LDLR) and HDL cholesterol (rs189679427 between GOT2 and APOOP5), and replicated previous associations of a splice variant in APOC3 (rs138326449) with triglycerides and HDL-C. All three variants are at well-replicated loci in GWAS but are independent from and have larger effect sizes than the known common variation in these regions. Candidate eQTL analyses in in LCLs in the Hutterites suggest that these rare non-coding variants are likely to mediate their effects on lipid traits by regulating gene expression.Founder populations are ideally suited for studies on the clinical effects of alleles that are rare in general populations but occur at higher frequencies in these isolated populations. Whole genome sequencing in 98 Hutterites, a founder population of European descent, and subsequent imputation revealed 660,238 single nucleotide polymorphisms that are rare (<1%) or absent in European populations, but occur at frequencies >1% in the Hutterites. We examined the effects of these rare in European variants on plasma lipid levels in 828 Hutterites and applied a Bayesian hierarchical framework to prioritize potentially causal variants based on functional annotations. We identified two novel non-coding rare variants associated with LDL cholesterol (rs17242388 in  These traits include plasma levels of low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), total cholesterol and triglycerides (TG) and have estimated heritabilities of 40–60% across populations4. Although genome-wide association studies (GWAS) of lipid traits have been successful in identifying hundreds of common variants with robust associations, the associated variants account for only about 10–14% of the total phenotypic variance6. While a portion of the unexplained genetic variance may result from overestimates of heritability and complex genetic architectures, such as those involving epistasis or gene-environment interactions7, the effect of rare loss-of-function variation in complex traits is understudied and likely contributes to the heritability of blood lipid traits and risk for cardiovascular disease.Blood lipid traits are under strong genetic control and are modifiable risk factors for cardiovascular disease, one of the leading causes of death10. These studies have supported earlier observations suggesting that rare variants in the general population contribute significantly to lipid traits and possibly more generally to common, complex phenotypes. For example, a resequencing study of genes that harbor causal mutations in monogenic lipid disorders identified an enrichment of nonsynonymous variants associated with lipid levels by sequencing unrelated individuals sampled from the tails of the HDL-C trait distribution11. This study demonstrated for the first time that rare variants contribute to population-level variation in blood lipids.In fact, sequencing studies in families or patients with rare monogenic lipid disorders have uncovered many novel genes harboring rare coding mutations of large effect and revealed critical pathways for lipid metabolism12. As a result, the non-coding portion of the genome has been largely unexplored. Similar to common non-coding variants, rare non-coding variants may also impact gene expression and protein abundance, but the sample sizes of most studies of gene expression are underpowered to identify the independent effects of rare variants. A few studies have provided strong support for the aggregated effect of multiple rare non-coding variants on nearby gene expression, and identified enrichments of rare non-coding variation for individuals showing extreme gene expression levels, as compared with the same genes in non-outlier individuals15, and shown cis eQTL effect sizes are significantly higher for SNPs with lower allele freuquencies16. Despite this evidence, the broader questions of what functional features characterize rare non-coding variants influencing gene expression and how different functional classes of rare non-coding variations influence disease are unknown.While providing valuable insight into the mechanisms regulating lipid traits, rare variant studies in complex traits have focused on coding regions of the genome, largely due to the recent explosion of exome sequencing studies and the relative ease in interpreting these findings19. In addition, their overall reduced genetic complexity and relatively homogenous environments and lifestyles can enhance the effects of rare genetic variants on phenotypic traits and thereby facilitate the detection of susceptibility loci that underlie complex disease, as elegantly illustrated in studies of the Amish and Icelandic populations22.Founder populations offer the opportunity to study the effects of variants that are rare in general populations but have reached higher frequencies in these isolated populations due to the effects of random genetic drift in the early generations after their foundingIn this study, we dissect the genetic architecture of plasma lipid traits in members of the South Dakota Hutterites, a founder population of European descent. The 828 individuals who participated in these studies are related to each other in a 13-generation pedigree with 64 founders. We performed GWAS using 660,238 “rare in European variants” (REVs) that occur at frequencies greater than 1% in the Hutterites, and integrated functional and regulatory annotations that allowed us to narrow down potential candidate variants despite the long-range linkage disequilibrium present in the population. Our studies revealed rare variants with large effects at two novel and three known blood lipid loci associated with LDL-C, HDL-C or TG, potentially yielding novel insights into the mechanisms regulating lipid traits and new therapeutic targets.23 to the 828 individual in our study. We selected 660,238 variants that were either absent or rare (<1%) in European databases (see Methods) and occurred at frequencies greater than 1% in the Hutterites (REVs) for association testing with fasting lipid measurements  or intronic (43.7%); the remaining 2.1% were exonic (1%), in the 3′ or 5′ UTR (1.1%) or predicted to affect splicing  identified through whole genome sequencing in 98 Hutterite individuals were imputed using the known identity by descent (IBD) structure of the Hutterite pedigreeor rare <% in Euroor rare <% in Euro25), including age and sex as fixed effects and kinship as a random effect. Quantile-quantile plots showed no inflation of test statistics for any of the lipid traits  associated with either increased LDL-C or with reduced TG levels and contained multiple rare variants of large effect in regions previously associated with lipid traits in GWAS6. These two loci include 78 REVs associated with increased LDL-C over a 7.6 Mb region flanking the LDL receptor gene (LDLR) on chromosome 19, and 39 REVs associated with reduced TG levels over a 3.8 Mb region flanking the Apolipoprotein C3 (APOC3) gene on chromosome 11. The latter includes a known rare splicing variant in APOC3 (rs138326449)27. No genome-wide significant associations with REVs were detected for HDL-C or total cholesterol.For each lipid trait, we performed association analyses in two stages. First, we performed single variant analyses using a linear mixed model GEMMA5, includ28 framework to further evaluate the GWAS results and prioritize candidate rare variants based on prior functional knowledge. We split the genome into blocks averaging 125Kb (~50 REVs) and performed forward selection to build models that combined effects from multiple annotations followed by a cross validation step to avoid overfitting while maximizing the likelihood of each model. Figure In second stage analyses, we annotated all variants discovered in the Hutterites using 25 sequence-based annotations and 332 functional annotations (see Methods) and applied the functional GWAS (fgwas)−8), and additional blocks associated with HDL-C in a novel region on chromosome 16 and one on chromosome 11. In addition, when we applied a slightly lower regional threshold of PPA > 0.75, additional blocks associated with LDL-C on chromosomes 1 and 3 were identified. We present all variants within regions with PPA > 0.75 and SNP PPA > 0.5 in Supplementary Table The joint model for each fgwas estimated both the probability that each block contains a causal variant and the posterior probability that a variant is causal conditional on the presence of one causal variant in the region. Variants with the largest posterior probabilities of causality will tend to have the smallest p-values at that locus and functional annotations that predict association genome-wide. We weighted our GWAS results based on the fgwas joint model and applied a regional prior probability of association (PPA) threshold of 0.9. Overall, we identified 63 consecutive blocks associated with LDL-C on chromosome 19 and 20 blocks on chromosome 11 associated with TG, all of which harbor REVs that passed genome-wide significance in the single variant GWAS p < 5 × 10 × 10−8, LDLR  was the fgwas candidate variant with the highest predictive functional score .The GWAS of LDL-C identified 78 genome-significant REVs associated with increased levels of LDL-C on a haplotype that spans 7.6 Mb on chromosome 19p13.2. Despite the high LD and resulting long haplotype, we were able to prioritize 35 candidate variants with SNP posterior probabilities greater than 0.75, 13 of which had posterior probabilities equal to one  and was associated with decreased LDL-C . The second variant was an intronic variant on chromosome 3 in the EPH Receptor A6 gene  associated with increased LDL-C .We identified two additional loci that were suggestively associated with LDL-C  is an intronic variant within the non-coding RNA (ncRNA) gene Transmembrane Protease Serine 4 Antisense RNA 1 (TMPRSS4-AS1) and is 25 Kb away from the most associated variant in the region . Chromosome 11q23 is a well replicated GWAS locus for multiple lipid traits31  is a previously reported splice variant in APOC3  but had a slightly lower posterior probability of association in our model  compared to other variants in the region. To our knowledge, the role of this potential splice donor APOC3 polymorphism (rs138326449) in regulation of plasma lipids has not been characterized thus far. Therefore, while there is be compelling evidence that reduced plasma levels of APOC3 protein results in lower TG levels26, there may be multiple rare variants within an extended haplotype influencing TG levels in the Hutterites.Out of the 39 REVs on a 3.8 Mb haplotype associated with TG levels, 14 were potentially causal with SNP posterior probabilities of association greater than 0.75. The SNP with the highest posterior probability . The variant with the highest posterior