Striga hermonthica is influenced by multiple genomic regions with moderate effects. It is possible to increase genetic gains from selection for Striga resistance using genomic prediction.Genome-wide association revealed that resistance to Striga hermonthica (Del.) Benth., commonly known as the purple witchweed or giant witchweed, is a serious problem for maize-dependent smallholder farmers in sub-Saharan Africa. Breeding for Striga resistance in maize is complicated due to limited genetic variation, complexity of resistance and challenges with phenotyping. This study was conducted to (i) evaluate a set of diverse tropical maize lines for their responses to Striga under artificial infestation in three environments in Kenya; (ii) detect quantitative trait loci associated with Striga resistance through genome-wide association study (GWAS); and (iii) evaluate the effectiveness of genomic prediction (GP) of Striga-related traits. An association mapping panel of 380 inbred lines was evaluated in three environments under artificial Striga infestation in replicated trials and genotyped with 278,810 single-nucleotide polymorphism (SNP) markers. Genotypic and genotype x environment variations were significant for measured traits associated with Striga resistance. Heritability estimates were moderate (0.42) to high (0.92) for measured traits. GWAS revealed 57 SNPs significantly associated with Striga resistance indicator traits and grain yield (GY) under artificial Striga infestation with low to moderate effect. A set of 32 candidate genes physically near the significant SNPs with roles in plant defense against biotic stresses were identified. GP with different cross-validations revealed that prediction of performance of lines in new environments is better than prediction of performance of new lines for all traits. Predictions across environments revealed high accuracy for all the traits, while inclusion of GWAS-detected SNPs led to slight increase in the accuracy. The item-based collaborative filtering approach that incorporates related traits evaluated in different environments to predict GY and Striga-related traits outperformed GP for Striga resistance indicator traits. The results demonstrated the polygenic nature of resistance to S. hermonthica, and that implementation of GP in Striga resistance breeding could potentially aid in increasing genetic gain for this important trait.The online version contains supplementary material available at (10.1007/s00122-020-03744-4). Striga hermonthica (Del.) Benth., is the most widespread parasitic weed posing a serious threat to maize production in sub-Saharan Africa (SSA)  lacks its own root system and survives by drawing water and nutrients from host plants like maize for its own growth and has a potent phytotoxic effect  enables genetic dissection of complex traits. GWAS offers high mapping resolution and could effectively identify favorable genomic regions associated with the trait of interest (Yu and Buckler Striga resistance. Genomic prediction (GP) facilitates prediction of best-performing lines and can potentially accelerate the breeding cycle with optimal resources  evaluate the diverse array of 380 tropical maize inbred lines for response to Striga under artificial infestation; (ii) detect main-effect QTL and putative candidate genes associated with Striga resistance; (iii) assess the utility of GP for Striga resistance with different cross-validation methods in the tropical maize panel; and (iv) evaluate multivariate predictions of Striga resistance indicator traits using other correlated agronomic traits based on the IBCF approach. We evaluated the IBCF approach for predicting a given target trait  by incorporating information from other traits that can affect the target trait(s). In this study, users represent the target trait while items represent other related traits evaluated in different environments, that are expected to have some correlation with the target trait.While several studies have reported the use of GWAS to understand the genetic architecture of some biotic stresses, e.g.,  association mapping panel . Both Kibos  and Alupe  have a bimodal rainfall distribution. Striga seeds collected from sorghum fields were stored for few months which helps to break the seed dormancy which was later used for the infestation. Artificial infestation of the S. hermonthica trials at Kibos and Alupe was carried out by adding viable Striga seeds to each planting hole. Each maize plant was exposed to a minimum of 2000 viable S. hermonthica seeds. A standard scoop calibrated to deliver specified amount of germinable Striga seeds per hill was used for the artificial infestation  under artificial tion Kim . Striga u et al.  to ensurStriga damage syndrome rating and emerged Striga plant counts were recorded in the field trials. Host plant Striga damage syndrome rating (SDR) was visually rated for each plot at 10 and 12 weeks after planting (WAP) using a scale of 1–9, where 1 to 3 = no visible or mild damage symptoms; 4 to 5 = some leaf blotching, wilting and stunting; 6 to 8 = extensive leaf scorching, noticeable stunting and reduction in plant growth; and 9 = all leaves completely scorched and dead , using the formula for “area under the disease progress curve” (AUDPC)  was calculated from ear weight and grain moisture, assuming a shelling percentage of 80% and adjusted to 12.5% grain moisture content. These agronomic traits data were collected to understand their correlations and their usefulness in the breeding for Striga resistance. Further other traits are also used in multivariate predictions for Striga-related traits and GY.Other agronomic traits including days to anthesis  and days to silking  were recorded. Plant height , ear height , number of ears per plant , husk cover , plant aspect  and grain moisture at the time of harvest were also recorded. All the ears harvested from each plot were weighed, and representative samples of ears were shelled to determine percentage moisture using a Dickey John moisture meter at all locations. Grain yield kj is the effect of the kth replication at the jth environment, B(R.E)ojk is the effect of the oth incomplete block in the kth replication at the jth environment and eijko is the residual. Environments and replications were treated as fixed effects and the other effects as random. Heritability on an entry-mean basis was estimated from the variance components on a progeny mean basis as described by Hallauer and Miranda (https://hdl.handle.net/11529/10201). Pairwise Pearson’s correlation coefficients (r) among the traits were calculated using R software version 3.2.5 (https://www.r-project.org/).All quantitative genetic parameters were estimated based on the performance of the 380 inbred lines in the IMAS association mapping panel. Residuals for all traits were normally distributed. Individual location analyses were performed, and data from locations with significant genotypic variation and good repeatability were selected for across location analyses. We removed the outliers and did the across location analyses. Analyses of variance within and across environments were undertaken by the restricted maximum likelihood method using the software ASREML-R  markers was described earlier , as well as population structure and kinship (Q + K) using the mixed linear model (MLM) algorithm .BLUPs calculated for Striga-associated traits in individual environments using its similarity to other traits measured at different environments. The detailed steps involved in IBCF approach implementation are described in earlier studies  denotes the items rated by user (line) j, We used the IBCF approach for multivariate prediction of GY and G-matrix) calculated from markers  scheme and was found very useful in breeding programs, this cross-validation called incomplete field trials (CV2) was the practical method when some lines were evaluated in some environments but not in others; the goal here was to predict the performance of these lines in environments where they had not been phenotyped  genotypic and genotype-by-environment (G × E) interaction variances  for Striga plant counts and AUSNPC, and high for SDR (0.84) and GY (0.70). The distribution of phenotypic values was unimodal for the number of emerged Striga plants at different intervals, AUSNPC and SDR indicating the quantitative nature of Striga resistance .The strongest positive and significant correlations were observed between number of emerged Striga infestation are shown in Table Striga plants at 10 and 12 WAP. Inbred lines with high GY and supporting a low number of emerged Striga plants in addition to lower SDR were considered as resistant/tolerant to Striga.The top 16 lines in terms of GY and their performance for other traits under Striga plants at 8, 10 and 12 WAP, AUSNPC, SDR and GY. The results for the six traits are shown in Manhattan and Q–Q plots of P values comparing the expected -log10p values to observed  − log10p values . Eleven, fourteen and six significant SNPs individually explained 8–10%, 8–11% and 8–10% of the total phenotypic variance for number of emerged Striga plants at 8, 10 and 12 WAP, respectively. For AUSNPC, SDR and GY, sets of 11, four and nine significant SNPs individually explaining 7–10%, 6–7% and 7–8% of the total phenotypic variance, respectively, were detected. The most significant SNP across the six traits was S5_56842787 on chromosome 5 which explained 10% of the total phenotypic variance for number of emerged Striga plants at 8 WAP. SNPs S5_56842787 on chromosome 5, S7_70368510, S7_7160192 and S7_144538472 on chromosome 7, S1_298988628 on chromosome 1 and S7_165944748 on chromosome 7 were found to be the most significantly associated for Striga count at 8, 10 and 12 WAP, AUSNPC, SDR and GY under Striga, respectively. A set of putative candidate genes associated with the significant markers was identified (Table r2) between adjacent pair of markers and distance in kb . The average physical distance was 6.53 kb and 18.82 kb at a cut-off value of r2 = 0.2 and 0.1, respectively.GWAS was performed for the number of emerged Striga plants at 8, 10 and 12 WAP and AUSNPC compared with the other environments, whereas for SDR and GY, Alupe2013 performed better. There were slight increases in the prediction correlations for models with the G × E interaction in cross-validation scenarios for most traits and environments. Prediction correlations were also obtained for the measured traits using BLUEs across locations which showed values similar to those observed for CV0 and CV2  and number of emerged Striga plants 12 WAP (0.68) observed in this study corroborate previous reports under artificial Striga infestation  indicating that the IMAS association mapping panel has significant diversity, mimicking a natural population, and thus was appropriate for conducting GWAS. Previous studies on the population structure of the IMAS panel used in this study showed confounding structure in the panel .Correcting for population structure is an important step in GWAS to reduce the false positives that could arise from it without overcorrecting and further causing false negatives , we identified a total of 57 marker trait associations distributed across the genome and controlled by few major and many minor effect QTL  suggesting the complexity of resistance to Striga infestation. For SDR and number of emerged Striga plants at 8 WAP, we observed four and 11 SNPs and Adewale et al  detected for number of Striga-emerged plants were overlapped with four QTL detected for SDR in an earlier study  involved in the post-translational targeting of the nuclear encoding light harvesting chlorophyll-binding proteins (LHCPs) to the thylakoid membrane Below is the link to the electronic supplementary material.


