CSMD3 is associated with better prognosis in Asian patients. Applying a robust computational strategy that adjusts for both technical and biological confounding factors, we find that TP53, EP300, and NFE2L2 show higher mutational frequencies in Asian patients. Moreover, NFE2L2 mutations correlate with the allele status of a nearby high-Fst SNP, suggesting their potential interaction. Our study provides insights into the molecular basis underlying the striking racial disparities of this disease, and represents a general computational framework for such a cross-population comparison.Esophageal squamous cell carcinoma is a major histological type of esophageal cancer, with distinct incidence and survival patterns among races. Although previous studies have characterized somatic mutations in this disease, a rigorous comparison between different patient populations has not been conducted. Here we sequence the samples of 316 Chinese patients, combine them with those from The Cancer Genome Atlas, and perform a comparative analysis between Asian and Caucasian patients. We find that mutated  Esophageal squamous cell carcinoma (ESCC) exhibits differences in incidence and survival patterns among races. Here, analysis of Chinese and TCGA ESCC patients reveals that Asian patients exhibit higher TP53, EP300 and NFE2L2 mutational frequencies, and mutated CSMD3 associates with better prognosis. Histologically, esophageal squamous cell carcinoma (ESCC) is the major type in Asian populations ; while esophageal adenocarcinoma is the dominant type in western countries3. Among American patients, the incidence rate for ESCC is 81% higher among Asian/Pacific Islander males compared with the rate for white males, but is 64% lower than the rate for black males4. Furthermore, there are substantial differences among races in terms of cancer survival6.Esophageal cancer disproportionately affects certain ethnic groups and races. More than half of global esophageal cancer cases occur in China, with 477.9 thousand new cases diagnosed in 20157, it is essential to investigate the molecular basis underlying the striking racial disparities in ESCC. Several recent studies12 have characterized significantly mutated genes in this disease, but a rigorous cross-population comparative analysis remains challenging for two notable reasons. First, mutation detection is largely affected by technical factors, including the sequencing platform, mapping pipeline, sequencing depths, and mutational calling algorithms, all of which should be carefully controlled in such an analysis. The effects of these factors are often large for mutation data collected from different studies13. Second, biological factors such as patient age, gender, and clinical stage also strongly impact the mutational status of a gene15, and these confounding effects should be considered when detecting race-specific mutational differences.Besides external factors such as environmental differences, behaviors, and treatment options8) and Caucasian patients (characterized by the TCGA)  studyGA) Fig. . Brieflyn = 39) and Vietnamese (n = 41) patients with ESCC in TCGA as a discovery cohort to identify race-specific mutational features in a genome-wide, unbiased way. Among the three populations surveyed, Vietnamese and Chinese populations are geographically close and belong to the same Mongolian race. We first examined the genetic relationship of the patients based on common single-nucleotide polymorphism (SNP) status of the 158 WES samples. The hierarchical clustering identified two major clusters  and ADH1B (rs1229984) in the three populations and found very similar patterns between Chinese and Vietnamese patients  with a mutation frequency of ≥5%  remains largely unknown in this disease . We therefore examined the correlation of CSMD3 mutation status with patient survival times, which is orthogonal to the mutation rate analysis. We found that among Chinese patients with WES data (n = 78), patients with mutated CSMD3 showed significantly better survival time than those with the wild-type allele , and we observed a similar pattern in Asian patient samples (n = 354) . However, there was no such pattern in Caucasian patients ; whereas before downsampling, there was no difference among these three populations   Fig. . This re44) Fig. .Fig. 3Co16 to adjust for the potential effects of these confounders. Importantly, samples with the same propensity score have the same distribution of measured confounders, so balancing the confounders can be achieved by simply balancing the propensity scores22. Using this algorithm, we identified six genes between Asian and Caucasian patients  <0.1). Specifically, TP53, NFE2L2, and EP300 showed a significantly higher mutation rate in Asian populations; while KRTAP9-1, LRFN5, and MAP2 showed the opposite patterns  is of particular interest: this gene is a transcription factor that regulates many proteins involved in response to injury and inflammation as well as cellular defense against oxidative stress; NFE2L2-knockout mice are more susceptible to esophageal carcinogenesis than wild-type mice23; and its mutations have been recently reported to enrich in Vietnamese patients8. We identified several mutation hotspots at the first 100 amino acids of the protein encoded by NFE2L2 and found that the most frequently mutated site resided in the coiled coil region , Fst for common SNPs of this gene by comparing European and Chinese populations using data from the 1000 Genomes Project25. We found a SNP (rs113671272) with a very high Fst score in the 5′UTR of NFE2L2  whole-genome sequencing and TCGA RNA-seq data to compare the mRNA expression level of NFE2L2 between cancer samples with and without this SNP and found that the presence of this SNP was associated with a significantly lower NFE2L2 expression across cancer types . Interestingly, the mutational status for Asian patients was strongly associated with the allele status of this SNP  with potential prognostic power and three race-biased mutated genes  in Asian patients. Additional efforts are needed to investigate their potential as biomarkers or therapeutic targets specific to Asian patients. The computational pipeline we developed can be readily applied to similar analyses for other cancers.Here we performed such an analysis of race-biased mutational features in ESCC patients by sequencing large patient cohorts and employing a rigorous analytic pipeline that explicitly considered various confounding factors. Compared to Caucasian patients, we identified one frequently mutated gene (Human primary ESCC and corresponding adjacent non-tumor tissues (5 cm from the tumor site) were collected from patients who were diagnosed and received surgery as primary treatment at Fudan University Cancer Center  from September 2007 to June 2011. The tumor tissues were snap-frozen in liquid nitrogen immediately after surgical resection and then stored at −80 °C until they were analyzed. The clinicopathological features of the patients were collected from in-patient medical records. The pathological features were evaluated by independent pathologists according to the TNM staging system of the American Joint Committee on Cancer (AJCC 7th edition). All patients were followed up after primary treatment at intervals that increased from 3 months to 1 year until death. The study protocol was approved by the hospital ethics committee and informed consent was obtained from all participants.18. Briefly, these genes included those recurrently mutated genes in gallbladder carcinoma, high-priority genes in Catalogue of Somatic Mutations in Cancer , genes related to drug sensitivity and highly mutated genes in gastrointestinal cancer. Targeted gene enrichment was performed with the TruSeq Custom Enrichment kits (Illumina).Genomic DNA was extracted from the tissue specimens using QIAamp DNA kit (Qiagen). The libraries were then prepared using protocols recommended by Illumina. Briefly, 1 μg DNA was sheared to short fragments (200–300 bp) using Covaris S220. DNA fragments were end repaired and an adenylate blocker was added at the 3′ ends. Adaptors with barcode sequences were then ligated to both ends of the fragments. E-Gel was then used to select DNA fragments of the targeted size. Afterward, 10 cycles of polymerase chain reaction (PCR) were performed, and the resulting mixture was purified. Whole-exome capture was performed using the TruSeqExome Enrichment kit (Illumina) according to the manufacturer’s protocol, with minor modifications. After the libraries were amplified with 10 cycles of PCR, the capture probes were added and incubated for 24 h at 65 °C. The hybridized mixtures were then amplified with another 10 cycles of PCR. Validated DNA libraries were then sequenced on the Illumina Sequencing System (IlluminaHiSeq 2500). We included 283 “cancer-related genes” in the target enrichment panel as previously describedhttps://github.com/ExpressionAnalysis/ea-utils). The resulting high-quality reads were aligned to the human reference genome (GRCh37) using Burrows-Wheeler Aligner (BWA 0.7.12)26. BAM files were processed by Genome Analysis Toolkit27 to improve alignment accuracy. Major steps included marking duplicates, local realignment around high-confidence insertion and deletions and base quality recalibration. We then used several popular callers, including Muse28, MuTect229, SomaticSniper30, Radia31, and VarScan232, to identify somatic point mutations. Only mutations reported by at least two callers were used in further analyses. Low coverage and strand-biased mutations were filtered out. To further reduce false positives and miscalled germline events, we used MuTect2 to call point mutations on all the normal samples. Any of these germline mutations, if found in more than one normal sample, were removed from our final list of somatic mutations. To assess the accuracy of our mutation calls, we obtained TCGA MC3 mutation data from Synapse  and calculated the (median) fraction of MC3 non-silent mutations  called in our mutation set across the same set of TCGA samples and vice versa. To further validate our somatic mutation calls, we performed targeted sequencing on the same Chinese samples (n = 75). For all somatic mutations called from WES data, 592 mutations positions had a depth of coverage ≥200 in the targeted sequencing data. Among them, 564 somatic mutations were also detected in the targeted sequencing data, resulting in a true positive validation rate of 95.3%.Read pairs (FASTQ format) were trimmed and filtered with fastq-mcf  for each exon across the three patient populations. We repeated downsampling for 10 times and called somatic mutations independently and used consensus mutations  for further analyses.https://github.com/genome/bam-readcount) for each normal sample. The minimum base quality was set to 15, and our analysis included 168,275 common SNPs with the coverage of ≥8 in each sample. We recoded the genotypes into 0, 1, 2 based on the wild-type/heterozygous/homozygous status of the SNPs. The WES samples were hierarchically clustered by the “dendextend” package in R33.For all TCGA and Chinese samples, we obtained the read coverage information of exonic common SNPs in the dbSNP (build 147) using bam-readcount (34. MutSigCV (V1.4)19 was applied to identify significantly mutated genes with default covariate tables. Genes with q (FDR) < 0.1 were considered to be significantly mutated. We performed survival analyses using the “survival” package in R35. Kaplan–Meier survival analysis curves and the univariate Cox regression model were used to test for survival differences between groups (capped at 3 years). We tested for the mutual exclusivity between two patient populations using CoMEt36, which uses a Markov chain Monte Carlo algorithm to compute the marginal probability of observing pairs of alterations. We used Genotype Query Tools37 to calculate the fixation index for each SNP site reported by the 1000 Genomes Project (phase 3) between southern Han Chinese (CHS) and the European super population (EUR). The SNP