probability of association was rs189679427  and 877 Kb from Apolipoprotein O Pseudogene 5 (APOOP5). While GOT2 has not been directly linked to HDL-C levels, its characterized function as a membrane associated fatty acid transporter highly expressed in the liver, the primary tissue for apolipoprotein metabolism34, makes it an interesting candidate.Although the HDL-C GWAS did not identify any genome-wide significant REVs, the fgwas revealed two loci associated with HDL-C with regional PPA greater than 90%. The first locus was associated with increased HDL-C levels and tags the same haplotype on chromosome 11q23.3 that is associated with lower TG levels. The selected variant at this locus with the highest probability was also the lead SNP in both the HDL-C GWAS , including all variants present in the Hutterites genomes that resided each of the associated regions regardless of their minor allele frequencies in Europeans. First, we conditioned on the lead rare variant in our analyses to assess whether other (rare or common) variants in the region either contribute to the observed association signal or are independently associated with the trait but whose effects were masked by the larger effect of the rare variant. Second, in regions with known associated variants from other GWAS, we also conditioned on the GWAS variant(s) to verify that the rare variant signal in our study is independent of known associations at this locus Table . We thenPDE4A; rs513663) that reached suggestive significance after removing the effects of the rare variant  and the LDLR rare variant (rs1724388) are associated with higher LDL-C but reside on different haplotypes in the Hutterites, with independent and additive effects of the minor alleles both on lowering LDLR gene expression (p = 0.008) and increasing plasma levels of LDL-C . We also performed conditional analysis with a commonly replicated variant in the LDLR gene that is associated with decreased LDL-C levels and lower risk for coronary heart disease (CHD)39  and confirmed that the identified rare variants in the Hutterites have independent and opposite effects compared to the common GWAS variant at this locus (Table Conditioning on the lead rare variant on chromosome 19 that is associated with LDL-C (rs557778817) revealed a novel common variant in the third intron of Phosphodiesterase 4 A (ZNF440), Dihydrouridine Synthase 3 Like (DUS3L) and Hook Microtubule Tethering Protein 2 (HOOK2), had expression levels associated with at least one candidate variant at a p < 10−4 (Supplementary Table ZNF439 (chr19:11978399) and decreased levels of Zinc Finger Protein 440 .We performed eQTL analyses with the 53 LDL-C candidate variants on chromosome 19 (SNP PPA > 0.5) and found three out of 245 genes tested, Zinc Finger Protein 440  effects of the rare variant in the Hutterites  that was associated with decreased TG levels uncovered associations with known common variation in the 33869 thaGOT2 and APOOP5 associated with lower HDL-C levels is associated with higher GOT2 expression in LCLs  by jointly incorporating functional data with our GWAS results. This allowed us to narrow the subset of likely causal variants at each locus.We performed GWAS with ~660 k rare in European variants that occur at higher frequencies in the Hutterites, and identified four novel associations with plasma lipid traits as well as replicating the effects of the known LDLR gene (rs17242388) was associated with increased LDL-C levels in our study. Other variants in the first intron of LDLR have been previously implicated in regulating LDL-C levels in two studies40, but in both studies the associations had opposite effects on LDL-C levels compared to the rare variant in our study. The known variants in this intron include a predominantly European variant that is associated with lower non-HDL-C levels  in the Icelandic population  and a common variant linked to multiple blood lipid traits40 . The LDLR variant in the Hutterites occurs at a frequency five times higher than that reported in 1000 genomes (Europeans), and is located within a predicted enhancer region in a number of digestive tract tissues, including liver, small intestine and stomach mucosa. Conditional analyses revealed that the rare rs17242388-A allele and the common rs513663-G occur on different haplotypes and have independent and additive effects on both lowering expression of the LDLR gene and raising plasma LDL-C levels.Three of the five rare variants identified by the GWAS in our study reside within known lipid loci identified by GWAS. However, even at those known loci, the associated rare variants in our study were independent of and had larger effects than known associated common variation in these regions. For example, a variant in the first intron of the GOT2 gene and pseudogene APOOP5 that is associated with lower levels of HDL-C in the Hutterites. While GOT2 and APOOP5 have not been directly linked to HDL-C levels, their characterized function thus far makes them interesting candidates. GOT2 is a membrane associated fatty acid transporter that is highly expressed in the liver and several apolipoprotein genes have been implicated in lipid metabolism34. Moreover, rs189679427 is located 1.9 Mb downstream of a well-established GWAS variant upstream of CETP , a lipid metabolism gene encoding a plasma protein that supports the transport of cholesteryl esters from HDL-C to apoB-containing particles in exchange for triglycerides41. The rs189679427 allele was associated with lower HDL-C and with higher expression of GOT2, but not with CETP even though CETP was expressed in Hutterite LCLs. This suggests that GOT2 may be directly involved in regulation of HDL-C levels, although gene expression studies in more relevant tissues are required to confirm this observation.A second association with a novel rare variant also resides within a known lipid trait locus on chromosome 16q13. This is an intergenic variant (rs189679427) located between the ARL6, a causative gene for Bardet-Bield syndrome42, a highly penetrant oligogenic disorder that results in a number of clinical phenotypes, including childhood obesity and hyperlipidemia in the majority of cases43. Many genes identified by GWAS of lipid traits also harbor loss of function mutations that underlie Mendelian disorders of lipid metabolism44. Mutations within these genes (coding and non-coding) provide complimentary viewpoints to the disease mechanisms influencing these traits, but further work in relevant tissues is necessary to understand the molecular basis for these associations. Overall, our results are consistent with previous findings that complex diseases are enriched for loci implicated in Mendelian traits45.The suggestive association between variants at 3q11.2 and higher LDL-C provide support for a shared genetic architecture between Mendelian and complex traits. The associated haplotype at this locus is centered around 6 are primarily loss-of-function coding variation and had no overlap with the variants in our study, 98.9% of which were non-coding. Nonetheless, the discoveries revealed in this study, even those that may be private to Hutterites, uncovered potentially novel disease genes, and highlight new clinically relevant pathways that could point toward novel therapeutic targets for hyperlipidemias and lowering the risk for cardiovascular disease.In summary, our findings further demonstrate the advantages of population isolates in the search for rare variants associated with complex traits. Importantly, all of the associated variation revealed in our study is within non-coding sequences and would have been missed had we focused just on exonic variation, and many were associated with gene expression, highlighting the importance of studying the effects rare non-coding variation on gene expression as well as their effects on common disease traits. An inherent limitation of this study, and most studies of rare variants, is the challenge of replicating findings due to the very low frequency of the alleles under investigation in most populations. For example, the rare and low-frequency variants surveyed in the Global Blood Lipids Consortium meta-analysiset al.35 for details). Briefly, blood samples were collected after an overnight fast from 828 Hutterites . Written informed consent was obtained from all individuals in our study. The study was approved by the institutional review boards at the University of Chicago and all methods were performed in accordance with relevant guidelines and regulations. Subjects receiving anti-hypercholesterimia medication, hormone replacement therapy, birth control, or diagnosed with sitosterolemia47 were excluded from the study. For subsequent analyses, we applied a cubed root transformation to absolute LDL-C and HDL-C levels, a natural log transformation to TG and total cholesterol.Standard fasting plasma lipid measurements were collected as part of a larger study of complex traits in the Hutterites  or absent in European populations in the ExAC% or abse35. The LCL RNA-seq data was processed as follows. Reads were trimmed for adaptors using Cutadapt (with reads <5 bp discarded) and remapped to hg19 using STAR indexed with gencode version 19 gene annotations55. To remove mapping bias, reads were processed using the WASP mapping pipeline56. Gene counts were collected using HTSeq-count57. VerifyBamID was used to identify potential sample swaps58. Genes mapping to the X and Y chromosome and genes with a Counts Per Million (CPM) value of 1 (expressed with less than 20 counts in the sample with lowest sequencing depth) were removed. Limma was used to normalize and convert counts to log transformed CPM values59. Technical covariates that showed a significant association with any of the top principal components were regressed out (RNA integrity number and RNA concentration).Candidate eQTL analyses in LCLs were performed in GEMMA and included gene expression for 441 Hutterite individuals (317 of which are in our lipid studies) that was collected as part of a separate study51, ESP52, 1000 genomes53) using Variant Effect Predictor (VEP)60. We downloaded promoter and enhancer annotations created by the Epigenomics Roadmap Project (−log10(p) ≥ 10; http://www.broadinstitute.org/~meuleman/reg2map/HoneyBadger2_release/) for 127 cell types or tissues. We directly annotated variants using the ClinVar database downloaded on 08/07/2016 and selected variants labelled as pathogenic or likely pathogenic. Lastly, we used 53 functional categories and 9 cell-type specific histone marks regions obtained from Finucane et al.61 . Briefly, the annotations include annotations for RefSeq, digital genomic footprint and transcription factor binding sites from ENCODE62, combined chromHMM and Segway annotations for six cell lines63, processed DHS data from ENCODE and Roadmap Epigenomics data and cell type specific H3K4me1, H3K4me3 and H3K9ac data from Roadmap Epigenomics64, H3K27ac from Roadmap Epigenomics and from Hnisz et al.65, super-enhancers from Hnisz et al.65, processed conserved regions in mammals from Lindblad-Toh et al.67 and FANTOM5 enhancers68. For each functional Finucane et al.61 annotation, a 500-bp window was added as an additional category. For each DHS, H3K4me1, H3K4me3, and H3K9ac sites, a 100-bp window around the ChIP-seq peak was added as an additional category.We obtained variant annotations from dbSNP, ENSEMBL, LOFTEE, conservation and functional scores , and allele frequencies from European populations 28. First, we divided the genome into ~14,000 blocks of approximately 120 Kb each (~50 variants/block) and applied forward selection to build step-wise models including the combined effects from multiple annotations. Second, we followed by a cross validation step to avoid over fitting while maximizing the likelihood of each model. We present the final best fitting models in Fig. Using the fgwas softwareThe datasets generated during and/or analyzed during the current study are available in the dbGaP repository, phs000185.Supplementary informationSupplementary Table 5