Apc) in leucine-rich repeats and immunoglobulin-like domains 1 (Lrig1)-expressing colonic progenitor cells induces rapid growth of adenomas in mice with high penetrance and multiplicity. The tumors lack functional APC, and a genetic loss of heterozygosity of Apc was previously observed.The loss of a single copy of adenomatous polyposis coli (n = 9 tumors) and mRNA (n = 5 tumors) sequences were compared with matched nontumoral colon tissue. Putative somatic mutations were called after stringent variant filtering. Somatic signatures of mutational processes were determined and splicing patterns were observed.To identify genomic features of early tumorigenesis, and to profile intertumoral genetic heterogeneity, tumor exome DNA (Apc was not observed, however, an epigenetic loss of heterozygosity was apparent in the tumor transcriptomes. Complex splicing patterns characterized by a loss of intron retention were observed uniformly across tumors.The adenomas were found to be genetically heterogeneous and unexpectedly hypermutated, displaying a strong bias toward G:C > A:T mutations. A genetic loss of heterozygosity of Lrig1 and Apc expression are highly mutated and genetically heterogeneous, with an inflammation-associated mutational signature and complex splicing patterns that are uniform across tumors.This study demonstrates that early tumors originating from intestinal stem cells with reduced  Human colorectal cancer (CRC) is the second leading cause of cancer death in the US, and ~ 25% of patients with CRC are incurable at the time of diagnosis , 2. GeneLgr5). Lgr5 is the downstream target of R-spondin in the canonical Wnt/β-catenin pathway. Mutations in the tumor-suppressor gene adenomatous polyposis coli (Apc) and other members of the canonical Wnt pathway are the hallmark of CRC [Apc tends to occur during the early stages of human CRC tumorigenesis.The cell-of-origin of CRC derives from a population of rapidly-dividing stem cells located at the base of the colonic epithelial crypts, which are identifiable based on the expression of Leucine-rich repeat containing G protein-coupled receptor 5 -induced inflammation increased the incidence of polyps in the distal colon of ApcMin/+ mice [ApcMin/+ mice shifted tumor development toward the distal colon. In 2009 Ritchie, K., et al. crossed a glutathione S-transferase Pi (Gstp) null allele into the standard ApcMin/+ mouse and reported a 6-fold increase in distal colorectal adenoma incidence and a 50-fold increase in adenoma multiplicity compared to ApcMin mice [Gstp) null ApcMin/+ mice expressed higher levels of inflammatory molecules interleukin 4 (IL4), interleukin 6 (IL6), and nitric oxide synthase. Taken together, these results indicate an important role for mucus in reducing inflammation-associated tumors in the distal colon [The standard mouse model used for CRC research, etic LOH . ApcMin/n/+ mice . SimilarMin mice . The autal colon .Lrig1) is a transmembrane feedback regulator of growth factor receptor tyrosine kinases that is expressed in the Lgr5+ stem cell population present at the base of colonic crypts [Lrig1 acts as a tumor-suppressor gene in several contexts [Lrig1-CreERT2/+;Apcfl/+ inducible mouse model of colonic adenoma is based on the conditional Cre-recombinase-driven loss of a single copy of Apc under the control of the Lrig1 promoter [. The colonic stem cells of these mice express one single wild type copy each of the Lrig1 and Apc genes after the engineered recombination of Apc in Lrig1+/−-expressing stem cells. Within 100 days of the loss of one copy of Apc, rapidly growing adenomas appear in the distal colon with extremely high tumor penetrance and multiplicity. The Lrig1-CreERT2/+;Apcfl/+ CRC model is very similar to the Gstp-null;ApcMin CRC model in terms of tumor onset, penetrance, multiplicity, anatomical location, and mortality. These findings imply that a common mechanism involving inflammation-induced tumor formation is responsible for tumorigenesis in the distal colon.Leucine-rich repeats and immunoglobulin-like domains 1  [Human tumors often exhibit distinct patterns of mutations that can provide clues into the origin and mechanism of tumorigenesis. The ‘somatic signature of mutations’ of a tumor is based on the specific nucleotide alterations present and the background sequence context of the mutations –28. Somaand RNS) .Lrig1-CreERT2/+;Apcfl/+ mice. Exomic DNA and mRNA sequences from the adenomas were analyzed in order to detect the presence of transcriptomic and genomic alterations. Specifically, the genetic heterogeneity across tumors and somatic signatures of mouse colonic adenomas tumors were assessed using exome DNA profiling, and the prevalence of differential gene expression and splicing defects was assessed using mRNA-Seq.This work sought to understand the genomic changes occurring during the early stages of tumorigenesis in rapidly-growing colonic adenomas in Lrig1-CreERT2/+;Apcfl/+ tumors was previously described [Lrig1-CreERT2/+ mice were crossed to Apc580S/+ mice  [Lrig1-CreERT2/+;Apcfl/+ mice [Lrig1-CreERT2/+;Apcfl/+ mice were intraperitoneally injected with 2 mg tamoxifen per mouse  in corn oil for 3 consecutive days and multiple dysplastic colonic adenomas were extracted 100 days later. For the DNA studies, the control is nontumor tissue parts of the same Lrig1-CreERT2/+;Apcfl/+ mouse, referred to as ‘nontumor’ in figures. For the RNA studies, the control is untreated wild type C57BL/6 J mice, referred to as ‘wild type’ in figures.Generation of escribed . Briefly, U.S.A)  to generl/+ mice . Adult (n = 9 tumors) was performed by HudsonAlpha Labs  Exome capture using NimbleGen v3.0 captured 64 megabasepair (Mbp) baits. A total of six gigabasepairs (Gbp) of exome data were sequenced per sample. Exomic tumor DNA of nine tumors from three mice was sequenced in parallel with adjacent normal colon (n = 3) to 12x average read depth. Adjacent normal colon tissue was used for the ‘nonutmor’ control.Exomic DNA sequencing (Mus musculus (house mouse) genome  using Bowtie2 (v2.2.1), in default-sensitive mode [Raw fastq reads were cleaned to remove low quality bases at the ends of the reads using Stacks v 1.35) process_shortreads . Cleanedive mode . Sam/bamive mode  and Pica process_n = 3, provided by Hui Zong Lab, University of Virginia Medical School) were compared to the adenoma dataset; hypermutation or a clear somatic signature were not observed in the negative controls.Somatic mutations were called using SeuratSomatic (v2.5), a GATK module –38. Soman = 5) as well as normal wild type colonic tissue from untreated C57BL/6 J mice (n = 3), were assayed. RNA was sequenced to 25x average read depth. Cleaned cDNA reads were aligned to the mouse mm10 genome (Accession GCA_000001635.2) with STAR (v1.0) [Whole tumors (R (v1.0) . ChangesR (v1.0) .Apc in Lrig1+/−-expressing colonic stem cells are hypermutated and genetically heterogeneous. Tumor exome DNA contained 930–1300 high-quality somatic mutations per tumor (~ 25–30 mutations per megabase) distributed uniformly throughout the genome. Variants were called only when present in both the forward and reverse DNA strand, with a minimum quality score of Q10 and a minimum variant coverage of 4 reads. The tumors are highly heterogeneous in terms of the specific genetic loci mutated, however nearly identical frequencies of mutated nucleotide motifs were observed across all nine tumors sequenced . Two genes were mutated in 4/9 tumors: mucin 4 (Muc4) and DEAH-Box Helicase 8 (Dhx8). Dhx8 is involved in alternative splicing. Eleven genes were independently mutated in >30% of the tumors , Neurofibromin 1 (Nf1), and Lrig1 were observed. The DNA repair genes MutS protein homolog 4 (Msh4), MutS protein homolog 6 (Msh6), and DNA polymerase delta 1 (Pold1) were also mutated. Unexpectedly, most tumors contained several additional mutations in genes of canonical and non-canonical Wnt pathways. The specific genes affected were different for each of the tumors.Mutated tumor-suppressor genes, such as phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha  and Msh4 (FDR = 0.026) were observed . The adenomas expressed exclusively Apc transcripts lacking codon 580 in exon 15  are a likely source of guanine adducts and C:G > A:T transversion point mutations in the colon. The high incidence of Lrig1-CreERT2/+;Apcfl/+ adenomas localized to the distal colon might be explained by the high levels of ROS inherent to the distal colon and rectum.The hypermutated phenotype of Genetic heterogeneity was observed in tumors from the same mouse , as well as from sibling mice . Interestingly, in contrast to their variable genomes, the tumor transcriptomes and splicing patterns were strikingly uniform across all samples. The uniformity of the tumor transcriptomes suggests that the adaptation of an invasive phenotype resulted from convergent evolution. The lack of uniformity in the tumor genomes compared to the transcriptomes implies that expressed RNA and proteins may represent better drug targets than mutations, due to the fact that tumor transcriptomes are more uniform and predictable than tumor genomes.Lrig1-CreERT2/+;Apcfl/+ mouse model of CRC is notable for its high penetrance and rapid onset after the loss of just a single copy of Apc. The extremely high tumor burden observed in Lrig1-CreERT2/+;Apcfl/+ mice is especially striking considering that prior to the spontaneous loss of an Apc allele, tamoxifen-induced Cre-recombination of the other Apc allele, must first occur in the same cell under the control of the Lrig1 promoter, which is also a rare event. Spontaneous LOH of Apc in humans and mice tends to occur much more slowly, generally in the order of several months to years. For example, human familial adenomatous polyposis intestinal tumors typically become malignant at around age 40 years.The Lrig1-CreERT2/+;Apcfl/+tumors. This fact is unsurprising when one considers the extremely low theoretical likelihood of multiple spontaneous somatic LOH events at a single genetic locus multiple times in one colon over the span of a few months. It is worth noting that in the original report on these tumors, a standard polymerase chain reaction genotyping assay was used to detect the LOH of Apc, which is an archaic and much less sensitive technique for genotyping compared to DNA sequencing.In contrast to previous reports, a genetic loss of heterozygosity (LOH) of Apc was not observed in the Apc alleles in the tumor exome DNA, there was clear evidence of an epigenetic (‘above the genes’) LOH of exon 15 of Apc observed in the tumor mRNA. Unlike the tumor DNA, the Apc mRNA transcripts expressed by the tumor cells clearly displayed a copy number loss of exon 15. The expressed Apc transcripts had a genetic sequence identical to that of the original Apc transgene following Cre-induced recombination, indicating that only the mutant transgene is being expressed by the tumor cells. The assumed mechanism responsible for the lack of wild type Apc expression is transcriptional silencing of the single wild type copy of Apc remaining after engineered recombination of the Apc transgene. It is possible that the double heterozygosity of Lrig1 and Apc act synergistically with Lrig1 haploinsufficiency to increase the possibility of transcriptional silencing of wild type Apc.Despite the normal appearance of the remaining Lrig1-CreERT2/+;Apcfl/+ model is the following: Transcriptional silencing of Apc leads to a decrease in goblet cells and a corresponding increase in inflammatory reactive oxygen species (ROS) in the distal colon. Somatic C > A mutations resulting from increased inflammation provide the raw genetic material required for adenoma formation and progression. Natural selection of growth-promoting mutations leads to convergent evolution towards a transcriptome marked by dysregulated splicing and altered gene expression resulting in an invasive phenotype.The proposed mechanism of colonic adenoma formation in the The results of this study demonstrate that the genomic events leading to tumor growth and invasion can happen early in tumorigenesis and that tumors can generate large pools of mutations early in the course of disease progression. The selection of advantageous mutations is a dynamic process, changing throughout the course of tumorigenesis. The ability of a tumor to create and store mutations in a reservoir for later use provides an advantage to the tumor by optimizing its ability to dynamically modulate the selection of oncogenic mutations in a context-dependent manner throughout disease progression. Mutational reservoir creation provides tumors with the ability to conditionally express somatic mutations which could ultimately leading to tumor metastasis and drug resistance.In order to develop therapeutic treatments that are less susceptible to drug resistance, it is important to understand the mechanisms underlying tumor formation and evolution. Going forward, mutational processing mechanisms altered during early tumorigeneses make attractive therapeutic targets since they should theoretically be present in a large proportion of tumor cells and they are the drivers of tumor growth. Additional studies are needed in order to understand the mechanisms underlying the processes of mutation generation in tumors.Lrig1-CreERT2/+;Apcfl/+ tumors exhibited transcriptional silencing of Apc and contained a high incidence of C > A mutations cause by increased inflammation. In contrast to their variable genomes, the tumor transcriptomes and splicing patterns were strikingly uniform across all tumors, and likely represent more predictable cancer biomarkers.Understanding the process of mutation generation during tumorigenesis can provide insights into the mechanisms underlying cancer cells’ ability to evolve and grow. Additional file 1. Somatic mutations identified in Lrig1-Cre/+;Apcfl/+ colonic adenomas.Additional file 2. Annotated RNA-Seq results for in Lrig1-Cre/+;Apcfl/+ colonic adenomas vs. wildtype colon.Additional file 3. Raw RNA-Seq results for in Lrig1-Cre/+;Apcfl/+ colonic adenomas vs. wildtype colon.


