure S2B). The TCR-switched tetramer+ T cells were predominantly enriched in cells expressing Vβ-chains that are associated with high avidity auto-antigen binding: Vβ2, Vβ8. 1/8. 2, and Vβ7 (Figure 4D, E) [3], [4], [42]. The exceptions were CD8+ TCR-switched tetramer+ T cells, in which Vβ7-expressing cells were not enriched. The bias toward tetramer+ CD8+ T cells (Figure 4C) is most likely due to more efficient Mx-Cre-mediated recombination in these cells [40]. Animals containing TCR-switched tetramer+ T cells, but not controls, displayed splenomegaly (Figure 5A, B), characterized by increased numbers of macrophages/monocytes, neutrophils, and Ter119+ erythroid progenitor cells, suggesting an inflammatory state (Figure 5C–E). In line with these findings, we could detect elevated serum TNF in more than half of these mice (Figure 5F). Elevated levels of other cytokines, such as IL-2, IL-4, IL-5, IL-6, IL-10, IL-17, and IFN-γ, were not found in the sera of these mice (unpublished data). Interestingly, we found that 6 (highlighted in red throughout the figure) of 17 spleens containing TCR-switched T cells were almost completely devoid of B cells (Figure 5G) as well as dendritic cells (DCs, Figure 5H), which present lipid antigens to NKT cells via CD1d [1]. Furthermore, tetramer- “conventional” T cells were also strongly reduced in these animals (unpublished data). Together, these results suggest that induced expression of the Vα14i-TCR on conventional naïve T cells causes sterile inflammation, possibly due to autoimmune activation. The appearance of tetramer+ cells displaying a Vβ bias similar to antigen-selected NKT cells, together with signs of inflammation upon TCR switch and the absence of CD1d-expressing B cell and DCs in some cases, suggested auto-antigen-mediated activation of TCR-switched cells. To verify that the newly assembled Vα14i-TCR on conventional T cells is functional, we injected recipients of Mx-Cre CαF/Vα14iStopF and control cells with α-GalCer or PBS 2 mo after switch induction. Ninety minutes after α-GalCer, but not PBS, injection, CD4+ and CD8+ tetramer+ T cells produced IFN-γ and TNF (Figure 6A), demonstrating the functionality of the newly assembled Vα14i-TCR. In comparison to NKT cells from wild-type or CD4-Cre Vα14iStopF/wt animals, a smaller proportion of tetramer+ T cells produced cytokines (Figures 6A and S2C). Tetramer+ TCR-switched T cells could also be activated in vitro through α-GalCer-pulsed A20 cells overexpressing CD1d (unpublished data) [43]. To study the consequences of Vα14i-TCR expression on tetramer+ TCR-switched T cells in more detail, we analyzed their surface phenotype and transcription factor expression. Absence of NK cell markers (Figures 6B and S2D) and PLZF expression (Figure 6C) indicated that the Vα14i-TCR signals are not sufficient to induce NKT cell differentiation of mature conventional T cells. However, the TCR-switched tetramer+ T cells expressed significantly higher levels of Egr2 in comparison to tetramer− T cells in the same animals (Figure 6D), suggesting that the switched cells receive stronger TCR signals [13]. TCR-switched T cells showed further signs of cellular activation, as they expressed elevated levels of CD69 (Figure 6E). Interestingly, these T cells displayed also significantly increased surface levels of PD-1, LAG-3, and less frequently, BTLA and TIM-3, which is typical of exhausted/anergic cells (Figure 6F–H and unpublished data) [44], [45]. To test whether exhaustion/anergy of tetramer+ TCR-switched T cells prevented a more dramatic form of autoimmune inflammation, we injected mice with PD-L1 and PD-L2 blocking or control antibodies twice a week for 4 consecutive weeks, starting 2 d before switch induction. The administration of these blocking antibodies has previously been shown to efficiently prevent anergy induction of conventional T as well as NKT cells, and to partially reverse the exhaustion of CD8+ T cells [44], [45]. However, we did not observe any dramatic differences in spleen weight or cellularity, or signs of increased inflammation, between animals receiving PD-L blocking or control antibodies (unpublished data). In response to PD-1 blockade, other inhibitory receptors such as LAG-3, BTLA, or TIM-3 might control the TCR-switched T cells. Taken together, our results showed that expression of the Vα14i-TCR on mature conventional T cells is not sufficient to induce a NKT cell differentiation program. Still, it is likely that Vα14i-TCR signals induce auto-antigen-mediated activation, possibly to the point of exhaustion. We therefore present strong evidence that the Vα14i-TCR can constitutively recognize self-lipids in the naïve steady state situation in vivo. The evidence for autoreactivity of the Vα14i-TCR on mature peripheral T cells raised the old but still not completely resolved question whether and to what extent interactions with self-lipid-presenting APCs are required for NKT cell maintenance, cellular identity, and function. In order to evaluate the importance of constitutive TCR expression and signaling for NKT cells directly in vivo and for long periods of time, we ablated the TCR on mature T cells using poly (I: C) injection of Mx-Cre CαF/F mice [40]. Two weeks after induced Cre-mediated recombination, around 30% of CD4 and 65% of CD8 T cells had lost functional TCR expression in these mice (Figure 7A and [40]). To unambiguously identify TCR-deficient NKT cells, we developed a robust staining strategy based on CD4, NK1. 1, CD5, and CD62L expression (Figure S3A). This limited us to CD4+ NKT cells, but our staining identified over 50% of the total NKT cell populations in thymus and spleen (Figure S3B). Around 65% of the thus identified NKT cells had lost TCR surface expression 2 wk after Cre induction (Figure 7A, B). Due to complete Cre-mediated recombination in lymphoid progenitors, T cell development is blocked at the double positive stage in Mx-Cre CαF/F mice after induction of Cre [40]. This allowed us to study the T cell decay in the absence of cellular efflux from the thymus. In agreement with previous studies [40], [46], we found that loss of the TCR leads to decay of naïve CD4+ CD44low and memory/effector-like CD4+ CD44high T cells with a half-life of 40 d and 297 d, respectively (Figure 7C, D). Interestingly, we observed essentially no decay of receptor-less NKT cells, with a calculated half-life of 322 d (Figure 7E), and could find significant numbers of TCR-deficient NKT cells even 45 wk after TCR deletion (unpublished data). To evaluate the role of TCR signals during in situ homeostatic proliferation, we administered BrdU for 4 wk via the drinking water, starting 2 wk after induced TCR ablation. Naïve CD4+ CD44low as well as CD4+ CD44high memory/effector-like T cells showed significantly decreased BrdU incorporation in TCR-deficient compared to TCR-expressing cells (Figure 7F, G). In contrast, TCR ablation did not affect NKT cell proliferation (Figure 7F, G). Interestingly, the BrdU incorporation was identical in TCR-deficient CD4+ CD44high T and NKT cells, indicating that in the absence of TCR signals the cytokine-driven expansion of CD4+ CD44high memory/effector-like T and NKT cells is similar (Figure 7F, G). Our results therefore indicate that long-term in situ NKT cell homeostasis is completely independent of TCR-induced signals. In absence of de novo T cell generation, we found elevated Egr2 expression in mature thymic, but not splenic, NKT cells compared to DP thymocytes and CD4+ T cells, respectively (Figure 8A). This indicates that NKT cells receive stronger TCR signals in the thymus, which is supported by the decreased Egr2 expression of mature thymic TCR-deficient NKT cells (Figure 8A). Surprisingly, in mature NKT cells in thymus and spleen, expression of the TCR-signal-induced key transcription factor PLZF is completely unaffected by TCR ablation (Figure 8B). In order to more generally evaluate to what extent NKT cell TCR-expression is required for the maintenance of characteristic lineage-specific gene expression (resembling recently activated T cells), we extensively analyzed the cell-surface phenotype of NKT cells 6 wk after TCR ablation. Of all the analyzed markers, the only significant changes that we observed on splenic NKT cells upon TCR ablation were downregulation of NK1. 1, CD4, CD5, and ICOS (Figures 8C, D and S3C–E). NK1. 1 expression was also reduced in thymic TCR-deficient NKT cells, in addition to CXCR6 expression (unpublished data). CD5 and ICOS expression were also reduced in TCR-deficient splenic naïve as well as CD62Llow CD4+ T cells (Figure S3C, D). CD4 was upregulated on TCR-deficient CD4+ naïve, but downregulated on NKT and CD4+ CD44high T cells (Figure S3E). Strikingly, all other cell surface markers characteristic for the NKT cell lineage, among them the transcription factors PLZF, GATA-3, T-bet, and Th-POK, as well as many cell surface markers whose expression is also induced upon TCR engagement, remained largely unaffected by loss of the NKT cell TCR (Figure 8D). Treatment of mice with LPS, a cell wall component of gram-negative bacteria, leads to release of IFN-γ by NKT cells via stimulation with IL-12 and IL-18 produced by innate immune cells. This does not require acute TCR engagement [21]. However, it has been proposed that the ability of NKT cells to rapidly release IFN-γ in this context critically requires continuous weak TCR activation in the steady state [25]. We therefore analyzed IFN-γ release of TCR+ and TCR- NKT cells after in vivo injection of LPS, α-GalCer, and PBS (Figure 9A, B). As expected, Egr2 expression could only be detected in NKT cells that were activated through their TCR (Figure 9A). Accordingly, 90 min after α-GalCer injection, the majority of TCR+ NKT cells, but virtually none of the TCR- NKT cells or the CD4+ conventional T cells, produced IFN-γ protein (Figure 9B). Interestingly, NKT cell activation through LPS injection in vivo was able to induce similar IFN-γ production by TCR- NKT cells in comparison to their TCR+ counterparts (Figure 9B). Our results thus clearly demonstrate that homeostasis and key features defining the nature of NKT cells, namely the unique activated cell-surface phenotype and the innate capacity for instant production of IFN-γ, do not require continuous auto-antigen recognition in the mouse. The elucidation of NKT cell function and their intriguing semi-invariant TCR benefited enormously from Vα14i-TCR transgenic mouse models [11], [32], [47], [48]. Over the last years, it became increasingly clear that premature expression of transgenic TCRα chains, including Vα14i [11], [32], leads to various unwanted side-effects such as impaired β-selection and the generation of large numbers of DN T cells both in the periphery and in the thymus [26], [27]. This drawback affects even TCR alleles generated through nuclear transfer of mature NKT cells [33]. For that reason, Baldwin et al. developed a system in which a transgenic CAGGS-promoter-driven TCRα-chain is expressed upon CD4-Cre-mediated excision of a loxP-flanked STOP cassette, mimicking the physiologic expression time point [26]. Likewise, Griewank and colleagues expressed the Vα14i-TCR under direct control of CD4 promoter and enhancer sequences [11]. These are clear improvements, but carry the inbuilt caveats of the respective heterologous expression construct. For example, it has been shown that a large proportion of activated mature T cells loses expression from such transgenic CD4 promoter enhancer constructs [49]. Here, we present a novel approach, in which the expression of the transgenic Vα14i-TCRα-chain, and in the future any other TCRα-chain of interest, can be initiated via CD4-Cre at the DP stage in the thymus, and is under endogenous control of the Tcrα locus throughout the lifespan of the cell. In these mice, large numbers of bona fide CD4+ and DN NKT cells were generated. The reduced proportions of fully mature stage 3 NKT cells (NK1. 1+, CD69high, T-bet+), as well as the reduced numbers of NK cells, are most likely a consequence of limiting amounts of common differentiation and maintenance factors, such as IL-15 [14], [37], [50]. In addition, attenuated TCR-signaling due to increased competition for self-antigen/CD1d-complexes might delay the full maturation of NKT cells in the transgenic animals. TCR signals have been proposed to play a role in the initiation of CD69 expression on NKT cells, as well as in the induction of IL-2Rβ, the β-chain of the IL-2 and IL-15 receptors [13]. Moreover, we observed the generation of tetramer+ CD8+ T cells. CD8+ NKT cells are found in the human, but not in wild-type mice. CD8 expression on Vα14i NKT cells does not interfere with negative selection, avidity for antigen presented by CD1d, or NKT cell function [28]. Instead, it was proposed that the absence of CD8+ NKT cells in the mouse is due to the constitutive expression of the transcription factor Th-Pok in all CD4+ as well as DN NKT cells [28]. Th-Pok has been shown to be crucial for the maturation and function of NKT cells, and directly represses CD8 expression [28]. This scenario fits well with the fact that the CD8+ tetramer+ T cells in the CD4-Cre Vα14iStopF/wt (as well as in the Vα11p-Vα14itg animals) did not express Th-Pok. These cells also lack many other characteristic features of NKT cells, including PLZF expression. Therefore, we refer to them as tetramer+ CD8+ T cells. Given the faithful recapitulation of endogenous TCRα-chain expression timing and strength in our knock-in mice, combined with the extremely high homologous recombination efficiency, we believe that our strategy should prove useful for the generation of further novel TCR-transgenic mouse models. By replacing RAG-mediated Vα14 to Jα18 recombination with Cre-mediated activation of Vα14i expression in CD4-Cre Vα14iStopF/wt mice, we can directly couple conditional gain or loss of gene function with Vα14i-TCR expression in NKT cells. NKT cell-specific gene targeting in mice with physiological NKT cell numbers could be achieved through the generation of mixed bone marrow chimeras with Jα18−/− bone marrow, which cannot give rise to Vα14i-NKT cells. Our studies were designed to elucidate whether or to what extent the expression of the autoreactive semi-invariant TCR would activate a peripheral mature naïve conventional T cell, convert it into an NKT cell, or induce gene expression typical of NKT cells. We took advantage of the conditional nature of the Vα14i-TCR knock-in transgene for a TCR switch experiment on conventional peripheral T cells. Naïve CD4+ T cells inherit a high plasticity [51]. Depending on TCR signaling strength and cytokine environment, they can differentiate in various subsets in periphery. This differentiation includes the induction of specific transcription factors, namely T-bet (Th1), GATA-3 (Th2), ROR-γt (Th17), and FoxP3 (peripherally derived regulatory T cells). For NKT cells, it is believed that strong TCR signaling, together with homotypic interactions involving the SLAM family (SLAMf) receptors 1 and 6, ultimately leads to PLZF induction during thymic development [11], [13]. DP thymocytes, presenting auto-antigen via CD1d and also expressing SLAMf members, are crucial for thymic NKT cell selection [11]. These SLAMf receptors are expressed on peripheral lymphocytes in comparable levels to double positive thymocytes (www. immgen. org). Therefore, lymphocytes, especially marginal zone B cells, which express CD1d to a similar level as DP thymocytes, should be able to present antigen and SLAMf-mediated co-stimulation, to naïve conventional T cells with a newly expressed Vα14i-TCR on their surface. The elevated levels of the TCR-induced transcription factor Egr2 in switched tetramer+ T cells suggest that they receive an (auto-) antigenic signal. This finding is in principle in agreement with our finding that tetramer+ TCR-switched T cells are enriched in cells that express Vβ2- and Vβ8. 1-/8. 2-containing Vα14i-TCRs. These TCRs were shown to have the highest avidity for NKT cell antigens [3]. Furthermore, Vβ7-containing Vα14i-TCRs were shown to be favored when endogenous ligand concentration are suboptimal in CD1d+/− mice [42]. In fact, in CD4+ tetramer+ TCR-switched T cells the relative enrichment for Vβ7-expressing cells was slightly higher than for Vβ2- and Vβ8. 1-/8. 