The comparison between Fe-deficient, Mn-deficient and control xylem sap samples using a multivariate statistical data analysis  is also included. Data included in this article are discussed in depth in the research article entitled “Effects of Fe and Mn deficiencies on the protein profiles of tomato (Solanum lycopersicum) xylem sap as revealed by shotgun analyses” [1]. This dataset is made available to support the cited study as well to extend analyses at a later stage.This article contains consolidated proteomic data obtained from xylem sap collected from tomato plants grown in Fe- and Mn-sufficient control, as well as Fe-deficient and Mn-deficient conditions. Data presented here cover proteins identified and quantified by shotgun proteomics and Progenesis LC-MS analyses: proteins identified with at least two peptides and showing changes statistically significant (ANOVA;  Specifications TableValue of the data•Tomato xylem sap proteins identified and quantified using a shotgun approach are presented herein, providing data on the protein composition of this fluid and facilitating comparisons with other plant species and different plant stresses.•Statistically significant and biologically relevant changes in the xylem sap protein composition upon Fe and Mn deficiencies provide information to assess the effects of these nutritional deficiencies on the metabolic pathways of tomato plants growing in controlled environmental conditions, and results could be compared to those found in other nutritional constraints.•Data would allow other researchers to assess the companion paper 1p ≤ 0.05) and above a biologically relevant threshold (fold ≥ 2) are shown in p < 0.05) are shown in via the Pride partner repository with the data set identifier PXD007517. The full description of Materials and Methods, Results and Discussion for this data set are presented in the associated research article The proteome data presented herein were collected from xylem sap fluid of tomato plants grown in two nutritional deficiencies that occur often in plants, Fe and Mn deficiency , Fig. 2.2n = 6). Xylem sap proteins were precipitated, resuspended in the appropriate buffer p ≤ 0.05) and a biologically relevant threshold level (fold ≥ 2) , the xylem sap fluid from six independent batches of plants were collected after eight days of treatment , Fig. 2.old ≥ 2) .Protein identification was carried out using the list of total peptides with the Mascot search engine p ≤ 0.05 and fold ≥ 2). Proteins achieving these thresholds are shown in Positive protein identification was assigned with at least two unique top-ranking peptides matched and with scores above the statistical and biological threshold levels (ANOVA;


Hepatocellular carcinoma (HCC) is the leading cause of cancer-related death worldwide, because recurrence often occurs in most HCC patients undergoing hepatectomy. It is necessary to identify patients with high risk for recurrence and adopt effective therapies. An obstacle to monitor patients at high risk for poor prognosis has been the lack of useful predictive biomarkers. Fortunately, recent progress in system biology allows to screen the biomarkers for HCC prognosis in a high-throughput manner. In this study, we performed systematic Kaplan-Meier survival analysis of the whole mRNA transcriptomics based on the Cancer Genome Atlas project (TCGA) and developed a three-gene prognostic signature composing of three genes UPB1, SOCS2 and RTN3. The model was validated in two independent microarray data sets retrieved from Gene Expression Omnibus (GEO) and the expression pattern of these three predictive genes in HCC was confirmed by western blot and immunohistochemistry with our HCC samples. In conclusion, our results showed that this three-gene signature has prognostic value for HCC patients. With the development of non-invasive radiological techniques and the novel advancements in the management of HCC, at-risk populations such as hepatitis virus infected patients are often under close surveillance5. As a result, HCC is increasingly diagnosed at earlier stages and treatment is likely to be successful. Nevertheless, recurrences eventually occur in most patients undergoing hepatectomy and the outcome of HCC patients is still unsatisfactory7. It is necessary to monitor patients at high risk for bad clinical outcome and adopt effective strategies to prevent recurrence. Therefore, identification of novel prognostic biomarkers is as important as early diagnostic molecular markers.Hepatocellular carcinoma (HCC) is estimated to be responsible for about 700,000 deaths globally in 2012, making it the leading cause of cancer-related death worldwide8 and AFP mRNA9 have been found to be prognostic. However, they rely on significant tumor burden making their usefulness in operable tumors questionable10. The past decades have seen the rapid development of predictive biomarkers with advances in understanding of tumor biology, and there is a large volume of published studies describing the role of tissue and serum markers such as tumor-associated antigens, oncogenes and tumor suppressor genes, enzymes and isoenzymes, adhesion molecules, angiogenic factors, growth factors and their receptors, tumor microenvironment, the microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) in HCC prognosis16. Our previous study has demonstrated that the expression of glutaminase is a sensitive and specific biomarker for the prognosis of HCC17. However, traditional screening methods focused on few genes, suffered from a lack of systematical evaluation and single biomarker for prognosis may lack sensitivity and specificity. Fortunately, recent advances in high-throughput technologies allow quantitative monitoring of various biological molecules and make it much easier to examine a large number of potential biomarkers at once, leading to an explosion of new biomarkers for HCC prediction. The innovative and seminal work of Hoshida discovered and validated a gene-expression signature associated with the survival of HCC patients18. The Cancer Genome Atlas (TCGA) program has profiled and analyzed molecular aberrations at the DNA, RNA, protein and epigenetic levels, facilitating the study of the expression of multiple genes simultaneously and offered an opportunity to screen prognostic genes on a global gene scale19. The microRNAs (miRNAs), long noncoding RNAs (lncRNAs) and mRNA have been proposed as potential prognostic predictor in HCC21. To date, several studies were designed to screen the miRNAs24 and lncRNAs25 related to the clinical outcome of HCC systematically. It is more likely to generate a prognostic signature from mRNAs than miRNAs and lncRNAs because of the considerable number of mRNAs, yet there is no study based on the whole transcriptomics to probe prognostic mRNA in HCC.Traditional serum markers particularly alpha-fetoprotein (AFP)TCGA liver hepatocellular carcinoma (LIHC) data sets allow us to correlate transcriptomic profiles to clinical outcomes. In this study, we performed systematic analysis of the whole transcriptomics using Kaplan-Meier survival analysis and revealed the association between the expression of thousands of genes with and survival time of HCC patients. Based on our whole-transcriptome survival analysis, we developed a three-gene prognostic signature consisting of UPB1, SOCS2 and RTN3, and validated this model in two independent microarray data sets retrieved from Gene Expression Omnibus (GEO). Finally, we validated the expression profiles of the outcome predictive genes by western blot and immunohistochemically staining with our HCC samples and performed functional analysis of prognostic genes with TCGA datasheet. To the best of our knowledge, this study is the first to make use of the whole mRNA sequencing data to investigate the cancer specific mRNAs expression patterns and their association with clinical outcome of patients with HCC.In order to comprehensively analyze the genomic prognostic genes in hepatocellular carcinoma, a workflow outlined the procedure for the analysis was developed Fig. . We acquTo rule out the bias caused by the survival-related clinical parameters in the following analysis, we obtained the clinical dataset from TCGA and screened for the survival-related clinical index by univariate and multivariate cox regression analysis Fig. . UnivariWe then stratified the patients with T stage, resulting in three different groups, with 176 patients in T1 group, 89 patients in T2 group, and 92 patients in T3/4 group. Three patients with unknown T stage were not included in this analysis Fig. . The medTo better understand which genes of the 15 candidates are more representative in evaluation of clinical outcome, we further assessed these 15 genes with univariate and multivariate Cox regression analysis in a total of 360 patients. All genes except HPR and SDHAP3 were of statistically significant correlation with survival time in univariate Cox regression analysis Table . Multiva26 and GSE5423613) which incorporated gene expression information and survival time of HCC patients from the GEO database. We applied these two datasets to validate our prognostic model respectively. In GSE54236, the survival time for the poor prognosis patients predicted by our model (n = 40) was significantly worse than that of the good prognosis (n = 40)  and SOCS2 (p = 0.03) was significantly lower in HCC patients with vascular invasion (n = 105), compared with patients without vascular invasion (n = 200), while no significant differences were found in RTN3 expression Fig. . FurtherHCC is one of the deadliest solid malignancies, because recurrence occurs in most patients even after undergoing potentially curative treatment. It’s important to predict clinical outcome and adopt novel interventional measures in time for the patients at high risk for recurrence. Recent progress in integrated “Omics” analysis shed new light on HCC biomarker screening and validation. In this study, we investigated the prognostic clinicopathological characteristics and the predictive genes based on RNA-seq data from the public databases, established and validated of a three-gene prognostic signature containing UPB1, SOCS2 and RTN3.18. However, we succeeded in developing a three-gene signature including UPB1, SOCS2 and RTN3 based on gene-expression profiles of tumor tissue in this study, and fortunately we have validated this model with two independent GEO microarray datasets produced from different platforms. There are several possible explanations for the inconsistency. Firstly, Hoshida’s study was accomplished by hybridization-based techniques (microarrays), and only 6000 oligonucleotide probes were included in the analyses, while TCGA datasets were generated by the next-generation sequencing technologies which do not use molecular hybridization to capture transcripts and therefore do not depend on the probe numbers. The limitation of the cross-platform analysis and the limited number of probes of Hoshida’s study might partly explained the inconsistency of the two studies. The second possible explanation for the discrepancy was the heterogeneity of HCC patients. The tissue samples of 80 HCC patients in the training set in Hoshida’s study were all from a single center in Japan with high hepatitis virus infection rate , while the patients in TCGA LIHC dataset were from multicenter consisting of different races including Asian, White and Black with low viral hepatitis rate , which failed to identify an