Human noroviruses are a major cause of diarrheal illness, but pathogenesis is poorly understood. Here, we investigate the cellular tropism of norovirus in specimens from four immunocompromised patients. Abundant norovirus antigen and RNA are detected throughout the small intestinal tract in jejunal and ileal tissue from one pediatric intestinal transplant recipient with severe gastroenteritis. Negative-sense viral RNA, a marker of active viral replication, is found predominantly in intestinal epithelial cells, with chromogranin A-positive enteroendocrine cells (EECs) identified as a permissive cell type in this patient. These findings are consistent with the detection of norovirus-positive EECs in the other three immunocompromised patients. Investigation of the signaling pathways induced in EECs that mediate communication between the gut and brain may clarify mechanisms of pathogenesis and lead to the development of in vitro model systems in which to evaluate norovirus vaccines and treatment. Human norovirus pathogenesis is incompletely understood due to a lack of appropriate animal disease models. Here, Green et al. show norovirus replication in chromogranin A-positive enteroendocrine cells and other epithelial cells in tissue from a pediatric intestinal transplant recipient with severe gastroenteritis. Caliciviridae, are highly infectious human pathogens with a global distribution. Clinical outcomes of infection range from severe, life-threatening gastroenteritis to asymptomatic shedding of virus in stool. Moreover, noroviruses can establish chronic infection of varying severity in patients with underlying immune deficiencies1. Norovirus vaccines and therapeutics are a recognized public health need, but the development of such strategies has been hampered by poor understanding of pathogenesis in the human host.Noroviruses, positive-sense single-stranded RNA viruses classified in the family 2. Murine norovirus (MNV) has been studied most extensively3, with macrophage and dendritic cells bearing the virus receptor CD300lf defined as major target cells in mice4. Supporting this, MNV replicates in primary macrophage and dendritic cells cultured in vitro as well as in immortalized macrophage-derived cell lines such as RAW264.75. Upon entry into the lamina propria via M cells6, MNV replicates in both myeloid and lymphoid immune cells, including T and B cells in the gut-associated lymphoid tissue (GALT)7. Permissive cells in mice have recently been extended to include chemosensory tuft epithelial cells, the only known epithelial cells that express the CD300lf receptor8.Experimental model systems in animals have largely informed investigations of norovirus pathogenesis, but no animal model has fully replaced human challenge studies9. Replication of human norovirus in an in vitro stem cell-derived intestinal enteroid cell culture system supported the identification of enterocytes as a permissive target cell10. Tissue from gnotobiotic piglets and calves challenged with human norovirus and bovine norovirus, respectively, showed the presence of viral antigen-positive enterocytes localized to the sides and tips of small intestinal villi12. Morphological changes to villi (blunting), including epithelial cell apoptosis, have been documented15. Human norovirus antigen has been detected in various immune cells of the lamina propria in both humans and animal models17. Evidence of replication in a human B cell line (BJAB)18 prompted a search for B cell targets, but the role of immune cells in replication has not been verified in vivo19.Target cells for human norovirus replication are less well defined. Active replication has been linked to human enterocytes in the epithelial layer by co-localization of viral structural and nonstructural protein expression within the same cell7, we show that human noroviruses target epithelial cells in this immunocompromised patient. Enteroendocrine cells (EECs), specialized epithelial cells in the intestine that secrete hormones and other signaling molecules integral to gut function, are identified as permissive target cells during acute and chronic infection in four immunocompromised patients. This opens avenues of investigation into the mechanisms of norovirus pathogenesis in diarrheal disease and in the development of optimized in vitro cell culture systems for the evaluation of vaccines and treatment.The purpose of this study is to identify target cells in the human enteric tract that support active norovirus replication. Biopsies obtained from different portions of the intestine of a pediatric intestinal transplant patient with acute norovirus diarrhea are analyzed for the presence of viral antigen and RNA. Moreover, the identities of norovirus-infected cells are investigated with antibodies directed against cell-specific surface markers. In contrast to the striking tropism of MNV for immune cells20 consisting of an en bloc stomach, duodenum, pancreas, liver, small intestine, and large intestine graft with splenectomy. Fourteen months later the patient presented with sudden profuse, nonbilious vomiting followed by equally sudden and profuse watery yellow-green diarrhea a few hours later resulting in severe dehydration. Gastrointestinal pan-endoscopy was performed 26 h after onset of vomiting and showed normal appearance of the duodenum-upper jejunum portion of the intestinal graft and the graft colon −1, presented with decompensated cirrhosis of unknown cause within the first months after birth and complicated by complete porto-mesenteric venous thrombosis precluding transplantation of the liver alone. At 9 months old the patient underwent multi-visceral transplantationImmunohistochemical chromogenic staining of the sectioned biopsies with a norovirus VP1-specific monoclonal antibody (TV19) showed the presence of abundant viral capsid antigen (staining brown) in localized regions of intestinal tissue Fig. . Three g22 , choline acetyltransferase (ChAT), and chromogranin A (CgA) as optimal for the identification of M cells, tuft cells, and enteroendocrine cells (EECs), respectively , a subtype of EECs27. There was evidence also that ECs could occasionally be infected directly by rotavirus27, and serotonin receptor antagonists have been explored for the treatment of rotavirus-associated vomiting and diarrhea28. Additional study will be needed to establish the role of EECs in both immunocompetent and immunocompromised human hosts with norovirus disease, but the wide-ranging roles of EECs in gut-brain signaling, digestion, and mucosal immunity could offer important insight into strategies to treat norovirus illness.A pediatric intestinal transplant recipient presenting with severe norovirus gastroenteritis provided a rare opportunity to study norovirus infection during acute, life-threatening illness. Norovirus has been identified as a potential risk factor for acute secondary organ rejection in this patient population9. Previous observations, coupled with our strand-specific viral RNA analysis presented here , indicate that intestinal epithelial cells in the small intestine are major primary target cells for human norovirus replication  and included written informed consent from patients and/or their legal representatives. Human biospecimens collection and analysis at NIH was approved by the NIH IRB and included written informed consent. Animal studies were approved by the Institutional Animal Care and Use Committee of an NIH-approved vendor and conducted adhering to the guidelines and basic principles in the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals, and the Guide for the Care and Use of Laboratory Animals by certified staff in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International accredited facility.The samples from four norovirus-positive and two norovirus-negative patients analyzed in the present study were obtained under IRB-approved protocols at two participating medical centers, MedStar Georgetown Transplant Institute of the MedStar Georgetown University Hospital  and the National Institutes of Health Clinical Research Center .31 were used in conjunction with a synthesized RNA standard (Bio-Synthesis) representing the consensus sequence of a highly conserved region in NS7Pol to calculate viral genome copy numbers  . Norovirus-positive stool was prepared as a 10% w/v suspension in PBS, and viral RNA was extracted from a 50 µl volume with the MagMAX-96 Viral RNA Isolation Kit (Thermo Fisher Scientific). The presence of norovirus RNA, presumptive genogroup (G) assignment, and viral genome copies per gram of stool were determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) with the TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher Scientific) according to the manufacturer’s instructions. Primer pair MGBGII_F/MGBGII_R and probe MGBGIIEndoscopic biopsies collected from jejunum, ileum, and colon were fixed in 10% neutral buffered formalin prior to embedding and processing in histological grade paraffin. All tissues were sectioned at 5 µm and stained with hematoxylin-eosin (H&E) for examination by light microscopy. Sections received at NIH were evaluated by a board-certified veterinary pathologist. Images were taken using an Olympus BX51 microscope (Olympus Corp.) and photomicrographs were taken using an Olympus DP73 camera.All chromogenic immunohistochemistry (IHC), immunofluorescence (IF), and in situ hybridization staining was performed on the Leica Bond RX automated system. All tissue sections were dewaxed for 30 min in Bond Dewax Solution (Leica Microsystems) heated to 72 °C. Tissues were then rehydrated with absolute ethanol washes and 1x ImmunoWash (StatLab). All antibodies were diluted in Background Reducing Antibody Diluent .34 and 30A1135 were used at dilutions of 1:100 and 1:500, respectively. For detection of norovirus nonstructural proteins, primers pairs were designed to amplify the NS5VPg, NS6Pro and NS7Pol regions of GII.4 norovirus Rockville/2012 (GenBank KY424328), each pair with an incorporated histidine tag and restriction endonuclease sites for cloning into the pET28b vector (EMD Biosciences) (Supplementary Table E. coli BL21 (DE3) cells and purified by immobilized metal (nickel) affinity chromatography prior to immunization of rabbits36. Rabbit immunizations were conducted according to an Animal Care and Use Committee-approved protocol at Pocono Rabbit Farm and Laboratory Inc., and the resulting three nonstructural protein-specific antisera were each used at a dilution of 1:50. Following incubation with primary antibodies for 15 min, the sections were rinsed with ImmunoWash and treated with an appropriate secondary antibody if needed. Primary antibodies with a rat host were treated for 8 min with an unconjugated rabbit anti-Rat IgG antibody . The Polymer Refine Detection System (Leica Microsystems) was used for DAB and the Polymer Refine Red Detection System (Leica Microsystems) for Fast Red detection. Colorization was achieved with DAB incubation for 10 min and Fast Red for 15 min. Counter-staining was achieved with hematoxylin. In the case of double IHC, the DAB protocol was applied first and was followed by Fast Red detection. Epitope retrieval was applied prior to the DAB protocol.Epitopes were retrieved through a 20 min treatment in Bond Epitope Retrieval Solution 1 (Leica Microsystems) heated to 100 °C. Only slides to be detected with 3,3′-Diaminobenzidine (DAB) tetrahydrochloride hydrate were exposed to peroxide block (Leica Microsystems) for 5 min. Sections were incubated with the following primary antibodies for the detection of cellular markers  was the same as that for IHC. Prior to primary antibody