The formation of a hierarchical vascular network is a complex process that requires precise temporal and spatial integration of several signaling pathways. Amongst those, Notch has emerged as a key regulator of multiple steps that expand from endothelial sprouting to arterial specification and remains relevant in the adult. This review aims to summarize major concepts and rising hypotheses on the role of Notch signaling in the endothelium.A wealth of new information has helped to clarify how Notch signaling cooperates with other pathways to orchestrate vascular morphogenesis, branching, and function. Endothelial vascular endothelial growth factor, C-X-C chemokine receptor type 4, and nicotinamide adenine dinucleotide phosphate oxidase 2 have been highlighted as key regulators of the pathway. Furthermore, blood flow forces during vascular development induce Notch1 signaling to suppress endothelial cell proliferation, enhance barrier function, and promote arterial specification. Importantly, Notch1 has been recently recognized as an endothelial mechanosensor that is highly responsive to the level of shear stress to enable differential Notch activation in distinct regions of the vessel wall and suppress inflammation.Although it is well accepted that the Notch signaling pathway is essential for vascular morphogenesis, its contributions to the homeostasis of adult endothelium were uncovered only recently. Furthermore, its exquisite regulation by flow and impressive interface with multiple signaling pathways indicates that Notch is at the center of a highly interactive web that integrates both physical and chemical signals to ensure vascular stability. The process of blood vessel formation requires strict coordination of proliferation, differentiation, and maturation. These are concurrent events in a vascular sprout and require the integration of multiple signaling pathways, including but not limited to: vascular endothelial growth factor (VEGF), bone morphogenetic proteins (BMPs), and Notch ,2.Highly conserved across vertebrate species, the Notch signaling pathway includes a group of four transmembrane receptors Notch1–4) that interact with five transmembrane ligands . Activation involves two sequential proteolytic events that require the enzymes a disintegrin and metalloproteinase domain-containing protein 10 and γ-secretase  that int,6.The influence of Notch signaling in differentiation and development cannot be overstated as Notch regulates fate decisions in cell types of all four major tissues: epithelium, muscle, connective , and nervous tissue . During ▪▪,9▪▪]. How Notch blocks proliferative signals has been unclear until recently when Notch-mediated inhibition of proliferation was found to require the well known tumor suppressor phosphatase and tensin homolog (PTEN)  , which stimulate endothelial sprouting and proliferation in the expanding vascular plexus [▪▪].Notch signaling is also required for proper formation of vascular branches. Recent work demonstrated that endothelial branching in response to BMP signaling and activation of mothers against decapentaplegic (SMAD)1/5/8 are Notch dependent. Specifically, SMAD6, an inhibitory SMAD that titrates input signals from SMAD1/5/8, is regulated cell intrinsically by Notch levels ▪▪  by Nox2 regulates Notch signaling [▪▪]. It is well accepted that flow initiates a series of events including activation of NADPH oxidases that promote generation of ROS . A role for Notch in endothelial cell cycle regulation and maintenance of quiescence was also supported by inducible deletion in adult mice. Here, Notch1 inactivation triggered cell cycle reentry in vivo[▪▪] indicating that Notch1 in adult vessels provides pressure to maintain quiescence. Combined, these findings suggest that, in the context of shear stress, Notch1 is absolutely essential to maintain endothelial quiescence -l-alanyl]-S-phenylglycine t-butyl ester, a γ-secretase inhibitor)-treated embryoid bodies and retinas, suggesting that Notch regulates cell–cell junctional rearrangements. Consistent with this finding, recent work from two independent groups has identified a role for Notch1 in promoting endothelial junctional integrity in stabilized blood vessels [▪▪] and discovered that the Notch1 transmembrane domain regulates adherens junctions in microvessels under flow [▪▪]. Furthermore, in the brain, Notch signaling limits vascular permeability in both resting and inflamed states . Notch receptor was initially recognized as an arterial marker overlapping with EphrinB2 . Similarly, in the endocardium, Notch signaling restricts inflammation in the regenerating heart and a loss of Notch leads to increased endothelial inflammatory gene expression and more infiltrating macrophages [et al.[et al.[The role of endothelial Notch signaling in inflammation has been difficult to ascertain because of conflicting findings. Work has shown that proinflammatory cytokines drive changes in Notch activity to elicit a reduction in hairy and enhanced of split 1 and Hey2 leading to increased endothelial apoptosis . An antiive role . This woive role ▪▪. Similrophages . In conts [et al. observedl.[et al.▪ showed Notch activity has been linked to arteriovenous malformations where forced expression of constitutively active Notch signaling resulted in non-arterial endothelial cells developing arterial characteristics and triggering arteriovenous shunts ,55. CereIn cancer, endothelial Notch1 activity was linked to metastasis by promoting a senescent, proinflammatory endothelium ▪. Yet, NFew signaling molecules exert the broad and influential effect on blood vessel formation as does the Notch pathway. From the onset of vascular development, Notch determines the identity of tip and stalk cell, promotes arterial specification, and ensures the recruitment of mural cells. However, expression of Notch is not exclusive to developmental stages of the cardiovascular system, indeed, receptors and ligands continue to be present in mature vessels to maintain vascular integrity and homeostasis. Future work will likely highlight the relevance of multiple accessory molecules that act as modulators of the Notch-signaling pathway affecting protein glycosylation, ligand-receptor recognition, proteolytic cleavage, and target gene selection.We would like to acknowledge the contribution of Patrick Lane in the final rendering ofFig. 1 and would like to apologize to all authors whose work could not be cited because of space limitations.We gratefully acknowledge funding by National Institutes of Health 2P01HL030568-31 (to MLIA).There are no conflicts of interest.▪ of special interest▪▪ of outstanding interestPapers of particular interest, published within the annual period of review, have been highlighted as:


Wolbachia pipientis is an intracellular endosymbiont known to confer host resistance against RNA viruses in insects. However, the causal mechanism underlying this antiviral defense remains poorly understood. To this end, we have established a robust arthropod model system to study the tripartite interaction involving Sindbis virus and Wolbachia strain wMel within its native host, Drosophila melanogaster. By leveraging the power of Drosophila genetics and a parallel, highly tractable D. melanogaster derived JW18 cell culture system, we determined that in addition to reducing infectious virus production, Wolbachia negatively influences Sindbis virus particle infectivity. This is further accompanied by reductions in viral transcript and protein levels. Interestingly, unchanged ratio of proteins to viral RNA copies suggest that Wolbachia likely does not influence the translational efficiency of viral transcripts. Additionally, expression analyses of candidate host genes revealed D. melanogaster methyltransferase gene Mt2 as an induced host factor in the presence of Wolbachia. Further characterization of viral resistance in Wolbachia–infected flies lacking functional Mt2 revealed partial recovery of virus titer relative to wild-type, accompanied by complete restoration of viral RNA and protein levels, suggesting that Mt2 acts at the stage of viral genome replication. Finally, knockdown of Mt2 in Wolbachia uninfected JW18 cells resulted in increased virus infectivity, thus demonstrating its previously unknown role as an antiviral factor against Sindbis virus. In conclusion, our findings provide evidence supporting the role of Wolbachia–modulated host factors towards RNA virus resistance in arthropods, alongside establishing Mt2’s novel antiviral function against Sindbis virus in D. melanogaster. Wolbachia pipientis as a novel biocontrol agent to prevent RNA virus transmission in mosquitoes. Previous work in the field suggests that the presence of this bacterium induces virus resistance within the host. However, the underlying mechanism of this antiviral phenotype is poorly understood, impeding its widespread use. Using the alphavirus, Sindbis as our model, we explored the tripartite interaction between the virus and the endosymbiont within its natural host, the fruit fly Drosophila melanogaster. In this study, we show that Wolbachia negatively influences multiple important aspects of the virus life cycle, extending our current understanding of the molecular nature of this interaction. We also provide evidence highlighting the role of a host gene, Mt2, in Wolbachia–mediated antiviral resistance, while uncovering its previously unknown role as an antiviral host factor against Sindbis virus.Effective