2-expressing cells (unpublished data). However, the interpretation that this advantage is due to antigenic selection is at odds with the fact that Vβ7-expressing cells are not enriched in tetramer+ TCR-switched CD8+ T cells. We currently have no satisfactory explanation for this discrepancy. Both CD4+ and CD8+ Vα14i-TCR-expressing conventional T cells show features of activation and exhaustion/anergy, but do not develop into NKT cells, judged by absent PLZF and NK cell marker expression. This indicates that either mature T cells have lost the ability to enter the NKT cell lineage, the peripheral Vα14i-TCR signal is not strong enough, or as yet unidentified components of the thymic microenvironment are required to induce an NKT cell fate. Indeed, the high Egr2 expression of mature NKT cells that matured in the periphery and migrated back to the thymus (Figure 8A) suggests that stronger self-antigens are presented at this location. Interestingly, unlike TCR-switched tetramer+ T cells, Egr2 expression in mature splenic NKT cells was similar to that of conventional mature CD4+ T cells. Our data therefore suggest that in the periphery, the Vα14i-TCR can recognize self-lipids, but maturing NKT cells undergo a developmental program that prevents an auto-reactive inflammatory response. At this point, we cannot exclude the possibility that the observed cellular activation was antigen-independent. The fact that the internal control cells, the co-transferred tetramer− T cells, show no or significantly less signs of activation strongly argues for an involvement of antigen recognition or tonic signaling by the Vα14i-TCR. It also remains possible that the transient immune activation caused by the poly (I: C) administration contributes to the observed phenotypes. In all likelihood, this contribution is small, as we never observed any significant immune activation, not to mention loss of CD1d-expressing antigen-presenting B cells and dendritic cells, in Mx-Cre CαF/wt control mice that received poly (I: C). Despite these caveats, our results clearly show that under our experimental conditions, Vα14i-TCR expression on conventional naïve T cells leads to their activation and general immune deregulation. These findings seemed to support notions that NKT cell maintenance [52], their activated surface phenotype, and especially their rapid cytokine expression abilities might depend on constant antigen recognition [25]. However, by ablating the TCR on mature NKT cells in situ, we unequivocally demonstrated that long-term mouse NKT cell homeostasis and gene expression are nearly completely independent of TCR signals. In this regard, they are similar to memory T and B cells, which can maintain their numbers, identity, and functional capabilities in the absence of antigen [53], [54]. Our results are hard to reconcile with a recent report suggesting that NKT cell maintenance requires lipid presentation by B cells [52]. While there might be some differences between mouse and man, a more likely scenario is that the observations of Bosma et al. reflect rather acute local activation than true homeostatic requirements. Most of the known functions of NKT cells critically depend on their ability to rapidly secrete large amounts of many different immune-modulatory cytokines shortly after their activation. Still, it is not fully understood how NKT cell activation is triggered in different disease settings, and especially to what extent signaling in response to TCR-mediated recognition of antigens versus activation by proinflammatory cytokines contributes to this. Various studies reported that CD1d-dependent signals were required for full NKT activation in vitro [19], [20], [55], although most of them contained the caveat of potentially incomplete blockade of CD1d function by blocking antibodies. Our experiments, in line with a recent report [21], show that even in the complete absence of TCR signaling for 4 wk, NKT cells can be robustly activated in vivo to produce IFN-γ upon LPS injection in similar amounts as their TCR+ counterparts. Thus, we demonstrate that in mouse NKT cells continuous steady-state TCR-signaling is not required to maintain the Ifng locus in a transcriptionally active state, as recently proposed for human NKT cells [25]. Therefore, our results clearly demonstrate that cellular identity and critical functional abilities of mature NKT cells, such as steady-state proliferation and innate cytokine secretion ability, although initially instructed by strong TCR signals, do not require further antigen recognition through their TCR. Collectively, our data strongly support the view that Vα14i-TCR expression on developing NKT cells triggers a program that makes them unresponsive to peripheral self-antigens, which can continuously be recognized by their auto-reactive TCR. NKT cells are extremely potent immune-modulatory cells that upon activation can instantly secrete a large array of cytokines. Although they are selected by high affinity to auto-antigens, similar to regulatory T cells, they are not mainly suppressive cells. Therefore, it seems plausible that NKT cells are rendered “blind” to peripheral auto-antigens, rather than depend on continuous stimulation by self-lipids to maintain their cellular identity and innate functions. By keeping their activated state independent of self-antigen recognition, NKT cells can stay poised to secrete immune-activating cytokines while minimizing the risk of causing damage to self during normal physiology. On the other hand, the presence of the auto-reactive Vα14i-TCR serves to detect pathogenic states when a stronger signal is generated by the enhanced presentation of potentially more potent self-antigens or foreign lipids. To generate Vα14iStopF mice, B6 ES cells (Artemis) were transfected, cultured, and selected as previously described for Bruce 4 ES cells [56]. Mx-Cre [39], CαF [40], CD4-Cre [57], Nestin-Cre [31], Vα11p-Vα14i-tg [32], and Vα14iStopF mice were kept on a C57BL/6 genetic background. As we did not observe any differences between CD4-Cre and Vα14iStopF/wt mice in NKT cell biology, they were sometimes grouped together as controls. Mice were housed in the specific pathogen-free animal facility of the MPIB. All animal procedures were approved by the Regierung of Oberbayern. At the age of 6–8 wk (or 2 wk after cell transfer for the TCR switch experiment), animals were given a single i. p. injection (400 µg) of poly (I: C) (Amersham). All mice were analyzed 6–8 wk after injection, unless otherwise indicated. Single-cell suspensions were prepared and stained with monoclonal antibodies: B220 (clone RA3-6B2), BTLA (8F4), CD11c (N418), CD122 (TM-b1), CD127 (A7R34), CD160 (eBioCNX46-3), CD25 (PC61. 5), CD28 (37. 51), CD38 (90), CD39 (24DMS1), CD4 (RM4-5), CD44 (IM7), CD45RB (C363. 16A), CD5 (53-7. 3), CD62L (MEL-14), CD69 (H1. 2-F3), CD8α (53-6. 7), CD8β (H35-17. 2), CD95 (15A7), DX5 (DX5), Egr2 (erongr2), GATA-3 (TWAJ), Gr1 (RB6-8C5), ICOS (7E. 17G9), IL-4 (11B11), IL-13 (eBio13A), IL-17A (eBio17B7), IFN-γ (XMG1. 2), LAG-3 (eBioC9B7W), LFA-1 (M17/4), Ly49A/D (eBio12A8), Ly49C/I (14B11), Ly49G2 (eBio4D11), Mac1 (M1/70), NKG2A (16A11), NKG2D (CX5), NK1. 1 (PK136), PD-1 (J43), ROR-γt (AFKJS-9), T-bet (eBio4B10), TCRβ (H57-597), Ter119 (TER-119), Th-POK (2POK), and TNF (MP6-XT22) (all from eBioscience). SiglecF (E50-2440) was from BD. TCRβ chains were stained with the mouse Vβ TCR screening panel (BD). PLZF antibody and the CXCL16-Fc fusion were generous gifts from Derek Sant' Angelo and Mehrdad Matloubian, respectively. mCD1d-tetramers were provided by the NIH tetramer core facility. For intracellular transcription factor stainings, cells were fixed and permeabilized with the FoxP3 staining kit (eBioscience). For intracellular cytokine stainings, mice were injected i. v. in the tail vein with 40 µg of LPS (Sigma) or 2 µg αGalCer (Funakoshi) in a total volume of 200 µl PBS. Afterwards, cells were treated according to manufacturer' s instructions with the Cytofix/Cytoperm kit (BD). For multiplex measurement of cytokines in the serum, we used the mouse Th1/Th2 10plex Cytomix kit according to manufacturer' s instructions (eBioscience). Samples were acquired on a FACSCanto2 (BD) machine, and analyzed with FlowJo software (Treestar). The heat map was generated using perseus (part of the MaxQuant software [58]). Mice were fed with 0. 5 mg/ml BrdU (Sigma) in the drinking water for 4 consecutive weeks. Directly afterwards, BrdU incorporation was analyzed with a BrdU Flow Kit (BD). Serum TNF levels were determined by ELISA as recommended by the manufacturer (BD). RNA was isolated (QIAGEN RNeasy Micro Kit) and reverse transcribed (Promega) for quantitative real-time polymerase chain reaction (PCR) using probes and primers from the Universal Probe Library (Roche Diagnostics) according to the manufacturer' s instructions. Statistical analysis of the results was performed by one-way ANOVA followed by Tukey' s test, or by student t test, in Prism software (GraphPad). The p values are presented in figure legends where a statistically significant difference was found.

Immune system natural killer T (NKT) cells help to protect against certain strains of bacteria and viruses, and suppress the development of autoimmune diseases and cancer. However, NKT cells are also central mediators of allergic responses. The recognition of one' s own glycolipid antigens (self-glycolipids) in the thymus via the unique Vα14i T cell receptor, Vα14i-TCR, triggers the NKT cell developmental program, which differs considerably from that of conventional T cells. We generated a mouse model to investigate whether the Vα14i-TCR on mature NKT cells constantly recognizes self-glycolipids and to assess whether this TCR is required for survival and continued NKT cell identity. Switching the peptide-recognizing TCR of a mature conventional T cell to a glycolipid-recognizing Vα14i-TCR led to activation of the T cells, indicating that this TCR is also autoreactive on peripheral T cells or can signal autonomously. But TCR ablation did not affect the half-life, characteristic gene expression or innate functions of mature NKT cells. Therefore, the inherently autoreactive Vα14i-TCR is dispensable for the functions of mature peripheral NKT cells after instructing thymic NKT cell development. Thus the Vα14i-TCR serves a similar function to pattern-recognition receptors, in mediating immune recognition of foreign invasion or diseased cells.

lay_plos

As emerging and re-emerging infectious arboviruses like dengue, chikungunya, and Zika threaten new populations worldwide, officials scramble to assess local severity and transmissibility, with little to no epidemiological history to draw upon. Indirect estimates of risk from vector habitat suitability maps are prone to great uncertainty, while direct estimates from epidemiological data are only possible after cases accumulate and, given environmental constraints on arbovirus transmission, cannot be widely generalized beyond the focal region. Combining these complementary methods, we use disease importation and transmission data to improve the accuracy and precision of a priori ecological risk estimates. We demonstrate this approach by estimating the spatiotemporal risks of Zika virus transmission throughout Texas, a high-risk region in the southern United States. Our estimates are, on average, 80% lower than published ecological estimates—with only six of 254 Texas counties deemed capable of sustaining a Zika epidemic—and they are consistent with the number of autochthonous cases detected in 2017. Importantly our method provides a framework for model comparison, as our mechanistic understanding of arbovirus transmission continues to improve. Real-time updating of prior risk estimates as importations and outbreaks arise can thereby provide critical, early insight into local transmission risks as emerging arboviruses expand their global reach. The explosive emergence of Zika virus (ZIKV) in the Americas in 2016 caught the global health community by surprise. Officials scrambled not only to control the disease at its source but also to anticipate and rapidly contain global transmission via infected travelers [1]. The rate at which a newly introduced pathogen spreads can vary enormously though, particularly for ZIKV and other pathogens with multi-faceted drivers of their transmission [2]. For example, serological surveys of human exposure to the ecologically similar dengue virus (DENV) on either side of the Texas-Mexico border indicated far higher DENV exposure in the Mexican community, despite virtually identical climatic conditions and even higher mosquito abundance in the Texan community [3]. This result suggests that a priori prediction of risk may be quite challenging. Epidemiological risk assessment—estimating the severity and transmissibility of a threatening disease—can be vital to successful mitigation with limited resources. Historical outbreak data can provide invaluable insight into future epidemic risk. However, for a pathogen that has yet to arrive or has just begun to spread, we are forced to borrow epidemiological data from other populations or related pathogens, or to indirectly assess risk based on environmental suitability. For example, as the first importations of ZIKV arrived in the US in 2016, early attempts to determine the likelihood and rate of local transmission relied primarily on dengue epidemiological data from regions with markedly different climatic and socioeconomic conditions [4–6]. These ecological risk assessments provide information regarding the basic reproduction number of a disease (R0) —the expected number of secondary human infections resulting from a single human infection—which provides a meaningful and predictive measure of local epidemiological risk. For ZIKV, R0 encompasses the combined effects of the multi-step transmission life cycle: a mosquito biting an infected individual, incubating the disease and becoming infected, and finally biting and infecting a new susceptible individual. In a naive population, R0 indicates whether importations can potentially lead to sustained local epidemics; if so, it also provides insight into the probability, magnitude, and speed of spread [7,8]. However, ecological estimates often carry considerable uncertainty stemming from model parameterization and regional extrapolation, and suggest a wide range of possible epidemic outcomes, from terminal importations to stuttering chains of transmission to full blown epidemics [4,9–11]. Once an outbreak is underway, early case data can be used to directly estimate R0 [12–14]. For arboviruses with environmentally constrained spatial heterogeneity, such case-based estimates cannot be easily extrapolated from one region to others. Here, we introduce a method for performing real-time updating of ecologically informed estimates of R0 that reconciles discrepancies between initial estimates of R0 and real-time data on local transmission of an emerging arbovirus. This approach is applicable to settings in which there is an ongoing threat of arbovirus importation and in which transmission has not precipitated a large-scale epidemic. In particular, this work was motivated by recent introductions of ZIKV into the continental US. As hundreds of cases arrived from affected regions throughout the Americas, officials sought to estimate risks of autochthonous (local) transmission and identify high-risk regions in the southern US. However, given the novelty of ZIKV and the large proportion of ZIKV cases that go undetected, early ecological estimates had high uncertainty [5,6, 15–17]. Our method combines indirect and direct estimation methods to reduce such uncertainty and increase accuracy at high spatiotemporal resolution. We first build prior ecological estimates of local R0 and then harness real-time importation data—cases that arrive in a naive location with or without subsequently infecting others—to update the estimates, while explicitly modeling case reporting uncertainty. As a case study, we use the almost complete absence of secondary transmission following 298 importations of ZIKV into the state of Texas in 2016 and 2017 to lower and narrow local estimates of R0. We analyzed all known ZIKV importations into Texas from January 2016 to September 2017, including the county and notification date; county-level purchasing power parity (PPP) in US dollars [18]; daily temperature data at a 5 km x 5 km resolution for 2016-2017 and historical averages from 1960-1990 [19,20]. For each county and month, we averaged daily temperatures across all 5 km x 5 km grid cells whose center fell within the county; we aggregated 5 km x 5 km mosquito (Aedes aegypti) occurrence probabilities similarly [21]. Those data are available at dx. doi. org/10. 18738/T8/HYZ53B. In all, six mosquito-borne, autochthonous cases of ZIKV were reported in Texas in 2016 and one was reported in 2017 [22]. For updating R0 estimates, we analyzed 2016 data where two autochthonous cases were detected in Cameron County from passive surveillance–one in November and one in December 2016; we excluded four nearby cases discovered during the November follow-up investigation, because our model does not incorporate active surveillance. As sensitivity analyses, we re-estimated R0 assuming that no cases were detected and that all six cases were detected (S1 Fig). Accounting for this worst-case scenario lead to increased estimates for risk across the state, but expected risk still remained well below epidemic potential. For validating our estimates, we analyzed 2017 data and considered only one of the two reported autochthonous cases, as the second case occurred outside the timeline of our 2017 importation data. For ZIKV to be locally transmitted in a location, a mosquito must bite an individual, the mosquito must be infectious with the virus, and then that mosquito must bite a susceptible individual and transmit that virus. We focus our analysis on a singular value, R0, which captures the individual contributions of these factors on the potential for human to human transmission. Following Perkins et al. [6], we estimated R0 using a temperature-dependent Ross-Macdonald formulation, R 0 (T) = m b c a 2 e − μ (T) n (T) μ (T) r, (1) with parameters as defined in Table 1. Although others have considered temperature dependence in parameters other than μ and n (e. g., [23,24]), we opted for our relatively simple formulation for a few reasons. First, effects of temperature on μ and n are likely to be the most critical to include, due to their relatively strong influence on R0 [25]. Second, whereas temperature can influence m in multiple ways [23], we elected to model m in an alternative fashion that incorporates influences from a wider range of factors than temperature alone. Third, our approach for scaling R0 through data assimilation—which is the primary goal of this work—is applicable to essentially any formulation of a priori county-month R0. The R0 prior used here is particularly germane to the real-time nature of this exercise, given that an early (but largely similar) version of it was available as a preprint near the very beginning of the time window of our analysis [26]. The ratio of mosquitoes to people, m, makes use of two spatially varying inputs: Ae. aegypti occurrence probability and an index of economic purchasing power. The first of these was based on a global collection of Ae. aegypti occurrence records used to inform an occurrence probability model that made use of a number of environmental variables, including a temperature suitability index, precipitation, enhanced vegetation index, and urbanicity [21]. The second of these features spatial estimates of purchasing power parity (PPP) [18], which offers a relative spatial index of economic wealth that other work [6,32] has shown can serve as a useful proxy for spatial variation in economic factors affecting mosquito-human contact. Here, we used gridded estimates of those two spatially variable inputs for Texas, but otherwise followed the methodology of Perkins et al. [6] for translating them into a gridded surface of m. In brief, this involved transforming mosquito occurrence probability into a proxy for mosquito abundance and multiplying it by a shape-constrained additive model of PPP, which resulted in a convex, monotonically decreasing relationship between PPP and m. We do not consider the near-term effects of precipitation on local ZIKV transmission risk, but one could be included in future iterations, given a validated a priori model describing the relationship. To derive a priori distributions of R0 for each county-month, we drew 1,000 Monte Carlo samples from each parameter underlying the R0 formulation described above. This accounted for uncertainty in μ, n, Ae. aegypti occurrence probability, and the relationship between PPP and m, consistent with previous descriptions of uncertainty for each of those parameters [6]. These parameter draws were applied to the appropriate county and month data for Texas. Finally, we fit gamma distributions to each probability distribution for local transmission risk for use as informative priors, see S2 Fig for comparisons between the samples and the fitted gamma distributions. We developed a likelihood function describing the expected outbreak size following an importation, using the approach from [13]. Assuming that the secondary case distribution for each infected individual is negative binomial with mean R0 and dispersion parameter, k, and that all cases are detected, then the probability of an outbreak of chain size, j, is can be described by [13,14] as s (j, R 0, k) = Γ (k j + j − 1) Γ (k j) Γ (j + 1) (R 0 / k) j − 1 (1 + (R 0 / k) ) k j + j − 1 (2) where Γ (n) = (n − 1)!. However, not all cases are detected and we assume the imported index case is always detected and correctly classified as an importation, so the probability of detecting a chain of size, j, from a given importation is given by s * (j, R 0, k, p d) = ∑ l = j ∞ s (l, R 0, k) · (l − 1 j − 1) · p d j − 1 · (1 − p d) l − j (3) where pd is the case detection probability [13,14]. This equation can be understood intuitively. If cases can be missed then there are infinitely many ways in which an outbreak of a certain size can be detected (e. g. 5 detected cases could stem from an outbreak of size 6 with 1 case missed, or size 7 with 2 missed etc). This equation enumerates those possibilities and sums their individual marginal probabilities. Importantly, this allows for local, undetected cases. We take the product of all likelihoods for each imported case as L (O → | α, R 0 →, k, p d) = ∏ i = 1 length (O →) s * (O i, α R 0 γ i, ω i, k, p d) (4) where O →, contains the observed outbreak sizes for each importation (terminal importations have an outbreak size of one), R 0 γ i, ω i denotes the county (γ) -month (ω) R0 for the location and time that the importation occurred, and α is a statewide scaling factor applied to each R 0 γ i, ω i. The introduction of the state-wide scaling factor allows for localized importations to inform statewide estimates, but assumes that biases in the a priori R0 estimation procedure are constant across counties and months. While our analysis does not assume that all imported cases are detected, we do not explicitly model missed imported cases and simply explore the consequences of these potentially missed cases in a sensitivity analysis. If systematic biases in the spatiotemporal detection of imported cases are discovered, our method should be updated accordingly. Details of simulations and validation of the likelihood can be found in S1 File and S3 Fig. The negative binomial dispersion parameter governs the variability in secondary cases following each importation, with values near zero meaning that most importations yield few or no cases while a few “superspreaders” produce many. Superspreading dynamics are known to occur with mosquito-borne diseases like ZIKV [33,34]. We assume that ZIKV secondary case distributions are the same as that of dengue virus (DENV) [35], though these estimates should be updated as ZIKV-specific secondary case distributions are identified. Padmanabha et al. describe the relationship between regional R0 and the percentage of DENV imported cases that generate over 20 secondary infections (p20), as R0 = 0. 