incubation, samples were blocked with Serum-Free Protein Block  for 30 min. Samples were incubated in a primary antibody solution for 60 min then rinsed with ImmunoWash. Secondary antibodies . All tissues were blocked with Serum-Free Protein Block for 30 min. Samples were subsequently incubated in a cocktail mixture of primary antibody solution for 60 min then rinsed with ImmunoWash. A cocktail mixture of secondary antibodies against the primary antibody host species was applied to the tissues for 30 min. The secondary antibodies (Supplementary Table RNAscope probes (ACDBio) were synthesized to detect the positive-sense and negative-sense RNA species of the GII.4 ORF2, based on sequence of the related norovirus Hu/USA/2015/ GII.4 Sydney[P16]/Pasadena 3477 (GenBank Accession Number KY947550). PPIB Positive Control (ACDBio) and dapB Negative Control (ACDBio) were used as controls in select cases. Epitope retrieval was performed on tissues with application of Bond Epitope Retrieval Solution 2 (Leica Microsystems) at 88 °C for 15 min followed by a Protease III (ACDBio) treatment at 40 °C. Tissues were hybridized with the probes for 120 min and visualized with either the RNAscope 2.5 LS Reagent Kit-RED or the RNAscope LS Multiplex Fluorescent Assay (ACDBio). The detection kits allowed for visualization of the probe through chromogenic and/or fluorescence methods. For ISH coupled with IF, the slides were treated with the ISH procedure prior to IF. After the ISH protocol, sections proceeded with the IF protocol detailed above without an additional epitope retrieval step. Slides were counterstained with DAPI.Images were captured using a Leica TCS SP8 DMI6000 inverted fluorescence confocal microscope (Leica Microsystems) equipped with a photomultiplier tube/hybrid detector and a Leica DFC345 Monochrome camera. Images were viewed with the 20×, 40x or 63x oil immersion objective  and data were collected using the Leica Application Suite X software platform. White light laser and specific emission and excitation range were applied depending on the fluorophore. The following spectra for excitation/ emission were used: 488/520 for IBA-1, DC-SIGN, VP1 and ChAT; 594/620 for viral RNA positive and negative-sense probes; and 680/700 for CK and CgA. DAPI was excited using a 405-nm diode laser. Images were taken using sequential acquisition and variable z-steps. Image processing was performed using Imaris 9.2.1 . Maximum intensity projection (MIP) is represented in xy pictures, and cross sections of between 5–7 μm were obtained with the ortho slicer feature from Imaris.Captured images were prepared for presentation in Photoshop CC 2019  and scale bar lengths were defined either directly on the figure or in the corresponding legend. Data are presented as representative images of multiple fields of view for each antibody or probe analyzed per biopsy section. Analyses were performed 3–5 times on each biopsy and staining patterns were reproducible within and between the four norovirus-positive patients in the study. Biopsy sections from two norovirus-negative patients were included as controls to monitor nonspecific signals with the antibodies and probes.Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummaryDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6


An emerging body of literature has indicated that moderate alcohol intake may be protective against Alzheimer disease (AD) dementia. However, little information is available regarding whether moderate alcohol intake is related to reductions in amyloid-beta (Aβ) deposition, or is protective via amyloid-independent mechanisms in the living human brain. Here we examined the associations of moderate alcohol intake with in vivo AD pathologies, including cerebral Aβ deposition, neurodegeneration of AD-signature regions, and cerebral white matter hyperintensities (WMHs) in the living human brain.n [%] = 180 [43.5]) were recruited from 4 sites  around Seoul, South Korea. All the participants underwent comprehensive clinical assessments comprising lifetime and current histories of alcohol intake and multimodal brain imaging, including [11C] Pittsburgh compound B positron emission tomography (PET), [18F] fluorodeoxyglucose (FDG) PET, and magnetic resonance imaging (MRI) scans. Lifetime and current alcohol intake were categorized as follows: no drinking, <1 standard drink (SD)/week, 1–13 SDs/week, and 14+ SDs/week. A moderate lifetime alcohol intake (1–13 SDs/week) was significantly associated with a lower Aβ positivity rate compared to the no drinking group, even after controlling for potential confounders . In contrast, current alcohol intake was not associated with amyloid deposition. Additionally, alcohol intake was not related to neurodegeneration of AD-signature regions or cerebral WMH volume. The present study had some limitations in that it had a cross-sectional design and depended on retrospective recall for alcohol drinking history.The present study was part of the Korean Brain Aging Study for Early Diagnosis and Prediction of Alzheimer’s Disease (KBASE), an ongoing prospective cohort study that started in 2014. As of November 2016, 414 community-dwelling individuals with neither dementia nor alcohol-related disorders  between 56 and 90 years of age  dementia.Preclinical studies from animal and cell culture models have demonstrated that moderate alcohol intake confers protection by attenuating molecular amyloid-beta (Aβ) pathology.Little information is available regarding whether moderate alcohol intake is related to decreased Aβ deposition, or is protective via amyloid-independent mechanisms in the living human brain.We performed cross-sectional analyses of the baseline data from 414 middle- and old-aged individuals with neither dementia nor alcohol-related disorders who participated in the Korean Brain Aging Study for Early Diagnosis and Prediction of Alzheimer’s Disease (KBASE), an ongoing prospective cohort study.All participants were systematically interviewed on current and past alcohol intake and underwent brain imaging to examine the associations of alcohol intake with various in vivo AD pathologies.We observed that moderate lifetime alcohol intake  was significantly associated with lower amyloid deposition compared to no drinking, whereas current alcohol intake did not affect amyloid deposition.In contrast, neither lifetime nor current alcohol intake was associated with neurodegeneration of the AD–signature cortical regions or cerebral white matter injury.The present findings from middle- and old-aged individuals with neither dementia nor alcohol-related disorders suggest that moderate lifetime alcohol intake may have a beneficial influence on AD by reducing pathological amyloid deposition.The findings should be cautiously interpreted because the present study had a cross-sectional design and depended on retrospective recall for alcohol drinking history. Debate remains regarding whether alcohol intake has protective or harmful effects with respect to the risk of dementia or cognitive decline. Although excessive alcohol intake is associated with an increased risk of dementia or cognitive decline –4, an emSeveral human studies have reported that moderate alcohol intake is associated with a lower risk of Alzheimer disease (AD) dementia ,9–13. NePrevious findings for the influence of moderate alcohol intake on neurodegeneration and cerebrovascular changes are controversial –30. Whil11C] Pittsburg compound B (PiB) positron emission tomography (PET). We also tested the hypothetical associations of moderate alcohol intake with AD-related neurodegeneration and cerebrovascular white matter injury. The neurodegeneration of AD-signature regions was measured by both MRI and [18F] fluorodeoxyglucose (FDG) PET imaging. Cerebral white matter hyperintensities (WMHs) on MRI were used as a measure of cerebrovascular injury  = 180 [43.5]) who were recruited as of 30 November 2016. Participants were recruited through 4 recruitment sites around Seoul, South Korea. Potentially eligible individuals who participated in a dementia screening program at 2 public centers for dementia prevention and management or visited memory clinics at 2 university hospitals  around Seoul, South Korea, were informed about study participation, and those who volunteered were invited for an assessment of eligibility. In addition, volunteers from the community were recruited through advertisements online, posters and brochures at the main recruitment sites, and word of mouth . More detailed information on KBASE study characteristics including recruitment has been published previously  versus older [≥75 years]) , sex , while lifetime alcohol intakes of <1 SD/week and 14+ SDs/week were not related to Aβ positivity ; Fig 1. No group differences were observed in AD-CM, AD-CT, or WMHs between the lifetime (or current) alcohol intake categories. The findings for the association between alcohol intake and neurodegeneration or WMHs were not changed even after additional controlling for all the covariates .Sensitivity analyses including only participants without binge drinking showed similar results  Tables. As shown in In this study, we observed that lifetime alcohol intake of 1–13 SDs/week (moderate drinking) was associated with lower cerebral Aβ deposition compared to the no drinking group in these middle- and old-aged individuals with neither dementia nor alcohol-related disorders.The present finding of an association between moderate alcohol intake and lower Aβ deposition is in line with results from previous studies using animal or cultured cell models, which indicated that moderate alcohol intake exerts a protective effect via attenuating Aβ accumulation ,20. ManyWhile moderate lifetime alcohol intake had a significant association with Aβ deposition, moderate current intake did not. This difference indicates that the protective effects of moderate alcohol intake against Aβ pathology involve the chronic effects associated with long-term exposure rather than an acute effect. The significant finding for lifetime intake only also suggests that the protective association for moderate alcohol intake is not due to the inclusion of forced abstainers, i.e., those who stopped using alcohol owing to other health concerns related to problem drinking, among the reference group . Forced abstainers were classified as drinkers for lifetime alcohol intake status, whereas they were classified as non-drinkers for current alcohol intake status.Unlike for Aβ deposition, there were no associations between moderate alcohol intake and neurodegeneration or WMHs. Similarly, previous human MRI studies did not observe an association between moderate alcohol intake and cerebral gray matter volume  or totalAlthough excessive alcohol intake has been related with an increased risk of cognitive decline –4, and Un = 62) in the younger age group and 37.3% (n = 57) in the older group. The relatively small proportion of Aβ+ individuals in the younger group might decrease the likelihood of detecting a significant difference. It is also possible, as mentioned above, that these age-related differences are related to the chronic effects associated with long-term alcohol exposure. In contrast, sex, APOE4, and clinical diagnosis did not have any moderating effect on the association between moderate alcohol intake and Aβ positivity.The investigation of the influence of age on the association between moderate alcohol intake and Aβ positivity revealed that the protective effect of moderate alcohol intake on Aβ positivity was more prominent in older individuals (≥75 years) than younger ones. This finding may be due in part to age-associated increases