vector control is critically important to reduce the incidence of diseases caused by arthropod transmitted viruses. One proposed strategy involves the use of endosymbiotic bacteria  Wolbachia pipientis is an alpha-proteobacterial, maternally transmitted endosymbiont that invades insect host populations by manipulating host reproduction, favoring infected females [Drosophila and important disease vectors such as Aedes albopictus and Culex species of common house mosquitoes [D. melanogaster, Wolbachia strain wMel exhibits weak reproductive manipulation [Wolbachia strain can also protect D. melanogaster from common viral pathogens such as Drosophila C virus (DCV), cricket paralysis virus (CrPV), and Flock House virus (FHV), as evidenced by increased survival and delay in virus accumulation [Wolbachia–mediated antiviral protection, so-called “pathogen blocking” is considered an exciting phenomenon that could be leveraged to reduce arthropod-transmitted diseases [Heritable symbioses are pervasive in nature and exceedingly common in the insect world, where many endosymbiotic associations have been described . Wolbach females . It is psquitoes . In D. mpulation , but recmulation , 6. Not diseases .A. albopictus infected with its native Wolbachia strain (wAlbB) has little to no effect on RNA virus replication (such as Dengue), transinfection of non-native Wolbachia strains like wMel into this mosquito species, as well as the naturally uninfected Aedes aegypti, have been shown to induce pronounced antiviral resistance [A. aegypti mosquitos can invade native A. aegypti mosquito populations, persisting over the seasons [Wolbachia’s pathogen-blocking ability is currently being implemented in vector control, we know little to nothing about the mechanism underlying Wolbachia-induced antiviral resistance. Mosquitos as a model suffer from a lack of widely accessible genetic tools, coupled with the added complexity of introducing Wolbachia transinfections. Conversely, the antiviral phenotype was originally characterized in Drosophila and the genetic tractability of this model organism makes it an ideal candidate platform for a mechanistic dissection of the tripartite interaction [Drosophila genetics, combined with the molecular tractability of a parallel tissue culture system, to probe previously uncharacterized aspects of Wolbachia-mediated resistance against the prototype alphavirus, Sindbis virus (SINV). In this study, we show that presence of Wolbachia in D. melanogaster results in reduced SINV infectivity, viral RNA replication and protein synthesis. Furthermore, we have identified the DNA/RNA methyltransferase gene, Mt2 as a potential host factor responsible for Wolbachia-mediated antiviral resistance. Based on these results, we conclude that resistance towards SINV occurs at an early stage of virus replication that further affects subsequent stages of the virus life cycle and show evidence supporting the hypothesis that Wolbachia modulates the expression of a host methyltransferase gene (Mt2) to target virus RNA synthesis.While sistance , 9. More seasons , 11. Howeraction , 6. To tWolbachia on SINV titer in wild-type flies infected or uninfected with Wolbachia strain wMel, which we refer to as Wolb+ and Wolb- respectively from this point onwards  and subgenome (quantified using primers to E1 coding sequence) RNA levels, suggesting that viral RNA synthesis is inhibited in the presence of the endosymbiont , to viral RNA (copies/ug), we failed to find any significant difference in the protein-to-RNA ratios between ficiency . Taken t18 cells .Wolbachia could be explained by modulation of host immune gene expression by the bacterium [Wolbachia on host gene expression was performed in the absence of a viral infection. We reasoned that it is important to consider the cellular environment that the virus is initially exposed to upon entry into the cell, not necessarily one that it encounters later during infection. In addition to canonical innate immune pathway components we examined the expression of Mt2, a gene encoding a nucleic acid methyl transferase previously shown to be required for DCV resistance in Drosophila [Drosophila in the absence or presence of Wolbachia.Based on evidence from other systems, we investigated whether the antiviral resistance mediated by acterium –19. We tosophila . Host geof Wolbachia  [Wolbachia infected and uninfected flies that were either wild-type or Mt2 -/- and the infection progressed for 48 hours before samples were collected, snap-frozen and virus titer was determined using end-point dilution assay on BHK cells  . SINV inHK cells . SINV tisistance –24. In lhibition .Mt2 expression in two genetically distinct Wolbachia–infected fly lines, with each of the two shRNAs targeting a different area of the Mt2 gene , relative to that observed in their wild-type counterparts  represent significant and imminent threats to public health across the globe due to the lack of commercially available vaccines or antiviral therapeutics. Several of the classic arboviral diseases are characterized by their long periods of absence, followed by sudden cases of re-emergence which may occur at unpredictable intervals, often decades apart , 26. TheWolbachia induced RNA virus resistance in insects is poorly understood. Despite being observed in both native and transiently established Wolbachia infections [Wolbachia strains, hosts and viruses [Wolbachia strains have been cited as a potential determining factor, while others have suggested a mechanistic divide between different host systems [Wolbachia is specific to RNA viruses, and this fact could help to narrow the underlying mechanism of virus inhibition. The data presented above demonstrate Wolbachia’s capacity to inhibit SINV replication in an arthropod host. We observed a significant decrease in the infectivity of SINV particles produced from Wolbachia infected cells. That is, while a significant number of virus particles were being produced in the presence of Wolbachia they were less infectious on a per particle basis than those produced in the absence of Wolbachia. This is important, as these results may help explain recent observations regarding the lack of detectable infectious virus particles present in salivary glands of wMel-infected A. aegypti mosquitoes harboring other RNA viruses [Mt2, plays a significant role in blocking SINV infection.At this time the causal mechanism behind fections , 28–33,  viruses , 28–33.  systems . However viruses –33. FinaWolbachia induces resistance against different RNA viruses, it would be logical to think that the intracellular endosymbiont alters cellular physiology, creating an antiviral state that consequently influences the titer of the progeny virus produced. If this is indeed the case, where the induced antiviral effect is cell autonomous, then virus resistance would unconditionally require the presence of Wolbachia inside the cell. Furthermore, varying densities of Wolbachia in different tissues would then affect the degree of antiviral effect from cell to cell [Wolbachia is known to influence host gene expression [Wolbachia–infected vector species such as A. aegypti and Anopheles gambiae [Wolbachia infection, the bacterium was found to inhibit translation of SFV in JW18 cells as early as 7hpi with an absence of active transcriptional change in Wolbachia genes in response to virus infection [Wolbachia’s antiviral mechanism is either fast acting or is already present upon virus infection [Wolbachia might prime the host immune system, including factors such as the induction of ROS and alterations of host miRNA profiles, to induce antiviral resistance [Drosophila innate immune components like RNAi as well as pathways such as Toll and Imd [Mt2 as a host factor that restricted SINV infection in Drosophila melanogaster and established a link between its antiviral function and Wolbachia–mediated host resistance against SINV.The physiology of the host cell harboring a virus influences the titer of the resulting virus progeny. Several factors, such as the stage of infection, for example, influence virus titer, whereby titer improves temporally as virus accumulates with the progress of infection. On the other hand, virus titer is also dependent on the physiological attributes of the cell from which the virus originates . Given t to cell . Indeed,pression , 36 and  gambiae , 16. In nfection . This lenfection . These rsistance , 37 – 39 and Imd , 39. In D. melanogaster Mt2 gene encodes for a 40 kDa Dnmt2 protein which belongs to the most widely conserved Dnmt protein family and is the denoted as the lone cytosine methyltransferase present in many arthropods, including flies and mosquitoes [Asp at the cytosine-5 position (m5C). Dnmt2-directed cytosine methylation of genomic DNA in D. melanogaster is limited to specific loci on selected retrotransposons [Asp and additional tRNAs [Drosophila and RNA immunoprecipitation experiments demonstrated interaction of Dnmt2 with DCV RNA during infection [Wolbachia. Our results show that Wolbachia infection leads to an increase in Mt2 expression prior to virus infection, thus the virus is being introduced to a system in which Mt2 expression is high. Interestingly SINV infection led to a reduction of Wolbachia-induced Mt2 expression. This is important to note as a prior study found no effect of Wolbachia on Mt2 expression, but in that study the analysis of expression levels was performed after virus infection [Mt2 results in a significant increase in SINV transcript levels as well as a concomitant increase in the synthesis of virus structural proteins and virus titer. We hypothesize that Mt2 may function to inhibit viral RNA synthesis, which is in line with its previously described RNA binding properties.squitoes . Its funnsposons –44. Howeal tRNAs , 45. Thinfection . Here, wnfection . AdditioDrosophila embryos and localization of fluorescently tagged Dnmt2 in DCV infected fly tissues have confirmed the presence of Dnmt2 in the cytoplasm [Wolbachia is correlated with antiviral resistance in both flies and mosquitoes [A. aegypti) Dnmt2 promoted dengue virus replication [Wolbachia. Although further research is required to determine the exact nature of interaction between SINV RNA and Dnmt2, previous work involving DCV revealed significantly higher virus induced mortality in flies carrying a catalytically inactive mutant of Dnmt2, which suggests that enzymatic activity of Dnmt2 is required for its function as an antiviral factor [Wolbachia and the relative importance of Dnmt2’s RNA methyltransferase activity in SINV inhibition.Unlike traditional DNA methyltransferases, whose subcellular localizations are predominantly in the nucleus, evidence has been provided that Dnmt2 is distributed in the nucleus