status of rs113671272 in Chinese and Vietnamese patients with WES was inferred from off-target reads with a minimum of 3 read coverage. To examine the SNP effects on the gene expression, we obtained the genotypes of rs113671272 in TCGA samples from ICGC whole-genome sequencing data, and obtained the mRNA expression level of NFE2L2 (based on the longest transcript uc002uli.3) from Fire Browser . Integrating both genotype and expression data, our analysis included 12 cancer types in which each comparison group (samples with or without the SNP) contained at least three samples, and the SNP effect on gene expression was assessed by paired Wilcoxon rank-sum test. The cancer samples with NFE2L2 somatic mutations were excluded from the analysis.To identify significantly mutated genes, somatic mutations were annotated using Oncotator16 continuously to assign weights for each sample based on the propensity scores to search for balance. When the standardized difference of the weighted propensity scores between two race groups was smaller than 10%, we considered the clinical characteristics balanced between the propensity score weighted samples. We then compared gene mutation frequencies between the two race groups by supplying the weights to a weighted chi-squared test, and calculated P-values and FDRs. Any gene with FDR ≤ 0.1 was considered biased in the two groups. We confirmed the statistical significance by randomly shuffling the race labels of the samples and repeated the above procedures 100 times. We calculated the statistical significance by comparing the number of significant features calculated from our real data to those from the permutated data.We collected and obtained clinical characteristics  for all ESCC samples. We then employed a propensity score analysis to identify genes that preferentially showed higher mutation frequencies in one race group. For a comparison between Asian and Caucasian patient groups, we first calculated the propensity score using logistic regression with “race” as the responsible variable. We used the matching weight schemehttps://portal.gdc.cancer.gov/). All relevant data sets for this study are available from the authors.The WES and targeted-sequencing data of Chinese ESCC samples have been deposited in the NCBI Sequence Read Archive (SRA) under Bioproject (accession number: PRJNA399748). The WES data of TCGA ESCC samples are available from NCI Genomic Data Commons (Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1


Tumor cells disseminate early in tumor development making metastasis-prevention strategies difficult. Identifying proteins that promote the outgrowth of disseminated tumor cells may provide opportunities for novel therapeutic strategies. Despite multiple studies demonstrating that the mesenchymal-to-epithelial transition (MET) is critical for metastatic colonization, key regulators that initiate this transition remain unknown. We serially passaged lung metastases from a primary triple negative breast cancer xenograft to the mammary fat pads of recipient mice to enrich for gene expression changes that drive metastasis. An unbiased transcriptomic signature of potential metastatic drivers was generated, and a high throughput gain-of-function screen was performed in vivo to validate candidates. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) was identified as a metastatic driver. CEACAM5 overproduction enriched for an epithelial gene expression pattern and facilitated tumor outgrowth at metastatic sites. Tissues from patients with metastatic breast cancer confirmed elevated levels of CEACAM5 in lung metastases relative to breast tumors, and an inverse correlation between CEACAM5 and the mesenchymal marker vimentin was demonstrated. Thus, CEACAM5 facilitates tumor outgrowth at metastatic sites by promoting MET, warranting its investigation as a therapeutic target and biomarker of aggressiveness in breast cancer. A screen for drivers of metastasis has revealed a key protein involved in the spread of breast cancer into lung tissues. A US research team led by Helen Piwnica-Worms from the University of Texas MD Anderson Cancer Center in Houston enriched cells for genes involved in metastasis by engrafting mice with breast tumor biopsies taken from women with metastatic triple negative breast cancer and then metastases of these mice to mammary fat pads of recipient mice. The researchers pinpointed the gene encoding CEACAM5—a protein known to play a role in cell invasion and spread—as a key promoter of the cellular transition associated with metastasis. Tissues samples from patients confirmed that CEACAM5 levels were elevated in metastatic lung tumors relative to primary breast tumors. The protein provides a potential therapeutic target for drug development and candidate biomarker for patient stratification. The development of novel strategies to prevent or treat metastatic TNBC will rely on defining properties of the tumor cell as well as the metastatic niche that enable dissemination, seeding, and outgrowth of metastatic tumor cells.6Metastatic breast cancer is incurable in most cases.10 and in some cases these gene expression changes predicted patient survival.12 Unbiased genetic screens are useful for simultaneously identifying and functionalizing metastatic drivers as well as suppressors. In vivo screening approaches including those that utilize shRNAs or CRISPR technology have been useful for identifying genes that suppress metastasis.13 However, metastasis suppressors are generally not candidate therapeutic targets. Gain-of-function forward genetic screens in orthotopically-engrafted cancer cell lines have identified a limited number of metastasis drivers.15 However, successful translation of these collective findings into the clinical setting has been limited.16 Furthermore, discordance between in vitro and in vivo screens has been reported, highlighting the importance of in vivo model systems.17 Orthotopic patient-derived xenograft (PDX) models are powerful tools for identifying metastatic drivers because the tumors retain much of the genomic heterogeneity of the original patient tumor and require metastatic cells to dynamically regulate gene expression programs as they metastasize in vivo.18Previous studies identified gene expression changes in metastatic subpopulations of breast cancer cells,20 Epithelial cells undergoing EMT down regulate expression of cell adhesion molecules, lose adhesive contacts with neighboring cells, and acquire an invasive migratory phenotype. Thus, EMT endows cells with the ability to escape the primary tumor, enter into the hematogeneous and lymphatic circulatory systems, extravasate, and invade the metastatic site. However, cells that have undergone EMT exhibit decreased rates of proliferation, and thus are not well equipped to populate the metastatic site.19 Therefore, upregulation of genes that promote MET may endow tumor cells with a selective advantage at the metastatic site. Despite findings from multiple studies that MET is critical for metastatic colonization,22 the key regulators that initiate the transition from the mesenchymal to the epithelial cellular state at the metastatic site remain largely unknown. These regulators may serve as clinical biomarkers for the aggressiveness of metastatic lesions that have already colonized the metastatic site. Indeed, numerous studies have revealed that tumor cells disseminate to metastatic sites early in tumor progression,25 and thus metastasis-prevention strategies may not be clinically feasible. Therefore, focusing preclinical efforts on characterizing and targeting tumor cells that have already seeded the secondary site may be a promising clinical intervention for patients with metastatic TNBC. Here, a functional genomic screen, performed in vivo using a PDX model derived from the primary breast tumor of a patient with metastatic TNBC, identified CEACAM5 as a metastatic driver, and functional studies demonstrated a role for CEACAM5 in late-stage metastasis.Epithelial-to-mesenchymal transition (EMT) and its reverse process mesenchymal-to-epithelial transition (MET) are dynamic developmental programs that contribute to metastasis.27 We engineered these tumor cells to stably express Click Beetle Red Luciferase (CBR-Luc), mCherry, and an shRNA specific for p53 (shA2) to create the PDX line BC3_A2 (formerly known as BC3-p53KD28)  were isolated from replicate mice, pooled, and engrafted into the MFPs of recipient mice. The resulting lung metastases (P1) were isolated, pooled, and engrafted into the MFPs of additional mice. The lung metastases from these mice (P2) were also collected and pooled  of NOD/SCID mice mice.To identify changes in gene expression patterns that accompany metastasis, we performed RNA sequencing (RNAseq) on lung metastases (P0 and P2) and MFP (P0 and P2) tumors Fig. . After sQuantitative real time (qRT-PCR) was performed to monitor expression of vimentin, a mesenchymal marker, in MFP tumors and lung metastases throughout serial passaging. Vimentin expression exhibited a dynamic pattern with higher expression in MFP tumors relative to corresponding lung metastases at each passage Fig.  and to l29 served as a positive control for screen optimization, and GFP was used as a negative control. BC3_A2 cells were transduced with lentivirus encoding either HOXA1 or GFP, and infected tumor cells were mixed in different ratios , but a pool of one driver with 19 non-drivers masked our ability to detect the driver. Based on these results we limited our pool size to 12 ORFs.A high throughput gain-of-function (GOF) screen was designed to identify functional drivers of metastasis from our P2 lung metastases signature. HOXA1, a known driver of metastasis,n = 10 mice per pool, Fig. ex vivo  were evaluated to determine if the increase in CEACAM5 in metastatic lesions was a general property of metastasis. PIM001-P was established from the treatment-naïve primary tumor of a patient with metastatic TNBC, whereas PIM001-M was established from the dermal metastasis from the same patient. PIM001-P tumor cells were engineered to express both CBR-Luc and mCherry and then engrafted in the MFPs of recipient mice. At time of necropsy, MFP tumors and lung metastases were isolated. CEACAM5 expression was increased at both the mRNA-levels Fig.  and prot31 Interestingly, phosphorylated (active) p38 correlated with high expression levels of vimentin in MFP tumors, and p38 phosphorylation decreased in lung metastasis where vimentin levels were low  were injected into the tail veins of recipient mice to monitor the effects of CEACAM5 overproduction on tumor growth in the lung. Overexpression of CEACAM5 led to increased tumor growth in lungs . RNA was isolated and subjected to RNAseq. Differential gene expression analysis was performed, and GSEA identified EMT as the only cancer hallmark pathway that was significantly downregulated both in vitro and in vivo by CEACAM5 overproduction Fig. . This re32 The majority of lung metastases expressed high levels of CEACAM5  screens in vivo to identify drivers of breast cancer metastasis  is necessary for the upregulation of proliferation to establish macrometastases.22 Studies have shown that the pre-metastatic niche can also influence whether tumor cells will efficiently seed and proliferate there,34 but what initiates the switch to MET at the metastatic site has not been determined. Our data demonstrate that CEACAM5 has a role in this process and suggest that perturbation of MET programs may be a therapeutic strategy to restrain tumor outgrowth at the metastatic site.We observed that EMT markers were dynamically regulated as tumors were passaged between the lung and MFP in vivo. EMT signatures were high in MFP tumors and low in lung metastases. Metastatic tumor cells are commonly thought to rely on EMT to escape primary tumors in the initial stages of metastasis by downregulating intercellular adhesion and proliferation and acquiring invasive and stem cell-like properties.Although MET programs may retard the growth of metastatic