in the Aβ positivity rate in individuals without dementia . In the The present study had a couple of strengths. To the best of our knowledge, this study is the first to show the association of moderate alcohol intake with Aβ accumulation in the living human brain. The study included a relatively large number of participants who were well-characterized through comprehensive clinical assessments including systematic interview for detailed alcohol drinking history and multimodal brain imaging for in vivo AD pathologies and WMHs. In addition, various potential confounders were systematically evaluated and controlled in the statistical models in order to reveal the association between alcohol intake and brain pathologies as clearly as possible. Even after controlling for all potential confounders, the findings did not change. The results were also confirmed by sensitivity analyses conducted after excluding binge or former drinkers.Nevertheless, the present study also had several limitations that should be considered. First, because this was a cross-sectional study, causal relationships cannot be inferred from the findings. Second, in terms of lifetime alcohol intake, underestimation of drinking or retrospective recall bias may have affected the results in older individuals. However, it is unlikely that underestimation of alcohol intake was significant because harmful drinkers and individuals with a history of alcohol use disorder were excluded from the analyses, and moderate drinkers have no reason to underestimate their alcohol intake. Moreover, to reduce recall bias, information was obtained from reliable informants as well as the study participants. Additionally, a review of self-report bias in the assessment of alcohol intake suggested that this recall bias is not greater in older individuals than in the general population . Third, Although further long-term follow-up investigations in larger populations with heterogeneous alcohol intake patterns are still needed, the association of moderate alcohol intake with reduced risk of pathological Aβ deposition (about one-third of the risk for no drinking) observed in the present study may suggest that moderate lifetime alcohol intake may be beneficial in preventing AD dementia or related cognitive decline.In conclusion, the present findings from middle- and old-aged individuals with neither dementia nor alcohol-related disorders suggest that moderate lifetime alcohol intake may have some beneficial influence on AD by reducing pathological amyloid deposition rather than amyloid-independent neurodegeneration or cerebrovascular injury.S1 STROBE Checklist(DOCX)Click here for additional data file.S1 Interview Form(DOCX)Click here for additional data file.S1 Method(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.


Our analytical framework includes: (1) conducting pairwise mediation for unique exposure-mediator combinations, (2) exposure dimension reduction by estimating environmental risk scores, and (3) multivariate mediator analysis using either Bayesian shrinkage mediation analysis, population value decomposition, or mediation pathway penalization. Dimension reduction demonstrates that a one-unit increase in phthalate risk score is associated with a total effect of 1.07 lower gestational age (in weeks) at delivery  and eicosanoids from the cytochrome p450 pathway mediated 26% of this effect . Eicosanoid products derived from the cytochrome p450 pathway may be important mediators of phthalate toxicity.Diverse toxicological mechanisms may mediate the impact of environmental toxicants  on pregnancy outcomes. In this study, we introduce an analytical framework for multivariate mediation analysis to identify mediation pathways ( Diverse toxicological mechanisms may mediate the impact of environmental toxicants on pregnancy outcomes. In this study the authors introduce an analytical framework for multivariate mediation analysis to identify mediation pathways in the relationship between environmental toxicants and gestational age at delivery. Phenol derived compounds are also widely used in plastics, as well as personal care and pharmaceutical products3. Polycyclic aromatic hydrocarbons commonly enter the environment through industrial and natural processes, such as the combustion of coal, fossil fuel, and organic debris4. Additional anthropogenic activities such as natural resource extraction and electronic waste recycling can yield environmental contamination of heavy metals 5. Biomonitoring analytes derived from these exposure classes can capture the magnitude of exposure in humans to inform health studies.Globally, humans are susceptible to widespread exposure to environmental contamination through several anthropogenic operations, including the manufacturing of industrial chemicals, natural resource extraction, and fossil fuel combustion. Pregnant women and developing fetuses are uniquely vulnerable to environmental exposures. The prenatal exposome encompasses the totality of environmental exposures that occur during preconception and pregnancy. Among the vast landscape of pollutants that exist in modern society, we focus the attention of this present study on four classes of environmental contaminants: phthalates, phenols, polycyclic aromatic hydrocarbons, and trace metals. Phthalates are high-production volume chemicals used as plasticizers in numerous consumer products such as vinyl flooring, children’s toys, and food packaging6. Based on extensive in vitro and animal models, there is evidence indicating inducible receptor activity attributable to exposure classes 8. Perturbations in receptor signaling and transcription factor activity can result in the altered production of signaling molecules responsible for inflammation and metabolism. Therefore, biomarker signals of toxicant exposures can help disentangle the mediating biological pathways linking toxicant mixtures to pregnancy outcomes, which is critical for early detection of disease and prevention. Mediation pathways can also inform causal inference in human observational studies and thus the need for policy-driven exposure reduction and prevention measures.Exposures to environmental toxicants are suspected risk factors for adverse pregnancy outcomes such as preterm delivery or altered gestational duration10. Additionally, we learned that cytochrome p450 and lipoxygenase-derived eicosanoids had the greatest relative predictive capacity for classifying preterm birth compared to a large panel of endogenous biomarkers11. Eicosanoids are potent signaling molecules derived from parent fatty acid compounds such as arachidonic acid and linoleic acid12. For example, epoxyeicosatrienoic acids (EETs) produced through the cytochrome p450 pathways are involved in vascular remodeling and regulation of inflammation13. Therefore these eicosanoids may be up-regulated in disease states involving cardiovascular irregularities, an important precursor for adverse pregnancy outcomes14. Among the environmental exposures, we also showed that phthalates have been associated with increased risk for preterm delivery and reduced gestational age at delivery within the LIFECODES cohort16. Based on these previous studies, there is circumstantial evidence of potential mediation between prenatal phthalate exposure and pregnancy outcomes through eicosanoids.In previous analyses in the LIFECODES cohort, we have found that biomarkers of inflammation (cytokines and c-reactive protein [CRP]) and oxidative stress (8-isoprostane) have been associated with increased risk of preterm birth18. With the expansion of technology to measure increasingly high-dimensional endogenous biomarkers, the mediation framework needs to evolve in order to handle the analysis of multiple mediators simultaneously. Theoretical advancements in the mediation analysis framework have afforded new applications of mediation analysis to account for multiple mediators. For example, Chen and colleagues have developed methodological applications for leveraging population value decomposition to reduce the mediator matrix and analyze multiple mediators simultaneously19. More recently, Song and colleagues developed a Bayesian approach for continuous shrinkage estimation of multiple predictors to identify the most important mediation pathways20. In another study, Zhang and colleagues sought to approach this high-dimensional mediation scenario through penalized regularization using the minimax concave penalty and joint significance testing21. Another regularization approach that has been applied is a lasso penalty on mediating pathways22. Altogether, these statistical methods have pushed the frontier of mediation analysis and created opportunities to make critical scientific discoveries using biomarkers from complex biological pathways.Precise risk profiles can be estimated through various inferential tools, including the implementation of causal mediation analysis. Early methodological development of mediation analysis enabled investigators to decompose the relationship between a predictor and an outcome, and test whether the total effect is partially mediated by an intermediate variable24 with the aforementioned multivariate mediation analysis methods21. In this framework, we provide a guided discussion on approaches that include one-at-a-time pairwise mediation, exposure dimension reduction, and mediator shrinkage, dimension reduction, and penalization.Currently, there is a critical knowledge gap for addressing a multivariate mediation setting where there are not only high-dimensional mediators, but also multiple toxicants with inherent collinearity and grouping structures. Our study aims to advance methodological applications to investigate a mixtures mediation setting with multiple exposure classes and groups of endogenous biomarker mediators. We propose an analytical framework Fig.  that int2 at their initial visit. There were very few participants who smoked (5.4%) or drank alcohol (5.0%) during pregnancy. Sample characteristics were also compared to the larger parent LIFECODES cohort in Supplemental Table The weighted characteristics of our study sample are reported in Table p > 0.05). Associations with phthalates have been previously explored in the larger LIFECODES cohort, where several of these associations were significant16. Three toxicants  were associated with decreased odds of overall preterm birth  associated with decreased gestational age at delivery , although those estimates were not robust to false discovery adjustment   that were observed for spontaneous preterm birth  observed for gestational age at delivery   and positive total effect , however the direction of the natural indirect effect was negative—indicating inconsistent mediation  Fig. . MECPP iContinuing to focus strictly on gestational age at delivery, we reduced the dimensionality of the exposure matrix by creating environmental risk scores for each exposure class: phthalates, phenols, metals, and polycyclic aromatic hydrocarbons. Prior to exposure dimension reduction, individual toxicants had high within-class correlation, with the highest correlation coefficient (ρ = 0.97) observed between the phthalate metabolites MEOHP and MEHHP  between almost all of the exposures and inflammatory markers, with the exception of one phthalate metabolite MECPP and the cytokine TNF-α (ρ = 0.45) (Supplementary Data ρ < 0.17) between metals and parent compounds  to IL-10 (inflammation) (Table In Table n) Table . Althougn) Table . The phen) Table . Last, tn) Table .Table 3PAn alternative approach to population value decomposition derived mediator group effects is the use of sparse principal component constructed