and the cytoplasm where SINV replication takes place. Indeed, fractionation studies of protein extracts from ytoplasm , 20. Thesquitoes , 46, 47.lication . It is nMt2 and Wolbachia’s pathogen blocking, the majority of the mechanistic details regarding this phenomenon remain unknown, and certainly other Wolbachia induced factors are likely to be involved. At this time, it is unknown whether Dnmt2 alters the methylation of SINV RNA. Given what is known about the functional implications of mRNA modifications in eukaryotic organisms, it is possible that the viral RNA acts as a target for cellular methyltransferases that subsequently influences the function of that RNA either positively negatively. Indeed, evidence of eukaryotic host methylation machinery acting as an either proviral or antiviral factor has been reported in the field [6A modified regions spanning the RNA genomes of multiple members of Flaviviridae family, including Hepatitis C Virus (HCV), Zika Virus (ZIKV), Dengue Virus (DENV), Yellow Fever Virus (YFV) and West Nile Virus (WNV) [Although we discovered a link between he field –53. Receus (WNV) , 52.Wolbachia infection in insects, various degrees of evidence support the role of the endosymbiont in modulating the cytosine methylation profile of the host. In D. melanogaster, presence of native Wolbachia wMel is correlated with increased methylation across all cytosine residues in male testes [wMelPop infected A. aegypti mosquitoes reveal mostly random, yet widespread changes in the host cytosine methylation profile compared to Wolbachia uninfected individuals [D. melanogaster, mosquitoes such as A. aegypti and A. gambiae possess a single Dnmt2 like cytosine methyltransferase [Wolbachia induced resistance towards viruses possessing RNA genomes, as well as growing evidence in the field that viral genome methylation regulates RNA virus infection, our data support the idea of Wolbachia induced modification of SINV RNA.In the context of e testes . In addiividuals . It is insferase , 57. TheWolbachia-induced Mt2 expression changes the methylation state of the viral RNA it is apparent that it has a biological effect on virus replication. Our data indicate this effect is exerted at an early time in infection and results in reduced levels of RNA synthesis. Future work fill focus on understanding how Dmnt2 interaction with viral RNA influences RNA function. The observation that infectious unit production is more severely inhibited than particle production Wolbachia infected hosts indicates that the genomes in the particles are less capable of initiating infection. It is known that methylation of RNA can change the stability of that RNA [Wolbachia-mediated Dmnt2 activity may lead to downstream alterations in the use of the genome as a template for translation and minus-strand RNA synthesis.Whether or not that RNA  which, iWolbachia as an emerging vector control agent requires better understanding of the molecular mechanism underlying its antiviral resistance in the context of a tractable arthropod system. We provide evidence that the presence of Wolbachia reduces SINV particle infectivity and results in reduced virus titer. Furthermore, our data indicate a reduction in viral RNA synthesis, accompanied by decreased viral protein synthesis. Further characterization of early infection events is required to determine the exact nature of Wolbachia–mediated inhibition of SINV genome replication. Importantly, our data clearly demonstrate antiviral activity of the host RNA methyltransferase gene Mt2 in the presence and absence of Wolbachia. Given the data provided in this study, it is likely that the effect of Mt2 lies at the stage of viral RNA synthesis/stability. Future work will therefore focus on characterizing the nature of interaction between Mt2 and SINV RNA. The fact that both Wolbachia mediated antiviral resistance and the antiviral effect of Mt2 extends to members of different single stranded RNA virus families is significant, suggesting that existence of a common mechanism [Continued use of echanism , 20, 22.Drosophila fly stock 6326 (of a W1118 genetic background), carrying Wolbachia was obtained from the Bloomington Stock Center (BDSC), and used as the infected wild-type background. A corresponding Wolbachia–uninfected line was created through tetracycline treatment (approx. 20 ug/mL fed in the fly media for 3 generations). Wolbachia infection status was subsequently confirmed through quantitative PCR using published primer sets  and 42906  were used for shRNA-targeted knock-down of Mt2 gene expression by driving Mt2 shRNA expression using previously described Act5C-Gal4 driver males  y1 w*; P{Act5C-GAL4}25FO1/CyO, y+  were maintained at 24°C in Shields and Sang media (Sigma), supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% Antibiotic-Antimycotic (Gibco). BHK-21 cells (American Type Culture Collection) were grown at 37°C under 5% CO2 in MEM (CellGro) supplemented with 1% L-Gln, 1% Antibiotic-Antimycotic (Gibco), 1% non-essential amino acids and 10% heat inactivated Fetal Bovine Serum (FBS). JW18 cells were seeded in a 6-well plate at a density of approximately 2.26x106 cells/well 24h prior to infection. Infection was carried out using virus derived from BHK-21 cells, titered using a standard plaque assay. Virus was diluted in Shields and Sang media (Sigma) and the infection was established at an MOI = 100. For doxycycline cleared JW18-dox cells, around 50–55% infection was observed at 96 hpi. Mock infections were carried out by treating the cells equivalently, without the addition of virus.Quantification of viral genome and subgenome translation was performed by introducing SINV luciferase reporter viruses nsP3-nLuc and cap-nLuc, respectively into 2-day old virgin female flies as described. The samples were homogenized in 1X Cell Culture Lysis Reagent (Promega) after they were collected 2-days post infection and clarified via centrifugation at 16,000 × g for 2 min. The samples were then mixed with luciferase reagent (Promega), and luminescence was recorded using a Synergy H1 microplate reader (BioTech instruments). In all cases, the luciferase readings were normalized to the levels of SINV genomic and sub-genomic RNA present in the assayed samples, determined by using qRT-PCR methods with ΔΔCT calculation of transcript levels.Wolbachia density from fly homogenates and tissue culture cells were determined via qPCR on whole DNA using an Applied Biosystems StepOne Real-time PCR system (Wolbachia density (and infection status) in JW18 cells was determined using DAPI staining, where Wolbachia were visualized in the form of cytoplasmic foci within the cells.R system  and SYBRMt2 expression was achieved in doxycycline treated JW18 cells using target dsRNA against the Mt2 gene. Mt2 dsRNA was synthesized from corresponding dsDNA generated using self-annealing primer sets. First, custom oligonucleotide was designed with a 5’ T7-Polymerase binding site (GAATTAATACGACTCACTATAG) followed by a 3’ target sequence specific to the Mt2 coding region. Similarly, another oligonucleotide was designed with a similar 5’ T7 polymerase binding site followed by a 3’ end complementary to the 3’ end of the previous oligo  with random hexamer primers (Integrated DNA Technologies). Negative (no RT) controls were performed for each target. Quantitative RT-PCR analyses were performed using Brilliant III SYBR green QPCR master mix (Agilent) with gene-specific primers according to the manufacturer's protocol and with the Applied Bioscience StepOnePlus qRT-PCR machine (Life Technologies). The expression levels were normalized to the endogenous 18S rRNA expression using the delta-delta comparative threshold method (ΔΔCT). Fold changes were determined using the comparative threshold cycle (CT) method .P-values were calculated as described in the individual Fig legends. The average fold change (FC) in each experiment was calculated using the variable bootstrapping method, measuring the fold change between each potential pair of flies to determine the variability of the mean [the mean . 95% conthe mean .S1 FigWolbachia. Total RNA was isolated from fly tissue homogenates 48 hours post infection and assayed for fold change in RNA synthesis using qRT-PCR as described in Materials and Methods. Primer sets used in the analyses can be found in the supplementary information (Wolbachia free flies (set at 1) and represent the mean of three independent biological replicates. P values were calculated using Mann-Whitney U tests. All error bars represent standard mean of error (SEM).Gene expression profile of candidate immune genes was examined in mock infected flies either in the presence or absence of ormation . Values (TIFF)Click here for additional data file.S2 FigWolbachia titer in Mt2 -/- mutant flies was quantified using qPCR. Reported values are relative to isogenic wild-type flies (set at 1) and are represented as the mean of three independent biological replicates. All error bars represent standard error of mean (SEM).Relative (TIFF)Click here for additional data file.S3 FigMt2 expression was knocked down in Wolbachia infected transgenic RNAi fly stocks 38224 (Mt2 shRNA 1) and 42906 (Mt2 shRNA 2) by driving Mt2 shRNA expression via chromosome III Act5C-Gal4 driver  as described in Materials and Methods. For each set of crosses, siblings lacking the expression of Mt2 targeting shRNA were used as the wild-type controls.  Flies were challenged with SINV as described previously. Infection was allowed to last for 48 hours before RNA was extracted from whole fly tissues, followed by quantification of viral nsP1 and Mt2 expression via qRT-PCR. Quantitative analyses of gene expression in TRiP mutant flies was performed relative to their respective wild-type sibling controls (set at 1). Values represent the mean of six and four independent biological replicates, respectively. P values were calculated using Mann-Whitney U tests . (C) Wolbachia titer was quantified using qPCR. Reported values are relative to respective wild-type sibling controls (set at 1) and are represented as the mean of three independent biological replicates. All error bars represent standard error of mean (SEM).(TIF)Click here for additional data file.S4 FigWolbachia–free flies overexpressing Mt2 was generated by crossing uninfected UAS-Mt2 males with uninfected virgin Act5C-Gal4 driver females. Siblings generated through these crosses were used as wild-type controls. Flies were challenged with SINV that lasted for 48 hours before whole fly tissues were harvested. (A) Infectious virus was quantified as described previously, using end-point-dilution assay on BHK-21 cells. Values represent the mean of three independent biological replicates. P value was calculated using Mann-Whitney U test (p = 0.0476). (B) SINV RNA was quantified using qRT-PCR by probing against the viral E1 gene. Values represent the mean of three independent biological replicates. P value was calculated using Mann-Whitney U test (p < 0.001). All error bars represent standard error of mean (SEM).(TIFF)Click here for additional data file.S5 FigD. melanogaster Wolbachia strain(s) present in the wild-type (W1118) and Mt2 -/- flies used in this study. (A) Genomic maps of common Wolbachia strains present in D. melanogaster indicate the location of distinct chromosomal IS5 transposon elements present or absent within either WD1310 or WD0516/7 loci. (B) Genomic region spanning the WD0516 loci was amplified using DNA isolated from W1118 and Mt2 -/- fly lysates (n = 3/sample) (Left). Controls obtained from flies infected with Wolbachia strains either wMel3, wMel2, wMelCS2 or wMelCS (Right). Increased shift in band migration indicate the presence of IS5 element at the WD0516 locus.PCR based genotypic analyses was carried out to identify native (TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