lesions, inhibition of MET in metastatic tumor cells may in turn lead to the upregulation of EMT-associated tumor cell migration, consequently resulting in secondary metastases. Thus, one limitation of this study is that clinical application of these findings may be a palliative, rather than curative, therapeutic strategy. Additionally, the mechanisms by which MET promotes lung metastasis have not been fully elucidated. Upregulation of MET may influence multiple steps in the metastatic cascade including increased cell survival in circulation, increased adhesion to blood vessels in secondary sites, or increased proliferation during metastatic colonization. Finally, it is important to note that these studies were conducted in immune-compromised mice, and thus, the contribution of the immune system to metastasis cannot be fully explored in this system. However, elevated expression of CEACAM5 was confirmed in metastatic lesions obtained directly from cancer patients, thus confirming the clinical relevance of our findings.35 High CEACAM5 expression in breast tumors correlates with reduced patient survival.38 Additionally, CEACAM5 is secreted into the blood of breast cancer patients, and elevated serum levels indicate progression to metastasis.39 CEACAM5 is also a well-recognized tumor and serum biomarker for the progression of colorectal and pancreatic cancers.41 We demonstrated that CEACAM5 expression is elevated in metastatic breast cancer cells compared with the corresponding primary breast tumors in PDX models of TNBC and in tissue sections obtained directly from breast cancer patients. In preclinical models, anti-CEACAM5 neutralizing antibodies have to been shown to affect cell adhesion, migration, and invasion in vitro, and pre-incubation of tumor cells with these antibodies resulted in decreased metastasis of tumor cells following intrasplenic injection.42 Collectively, these results suggest that CEACAM5 may serve as a tumor and serum biomarker for metastatic progression and may also be a viable therapeutic target.CEACAM5 is not expressed on most normal adult tissues including the breast; its expression is restricted to the apical surface of the gastrointestinal tract.43 However, the ability of these agents to target tumors and suppress their growth is limited, and relatively few drugs that directly target CEACAM5 have been developed. Accordingly, the most effective treatments are based on stimulation of immune responses against CEACAM5.43 Our results suggest that the development of novel therapeutic agents directly targeting CEACAM5 could be effective in inhibiting tumor outgrowth in the metastatic setting. However, one concern is that this could also promote migratory properties of tumor cells through expression of EMT markers thereby favoring mesenchymal (migratory)-like properties and secondary metastatic seeding events. Our finding that CEACAM5 can inhibit p38 activity and promote the growth of metastatic lesions agrees with previous studies showing that pharmacologic inhibition of p38 promotes the outgrowth of micrometastatic tumor cells, likely through induction of MET.44 Thus, metastatic stage should be carefully considered before clinical application of pharmacologic agents targeting these pathways.Attempts to target CEACAM5 with anticancer therapeutic approaches have included the development and clinical implementation of vaccines, immunotherapies, and antibodies.Despite remarkable recent advances in pharmacologic development, outcomes for patients with metastatic breast cancer remain poor. Therefore, there is a clear need to elucidate the molecular mechanisms governing the progression of established metastatic tumors. Identifying molecules that promote macrometastasis, such as CEACAM5, is expected to lead to the development of new therapeutic targets and the application of existing therapies for patients with advanced metastatic disease.28 this study was carried out in accordance with the sanctions in the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health (NIH) Institutional Animal Care and use Committee (IACUC). The IACUC protocol was approved by the Committee on the Ethics of Animal Experiments of Washington University and the IACUC at MD Anderson Cancer Center. Mice were humanely euthanized as dictated by the Association for Assessment and Accreditation of Laboratory Animal Care International and IACUC euthanasia endpoints when they became moribund or when they reached defined study end points. Animals were euthanized as dictated by the Association for Assessment and Accreditation of Laboratory Animal Care International and IACUC euthanasia end points. Informed consent was obtained from all living patients from whom data was included in this study.Similar to our previous studies,45 Briefly, GFP-labeled human stromal fibroblasts were irradiated with 400 Rads and mixed with an equal number of non-irradiated fibroblasts (2.5 × 105 irradiated cells and 2.5 × 105 non-irradiated cells). Fibroblasts were then mixed with 1 × 106 BC3_A2 tumor cells and 1/3 volume Matrigel. This mixture was injected into the fourth mammary fat pads of 4 to 5-week-old female nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice . PIM001-P and PIM001-M tumor cells were not mixed with fibroblasts prior to implantation. Tumors were resected in a survival surgery when they reached approximately 1 cm in diameter. Animals were euthanized when they became moribund or a maximum of 30 weeks post-tumor-implantation.PDX models were established according to published methods.oC on a rotator. Tumor cells were washed in DMEM/F12 with 5% bovine calf serum, and red blood cells were lysed for 5 min at room temperature . Cells were passed through a 70 μm filter and counted using a fluorescence-coupled microscope (only the mCherry-positive cells were counted). A maximum of 1 × 106 mCherry-positive tumor cells were then implanted into the fourth mammary fat pads of recipient mice, along with irradiated and non-irradiated human stromal fibroblasts and 1/3 volume Matrigel.Metastases were isolated at animal necropsy using bioluminescence-guided macro-dissection. Tissues were digested to single cells and organoids in collagenase and hyaluronidase for 4 h at 37 46 First, barcode quantification cycle (Cq) values were normalized to total DNA with the Cq value for DNA reference probe. Next, tumor sample barcode values were converted into a fold change relative to the respective pooled reference sample. ΔΔCt of the lung vs. the mammary was also calculated for each matched pair, and samples with three or more matched pairs were averaged to rank hits. Samples with fewer than three data points were not considered for analysis. All genes with an average depletion of at least two-fold in the mammary relative to the reference pool were considered as putative metastatic drivers (hits).Barcode quantification was carried out using the DDCt method.28 tumors were snap frozen to preserve RNA integrity and avoid experimental manipulations that could lead to alterations in gene expression. Tumors were stored at −80 °C, thawed on ice in Trizol, and homogenized with a pedestal homogenizer. Total RNA was extracted with chloroform followed by purification using the RNEASY kit . Total RNA was treated with DNase  followed by column purification . mRNA was isolated by poly-A selection. RNA Integrity Number (RIN) was assessed on a Bioanalyzer . RNAseq libraries were prepared and sequencing was performed by the Genome Technology Access Center . RNAseq reads were first mapped to the mouse reference genome (UCSC mm10) using Tophat2 (v2.0.14) and Bowtie2 (v2.2.2), allowing a maximum of two mismatches per 75 bp read end. The unmapped reads were then aligned to the human genome (UCSC hg19) using the same tool and parameters. Gene-level read counts were quantified using HTseq (http://www.huber.embl.de/users/anders/HTSeq/doc/overview.html). Normalization and differential expression analyses for gene/isoform expression were performed using DESeq2.47 The log2 fold-change 1.5 and adjusted p-value cutoff <0.05 were used to identify differentially expressed genes. log10 p-values from the differential analysis were used to build the rank file for pre-ranked GSEA against the Cancer Hallmark pathways.49 Numbers of mice included in each analysis were as follows: P0 MFP tumors, n = 3; P0 lung metastases n = 4; P2 MFP tumors, n = 3; P2 lung metastases, n = 3; BC3_A2 cells expressing GFP and injected into tail veins, n = 3; BC3_A2 cells expressing CEACAM5 and injected into tail veins, n = 3.Tumors were harvested in a survival surgery when they reached 1 cm and were immediately snap frozen in liquid nitrogen. Metastatic lesions were harvested by bioluminescence-guided macro-dissection and immediately snap frozen in liquid nitrogen. Mice with the largest metastatic lesions were selected for RNAseq. Similar to our previous studies,28 Wilcoxon rank sum tests were used to evaluate the mean differences in photon flux in each organ due to relatively small sample size in most groups. Hierarchical clustering of RNAseq data was performed using Euclidean distance and complete linkage. PCA plots and heat maps, and statistical analyses were performed using R (http://www.r-project.org/).Photon fluxes were normalized to the time of study end point as described previously.28 the univariate Cox proportional hazard model was used to assess the correlation of gene expression with patient overall survival, and the log-rank p-value was reported. The dataset from The Cancer Genome Atlas (TCGA)50 was used for determining overall survival of breast cancer patients. Log-rank tests were used to estimate the p-values between high and low expression groups using the top and bottom 10% as the cutoff for grouping.Similar to our previous studies,RNAseq data are publically available at Sequence Read Archive (SRA), accession numbers SRP133840.Supplementary FiguresSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary methods


Opithancatus Madagascariensis—have been synthesized and tested to compare their antibacterial and hemolytic activity against natural peptides. In general, results show that increasing hydrophobicity by substituting positions 5 and 9 of the sequences with alanine, valine, and leucine, enhances antibacterial activity. However, this also increases hemolytic activity. The analog with an increased net positive charge from +1 to +3 produces moderate bacterial growth inhibition but also has high hemolytic activity. On the other hand, the analog with a negative net charge (−1) has low antibacterial properties but also no cytotoxicity under the tested conditions, a similar result was found for five of the seven studied analogs.Seven analogs of the natural, α-helix peptides IsCT1 and IsCT2—found in the venom of scorpion  In recent years, pathogenic organisms have developed resistance to highly utilized drugs, not only for humans, but also for animals . This inOpithancatus madagascariensis , is given in deg·cmeviously . Values Staphylococcus aureus (ATCC# 29213) and Bacillus cereus (ATCC# 11778); and three Gram-negative bacteria: Salmonella typhimurium (ATCC# 14028), Enterobacter aerogenes (ATCC# 13048), and Escherichia coli (ATCC# 25922) were tested.Two Gram-positive bacteria: Activity was determined by the standard liquid dilution method described previously . All resHemolytic activity was carried out as described previously . BrieflyOur study with analogs of IsCT peptides found in scorpion venoms has shown that natural or wild type peptides have the highest bacterial growth inhibition compared to their synthetic analogs here studied. Of these, leucine substituted analog IsCT1L1, shows higher antibacterial activity but also toxicity similar to the wild type peptides. An increase in the net positive charge in IsCT1K7 or the net negative charge in IsCT1E7 does not improve the inhibition much. However, the fact that analog IsCT1E7 along with others have no apparent cytotoxicity for the concentrations tested is a promising starting point to design future potential antibacterial peptides.