outcome associated signature based on gene-expression profiles of tumor tissue, but discovered and validated a gene-expression signature associated with survival based on the profiles of the surrounding nontumoral liver tissue31. Additionally, RTN3 is reported to interact directly with a viral nonstructural protein and is important for viral replication32. However, RTN3 was found to limit the replication of HCV33. It has been observed that RTN3 was overexpressed in astrocytoma compared to non-cancerous tissue34. It’s reported that RTN3 could interact with the oncogene Ras at endoplasmic reticulum35. Further work needs to be done to assess the role of RTN3 in tumorigenesis especially in HCC.The role of RTN3 in Alzheimer’s disease (AD) has been widely investigated. Transgenic mice overexpressing RTN3 developed neuritic abnormalitiesin vitro and in vivo36 and was identified as an important regulator of hepatic homeostasis in response to high-fat dietary stress37. In addition, SOCS2 was a crucial intracellular mediator of the anti-inflammatory actions. SOCS2 deficient mice had uncontrolled production of proinflammatory cytokines upon infection with an intracellular pathogen38. Livers from high-fat-diet-fed SOCS2−/− mice showed increased Nuclear Factor κB (NF-κB) activity as well as elevated expression of genes for the inflammatory cytokines Interferon (IFN)-γ and Interleukin (IL)-637. Over the last years, SOCS2 has emerged as potential tumor suppressor-like gene in several tumors. Previous studied have revealed that SOCS2 was downregulated in ovarian, breast carcinomas, pulmonary adenocarcinoma and HCC42. Consistent with our study, it has been suggested that SOCS2 was an independent predictor for survival of patients with breast cancer40, colorectal cancer43, and HCC42, and patients with low expression of SOCS2 displayed shorter survival time. SOCS2 deficiency promotes spontaneous intestinal tumorigenesis in a specific mice model driven by mutations in the APC/beta-catenin pathway44. Although conflicting results were reported that SOCS2 expression was upregulated in prostate cancer46, these findings provide insights into the role of SOCS2 in tumor suppression and highlight the potential usefulness of SOCS2 as a prognostic prediction biomarker.SOCS2 was reported to negatively regulate the growth hormone signaling 47. Further experimental investigations are needed to estimate the function of UPB1 in tumors.UPB1 catalyzes the last step in the pyrimidine degradation pathway. Thus far, few studies have explored the relationship between UPB1 and cancer. In a study aimed to discovery novel circulation biomarkers for HCC by integrating various bioinformatics tools, they identified a seven-protein predictive signature including UPB1The initial aim of our study was to seek out several prognostic genes from the RNA-seq data, and validate the expression pattern and the prognostic value in protein level and then establish a predictive immunohistochemical staining signature and finally apply to clinical research and practice because of the easy accessibility of immunohistochemistry to patients. Though we validated the expression pattern of UPB1, SOCS2 and RTN3 by western blot and immunohistochemistry, we didn’t associate the expression of these genes to the survival of patients, because the subsequent validation process is difficult and costly. We acquired three survival-related genes and it was reported that these three genes played important roles in tumors. However, further research should be undertaken to investigate the function of UPB1, SOCS2 and RTN3 in HCC. The three-gene signature is promising, but the patient cohorts from both TCGA and GEO were retrospective, and prospective cohorts were needed to validate this signature. Although some limitations remain in our study, we have performed the first whole mRNA transcriptome survival analysis in HCC to search for predictive biomarkers, and we think our method can be extended to other cancer type.In conclusion, we identified a three-gene predictive signature comprised of UPB1, SOCS2 and RTN3 for hepatocellular carcinoma and validated these three genes expression pattern in HCC tissues. This study may contribute to provide significant clinical implications for the prognosis prediction of HCC patients. However, what remains unclear is how these genes impact survival and it is beyond the scope of this study. Further investigation and experimentation are needed to elucidate the biological mechanisms of UPB1, SOCS2 and RTN3 in HCC development and progression.All experimental protocols were approved by the research ethics committee of Drum Tower hospital and research ethics approval for this project was granted from the same institution. Utilization of data was conducted in accordance with TCGA and GEO data access policies. All analyses were performed in accordance with relevant guidelines and regulations.http://www.ncbi.nlm.nih.gov/geo/), in which outcome and survival time of the patients were incorporated. The survival time of HCC patients in microarrays was downloaded from ArrayExpress (http://www.ebi.ac.uk/arrayexpress/). GSE14520 (based on GPL3921 platform) and GSE54236 (based on GPL6480) enrolled 209 and 80 HCC patients with survival time, respectively.TCGA LIHC level 3 RNAseqv2 data  and clinical datasheet were obtained from the TCGA data portal . 371 patients containing 49 patients with paired normal liver tissues and 20531 genes were included. 10 patients whose pathological diagnoses were not hepatocellular carcinoma were ruled out. The gene abundance was filtered by requiring the RSEM normalized count to be ≥3 present in no less than 60% samples of the 49-paired tumor or normal tissues. The log2 transformation of RSEM was adopted in all plots. Two independent expression microarray HCC data sets (GSE14520 and GSE54236) were downloaded from the Gene Expression Omnibus (GEO) database  analysis was performed on 49 paired HCC samples. The Shapiro-Wilk test was applied to determine whether the difference of normal/tumor RSEM followed a normal distribution. The paired t-test was applied to normally distributed data otherwise the Wilcoxon rank test for paired data was applied to assess whether a gene was differentially expressed between tumor and normal samples. Statistical significance was determined using p < 0.05.For survival analysis, we used the Kaplan-Meier method to analyze the correlation between overall survival time and gene expression. The statistical significances of overall survival were determined using the Log-Rank test. Survival analysis was performed in R (version 3.3.1) and the survival curve was generated by R (version 3.3.1). The median was used as a cutoff value for classification of patients into high and low expression groups. Likewise, we use two tertiles to split patients into three groups with each gene expression, trisected patients into low expression group, middle expression group, and high expression group. We used R software version 3.3.1 and the “survivalROC” package to do the time-dependent ROC curve analysis.Prognostic index model was created using the three-gene signature based on multivariate Cox survival analysis. This algorithm is based on an importance score assigned to each gene. Using the Cox regression models, we calculated a risk score for each patient based on their individual expression levels of the three genes, where prognostic index (PI) =  −  − . The median expression was used as a cutoff value, and the relative expression value was defined as follows: score 1, <median; score 2, >median.Liver tissues were collected from patients undergoing hepatectomy with confirmed diagnosis of HCC at Department of Hepatobiliary Surgery of the Affliated Drum Tower Hospital of Nanjing University Medical School. Written informed consent was obtained from all patients or their guardians for the use of the biospecimens for research purposes. The samples were frozen in liquid nitrogen immediately after surgical resection and stored at −80 °C until further analysis.Cells were lysed in RIPA buffer containing a protease inhibitor cocktail . Protein concentration was determined. Equal amounts (20 ug/lane) of proteins were separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane . After blocking with 5% nonfat milk in Tris-buffered saline for 1 h, the membrane was incubated overnight at 4 °C with specific primary antibodies, followed by incubation with appropriate HRP conjugated secondary antibodies. Signals were detected using an enhanced chemiluminescence reagent  and subjected to Alpha Innotech Flour Chem-FC2 imaging system . The antibodies used in WB were as follows: anti-RTN3 , anti-SOCS2 , anti-UPB1 , anti-β-Actin , horseradish peroxidase (HRP)-conjugated secondary antibodies . Quantitative densitometry was performed using Photoshop CC .Formalin-fixed and paraffin-embedded HCC sections with a thickness of 4 μm were dewaxed in xylene and graded alcohols, hydrated and washed in phosphate buffered saline (PBS). Antigen retrieval was done by heat treatment of the deparaffinized sections in a pressure cooker in EDTA-TRIS (PH9.0) for 2 minutes. After the initial processing steps, sections were incubated overnight with primary antibody at 4 °C. The primary antibodies used in IHC were as follows: anti-RTN3 , anti-SOCS2 , anti-UPB1 . A subsequent reaction was performed with biotin-free HRP enzyme-labeled polymer from an EnVision plus detection system . Positive reactions were visualized with diaminobenzidine (DAB) solution followed by counterstaining with hematoxylin. Negative controls were performed using non-immune goat serum instead of the primary antibodies. The protein expression was assessed by a certified pathologist who was blind to all clinical and biological variables using a semi-quantitative scoring consisting of an assessment of both staining intensity  and the percentage of positive cells , yielding an overall score.Univariate and multivariate survival analysis were performed using the Cox proportional hazards regression model. Only variables with p < 0.1 on univariate analysis were incorporated into the multivariate Cox regression analysis. The paired t-test or Wilcoxon signed-rank test were used for paired samples to make a statistical comparison between groups. The Kruskal-Wallis test and Mann-Whitney-Wilcoxon test were used for ranked data as appropriate. All testing was carried out using Prism 6.0  or SPSS  or R . The two-sided p value less than 0.05 was defined as statistically significant for all statistical analyses. The data were plotted as mean ± standard deviation (SD).Supplemental Table s1Supplementary Info 2