In the agenda towards malaria eradication, assessment of both malaria exposure and efficacy of anti-vectorial and therapeutic strategies is a key component of management and the follow-up of field interventions. The simultaneous use of several antigens (Ags) as serological markers has the potential for accurate evaluation of malaria exposure. Here we aimed to measure the longitudinal evolution of the background levels of immunity in an urban setting in confirmed clinical cases of malaria.Plasmodium falciparum , one P. malariae (PmCSP) and one Anopheles gambiae salivary (gSG6-P1) antigens were measured using magnetic bead-based multiplex immunoassay (MBA). Total anti- P. falciparum IgG responses against schizont lysate from african 07/03 strain (adapted to culture) and 3D7 strain was measured by ELISA.A retrospective serological cross-sectional study on was carried out using 234 samples taken from 2010 to 2013 in peri-urban sentinel facility of Cote d’Ivoire. Antibody responses to recombinant proteins or BSA-peptides, 8 −3, rho = 0.3). The particular recruitment of asymptomatic individuals in 2011 underlined a high background level of immunity almost equivalent to symptomatic patients, possibly obscuring observable yearly variations.High prevalence (7–93%) and levels of antibody responses to most of the antigens were evidenced. However, analysis showed only marginal decreasing trend of Ab responses from 2010 to 2013 that did not parallel the reduction of clinical malaria prevalence following the implementation of intervention in this area. There was a significant inverse correlation between Ab responses and parasitaemia (P<10The use of cross-sectional clinical malaria surveys and MBA can help to identify endemic sites where control measures have unequal impact providing relevant information about population immunity and possible decrease of transmission. However, when immunity is substantially boosted despite observable clinical decline, a larger cohort including asymptomatic recruitment is needed to monitor the impact of control measures on level of immunity. Plasmodium falciparum malaria remains a major threat in tropical and sub-tropical regions, with nearly 50% of the world population exposed to infective bites by Anopheles mosquitoes and almost half million deaths annually .The Magnetic Bead-based MAGPIXescribed ,12,17. 2ie a pool of 25 sera from clinically immune adults living in the village of Dielmo  and a pool of European and/or African non-immune, respectively. Results were expressed as OD ratio = OD sample / OD naive serum pool ; (ii) elevated [104–5x104]; (iii) high [5x104–105]; (iv) very high [>105] trophozoite per μl. Levels of Ab responses were not significantly different as function of levels of parasitemia for any Ag in 2010. Analysis of 2012 cohort showed significant high Ab responses against CSP and LSA141 (P<0.01) in individual with the lowest level of parasitemia compared to the highest one (<104vs 5x104-105 trophozoite per μl). For patients recruited in 2013, similar result was found for Ab responses against schizont extract Ag (P<0.01).Further analysis was done by stratifying levels of parasitemia in 4 classes: (i) positive [<10Analysis for the group of individuals recruited in 2011 was done after stratification according to RDT: negative, positive with/without circulating parasite. As illustrated on Multiple linear regression adjusted on age-groups including all years but 2011 showed no Ab responses significantly (P<0.05) associated with lower parasitemia level .When analyzing year 2011 only, Ab response to GLURP were significantly associated with lower parasitemia level (P = 0.03).When regression model included all years and was adjusted on age and years of follow-up, Ag targets associated with lower parasitemia were: GLURP (P = 0.03), MSP4p20 and PF13 (P = 0.01).Analysis of interrelation between antibody responses and age of individuals showed significant positive correlations  for schizont extract CSP, SALSA, GLURP and PF13 Ags (rho from 0.2 to 0.3).Profiles of Ab responses by age-groups are shown on Figs 41, LSA3 and gSG6 salivary peptide.Results from systematic year by year dichotomy comparisons (Mann Whitney test) are summarized in There is a high level of malaria endemicity in Côte d’Ivoire  and a coThe scaling up of integrated interventions strategies, as reported for Abobo, showed clearly a substantial positive result between 2010 and 2013 in Abobo , underliPlasmodium species-specific antibodies as indicators for exposure when analyzing multi-target Ab responses in acute malaria patients from sentinel health center. Malaria transmission in these settings including Abobo was high; profile of antibody responses was found different according to the location of the setting, accurately reflecting the immune background [A pilot study conducted in 3 different settings of Côte d’Ivoire highlighted the potential of ckground . A similDespite the observed decrease of malaria burden, analysis of the antibody responses to such array of antigens in this study could not sustain any substantial decrease of immune background level in those patients. Indeed, antibody responses were high as expected in individuals where parasite invasion induced clinical symptoms. The age-related significant increase of antibody responses found here witnesses a profile of a site with high ongoing endemic transmission, whose actual decrease is not yet reflected by detectable variation of the immune background. Several factors can contribute to this observation: firstly the recruitment of symptomatic individuals with high antibody boosting, secondly the short time frame of only two years between the strong implementation of prevention measures conducted in 2010–2011 and the latest measure in 2013 and thirdly the lack of biomarkers adapted to precise exposure estimates .Studies using serological analyses as indicators of malaria transmission generally involve recruitments in a relatively stable state for host-parasite interaction, usually of asymptomatic individuals in different settings and/or in longitudinal manner. Several antigens have been used as markers of exposure and malaria risk ,26, inclNumerous results and data underline antibodies to recombinant merozoite antigens as valuable biomarkers of immunity ,31. TherAnopheles salivary peptide gSG6-P1 antigen was the only indicator of a significant trend to decrease, a relevant marker of risk for malaria transmission already underlined [Altogether, analysis of antibody responses in case of longitudinal symptomatic malaria recruitment would rather require testing a multiplex array of antigens as a potential valuable tool. Antibody responses to different parasite targets and other plasmodium species were useful: in this study, IgG response to derlined ,35.vs 88%, P<10−3 by fisher exact test). Whether this observation relates with a lower efficient level of immunity for parasite clearance requires further longitudinal investigation on a larger cohort of individuals.Regarding parasitemia, no trend of differential susceptibility was evidenced in the different groups. Levels of parasitemia were comparable , in line with the almost similar levels of immunity measured in 2010, 2012 and 2013. Interestingly, young individuals harbored higher parasitemia and a significant relationship between high levels of Ab to GLURP, PF13 and MSP4p20 and lower parasitaemia was found. These results confirm that antimalarial antibodies may play an important supportive role in the therapeutic response to antimalarial drugs during acute falciparum malaria as already observed ,37,38. I5 trophozoite per micoliter of blood without any clinical symptoms. In these children, elevated antibody levels and prevalence against the array of antigens were found reaching higher levels than symptomatic individuals for SALSA, GLURP, PF13 and MSP4p20. In non-parasitized RDT positive individuals, there was a trend towards highest levels of antibody (Taken together, this study lacked of power for analyzing potential decrease of immunity expected to parallel the observable decrease of malaria burden due to effectiveness of control measures. However, this study underlined several interesting information regarding analysis of immune responses in high endemic setting. The 2011 particular recruitment shows clearly that immune background and natural immunity are substantially high. A proportion of 27% of young schoolchildren had positive RDT, in these individuals 16% had circulating parasitemia over 10antibody , indicatIndeed, recruitment of symptomatic patients in sentinel centers is a convenient possibility for investigation and follow-up of malaria immune responses which is a mandatory public health structure that supports patients and relevant bio-clinical follow-up. However, this study showed clearly that active additional recruitment of asymptomatic individuals is needed for accurate analysis in such areas where malaria remains strongly endemic.This longitudinal immuno-epidemiological study conducted in peri-urban endemic setting of Abobo shows that immunity profiling using biomarker has the potential for immunological mapping of intervention and help for prevention measures towards malaria elimination. However, analysis of longitudinal variations of immunity in high endemic setting requires further studies delineating the relationship between seroprevalence and transmission intensity. The limited but useful snapshot picture of the high immune background from this study underlines the need for multiple sero-surveys in enlarged cohort of individual among populations in Côte d’Ivoire with different levels of exposure for accurate immune profiling.