These data suggest that cerebral hyperperfusion after revascularization inhibits development of cerebral ischemic lesions due to artery-to-artery emboli during carotid exposure in CEA, supporting the “impaired clearance of emboli” concept. Blood pressure elevation following carotid declamping would be effective when embolism not accompanied by cerebral hyperperfusion occurs during CEA.The purpose of the present study was to determine whether cerebral hyperperfusion after revascularization inhibits development of cerebral ischemic lesions due to artery-to-artery emboli during exposure of the carotid arteries in carotid endarterectomy (CEA). In patients undergoing CEA for internal carotid artery stenosis (≥70%), cerebral blood flow (CBF) was measured using single-photon emission computed tomography (SPECT) before and immediately after CEA. Microembolic signals (MES) were identified using transcranial Doppler during carotid exposure. Diffusion-weighted magnetic resonance imaging (DWI) was performed within 24 h after surgery. Of 32 patients with a combination of reduced cerebrovascular reactivity to acetazolamide on preoperative brain perfusion SPECT and MES during carotid exposure, 14 (44%) showed cerebral hyperperfusion , and 16 (50%) developed DWI-characterized postoperative cerebral ischemic lesions. Postoperative cerebral hyperperfusion was significantly associated with the absence of DWI-characterized postoperative cerebral ischemic lesions (95% confidence interval, 0.001–0.179;  It has been previously reported that hemodynamic and embolic mechanisms are closely linked, and they may act together to cause cerebral ischemia ,2. The aMore than 70% of the intraoperative procedure-related strokes that occur during carotid endarterectomy (CEA) are due to surgical site embolisms . When inCerebral hyperperfusion is defined as a major increase in ipsilateral cerebral blood flow (CBF) after surgical repair of carotid stenosis that is well above the metabolic demands of the brain tissue; it is another adverse event following CEA . It occaThus, both cerebral ischemic lesions due to artery-to-artery embolism and cerebral hyperperfusion may develop simultaneously during CEA in patients with preoperative cerebral hemodynamic impairment. When broadening the interpretation of the “impaired clearance of emboli” concept, blood flow greater than the normal level can inhibit development of ischemic lesions due to emboli in the brain, and research regarding the influence of cerebral hyperperfusion on the development of cerebral ischemic lesions due to artery-to-artery embolism seems interesting from the standpoint of this concept.The aim of the present study was to determine whether broadening the interpretation of the “impaired clearance of emboli” concept is correct, namely cerebral hyperperfusion after revascularization inhibits development of cerebral ischemic lesions due to intraoperative artery-to-artery emboli. In order to do this, the relationship between development of DWI-characterized postoperative cerebral ischemic lesions and cerebral hyperperfusion was investigated in patients with a combination of preoperatively reduced CVR to acetazolamide on brain perfusion single-photon emission computed tomography (SPECT) and MES on TCD during carotid artery exposure in CEA.The mean age of the 32 patients  was 71.7 ± 4.5 (mean ± standard deviation (SD)) years (range of 63–85 years). Twenty-six patients had preoperative hypertension, and 23 patients received antihypertensive drugs . Thirteen patients had preoperative diabetes mellitus, and all these patients received antidiabetic drugs. Fifteen patients had preoperative dyslipidemia, and 12 patients received a statin (strong statin for six). Seven patients had ischemic heart or valvular disease that did not satisfy the criteria for high-risk factors for CEA in the Stenting and Angioplasty with Protection in Patients at High Risk for Endarterectomy (SAPPHIRE) study  . None ofIn the 32 patients studied, postoperative CBF was 52.9 ± 17.5 mL/100 g/min (range of 35.8–90.5 mL/100 g/min); 14 patients (44%) met CBF criteria for cerebral hyperperfusion. Sixteen patients (50%) developed new postoperative ischemic lesions on DWI 24 h after surgery in the cortex and/or white matter in the cerebral hemisphere ipsilateral to CEA. All new ischemic lesions were spotty, and their diameters were 1 cm or less. Five (16%) of 32 patients studied developed new neurological deficits after recovery from general anesthesia. All deficits included slight hemiparesis contralateral to the CEA. These deficits resolved completely within 12 h in these five patients, and they underwent additional DWI between 6 and 8 h after surgery and had new postoperative ischemic lesions on both the first (6 to 8 h after surgery) and second (24 h after surgery) postoperative DWI examinations.p < 0.2 in univariate analyses were adopted as confounders in the logistic regression model for multivariate analysis: degree of ICA stenosis and postoperative CBF or postoperative cerebral hyperperfusion . This analysis showed that greater postoperative CBF  or postoperative cerebral hyperperfusion  was significantly associated with the absence of DWI-characterized postoperative cerebral ischemic lesions.Results of univariate analyses of factors related to the development of DWI-characterized postoperative cerebral ischemic lesions are shown in The present study demonstrated that cerebral hyperperfusion after revascularization inhibits the development of cerebral ischemic lesions due to artery-to-artery emboli during carotid exposure in CEA for patients with preoperatively impaired cerebral hemodynamics, supporting the “impaired clearance of emboli” concept when broadening its interpretation.Hemodynamic cerebral ischemia due to hemispheric cerebral hypoperfusion during ICA clamping, as well as emboli from the surgical site, plays a significant role in the development of new ischemic lesions after CEA ,23. IntrIn the present study, 44% of patients with preoperatively reduced CVR to acetazolamide showed cerebral hyperperfusion immediately after surgery. This incidence was comparable to a previous study ,20,21. AIn the present study, preoperative CBF, preoperative CVR to acetazolamide, mean systolic blood pressure during carotid exposure, and interval from the first or last MES to ICA declamping did not differ between patients with and without DWI-characterized postoperative cerebral ischemic lesions. Thus, perfusion in the cerebral hemisphere ipsilateral to surgery during carotid exposure and the duration of cerebral ischemia caused by emboli until ICA declamping might be equivalent between these two subgroups of patients. The duration of ICA clamping also did not differ between them. Nevertheless, greater postoperative CBF or postoperative cerebral hyperperfusion  was associated with the absence of DWI-characterized postoperative cerebral ischemic lesions, and only 7% of patients with a combination of MES during carotid exposure and postoperative cerebral hyperperfusion developed these lesions. These findings suggested that blood flow that increased far beyond the normal level might clear cerebral emboli generated from the surgical site, inhibiting the development of ischemic lesions. These support the “impaired clearance of emboli” concept if the interpretation of this concept is broadened.We have another hypothesis regarding the correlation between postoperative cerebral hyperperfusion and the development of ischemic lesions by emboli in patients with reduced CVR to acetazolamide. Reduced CVR to acetazolamide implies a chronic reduction in cerebral perfusion pressure and poor collateral blood flow ,26,27. WThe present study suggests that CBF greater than the normal level after declamping of the ICA can inhibit development of cerebral ischemic lesions due to emboli from the surgical site during exposure of the carotid arteries. On the other hand, postoperative cerebral hyperperfusion, which is defined as a postoperative CBF increase of ≥100% when compared to preoperative values, occasionally evolves into cerebral hyperperfusion syndrome, leading to intracerebral hemorrhage ,16,17,18Although TCD detects emboli generated from the surgical site of the carotid arteries as MES, it cannot provide information about the size and characteristics of each embolus, which may affect the development of postoperative cerebral ischemic lesions. The present results did not take into account these two factors.The present study was designed as a prospective, observational study. This study was approved by the Regional Ethical Board in Iwate Medical University (H22-3) and was in compliance with the Helsinki Declaration, and written, informed consent was obtained from all patients or their next of kin prior to participation.4.2.CBF Measurements” section); undergoing CEA; and having MES during exposure of the carotid arteries under reliable TCD monitoring according to the methods described below (see “4.3.TCD Monitoring” section). Patients who showed electroencephalography (EEG)-defined cerebral hemispheric ischemia during ICA clamping according to the methods described below  were excluded from the present study.Of symptomatic or asymptomatic patients with ipsilateral ICA stenosis ≥70%, as per the North American Symptomatic Carotid Endarterectomy Trial , on angi123I]N-isopropyl-p-iodoamphetamine (IMP) and SPECT with a ring-type scanner  within 14 days before and immediately after CEA. CBF measurement with acetazolamide challenge was also performed before CEA. The [123I]IMP SPECT study with and without acetazolamide challenge was performed as described previously [123I]IMP (5 mL volume) at a constant rate of 5 mL/min and a 1 min infusion of physiologic saline at the same rate, data acquisition was performed at a midscan time of 30 min after the [123I]IMP administration for a scan duration of 20 min. At 10 min after the beginning of the [123I]IMP infusion, arterial blood (1 mL) was taken from the brachial artery. The whole-blood radioactivity of each blood sample obtained was measured using a well counter that was cross-calibrated to the SPECT scanner. All reconstructed SPECT images were corrected for the radioactive decay of 123I back to the [123I]IMP injection start time, normalized by the data collection time and cross-calibrated to the well counter system. The CBF images were calculated according to the [123I]IMP-autoradiography method  × 100. For CBF in the resting state and CVR to acetazolamide, data described previously ((mean ± SD), 35.9 ± 4.4 mL/100 g/min and 36.8% ± 9.2%, respectively) were used as control values, and decreased CVR to acetazolamide was defined as less than mean − 2 SD of the control value (18.4%) . In eachTCD was performed using a PIONEER TC2020 system  for insonation of the MCA ipsilateral to the carotid artery undergoing CEA. TCD data were stored on a hard disk using a coding system and later analyzed manually by a clinical neurophysiologist who was blinded to patient information. MES were identified during exposure of the carotid arteries (from skin incision to ICA clamping) according to the recommended guidelines .DWI was performed using a 1.5 T whole-body imaging system  within three days before and 24 h after surgery.A neuroradiologist who was blinded to patient clinical information analyzed the images and determined whether new ischemic lesions had developed postoperatively.2/propofol. A bolus of heparin  was given prior to ICA clamping. Blood pressure was measured in the same fashion as preoperatively every 5 min throughout surgery. The EEG was recorded, and a clinical neurophysiologist monitored the recordings continuously during the surgical procedure. The presence of unilateral or bilateral decreases of alpha and beta activity during ICA clamping, with or without simultaneous increases of theta or delta activity, was defined as development of cerebral hemispheric ischemia by the clinical neurophysiologist [Blood pressure was measured at the upper arm using an automatic sphygmomanometer with the oscillometric method, and mean systolic blood pressure in the morning for the three days before surgery was defined as the preoperative value for each patient. Patients received medications including antihypertensive and antidiabetic drugs and statins until the evening of the day before CEA was performed. All patients received a single antiplatelet drug until the morning of the day on which CEA was performed. For all patients, surgery was conducted under general anesthesia, which was induced with etomidate/fentanyl and maintained with Oiologist . In this2 test. Hypertension was defined as preoperative systolic blood pressure ≥140 mmHg, preoperative diastolic blood pressure ≥90 mmHg or preoperatively receiving antihypertensive drugs; diabetes mellitus was defined as preoperative hemoglobin A1c ≥6.5% or preoperatively receiving antidiabetic drugs; dyslipidemia was defined as preoperative plasma low density lipoprotein (LDL) cholesterol ≥140 mg/dL, preoperative plasma high density lipoprotein (HDL) cholesterol <40 mg/dL, preoperative plasma triglyceride ≥150 mg/dL, or preoperatively receiving statins. Multivariate statistical analysis of factors related to DWI-characterized postoperative cerebral ischemic lesions was also performed using a logistic regression model. Variables with p < 0.2 on univariate analyses were selected for analysis in the final model. Differences were deemed significant for values of p < 0.05.Data are expressed as means ± SD. The relationship between each variable and DWI-characterized postoperative cerebral ischemic lesions was evaluated by univariate analysis using the Mann-Whitney U test or the χThe present study demonstrated that cerebral hyperperfusion after revascularization inhibits the development of cerebral ischemic lesions due to artery-to-artery emboli during carotid exposure in CEA for patients with preoperatively impaired cerebral hemodynamics, supporting the “impaired clearance of emboli” concept when its interpretation is broadened.