BRCA1/2 mutation-negative breast cancer patients from the same family for WES. Homozygosity for the MTHFR 677 C>T mutation detected during this PSGT pre-screen step was considered insufficient to cause bilateral breast cancer in the index case and her daughter diagnosed with early-onset breast cancer (<30 years). Extended genetic testing using WES identified the RAD50 R385C missense mutation in both cases. This rare variant with a minor allele frequency (MAF) of <0.001 was classified as a VUS after exclusion in an affected cousin and extended genotyping in 164 unrelated breast cancer patients and 160 controls. Detection of functional polymorphisms (MAF > 5%) in the folate pathway in all three affected family members is consistent with inheritance of the luminal-type breast cancer in the family. PSGT assisted with the decision to pursue extended genetic testing and facilitated clinical interpretation of WES aimed at reduction of recurrence risk.Panel-based next generation sequencing (NGS) is currently preferred over whole exome sequencing (WES) for diagnosis of familial breast cancer, due to interpretation challenges caused by variants of uncertain clinical significance (VUS). There is also no consensus on the selection criteria for WES. In this study, a pathology-supported genetic testing (PSGT) approach was used to select two  BRCA1 and BRCA2 [Oncogenomics is a rapidly evolving integrative discipline that enables a novel approach to the molecular characterization of breast cancer pathology. Understanding the relationship between germline and tumour genetics is important in patients where early-onset familial breast cancer or differences in drug response and survival outcome cannot be explained by mutations in the major tumour suppressor genes, nd BRCA2 . In thesnd BRCA2 . UtilizaBRCA1/2 mutation screening. There is currently no consensus on eligibility criteria for WES in BRCA1/2 mutation-negative familial breast cancer patients.NGS includes targeted sequencing of preselected gene panels as well as whole genome/exome sequencing, RNA sequencing, and bisulfite sequencing for assessment of DNA methylation patterns ,6. AlthoGrant et al.  demonstrBRCA1 familial breast cancer [Germline genotyping interpreted in the context of tumour subtype may help to determine whether breast cancer is caused by genetic or environmental factors, or a combination of both, in order to direct treatment accordingly. Naushad et al.  found tht cancer . In linet cancer . Risk abBRCA1 and BRCA2 account for approximately 20% of familial risk, with the remaining variation caused by mutations with low-moderate penetrance that may be inherited in a subtype specific manner [It is estimated that known breast cancer susceptibility loci including c manner ,18. Failc manner  may partc manner ,22. Xu eMTHFR) gene support individualised dietary recommendations to reduce cancer risk in the general population [MTHFR mutation-positive cases, yet high doses of the synthetic form (folic acid) are suspected to promote tumour growth when administered after diagnosis of cancer. It is therefore reassuring that recent evidence obtained in postmenopausal patients with increased folate intake through diet and/or supplementation showed significantly higher circulating folate concentrations associated with improved survival after breast cancer diagnosis [Germline variation in folate pathway genes is associated with aberrant patterns of tumour pathology in cancer patients , with dypulation ,25. Thesiagnosis . Since tMTHFR 677 C>T mutation found to be associated with hormone receptor positive breast cancer [MTHFR 677 C>T mutation. Depression is a major comorbidity in breast cancer survivors and impacts on both the quality of life and treatment decision making. In this context, Van der Merwe et al. [CYP2D6 genotyping in breast cancer patients who (1) receive tamoxifen treatment; and (2) are at increased recurrence risk due to a family history of breast cancer/BRCA-mutation positivity; or (3) are required to take potential competing antidepressants that may inhibit CYP2D6 enzyme function. CYP2D6 genotyping combined with the unique spectrum of BRCA1/2 founder mutations identified in the South African population [The low-penetrance t cancer , has pret cancer . A low ft cancer , correlae et al.  recommenpulation ,30, formpulation  for imprpulation ,33, who BRCA1/2 mutation-negative breast cancer patients were selected from the same family for WES, followed by extended mutation screening in 164 unrelated cases stratified according to ER status. Lifestyle risk factors such as BMI were assessed in study participants due to the link with cardiovascular disease (CVD), predicted to equal or exceed recurrence risk of breast cancer in ER-positive postmenopausal women [MTHFR 677 C>T as an invariant marker of increased risk for many chronic diseases associated with dysfunctional methylation may add value beyond biochemical analysis of homocysteine and folate levels, which are influenced by various environmental factors. For this reason, MTHFR is not only a feasible anticancer drug target, but may also be considered an important factor in prevention of cumulative disease risk across clinical entities.In this study, two al women . This apal women . These lBRCA1 and BRCA2 genes. She was initially referred for the MammaPrint test and could safely forego chemotherapy based on a low genomic risk for breast cancer recurrence. Microarray analysis was preceded by immunohistochemistry (IHC) testing indicating ER-positive (ER+), progesterone receptor-positive (PR+) and HER2-negative (HER2−) status. The index case remained disease-free since 2008 despite termination of tamoxifen after one year due to severe side effects, including. Use of the CVD multi-gene test excluded the most prevalent CYP2D6 null-allele in Caucasians (CYP2D6*4), while homozygosity of MTHFR 677 C>T was detected in both the index case and her daughter with the luminal-type breast cancer. It was uncertain whether this functional single nucleotide polymorphism (SNP) expressed in a high-risk environment, as reflected by high BMI (>27 kg/m2) and inadequate folate intake (score <14) in both cases, was sufficient for development of breast cancer in this family.RAD50 R385C, Refseq mRNA accession NM_005732.3) and mucin 1  genes were identified as potential causative mutations. These missense mutations were confirmed using Sanger sequencing . Rare variants in the double strand break repair protein  of <0.001 is shown in The RAD50 R385C using allele-specific real-time polymerase chain reaction (PCR) methodology excluded this rare variant in an additional 163 unrelated breast cancer patients stratified according to ER status . This missense mutation was however detected in one (aged 36 years without a family history of cancer) of the 160 control individuals screened, but absent in the cousin of the index case diagnosed with lobular invasive carcinoma at the age of 42 years.Extended mutation screening for MTR) and methionine synthase reductase (MTRR). These genes are included in the Ion AmpliSeq™ Comprehensive Cancer Panel used to identify the RAD50 and MUC1 rare variants. Two functional SNPs were detected in the MTR  and MTRR  genes, in addition to the low-penetrance MTHFR 677 C>T (rs1801133) mutation previously identified using the testing algorithm illustrated in MTHFR 1298 A>C (rs1801131) known to be in linkage disequilibrium with MTHFR 677 C>T was not detected. Extended analysis of the CYP2D6 gene in the index case due to premature termination of tamoxifen, confirmed a fully functional enzyme based on the gene region covered by WES. Failure to identify high impact pathogenic mutations in all affected family members prompted extended analysis for functional polymorphisms  in the folate pathway genes, methionine synthase  and drug metabolism (CYP2D6*4), WES was performed in the index patient and her daughter. An important aim was to determine whether homozygosity for the MTHFR 677 C>T mutation may be sufficient to cause ER-positive breast cancer in a high-risk environment as reflected by high BMI in both patients. WES identified a rare missense mutation R385C in the DNA mismatch repair gene RAD50, in both the mother and daughter. This rare variant was excluded in a cousin diagnosed with invasive lobular carcinoma at the age of 42 years, as well as a further 163 unrelated breast cancer patients selected from the genomics database. The index case was diagnosed with bilateral breast cancer, invasive ductal and lobular carcinoma, while the daughter was diagnosed with invasive ductal carcinoma. It is therefore possible that RAD50 R385C may only be associated with invasive ductal carcinoma. However, detection of RAD50 R385C in one of 160 controls screened and conflicting results obtained with use of several bioinformatics tools to predict functionality, classifies this missense mutation as a VUS.After excluding high-penetrance mutations in the coding regions of the RAD50 mutations in approximately 3% of genetically uncharacterised breast cancer in a cohort of non-BRCA cases analysed. These authors reported potentially pathogenic variants that may explain 35% of the missing heritability. Bodmer and Tomlinson [RAD50 has been documented as an intermediate-risk gene, attributable to its critical role in the repair of DNA double strand breaks [RAD50 variant (rs139372231) is a C to T base change at nucleotide position 1153 that results in an amino acid change from arginine to cysteine at codon 385. The RAD50 protein forms a complex with the meiotic recombination 11 (MRE11) and nibrin (NBN) double-strand break repair proteins, which bind to strands of damaged DNA and hold broken ends together during the repair process. This MRE11-RAD50-NBN complex is important to prevent accumulation of DNA damage that may trigger uncontrolled cellular division, in order to promote maintenance of genome stability [RAD50 gene may lead to the production of an abnormally small, nonfunctional or missing RAD50 protein that may fail to respond effectively to DNA damage [Aloraifi et al.  identifiomlinson  suggested breaks . The RADtability . PathogeA damage .MUC1 gene in the daughter of the index case with early-onset breast cancer, was absent in the mother and her affected cousin. MUC1 is a transmembrane heterodimeric oncoprotein that is highly expressed in various cancers and correlates with potential for malignancy [MUC1 gene expression is regulated by DNA methylation and histone modification of the MUC1 promoter. They postulated that an understanding of the epigenetic changes of MUC1 may be of importance for prediction of risk and outcome for patients diagnosed with breast cancer. Deleterious germline mutations in the MUC1 gene is a well-documented cause of medullary cystic kidney disease, also known as mucin-1 kidney disease [MUC1 gene may only present as somatic changes within tumour DNA, and are unlikely to be clinically useful in prediction of familial risk. Thus, detection of this germline MUC1 mutation in the daughter may be considered an incidental finding in relation to kidney disease, if proven to be pathogenic in future studies.This study has demonstrated the feasibility of WES to achieve a high level of comprehensive genomic testing. The rare missense mutation Q85E, identified in the lignancy . Yamada lignancy  reported disease . In breaMTHFR gene differ significantly between population groups in South Africa [MTHFR 677 C>T is not only of concern for cancer risk, but also in development of other chronic diseases such as CVD. The SNP modifies the effect of hormone replacement therapy (HRT) on lipid and metabolic parameters in postmenopausal women [MTHFR 677 C>T is associated with a statistically significant increase in both total and low-density lipoprotein (LDL) cholesterol levels, while presence of the C-allele is associated with a reduction in these lipid levels after one year of HRT. The favourable effect of HRT on lipid levels was reversed in MTHFR TT homozygotes.A shift in our focus to low-penetrance gene variants highlighted the potential significance of folate pathway SNPs as a risk determinant for hormone receptor-positive breast cancer. Previous studies have demonstrated that the genotype distribution and allele frequencies of SNPs in the h Africa . The relal women . HomozygMTHFR 677 C>T mutation has previously been linked