Exposures to various DNA damaging agents can deregulate a wide array of critical mechanisms that maintain genome integrity. It is unclear how these processes are impacted by one's age at the time of exposure and the complexity of the DNA lesion. To clarify this, we employed radiation as a tool to generate simple and complex lesions in normal primary human mammary epithelial cells derived from women of various ages. We hypothesized that genomic instability in the progeny of older cells exposed to complex damages will be exacerbated by age-associated deterioration in function and accentuate age-related cancer predisposition. Centrosome aberrations and changes in stem cell numbers were examined to assess cancer susceptibility. Our data show that the frequency of centrosome aberrations proportionately increases with age following complex damage causing exposures. However, a dose-dependent increase in stem cell numbers was independent of both age and the nature of the insult. Phospho-protein signatures provide mechanistic clues to signaling networks implicated in these effects. Together these studies suggest that complex damage can threaten the genome stability of the stem cell population in older people. Propagation of this instability is subject to influence by the microenvironment and will ultimately define cancer risk in the older population. DNA damage is believed to be the initial insult that underlies carcinogenesis and the process of aging. In addition to endogenous lesions caused by reactive oxygen species (ROS), cells are subject to a variety of environmental stresses that can damage their DNA. While oxidative radicals can cause simple lesions such as base damages or strand breaks, additional damages occurring in close proximity within the DNA can result in complex lesions that consist of two or more types of DNA damages within a single turn of the helix. The ability to effectively repair all these lesions in an error-free manner influences cancer susceptibility. Interestingly, the consequence of stochastic accumulation of deleterious lesions in post-mitotic cells over the lifetime of an individual also contributes to aging, a life stage characterized by gradual deterioration of function and increased risk of diseases such as cancer . ProgresRadiation is a well-known carcinogen that can cause both simple and complex DNA lesions. Environmental exposures range from mGys from high background radiation to 60-80 Gy received during fractionated radiotherapy. Given the dramatic increase in diagnostic and therapeutic radiation exposures in the past few decades it is essential to understand its carcinogenic potential, especially in radiation-sensitive organs such as the human mammary gland. Older individuals, who typically have a higher incidence of cancer also coincidentally, receive higher cumulative exposures. If carcinogenesis were proportional to exposure, then risk would be significantly higher in older women. However epidemiological data from two different radiation-exposed populations, the Japanese who survived the nuclear explosions in 1945 and children who are clinically exposed to radiation, contradict this postulate. They reveal that individuals exposed at an early age to low non-lethal doses of gamma rays, which primarily cause simple damages, exhibit higher excess relative risk of developing radiological cancers . The mecCentrosomes are microtubule-organizing centers that orchestrate a wide variety of cellular processes such as chromosome segregation, cell motility, adhesion, signaling and acquisition of polarity . They arAdult stem/progenitor (S/P) cells have the ability to renew and repopulate the cellular niche with mature differentiated cell types specific to the tissue of origin. Thus they play a significant role in regeneration, repair and maintenance of tissue homeostasis. Conversely, mutations that compromise their genetic integrity can potentially give rise to cancer. One subpopulation, coined the “cancer stem cell”, shares attributes of the adult stem cell, but is thought to have tumor initiating properties. While the validity of the cancer stem cell hypothesis as the causative tool for carcinogenesis is still under debate, a growing body of evidence from tumors of different tissue origin, including the breast, strongly supports the existence of a sub-population of stem cells within cancers with tumorigenic potential . StudiesIn order to understand the combined effects of age and damage complexity on centrosome number and stem cells, we exposed a panel of 15 well-characterized finite-lifespan normal human primary mammary epithelial strains from a wide age-range  to two types of radiation; Titanium ions (Ti) that primarily generate complex lesions and Cesium (Cs) that predominantly causes simple lesions. These strains were previously reported by Garbe et al. . Cells wCentrosome aberrations were assayed as an indicator of genomic instability and a potential surrogate marker for increased cancer risk. Previous studies have noted an increase in centrosome aberrations following radiation exposure -26. HoweTo assess age as a prognostic factor in radiation induced cancer risk, we next investigated the relationship between the age of the individual who provided the mammary tissue and the frequency of aberrations induced using linear regression Fig. . For cla>4P subgroup. For both radiation types, the 3P subgroup was significantly increased with high dose as compared to cells exposed to low dose  identified while scoring a minimum of 400 cells were classified into two sub-groups; cells with 3 pericentrin foci (3P) and those containing 4 or more pericentrin foci , middle (31-50 yo) and old (51-72 yo). The mean fold change in 3P and ≥4P frequency with Ti exposure was plotted for all three aged cohorts  were plotted for various days. These time course experiments confirm that the numbers of cells with aberrant centrosomes increase as early as D3, but the level of increase relative to control was similar in all age groups. Fitting these data to a linear model showed that the centrosome aberration frequency shows a linear increase with age at D9, as noted in Fig. There is strong evidence that centrosome aberrations can cause multipolar spindles that retard mitosis. The inherent growth advantage of normal healthy cells compared to cells containing centrosome aberrations thus poses a serious limitation in assessing frequency of this population at long times post-exposure. Centrosome aberrations have been observed as early as 3 days after radiation exposure  and can equency >P were plStem/progenitor (S/P) cells have been shown to express higher levels of aldehyde dehydrogenase (ALDH). This population described as ALDH positive (ALDH+) can be used to quantify S/P cell numbers by flow cytometry. Samples were exposed to both a low and high dose of Cs and Ti; subsequently cultured and processed 9 days post-exposure. Single cell suspensions were analyzed for ALDH+ cells to assess radiation-dependent changes in S/P cells. Only eleven HMEC strains were used for ALDH analysis, as we did not have sufficient numbers of viable cells for 4 strains . In analyzing the entire cohort, we noted a radiation-induced increase in the mean proportion of ALDH+ cells relative to control for both types of radiation  with age , pCREB (Ser133) and total TGFβRII protein levels remain unchanged with Cs dose and did not show an association with age , pAKT (Ser473) and pCREB (Ser133) following both doses , pSmad3 (Ser423/Ser425), as well as total protein levels of TGFβRII and SMAD4 were notably higher in the strain from the 66 y old exposed to low dose Ti relative to high dose Ti compared to inductions in a strain of a woman of similar age (68 y old)  Fig. . These dGenome stability is a major factor that contributes to assessment of cancer susceptibility. Amongst different age groups, the mechanisms that disrupt genomic stability and initiate and/or promote a neoplastic phenotype could vary in nature and/or magnitude based on the type of damage incurred. We used radiation as a tool to provide insight into these processes.Epidemiological studies in radiation exposed populations suggest that individuals exposed at an early age to simple damages, have an increased cancer risk . Our goaStudies have shown that complex damages overwhelm the cellular repair mechanisms and result in lesions that are either mis-repaired or remain unrepaired. This loss of DNA repair efficacy has been shown to cause genome instability by increasing the mutation load. However, the high incidence of non-clonal genomic instability in the progeny of irradiated cells argues against mutation frequency being the primary driver of radiation carcinogenesis. Increasing evidence suggests that centrosome aberrations could fulfill this role. As aneuploidy is a prominent feature of genomic instability observed in early stages of cancer, the ability of centrosome aberrations to transmit information regarding structural and positional alterations through generations and generate aneuploidy is thought to have important implications for regulation of phenotypes that impact breast cancer development . Our datWhat could be causing the increase in centrosome numbers in the older population? Centrosomes typically duplicate once per cell cycle in a semi conservative fashion starting at the G1/S boundary and ending in the G2 phase, prior to mitosis . The higThe magnitude of the age-related increase in centrosome aberrations with high doses of Ti highlights dose effects. We noted an approximate 1.8-fold rise in the frequency of centrosome aberrations with a 10-fold increase in dose, revealing centrosome aberration induction following complex damages elicited by Ti not proportional to dose. This lack of linearity suggests that the centrosome aberration frequency is not proportional to the initial damage but instead could reflect chronic phenotypes that persist weeks post-exposure. One likely candidate is chronic oxidative stress caused by ROS that are both endogenously generated as a product of cellular metabolism or by exposure to factors such a radiation . These fWe also evaluated stem cell frequency in relation to age and radiation exposure. Our studies show that S/P cell numbers increase with dose for both radiation qualities. This is consistent with previous studies, which have shown that a fraction of S/P cells increase with radiation exposure , 44. SimWhile our results reveal that the relative fraction of S/P cells does not universally increase with age following all dose and radiation quality exposures, we cannot exclude the possibility that their ability to differentiate is impacted by the complexity of the lesion induced. Aging has been associated with increased stem cell dysfunction. Under conditions of stress, while a fraction of progenitors decrease due to increased apoptosis with stress, the remaining stem cells are thought to leave their quiescent stage and divide more rapidly to undergo self-renewal and expansion to compensate for the cell loss and prevent premature exhaustion of the stem cell niche . As bothAssessment of global phospho-protein signatures has provided some clues as to the possible signaling mechanisms that underlie the age-dependent effects we observed. They also reveal signaling can influence strain specific differences in biological response. Further characterization of these pathways in isolated S/P cells would be essential to better understand how aberrations in these