LPA) have been implicated in cardiovascular disease (CVD), presumably through their association with lipoprotein (a) [Lp(a)] levels. While Lp(a) is recognized as a lipoprotein with atherogenic and thrombogenic characteristics, it is unclear whether or not the two Lp(a)-associated genetic variants are also associated with markers of thrombosis . In the present study, we genotyped the two genetic variants in 2919 subjects of the Old Order Amish (OOA) and recruited 146 subjects according to the carrier and noncarrier status for rs3798220 and rs10455872, and also matched for gender and age. We measured plasma Lp(a) and plasminogen levels in these subjects, and found that the concentrations of plasma Lp(a) were 2.62- and 1.73-fold higher in minor allele carriers of rs3798220 and rs10455872, respectively, compared with noncarriers . By contrast, there was no difference in plasminogen concentrations between carriers and noncarriers of rs3798220 and rs10455872. Furthermore, we observed no association between carrier status of rs3798220 or rs10455872 with clot lysis time. Finally, plasminogen mRNA expression in liver samples derived from 76 Caucasian subjects was not significantly different between carriers and noncarriers of these two genetic variants. Our results provide further insight into the mechanism of action behind two genetic variants previously implicated in CVD risk and show that these polymorphisms are not major modulating factors for plasma plasminogen levels and fibrinolysis.Two genetic variants (rs3798220 and rs10455872) in the apolipoprotein (a) gene ( LPA gene located on chromosome 6q26–27, which encodes for apo(a) (LPA gene are associated with Lp(a) levels. Among them, a nonsynonymous SNP that results in an isoleucine to methionine substitution at position 1891 (rs3798220) and an intronic variant (rs10455872) have been confirmed to be strongly associated with increased levels of plasma Lp(a) and the risk of cardiovascular disease  [Lp(a)] is a significant and independent risk factor in the development of cardiovascular diseases . Levels PLG) and LPA gene are located on chromosome 6q26 within ∼40 kb of each other (LPA and PLG gene region on chr6q26 to be significantly associated with plasminogen levels (LPA and PLG mRNA expressions are likely to be coregulated in the liver. The genetic variants within the LPA and PLG gene region on chr6q26 may regulate Lp(a) and plasminogen levels to contribute to thrombogenicity. This promotes persistence of the clot and the thrombotic process that may contribute to thrombo-atherogenic diseases.Lp(a) is recognized as a lipoprotein with atherogenic and thrombogenic characteristics . StructuLPA SNPs rs3798220 and rs10455872 are consistently associated with Lp(a) levels and result in increased risk for cardiovascular diseases  for rs10455872. Likewise, all of the rs10455872 individuals had the same genotype (TT) for rs3798220. The study was approved by the institutional review board of the University of Maryland, Baltimore, and all participants provided written informed consent. The methods were carried out in accordance with the approved guidelines.The subjects were from the Old Order Amish community (OOA) of Lancaster, PA and were drawn from participants of our previous Lp(a) study . Since cg for 15 min at 4°. The plasma supernatant for ECLA measurement was recentrifuged at 2000 × g for 15 min at 4° to remove any residual platelets. Multiple aliquots of plasma were stored at −80° until assays were performed.Venous blood was drawn after an overnight fast. Four milliliters of blood was collected from each individual in vacutainer tubes containing EDTA [for Lp(a) and plasminogen measurement] or 3.2% sodium citrate (for ECLA), respectively. Plasma samples were subsequently separated by centrifugation at 2000 × Quantitative determination of Lp(a) and plasminogen antigen levels in human plasma samples was performed by an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instruction . Fifty microliters of standard or plasma using EDTA as an anticoagulant, diluted with dilution buffer (1:8000 dilution for Lp(a); 1:20,000 dilution for plasminogen), were added into each well of 96-well plates precoated with a polyclonal antibody specific for human Lp(a)/plasminogen. Wells were then incubated with the biotinylated polyclonal antibody specific for human Lp(a)/plasminogen, and was recognized by a streptavidin-peroxidase conjugate, and then a peroxidase enzyme substrate. After the reaction was stopped, the absorbance at 450 nm was read on a VICTOR X3 Multilabel Plate Reader . The standard curves were generated by polynomial regression analysis. Lp(a) and plasminogen levels were expressed in micrograms per milliliter.2 to each well (100 µl of ddH2O added to the blank wells). The plate was read on a VICTOR X3 Multilabel Plate Reader (PerkinElmer) at 405 nm at 3-min intervals for 10 hr and the temperature was maintained at 37°.ECLA was modified from the assay described by Manco-Johnson (N = 19: 19 for genotypes TT and CT) and rs10455872 (N = 20: 20: 3 for AA: AG: GG), and also matched for gender, age, and BMI ]. Steady-state mRNA levels were determined by two-step quantitative real-time PCR (qRT-PCR) using the LightCycler 480 (Roche) and TaqMan probe/primer sets . Relative expression of mRNAs was determined after normalization with the reference gene ACTB levels using LightCycler 480 software 1.5.Total liver RNA was extracted by TRIzol  according to the manufacturer’s instructions. The resulting RNA was subjected to DNAse digestion using RNase-free DNase Set  and purification using RNeasy MinElute Cleanup Kit (Qiagen). The RNA quantity and quality were determined using a ND1000 nanodrop spectrophotometer, and 1 µg of total RNA was reverse-transcribed using a Transcriptor First Strand cDNA Synthesis Kit . Real-time PCR was performed using TaqMan gene expression assay primers and probes .PLG gene, we tested whether these variants had any influence on PLG mRNA expression in the liver. Total RNA was extracted from liver samples derived from Caucasian patients undergoing bariatric weight loss procedures for extreme obesity or related comorbid medical conditions  and rs10455872  and matched for age and gender. We measured the PLG mRNA expression by qRT-PCR and found that there was no statistically significant difference in expression between carriers of rs3798220 and noncarriers   (Given the close proximately of rs3798220 and rs10455872 with the  = 0.12) . Similar = 0.15) .r2 from 0 to 0.227, We performed LD analysis for rs3798220, rs10455872, and the top SNPs associated with plasminogen on chr.6q26 in previous GWAS (LPA SNPs rs3798220 and rs10455872 are consistently associated with Lp(a) levels and result in increased risk for cardiovascular diseases (LPA variants confer CVD susceptibility. To our knowledge this study represents the first investigation to simultaneously assess the impact of these variants on both Lp(a) levels and markers of thrombosis . Consistent with previous results, we observed that both of these SNPs significantly impact Lp(a) levels; however, we observed no evidence to suggest that these variants influence plasminogen levels. In addition, we extend these finding to show that neither rs3798220 nor rs10455872 impact fibrinolytic activity or PLG mRNA expression in the liver.Lp(a) has a causal role in the development of multiple cardiovascular disorders . LPA SNPThe OOA community is a genetically well-defined Caucasian founder population. More than 95% of the current Lancaster Amish population can trace their ancestry to one of seven founder couples . The AmiLPA gene were identified as significant contributors to plasminogen levels by GWAS  influences fibrinolysis and found no difference in clot lysis time and maximum absorbance between carriers and noncarriers of both rs3798220 and rs10455872, indicating that clot formation and clot lysis are not affected by these two SNPs. Of note, however, Undas et al. reported decreased clot permeability and longer clot lysis time in both healthy subjects and patients with myocardial infarction that have increased Lp(a) levels [a cutoff value of Lp(a) 300 µg/ml] as a result of small apo(a) size isoforms  plasma levels had no effect on clot lysis time in diabetic subjects and normal controls (Since apo(a) can bind to fibrin but has no proteolytic activity, it has been hypothesized that apo(a) competes with plasminogen in circulation and may attenuate fibrinolytic function  concentration, but not significantly associated with variation in plasma plasminogen concentration. The increased levels of plasma Lp(a) with these genetic variants do not affect fibrinolysis in healthy subjects. Moreover, these two Lp(a)-associated genetic variants were not associated with PLG mRNA expression in the liver. These two Lp(a)-associated variants of rs3798220 and rs10455872 in the LPA gene are not the major genetic determinants of fibrinolysis, PLG mRNA expression, and plasminogen levels.In conclusion, our data indicate that two genetic variants in the


Mycoplasma gallisepticum (MG) in free-living house finches (Haemorhous mexicanus), which causes mycoplasmal conjunctivitis in this passerine bird species, resulted in a rapid coevolutionary arms-race between MG and its novel avian host. Despite extensive research on the ecological and evolutionary dynamics of this host–pathogen system over the past two decades, the immunological responses of house finches to MG infection remain poorly understood. We developed seven new probe-based one-step quantitative reverse transcription polymerase chain reaction assays to investigate mRNA expression of house finch cytokine genes . These assays were then used to describe cytokine transcription profiles in a panel of 15 house finch tissues collected at three distinct time points during MG infection. Based on initial screening that indicated strong pro-inflammatory cytokine expression during MG infection at the periorbital sites in particular, we selected two key house finch tissues for further characterization: the nictitating membrane, i.e., the internal eyelid in direct contact with MG, and the Harderian gland, the secondary lymphoid tissue responsible for regulation of periorbital immunity. We characterized cytokine responses in these two tissues for 60 house finches experimentally inoculated either with media alone (sham) or one of two MG isolates: the earliest known pathogen isolate from house finches (VA1994) or an evolutionarily more derived isolate collected in 2006 (NC2006), which is known to be more virulent. We show that the more derived and virulent isolate NC2006, relative to VA1994, triggers stronger local inflammatory cytokine signaling, with peak cytokine expression generally occurring 3–6 days following MG inoculation. We also found that the extent of pro-inflammatory interleukin 1 beta signaling was correlated with conjunctival MG loads and the extent of clinical signs of conjunctivitis, the main pathological effect of MG in house finches. These results suggest that the pathogenicity caused by MG infection in house finches is largely mediated by host pro-inflammatory immune responses, with important implications for the dynamics of host–pathogen coevolution.The recent emergence of the poultry bacterial pathogen  Emerging infectious diseases apply novel and powerful selection pressures on wildlife host immune responses. There is a growing list of examples of wildlife hosts that have rapidly evolved resistance or tolerance to recently emerged infectious diseases . For oun = 12), (2) NC2006-inoculated experimental group (n = 24), and (3) VA1994-inoculated experimental group (n = 24). To capture the temporal dynamics of cytokine expression, birds were further divided within each group into three equally sized time-point batches . All