63 × 100 × p20 + 0. 58. As we require p20 > 0 for all values of R0, we set p20 to an arbitrarily low, but non-zero value (p20 = 1 × 10−8) for values of R0 < 0. 58. We then found the dispersion parameter for every value of R0 that lead to the expected p20 value from that previously estimated relationship. The result was that a single dispersion parameter captured the relationship well for all R0 values and thus we set the dispersion parameter, k = 0. 12, for all analyses (S4 Fig). The highly dispersed nature of the secondary case distribution suggests that most importations will not lead to epidemics, even if R0 > 1. For example, even with R0 = 1. 5, we would only expect to see an epidemic stemming from ∼8% of imported cases. Given our set of prior distributions for county-month transmission risk, we set out to estimate the statewide scaling factor, α, as a means to update our a priori transmission risk estimates. We estimate the posterior distribution for this scaling factor for each day with a new importation between January 2016 and January 2017 (our method also explicitly updates county-month R0 estimates for county-month combinations with imported cases). Importantly, this doesn’t indicate that α is changing through time, but rather that we refine our estimate for it as more data accumulate. We assumed a uniform prior for α of U ∼ (0,2), and used a blocked Gibbs sampling algorithm of MCMC. For each MCMC step we provide the detected imported cases to date and propose each county-month R0, a single α, and a probability of case detection, pd. County-month R0 proposals were normally distributed around the previous sample with standard deviation of 0. 1, α proposals were distributed U ∼ (0,2). We used a previously estimated distribution for pd as a strong, informative prior, pd ∼ N (5. 74%, sd = 1. 49%), as there are identifiability issues given its relationship with α, and assumed it to not vary spatiotemporally [32]. There were no differences between the posterior and prior distributions for the reporting rate S5 Fig. We used the Metropolis-Hastings probability to accept or reject proposals. Our chains consisted of 200,000 samples with a burn-in duration of 100,000, thinning every 10 steps. We ran a single chain for each parameter set, and assessed convergence through visual inspection of the trace plots (S6 Fig). Further algorithmic details and code are available on Github (https: //github. com/sjfox/rnot_updater). To help with an intuitive understanding of the method we also present a hypothetical situation (S1 File and S7 Fig). We derived the expected number of autochthonous cases from the importation data through September 2017 (at that time, the most recent importation was detected in mid-May) and compared the estimates to the actual reported autochthonous cases. We incorporated uncertainty into our estimates by sampling from the posterior county-month R0 distributions and simulating outbreaks accordingly (full details in S1 File). Prior to making importation-based updates, our initial median estimates of R0 across Texas’ 254 counties in 2016 range from approximately 0 to 1. 5 throughout the year with July and August having the highest transmission risk (Fig 1A). Given the potential implications for determining the epidemic potential of a county, throughout the manuscript, we conduct a one-sided test at a 1% significance level of the resultant probability distributions. We thus take a conservative approach and only consider counties whose probability distribution’s 99th percentile (upper bound) is above one to be at risk for an epidemic (R0 > 1). Simply put, we only classify counties as no epidemic risk if there is less than a 1% chance of R0 > 1. Initial upper bound estimates reach as high as three, and 119 (47%) of Texas counties are expected to be at risk of a local epidemic in at least one month of the year (Fig 1A, S8 Fig). When we considered historic average temperatures rather than 2016 temperatures, the projected 2017 risks were consistently lower, with the largest differences occurring during the unseasonably warm 2017 winter (S9 Fig). Case importations peaked in July, August, and September of 2016, with 164 (55%) of the 298 total 2016 importations arriving then (Fig 1B). The few detected autochthonous cases occurred in November and December, when expected risk was relatively low but not negligible. Based on all importations and autochthonous cases that occurred in Texas prior to January 2017, we estimate that all Texas counties have a median posterior R0 below 1 (Fig 2). Median estimates range from 0 to 0. 29; upper-bound estimates range from 0 to 1. 12, with only six (5%) of the original 119 high-risk counties maintaining epidemic potential (S10 Fig). The six counties with remaining epidemic risk are all contained within the greater Houston metropolitan area, and all have relatively low estimated transmission risk estimates ranging from 0. 25-0. 29 (Grimes, Houston, Madison, Montgomery, Walker, and Waller counties). When we assume historic averages rather than 2016 temperatures, we obtain similar results (S11 Fig). In a sensitivity analysis that assumes ∼20 times more undetected importations, we found that the estimated risks decreased further (S1 Fig). We also varied the number of detected autochthonous cases in November: as they decrease from one to zero, the estimated risks decrease considerably; as they increase to five, estimated risks increase, with 83 counties becoming at risk for a local outbreak (S1 Fig). However expected transmission risk for these counties is still well below 1, ranging from 0. 3-0. 6. Importation events had variable impacts on the posterior estimates, depending on their timing and location (Fig 3). Terminal importations early in the year, when a priori R0 estimates were low, had little effect; those arriving in the summer months, when high a priori R0 estimates suggested that transmission should have occurred, led to sharp decreases and a shrinking confidence interval. By early November, the median α decreased from 1. 0 to 0. 06 with a 95% CI of 0. 002-0. 30. The two secondary transmission events detected in November and December increased the posterior R0 estimates and widen the confidence interval slightly, but did not provide enough evidence to qualitatively change the transmission risk estimates. Incorporating all data up to January 2017, our best estimate is that R0 values across the state are roughly one fifth the original estimates (median: 0. 19,95% CI: 0. 05-0. 48). Interestingly, based on the a priori R0 estimates, we would have predicted local transmission to occur during the summer when importations and transmission risk were high. In fact, these initial expectations for transmission were lowered even further prior to the detected locally acquired case in November. Since we estimated the posterior distributions for transmission risk and the statewide scaling factor jointly, we were able to compare the prior and posterior estimates to understand if specific counties were downgraded more or less than expected by the statewide average. We find that the ratio between prior and posterior estimates tends to average near the statewide scaling factor of 0. 19, except for counties that experienced many reported importations during the year (S12 Fig). These counties tended to have lower than average ratios like Harris, Dallas, Tarrant, Bexar, and Cameron counties (S12 Fig). While the timing of the autochthonous transmission may not have been predicted using our initial risk estimates, the transmission events both occurred in the southern tip of Texas (Cameron county), which was predicted to be the highest risk county during those months. The temporal mismatch between the model predictions and the observed ZIKV local transmission in November and December might stem from the a priori transmission risk model, which constrains posterior estimates. Specifically, the model does not account for potential time lags between ZIKV environmental suitability and eventual emergence of locally acquired cases. Environmental determinants of mosquito population growth may take several weeks or months to impact arbovirus risk. Furthermore, the timing of case reports following infection will depend on the timing and severity of symptoms, healthcare seeking behavior and clinical reporting. To assess how this effect might alter results, we incorporated a two month lag in our a priori suitability model as estimated in [36]. In essence we shifted transmission risk estimates two months later than our baseline predictions suggested. For example importations occurring in November are matched with baseline transmission risk estimates from September. The resulting model yielded estimates that are more consistent with the observed ZIKV local transmission, although posterior risk estimates were remarkably consistent with our baseline results, with the highest risk period shifted by two months (S13 and S14 Figs). In comparing our baseline model with the lagged one, we calculate a BIC of 2. 9 in favor of the lagged model, suggesting a moderate improvement to model fit. We use transmission risk estimates based on importations through December 2016 to estimate the number of autochthonous cases we would expect to detect in Texas in 2017. Based on our posterior estimates we estimate a 1% probability of at least one single detected case in Hidalgo county at the time the secondary transmission occurred. Since the sample size is low, we average the expected number of secondary cases over the 2017 time period where importation data were available. Assuming first that only the reported importations occurred in 2017 (26 total), we estimate that there should have been 0. 08 (95%CI: 0-1) detected autochthonous cases in the state; assuming that many importations went undetected, according to the reporting probability (26 p d ≈ 453 cases), we estimate 1. 3 (95% CI: 0-7) detected autochthonous cases. Both of these estimates are consistent with the single autochthonous case detected in Texas in 2017, though our results best fit a scenario with many undetected importations (Fig 4). The global expansion of ZIKV was declared a Public Health Emergency of International Concern in February 2016, and caused more than 565,000 confirmed or probable cases and over 3,352 documented cases of congenital Zika syndrome. Although it is receding in most regions of the world, ecological risk assessments suggest that previously unaffected or minimally affected areas may remain at risk for future emergence, including parts of Asia and South America [37–39]. Differentiating regions that can sustain a ZIKV epidemic (R0 > 1) from those that cannot is vital to effective planning and resource allocation for future preparedness plans. To address this challenge, we have developed a simple method for refining uncertain risk assessments with readily available data on disease importations. Using previously validated ecological suitability estimates as a starting point, we applied the method to update ZIKV R0 estimates for each of the 254 counties in Texas, and found that only six counties have non-negligible probabilities of sustained local transmission, though an additional 77 counties may also be at low risk if our assumptions about local case data are too liberal. This is a substantial downgrade in expected risk, given that 43% of the 254 counties were previously thought to be vulnerable to ZIKV outbreaks, and even counties still at risk for epidemics on average had R0 estimates downgraded by 80% [4]. Based on these estimates and the number of importations that occurred in the state, our refined model suggests that there should have been between zero and one detected case of locally acquired ZIKV between January and September of 2017, which corresponds to the single transmission event actually detected in Cameron County in July 2017. Interestingly, a model that incorporates the possibility of many more missed imported cases into the state of Texas fits the data better, with an expected number of cases close to one (Fig 4). There is a discrepancy between our model predictions and the ZIKV case data: our final model predicts the highest local transmission risk to be in the summer across the state, but most cases of local ZIKV transmission occurred in the winter (6 out of 7). While these case numbers are low and may simply reflect random variation, the most recent dengue outbreak in South Texas peaked in November, concurrent with a large dengue epidemic in Northern Mexico, suggesting that fall may be a higher risk season for arbovirus transmission than our final predictions indicate [40]. The discrepancy likely stem from errors in our initial risk estimates, which serve as informative priors for our analysis. Our model does not incorporate a time lag between case importation, local transmission, and case detection, which has been previously estimated around two months [23,36,37]. Incorporating a two month lag in our model slightly increased the fit of the resulting predictions (BIC = 2. 9), but does not produce qualitatively different results in the magnitude of county risk other than the temporal shift S14 Fig. While these results were promising, we chose to present our baseline results instead of those from the lagged model, because the baseline model has been previously validated [6]. Importantly our simplistic lagging procedure does not properly encapsulate the mechanistic intricacies determining the relationship between environmental suitability, mosquito population growth, case importation, local transmission, and subsequent detection that account for the lags outlined in [36]. Our results support these time lags as a crucial element for predicting ZIKV risk, and highlight the need for future research into the mechanistic underpinnings to reduce bias and improve model fit and predictive accuracy even further. The factors driving county transmission risk were primarily temperature and mosquito population numbers (occurrence probability). Remaining uncertainty in county risk stems from a lack of detected imported cases, as imported cases primarily helped to downgrade Zika risk in the absence of local transmission. This provides the counterintuitive explanation for why our model suggests low epidemic risk for Harris county (containing the city of Houston) even though it was by far the county with the most imported cases. Given comparable importation and local case data, this approach could readily update ZIKV transmission risk estimates for any county in the continental US. Our estimation method relies on several simplifying assumptions, which we divide into those concerning this ZIKV test case of the updating procedure, and those concerning the updating procedure itself. For this specific implementation, we assumed that the shape of the secondary case distribution resembles that of dengue. Although we have no evidence to the contrary, this should be updated as ZIKV-specific estimates become available [35]. We also assumed that transmission is equally likely from imported and locally acquired cases. Imported cases may be less infectious than locally acquired cases for two reasons, leading us to underestimate local transmission risks. First, they may be more likely to receive care that limits transmission, although most ZIKV cases are inapparent or mild and do not require medical care [16], and second their local infectious periods may be shorter than those of autochthonous cases. This assumption could be relaxed in cases where data are available on the relative detection rates. In this case, we also do not consider the possibility of sexual transmission of ZIKV. While sexual transmission has occurred and may be important for specific populations [41], we assumed that mosquito-borne transmission is the dominant mode of infection. Our current iteration of the model makes several simplifying assumptions related to how we handle the temporal dynamics of both the a priori risk estimation and the updating procedure. Our baseline transmission risk model does not take any outside events that could modulate future risk into consideration (e. g. rainfall in the previous month causing upticks in mosquito populations), and future model development will be necessary to account for these important temporal dynamics [23,36,37]. For example, approaches that account for variability in the serial interval between successive cases and distributed