cells could foster cellular transformation.The etiology of breast cancer involves a complex interplay amongst various factors such as repair capability, oxidative stress, genomic instability, stem cell effects and integrity of tissue architecture that contribute to susceptibility in all age groups. We postulate that when HMECs from older individuals are exposed to complex damages, this exposure unfolds a cascade of interconnected events Fig. . Poor reConventional view suggests that aneuploidy caused by centrosome aberrations could be the predominant causal factor in increasing genomic instability within stem cells in the older population . HoweverLastly, though we have centered our analysis on epithelial cells, in the broader context, we cannot ignore the fact that the neoplastic phenotype is context dependent. While centrosome aberrations can destabilize the genome of stem cells, the proliferative advantage required for propagation of this genomic instability requires additional impetus. Senescence, stromal effects and inflammation are key candidates that can provide this stimulus and are known to exhibit age related changes. Although senescence is part of the cellular arsenal to maintain genomic integrity, groundbreaking studies by the Campisi group have shown that senescence accompanied by the SASP phenotype can prime transformation of genomically unstable cells , 55. ThuIn conclusion, to our knowledge this is the first report revealing an age related increase in genomic instability with increasing dose and lesion complexity in normal primary HMECs. Given stem cell numbers are increasing with radiation, the age-dependent global increase in genomic instability could also impact the S/P cell population. However, potentiation of this genomic instability will be subject to other non-targeted effects such as senescence, stroma and inflammation. Further investigation of the interplay between these factors is necessary to better define cancer risk from these exposures in the context of aging.http://hmec.lbl.gov. For some analyses we have classified strains into three age-defined cohorts based on the age of the individual from which the strain was derived. Four to five strains were chosen from various age ranges. The young cohort contained strains from women 16-27 years old , the middle-aged cohort contained strains from women 40 to 49 years old  and the old cohort contained strains from women 51-72 years old . These strains were previously used as part of a wider cohort in a study by Garbe et al who reported age dependent defects in the functional differentiation of multi potent progenitors [We have chosen fifteen cell strains from the Human Mammary Epithelial Cell Bank at LBNL to test from a cohort of well-characterized pre-stasis finite-lifespan normal human mammary epithelial cells (HMEC) derived from a wide range of aged individuals. These HMEC strains were produced in Dr. Martha Stampfer's laboratory at LBNL, Berkeley CA, and originated from a large bank of uncultured organoids derived from reduction mammoplasty of normal breast tissue. Details on the derivation and culture of these HMECs can be found on genitors .Passage 2 (P2) cells were cultured and passaged to generate adequate P4 cell stocks for all experiments. These early passage (P4) mammary strains were grown to sub-confluence (~85%) in T75 flasks and exposed to radiation. Cell strains were maintained in serum-free M87A medium supplemented with 0.5 ng/ml cholera toxin (CT), 0.1 nM oxytoxin (Bachem) and 1X antibiotic-antimycotic (Invitrogen) . Culture2 (recovered viable cell number/number of viable cells seeded)/0.301. Population doublings ranged from ~1-6, the proliferative potential of these strains is 10-15 PDs .Strains plated in T25 flasks were exposed to two different radiation qualities at room temperature. Heavy ion exposures with Ti 300 MeV/n (nucleon) were carried out at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory, Upton, NY. To prevent particle fragmentation, cell culture flasks were placed perpendicular to the beam and oriented with the cell monolayer side of the flask facing the beam. Two to four independent experiments were carried out over the course of a two-year period at two different NSRL campaigns (NSRL14C and NSRL15B). At the NSRL14C campaign cells were exposed to just one dose (0.5 Gy), and centrosome aberrations were assessed at three different time points; day 3, day 9 and day 12 post-exposure. In the subsequent NSRL15B campaign, to assess low dose effects, we included a low dose (0.05 Gy) along with the high dose exposure and processed samples only at day 9. Dose rates ranging from (5.5-6.4) cGy/min for low dose exposures and (22.23-32.5) cGy/min for high dose exposures were used to maintain short exposure times (0.5-2.0 minutes). Cells were grown for 24 h following irradiation and either shipped back to LBNL as live cultures or frozen down at BNL for analysis at a later date. Upon arrival at LBNL, cells were allowed to acclimatize for a day and subsequently trypsinized, viability checked and reseeded at low concentrations in either 60 mm dishes or in 100 mm dishes for long-term culture to subsequently define centrosome analysis or to assess stem cell numbers. Cells were exposed to Cesium radiation at Lawrence Livermore National Laboratory at dose rates of ~25 cGy/min for low doses and ~100 cGy/min for high doses and processed similar to the Ti samples. Previous data, evaluating double strand break induction using ϒH2AX foci in a primary HMEC strain was used to calculate Cs doses that generate the same amount of initial damage as Ti (unpublished results). Based on these results, dose delivered with Cs exposures were ~1.5 fold higher than Ti for both the low and high doses to compare endpoints in response to approximately equitoxic doses. Two independent experiments were carried out for cesium exposures. Total population doublings for each culture was calculated beginning at passage 5 prior to the radiation exposure, using the formula PD= logoC, washed in PBS and subsequently stained for pericentrin. Coverslips were blocked in 1% BSA for 1 h at RT and centrosomes were detected by indirect immunofluorescence, using a primary rabbit antibody against pericentrin (1:1000) . Following primary antibody incubation (1 h at RT), the cells were washed three times in blocking buffer, and subsequently incubated for 1 h with goat anti-rabbit Alexa Fluor 594 . Following secondary antibody incubation, cover slips were washed twice in PBS, counterstained with 0.1 μg/ml 4′, 6-diamidino-2-phenylindole (DAPI) in PBS, air-dried and mounted with Vectashield . Stained cells were imaged with a Zeiss Axiovert 200 M inverted fluorescence microscope equipped with a cooled CCD camera and Image-Pro®Plus software . Image acquisition was carried out within a week of staining.For centrosome aberration analysis, cells were trypsinized 48 h after radiation exposure, and seeded at low density in 60mm dishes. In NSRL 14C campaign, Ti exposed cells were trypsinized on D2, and D8 and re-plated onto coverslips to analyze samples at D3, D9 and D12 after radiation exposure. Cesium samples were analyzed only at D9 post-exposure to compare with D9 Ti exposed samples. Coverslips were fixed in 100% methanol for 10 min at −20>4P). Between 200-400 cells were examined for each sample.Centrosome aberrations were scored blind and at representative areas throughout the slide. For each region we assessed the total number of cells based on DAPI stain, and the number of cells with aberrant centrosomes (>2). Images of cells with aberrant numbers of centrosomes were taken at 40 X and scored by two independent observers to evaluate the number of centrosomes in each aberrant cell. The type of centrosome aberration was characterized as either 3 centrosomes/cell (3P) or greater than 3 centrosomes/cell . Ploidy was determined by flow cytometry based on DNA content and PI staining. DNA content of 10,000 cells was assessed on the FL2 channel using a BD FACS Calibur. Data was analyzed using Flowjo software Ver 8.6 (Tree Star Inc. OR).1x10For assessing the proportion of stem cells we have used ALDEFLUOR reagent (STEMCELL Technologies), which has been shown to be specific for stem/progenitor cells in mammary epithelial cell populations. A previous study  using a 490/498) (1:1000) . Following primary antibody incubation (1 h at RT), the cells were washed three times in blocking buffer, and subsequently incubated for 1 h with goat anti-mouse Alexa Fluor 594  and goat anti-rabbit Alexa Fluor 488 . Following secondary antibody incubation, cells were washed twice in PBS, counterstained with 0.1 μg/ml 4′, 6-diamidino-2-phenylindole (DAPI) in PBS, air-dried and mounted with Vectashield . Image acquisition was carried out within a week of staining. Data from two independent experiments were analyzed. Cells with >3 pATF2 foci were stratified as aberrant cells exhibiting persistent pATF2 signal. 200 cells were scored for each sample.For analysis of persistent pATF2 foci, coverslips processed on D12 following Ti exposure were co-stained with a mouse monoclonal antibody specific for pericentrin  and a rabbit polyclonal antibody against pATF2  signaling pathway and TNF alpha signaling pathway. This kit was used to detect changes in phosphorylated ERK/MAP kinase ½ (Thr185/Tyr187), AKT (Ser473), STAT3 (Ser727), JNK (Thr183/Tyr185), p70 S6 kinase (Thr412), NF-κB (Ser536), STAT5A/B (Tyr694/699), CREB (Ser133), and p38 (Thr180/Tyr182) in cell lysates using the Lumina® system. The MILLIPLEX MAP Human TGFβ Signaling Magnetic Bead Panel 6-plex, was used to detect changes in phosphorylated Smad2 (Ser465/Ser467), Smad3 (Ser423/Ser425), as well as total protein levels of TGFRβII and Smad4 in cell lysates using the Luminex® system. 25 ugs of sample was used in each well and the immunoassay was carried out according to the published protocol for this kit  and analyzed using the Luminex® system.Two sets of samples were exposed to Ti  and Cs . Both irradiated and sham samples were trypsinized and grown in 100 mm dishes and processed 9 days post-plating. Six plates were processed in each batch. After a wash with cold PBS, 300 ul of ice-cold 1X Milliplex lysis buffer with protease inhibitors (Sigma) and phosphatase inhibitors  was added to each plate on ice. Cells were scraped off the plate, and cell suspensions were transferred to a centrifuge tube and rocked for 15 mins at 4Linear regression was used to determine trends in surrogate endpoints for cancer relative to age. One-way ANOVA were used for multiple comparisons. Statistical significance of data was determined with p values <0.05. Pair wise comparison of differences in the percentage of cells with centrosome aberrations among dosed and unexposed controls were carried out using the Wilcoxon test. Differences in centrosome aberration frequency and stem cells amongst age-grouped cohorts were analyzed by the t test for independent samples. Pearson's correlation was used to examine the correlation between frequency of centrosome aberrations, ALDH+, pATF2 persistent signal and the relationship of these parameters to the level of various phospho-proteins in the stress activated signaling pathways. Analysis of variance (ANOVA) was used to estimate the 95% confidence interval of the mean. All statistical analysis was performed using GraphPad Prism 6.0.Research was conducted under Lawrence Berkeley National Laboratory Human Subjects Committee IRB protocols 305H002 and 108H004.