to the luminal subtype of breast cancer [BRCA1 mutations predominate in patients with hormone receptor-negative breast cancer of the basal-type. MTHFR 677 C>T has also been implicated as a causative genetic determinant in altered expression of microRNAs (miRNAs) [of RAD50 R385C, in combination with MTHFR 677 C>T previously identified as a BRCA-modifier gene [The correlation of a given genomic risk profile with clinical and therapeutic outcomes as well as risk for drug toxicity is complicated by clinico-pathological heterogeneity as observed in the family studied here. The low-penetrance t cancer , in a si(miRNAs) . These s(miRNAs) . The ove(miRNAs) ,49,50. Tier gene , is consBRCA familial disease [MTHFR, MTR and MTRR may disrupt homeostasis and confer significant risk of breast cancer, attributable to impaired DNA repair [MTR and MTRR genes in relation to menopausal status of breast cancer patients. Co-inheritance of MTHFR 677C>T with high-penetrance causative mutations in the BRCA1/2 genes may increase the risk of early-onset breast/ovarian cancer, while mutation 1298 A>C in the same gene is associated with sporadic breast cancer [MTHFR as a modifier gene in familial breast cancer is of particular relevance in South Africa, given the high frequency of founder BRCA1/2 mutations in Afrikaner, Coloured and Xhosa breast cancer patients [Although functional SNPs do not confer significant individual risks for breast cancer, cumulative data supported by WES suggest that co-inheritance of multiple low-penetrance mutations underlying cancer pathways and rare variants in driver genes may explain the majority of non- disease ,53. VariA repair ,55. SangA repair  highlight cancer . Elucidapatients .BRCA founder mutation screening where appropriate, may provide a viable risk reduction strategy for non-communicable diseases characterized by shared disease pathways [CYP2D6 pharmacogenetics [Current literature does not suggest a particular strategy with regards to surveillance and prophylactic management in patients with low-moderate risk variants . Howeverpathways ,7. Due tgenetics ,31, causgenetics . ImproveCYP2D6 gene, which could be addressed by using a combination of different mutation detection methods to obtain reliable results [Limitations of the study include the fact that WES evaluates only the subset of DNA regions that encode proteins , which constitutes about 1% of the human genome. There are approximately 20,000 genes encoded by an estimated 180,000 exons, but due to the technological limitations of NGS previously reviewed , it is n results . A simil results . For thiMTHFR gene that could be further explored by WES to confirm or support clinical relevance.The strength of this study lies in capturing genetic, pathology and lifestyle information before and after WES to formulate pathway-based algorithms for breast cancer treatment and prevention. The important role of gene–gene and gene–environment interaction has been emphasised previously to explain the missing heritability in breast cancer. However, to our knowledge, this is the first study to demonstrate the importance of functional SNPs in the In response to a needs assessment survey amongst healthcare professionals in South Africa, the PSGT platform was established to combine service delivery with health outcome studies . ApprovaThree members of the pedigree of the index case were included in this study. The index patient diagnosed with breast cancer at the age of 57 years was referred for the MammaPrint test performed in 2008. She forms part of the cases included in the study of Grant et al. , who madA total of 164 unrelated female breast cancer patients  and 160 CYP2D6 pharmacogenetics [MTHFR 677 C>T, investigated in this study as a potential contributing factor for breast cancer development against a possible genetic background of rare variants that may be uncovered by WES. In this family with inherited breast cancer, both the daughter (BMI 27.5 kg/m2) and her mother (BMI 27.8 kg/m2) were overweight with suboptimal intake of folate-rich food in the diet (folate score < 14) according to the nutrition and lifestyle assessment performed as previously described by Delport et al. [BRCA1 and BRCA2 mutations was largely excluded as the cause of familial breast cancer using NGS and multiplex ligation-dependent probe amplification (MLPA) at a commercial laboratory .Patients with benign neoplasms of the breast or other malignancies were excluded from the study. Only unrelated breast cancer patients with known ER status who signed informed consent for genetic studies were selected for high-throughput genotyping, comprising 60 (37%) Coloured patients of mixed ancestry and 104 (63%) Caucasians, including the index case. Patients with histopathology confirmed breast cancer were recruited at the Tygerberg Hospital Breast Cancer Clinic or referred from private practicing clinicians, as previously described by van der Merwe et al. . Patientgenetics . An onlihter BMI .5 kg/m2 ® DNA Blood Mini Kit  or Oragene reagents  for saliva specimens, according to the instructions provided with these commercially available kits. The NanoDrop® ND-1000 Spectrophotometer  and its v3.5.2 software package was used to measure the DNA quality and quantity. All genomic DNA samples were diluted to a final concentration of 10 ng/µL using nuclease-free water.DNA was extracted from whole blood using the spin protocol of QIAGEN QIAampWES was performed at the Central Analytical Facility  using the Ion Proton™ System  for NGS. The Ion AmpliSeq™ Exome RDY Library Preparation protocol was used (Publication Number MAN0010084) and template amplification was performed using the Ion PI™ Template OT2 200 Kit v3  (Publication Number MAN0009133). Semi-conductor sequencing on the Ion Proton system was performed using the Ion PI™ Sequencing 200 Kit v3 (Publication Number MAN0009136) with the Ion PI™ Chip Kit v2. This method is designed to target all human exons. The sequencing run time on the Ion Proton™ System was approximately 4 h which enabled the entire workflow from library construction to primary data analysis and result generation to be done in under 24 h. The Torrent Suite™  proprietary software is the standard program through which this NGS apparatus operates and offered simple run setup using predesigned workflows and run monitoring in real-time. WES analysis comprised several intermediate steps involved in the read-processing pipeline, as illustrated in First, sequence reads were aligned to the human reference genome (hg19) using the torrent mapping alignment (TMAP) program . This waA coverage depth of 15× was used for detection of potentially causative gene variants, prior to confirmation by Sanger sequencing. Coverage provides counts of read depth (DP) at two different levels; the sample level where the DP value is the count of reads that passed the caller’s internal quality control metrics and at the site level where this value is the unfiltered depth over all samples. In addition to the aforementioned quality filters applied, rare variants were filtered on a population frequency of <0.01% with variant function set to exclude all synonymous variants. A list containing 409 genes from the Ion AmpliSeq™ Comprehensive Cancer Panel  was used to search for high impact mutations as well as polymorphic variation in folate-related genes included in the panel. Bioinformatics tools freely available on the internet  were usen = 160) and the total breast cancer sample (n = 164) using real-time PCR. Two microliters of template DNA (10 ng/µL) was used in a total reaction volume of 10 µL. Applied Biosystems® (ABI™)  TaqMan® SNP Genotyping Assays were used for the real-time PCR runs according to standard TaqMan® SNP Genotyping protocols. Pre-designed 40× assay mix consisted of unlabelled primers and TaqMan® Minor Groove Binder (MGB) probes (VIC® and FAM™ dye-labelled). These assays were used for end-point genotyping by allelic discrimination analysis for SNPs on the Corbett Rotor-Gene™ 6000  or Roche LightCycler® 480 II . The oligonucleotide primer sequences used for Sanger sequencing and the TaqMan assays employed in this study are presented in Following the identification of potentially causative variants or functional SNPs involved in the folate pathway with use of WES, confirmation was performed by Sanger sequencing. Selected variants were screened for in controls (BRCA1/2 founder mutations in the high-risk South African population to multi-gene panel testing, meant to assist clinicians in the decision to pursue WES. The selection of the index patient was primarily based on the need to establish the role of MTHFR 677 C>T in familial risk, due to the potential for cancer prevention at the population level. Our results support previous findings indicating that the majority of genetically uncharacterised breast cancer cases may be caused by a combination of low–moderate penetrance mutations exerting their effect against a high-risk clinical background. To our knowledge, this is the first study using WES to investigate the significance of folate pathway SNPs as risk reduction targets beyond BRCA1/2 in familial risk. WES preceded by PSGT facilitated the identification and clinical interpretation of genetic risk factors of relevance to both cancer development and tailored therapeutic intervention in a single test.This study describes a framework for WES performed alongside clinical and pathology assessments to provide a clear strategy for the way forward. We moved from single-gene analysis of


PhIP and PhIP@OA did not show significant cytotoxic effects on SHSY5Y, MRC5, and human dermal fibroblast cells compared with the dissolved PhIP but clearly induced premature senescence activities that may be caused by a limited release of PhIP molecules from the particulate PhIP.The heterocyclic amine 2-amino-1-methyl-6-phenylimidazopyridine (PhIP) is considered as a human carcinogenic or mutagenic compound that is produced from the co-condensation of creatinine and amino acids as meats cook at high temperatures. The cooking of meats at high temperatures produces fumes, and these fumes can be suspended as aerosols via the vapor-to-particle (or -droplet) process in a temperature gradient field. Size distributions of the aerosols included a significant portion of nano- and submicron-sized particles, and these can be directly deposited in the lungs and on skin by particle transport phenomena near cooking areas. In this study, for the first time, PhIP-incorporated oleic acid  (PhIP@OA) particles, including individual particulate PhIP as simulated fumes from meat cooking, were constantly produced via collison atomization and subsequent drying processes. The aerosol particles were then dispersed in phosphate-buffered saline for cytotoxicity and senescence-associated  The adverse effects of fine particulate matters on human health in indoor environments also are receiving much attention because people are spending more time indoors (up to 90%)2. In particular, cooking fumes have been introduced as a major indoor source of fine-particle organic aerosols containing hazardous chemical substances 4; thus, human exposure to these substances could increase carcinogenic risks5. When meats cook at high temperatures, a deposit of these organic aerosol forms on the surfaces of kitchen walls and ventilation fans, which then leads to potential adverse effects on indoor air quality and inhabitant health6. People are at a significant risk of exposure  to these aerosols