groups were designed to contain approximately equal proportions of males and females, and hatch-year birds and individuals of unknown age , but otherwise assignments to treatments were random. We tested for pretreatment differences in size or mass and no significant differences in these traits were present .To quantify house finch cytokine responses to an original and more evolved isolate of MG, we used three treatment groups Table : (1) shavia micropipette , while sham control individuals were given the same volume of FMS alone. Afterward, birds were held in individual paper lunchbags for approximately 5 min to allow full absorption of inoculum, and then released back into their home cages and left undisturbed. On day 3 post inoculation (DPI 3), birds assigned to the first time point batch were eye scored (see below), conjunctival swabs were collected (see below), and the birds were euthanized via rapid decapitation to collect fresh tissue samples and prevent confounding effects of chemical inhalants on gene expression. The same procedure was performed with birds assigned to the second time point batch on DPI 6 and to the third time point batch on DPI 13. All manipulation with experimental animals (including euthanasia) was performed in the quickest and most humane way possible to minimize pain and distress. Latex gloves were changed between manipulations with each individual to prevent any inadvertent MG transmission. Because MG requires direct contact between individuals or contact with a highly contaminated surface . Thereafter, all birds were treated with the respective inoculum: experimental birds were inoculated with 35 µL of MG suspended in FMS (either isolate NC2006 or VA1994) administered directly into the palpebral conjunctiva of each eye Eye lesions characteristic of mycoplasmal conjunctivitis were visually scored on a 0–3 scale as previously described . Brieflymgc2 gene of MG using primers and a probe previously described . Swabs were swirled in 300 µL of sterile TPB and then wrung out into the sample collection tube. Samples from both eyes were pooled within sampling date for a given individual and frozen at −20°C until further processing. Genomic DNA was extracted with Qiagen DNeasy 96 Blood and Tissue kits . Extracted DNA was used to measure overall numbers of MG in the conjunctivae using a qPCR assay targeting the escribed  and qPCRescribed .later : conjunctiva , nictitating membrane , Harderian gland (HG), upper respiratory tract and choana, brain, bone marrow, liver, spleen, trachea, lungs, kidney, pancreas, duodenum, and ileum . Dissection and sample collection were performed simultaneously by three persons, and all tissues were collected within 15 min after euthanasia. The collected tissue samples were stored at −80°C until total RNA extraction. RNA extraction was performed using High Pure RNA Tissue Kit v. 09  according to the manufacturer’s instructions. The total RNA concentration in each extracted sample was then measured using NanoDrop 2000 (Thermo Scientific): range 2.7–1,335.8 ng/µL, average 197.6 ng/µL. We checked for the quality of the RNA extracted from different tissues in different batches using TapeStation (Agilent) as a service of the Genomics Research Laboratory, Biocomplexity Institute, Virginia Tech. In most tissue samples, the RNA quality was good [RNA integrity number (RIN) generally ranged between 8.4 and 9.5]. RIN was <8.0 in some lung, liver, and tracheal samples, and these tissues were thus excluded from further analysis.Immediately after decapitation, blood from the disconnected carotids was collected from each bird into a microcentrifuge tube containing RLT lysis buffer . Then, the following tissues were collected separately into RNAIL1B, IL6, IL10, IL18, TGFB2, CXCLi2 (IL8L), and TNFSF15. Pro-inflammatory IL1B and anti-inflammatory IL10 were selected as key target genes based on the results of previous studies (ACTB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and 28S rRNA.In this study, we focused on a set of 7 selected pro-inflammatory and anti-inflammatory cytokine genes:  studies , 38. To Serinus canaria) sequences  as references. The filtered reads were then quality trimmed and mapped to reference using Geneious v. 9.1.8-implemented tools. While this approach yielded good data on sequence variation for the reference genes , the sequence data were scarce for several cytokine genes . Therefore, to improve our information on the sequence variation in our genes of interest, we Sanger-sequenced the partial coding regions in mRNA of the cytokines in seven additional individuals and of ACTB and GAPDH in two additional individuals .Because allelic variation in primer/probe-annealing regions would importantly bias the accuracy of our RT-qPCR data, we first evaluated the sequence variation in the genes of interest in a sample of six individuals (three control and three MG infected) with spleen and liver RNA-seq data available (sequences obtained through Illumina HiSeq 2500) to check for common SNPs. The reads were filtered for hits to the genes of interest using blast with Atlantic canary (28S rRNA qPCR); cycling conditions (1) 50°C 10 min, (2) 95°C 3 min, (3)  × 40. All assays were performed with a template-free negative control and gBlock positive controls  in a freshly prepared dilution series. Calibrator samples were included in all assays to check for interplate RT-qPCR variation. Based on the results of GeNorm and RefFinder analyses . PCR with these primers was tested using cDNA (electrophoresis and Sanger-sequencing of amplicons was performed for assay specificity verification). Next, qPCR with synthesized standard DNA sequences  was done using the iTaq™ Universal Probes One-Step Kit  with cycling conditions following manufacturer’s instructions. Once the assay efficiency was estimated, all further RT-qPCR were performed with the same kit  and set up: final primer concentration 0.6 µM, final probe concentration 0.125 µM and mixed RNA template diluted in carrier tRNA  enriched molecular water 1:5  is calculated as R = (ET)ΔCqT/(ER)ΔCqR, where ET is the mean amplification efficiency of the particular assay for a target gene (cytokine), ER is the mean amplification efficiency of the particular assay for a reference gene (28S rRNA), the ΔCqT is the difference in Cq values between control mean and the treatment mean in the target gene (cytokine), and ΔCqR is the difference in Cq values between control mean and the treatment mean in the reference gene (28S rRNA). Then, for the purpose of statistical testing of the differences in cytokine expression between controls and treatments in the selected tissues, we calculated the relative RNA quantities (Q) as Q = EΔCq, where E is the mean amplification efficiency of the particular assay and ΔCq is the difference between an arbitrary Cq value chosen for the gene  and the sample Cq  was calculated as number of target gene copies per nanogram of the total extracted RNA. Target gene copy number was estimated based on the average standard curve equation obtained from a dilution series of a calibrated synthetic standard (gBlock sequences of known DNA copy number in the solution).Before data analysis, any technical replicates with Cq values highly deviating from the other two measurements in the triplicate  were excluded from the calculation of Cq means. To select candidate tissues of interest, we first performed an initial screening of the qPCR data on y Pfaffl . Here, tample Cq , 43. TheR (28S rRNA). Similarly, absolute quantification ratio was calculated as A = (CNT/CNR)Treatment/(CNT/CNR)Control, where CNT is target gene copy number, CNR is reference gene (28S rRNA) copy number; in controls we used mean copy number values for the particular sampling time point. Log base 2 values of R and A were used. A general overview of the gene expression data analysis procedure is shown in Figure Finally, to compare the cytokine expression profiles of the response to VA1994 and NC2006 MG isolates in the data set of all experimental individuals, we again used the relative quantification based on relative expression ratio R , where, P = 0.05. Because cytokine expression data (both absolute and relative) often deviate from a normal distribution , we used Spearman’s rank correlation tests to reveal correlations between relative (stQ) and absolute (A) expression data. As the relative and aQ data were highly correlated , we show only the results obtained for the relative expression data in the main text of the article and report analogous results obtained using absolute quantification in the Supplementary Material to provide quantification method-independent confirmation to our main results.All statistical analysis was performed using R software v. 3.4.0  with a s2-transformation. The first three principal component scores were added to a correlation matrix prepared using Pearson’s correlations with Holm’s adjusted P-values. A heatmap showing variation in cytokine gene expression was constructed based on log2-transformed stQ and aQ quantities in R software using the “heatmap” function of the “stats” library, with the Unweighted Pair Group Method with Arithmetic Mean selected as the clustering algorithm.We used the non-parametric Wilcoxon rank sum test to test for differential gene expression (stQ or aQ) between control and NC2006-infected individuals screened across 7 tissues selected for further investigation after the initial cursory screening of 15 tissues in 2 individuals per treatment. To indicate consistency in the expression of the cytokine genes, principal component analysis (PCA) was done for the stQ and aQ data after logR or A as the response variable. For these GLMs, the non-Gaussian response variables were log2-transformed to achieve residual normality and tested against selected factors as explanatory variables: tissue or MG isolate , time point, sex, and all two-way interactions. Because there was little variability in size and mass and the treatment groups and time point groups did not show any significant differences with respect to size or mass or mass change over the experiment , these variables were excluded from the full models tested in this study. We tested the significances of the explanatory variables both in full models and in minimum adequate models  that were obtained by backward eliminations of particular terms from the full model. Candidate models were compared based on the change in deviance with an accompanied change in degrees of freedom (ANOVA) using F statistics. Tukey’s post hoc tests were used to test for differences between individual time points or MG isolates.After this second and more in-depth round of screening of seven house finch tissues, we focused on a subset of two selected tissues  to examine the temporal dynamics of cytokine expression responses to MG and the differences between MG isolates. We used generalized linear models (GLMs) with cytokine expression ratio t-test was used to compare relative IL1B and IL10 expression (log2R) induced by the two MG isolates in upper respiratory tract at DPI 6. Relative IL1B and IL10 expression (log2R) comparisons between upper respiratory tract, internal eyelid and HG were done using linear mixed-effects models (LMMs), where log2R data (response variable) were tested against MG isolate, tissue type, sex and all two-way interactions (explanatory variables with fixed effects). Individual identity was included into the models as a random effect and we used backward elimination of fixed effects to obtain the minimum adequate models.A two-sample IL1B expression and conjunctival MG loads, IL1B log2R data were used as the response variable in a GLM containing endpoint MG quantities (log10MG), MG isolate, sampling time point (DPI), sex, and two-way interactions of the factors as explanatory