ESCC) and to provide valuable information for the identification of potential diagnostic biomarkers in ESCC. Tumor samples of ESCC patients retrieved from The Cancer Genome Atlas were divided into Grade 1 , Grade 2  and Grade 3  groups in accordance with the clinical record of the tumor grade of ESCC patients. The genes associated with tumor grade were identified. The signaling pathways of identified genes were enriched from the Kyoto Encyclopedia of Genes and Genomes (KEGG). The diagnostic value of candidate genes was assessed by receiver operating characteristic analysis. We used the GSE23400 dataset generated from the Gene Expression Omnibus to examine the expression levels of candidate genes in ESCC tissues compared to matched mucosa tissues. In total, 440 genes positively correlated with tumor grade and 882 genes negatively correlated with tumor grade were identified. There were 310 differentially expressed genes (DEGs) between G1 and G2, 184 DEGs between G2 and G3, and 710 DEGs between G1 and G3. There were 1322 genes associated with tumor grade that were significantly enriched in pathways in cancer and the phospholipase D signaling pathway. Cyclin‐dependent kinase inhibitor 1A, golgin A7 family member B and transforming growth factor B1‐induced anti‐apoptotic factor 1 (TIAF1) had potential diagnostic value for discriminating ESCC patients with G1 from those with G3. TIAF1 was significantly down‐regulated in ESCC. The results of the present study comprise useful groundwork with respect to determining the tumorigenesis mechanism in ESCC and discovering potential diagnostic biomarkers for ESCC.The present study aimed to identify the genes associated with the histologic tumor grade of patients with esophageal squamous cell carcinoma ( ARNT2aryl hydrocarbon receptor nuclear translocator 2AUCarea under the curveBCRbiospecimen core resourceBDKRB2bradykinin receptor B2BEBarrett's esophagusCA12carbonic anhydrase 12CABC1coenzyme Q8ACDKN1Acyclin‐dependent kinase inhibitor 1ADEGdifferentially expressed geneEACesophageal adenocarcinomaECesophageal carcinomaESCCesophageal squamous cell carcinomaGEOGene Expression OmnibusGERDgastroesophageal reflux diseaseGNA15G protein subunit α15GOLGgolginHCChepatocellular carcinomaJUPjunction plakoglobinKEGGKyoto Encyclopedia of Genes and GenomesLAD1ladinin 1LAMB3laminin subunit β3NSCLCnonsmall cell lung cancerPHLPPPH domain and leucine rich repeat protein phosphatasePKP1plakophilin 1PPARperoxisome proliferator activated receptorPRSS8protease, serine 8ROCreceiver operating characteristicTCGAThe Cancer Genome AtlasTIAF1transforming growth factor B1‐induced anti‐apoptotic factor 1TMEM108transmembrane protein 108TNMtumor, node and metastasisTSStissue source siteEsophageal carcinoma (EC) is a common malignancy of digestive tract carcinoma worldwide, with an estimated 455 000 new cases and 400 000 deaths each year Smoking, a low intake of vegetables and fruits, being overweight, obesity, chronic gastroesophageal reflux disease (GERD) and Barrett's esophagus (BE) are risk factors in EAC The histologic tumor grade of ESCC is segregated into Grade 1 (well‐differentiated), Grade 2 (moderately‐differentiated) and Grade 3 (poorly‐differentiated) according into the World Health Organization classification. Tumor grade is an independent prognostic factor of EC PHLPP2PRSS8) with hypermethylation is significantly decreased in ESCC, which predicts a shorter overall survival of patients with ESCC. Demethylation of PRSS8 contributes to the inhibition of tumor progression, including cell proliferation, migration and cell cycle arrest It is acknowledged that the intersection of etiological factors, including dysregulated genes, aberrant expression of micorRNAs and abnormal DNA methylation, contributes to the initiation and development of ESCC. Over‐expression of miR‐483‐3p results in a poor prognosis for patients through promoting ESCC progression as a result of targeting EI24 The Cancer Genome Atlas (TCGA) database. We aimed to provide the groundwork with respect to determining the tumorigenesis mechanism in ESCC, as well as discovering potential diagnostic biomarkers.In the present study, bioinformatics analyses were performed to identify the dysregulated genes and pathways correlated with histologic tumor grade based on the expression profiling of ESCC in The present study used sequence‐related data from ESCC tissues from the TCGA database. ESCC tissues and clinical metadata of eligible ESCC patients were collected by Tissue Source Sites (TSSs), such as the University of Alabama, the Technical University of Munich and the University of Kansas Medical Center. After a preliminary pathological review, TSSs deliver ESCC tissue samples and metadata to the Biospecimen Core Resource (BCR). Next, the BCR verifies the quality and quantity of the pathological diagnosis of ESCC tissues. The RNA is then extracted from ESCC tissues and also by BCR for genomic characterization and high‐throughput sequencing. Sequence‐related data are deposited in the TCGA database A total of 188 esophageal squamous cell carcinoma patients with clinical records (collected from 26 June 2012 to 28 January 2015) were available in the TCGA database. The tumor grade of ESCC samples is recorded, which was divided into five grade groups, such as GX (unknown), G1 (well‐differentiated), G2 (moderately‐differentiated), G3 (poorly‐differentiated) and G4 (undifferentiated), in