because the aerosol emissions from meat cooking have been measured at 40 g per kg meat7.By the year 2050, premature deaths from gaseous and particulate air pollutants will be significantly higher than those from human immunodeficiency virus and malaria9. The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo pyridine (PhIP) is one of the representative HCAs and a by-product of Maillard or browning reactions at high temperatures among free amino acids, creatine/creatinine, and sugars during meat cooking9. PhIP is known to react with DNA to produce carcinogenic adducts10; thus, it can cause an increase in the incidence of cancers from dietary exposure12. Recently, the neurotoxic effects of PhIP were investigated in vitro, and results showed toxic effects to dopaminergic neurons due to oxidative stresses13. Expression of cytochromes regarding metabolic and toxicological aspects was also introduced for different cell lines  upon the cell exposure to PhIP15. PhIP was detected in aerosols  produced during the frying of beef patties and bacon16. Meat cooking at high temperatures generates oil mist aerosols from the cooking oil and/or animal fat, and PhIP aerosols may be incorporated in the oil mist as well as in carbonaceous particles 18. However, there are no appropriate test protocols or relevant data for the biological effects of pulmonary and dermal exposures to these aerosol particles.Emissions from the thermal cooking of meats contain polycyclic aromatic hydrocarbons (PAHs) and heterocyclic aromatic amines (HCAs) because both compounds are produced during high temperature treatments of proteinaceous materials20. The collected PhIP aerosols were dispersed in phophate-buffered saline (PBS) at the chosen mass concentration for biological assessments. Cytotoxicity measurements of the dispersion were conducted using the colorimetric 3--2,5-diphenyltetrazolium bromide (MTT) assay for cell metabolic activities in SHSY5Y (human neuroblastoma), MRC5 (human lung fibroblast), and human dermal fibroblast (HDF) cells, which then were compared with dissolved PhIP , as shown in Fig. β-gal) and reactive oxygen species (ROS) generation assays were further conducted on HDF cells to correlate with the cytotoxicity assays. Dioctyl sebacate (DOS) was also employed instead of OA to produce PhIP@DOS aerosol particles using the atomizer. Spark-produced carbon black  particle-laden flow was employed as an operating gas to atomize the PhIP@OA solution as an effort to fabricate another realistic configuration (PhIP-CB@OA) of PhIP-containing aerosol particles -incorporated PhIP (PhIP@OA) aerosol particles using a serial aerosol reactor comprising a collison atomizer and a diffusion dryer. PhIP (or PhIP@OA) was dissolved in dimethyl sulfoxide (DMSO) to form aerosol droplets, and the droplets were then passed through a diffusion dryer to extract DMSO, thereby forming particulate PhIP aerosols. OA was frequently employed as a representative aerosol component from meat cooking because organic aerosols emitted from meat cooking contain a significant fraction of OA22, aerosol particle sizes (Dp) from meat cooking at high temperatures were within nano- and submicron-ranges even with cooking oils. The number concentrations (Np) of aerosols from meat cooking processes ranged from 105 to 106 particles cm−3 23. To prepare simulated PhIP (or PhIP@OA) aerosols with size distributions similar to previous reports, the concentration of PhIP (or PhIP@OA) dissolved in DMSO solution was controlled based on the following equations:ρp and ρ* are the densities of particles and reference (1 g cm−3), respectively; Cs is the concentration of PhIP (or PhIP@OA) dissolved in DMSO; Dd is the droplet diameter [measured using aerodynamic particle sizer ]; Q is the gas flow rate. By modulating the solution concentration, appropriate size distributions  than graphitic carbon. Anisotropic morphologies were also found in case of PhIP@OA, and the anisotropic structures were covered by a lighter contrast layer that was caused by OA incorporation on particulate PhIP. Lattice fringes also existed in the core region  of the particulate PhIP@OA. The lattice distance was 0.553 nm, and this was different from that measured in particulate PhIP alone, implying that the polycarbonate crystallization could be affected by co-existing compounds during the solidification process into particles. This tendency could also be seen in other configurations  was incorporated; only the bands for OA (or DOS) remained for the PhIP@OA (or PhIP@DOS) sample and for the PhIP-CB@OA sample. In PhIP@OA and PhIP-CB@OA samples, for example, the bands at around 1470 cm−1 were attributed to the in-plane –O–H groups of OA, and an intense band at 1710 cm−1 was related to the carbonyl stretching mode. PhIP@DOS showed an intense band at 1720 cm−1 corresponding to the carbonyl stretching vibration. These findings suggest that OA (or DOS) was deposited uniformly over the entire surface of all particulate PhIPs.To validate the surface chemistry of particulate PhIP, FTIR measurements were performed using directly sampled aerosol PhIP particles on polytetrafluoroethylene substrates . Successive measurements of zeta potentials showed negative polarities for the dispersed simulated particles in PBS (pH 7.4), and there were no significant differences among the samples (Supplementary Table −1) was identical. Dissolved PhIP showed greater cytotoxicities at all concentrations after 24 h  produced similar results even after extended incubations (96 h)  was employed as a control in SA-β-Gal assays to compare its activity with that of the particulate PhIP samples because low-concentration DOX is known to induce premature senescence in HDF cells29. DOX and particulate PhIP samples (50 µg mL−1) induced SA-β-Gal positive cells, showing bluish stains on the cells  than DOX. On the other hand, dissolved PhIP (10 µg mL−1) showed an activity  with nano and submicron size distributions (~106 particles cm−3); this is well-matched to the distributions of fumes from the actual meat cooking processes. The TEM measurements of particulate PhIP presented polycarbonate crystallites, and the lattice fringes of the crystallites also were found for the cases where OA and DOS were incorporated into the molecules. Nevertheless, particulate PhIP maintained the characteristic IR bands of standard PhIP, and these bands disappeared when OA or DOS were included in the PhIP solution  for atomization. This implies that the surfaces of particulate PhIP were uniformly covered by OA or DOS component during the atomization and subsequent drying processes; this was confirmed by TEM measurements. The same was true for PhIP-CB@OA samples in which CB was introduced to the molecules.PhIP is one of the HCAs found in meat cooked at high temperatures and has carcinogenicity and mutagenicity; thus many studies have been conducted to evaluate its adverse effects at β-Gal assays, and these were compared with those of 500 nM DOX releases. Unlike MTT assays, particulate PhIP and PhIP@OA induced senescence activities in HDF cells that were comparable to those induced by DOX as a control. The dissolved PhIP (10 µg mL−1) did not show remarkable senescence activities, and this supports the premise that different cellular responses could result from different PhIP exposure levels . Western blot analysis, the DCFDA assay, and fluorescence microscopy further supported the results from SA-β-Gal activities for low-concentration DOX and particulate PhIP. From these in vitro assessments for simulated meat cooking particles, further studies will be required to evaluate the adverse effects regarding acute and chronic exposures at in vivo levels. This is necessary to secure more useful information from more efficient analyses of PhIP aerosol exposures and its related configurations.There were significant differences between the dissolved and particulate PhIP samples in cytotoxicity profiles that may be caused by slow releases of PhIP molecules from the particulate PhIP that has a polycarbonate crystalline structure. The quasi-crystalline nature induced a significantly smaller elution of PhIP molecules into PBS, which was proven by HPLC measurements. Adverse effects from the low-level PhIP releases were evaluated through SA-β-Gal expression, western blotting, DCFDA assay, and fluorescence microscopy corroborated the adverse effects of low-level PhIP releases from the particulate PhIP. Therefore, these results may offer an early assessment platform/model to efficiently analyze the acute and chronic biological effects of aerosol exposures of PhIP and its related particles.Collison atomization-based processing was designed and employed to produce simulated PhIP and its related aerosol particles with size distributions similar to fumes from high temperature meat cooking. The particulate PhIP from the aerosol process showed a quasi-crystalline structure of polycarbonate, and this structure induced significantly smaller releases of PhIP molecules from particulate PhIP surfaces. The crystalline nature of particulate PhIP was retained when other components  were introduced during the aerosolization. This property resulted in remarkable differences between the dissolved and particulate forms of PhIP in cytotoxic profiles for SHSY5Y, MRC5, and HDF cells. As a result, biological assays for low-concentration PhIP exposures were required. SA-−1) was injected into a collison atomizer containing a 100 mL DMSO solution  with 0.09 g PhIP . DMSO in the PhIP-containing droplets from the atomizer was extracted in a diffusion dryer; thus, particulate PhIP aerosols were made. In case of PhIP@OA, 0.01 g OA  was further added to the PhIP-containing solution. The fabricated PhIP or PhIP@OA aerosols were then electrostatically charged  in a corona discharge reactor with pin-ring electrodes, and the aerosols were collected on a cylindrical rod with negative potential  in an electrostatic precipitator. The aerosol collection rod was immersed in the PBS solution under ultrasonic irradiation for 10 min to detach the aerosols from the rod. The rod was then removed from the solution and rinsed with PBS, and additional PBS was further injected into the solution to disperse PhIP or PhIP@OA with a desired mass concentration  gas (3 L min−1. A highly sensitive mercury cadmium telluride (MCT) detector was employed for microanalysis of the particles on a PTFE substrate, and the detector was cooled using liquid nitrogen during the measurements to maintain detection sensitivity.A scanning mobility particle sizer  and a dynamic light scattering (DLS) particle sizer  were employed to measure particle size distributions of PhIP and PhIP@OA particles in the aerosol and aqueous states, respectively. Morphologies of the particles were analyzed using a transmission electron microscope . To analyze chemical characteristics, PhIP and PhIP@OA aerosols were directly deposited onto polytetrafluoroethylene substrates , and the particle-deposited substrates were transferred to Fourier transform infrared spectroscopy  for absorbance detection within the range of 3500-650 cm−1 MTT reagent for 2 h in the dark. Subsequently, the formazan was completely dissolved by adding isopropyl alcohol containing 40 mM HCl, and the absorbance of samples was measured at 570 nm using an ELISA plate reader . SA-β-gal activity in HDF cells was measured using the chromogenic cytochemical assay32. The cells treated with the samples including doxorubicin (DOX) and paraformaldehyde were washed with PBS and stained with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside solution . The increased level of lysosomal β-gal was indicated by blue-green stains, as visualized under inverted bright-field microscopy , and SA-β-gal positive cells were represented as a percentage of the total cell number. For western blot analysis, the proteins on a polyvinylidene fluoride membrane from particle-exposed HDF cells were incubated with primary antibodies against glyceraldehyde-3-phosphate dehydrogenase, p21, and p53 . The proteins were then detected using enhanced chemiluminescence agents on a luminescent image analyzer . To measure ROS, the 2′,7′-dichlorodihydrofluorescein diacetate assay (DCFDA)  was employed in HDF cells after treatment with the samples including DOX . The 2′,7′-dichlorofluorescein probe was visualized using fluorescence microscopy  and analyzed using a flow cytometer .Cytotoxicity measurements of PhIP and PhIP@OA particles, including dissolved PhIP, on SHSY5Y, MRC5, and HDF cells were conducted using MTT assays. After particle exposures, cells were incubated with 0.5 mg mLAll data generated or analysed during this study are included in this published article (and its Supplementary Information file).Supplementary Information