variables. In an analogous GLM, we tested for an association between total eye scores and IL1B expression using the quasipoisson residual distribution. Here, the endpoint total eye scores were used as the response variable, and we included IL1B log2R data, MG isolate, sampling time point (DPI), sex and two-way interactions of the factors as explanatory variables in the model. Again, we used a backward elimination method (as described earlier) to obtain the minimum adequate models.For testing the association between R based on two NC2006-infected and two control individuals for two selected genes (IL1B and TGFB2) across the whole panel of 15 tissues. Our results  show highest R in at least one of these genes in blood, brain, conjunctiva, HG, choana and internal lid; we thus selected these six tissues for further analysis. Spleen was also included in further analysis as a standardly investigated lymphatic tissue for the purpose of comparison.To identify candidate tissues for further research, we first compared the relative expression ratios IL1B in brain, conjunctiva, HG and internal eyelid, of IL6 in conjunctiva and internal eyelid, of IL10 in conjunctiva, HG and internal eyelid, and of CXCLi2 and TNFSF15 in conjunctiva and internal eyelid . No differential expression was indicated for spleen and only marginally non-significant changes in inflammatory cytokine expression were revealed in blood and upper respiratory tract. No differential expression was revealed for IL18 or TGFB2 in any tissue. While there was low consistency in the cytokine expression patterns across distinct tissues  show significant differential expression of IL1B and IL10 expression changes in NC2006 isolate-inoculated birds. Our results show significant effects of both tissue type and DPI for both genes , while in HG, DPI 6 was significantly different from the other sampling time points for both genes .To initially compare temporal patterns of cytokine expression in internal eyelid and HG in MG infected birds, we compared IL1B, IL10, IL6, CXCLi2, and TNFSF15, we found significant effects of MG isolate on cytokine expression quantified using both relative .To compare the immune responses triggered by distinct MG isolates (VA1994 and NC2006), we selected the internal eyelid as a primary model tissue, because here the strength of cytokine expression relative to controls was most prominent. In IL1B and IL10) in HG. Similar to the results from internal eyelid, the NC2006 isolate triggered stronger IL1B responses than the original VA1994 isolate in HG  for a subset of cytokines (IL1B and IL10 stimulation in the upper respiratory tract at the time of the peak cytokine expression response (DPI 6) and compared the cytokine expression in this tissue with cytokine expression in HG and internal eyelid. Although we found significant effects of both tissue and MG isolate on IL1B and IL10 expression across the three tissues , we did not detect any significant differences between the MG isolates in either IL1B or IL10 in the upper respiratory tract in particular  than VA1994 ; however, in the case of NC2006, cytokine expression responses are significantly stronger in internal eyelid/HG compared with the upper respiratory tract .Because MG infection in chickens induces cytokine responses in the upper respiratory tract , we also4 Figure A. When aIL1B as the cytokine that showed the most consistent differences between isolates. We found strong effects of conjunctival MG load on internal eyelid IL1B relative expression , with higher IL1B relative expression in birds with higher conjunctival MG burdens , with higher average pathogen loads in birds inoculated with NC2006  and cytokine expression in periorbital tissues, we focused on IL1B and total eye scores  at the time of sampling. Our results show significant associations between IL1B expression , sampling time point , and total eye scores .profiles  and antiprofiles . In thisIL1B, IL10, IL6, CXCLi2, and TNFSF15) of the seven examined cytokines in conjunctiva and nictitating membrane , and significant upregulation of IL1B and IL10 in HG. In birds, the conjunctiva and the HG are parts of the eye-associated secondary lymphoid tissue , overall, our results clearly indicate that differential cytokine expression responses during MG infection are strongest in periorbital tissues . Finally, although the expression of most inflammatory cytokine genes was highly correlated within individual tissues, we did not detect differential expression of IL18 or TGFB2 in any tissue examined, suggesting that these cytokines do not respond to MG infection. This pattern is consistent with house finch cytokine regulation toward a type 1 adaptive immune response that is mediated by non-specific inflammation leading to induction of Th1 cell activation.There was only low consistency in the cytokine expression patterns across distinct tissues, and we found no evidence for significant differential cytokine expression in any other tissues aside from the brain and periocular tissues, including spleen, upper respiratory tract, or blood. Although this result could be affected by the limited sample size used for the investigation of the tissue distribution of cytokine expression , where the expression in internal eyelid exhibits similar patterns of early upregulation (DPI 3–6) followed by a decline in cytokine expression at our latest sampling point examined (DPI 13). Nevertheless, our multiple-isolate analysis showed that the temporal dynamics of responses varied to some extent with the strain of MG used. Future work with higher temporal sampling resolution and larger sample sizes will help to shed further light on strain-specific temporal dynamics of cytokine expression in house finches.After determining which genes and tissues would be most relevant for further study, we examined the temporal dynamics of cytokine expression in internal eyelid and HG , focusing on pro-inflammatory day 8 PI . This isIL1B, IL6, IL10, CXCLi2, and TNFSF15), we found significantly stronger cytokine responses in birds inoculated with NC2006 relative to those inoculated with VA1994 in internal eyelid (and for IL1B in HG). These results are consistent with the stronger stimulation of humoral responses by the NC2006 isolate relative to the VA1994 isolate detected in prior work  and infection intensity  at the time of euthanasia to further link host immune responsiveness to virulence, as has been done in prior studies with humoral responses . We show finches , 26, 57,volution . ExperimThere are several documented examples of animal diseases where overactivation of immune cytokine signaling is responsible for immunopathology –61. In pPathogens have been shown to use many different means to manipulate host immunity for the purpose of increasing their transmission rate , 66. ThiAltogether, our results show that increased virulence of an evolutionarily derived MG isolate is associated with increased periocular expression of pro-inflammatory cytokines. Although our experiment cannot confirm the direction of causality underling this association, immunopathology induced by this inflammation might explain the mechanism of maladaptation of house finch immunity to MG. Given the demonstrated fitness costs of conjunctivitis for free-living house finches , future This study was carried out in accordance with the recommendations of federal (USFWS permit MB158404-1) and state (VDGIF permit 050352) agencies. The protocol was approved by the Virginia Tech’s Institutional Animal Care and Use Committees.MV and DH designed the research; MV, AL, LK, and DH performed the research procedures; RD provided RNA-seq data; MV and DH analyzed the data; and MV, RD, and DH prepared the manuscript. All the authors contributed with their comments to the conception of the work and final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Anopheles gambiae, remains a significant threat to global health. A critical component in the transmission of malaria is the ability of An. gambiae females to detect and respond to human-derived chemical kairomones in their search for blood meal hosts. The basis for host odor responses resides in olfactory receptor neurons (ORNs) that express chemoreceptors encoded by large gene families, including the odorant receptors (ORs) and the variant ionotropic receptors (IRs). While ORs have been the focus of extensive investigation, functional IR complexes and the chemical compounds that activate them have not been identified in An. gambiae. Here we report the transcriptional profiles and functional characterization of three An. gambiae IR (AgIr) complexes that specifically respond to amines or carboxylic acids - two classes of semiochemicals that have been implicated in mediating host-seeking by adult females but are not known to activate An. gambiae ORs (AgOrs). Our results suggest that AgIrs play critical roles in the detection and behavioral responses to important classes of host odors that are underrepresented in the AgOr chemical space.The principal Afrotropical human malaria vector mosquito,  An. gambiae s.l. Giles species complex, including An. gambiae s.s. (S form) and An. coluzzii (M form)4Drosophila melanogaster and have been shown to respond to amines, carboxylic acids and other environmental cues91011121314An. gambiaeAt least half the world’s population is at risk for malaria, while an even larger proportion of people are at risk for many other mosquito-borne diseases, including the arboviruses that cause dengue fever, yellow fevers and ZikaAn. gambiae (AgIrs) have not been functionally characterizedD. melanogaster (DmIrs), where they are expressed in olfactory neurons of sensilla that are morphologically analogous to grooved peg sensilla found in An. gambiaeDmIr derived response profile that is distinct from the odor space encoded by DmOrs910111291011121314DmIrs utilize DmIr8a as a co-receptor while others require both DmIr25a and DmIr76b, but not all three91011121314DmIr25a has recently been shown to function as a thermosensor and play a role control in establishing circadian rhythm in D. melanogasterDmIr25a and DmIr21a have been shown to mediate exquisite cool temperature avoidance in Drosophila larvaeIrs have diverged in terms of gene numbers and coding (i.e. amino acid) sequence within and across species, the candidate co-receptors are highly conserved among insects1113Irs will be analogous across lineages. We have observed that the AgIrs function independently of AgOrs at the larval stage where AgIr76b, a potential Ir coreceptor, is required for An. gambiae larval responses to butylamineAn. gambiae also requires the expression of AgIr76b, plus the coreceptor AgIr25a and an as yet identified “tuning” AgIr.With a single exception, the family of 46 variant ionotropic receptor genes identified in AgOr family of chemoreceptors, very little is known about the roles of AgIrs in odor coding  and function. Importantly, butylamine, lactic acid, and other carboxylic acids are activators of olfactory sensory neurons in An. gambiae17181920212223An. gambiae and other mosquitoes22526AgOr sensitivities revealed a relative lack of responses to these classes of compounds, suggesting that non-AgOr mediated mechanisms likely underlie sensitivity to these important kairomones in An. gambiae4Although several of functional studies have been carried out on the AgIrs that are expressed in the An. coluzzii (hereafter An. gambiae for consistency with gene annotations and the literature) antennae and maxillary palpsAgIrs are expressed in subset(s) of antennal sensilla in An. gambiae that are distinct from AgOr-containing sensilla. In addition, we have characterized AgIr complexes comprised of specific co-receptors in the Xenopus laevis oocyte heterologous system that confer sensitivities to a narrow range of amines and