mediators. This approach assumes sparse effects on the outcome variable among individual mediators. Sparse principal component mediation analysis yielded 24 principal components that accounted for 80% of the variance in the mediator matrix. There was no single sparse principal component that exhibited a significant mediation signal between the phthalate risk score and gestational age at delivery, however, a majority (66%) of the components had natural indirect effect estimates in the same direction as what we observed when evaluating all biomarkers simultaneously using population value decomposition  by those confounders equals to 0.187 20, population value decomposition19, pathway lasso22, and application of minimax concave penalty and joint significance testing21. The theoretical basis for each of these methods applies unique penalties and estimation algorithms; therefore we expect a substantive degree of non-uniformity in results across methods. Nonetheless, findings that appear repetitively across methods materialize relationships that indicate importance, afford greater confidence of a true mediation pathway, and warrants deeper investigation through replication in independent samples. Chiefly, we learned that the phthalate risk score appears to be linked with the whole cytochrome p450 pathway effect using population value decomposition. When comparing this finding with the other multivariate mediation methods, individual cytochrome p450 metabolites were identified in both the pathway lasso penalty and the minimax concave penalty with joint significance testing. Furthermore, there are suggestive signals of lipoxygenase pathway and metabolites being linked to the phthalate risk score across each of these multivariate mediation methods. Another important finding was the lack of a mediation effect observed between the phthalate risk score and cyclooxygenase pathway or its metabolites when applying each of the high-dimensional mediation methods. Our study provides consistent evidence that phthalates may impact gestational age at delivery mediated through the cytochrome p450 pathway – and to a lesser degree the lipoxygenase pathway.Throughout our analytical framework, we explored a sophisticated ensemble of contemporary approaches for high-dimensional mediation analysis: Bayesian continuous shrinkage mediation27. Transformation of endogenous fatty acid compounds such as arachidonic acid results in the production of bioactive signaling molecules, which can influence cellular biology through altering membrane permeability and transcription factor activity28. For example, cytochrome p450 derived HETEs can influence renal vascular changes and impact other enzymes, such as inhibiting adenosine triphosphatase [ATPase] activity or stimulating peroxisome proliferator-activated receptor-gamma [PPARγ]30. Additionally, EETs can mediate anti-inflammatory effects by inhibiting NF-κB activity and increasing MAP kinase activity, thereby influencing cyclic AMP concentrations13. Cytochrome p450 activity is partially stimulated by elevated pro-inflammatory cytokines such as TNF-α28. As such, we expect maternal biomarker profiles to reflect observable differences in cytochrome p450 products in disease states. Furthermore, the circulation of cytochrome p450 products is critically important for regulating cardiovascular functions, such as vasodilatory changes and vascular inflammation partly through the expression of endothelial adhesion molecules for chemotaxis and leukocytic migration28. The underlying mechanisms associated with reduced gestational age duration and preterm delivery include maternal inflammation and systemic oxidative stress32. Therefore, the cardiovascular and inflammatory responsiveness of cytochrome p450 activity underlines the critical importance of these enzymes for pregnancy outcomes.The cytochrome p450 enzymes are a conserved superfamily of enzymes that function to metabolize environmental toxicants, drugs, and endogenous compounds33. However, another in vitro study demonstrated that cytochrome p450 activity is enhanced with di-2-ethylhexyl phthalate exposure, and this relationship depended on both the expression levels of the pregnane X receptor as well as concentrations of glucocorticoids34. Given that cytochrome p450 enzymes are involved in the metabolism of xenobiotics, they may be sensitive to phthalate exposures. Furthermore, since cytochrome p450 enzymes also metabolize endogenous signaling molecules, an increased burden in phthalate exposure may impose adverse perturbations in the normal functions of these enzymes.The mediation signal between phthalates and cytochrome p450 pathway underline an important toxicological mechanism. Several phthalate parent compounds  have been previously shown to inhibit cytochrome p450 enzymatic activity in vitro37. In the larger LIFECODES cohort, Ferguson and colleagues conducted mediation analysis using the oxidative stress biomarker 8-IP as a potential mediator in the relationship between phthalate metabolites and preterm birth 37. This study demonstrated that 8-IP mediated the relationship between preterm birth and MEHP, MECPP, and mono-n-butyl phthalate (MBP)37. A pilot study based in China (n = 115) investigated DNA methylation signatures as potential mediators but did not find evidence of mediation in the relationship between prenatal phthalates and gestational age35. Extending to outcomes beyond the pregnancy, a study based in Norway investigated maternal thyroid hormones as potential mediators in the relationship between prenatal phthalate exposure and attention-deficit hyperactivity disorder , however, there was no evidence of mediation38. The lack of studies focused on high-dimensional mediation pathways in the relationships between toxicant mixtures and pregnancy outcomes signifies a major knowledge gap in environmental epidemiology research. Identification of high-dimensional mediation pathways helps prioritize biomonitoring efforts for early interventions. By further validating the mediation effect of the cytochrome p450 pathway in independent samples, we may potentially be able to utilize eicosanoid biomarkers of this pathway as early signals of disease states in pregnancy attribute to phthalate exposure.Studies that have assessed mediation pathways for phthalates in the context of pregnancy outcomes focused on limited mediators and did not assess the whole biomarker groups39. Another study has also shown that fetal expression levels of cytochrome p450 enzymes vary significantly from gestation to neonatal years27. Additionally, the cross-sectional design of our study also underlines the potential for reverse causation. For example, elevated inflammatory disease states from other causative agents may be linked to increased cytochrome p450 products, in turn impacting renal clearance of phthalate metabolites measured in urine samples. The cross-sectional nature of our study is also vulnerable to exposure measurement misclassification given that environmental toxicants such as phthalates, phenols, and polycyclic aromatic hydrocarbons have short half-lives in humans. However, when we previously investigated phthalates and preterm birth using four time points15 and evaluated single time points cross-sectionally40, we observed consistent associations between the repeated measures analysis and the time point that was used for this present study (26 weeks gestation). Single time point measurements of endogenous biomarkers further limit the inference of long-term biological effects. However, we can evaluate previous repeated measures studies of endogenous biomarkers and assess their intra-class correlation coefficients to quantify the reliability of single measurements. For example, a previous repeated measures study of select eicosanoids in women (n = 9) observed low intra-individual variability across eight repeated blood measurements41. In the LIFECODES cohort, we have also leveraged repeated measurements of the inflammatory biomarkers (CRP and cytokines), and observed moderate intra-individual variability10. These results provide some indication that although single time point measurements of eicosanoids and inflammatory markers are not ideal, they may have moderately good utility to assess biological effects. Future studies should build upon these findings to investigate environmental exposures and cytochrome p450 products as mediators in a longitudinal design in order to evaluate other vulnerable windows of development during pregnancy.There are features of our study that can be improved upon in order to advance knowledge of mediating biological pathways for pregnancy outcomes. Our study was not only limited in sample size, but also by a single cross-sectional assessment of exogenous and endogenous biomarkers. One previous study demonstrated significant changes in the expression of cytochrome p450 enzymes across pregnancy in placental tissuemedsens function and E-value estimation).In regard to the methodological framework, multivariate mediator shrinkage, dimension reduction, and penalization were limited to a continuous outcome variable, and future innovations in statistical modeling will need to be developed in order to adapt our analytical framework for case-control settings with preterm birth. Another limitation of our study is the lack of potential confounders that may contribute to residual confounding. Specifically, dietary information may be an important confounder, given that some toxicants are associated with the type of food products that are consumed. To evaluate this, we conducted sensitivity analyses to quantify the magnitude of association required by unmeasured confounders using two separate techniques . In total, this sample includes 52 cases of preterm birth (defined as delivery <37 weeks gestation), and 109 controls. Spontaneous preterm birth  was defined as either having a preterm premature rupture of the membranes, or spontaneous preterm labor43. Questionnaires and physical exams were administered at study visits to collect data on key covariates . Participants provided biological specimens (urine and blood) at a clinic visit occurring between 23.1 and 28.9 weeks gestation. Maternal urine samples were collected at a median of 26 weeks gestation and subsequently frozen at −80 °C. Plasma was collected from 10 mL of blood draw using ethylenediaminetetraacetic acid plasma tubes. After collection, blood was stored at +4 °C for less than 4 h and subsequently centrifuged for 20 min before being stored at −80 °C.The LIFECODES pregnancy cohort was developed at the Brigham and Women’s Hospital in Boston, MA47. We used high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) to quantify a total of nine phthalate metabolites. We used isotope dilution liquid chromatography with tandem mass spectrometry (ID-LC–MS/MS) to quantify a total of eight polycyclic aromatic hydrocarbon metabolites and ten phenolic analytes. Finally, a total of 17 trace metals were measured using a Thermo Fisher  iCAP RQ inductively coupled plasma mass spectrometer (ICPMS) with Teledyne CETAC Technologies  ASX-520 autosampler.All exposure analytes were measured in urine samples by NSF International in Ann Arbor, MI. Quantification of individual toxicants and analytes followed protocols developed by the Centers for Disease Control and Prevention12. We used the Milliplex MAP High Sensitivity Human Cytokine Magnetic Bead Panel  to measure four cytokines: IL-1β, IL-6, TNF-α, and IL-10. An additional inflammation marker, CRP, was also measured using a DuoSet enzyme-linked immunosorbent assay (ELISA) 10.A panel of 51 eicosanoids and lipid metabolites were measured in plasma samples using a 6490 Triple Quadrupole mass spectrometer . The individual eicosanoids were identified using metabolite-specific fragmentation and retention timeso,o’-dityrosine [DY]. To quantify these biomarkers, total plasma protein was first precipitated and diluted with a phosphate buffer. The samples were then delipidated, injected with isotopically labeled standards, and hydrolyzed for 24 h. Subsequently, the processed plasma samples underwent liquid chromatography-electrospray ionization tandem mass spectrometry. Additional oxidative stress markers 8-IP and 8-OHdG were measured in urine samples at Cayman Chemical . 