lagged effects of environmental variables on transmission potential may hold promise for addressing this limitation [42]. The updating method itself also requires several simplifying assumptions. First, it relies on informative Bayesian priors for the relative spatiotemporal risk of a region. Posterior estimates could be biased by errors in these priors. At this point, we lack sufficient data from Texas to improve our estimates of the component parameters of a priori R0. The lack of such data, in fact, motivated our more modest goal of refining estimates of relative risk across counties. We could potentially improve model accuracy by incorporating smoothing techniques that more realistically capture spatiotemporal correlations and heterogeneity in transmission risk across Texas. This method also treats all importations as independent. However, spatiotemporal heterogeneity in case detection probabilities or clustering of cases (e. g., testing of travel companions) could bias risk estimates. When secondary clusters are detected, we assume they share a transmission tree stemming from a single detected importation. In this case, the low ZIKV detection rate suggests that both primary importations and secondary cases are likely to be missed. If the detection rates between the two types of cases are roughly similar our results hold. If importations are detected at higher rates than secondary cases, then the resulting risk estimates will be higher; when we assume the reverse, meaning locally transmitted cases are more likely to be detected than imported cases, the posterior risk estimates will be reduced. The additional assumption, that clusters are epidemiologically connected, seems reasonable for the small contained outbreaks detected in Texas, but may not be appropriate for importation-fueled arbovirus outbreaks in Florida, for example. In such cases, molecular data might enable estimation of transmission clusters [43,44]. Furthermore, there are identifiability issues associated with estimating the statewide scaling factor alongside the case detection probability, as both parameters modulate the expected number of secondary cases in the same fashion. We therefore used a strong, informative prior for the case detection probability from a previous study, and found no changes in the posterior distribution S5 Fig. If case detection is lower than we assumed, then the detected cases in Texas would have had to arise from larger outbreaks, so our final risk estimates would be closer to the prior (larger). Alternatively, if case detection in Texas is higher than we assumed, there would be an even larger discrepancy between our prior and resultant posterior risk estimates S15 Fig. During the height of the ZIKV threat, public health agencies in the US rapidly implemented both preventative measures (e. g., vector control and educational campaigns) and response measures (e. g., laboratory testing and epidemic trigger plans), particularly in high-risk southern states. Decision makers sought to identify and narrow the spatiotemporal scope of outbreak risk to enable targeted responses, efficiently allocate resources, and avoid false alarms [15,45]. Our method facilitates such rapid, real-time geographic risk estimation from typical early outbreak data, and allows for real-time updating of estimates as new data arise. In interpreting our results from 2016 in retrospect, they would suggest that the baseline transmission risk combined with the public health response was sufficient to mitigate the threat of a ZIKV epidemic across the state. Our method cannot disentangle the impact of public health response from the underlying transmission risk, so we cannot estimate the impact of the public health interventions. In real time, our results would have validated the magnitude of the public health response through the summer of 2016, and likely alleviated some of the epidemic concerns that arose in the winter when a handful of locally transmitted cases were detected in the state. Critically, we can conclude neither that all initial ecological risk assessments for ZIKV will overestimate risk, although this seems to be the case for ZIKV in Texas and elsewhere in the US [9], nor that public health preparations and interventions for ZIKV are no longer necessary in Texas or the southern US. Rather, our results suggest that sustained ZIKV outbreaks are unlikely, but not impossible (3% of the state remains at low epidemic risk), and provide more robust and localized estimates of ZIKV risk that can inform more targeted surveillance and reactions to future ZIKV importations. While our method provides an avenue to improve imperfect a priori risk estimates using real-time data, future predictions and estimates could be improved with advances to our mechanistic understanding of transmission risk (e. g., incorporating rainfall or improved understanding of mosquito-human contact patterns) [2]. These estimates should also only be taken as the baseline level of risk for the state. Climate change, anomalous weather events, or unforeseen circumstances that alter mosquito populations or mosquito-human interactions could raise or lower the Zika risk in Texas [3,46]. This framework is novel in its integration of a priori ecological transmission risk estimates with updating directly from real-time case reports [13,14]. It thereby provides increasingly accurate and precise risk assessments to support public health decision making, and can be generalized to update R0 estimates from importation data, regardless of the a priori method of estimation. For example, a new approach combining epidemiological and molecular analyses suggests that transmission risk in Florida is subcritical (i. e., R0 < 1) [44,47]. Given that Florida experienced thousands of introductions, only a few of which sparked large outbreaks, coupling such outbreak-driven estimation with our terminal importation method may provide a powerful real-time risk assessment framework for exploiting all available data. This method resembles those used to assess disease transmission risk during elimination efforts, including malaria in non-endemic regions [48]. The key innovation is that, by starting with ecological suitability maps, we simultaneously identify important transmission hotspots and leverage case data from one region to inform risk estimates elsewhere. We presented a simple and rational method for continuously updating transmission risk estimates for populations experiencing infectious disease importations, with or without secondary transmission. As we demonstrated for ZIKV in Texas, large numbers of terminal importations can profoundly lower both estimated risks of transmission and uncertainty in prior estimates, particularly those derived from ecological suitability or other models that borrow inputs from related pathogens in other parts of the world. Expanding our model to other regions could sharpen transmission risk estimates, and allow for more targeted public health interventions in the remaining hot spot locations [5,9, 17]. Transmission risk estimates always include uncertainty, but by assimilating data in real-time, our method can help confirm or revise public health understanding in the midst of an outbreak. Although the threat of ZIKV emergence in the continental US motivated this study, this new framework can be applied to improve transmission risk assessments when a disease newly threatens a population via regular introductions with minimal secondary transmission.

When novel infectious diseases like chikungunya or Zika emerge and threaten global spread, public health officials worldwide must assess the risk for local introductions and outbreaks. These assessments are made in anticipation of local case data, and officials must draw upon historic evidence from similar diseases or locations. Thus, accurate local risk assessments have most often been limited to retrospective analyses and have been unavailable in real-time during an emerging epidemic. Here we present a method that can harness both historic and current data to produce early risk assessments and update projections in real-time. We demonstrate our approach by estimating local transmission risk for Zika throughout the state of Texas. Our findings suggest that the majority of Texas counties face little risk for sustaining a Zika epidemic, and successfully predict the number of locally transmitted cases across the state. Real-time updating of local transmission risk estimates during an emerging epidemic can thus provide actionable, early insight for public health response as emerging arboviruses expand their global reach.

lay_plos

Gene conversion, the non-reciprocal exchange of genetic information, is one of the potential products of meiotic recombination. It can shape genome structure by acting on repetitive DNA elements, influence allele frequencies at the population level, and is known to be implicated in human disease. But gene conversion is hard to detect directly except in organisms, like fungi, that group their gametes following meiosis. We have developed a novel visual assay that enables us to detect gene conversion events directly in the gametes of the flowering plant Arabidopsis thaliana. Using this assay we measured gene conversion events across the genome of more than one million meioses and determined that the genome-wide average frequency is 3. 5×10−4 conversions per locus per meiosis. We also detected significant locus-to-locus variation in conversion frequency but no intra-locus variation. Significantly, we found one locus on the short arm of chromosome 4 that experienced 3-fold to 6-fold more gene conversions than the other loci tested. Finally, we demonstrated that we could modulate conversion frequency by varying experimental conditions. Gene conversion is the replacement of DNA sequence at one locus using a second locus as a template. It can occur between alleles or between non-allelic sequences that share homology and during mitotic or meiotic recombination, but unlike crossovers it does not result in the reciprocal exchange of DNA [1],. Research interest in gene conversion is motivated by its roles in human pathogenesis, genome dynamics and evolution as well as its usefulness in evaluating mechanistic models of recombination [3]. Despite its importance, gene conversion has been difficult to study except in organisms, like many fungi, that retain their meiotic products in distinct groupings. We have used a novel gene conversion reporter system in the flowering plant Arabidopsis thaliana to measure allelic gene conversion frequencies in over a million meioses. Meiotic recombination is initiated by the formation of DNA double-strand breaks (DSBs) catalyzed by the phylogenetically conserved protein SPO11 (Figure 1) [4]. The canonical model for recombination, called Double Strand Break Repair (DSBR), begins with release of SPO11 from either side of the break leaving 3′ overhangs that are further resected to form 3′ tails [5]. A 3′ tail can invade a homologous chromosome by annealing to its complement and displacing the other strand to form a D-loop. This annealing process results in heteroduplex DNA [6]. As a result, any sequence polymorphisms between the homologous chromosomes will yield mismatches in the heteroduplexed region. DNA polymerase can extend the invading 3′ end using the homologous chromosome as a template thereby expanding the D-loop until it can capture the second 3′ end of the break. Annealing of the D-loop to the second 3′ end also produces heteroduplex DNA, and polymorphisms will again yield mismatches. DNA synthesis primed by the second 3′ end, followed by ligation results in the recombination intermediate known as a double Holliday Junction (dHJ). Theoretically, dHJs can be enzymatically resolved to produce either crossovers (COs) or non-crossovers (NCOs). However, biochemical studies in Saccharomyces cerevisiae examining the temporal sequence of DSBs, dHJs, COs and NCOs suggest that dHJs are preferentially resolved to produce COs [7]. While the origin of NCOs in plants has yet to be determined, in S. cerevisiae they are thought to be produced primarily by an alternative pathway called Synthesis Dependent Strand Annealing (SDSA) [8]. SDSA diverges from the DSBR pathway at the point of second-end capture. The D-loop does not capture the second 3′ end. Instead, the invading strand dissociates from the homologous chromosome after extension by DNA polymerase and anneals to the second end. Gap filling and ligation can then produce a NCO. Because extension of the invading end uses a homologous chromosome as a template, re-annealing to the second end will also produce mismatches if polymorphisms are present. The DSBR and SDSA pathways both generate heteroduplex DNA making DNA mismatches possible. The Mismatch Repair (MMR) system can recognize and mend such lesions [9]. If MMR restores the original allelic state, the expected Mendelian 2∶2 ratio of alleles is maintained at the locus. The alternative is for MMR to repair the mismatch in favor of the homologous allele, resulting in gene conversion and a tell-tale non-Mendelian 3∶1 ratio of alleles. Absent any other considerations, MMR should produce conversions and restorations in a 50∶50 ratio. There is evidence, however, for biased gene conversion that favors alleles on the chromatid that did not experience the initiating DSB, as well as bias for the generation of G/C base pairs [10]–[12]. The term “gene conversion” was coined by Hans Winkler, who saw it as the basis for all recombination [13]. Gene conversion as an event resulting in the non-reciprocal exchange of genetic information was first observed by H. Zickler in the octads of the ascomycete Bombardia lunata [14]. In S. cerevisiae, where it has been studied most comprehensively, gene conversion converts up to 1% of the genome (92–320 kb from COs and 62–148 kb from NCOs) in each meiosis [15]. Gene conversion is more difficult to measure directly in multicellular eukaryotes, including humans and plants, because their gametes do not typically remain grouped after meiosis, so the classic 3∶1 signature cannot be observed. Instead, gene conversion is assumed to have occurred in these systems when a polymorphic marker switches parental type, but closely spaced flanking markers don' t experience exchange. Similar analyses of haplotypes at the population level enable estimation of historic gene conversion frequencies [16]. Formally, these indirect observations could also result from closely spaced double crossovers, but crossover interference is assumed to limit such events making their influence negligible [17]. Despite these detection difficulties, gene conversion has been implicated in at least 18 human diseases and is important in shaping linkage disequilibrium in the genomes of most eukaryotes [3]. Gene conversion in plants has been measured primarily at specific loci or limited regions. For example, Shi et al. used indirect measurement techniques similar to those described above and estimated that there are between 1×10−5 and 3. 7×10−5 gene conversions per marker per generation in the functional centromeres of maize [18]. This observation is a particularly interesting because in maize, like most eukaryotes, COs are thought to be essentially absent from centromeres [19], [20]. Thus, the detection of gene conversion events implies that these regions still experience DSBs that are presumably repaired by NCOs or by sister repair. More recently, Lu et al. sequenced the products of two meioses from an F1 hybrid between the Landsberg and Columbia ecotypes of Arabidopsis [21]. They observed 18 COs, six of which were associated with conversion tracts. They also observed 4 NCO gene conversions for a total of 10 conversions in two meioses. This analysis is powerful in that it has nucleotide resolution and is limited only by the density of polymorphisms between the two parental ecotypes and the number of tetrads that can be feasibly sequenced. Our analysis takes a different approach – we measured GC at a limited number of loci across the genome but examined over a million meioses. We developed a novel visual assay that enables us to measure COs, NCOs and GCs directly in the gametes of Arabidopsis [22]. Previously, we had generated a collection of transgenic plants with transgenes encoding fluorescent proteins expressed by a pollen-specific, post-meiotic promoter called LAT52 in a quartet1–2 (qrt1–2) mutant background that produces persistent pollen tetrads (we call these plants Fluorescent Tagged Lines or FTLs) [23]–[26]. The segregation of the fluorescent proteins in the pollen tetrads is a direct reflection of the segregation of the transgenes that encode them. As a result, when transgenes encoding fluorescent proteins of different colors are linked on the same chromosome, their expression patterns will differ in pollen tetrads if a CO occurs in the interval between them [27]. To detect GCs, we modified the FTL system by generating non-fluorescent mutant alleles of our existing transgenic lines. Pollen tetrads from plants that have heterozygous fluorescent and non-fluorescent alleles at a transgene locus will typically segregate fluorescence in a 2∶2 ratio (Figure 2). If GC occurs at the test locus, however, a non-Mendelian 3∶1 ratio is observed. This system has two significant advantages. Large numbers of tetrads (meioses) can be scored by visual analysis in a relatively short time, and the analysis can occur in an otherwise isogenic background that limits the influence of sequence heterogeneities between polymorphic parents [28], [29]. Using this system, we scored the frequency of gene conversion at 7 test loci distributed among the 5 Arabidopsis chromosomes in over a million tetrads to provide a genome-wide estimate of GC frequency and locus to locus variation. We also measured the ratio of CO∶NCO associated GCs as well as how environmental queues and developmental status influence GC frequencies. To measure meiotic COs in Arabidopsis, we had previously generated a collection of qrt1–2−/− plants with transgenes encoding fluorescent proteins (eYFP, DsRed2 and AmCyan) expressed under the control of the post-meiotic, pollen-specific promoter LAT52 [23]. To modify this system so that it could also be used to measure gene conversion, we created non-fluorescent mutant alleles for a subset of the collection, using ethyl methanesulfonate (EMS) mutagenesis. The Agrobacterium tumefaciens transformation method used to create the original FTL lines is known to sometimes insert tandem copies of transgene cassettes [30]. Since tandem transgene copies would reduce the efficiency of our EMS mutagenesis, we first screened our FTL lines by using PCR to amplify the whole transgene cassette from primers in the flanking genomic DNA, and by using ligation mediated suppression PCR to detect which lines carried single-copy transgenes. The FTL lines that passed the PCR screen were verified by hybridizing Southern blots of FTL genomic DNA digested with EcoRI (single recognition site within the transgene cassette) with a probe corresponding to the LAT52 promoter. Of the 66 FTL lines screened, we identified 10 with single copy transgenes. It should be noted that Agrobacterium mediated transgene insertion is nonrandom with respect to gene density and therefore the distribution of our test loci may be similarly nonrandom [31], [32]. Seed from FTLs homozygous for single copy transgenes were subjected to EMS mutagenesis. Pollen tetrads from the M1 plants were observed using epi-fluorescence microscopy to identify individuals expressing a 2∶2 fluorescent: non-fluorescent phenotype. Normally, plants homozygous for the fluorescent transgene will express a 4∶0 fluorescent pollen phenotype; hence, a 2∶2 phenotype indicates that one of the fluorescent protein alleles has suffered a mutation. We identified a total of 17 non-fluorescent alleles in 7 of the 10 FTLs on which we attempted mutagenesis (Table 1, Figure S1). At each locus the non-fluorescent alleles (with the exception of 2 alleles which were not used further in the study) were sequenced and the non-fluorescent FTL (NFTL) was backcrossed three or four times to the parental FTL line to eliminate background mutations. Of the 15 alleles sequenced, 10 were G→A changes and 5 were C→T changes in the coding strand, consistent with a strong bias for G/C→A/T transitions by EMS in Arabidopsis and other organisms (Table 1) [33]. To determine how frequently gene conversion occurs in the Arabidopsis genome, we used epi-fluorescence microscopy to examine pollen tetrads from plants heterozygous for the fluorescent and non-fluorescent alleles at one of each of the seven test loci described above. 