Atherosclerotic plaque rupture is the culprit event which underpins most acute vascular syndromes such as acute myocardial infarction. Novel biomarkers of plaque rupture could improve biological understanding and clinical management of patients presenting with possible acute vascular syndromes but such biomarker(s) remain elusive. Investigation of biomarkers in the context of de novo plaque rupture in humans is confounded by the inability to attribute the plaque rupture as the source of biomarker release, as plaque ruptures are typically associated with prompt down-stream events of myocardial necrosis and systemic inflammation.We developed a novel approach to identify potential biomarkers of plaque rupture by integrating plaque imaging, using optical coherence tomography, with both plaque and plasma proteomic analysis in a human model of angioplasty-induced plaque disruption.We compared two pairs of coronary plaque debris, captured by a FilterWire Device, and their corresponding control samples and found matrix metalloproteinase 9 (MMP9) to be significantly enriched in plaque. Plaque contents, as defined by optical coherence tomography, affect the systemic changes of MMP9. Disruption of lipid-rich plaque led to prompt elevation of plasma MMP9, whereas disruption of non-lipid-rich plaque resulted in delayed elevation of plasma MMP9. Systemic MMP9 elevation is independent of the associated myocardial necrosis and systemic inflammation . This information guided the selection of a subset of subjects of for further label free proteomics analysis by liquid chromatography tandem mass spectrometry (LC–MS/MS). We discovered five novel, plaque-enriched proteins  to be significantly elevated in systemic circulation at 5 min after plaque disruption.This novel approach for biomarker discovery in human coronary artery plaque disruption can identify new biomarkers related to human coronary artery plaque composition and disruption.The online version of this article (doi:10.1186/s12014-017-9157-x) contains supplementary material, which is available to authorized users. Atherosclerotic plaque rupture is the culprit event which underpins a majority of acute vascular syndromes such as acute myocardial infarction , yet bioOne of the challenges in the investigations of plaque rupture in humans lies in the difficulty to establish the time course of a de novo plaque rupture event. In this regard, plaque disruption by percutaneous coronary intervention (PCI) induces controlled plaque rupture in humans and allows profiling of biomarker changes timed precisely to a plaque event –6. FurthHowever, even in such controlled settings, characterisation of biomarker of plaque disruption are further complicated by the presence of downstream effects such as myocardial necrosis and systemic inflammation after PCI , 10, 11,In this study, we applied a novel discovery workflow to the human model of PCI-induced plaque disruption. We hypothesised that by combining the information from coronary plaque imaging analysis, proteomics analysis of coronary plaque material, and proteomics analyses of serial plasma samples collected before and after plaque disruption, this would enable identification plaque proteins acutely elevated in systemic circulation that are specific markers of human coronary artery plaque disruption.In a prospective study at the Oxford Radcliffe Hospital (ORH), Oxford, UK, patients between the age of 30–90 who were referred for non-emergency coronary angiography with the view to proceed to PCI were invited to take part. Patients who underwent diagnostic angiography without stenting served as control subjects in this experimental model as the steps involved in diagnostic angiography  were the same as PCI except deployment of stents. We excluded patients who were haemodynamically unstable, or when there was a known history of renal impairment or anaemia. Patients undergoing left main coronary artery angioplasty were also excluded. The study was approved by the Oxford Regional Ethics Committee (Ethics Ref: 08/H0603/41) and complies with the Helsinki Declaration. Written informed consent was obtained from every participant.OCT images were acquired using the Lightlab system  prior to disruption of plaque by stent. Image analyses were performed by assessors who were blinded to the clinical status and other biomarker measurements of the patient. The culprit lesion was categorised into two subtypes (lipid-rich and non-lipid rich) using the pre-defined scoring system.  was deployed downstream of the lesion in a standard manner. The umbrella-like polyurethane mesh contains 110 µm pores to allow passage of blood components while trapping the plaque material released during angioplasty. To assess if blood components  would also adhere to the FW during the PCI procedure, therefore giving rise to artefactual signals unrelated to coronary plaque material, a paired FW device was prepared concurrently as the matched control sample in the following manner: after the diagnostic angiography confirmed suitability for stenting with FW distal protection, an additional 10 ml of arterial blood was aspirated from the coronary guide catheter and transferred into a 15 ml Falcon tube. A FW was then positioned downstream of the coronary lesion followed by the PCI procedure. Concurrently, an unused FW was placed in the Falcon tube containing the individual’s blood. This was then immediately transferred to a water bath and incubated at 37.5 °C for the duration of the stenting procedure . The plasma portion was then transferred to a second BD Vacutainer® without additive for the second spin (15 min × 2500 g). Confirmation of platelet-poor plasma was confirmed by platelet counts  and plaque morphology [lipid-rich (n = 23) vs. non-lipid-rich (n = 13)]. More prompt elevation in plasma MMP9 was observed in patients who presented with ACS  or CRP (P = ~1) during this time course  and C-reactive protein (CRP) in all participants  after plaque disruption, compared with a control group who underwent diagnostic angiography only. In these PCI patients with the highest MMP9P < 0.05) at 5 min after plaque disruption. These were compared against the plaque protein library determined from plaque debris. Five of these proteins were also enriched in plaque debris , highlighted the key pathways represented by these plasma proteins, with the highest representation being the Liver X receptor (LXR)/retinoic X receptor (RXR) pathway Fig. . After eis Table .Fig. 3CaAUC and TnIAUC or between LBPAUC and CRPAUC (data not shown).Lipopolysaccharide binding protein (LBP) is one of the protein that is present in human coronary artery plaque and is significantly elevated in plasma immediately after plaque disruption. Elevation of plasma LBP levels after PCI  was validated by an enzyme immunoassay in individual plasma samples, but remained unchanged in the control group who underwent diagnostic angiography without plaque disruption  are the substrates for spontaneous plaque rupture that leads to acute coronary events. We observed that the pattern of biomarker release after plaque disruption (exemplified by MMP9) was independent of the clinical presentation of the patient (i.e. ACS vs. SA), but was significantly different between lipid-rich plaques compared with non-lipid plaques, demonstrating that plaque composition directly influences biomarker release after plaque disruption.Our finding of systemic elevation in MMP9 within an hour after coronary plaque disruption is consistent with its reported role in atherosclerotic plaque destabilisation , 20. ThaThe potential discovery utility of the integrated plaque imaging proteomic analysis approach highlights the power of combining proteomic analysis of both plaque-derived particulate debris and plasma proteins, in a rigorous and highly controlled experimental design. The plaque debris collection and analyses were conducted using a paired FilterWire device incubated in patient-derived blood ex vivo to control for proteins in circulating blood that might have otherwise adhered to the device during its deployment. This enabled identification of a highly selected list of proteins that can be specifically attributed to liberation from the plaque, after plaque disruption, and do not represent circulating plasma proteins.We identified and validated LBP as a novel, plaque-derived biomarker of coronary atherosclerotic plaques. LBP is a secretory class 1 acute phase protein, previously associated with the response to bacterial sepsis. Various pathogen associated molecular patterns bind to LBP, and these complexes interact with Toll Like Receptor 4 (TLR4) associated pathways . There iGiven the role of inflammatory activation by TLR4 in atherogenesis , our finIn addition, our workflow also identified other plasma proteins which were acutely elevated after plaque disruption that have plausible biological associations. For example, plasma Annexin A5 has previously been shown to be elevated in patient with acute myocardial infarction  and is aFurther validation by larger cohort(s) will improve the external validity of our findings and to confirm the role of these proteins as novel biomarkers. It will also be important to establish the time course profile of these proteins of plaque disruption, and the range of plasma concentration of these proteins in physiological conditions in large cohorts.We demonstrate a novel approach to discover novel biomarkers of human coronary artery plaque disruption, by combining plaque imaging with proteomic analysis of plaque-derived material, and plasma proteins. We identify the LXR/RXR pathway, and, specifically LBP, as a novel biomarker signature of human coronary artery plaques and plaque disruption. Highly-stratified proteomic analyses of plaque-derived material and plasma can identify new targets of potential biological importance in human coronary artery disease.Additional file 1. Supplemental method and data.Additional file 2: Table S1. Proteins present in the plaque debris. We identified a total of 423 proteins that were derived from the plaque debris, captured by a FilterWire positioned downstream of the disrupted plaque.Additional file 3. Changes in lipopolysaccharide binding protein after plaque disruption.