8-IP was quantified using affinity column chromatography followed by an enzyme immunoassay, and 8-OHdG was measured directly through an enzyme immunoassay48.We measured three protein oxidation markers in plasma samples: 3-nitrotyrosine [NY], 3-chlorotyrosine [CY], and survey package (version 4.0) was used to account for inverse probability weights in all downstream analyses.All statistical analyses were conducted using R (version 3.5.1). Descriptive statistics were weighted based on calculated inverse probability weights that were corrected for over-representation of preterm birth cases in the sample compared to the larger LIFECODES cohort. The There is no unmeasured confounding for exposure-outcome relationship;There is no unmeasured confounding for the mediator-outcome relationship after controlling for the exposure variable;There is no unmeasured confounding for the exposure effect on all of the mediators;There is no downstream effect of the exposure that confounds any mediator-outcome relationshipIn Fig. The above four assumptions are required to hold with respect to the whole set of mediators. Finally, as in all mediation analysis, the temporal ordering assumption also needs to be satisfied, i.e., the exposure precedes the mediators, and the mediators precede the outcome. Additional information on notations, definitions, and assumptions that we draw upon for our study are outlined in detail in supplemental materials, section 1.Y and every possible unique combination of analytes from the p-by-n exposure matrix, A , and endogenous biomarkers from the q-by-n mediator matrix, M . This results in a total of 2,318 unique mediation models. The goal of this initial step is to conduct an exhaustive screen of all possible combinations of mediation pathways between each toxicant and endogenous biomarker measured in our study. The general framework for the mediation models is represented by:The first approach in our analytical framework is a one-at-a-time pairwise mediation analysis with outcome r-by-1  for the i-th subject. Each individual mediator is log-transformed and denoted one-at-a-time as Mi. The exposure variables are log-transformed and denoted one-at-a-time as Ai. In models where gestational age at delivery was the outcome variable, the conditional model for In these models, To account for the nested case-control sampling approach, we constructed inverse probability weights so that the subset sample was more representative of the proportion of cases and controls in the larger LIFECDOES cohort. Models in Eqs.  and 2)  2) were This first pairwise mediation analysis approach represents the baseline status quo in environmental epidemiology research, given that a majority of existing studies deploy a one-at-a-time assessment of mediation pathways. This approach may be most useful when there are few exposure variables with a targeted mediation hypothesis. However, it is susceptible to biased results when multiple exposure variables are highly correlated but are analyzed separately. The bias and hence inflated type 1 error result from possible confounding due to co-exposures missing from the analytic model. In addition, users can modify Approach 1a and utilize it as a screening step in multiple exposure settings. For example, mediation pairs that yield modest signals with relaxed significance thresholds estimated from Approach 1a may then be prioritized and passed onto Approach 2.23, thus reducing the dimensionality of the exposure matrix from 38 to 4, as well as reducing collinearity across the exposure classes by using a summary risk score . In this setting, we reduce the total number of pairwise mediation models to 244. We first estimate weights to construct environmental risk scores by applying adaptive elastic net regularization (gcdnet [version 1.0.5]) on toxicants based on their classes:In our analytical framework, we introduce an alternative approach for mediation analysis by proposing a method to construct environmental risk scores49. In this setting, Yi is the continuous outcome variable gestational age at delivery. There are pk exposure analytes in the k-th exposure class, and λ1 is a tuning parameter that performs shrinkage of coefficients for each exposure variable j, and λ2 operates as a tuning parameter used to stabilize solutions paths that navigate multi-collinearity in the exposure matrix. We utilized 5-fold cross-validations and optimization of prediction errors in order to estimate λ1 and λ2.The adaptive elastic net combines the least absolute shrinkage and selection operator penalty with ridge regressionk-th exposure class, the scalar To construct environmental risk scores for each exposure class, we first extract Next, we apply Eqs.  and 2)  2) to es49. This approach may be most useful when users have several exposure variables and are interested in evaluating whole exposure classes to inform mixtures-based risk assessment rather than an individual toxicant analyte.Approach 1b overcomes issues of confounding due to multiple co-exposures and is less susceptible to bias from the correlation between exposure variables, chiefly because all exposures are included in a single multivariate model and the adaptive elastic net is robust to collinearityq × n mediator matrix by either conducting Bayesian shrinkage estimation, reducing the dimensionality of the mediator matrix, or penalizing parameters in the mediation model. For shrinkage estimation, we applied Bayesian shrinkage estimation. In this setting, we deployed a high-dimensional shrinkage mediation analysis, one-at-a-time, for each exposure variable, Ai, as individual toxicants (p = 38) and in the dimensionally reduced exposure matrix using environmental risk scores for the k-th exposure class, The second approach of our analytical framework provides options for evaluating the mentclass2pt{minimbama package (version 0.9.1)20. In this application, bama modifies Eq. 19. In this application, mediators are again organized into five groups, (1-by-) under the consideration of each exposure, Ai, thus when using the first direction of mediation for each group, we reduce the total dimension of the mediator matrix . Specifically, we constructed directions of mediation  in Eqs. . The coes + 1) pair-wise single mediation analyses for exposures . We leverage Eqs.  high-dimensional mediation analysis using a joint significance test for mediation effect21. These methods evaluate specific pairs of Finally, using each  in Eqs. .Pathway lasso introduces a penalty parameter on the product of indirect effects and performs shrinkage and selection to identify pairs of hima package (version 1.0.7). Hima applies the minimax concave penalty (MCP) to regularize the coefficients estimated in Eqs.   hima pac52. In this approach, all mediators are evaluated simultaneously to construct individual mediator vectors similar to Eq. ..52. In tmedsens function within the mediation package (version 4.5.0). This sensitivity analysis simulates the change in the natural indirect effect as a function of a sensitivity parameter, ρ, which is representative of unmeasured confounders influencing the variation in the mediator and outcome models53. Another sensitivity analysis that we recommend is the calculation of an E-value to evaluate the vulnerability of adjusted mediation models to unmeasured confounding. To calculate this parameter, we used the EValue package (version 3.0.9) and focused only on our final model of phthalate risk score and the 54. A third sensitivity analysis that can be leveraged is the evaluation of the difference in mediation effect signal with the agnostic combination of all covariates in every unique combination. For six covariates in our study, that results in the evaluation of 63 unique covariate combinations.To evaluate the robustness of our proposed analytical approach toward the assumptions that we defined earlier, including sequential ignorability, we recommend deploying three additional sensitivity analyses. First, we leverage the Supplementary InformationSupplementary Data 1ASupplementary Data 1BSupplementary Data 1CSupplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Description of Additional Supplementary FilesPeer Review File


Acute rejection (AR) remains a significant problem that negatively impacts long-term renal allograft survival. Numerous therapies are used to prevent AR that differ by center and recipient age. This variability confounds diagnostic methods.Methods: To develop an age-independent gene signature for AR effective across a broad array of immunosuppressive regimens, we compiled kidney transplant biopsy (n=1091) and peripheral blood (n=392) gene expression profiles from 12 independent public datasets. After removing genes differentially expressed in pediatric and adult patients, we compared gene expression profiles from biopsy and peripheral blood samples of patients with AR to those who were stable (STA), using Mann-Whitney U Tests with validation in independent testing datasets. We confirmed this signature in pediatric and adult patients (42 AR and 47 STA) from our institutional biorepository.Results: We identified a novel age-independent gene network that identified AR from both kidney and blood samples. We developed a 90-probe set signature targeting 76 genes that differentiated AR from STA and found an 8 gene subset  that was associated with AR.Conclusion: We used publicly available datasets to create a gene signature of AR that identified AR irrespective of immunosuppression regimen or recipient age. This study highlights a novel model to screen and validate biomarkers across multiple treatment regimens. Examination of the transcriptome from renal parenchymal tissue has more fully characterized events occurring within the kidney in order to subclassify acute rejection events and help adjudicate difficult to interpret biopsies Interestingly, the combination of data from renal parenchymal and peripheral blood signatures to define a more complete signature of AR has been infrequently pursued A total of 1091 renal gene expression profiles were collected from 7 independent NCBI Gene Expression Omnibus datasets: GSE21374, GSE22459, GSE36059, GSE50058, GSE7392, GSE9493, and GSE25902 (pediatric) Gene expression profiles of all datasets were measured using Affymetrix U133A or U133 Plus 2.0 expression array. Each dataset selected for this study contained clinical outcome data and patients' unique IDs were also collected from series matrix files (GEO) to ensure there was no redundancy in the sample set. Raw Affymetrix expression CEL files from each dataset were robust multi-array average normalized independently using Expression Console Version 1.1 . All data were filtered to include those probes on the HG-U133A platform. Batch effects were mitigated using surrogate variable analysis (SVA) To further develop a gene signature of early AR, a total of 89 pediatric and adult patients age 1 to 78 transplanted between July 2009 to July 2017 were selected from our institutional biorepository. They were