1,054,024 tetrads were scored (an average of 150,574 per locus), and 186 tetrads with a 3∶1 segregation pattern were observed (Table 2). We also observed tetrads with a 1∶3 segregation pattern, but these were not included in our gene conversion counts since non-fluorescence could be attributed to pollen development defects or other causes. To account for both classes of conversion, we doubled the number of 3∶1 tetrads in all of our calculations. With this adjustment factor, the genome-wide average is 3. 5×10−4 conversions per locus per meiosis (or 1 conversion per locus in every 2,833 meioses). A 3∶1 segregation pattern could also be obtained if one of the non-fluorescent alleles experienced a mutation that restored fluorescence (a reversion). To control for this possibility, we scored 45,000 pollen tetrads from a plant that was homozygous for the NFTL 3282-GC1 allele and observed only 0∶4 tetrads (plant genotypes were confirmed using allele-specific PCR). Since no reversion events were observed, the reversion frequency under our experimental conditions is significantly lower than the GC frequency observed (P = 0. 008998). Consistent with these findings, mitotic reversion rates at transgene loci in Arabidopsis have been independently measured between 10−7 to 10−8 [34]. Another source of false-positives is the mechanical disruption and subsequent random re-association of pollen tetrads to yield a grouping of pollen grains that are not meiotically related. To control for this possibility we scored 45,000 tetrads from a plant that was hemizygous for the FTL 567 locus and observed no 3∶1 tetrads. Since no spurious 3∶1 tetrads were observed the frequency of this type of physical re-association is significantly lower than the observed GC frequency (P = 0. 008998). We conclude that most, if not all, the events we observed were due to gene conversion rather than reversion or false-positives. Gene conversion frequencies might be expected to vary depending on DSB distribution, CO/NCO balance, bias in restoration versus conversion by MMR, or through the indirect effects of sequence context or epigenetic influences. Alternatively, since gene conversion can alter the genome, there might be regulatory mechanisms constraining its action. To determine if there is locus to locus variation in gene conversion frequency in Arabidopsis, we looked for statistical differences in all pairwise combinations of our seven test loci (Table 3). NFTL 424-GC1 experienced significantly more gene conversion than all other test loci (P<2×10−11 to 2×10−15). NFTL 3411-GC1 also had a significantly higher gene conversion frequency when compared to 4 of the 6 other loci (P<1×10−6 to 0. 04). NFTL 1659-GC1 and 1273 GC-1 experienced the lowest frequency of gene conversions and were significantly lower than the genomic average (P<0. 02 and 0. 01 respectively), as was NFTL 1369 GC-1 (P<0. 04). We conclude that there is significant locus-to-locus variation in gene conversion in Arabidopsis. Given the locus-to-locus variation observed, we asked whether there was intra-locus variation as well by comparing the gene conversion frequency of two different alleles, NFTL 1369-GC1 and 1369-GC2 (the SNPs in each allele are separated by 120 bp). After counting 155,280 and 150,916 tetrads respectively, we observed 32 (adjusted) conversions for each allele indicating - at least for this locus - a lack of intra-locus variation (P<0. 94, Table 3). Evidence from S. cerevisiae has demonstrated that the frequency of gene conversion can exhibit polarity, typically (though not always) with a higher level at the 5′ end of genes [35], [36]. This polarity is thought to result from either a gradient in DSB formation, with a preference for promoter regions, or modulation of the direction of mismatch repair (conversion versus restoration) [37]–[39]. To see if we could detect a similar polarity for gene conversion in Arabidopsis we plotted the position of the mutation at each of our test loci relative to the transcriptional start site (Table 1) against the frequency of gene conversion observed at each locus (Table 2, Figure 3). The linear regression of position of the polymorphism on conversion frequency was not significant (r2 = 0. 038, P = 0. 645). The result of this limited analysis (seven loci) supports the prior suggestion that Arabidopsis lacks strong intra-locus variation in gene conversion frequency. As described above, COs produced by DSBR and NCOs produced by SDSA can both be accompanied by GC. To determine what proportion of the conversions we observed can be attributed to either pathway, we constructed a combination of markers that enables us to monitor both conversions and COs simultaneously. We flanked our NFTL 1659-GC1 test locus on chromosome 5 with FTL 1273 (DsRed2) and FTL 993 (AmCyan) on either side (Figure 4). When conversions at NFTL 1659-GC1 were observed, they could be categorized as CO-GC or NCO-GC by scoring the segregation of the flanking markers. We scored 149,965 tetrads and observed 13 conversions. 11 were associated with COs and 1 was associated with a NCO (the flanking AmCyan signal in the remaining tetrad was too weak to score unambiguously). These results demonstrate the utility of our system for measuring the balance between CO and NCO associated GCs, however measurements at similar triple-color test intervals across the genome will be necessary before broad conclusions can be drawn. Using our FTL system, we had previously demonstrated that CO frequencies can be significantly elevated in Arabidopsis either by growth at high temperatures or by sampling meioses from 2° or 3° axes (branches) rather than the 1° shoot (i. e. different developmental contexts) [23]. To determine whether gene conversion is also influenced by these environmental and developmental queues, we scored tetrads from plants that were heterozygous for the NFTL 424-GC1 marker under the same experimental conditions. Surprisingly, unlike their influence on CO frequency the treatments showed divergent effects on gene conversion. The elevated temperature treatment increased conversion frequencies significantly. Control plants grown at 20°C experienced 1 conversion in every 936 meioses (Table 2; n = 147,848 tetrads), while plants grown at 28°C had 1 conversion in every 321 meioses (P<8×10−4; n = 9,624 tetrads). By contrast, tetrads (n = 160,581) collected from branches showed no significant change in conversions with one event per 1,147 meioses (P<0. 21) compared to controls. To understand the molecular mechanisms that facilitate and regulate meiotic recombination, a useful experimental system permits easy and rapid analysis of COs, NCOs and GCs. We had previously used our FTL system to assay crossing-over, crossover interference and mutants influencing those processes [23], [40]. Here we' ve adapted the FTL system to enable measurement of GC frequencies and detection of NCOs, and we' ve established a baseline description of the meiotic gene conversion landscape in Arabidopsis. A limitation of the current version of this system is that it detects the conversion of a single SNP at the test locus. As a result, direct measurement of the length of DNA that is converted in a given event (i. e. the “conversion tract” length) is not possible. Nonetheless, our data can provide a tentative tract length estimate. The sequenced portion of the Arabidopsis genome comprises 119,146,348 nucleotides. The five centromeres and two ribosomal RNA gene arrays have not been sequenced and may contain an additional 15 Mb and 7 Mb of DNA respectively for an upper genome estimate of 141,146,348 bases (GenBank Assembly ID: GCA 000001735. 1) [41]–[44]. Unlike S. cerevisiae, the number of DSBs per meiosis has not been measured directly in Arabidopsis, but several studies using counts of RAD51 foci at mid-prophase as a proxy for DSBs suggest that there are likely between 120 and 222 breaks [45]–[47]. Assuming each of those breaks is repaired by either the DSBR or the SDSA pathway, associated conversion tracts should be possible for all of them (Figure 1). If MMR restores 50% of those events to their original allelic state, we would expect 60–111 conversions across the genome. Dividing the high and low estimates of the genome size by the low and high estimates of the number of conversions yields an expected frequency of 4. 25×10−7 to 9. 32×10−7 gene conversion events per nucleotide per meiosis. But this is several orders of magnitude lower than our observed frequency of 3. 5×10−4. The observed and expected frequencies can be reconciled if a tract length of 379–830 bp (average 605 bp) is assumed. This estimate is tenuous since it rests on several assumptions (numbers of DSBs, frequency of homolog versus sister repair, efficiency and directionality of MMR during meiosis), but it corresponds nicely to the median midpoint length of 558 bp that Lu et al. provided, based on their analysis of a limited number of conversions in Arabidopsis [21]. It may be possible in the future to modify our system further by incorporating additional SNPs at the test loci to enable direct measurement of tract lengths. As outlined above, the roughly 120–222 DSBs (as estimated by RAD51 foci) that occur in each Arabidopsis meiosis are thought to be repaired by either the DSBR or the SDSA pathways. Only a small fraction of these are repaired as COs (DSBR pathway): numerous studies have demonstrated that Arabidopsis experiences ∼9 COs per meiosis [21], ; so the remaining 111–213 breaks should be repaired as NCOs (SDSA pathway). This implies that the CO∶NCO balance in Arabidopsis should be ∼1∶12 to 1∶20 - in stark contrast to our observed ratio of 11 COs for each NCO. As described earlier, Lu et al. also observed more COs than NCOs after sequencing the equivalent of two tetrads. There are several ways to explain this. Most trivially, the particular locus we measured could be anomalous. A more interesting possibility is that most meiotic DSBs in Arabidopsis may be repaired using a sister chromatid rather than a non-sister chromatid. Sturtevant' s discovery of unequal crossing over at the Bar locus in Drosophila initiated the idea that there is a strong bias for meiotic inter-homolog rather than sister exchange [50]. The possibility of inter-homolog bias was bolstered by experiments in S. cerevisiae and Drosophila measuring recombination between structurally heterozygous chromosomes (ring/rod heterozygotes) [51], [52]. These findings are consistent with more recent experiments showing that the majority of the ∼160 DSBs/meiosis in yeast are repaired as inter-homolg COs or NCOs (∼137 total/meiosis) [53]. However, similar experiments looking at recombination in ring/rod heterozygotes in maize and Antirrhinum majus (snapdragons) suggest that meiotic sister chromatid exchange may occur more frequently in plants [54], [55]. Indeed, even in yeast where inter-homolog bias is thought to be robust, when DSBs occur in regions lacking a homologous locus the breaks are efficiently repaired from the sister [56]. Alternatively, most DSBs in plants could be repaired as inter-homolog COs or NCOs but MMR in the SDSA pathway may be disproportionately biased in favor of restoration rather than conversion, resulting in fewer detectable NCOs. Another possibility is that conversion tracts associated with COs may be longer than those associated with NCOs and are detected at a given test locus more frequently. This idea is supported by data from S. cerevisiae, which has an average conversion tract length of 2. 0 kb and 1. 8 kb for COs and NCOs respectively (P<0. 0001) [15]. The balance between inter-homolog versus sister exchange or bias in restoration versus conversion by the MMR machinery may also contribute to our results when trying to modulate gene conversion frequency. An increase in both COs and conversions may indicate that more DSBs are formed in Arabidopsis at elevated temperatures. Alternatively, DSBs may be held constant, but under normal growth conditions some breaks are repaired from sister chromatids while under elevated temperatures sister-repair is directed instead to homologous repair and manifests as COs and NCOs associated with conversions. To differentiate these possibilities, it will be necessary to use mutant analysis. We' ve built on our previous visual assay for COs in Arabidopsis and expanded it so that it can now measure both GCs and NCOs as well. Using this system, we' ve characterized the baseline gene conversion landscape during meiosis to serve as a useful reference point for future analyses. We' ve also demonstrated that the system can be used to detected experimentally induced changes in gene conversion frequencies. This provides a proof of principle that will enable the system to be used to investigate which proteins mediate and regulate meiotic recombination in plants. All FTL lines have been described previously and are available from G. P. Copenhaver upon request, as are all the lines generated in this study [23]. Seeds were sown on a pre-wetted 5∶1 mix of Metro-Mix 360 soil (Sun Gro Horticulture, http: //www. sungro. com/) and Horticultural Perlite (Carolina Perlite Company, Inc., Gold Hill, NC, USA) and stratified for 3–4 days at 4°C. Plants were grown under long-day conditions (18 h day, 6 h night) at 20°C unless otherwise noted. Genomic DNA was extracted from ∼100 mg of fresh cauline leaves using Plant DNAzol (Live Technologies, www. invitrogen. com) per the manufacturer' s instructions. To identify single-copy FTL lines, PCR primers corresponding to genomic sequences flanking the transgene (P1 and P2 – specific to each FTL line) were used in combination with primers corresponding to the left or right T-DNA sequence (L1 or R1) to amplify genomic-T-DNA junctions from FTL homozygotes (see Table S1 for primer sequences). PCR amplification using P1/P2/L1 or R1 will yield a single PCR product from single-copy FTL lines. The same primers (P1, P2, L1 or P1, P2, R1) amplify only genomic sequence from control wild-type plants. Amplification of whole transgene cassettes from putative single-copy FTL lines was achieved using LongAmp DNA polymerase (New England BioLabs, www. neb. com) with primers P1 and P2. Allele-specific genotyping was conducted using a P1 and L1 or R1, or P2 and L1 or R1. Ligation mediate suppression (LMS) PCR, as described previously, was used to verify single-copy transgene cassettes in FTL lines [23], [57]. Single copy transgenes were verified using Southern blotting as described by Forsbach et al. [58]. Genomic DNA was digested with EcoRI, which targets a single site within the transgene cassette, and separated by electrophoresis using an 0. 8% agarose gel. Gels were denatured with 0. 4 M NaOH and transferred to Zeta-Probe GT nylon membranes (BioRad, www. bio-rad. com). Probes were generated by PCR using primers for the LAT52 promoter (see Table S1 for primers), labeled with 32P dATP or dCTP using the DECAprime II labeling kit (Life Technologies), and purified with NucAway spin columns (Life Technologies). Blots were pre-hybridized in 0. 25 M Na3PO4 (pH 7. 2), 7% SDS for 1 hour at 65°C and then hybridized with the probe in 15 ml of the same solution overnight at 65°C with agitation. Hybridized blots were washed twice in 20 mM Na3PO4 (pH 7. 2), 5% SDS for 30 minutes at 65°C followed by a 20 mM Na3PO4 (pH 7. 2), 1% SDS wash for 30 minutes at 65°C. Hybridization patterns were visualized using a phosphorimager. 120 mg of seed were incubated with gentle agitation at room temperature for 16 hours in 45 ml ddH2O with 0. 27% ethylmethane sulfonate (EMS). Mutagenized seed were rinsed twice with 45 ml water for 4 hours followed by 9 additional 45 ml rapid rinses. Rinsed seed were suspended in 45 ml of 0. 05% agarose and incubated at 4°C for 3 days. The cold treated seeds were transferred to 100 ml of fresh 0. 05% agarose solution and planted on soil. The segregation of fluorescent marker protein expression in pollen tetrads was measured with either a Nikon (www. nikon. com) E1000 or Eclipse 80i epifluorescence microscope equipped with a Nikon Intensilight C-HGFI light source and filters from Chroma Technology (www. chroma. com). To collect pollen tetrads, flowers were dipped in a 10 µl drop of PGM media (34% sucrose, 4 mM CaCl2,3. 25 mM boric acid, 0. 1% Triton-X-100, pH 7. 5) on a glass microscope slide and covered with a glass coverslip. Photographs were taken with either a Nikon Coolpix5000 digital camera or a Nikon Digital Sight DS-Qi1MC cooled CCD camera. Figures were prepared using Adobe Photoshop CS2 (www. photoshop. com) and Canvas X (www. acdsee. com). To test whether the frequency of gene conversion events is independent of genomic position, experimental condition, mutation frequency, or false-positives from physical re-association 2×2 contingencies tables were constructed for each possible pair of test loci (or experimental and control conditions) and the G-Test of Independence was used to generate a P-value with the tools provided at the online version of the Handbook of Biological Statistics (http: //udel. edu/~mcdonald/statgtestind. html) [59]. To test for a correlation between the position of the SNP within the test locus (relative to the transcriptional start site) and gene conversion frequency tools from the same site were used to generate a regression line, calculate a correlation coefficient (r2) and a P-value.