A chronic inflammatory and fibrotic response was observed at all time points following VML. These results suggest that the pathological response to VML injury during the acute stage of the healing response overwhelms endogenous and therapeutic regenerative processes. Overall, the data presented delineate key molecular characteristics of the pathobiological response to VML injury that are critical effectors of effective regenerative treatment paradigms.Skeletal muscle possesses a remarkable capacity to regenerate when injured, but when confronted with major traumatic injury resulting in volumetric muscle loss (VML), the regenerative process consistently fails. The loss of muscle tissue and function from VML injury has prompted development of a suite of therapeutic approaches but these strategies have proceeded without a comprehensive understanding of the molecular landscape that drives the injury response. Herein, we administered a VML injury in an established rodent model and monitored the evolution of the healing phenomenology over multiple time points using muscle function testing, histology, and expression profiling by RNA sequencing. The injury response was then compared to a regenerative medicine treatment using orthotopic transplantation of autologous minced muscle grafts (~1 mm However, after volumetric muscle loss (VML) injury2, which is a type of severe trauma that ablates resident cells and structures primarily responsible for regeneration, the intrinsic muscle regenerative process fails3. Instead, gross compartmental tissue fibrosis manifests5 and chronic functional deficits result. VML is a common clinical outcome after open or closed  extremity trauma, for which there are currently no regenerative standards of care.Skeletal muscle comprises over 40% of body mass in lean individuals, is primarily responsible for coordinating voluntary movements and can readily adapt to its environment. After injury, skeletal muscle is capable of repairing and regenerating through a pool of resident stem cells (satellite cells) that activate, proliferate, differentiate, and fuse to form new or repair existing multinucleated myofibers14 but require significant further advancement to be of therapeutic benefit to patients presenting acute or chronic VML injury.The magnitude and location of VML injuries result in considerable heterogeneity and necessitate a cadre of therapies. For instance, a small VML defect isolated to a peripheral portion of a muscle unit may be best compensated by synergist hypertrophy mediated through physical therapy. Whereas, massive VML defects have been shown to not respond to conventional physical therapy and may require regenerative medicine augmentation. Regenerative treatments for VML have utilized various strategies16 a dysregulated immune response that coincides with aberrant or muted muscle fiber regeneration and fibrosis, although the temporal coordination of these events and drivers of this response trajectory have not been elucidated. Without determination of a clearly effective therapy and lack of knowledge of the molecular phenomenology-mediating injury repair, further advancement of the field depends on understanding and mitigation of the host pathobiological response. Thus, a key step to advance translational medicine in this domain is to establish a comprehensive reference map of the various molecular patterns that drive the diverse sub-processes through time and identify critical factors that influence and modulate gene expression amplitude and dynamics. The formulated unbiased views of the pathophysiology can then serve as a guide to assess and enhance new therapeutic modalities for VML.The fairly extensive number of regenerative therapies that have been tested for VML repair have proceeded to date without a comprehensive understanding of the pathophysiology of the injury response. Animal models of VML injury have describedHerein, we administer a VML injury to a rodent model and track the molecular phenomenology and etilogy through multiple time points using muscle function testing, histology, and expression profiling by RNA sequencing (RNA-Seq) Fig. . Next, w+ cells) persisted in the defect for weeks to months after injury  muscle and assessed for in vivo neural-evoked strength deficits intermittently post injury Fig. . At 3, 7ury Fig. . ConsistTo measure the molecular mechanisms contributing to poor muscle regeneration induced by VML, we performed expression profiling by RNA-Seq for unoperated controls, VML-affected muscles and unaffected contralateral muscle for multiple time points in the acute response phase . Biological and technical replicates for all three sample types demonstrated excellent correlations and reproducibility across all time points. Samples that did not meet adequate thresholds were discarded. In total, 16,020 mRNAs were detected (TPM > 1) for at least one time point; and 4,897 genes were observed to be dynamic (fold change > 2) at one or more time points Fig. . Princip19 have shown inflammation -related programs subsided after several days, but many of these genes remained upregulated over the entire time course  into the injured site was observed 14 and 28 days post injury  along with extracellular matrix (ECM) deposition and remodeling such as structural components (collagen and elastin) were detected. This result was also in agreement with histological observations of fibrotic supplantation of muscle tissue  Lewis rats were surgically implanted in a collagen gel in the VML defect site and progenitor cells derived from the minced grafts were observed to contribute to de novo muscle fiber regeneration, evidenced by the presence of GFP+ fibers 56 days post injury  analysis and hierarchical clustering of the data sets through time revealed highly similar patterns for the non-repaired samples with those that were treated with MMGs Fig. . This ob16 and observed upregulation of inflammatory and fibrotic transcriptional programs at 3 months post injury. Understanding the molecular signals that induce the response of skeletal muscle to adopt a non-regenerative phenotype and when these signals are presented is paramount in advancing our understanding of VML pathogenesis and therapeutic efficacy. In this study, we performed multi-dimensional profiling of VML-injured tissues during the healing process using muscle function, histology, and transcriptomics and compared the response to treatment with autologous MMGs. Profiling of unfractionated muscle tissue from the VML injured site enabled detection of transcripts from multiple cell types and insights into tissue-level responses.Currently, very little is known about the global molecular response after VML and why this injury exceeds skeletal muscle’s endogenous regenerative capabilities through satellite cells. Recently, we administered a VML injury to a swine model20 and promote fibrotic deposition. In contrast to muscle regenerative studies, whereby the cellular infiltrate transitions25 from a pro-inflammatory population to a primarily anti-inflammatory phenotype after several days, many of the inflammatory and immune-related signaling pathways remained upregulated over the entire time course. Persistent inflammation and inadequate debris clearance during muscle regeneration have been shown to influence fibrosis by inducing fibro-adipogenic progenitors (FAPs)27 to proliferate and differentiate into fibroblasts or adipocytes. We viewed sustained transcriptional overexpression of PDGFRα, a marker for FAPs, in parallel to sustained increases in expression of TGF-β1. Increases in TGF-β1 signaling have been shown to block FAP apoptosis and induce fibrogenic differentiation28, which our current findings suggest. Moreover, elevated TGF-β1 has been shown to suppress the regenerative functions of satellite cells29. The collective integration of these gene expression programs for non-repaired tissues suggests a model of continuous infiltration of immune cells and perpetual inflammation that in turn regulates mesenchymal progenitors and cells to produce excess matrix30. A similar model of chronic inflammation that contains both pro-inflammatory and anti-inflammatory immune cells has been shown for Duchenne muscular dystrophy, which also uniquely exhibits fibrosis and FAP infiltration and differentiation31.For muscles that were non-repaired after VML, in all time points the tissue appeared to be overwhelmed by persistent inflammatory and fibrogenic signals. Histological observation of the non-repaired muscles highlighted inadequate muscle fiber regeneration and a dense population of macrophages in the defect area. This result is consistent with sustained upregulation of multiple components of the Complement system, which has previously been shown to impair muscle regeneration33, MMGs were found to promote muscle regeneration after extended post-surgical times (8–16 weeks), but in the early time periods sampled here (3–28 days) myogenic regenerative expression patterns did not vary considerably compared to non-repaired samples. These early time periods were deterministically sampled, as immediate interventions have been shown to favorably promote long-term outcomes for VML. However, multiple factors may be contributing to prevention of regenerative pathways from these treatments during the early time periods sampled such as the strong upregulation of negative regulators of myogenic differentiation such as TGF-β, SMAD2/3, Snai, musculin (MSC), myostatin (MSTN), and inhibitor of differentiation (Id) proteins (Id2 and Id3). Since muscle repair and regeneration canonically utilizes the sequential activation of specific basic helix-loop-helix transcription factors34 , potent overexpression of negative regulators may block these factors from appropriately activating myogenic