carboxylic acids. Our results confirm that AgIrs function in adult peripheral chemosensory organs where they are likely to form the basis for An. gambiae behavioral responses to these two compound classes, which are critical for mosquito host seeking. Future studies will continue to illuminate the molecular foundations for host odor sensitivity, and specifically the roles that AgIrs play in this process.In the current study we have characterized of a subset of AgIr family in An. gambiae adult antennae and maxillary palps based on RNA sequencingAgIr transcript profile information for adult female bodies, antennae and maxillary palps  sensillaAgIrs. We therefore elected to further examine the relationship between Ir and Orco expression within the antennae of adult female An. gambiae. In order to evaluate the co-expression of AgIr76b and AgOrco in female antenna, double fluorescence in-situ hybridization (FISH) and single FISH, combined with immunohistochemistry (IHC) were conducted. Antennae were probed with pairs of differentially labeled AgIr76b- and AgOrco-specific riboprobes or, alternatively an anti-Orco antibodyAgIr76b and AgOrco labeled cell bodies were identified in the female antenna .While  subtend  sensillaAgIr odor coding, we utilized oocytes to heterologously express functional OR and other chemoreceptor complexes. While the majority of AgIr cRNA combinations did not elicit oocyte activation against a panel of over 400 unitary odorants encompassing a broad survey of chemical classes (AgIr41a plus the candidate co-receptors AgIr25a and AgIr76b (hereafter AgIR41a complex) into oocytes conferred responses to amine/imine compounds  in ac2 coeloconic sensillaDmIr41a and DmIr76b are required for behavioral attraction of fruitflies to putrescine and cadaverineIn order to examine  classes , two rec classes  respondeompounds , but notompounds . Controlompounds . These dAgIr complex containing AgIr41c, a paralog of AgIr41a  responded with high inward current amplitudes to carboxylic acids  elicit more potent responses in one or both complexes. It is noteworthy that gravid Ae. aegypti females have recently been shown to be attracted to the polyamines putrescine and cadverine in oviposition assaysIn this study, we have defined and functionally characterized the first three AgIrs reported and together with a previous study defining a role for AgIr76b in mediating larval responses to butylamineAgIr complex is responsible for this sensitivity in larvae.The stimuli that activate the AgIR75k complex are of particular interest as carboxylic acid-containing compounds are a major component of human sweat222324An. gambiae is an important step forward in our understanding of chemosensation in an insect that continues to have a dramatically negative impact on human health. Of particular significance in this work is the identification of receptor complexes that likely underlie mosquito sensitivities to some of the most important chemical classes that are known to elicit host-seeking behavior. For example, ammonia and lactic acid act synergistically to increase the attractiveness of either odor blends or live hosts to An. gambiae and other vector mosquitoes262741424344454647484950515253DmIrs have been implicated in ammonia and acid sensing in D. melanogaster91213Ir function are conserved, the potential differences, subtle or pronounced, would be of interest to many different research communities including evolutionary biology, medical entomology/vector biology, and neurobiology, among others.The ongoing functional characterization of a second major family of chemoreceptors in Molecular approaches have been successfully used to identify compounds that hyperstimulate ORNs in mosquitoes, thus providing a basis for the development of next generation repellents565759All research was conducted in accordance with the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules and the Vanderbilt University Environmental Health and Safety policies. Lab containment practices, equipment and facilities were reviewed and approved by the Vanderbilt Institutional Biosafety Committee (IBC).An. coluzzii (SUA 2La/2La) originating from Suakoko, Liberia, previously known as Anopheles gambiae s.s. M formAn. coluzziiAn. gambiae throughout this report in order to maintain consistency with the previously published literature as well as with pre-existing gene annotations.AgIr transcripts for adult female antennae and maxillary palps were derived from previously published dataIllumina mRNA sequencing kit . Libraries were sequenced using an Illumina HiSeq2000. Raw read files were quality checked, uniformly trimmed to account for spurious adapter incorporation (5′end) and for sequencing reaction degeneration (3′-end) and mapped to the An. gambiae genome (http://www.VectorBase.org). Mapping was carried out using seqmap software, configured to allow for a maximum of three mismatches per read. Processed mapping data was then consolidated based upon AGAP number and the results summarized by rseq software. Total weighted reads and AGAP transcript lengths were used to calculate a normalized transcript abundance level in units of Reads Per Kilobase per Million reads mapped (RPKMs)Quantification of AgIr amino acid sequences were aligned using MUltiple Sequence Comparison by Log-Expectation  and subsequently subcloned TOPO-TA dual promoter vector . Digoxigenin (DIG) or fluorescein (FITC)-labeled fluorescence in situ hybridization (FISH) riboprobes were transcribed from linearized plasmids containing AgIr gene fragments using an RNA labelling mix  and T7/SP6 RNA polymerase  under the manufacture’s recommended protocols. Antisense experimental, and sense control, probes were conducted in parallel.Full length or partial Single riboprobe and antibody studies were performed as described previouslyFollowing riboprobe hybridization and washing, slides were incubated with 1:500 anti-Digoxigenin-POD  and 1:500 anti-Orco antibodyThe protocol for double FISH was unchanged with the exception of the inclusion of an additional set of antisense and sense probes to the hybridization mixtures. After riboprobe hybridization and stringency washes as detailed above, slides were incubated with 1:500 anti-Digoxigenin-Peroxidase (POD)  diluted in TNB buffer in a humidified chamber for 1–3 hours at room temperature and then washed 3 times in TNT buffer with light agitation for 5 minutes each at room temperature. Anti-Digoxigenin-POD signal was developed using Tyramide Signal Amplification (TSA) reagents  according to the manufacturer’s instructions for approximately 10 minutes at room temperature in the dark, then washed 3 times in TNT buffer for 5–10 minutes each with light agitation. The POD enzyme was quenched by placing slides in a 3% H2O2 solution diluted in TNT for 30 minutes at room temperature. The slides were washed 3 times in TNT buffer for 5–10 minutes each with light agitation, placed in TNB for 30 minutes at room temperature and thereafter incubated with anti-fluorescein-POD diluted 1:500 in TNB buffer in a humidified chamber for 1–3 hours at room temperature followed by washing 3 times in TNT buffer with light agitation for 5 minutes each at room temperature. Anti-fluorescein-POD signal was developed using TSA reagents  according to the manufacturer’s instructions for approximately 10 minutes at room temperature in the dark, then washed 3 times in TNT buffer for 5–10 minutes each with light agitation. Slides were mounted with Vectashield and visualized using either an Olympus BX-60 fluorescence microscope or a Zeiss LSM 510 META Inverted Confocal microscope.AgIr cDNAs, based on current gene annotationsAn. gambiae antennae. Primers specific for the coding sequences of the AgIrs used were constructed using the predicted sequences as describedAgIr cloning and plasmids were verified by sequencing prior to subcloning into the destination vector, pSP64T, for in vitro transcription and heterologous expression in Xenopus oocytes. In vitro synthesis of 5′ capped complementary RNAs (cRNAs) for AgIr constructs were carried out using the mMESSAGE mMACHINE in vitro expression kits according to the manufacturer’s protocol .Full-length coding sequences of Xenopus laevis protocols were carried out with the approval of the Vanderbilt University Institutional Animal Care and Use Committee. Expression and functional characterization of AgIr complexes were conducted essentially as described for AgOr proteinsX. laevis ovarian lobes were extracted from fully mature females via laparotomy. An incision less than 1 cm was made ventrally, lateral of the body midline, and along the ovarian wall. Ovarian lobes were extracted and placed in a washing buffer  supplemented with 0.1% gentamycin. The ovarian lobes were gently separated via forceps and subsequently treated with 2 mg/mL collagenase S-1 in washing buffer for 1.5 hours at room temperature while on a laboratory shaker. Individual oocytes were incubated in ND96 buffer  supplemented with 5% dialyzed horse serum, 50 μg/mL tetracycline, 100 μg/mL streptomycin, and 550 μg/mL sodium pyruvate at 18 °C until microinjection. Each oocyte was injected with 27.6 nL of an AgIr cRNA mixture containing a tuning AgIr and one or more potential co-receptor subunits (i.e. AgIrs 25a + 76b or AgIr8a). After injection, oocytes were incubated separately at 18 °C in supplemented ND96 buffer. Oocytes were allowed to incubate for 3 to 5 days until assayed by two-electrode voltage clamp electrophysiology.All Whole-cell currents were recorded from injected oocytes using the OC-725C feedback amplifier (Warner Instruments). This amplifier was connected to a computer interface through the Axon Digidata 1440 A Digitizer; data acquisition and analysis were carried out using pCLAMP 10 software (Axon Instruments). The current-injecting and voltage-sensing electrodes were made of a Ag/AgCl wire housed in a pulled borosilicate glass capillary containing 3 M KCl. Each oocyte was clamped at a holding potential of −80 mV while being perfused with buffer.−4 M each and pipetted in the TEVC perfusion system. Compounds from mixtures that elicited oocyte responses were tested individually to identify ligand-receptor pairings. A minimum threshold of 50 nA was used as a cutoff for positive responses to screened compounds, roughly equal to the noise level in the system. For the most efficacious compounds, concentration response profiles were generated using multiple oocytes.Approximately 400 unitary compounds of the highest commercial grade were tested in this study . One molMicrosoft Excel. The R statistical package (www.r-project.org) was used for subsequent analyses, except where otherwise indicated. Oocyte inward currents were normalized by dividing individual responses by the maximum response per trial, followed by calculating the arithmetic mean of normalized responses for each compound. Assumptions of normality and equal variance were confirmed with the Shapiro-Wilk and F-tests, respectively, and arithmetic means for each treatment in the oocyte TEVC functional assays were analyzed for statistical significance by single-factor ANOVA; significant results were followed up with Tukey-Kramer post-tests to distinguish among group means. Concentration response curves and EC50 values were calculated using Prism 4 . The Student’s t-test was applied to mean EC50 values for AgIr41a and AgIr41c pyrrolidine responses to evaluate statistical significance.Descriptive statistics, including arithmetic means, standard deviations and standard errors of the mean were calculated using How to cite this article: Pitts, R. J. et al. Variant Ionotropic Receptors in the Malaria Vector Mosquito Anopheles gambiae Tuned to Amines and Carboxylic Acids. Sci. Rep.7, 40297; doi: 10.1038/srep40297 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.