During the production of gametes, most sexually reproducing organisms undergo meiotic recombination. The most familiar form of meiotic recombination is crossing-over, which results in the reciprocal exchange of DNA between parental chromosomes and is important for chromosome segregation as well as generating new allelic combinations in progeny. The same molecular mechanisms that facilitate crossing-over can also enable the non-reciprocal exchange of genetic information between chromosomes in the process called gene conversion. Understanding gene conversion is important because it influences allele frequencies and has been implicated in human diseases. Unfortunately, it has been difficult until now to measure directly except in organisms, like fungi, that group their gametes after meiosis. In this study we have developed a novel assay system that enables us to measure gene conversion directly in the model multi-cellular eukaryote A. thaliana (a flowering plant). Using this assay system we measured gene conversion frequencies across the Arabidopsis genome in more than 1 million meioses and also demonstrated that we can manipulate those frequencies by varying experimental conditions.

lay_plos

The expression of the human papillomavirus (HPV) E6/E7 oncogenes is crucial for HPV-induced malignant cell transformation. The identification of cellular targets attacked by the HPV oncogenes is critical for our understanding of the molecular mechanisms of HPV-associated carcinogenesis and may open novel therapeutic opportunities. Here, we identify the Lens Epithelial-Derived Growth Factor (LEDGF) gene as a novel cellular target gene for the HPV oncogenes. Elevated LEDGF expression has been recently linked to human carcinogenesis and can protect tumor cells towards different forms of cellular stress. We show that intracellular LEDGF mRNA and protein levels in HPV-positive cancer cells are critically dependent on the maintenance of viral oncogene expression. Ectopic E6/E7 expression stimulates LEDGF transcription in primary keratinocytes, at least in part via activation of the LEDGF promoter. Repression of endogenous LEDGF expression by RNA interference results in an increased sensitivity of HPV-positive cancer cells towards genotoxic agents. Immunohistochemical analyses of cervical tissue specimens reveal a highly significant increase of LEDGF protein levels in HPV-positive lesions compared to histologically normal cervical epithelium. Taken together, these results indicate that the E6/E7-dependent maintenance of intracellular LEDGF expression is critical for protecting HPV-positive cancer cells against various forms of cellular stress, including DNA damage. This could support tumor cell survival and contribute to the therapeutic resistance of cervical cancers towards genotoxic treatment strategies in the clinic. Oncogenic types of human papillomaviruses (HPVs), such as HPV16 and HPV18, are major human carcinogens. They cause cervical carcinoma, the second most common cancer in females worldwide and are closely linked to the development of other malignancies, including a subset of additional anogenital (e. g. anal, vulvar and penile) and oropharyngeal (e. g. tonsillar) cancers [1]. Two viral oncogenes, E6 and E7, are crucial for both the induction and the maintenance of the malignant phenotype of HPV-positive cervical cancer cells, indicating that cervical cancer cells display features of a phenomenon termed “oncogene addiction” [2]. On the basis of many mechanistic studies, the picture emerges that the two HPV oncogenes inactivate crucial tumorsuppressive responses of the cell, such as induction of senescence or apoptosis [3]–[6]. Importantly, at least some of these pathways are not irreversibly deregulated by HPVs. Rather, inhibition of viral E6/E7 activities in HPV-positive cancer cells leads to the reactivation of dormant tumor suppressor pathways and can eventually result in efficient growth arrest, senescence, and/or cell death [7]–[12]. These latter observations are significant for therapeutic considerations since it should be principally possible to revert the malignant phenotype of HPV-positive cancer cells. In general, this could be achieved by therapeutically blocking the E6/E7 oncogenes or, alternatively, by correcting the cellular pathways which are deregulated by the viral oncogenes. Thus, it is important to delineate critical cellular targets that are affected by viral E6/E7 oncogene expression and thereby contribute to the malignant phenotype of HPV-positive cancer cells. In order to search for cellular genes targeted by the viral E6/E7 oncogenes, we silenced endogenous HPV18 E6/E7 expression in HeLa cervical carcinoma cells by RNA interference (RNAi) and performed a genomewide transcriptome analysis. Data from this array suggested that the expression of the “Lens Epithelial-Derived Growth Factor/p75 (LEDGF) ” gene (alternatively called PSIP1) is reduced upon E6/E7 repression [13]. Its major splice product codes for the 530-amino acid LEDGF/p75 protein (in the following called LEDGF), a chromatin-associated factor that is best known for its important role during the human immunodeficiency virus-1 (HIV-1) life cycle. In this context, LEDGF interacts with the viral integrase (IN) and directs integration of the HIV-1 genome into the host cell chromosome [14]–[17]. More recently, however, there is emerging data indicating that LEDGF could also play an important role for human carcinogenesis. This notion is supported by the observations that: (i) LEDGF is overexpressed in several human cancers when compared with corresponding normal tissue [18]–[20]; (ii) the LEDGF gene is a target for chromosomal translocations in leukemias, leading to LEDGF/NUP98 fusion proteins [21] that protect leukemia cells against cell death [22]; (iii) the LEDGF protein contributes to leukemogenesis by tethering the mixed-lineage leukemia (MLL1) complex to cancer-associated target genes [23]; (iv) ectopically overexpressed LEDGF increases the tumorigenicity of different cancer cell lines in vivo [19], [24], [25]; (v) LEDGF can enhance angiogenesis and lymphangiogenesis, thereby possibly contributing to cancer metastasis [24], [26]; (vi) LEDGF can act as a survival factor in tumor cells towards different forms of cellular stress [22], [27]–[32], and (vii) LEDGF plays an important role for the repair of DNA damage [33], consistent with its genoprotective potential [19], [22], [33], [34]. Here, we investigated the connection between HPV E6/E7 oncogene and LEDGF expression, analyzed the contribution of LEDGF to the growth and to the DNA damage response of HPV-positive cancer cells, and examined the in vivo expression of the LEDGF protein in biopsies from premalignant lesions and cervical cancer. We show that (i) the maintenance of intracellular LEDGF amounts in HPV-positive tumor cells is critically dependent on continuous HPV E6/E7 expression, (ii) HPVs can transcriptionally stimulate LEDGF gene expression via LEDGF promoter activation, (iii) LEDGF is crucial for the growth and survival of HPV-positive cancer cells following DNA damage, and (iv) LEDGF levels are significantly elevated in cervical dysplasias and cancers. We propose that the E6/E7-dependent intracellular LEDGF expression could be an important determinant for the survival of HPV-positive cancer cells under different forms of cellular stress and for their resistance towards radio-and chemotherapy. Previous data from a genomewide transcriptome array in HeLa cells indicated that LEDGF transcript levels are significantly reduced upon silencing of endogenous HPV18 E6/E7 expression [13]. To confirm this result by independent methods, we tested the effects of HPV oncogene silencing on LEDGF expression by both qRT-PCR and immunoblot. We employed different siRNAs that either selectively block HPV E6 expression or concomitantly block E6 and E7 expression from the polycistronic E6/E7 transcripts [11]. As shown in Fig. 1A, these siRNAs efficiently reduced HPV18 mRNA amounts in HeLa cells. Inhibition of viral oncogene expression in HeLa cells was linked to a substantial reduction of LEDGF transcript levels upon combined E6/E7 silencing whereas E6 silencing alone inhibited LEDGF expression less strongly (Fig. 1A). LEDGF repression upon silencing of HPV E6/E7 expression was neither specific for HPV18 nor a peculiarity of HeLa cells, since inhibition of endogenous E6/E7 expression in HPV16-positive SiHa cells led to corresponding results as those observed in HPV18-positive HeLa cells (Fig. 1B). In order to investigate whether the E6/E7-dependent LEDGF mRNA modulation translates into alterations of LEDGF protein levels, we performed Western blot analyses of siRNA-treated cells. In agreement with the E6 property to induce degradation of p53 [35], treatment of HeLa cells with siRNAs blocking E6 or E6/E7 expression led to an increase of p53 protein levels, and siRNAs blocking E6/E7 expression additionally reduced E7 protein levels (Fig. 1C). Corresponding to the mRNA data, E6/E7 silencing substantially reduced LEDGF protein levels whereas inhibition of E6 expression alone reduced it less strongly. Taken together, these results show that continuous viral E6/E7 oncogene expression is a crucial determinant for the maintenance of LEDGF expression in HPV-positive cancer cells. The strong LEDGF repression observed upon E6/E7 silencing raises the possibility that the viral oncogenes can activate LEDGF expression. To test this issue, we transduced primary human keratinocytes with retroviral vectors coding for HPV16 E6, E7 or E6/E7. Compared to control-transduced keratinocytes, both E6 and E7 alone activated endogenous LEDGF expression and the effect was enhanced when both viral genes were co-expressed (Fig. 2A). Activation of LEDGF expression by the HPV oncogenes occurred, at least in part, at the transcriptional level, as indicated by Luciferase-reporter assays. Both E6 and E7 alone weakly activated the LEDGF promoter upon ectopic expression in primary human keratinocytes and the stimulatory effect was enhanced when both viral oncogenes were co-expressed (Fig. 2B). Activation of the LEDGF promoter by co-expressed HPV16 E6 and E7 was not limited to primary keratinocytes but was also detectable in different tested epithelial cell lines (Fig. 2B). Moreover, the potential of E6/E7 expression to significantly activate the LEDGF promoter was not restricted to HPV16, but also observed for high risk HPV18 and for low risk HPV6 or HPV11 (Fig. 2B). Vice versa, inhibition of endogenous E6 or E6/E7 expression by RNAi reduced LEDGF promoter activity in HeLa cells, with a stronger repression observed upon combined E6/E7 silencing (Fig. 2C). In line with the notion that the enhancement of LEDGF gene expression in HPV-positive cancer cells occurs, at least in part, at the transcriptional level, HeLa and SiHa cells exhibit substantially higher basal LEDGF promoter activities than primary cervical keratinocytes (Fig. 2D). If HPVs activate endogenous LEDGF expression, one would expect higher levels of LEDGF in HPV-positive cancer cells than in human keratinocytes, the natural target cells for HPV infection. To investigate this issue, we measured basal LEDGF mRNA and LEDGF protein levels in different isolates of primary human keratinocytes (from different donors), in a series of HPV16- and HPV18-positive cervical cancer cell lines, and in HPV-negative cell lines. Compared to primary foreskin or cervical keratinocytes, HPV18-positive HeLa and HPV16-positive SiHa, CaSki, and MRI-H-186 cells all exhibited elevated LEDGF expression levels, both at the transcript and protein level (Fig. 3). This increase in LEDGF mRNA and protein expression was not limited to HPV-positive cells and was quantitatively within the range of LEDGF expression levels in other, HPV-negative cell lines, e. g. lower than in C33A and higher than in HepG2 or MCF-7 (Fig. 3). Taken together, these results show that LEDGF expression levels in HPV-positive cancer cells, as well as in other cancer cells, are higher than in primary keratinocytes. These observations are in line with the LEDGF upregulation reported for cancer biopsies from several different tumor types [18]–[20]. Importantly, in HPV-positive cancer cells, LEDGF expression is critically dependent on the maintenance of viral E6/E7 oncogene expression. It is known that combined E6/E7 silencing, either by the viral E2 transcriptional repressor [36], [37] or by RNAi [38], blocks proliferation of HPV-positive cancer cells by inducing a G1 cell cycle arrest. This raises the question whether the strong reduction of LEDGF expression upon E6/E7 inhibition might be generally linked to an inhibition of cell cycle progression. In order to test this issue, we treated HeLa cells with chemical compounds that induce blocks in different cell cycle phases. Mimosine, thymidine, and nocodazole arrested the cells in the G1-phase, S-phase, and G2 phase, respectively (Fig. 4A), as expected for these drugs [39], [40]. However, none of the compounds significantly reduced LEDGF expression, neither at the transcript nor at the protein level (Figs. 4B and 4C). HPV E6/E7 mRNA and E7 protein expression levels were also not significantly changed by the compounds (Figs. 4B and 4C). Thus, LEDGF expression was not decreased by different cell cycle inhibitory drugs, indicating that the reduction of LEDGF expression is not a secondary effect of the cell cycle arrest induced by E6/E7 silencing. Next, we tested the phenotypic consequences of LEDGF modulation in HPV-positive cancer cells. We silenced endogenous LEDGF expression by stable transfection of plasmids expressing short-hairpin (sh) RNAs and performed colony formation assays (CFAs). For the shRNAs, we chose three different target sequences within the LEDGF mRNA, one of them (targeted by shLEDGF-3) being also present in the mRNA coding for the alternatively spliced LEDGF/p52 isoform [41]. All three shRNAs efficiently blocked LEDGF expression at the RNA and protein level (Fig. 5A). Compared to empty vector-transfected cells or cells transfected with vectors expressing control shRNAs (shContr-1, shNeg), HPV18-positive (HeLa) and HPV16-positive (SiHa, CaSki) cell lines all showed strongly reduced colony formation capacities upon silencing of endogenous LEDGF expression by each of the three different shRNAs (shLEDGF-1, -2, -3) (Fig. 5B). This effect was not limited to HPV-positive cells and was not linked to the p53 mutational status since LEDGF repression also resulted in a reduction of the colony formation capacity in HPV-negative C33A cervical carcinoma (mutant p53), H1299 lung cancer (p53 null) and HCT-116 colon carcinoma (wildtype p53) cells (Fig. 5B). To corroborate that the reduction in colony numbers of HPV-positive cells was specifically due to LEDGF gene silencing, we performed LEDGF reconstitution experiments. We ectopically expressed the wildtype LEDGF protein from a cDNA in which we introduced silent mutations that confer resistance to the employed LEDGF-targeting shRNA (shLEDGF-1). This cDNA efficiently rescued the capacity of HeLa and SiHa cells to form colonies (Fig. 5C), confirming that the strong inhibitory effect of the LEDGF-targeting shRNAs on the growth of HPV-positive cell lines is due to the silencing of endogenous LEDGF expression. These findings indicate that LEDGF silencing substantially inhibits the growth of HPV-positive cancer cells, as well as of other cancer cells, in CFAs. In order to get more insight into the underlying mechanism, we transiently transfected HeLa cells with synthetic siRNAs targeting LEDGF and tested possible effects of LEDGF depletion on cellular growth or apoptosis control. Surprisingly, and in apparent discrepancy to the prominent effects seen in CFAs (Fig. 5), we observed only a relatively modest