characterized as AR —with samples within the 30 days preceding the rejection event—or STA without rejection during the first year after transplantation. Immunosuppression protocols included induction with basiliximab, daclizumab, or rabbit anti-thymocyte globulin, while maintenance regimens included use of tacrolimus, cyclosporine, azathioprine, belatacept, sirolimus, and/or mycophenolate mofetil with or without steroids, including some patients on full steroid withdrawal regimens. Cryopreserved peripheral blood mononuclear cell mRNA expression of genes identified in our microarray data was measured using Applied Biosystems™ TaqMan™ Array Cards and Plates . All samples were collected from patients with informed consent and all related procedures were performed with the approval of the Duke Institutional Review Board (Pro00093938).EnrichrSTRING v11Reactome pathway analysis and GO Biological Process analysis were also completed Mann-Whitney U Tests were used to identify genes that were differentially expressed between AR and STA groups. We also used a multivariable Cox-regression survival analysis for risk of AR  to identify genes associated with freedom from AR. Shotgun Stochastic Search in Regression (SSS) was used for assigning coefficients to genes that were identified in our previous step Figure ). All the gene expression data were combined and batch effects in the combined data were corrected using SVA. .To capture the heterogeneity of renal allograft rejection, we compiled a large collection of gene expression profile data from either kidney allograft parenchymal biopsy specimens (n=1091) or peripheral blood (n=392) obtained from 12 independent public datasets. Allograft and peripheral blood gene expression profiles showed expression differences among samples obtained from different data sets .Using all patients (pediatric or adult) we utilized 45,782, probe sets to define expression levels in one of three clinical phenotypes—T Cell Mediated Rejection (TCMR), Borderline rejection, or Chronic Allograft Nephropathy (CAN)—as compared to STA patients . For each of these phenotypes, we plotted both adult and pediatric samples using the first two principal components of differentially expressed probe sets. We observed that adult and pediatric gene expression was significantly different within the TCMR and CAN clinical groups, but not in the borderline group .To define the differences between age groups, we subsequently compared expression profiles between adult and pediatric samples and identified 25,043 probe sets whose expressions in TCMR and/or CAN were significantly different between adult and pediatric samples . After removing these age-group related probe sets, we re-built the principal components of TCMR, CAN and borderline using the remaining of 20,739 probe sets. In doing so, we saw a minimization of differences between adult and pediatric samples within the same histologic subtype, indicated by clustering of the points for adult and pediatric samples of the same histologic type  or by Cox-regression survival analysis (p<0.001) (Table S1). This probe set group (AR90sig) was then utilized to train and test multiple models across sample groups.To develop an age-independent AR signature, we first identified AR associated genes in adult samples using the 20,739 probe sets whose expression was not significantly different between adult and pediatric samples. We compared allograft gene expression differences between samples with AR within 5 years after kidney transplant to samples without any rejection over five years (STA). We also determined differences in gene expression between adult and pediatric TCMR and CAN. We further identified genes whose expression patterns were significantly associated with AR-free survival using Cox-regression survival analysis. Because there was limited long-term follow-up for patients with peripheral blood expression data available in public databases, we determined differences between patients with AR and those that were stable over 2 years. As shown in Figure ). From the 90 AR associated probe sets, we identified 76 genes whose expressions were significantly changed in AR. To determine the biologic basis of these 76 genes, we performed gene set enrichment analysis and found our gene signature was significantly associated with immune system and interferon signaling . Furthermore, we defined a novel gene network using STRING v11 that included the pathways noted above as well as others .We plotted these genes on a heatmap to define their expression between groups which confirmed good segregation between AR and STA groups . All three analyses showed high sensitivity and specificity for the signature to identify AR. Because the kidney tissue and peripheral blood samples were normalized differently (as they are from different tissue compartments with different background variability), we built and validated renal tissue and peripheral blood models independently. Therefore, we next created a separate signature in adult peripheral blood (n=196) samples, and validated this model using an independent set of pediatric peripheral blood (n=24) samples . Though the two signatures contained the same genes, SSS was run on allograft and peripheral blood samples independently, yielding different coefficients. Of note, the 90-probe set signature performed well on ROC analysis with a minimum area under the curve (AUC) of 0.79 when considering both analyses.Using SSS modeling, we next created a 90-probe set predictor using a training set of 298 adult kidney allograft samples and validated this in independent sets of adult (n=316) and pediatric (n=33) samples . In peripheral blood, a similar analysis was performed which showed a PPV of 85% and NPV of 70% in the pediatric validation dataset (Table S3).Furthermore, we created a cut-off gene expression level at maximum sensitivity and specificity in training data to define high vs. low risk of AR and applied this cut-off to validation sets. The positive predictive value (PPV) in the adult renal validation set was 30%, while the negative predictive value (NPV) was at 98%. The model also successfully delineated AR event-free survival between high vs. low risk cases  (Table 1).In order to monitor early AR events, we obtained blood samples from AR (n=42) and STA (n=47) patients available from our institutional biorepository. All samples were from patients monitored for one year post transplant, with STA defined as no rejection during that time. All AR samples were obtained within 30 days prior to an AR event. Patients were excluded from the AR group if they experienced another event (e.g. an infection) up to 14 days after the rejection event. Patients in the two groups were demographically similar except with regards to immunosuppressive management. More patients in the AR group received basiliximab induction and/or belatacept maintenance, while patients in STA group received tacrolimus (Table S4). Using these 8 genes, PCA was again employed to create a model to identify AR. The ROC curve AUC was found to be 0.71. Finally, we applied this signature of early AR events to patients the microarray data that we samples in the GEO we had initially queried, including 110 patients (adult and pediatric) that either experienced AR within 1 year or were stable for at least 6 months. Utilizing the PCA we created with our institutional cohort, we applied the 8 gene signature which yielded an AUC of 0.77 in this cohort . The NPV and PPV for the institutional dataset were 74.5% and 70.6% respectively. The NPV and PPV for the validation in the public dataset were 83.2% and 66.7% respectively (Table S3).After removing genes located on the X chromosome and probe sets related to microRNA, a total of 76 genes corresponding to our original 90 probe sets were interrogated by Real Time-Polymerase Chain Reaction (RT-PCR). 8 genes  were found to be differentially expressed in AR and this signature retained its significance after adjusting for multiple clinical variables, including as race, gender, age, and treatment   and pediatric (1.00) datasets. In peripheral blood, our validation AUC in a pediatric cohort was moderate at 0.79. Examination of our institutional cohort identified a subset of 8 differentially expressed genes. We confirmed this 8 gene signature in a cohort of 110 patients from public databases and again demonstrated a reasonable AUC for identifying early acute rejection (0.77). Overall, our analysis demonstrates an effective method for biomarker discovery utilizing a combination of publicly available data and single center resources. We report an age-independent signature of AR that performs well in a peripheral blood assay despite diverse and non-standardized immunosuppressive regimens.Though there is considerable excitement regarding the ability of peripheral blood-based biomarkers to advance the diagnosis and treatment of disease, there have been great challenges in moving from the research setting into clinical care Mechanistically, our 90-probe set signature contained 76 genes, many of which are important in immune regulation. One central pathway in our signature is that of Tumor Necrosis Factor-α (TNF-α) and the nuclear factor κ-light-chain-enhancer of activated B-Cells (NFκB) signaling. This multifaceted pathway is important in pro-inflammatory and apoptotic mechanisms depending on the context Consistent with our initial analysis, we saw upregulation of Human Leukocyte Antigen (HLA)-E in our subset of 8 genes that were associated AR arising within 1-year post-transplant. HLA-E interacts with CD159c/NKG2C, which activates NK cells. This HLA-E mediated signaling has been shown to occur in the kidney during AR Although our study provides an age-independent gene signature that was validated in multiple pediatric and adult cohorts, our study has limitations. First, the data from which the initial signature was created are heterogenous. It is possible that there is unmeasured methodologic variance for which we cannot correct with our normalization methods. Definitions of endpoints between studies are also various, which could contribute to miscategorizations of rejection or stability. However, we favor these differences to be small and likely randomly distributed. We also note that the PPV of the validation set among adult renal tissue samples is low (31%). This may be because the differentially expressed genes with the most predictive power were identified initially in the peripheral blood datasets. Though this presents difficulty with pursuing this gene signature in biopsy tissue, it bodes favorably for continued investigation of this signature in peripheral blood. Moreover, we cautiously interpret our positive results as previous investigators have failed to corroborate gene signatures between peripheral blood and kidney biopsy tissue With regards to the corroboration of the microarray data with our newly generated RT-PCR data, there is a well-known and reported discordance between the two assays Acute rejection remains a significant problem after kidney transplantation. Less invasive methods of identifying acute rejection are important to maximize graft survival and minimize patient morbidity. We identified and validated an age-independent peripheral signature of acute rejection that is effective in the setting of diverse, non-standardized immunosuppressive therapies. This was done efficiently by utilizing prior datasets to define our candidate signature before validating in an institutional cohort. The conduct of a prospective trial to further validate this signature is warranted.Supplementary figures and tables.Click here for additional data file.