genes. Thus, even if satellite cells or differentiated progenitors migrate or are transplanted into the defect site, their differentiation is initially obstructed by overexpression of negative regulators.The similar gene expression programs generated from VML-injured tissues without and treated with MMGs suggests unrelenting immune and fibrotic responses following VML injury. These results support the hypothesis that persistent inflammation and strong fibrosis overwhelm and dampen myogenic repair. Previously35 for satellite cells or myogenic progenitors in the defect site, which in turn would augment their differentiation and fusion. Loss of adhesive cues and increases in integrins36  has previously been shown to alter muscle stem cell dynamics in aged tissues37 and produce weakened healing responses. Thus, the coupled action of negative regulators and dysregulated ECM produce an environment that is unfavorable for resident or transplanted myogenic cells to mediate optimal myogenic outcomes.Another factor that may be contributing to suboptimal regenerative outcomes and inhibiting treatment efficacy at the time periods sampled is the aberrant deposition and remodeling of ECM in the defect area. Multiple ECM transcripts were strongly upregulated after VML such as fibrillar collagens, lysyl oxidase, and proteoglycans in comparison to proteases that breakdown matrix. The resulting dense composition of the deposited ECM may confer differential integrin signaling38 that effectively limits therapies such as autologous grafts during these early periods  signaling that perpetuate the feedback loop. Anti-fibrotic agents can induce apoptosis of FAPs and differentiated fibrogenic cells and when combined with inhibition of pro-inflammatory signals may permit a more favorable environment for myogenic regenerative cells to “break” the feedback loop. Combinatory treatment of inflammatory and fibrotic signals has previously been shown to attenuate muscular dystrophy pathologies43 and requires further investigation for treatment of VML. The generation of a more favorable microenvironment would then provide invading or transplanted myogenic progenitors to properly differentiate and regenerate tissue. In addition to encountering a more favorable environment, it was shown myogenic progenitors in fast twitch muscles protect against fibrosis via emission of exosomes containing microRNA-206 (miR-206)44, which represses ribosome-binding protein 1, a regulator of collagen biosynthesis. Thus, restoring the lack of or reduced capacity of satellite cells in the defect area may simultaneously attenuate fibrosis45 and promote regeneration.Integrating these results implies that VML injury drives formation of an inhibitive feedback loopods Fig. . Thus, iIn summary, our work provides definitive evidence of the molecular networks that underpin the VML response and progressive degeneration and fibrosis. We used a large-scale data approach to understand how these networks are adjusted when treated with a regenerative treatment strategy. The results of a common response behavior for both treated and unrepaired samples resemble muscular dystrophies, non-dystrophic myopathies, and amyotrophic lateral sclerosis, where continuous cycles of myofiber degeneration and incessant inflammation drive fibrosis. Accordingly, the mechanisms that ultimately drive impaired tissue regeneration after VML suggest this injury may be effectively treated with therapies for dystrophic muscle or other chronic pathologies.n = 4) were purchased from the Rat Resource Center, all rats were housed in a specific pathogen-free animal facility and given food and water ad libitium. All rats received a pre-surgical (~30 min prior) administration of buprenorphine-SR  for pain management. No adverse events were observed.A total of 76 Lewis rats were used in this study. All animal procedures were approved by the Institutional Animal Care and Use Committee and were conducted in compliance with the Animal Welfare Act and in accordance with the principles of the Guide for the Care and Use of Laboratory Animals at the United States Institute of Surgical Research. Inbred male Lewis rats  were purchased from Harlan Laboratories (Envigo) and a subset of Lewis GFP rats (3 pieces of muscle tissue). The minced grafts were prepared from the excised tissue using Vanna scissors. The fascia was closed using 6-0 Vicryl and the skin was closed using surgical staples.Rats underwent surgical creation of a VML injury in the left TA muscle using aseptic technique. Briefly, the lateral incision was made along the lower leg and the skin and fascia overlying the TA muscle were reflected. A spatula tool was placed under the middle third of the TA muscle and a 6-mm biopsy punch was used partially ablate muscle tissue from the region of the muscle. Approximately, 1-mm width of tissue remained on either side of the defect, which provided continuity of proximal and distal regions of the remaining muscle mass. The wet weight of the excised tissue was measured (~86 mg). A subset of rats underwent VML injury repair with autologous MMGs . Composite brightfield images of the complete TA muscle were acquired using Axio Scan.Z1 microscope and ZEN imaging software (Carl Zeiss Microscopy). Additional labeling of muscle sections was completed by combinations of antibodies and probes including DAPI , monoclonal anti-mouse CD68 , monoclonal anti-rat laminin , and polyclonal anti-goat GFP . Appropriate isotype-specific-conjugated secondary antibodies  were then applied. Fluorescent imaging of the TA muscle was conducted with an Olympus FluoView FV1000 laser scanning confocal microscope  mounted on an inverted Olympus IX81 microscope and equipped with Multi-line Argon , HeNeG (543 nm), diode (405 nm), and diode (635 nm) lasers using an Olympus UPLSAPO 20×/0.85 N.A. oil immersion lens. All images were acquired as 12-bit multi-TIFF files.TA muscle tissue isolation and freezing were performed as described previously11. Briefly, TA muscle in vivo physiological properties were measured in anesthetized rats (isoflurane 1.5–2.0%) using a dual-mode muscle lever system . Subcutaneous needle electrodes were inserted in the posterior compartment of the lower limb on each side of the common peroneal nerve. Optimal voltage (2–5 V) was set with a series of tetanic contractions . Then, a skin incision was made at the antero-lateral aspect of the ankle and the distal tendons of the extensor digitorum longus and extensor hallicus longus muscles was isolated and severed above the retinaculum11. TA muscle maximal isometric tetanic torque was measured as a function of stimulation frequency (10–200 Hz) with the ankle at a right angle. The servomotor input and force and displacement transducer outputs are controlled and acquired, respectively, using a PC equipped with a data acquisition board  and custom designed Lab View -based software program.In vivo functional testing of TA muscles was performed using methodology previously describedTA muscles were harvested from euthanized rats, immersed in Trizol (Thermo Fisher) and snap frozen on liquid nitrogen and stored at −80 °C for subsequent use. The tissues were then thawed, homogenized  for 30 s at room temperature and total RNA was isolated from the slurry using the miRNeasy Mini Kit (Qiagen) as per the manufacturer’s instructions. RNA concentration and integrity were measured with a Nanodrop spectrophotometer (Nanodrop 2000c) and Bioanalyzer (Agilent 2100), respectively. Samples that did not pass quality metrics (RIN > 7) were precluded from further processing. 500 ng of isolated total RNA was used to produce cDNA libraries using the Truseq protocol (Illumina), as per the manufacturer’s instructions. Individual libraries were pooled and sequenced using 76 base-pair paired-end reads on a Hi-Seq 2500 in high output mode to an average depth of 35 M reads per library.Rattus norvegicus (Rat) genome (build 6.0), transcript abundances were quantified and gene counts were generated using Kallisto46 with default parameters. To determine sample quality for downstream analysis, per sample read depth was measured, and outliers were detected by conducting PCA on the log2-transformed gene counts. Samples that contained poor read depth (less than 5 million reads) or were determined to be outliers by PCA were removed from downstream analysis.High-throughput RNA sequencing reads were pseudo-aligned to the Genbank-annotated 47. Analysis of expression data, such as set analysis based on treatment-specific expression profiles over time, PCA of treatment by time gene profiles, and correlation of inter-treatment gene expression were performed with the R base package. DE of genes in treated samples relative to uninjured controls was determined using a log2 fold-change threshold greater than 1 or less than −1 and significance was measured using an adjusted p-value less than 0.05 (Benjamini–Hochberg correction for multiple testing). PCA plots from significant DE genes were generated using base R libraries and ggplot2 (version 2.1.0).DE of gene counts was calculated using the R/Bioconductor implementation of DESeq248. Pathway activation/repression was determined by gene set enrichment analysis performed by GAGE with pathway annotations from Gene Ontology Consortium49, KEGG and Reactome50 databases. Significance was measured using an adjusted p-value less than 0.05 (Benjamini–Hochberg correction for multiple testing).Pathway analysis of significant gene sets was performed using the GAGE R/Bioconductor packageα < 0.05 level and data are reported as mean ± SE.Data were analyzed using Prism 6 for Mac OSX . All dependent variables were analyzed separately using repeated measures ANVOAs, when appropriate Tukey HSD analysis was performed. Significance was accepted at the