influence on cell growth, cell cycle distribution, or apoptosis rate (data not shown), although the transiently transfected siRNAs led to efficient silencing of endogenous LEDGF expression, both at the RNA and protein level (Fig. 6A). An experimental difference between the stable and transient transfection studies performed here is the presence of hygromycin B in the cell culture medium for the former, in order to select for the maintenance of the shRNA-expressing plasmid vectors. Hygromycin B is an aminoglycoside antibiotic that is classically known for its inhibitory activity on protein biosynthesis [42]. However, hygromycin B has also been reported to possess DNA-damaging potential [43]. Therefore, we treated HeLa and SiHa cells with hygromycin B and modulated endogenous LEDGF expression by siRNAs. Interestingly, a significant induction of the DNA damage marker γH2AX (phosphorylated form of H2A histone family member X) [44] was observed when hygromycin B-treated HeLa or SiHa cells were depleted for LEDGF (Fig. 6B), indicating that LEDGF silencing increases the genotoxic potential of hygromycin B. On the basis of these experiments, we hypothesized that the reduced colony formation capacity observed in stable transfection experiments (i. e. in the presence of hygromycin B) could be linked to a reduced protection of LEDGF-depleted cells against DNA damage. We therefore performed CFAs upon transient transfection with synthetic siRNAs and treatment with well-characterized DNA damaging agents. We found that LEDGF silencing in HeLa cells led to an increased sensitivity towards both the topoisomerase inhibitor camptothecin (CPT) and γ-irradiation, leading to significant reductions of colony formation capacities (Fig. 7A). This effect was linked to a strong γH2AX increase when cells were depleted for LEDGF (Fig. 7B). These data indicate that LEDGF plays an important role for protecting HPV-positive cells against DNA damage exerted by genotoxic drugs (CPT, hygromycin B) or γ-irradiation which is also supported by a recent study showing that LEDGF is involved in DNA repair [33]. In line, ectopic expression of a mutant LEDGF protein (LEDGF-W21A) which has lost its genoprotective activity [33] no longer could revert the inhibitory effect of endogenous LEDGF depletion on the colony formation capacity of HeLa cells, in the presence of hygromycin B (Fig. 7C). Taken together, these results indicate that the activation of LEDGF expression by the HPV E6/E7 oncogenes plays an important role for the resistance of HPV-positive cancer cells towards genotoxic agents. Finally, we tested whether the observed positive correlation between HPV E6/E7 and LEDGF expression in vitro is also found in vivo. To this end, we analyzed LEDGF protein expression by immunohistochemistry in patient biopsies representing different degrees of premalignant cervical lesions (cervical intraepithelial neoplasia, CIN): CIN I (n = 16), CIN II (n = 7), CIN III (n = 13), and established squamous cell carcinomas (n = 7). The concomitant assessment of p16 protein expression served as a surrogate marker for HPV oncogene expression [45]. First, we tested the specificity of anti-LEDGF antibody (6E4) to be employed for LEDGF detection. Untreated HeLa cells, HeLa cells transfected with an siRNA silencing endogenous LEDGF expression, and HeLa cells in which LEDGF was ectopically overexpressed were prepared on thin-layer cytology slides. The cells were subsequently analyzed for LEDGF protein expression, employing our immunohistochemistry staining protocol. LEDGF protein was readily detectable in the nuclei of untreated HeLa cells (Supplemental Fig. S1). RNAi-mediated LEDGF gene silencing virtually completely extinguished the LEDGF signals whereas ectopic LEDGF overexpression resulted in augmented LEDGF signals when compared with untreated HeLa cells (Supplemental Fig. S1). These experiments indicate that the antibody is specific for LEDGF and suitable for LEDGF detection by immunohistochemistry. Analysis of histologically normal, p16-negative cervical epithelium revealed that LEDGF protein expression mainly localized to the basal and suprabasal cell layers (Fig. 8A and 8B). In comparison, epithelial LEDGF levels were clearly increased both in HPV-positive preneoplastic lesions and in established cervical cancers, overlapping with p16 signals in serial tissue sections (Fig. 8A). This finding is consistent with the positive correlation between HPV E6/E7 and LEDGF expression found in vitro. As observed for HeLa cells (Supplemental Fig. S1), LEDGF located primarily to the nuclei of the cells in the tissue sections (Fig. 8B). Notably, the strong LEDGF signals in the basal cell layers of both histologically normal and dysplastic HPV-positive cervical epithelium differed markedly from the expression of the Ki67 proliferation marker which was largely absent in the basal cell layer but was readily detectable in suprabasal cells (Fig. 8B). In order to quantitatively assess LEDGF expression, we employed a score that considers both the percentage of cells positive for LEDGF as well as LEDGF staining intensity (Supplemental Table S1). Box plot analyses showed that epithelial LEDGF levels were statistically highly significant increased both in HPV-positive preneoplastic lesions and in established cervical cancers when compared with histologically normal, p16-negative epithelium (Fig. 9). In addition, there was a trend that LEDGF expression in cervical epithelium levels increases from mild to severe dysplasias to cancer (CIN I vs. CIN II, p = 0. 021; CIN II vs. CIN III, p = 0. 17; CIN III vs. cervical cancer p = 0. 7). Taken together, these findings reveal a highly significant correlation between HPV-positivity and LEDGF expression levels in vivo, consistent with the in vitro data indicating activation and maintenance of LEDGF expression by the HPV oncogenes. In this study, we identify the cellular LEDGF gene as a novel target for oncogenic HPVs. We show that continuous E6/E7 oncogene expression is required to maintain intracellular LEDGF expression in HPV-positive cancer cells and that HPVs can transcriptionally stimulate the LEDGF gene via LEDGF promoter activation. Further, LEDGF expression is crucial for the resistance of HPV-positive cancer cells towards genotoxic stress. In line with the in vitro data demonstrating a positive correlation between HPV oncogene and LEDGF expression, we found that HPV-positive preneoplastic and neoplastic lesions exhibit significantly enhanced levels of LEDGF. We propose that stimulation of LEDGF expression by the viral E6/E7 oncogenes is a crucial mechanism to protect HPV-positive cancer cells towards different forms of cellular stress, including DNA damage. LEDGF is increasingly recognized as a factor involved in human tumorigenesis (see Introduction). Despite of the term “growth factor” in its name - which is based on its structural relatedness to hepatoma-derived growth factors [46] - it is currently uncertain whether LEDGF is secreted and serves as a classical growth factor [27], [47]. LEDGF possesses a nuclear localization signal [48] and is tightly bound to chromatin [49], [50]. The protein has been originally identified as a transcriptional coactivator interacting with components of the basal transcriptional machinery [41] and subsequently has been reported to stimulate expression of stress-related and cytoprotective genes, including the heat shock protein HSP27 and the antioxidant protein-2 (AOP2) genes [51], [52]. LEDGF has been reported to undergo several protein-protein interactions that could be significant for tumorigenesis. For example, LEDGF acts as a chromatin tether for a trimeric complex with Menin and the MLL (mixed-lineage leukemia) histone methyltransferase which is essential for leukemic transformation by MLL oncoproteins [23]. In addition, LEDGF can bind to and tether the Myc-interacting protein JPO2 to chromatin [53], a factor that may possess transforming potential by augmenting the oncogenicity of c-Myc [54]. Recently, LEDGF has been found to associate with CtIP (C-terminal binding protein interacting protein) [33], a multifunctional adaptor protein with tumor suppressive potential [55]. Among other functions, such as cell cycle control [55], CtIP plays an important role for the repair of DNA double strand breaks (DSBs) by homologous recombination [56]. The important observation that LEDGF is critical for the access of CtIP to DNA DSBs [33] could provide a mechanistic explanation for the genoprotective activity of LEDGF. This latter activity is also likely to account for the pronounced inhibition of LEDGF-depleted tumor cells in CFAs, in the presence of hygromycin B to select for stably transfected shRNA plasmids. In line with reports that aminoglycosides can induce single and double strand DNA breaks [43], [57], [58], we observed induction of the DNA damage marker γH2AX [44] when hygromycin B-treated HeLa and SiHa cells were depleted for LEDGF, although we cannot formally exclude that impurities in commercially available hygromycin B solutions may contribute to genotoxicity. In addition, inhibition of colony formation capacity in these HPV-positive tumor cells could be reverted by ectopic expression of wildtype LEDGF protein but not by a mutant LEDGF protein that has lost its genoprotective function. Taken together, our results indicate that the maintenance of LEDGF expression by the HPV oncogenes is an important determinant to allow growth of HPV-positive cells in the presence of genotoxic stress. It is unlikely that LEDGF repression upon E6/E7 inhibition is a secondary result of the accompanying cell cycle arrest, since LEDGF expression levels remained largely unchanged upon treatment of HPV-positive cervical cancer cells with different chemical compounds that block cell cycle progression. The notion that LEDGF expression levels are not simply proliferation-linked is further supported by the immunohistochemistry data. We found substantial LEDGF expression in the basal cell layers of both histologically normal and dysplastic cervical epithelium (Fig. 8C). This markedly differed from the expression of the Ki67 proliferation marker which was largely absent in the basal cell layer but was strongly expressed in the suprabasal layer, in line with the notion that suprabasal cells represent the main proliferative pool whereas basal cells contribute only little to the proliferative activity of the cervical epithelium [59]. Thus, the high levels of LEDGF protein in the basal cells that stain negative for Ki67 also suggest that pronounced LEDGF expression is not necessarily linked to a high proliferative index. Several lines of experimental evidence indicate that HPVs can activate the LEDGF gene. Ectopic E6/E7 expression in primary human keratinocytes, the natural target cells for HPVs, increased LEDGF mRNA levels. This was linked to enhanced activities of the LEDGF transcriptional promoter, as shown in reporter gene assays. Vice versa, silencing of endogenous E6/E7 expression in HeLa cells repressed the LEDGF promoter but did not lead to alterations in the half-life of the LEDGF mRNA (data not shown). Expression of E6 or E7 alone less strongly stimulated LEDGF expression than the combined expression of both viral oncogenes, suggesting some degree of functional cooperativity during stimulation of LEDGF transcription. Unfortunately, the understanding of the transcriptional control of the LEDGF gene is still at an early stage. Somewhat discrepant results concerning the LEDGF/p75 promoter have been reported by two research groups who mapped by transcriptional start site analyses a TATA-less promoter to two different genomic sites that are separated by 208 nucleotides [60], [61]. In our reporter gene assays, we employed a 782 bp fragment 5′ of the LEDGF gene which encompasses both putative promoters (fragment −723/+59; [60]). Activation of the LEDGF promoter by E6/E7 was not limited to the oncogenic types HPV16 and HPV18, but also detectable for HPV6 and HPV11, two HPV types that are rarely associated with malignancy. It will be interesting to investigate a possible role for LEDGF in the viral life cycle of HPVs. Conceivably, HPVs may profit from upregulating stress-protective and pro-survival genes like LEDGF, thereby protecting the infected host cell during virus replication and synthesis. Little is known about the specific transcriptional regulators involved in LEDGF promoter control, except for a stimulatory role found for the ubiquitous transcription factor SP1 [60], [61] and the observation that putative SP1 recognition sites within the LEDGF promoter can be targeted for epigenetic repression [62]. That LEDGF gene expression is indeed considerably regulated at the promoter level is also supported by the observation that basal LEDGF promoter activities were substantially enhanced in HPV-positive cancer cells above those in primary cervical keratinocytes, concomitantly with increased LEDGF mRNA and LEDGF protein levels in the former cells. The exact intracellular localization of the LEDGF protein is still under some controversy. It has been described by some researchers to be predominantly nuclear [48], [63]–[65] whereas others additionally reported varying degrees of a cytoplasmic distribution [18], [20], [66], or a differentiation-dependent localization with nuclear LEDGF in the basal cell layer and cytoplasmic LEDGF in more differentiated cells of the epidermis [67]. The investigation of tissue sections of cervical epithelium revealed a predominantly nuclear LEDGF localization. Importantly, and consistent with the positive correlation between HPV oncogene and LEDGF expression levels observed in vitro, we found that p16-positive regions in patient biopsies exhibited statistically highly significant increased LEDGF expression levels when compared with p16-negative, histologically normal areas from the same tissue sections. In addition, there was a non-significant trend that LEDGF levels increased with increasing severity of dysplastic lesions to established cervical cancer. The latter finding is reminiscent of a study in bladder cancer, reporting a tendency for increasing LEDGF levels during tumor progression [19]. It is interesting that the basal cell layer - in both histologically normal cervical epithelium as well as in dysplastic lesions - exhibited prominent LEDGF staining. This cell layer also harbors the stem cells of the cervix [68]. Notably, a study in brain tissue reported LEDGF staining in neuroepithelial stem cells [66]. It is tempting to speculate that LEDGF may play a role for protecting stem cells, including stem cells of the cervical epithelium, against various forms of cellular stress. Stress factors that have been shown to be counteracted by LEDGF include serum starvation [28], [29], [31], oxidative stress [20], [27], [30], [51], [69], alcohol toxicity [32], thermal stress [27], [29], [69], and DNA damage [19], [22], [30], [33], [34]. In view of these multiple pro-survival activities of LEDGF, tumor cells should benefit from upregulating LEDGF expression. Indeed, the increased LEDGF levels in many different tumor entities, despite of their genetic heterogenicity, suggests a broadly relevant role for LEDGF in human carcinogenesis. The mechanisms of how tumor cells achieve upregulation of LEDGF expression are not understood. Our results provide the first evidence that the HPV oncogenes stimulate and maintain LEDGF expression in cervical cancer cells. It will be interesting for future studies to investigate whether the capacity to increase LEDGF expression is also shared by other viral and cellular oncogenes. Under clinical aspects, the E6/E7-dependent maintenance of LEDGF expression could play a role for the therapeutic resistance of HPV-positive cancers, by protecting against the genotoxic effects of chemo- and radiotherapy. This raises the possibility that a combination of chemo- and/or radiotherapeutic agents with LEDGF inhibitors could increase the therapeutic sensitivity of cervical cancer cells and other tumor cells. Finally, the E6/E7-dependent LEDGF expression may not only promote tumor growth by protecting HPV-positive cancer cells against different forms of cellular stress, but also could contribute to tumor progression and metastasis more directly, e. g. by enhancing the formation of blood and lymph vessels, as reported for the LEDGF-mediated activation of VEGF-C in glioma, lung cancer and ovarial cancer models [24], [26]. HPV18-positive HeLa cervical carcinoma cells, HPV16-positive CaSki, MRI-H-186 and SiHa cervical carcinoma cells, HPV-negative C33A cervical carcinoma cells, HaCaT human immortal keratinocytes, H1299 and A549 lung cancer, RKO and HCT116 colon cancer, MCF-7 breast cancer, SH-SY5Y neuroblastoma and HepG2 hepatoma cells were maintained either in DMEM (pH 7. 2), McCoy' s, or RPMI medium (Gibco, Life Technologies, Carlsbad, CA), supplemented with 10% fetal bovine serum (FBS; Gibco Life Technologies), 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich, Saint Lois, MO). Primary human cervical keratinocytes (CxK) or primary human foreskin keratinocytes (HFK) were grown in keratinocyte growth medium 2, supplemented with 0. 06 mM CaCl2 and SupplementMix (PromoCell, Heidelberg, Germany). Plasmids were transfected by calcium phosphate co-precipitation into cell lines, as described [11], or with fugene HD (Roche Diagnostics, Mannheim, Germany) into primary human cervical keratinocytes, following the manufacturer' s protocol. Synthetic siRNAs were transfected with DharmaFECT (Dharmacon, Thermo Fisher Scientific, Waltham, MA) into HeLa cells or with Lipofectamine RNAimax (Invitrogen, Life Technologies) into SiHa cells at a final concentration of 10 nM, according to the manufacturer' s protocol. For DNA damage treatment with drugs, cells were plated on 6-cm dishes, transfected with siRNAs, and treated 24 h later with 1 µM camptothecin (CPT, Enzo Life Science, Lörrach, Germany) or 200 µg/ml hygromycin B (Invitrogen, cat. # 10687-010, LOT # HY064-L6, purity >90%, please refer to: http: //tools. lifetechnologies. com/content/sfs/COAPDFS/2013/HY064-L6_10687010. pdf), for the indicated time periods. For ionizing radiation treatment, a Gammacell 1000 137Cs source was employed (dose rate 5. 85 Gy/min, Atomic Energy of Canada Ltd., Edmonton, Canada). Primary human foreskin keratinocytes (HFK) stably expressing HPV16 E6 and/or E7 were generated as previously described [70], by transduction with the retroviral vector pLXSN carrying either the HPV16 E6 or E7 open reading frames or the HPV16 E6/E7 bicistronic sequence. As negative control, HFK were retro-transduced with empty vector pLXSN. siRNAs were either chemically synthesized (Ambion, Life Technologies) or expressed as shRNAs from vectors pCEPsh (selectable via its hygromycin B resistance) or pSUPER, as previously described [71]. The si/shRNA target sequences were as follows: 16E6-1 5′-ACCGUUGUGUGAUUUGUUA-3′, 16E6-2 5′-GGGAUUUAUGCAUAGUAUA-3′, 16E6-3 5′-UUAGUGAGUAUAGACAUUA-3′, 16E6/E7-1 5′-CCGGACAGAGCCCAUUACA-3′, 16E6/E7-2 5′-CACCUACAUUGCAUGAAUA-3′, 16E6/E7-3 5′-CAACUGAUCUCUACUGUUA-3′, 18E6-1 5′-GACAUUAUUCAGACUCTGU-3′, 18E6-2 5′-CAGACUCUGUGUAUGGAGA-3′, 18E6-3 5′-CUCUGUGUAUGGAGACACA-3′, 18E6/E7-1 5′-CCACAACGUCACACAAUGU-3′, 18E6/E7-2 5′-CAGAGAAACACAAGUAUAA-3′, 18E6/E7-3 5′-UCCAGCAGCUGUUUCUGAA-3′, LEDGF-1 5′-AGACAGCAUGAGGAAGCGA-3′ [72], LEDGF-2 5′-GGTAATCAGCCACAACATA-3′ [19], LEDGF-3 5′-GCAATGAGGATGTGACTAA-3′ [19]. The control si/shRNAs (Contr-1 5′-CAGUCGCGUUUGCGACUGG-3′ [73] and Neg 5′-UACGACCGGUCUAUCGUAG-3′ [74]) contain at least four mismatches to all known human genes. For LEDGF reconstitution experiments, a LEDGF cDNA containing seven synonymous mutations in the shLEDGF-1 target site [75] was cloned into pCEP4 vector, yielding pLEDGF. A derivative of pLEDGF containing a point mutation in the PWWP-domain (pLEDGF-W21A) was generated by site-directed mutagenesis. Both wildtype LEDGF and LEDGF-W21A protein were Flag-tagged. The luciferase-reporter plasmid containing the LEDGF/p75 promoter (pGL4. 10LEDGFp75 −723/+59) was kindly provided by Drs. S. Desfarges and A. Ciuffi (University Hospital Center and University of Lausanne, Switzerland) [60]. HPV16, HPV18, HPV6 and HPV11 E6 and E7 expressing plasmids have been described previously [38], [71], [76]. All luciferase assays were performed independently at least thrice, as double or triple values, following a previously described protocol [38]. In brief, cells were transfected with the LEDGF luciferase reporter plasmid, together with the indicated E6 or E7 expression vectors or pSUPER constructs. As an internal standard, each transfection also included a β-galactosidase reporter plasmid (pCMV-Gal) in order to correct for variations in transfection efficiencies [7]. Cells were harvested 48–72 h post transfection and processed as described [7]. Luciferase activities were quantified using a Lucy 1 microplate luminometer (Anthos, Krefeld, Germany). All qRT-PCR analyses were independently performed at least thrice, in duplicates. Total RNA from cells was isolated with the Pure Link RNA Mini Kit (Ambion, Life Technologies). Reverse transcription of 1 µg RNA was performed by using oligo-dT or random primers of the ProtoScript M-MuLV Taq RT-PCR Kit (New England Biolabs, Ipswich, MA), according to the manufacturer' s instructions. For HFKs, total cellular RNA was extracted using the Absolutely RNA Miniprep kit (Agilent Technologies, Santa Clara, CA). One µg of RNA from each sample was reverse transcribed to cDNA by using RevertAid H Minus M-MuLV Reverse Transcriptase (MBI Fermentas, Thermo Fisher Scientific). Expression levels were determined by real-time PCR with a 7300 Real-Time PCR System detector (Applied Biosystems, Carlsbad, CA), using the SYBR green PCR Master Mix (Applied Biosystems). The forward (fwd) and reverse (rev) primer sequences (Eurofins MWG, Ebersberg, Germany) were as follows: 16E6 fwd 5′-AGCAATACAACAAACCGTTGTGT-3′, 16E6 rev 5′-CCGGTCCACCGACCCCTTAT-3′, 18E6 fwd 5′-AGACAGTATACCCCATGCTGCAT-3′, 18E6 rev 5′-TCCAATGTGTCTCCATACACAGA-3′, 18E6/E7 fwd 5′-ATGCATGGACCTAAGGCAAC-3′, 18E6/E7 rev 5′-AGGTCGTCTGCTGAGCTTTC-3′, LEDGF fwd 5′-CAAGGGAAGAAAGGGCCAAACA-3′, LEDGF rev 5′-CGTGCTGGCTTCATGGTTGT-3′, β-Actin fwd 5′-GGACTTCGAGCAAGAGATGGC-3′, β-Actin rev 5′-GCAGTGATCTCCTTCTGCATC-3′, GAPDH fwd 5′-GAAGGTGAAGGTCGGAGTC-3′, GAPDH rev 5′- GAAGATGGTGATGGGATTTC-3′, 18S RNA fwd 5′-CATGGCCGTTCTTAGTTGGT-3′, 18S RNA rev 5′-ATGCCAGAGTCTCGTTCGTT-3′. Cycling conditions have been described previously [77]. The sizes of the PCR products were initially verified by agarose gel electrophoresis and subsequently checked by melting point analysis after each reaction. Relative quantification was performed using the comparative Ct (2−ΔΔCt) method [78]. Data are presented as the fold difference in gene expression normalized to a reference gene (β-Actin, GAPDH or 18S RNA) and relative to a calibrator sample. All immunoblot experiments were performed at least three times, if not otherwise indicated. Total protein extracts were prepared 48–96 h after transfection. Cell pellets were lysed in CSK-1 buffer (10 nM Pipes pH 6. 8,300 mM NaCl, 1 m M EDTA, 300 mM Sucrose, 1 mM MgCl2,0. 5% TritonX-100), supplemented with Pefabloc (Merck, Whitehouse Station, NJ), Inhibitor Cocktail (Sigma-Aldrich) and phosphatase inhibitor cocktail (Roche Diagnostics) for 30 min on ice. Proteins were collected by centrifugation at 12,000 g for 10 min and protein concentrations were determined by the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). For Western blot analyses, protein extracts were separated on NuPAGE Novex 4–12% Bis-Tris Mini Gels (Life Technologies). Proteins were electrotransferred to an Immobilon-P membrane (Millipore, Bedford, MA), using the Trans-Blot Semi-Dry Transfer Cell (Bio-Rad). Membranes were blocked with 5% skim milk powder (Saliter, Obergrünzburg, Germany) and 1% bovine serum albumin (BSA, Sigma-Aldrich) in PBS-T (PBS/0. 1% Tween-20) for 1 h at room temperature. Membranes were incubated with primary antibodies overnight at 4°C in PBS-T/5% skim milk powder/1% BSA, followed by incubation with the corresponding HRP-conjugated secondary antibody for 1 h at room temperature. Proteins were visualized using ECL Prime Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK). Images were monitored using Fusion SL Gel Detection System (Vilber Lourmat, Marne-la-Vallée, France). Band densities were determined by BioID image analysis software (Vilber Lourmat), relative to the respective loading controls. The following primary antibodies were used: mouse anti-α-Tubulin (Merck), mouse anti-β-Actin (Sigma-Aldrich), chicken-anti-HPV18 E7 (E7C) [11], mouse-anti-p53 (BD Biosciences, Heidelberg, Germany), rabbit-anti-LEDGFp75 (Bethyl Laboratories, Montgomery, TX), rabbit-anti-Flag (Sigma-Aldrich) and mouse-anti-γH2AX (Ser139, Millipore). The following secondary HRP-conjugated antibodies were used: anti-mouse IgG (W3021, Promega, Madison, WI), anti-rabbit IgG (W4011, Promega), and anti-chicken IgY (G1351, Promega). For blocking HeLa cells in different cell cycle phases, cells were treated with 400 µM mimosine, 2 mM thymidine or 0. 04 µg/ml nocodazole (all from Sigma-Aldrich), respectively, for 16 h. Cells were trypsinized, washed in ice-cold PBS and fixed in 70% cold ethanol overnight at −20°C. Subsequently cells were pelleted, resuspended in PBS containing 1 mg/ml RNase A (Roche Diagnostics) and 25 µg/ml propidium iodide (Sigma-Aldrich) and incubated for 30 min at room temperature. Cell cycle analyses were performed by fluorescence-activated cell sorting (FACS) using a FACSCalibur (BD Biosciences) with CellQuest Pro software provided by the manufacturer. Apoptotic cells were excluded and quantitation of the percentage of cells in the individual phases was performed using FlowJo software (Tree Star, Ashland, OR), applying the Watson model [79]. For CFAs with pCEPsh vectors, cells were transfected and selected for hygromycin B resistance. Colonies were fixed and stained with formaldehyde-crystal violet, 10 to 13 days after transfection. For CFAs using synthetic siRNAs, cell numbers were determined with a Countess Cell Counter (Invitrogen) at 24 h post transfection. Cells were plated on 6-cm dishes (1,000 cells/dish) and treated the next day with 10 nM or 100 nM CPT for 1 h or with ionizing radiation (6 Gy). Colonies were fixed and stained with formaldehyde-crystal violet, 6 to 8 days following DNA damage treatment, and cell clones were counted. For validating anti-LEDGF antibody 6E4 (anti-PSIP1, Thermo Fisher Scientific; detects the LEDGF/p75 but not the LEDGF/p52 isoform), HeLa cells were plated on 6-cm dishes and transfected with either siLEDGF-1 or pLEDGF, or left untreated. Cells were trypsinized 72 h post transfection, washed in ice-cold PBS and pelleted. Cell pellets containing at least 2×106 cells were suspended in a methanol-based preservation solution (PreservCyt, Hologic, Wiesbaden, Germany) and prepared as thin-layer cytology slides (ThinPrep, Hologic). Specimens were analyzed for LEDGF expression, using the staining protocol for immunohistochemistry detailed below. For immunohistochemistry analyses, serial sections of formalin-fixed, paraffin-embedded cervical cancer cone biopsy specimens were dewaxed and rehydrated using xylene and a series of graded alcohols. Heat-induced antigen retrieval was performed by immersing the sections in a 10 mM citrate buffer solution (pH 6. 0) and microwaving them for 3×5 min at 550 W. Slides were cooled in the antigen retrieval solution for 20 min. Endogenous peroxidase activity was blocked by incubating the sections in 1% hydrogen peroxide in methanol for 20 min at room temperature. Non-specific protein binding sites were blocked by incubating the slides in 10% horse serum diluted in PBS for 30 min. Sections were incubated over night at 4°C with primary antibodies diluted in PBS supplemented with 1% horse serum. The following primary antibodies were used: mouse-anti-p16INK4a (CINtec Histology, Roche mtm Laboratories, Mannheim, Germany), mouse-anti-Ki67 (MIB-1, Dako, Hamburg, Germany), and mouse-anti-LEDGF (anti-PSIP1,6E4, Thermo Fisher Scientific). Subsequent thorough washing in 0. 1% PBS-T was performed. Sections were then incubated with a biotinylated secondary anti-mouse antibody (Vectastain Elite ABC, Vector Labs, Burlingame, CA) for 30 min at room temperature, followed by incubation with an avidin-biotin complex peroxidase (Vectastain Elite ABC) for 20 min at room temperature. LEDGF, p16 and Ki67 expression were visualized by a brown 3,3′-diaminobenzidine and abbreviation (DAB) reaction. Sections were glass-covered and analyzed by light microscopy (Olypmus Vanox-T, Hamburg, Germany) using a magnification up to ×400. For immunohistochemical assessment of LEDGF expression, the product of the scores of staining frequency and intensity of immunoreactive cells was calculated as described [80]: the frequency ranged from 5% to 100% of LEDGF-positive cells, and the intensity comprised 1 = low to 3 = high. The final immunohistochemical score (ranging from 5 to 300) was obtained by multiplication of the intensity score and the frequency score. All sections were independently reviewed in random order by two researchers (JL and MR). For the few instances of discrepant scoring, a consensus score was determined. Formalin-fixed, paraffin-embedded tissue blocks were used anonymized without linked personal data according to the regional ethical regulations. Statistical significance of differences in measured variables between controls and treated samples was evaluated by a two-sided paired t-test using the Sigma Plot software (Systat Software Inc., San Jose, CA). For immunohistochemical analyses statistical significance of differences in calculated scores between histologically normal and HPV-positive samples was determined by two-sided t-test using the SPSS software version 21 (Armonk, NY: IBM Corp.). p-values of ≤0. 05 (*), ≤0. 01 (**), or ≤0. 001 (***) were considered statistically significant. Genebank accession numbers according to the National Center for Biotechnology Information (http: //www. ncbi. nlm. nih. gov/genbank) for genes and proteins discussed in this paper are as follows: PSIP1 (Gene ID 11168), LEDGF (NP_150091. 2), HPV16 E6 (Gene ID 1489078), HVP16 E6 (NP_041325. 1), HPV16 E7 (Gene ID 1489079), HPV16 E7 (NP_041326. 1), H2AX (Gene ID 3014), H2AX (NP_002096. 1).

Specific types of human papillomaviruses (HPVs) are closely linked to the development of malignant tumors, such as cervical cancer. Virtually all cervical cancers contain HPV DNA and the tumorigenic growth behavior of cervical cancer cells is dependent on the activity of two viral oncogenes, called E6 and E7. It is important to study the activities by which the HPV oncogenes can support the growth of tumor cells. This should allow new insights into the molecular mechanisms of virus-induced carcinogenesis and could also be useful for developing novel approaches for cancer therapy. We here show that the HPV oncogenes stimulate and maintain expression of the cellular LEDGF gene in HPV-positive cancer cells. Consistently, pre-malignant and malignant lesions of the cervix exhibit significantly increased LEDGF protein levels. LEDGF is crucial for the protection of tumor cells against various forms of cellular stress, including DNA damage. LEDGF stimulation by the viral oncogenes could be a critical survival mechanism by which HPVs support the growth of cervical cancer cells and provide resistance towards chemo- and radiotherapy in the clinic.

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