accordance with the World Health Organization classification.The inclusion criteria of patients were patients: (a) with a subtype of esophageal squamous cell carcinoma; (b) without a history of other malignancy; (c) without a history of neoadjuvant treatment; (d) for whom the expression profiling of mRNA was available; and (e) for whom the record of histologic tumor grade was G1–G3. In the present study, ESCC patients were separated into G1, G2 and G3 groups in accordance with the recorded tumor grade. Level 3 mRNA sequence data of ESCC patients were downloaded from the TCGA data portal, which is based on UNC Illumina Hiseq_RNASeqV2.rP < 0.05 was considered statistically significant.Those mRNAs with a 0 reads count were excluded from the study. A linear by linear association test rrThe significant correlations between expression levels of genes and tumor grade were visualized via a Box‐plot analysis in P < 0.05 was considered statistically significant.The genes associated with tumor grade were identified. To clarify the expression specificity of those genes, the significance analyses of differentially expressed genes between G1 and G2 groups, between G2 and G3 groups, and between G3 and G3 groups were subjected to Tukey's honest significant difference genecodis3 P < 0.05 was considered statistically significant.To obtain insights into the signaling pathways of genes associated with the tumor grade of ESCC, KEGG pathway enrichment was performed using ARNT2), GOLGA7B, transforming growth factor B1‐induced anti‐apoptotic factor 1 (TIAF1), cyclin‐dependent kinase inhibitor 1A (CDKN1A), peroxisome proliferator activated receptor (PPAR)G and laminin subunit β3 (LAMB3) in ESCC via the pROC package in r.To assess the diagnostic value of candidate genes in ESCC (G1 and G3 groups), the ROC curve was deciphered and the area under the curve (AUC) was calculated to assess the diagnostic value of aryl hydrocarbon receptor nuclear translocator 2 , which is a freely public data repository that archives microarray, next‐generation sequencing and other forms of high‐throughput functional genomics data submitted by the research community.To examine the expression levels of candidate genes in ESCC tissues, the microarray expression profiling of ESCC tissues and adjacent nontumor tissues were obtained from GEO database (GSE23400 (53 ESCC versus 53 adjacent nontumor mucosa tissues) were incorporated in the present study.The microarray expression profiling of ESCC issues was searched and the inclusion criteria of datasets required that: (a) the dataset was generated from the mRNA expression profiling of ESCC patients; (b) both the ESCC and adjacent nonumor mucosa tissues samples were available in the dataset; (c) the sample size of dataset was greater than 50; and (d) sequence data of the candidate genes were available in the dataset. Finally, P‐value indicating the difference between two group was calculated. P < 0.05 was considered statistically significant.Box‐plot analyses were performed to describe the expression of candidate gene both in ESCC and matched mucosa tissues. The A total of 83 ESCC patients were incorporated into the present study based on the exclusion criteria. In total, 16, 47 and 20 patients with ESCC were assigned to the G1, G2 and G3 groups, respectively. There were 20 531 genes available in the mRNA sequence data of the ESCC tumor based on the Illumina Hiseq_RNASeqV2 in TCGA database. After trimming the 6026 genes with a 0 reads count, 14 506 genes were included in the present study.TMEM108), C3orf47 and coenzyme Q8A (CABC1) were the top three genes positively correlated with tumor grade, whereas ladinin 1 (LAD1), TIAF1 and FGF11 were the top three genes negatively correlated with the tumor grade of ESCC patients. The respective expression status of genes in the G1, G2 and G3 groups is shown in Fig. TMEM108 and C3orf47 were positively correlated with tumor grade and eight genes, including LAD1, TIF1, FGF11, carbonic anhydrase 12 (CA12), G protein subunit α15 (GNA15), junction plakoglobin (JUP), plakophilin 1 (PKP1) and PPARD, were negatively correlated with the tumor grade of ESCC patients.Correlations between the expression of genes and tumor grade of ESCC patients were analyzed by a linear by linear association test. Eventually, 1322 genes associated with tumor grade were identified, including 440 genes positively correlated with tumor grade and 882 genes negatively correlated with tumor grade Table . As showIn total, 1322 genes associated with the tumor grade of ESCC were significantly enriched in 14 signaling pathways, including pathways in cancer (hsa05200), the phospholipase D signaling pathway (hsa04072), small cell lung cancer (hsa05222) and gastric acid secretion (hsa04971)  were identified as being differentially expressed genes. There were 310, 184 and 710 differentially expressed genes, respectively, identified in G1 versus G2, G2 versus G3 and G3 versus G1 , between the G2 and G3 groups (G2 versus G3) and between the G1 and G3 (G3 versus G1) groups was analyzed. Genes showing a significant difference (G1 Table . As showTIAF1 (0.862), GOLGA7B (0.869), LAMB3 (0.812), ARNT2 (0.819) and CDKN1A (0.878) was greater than 0.8; the AUC of GOLGA1 (0.762) and PPARG (0.766) was less than 0.8. CDKN1A, GOLGA7B and TIAF1 had a larger AUC than the other four candidate genes. The sensitivity and specificity of CDKN1A was 90% and 68.8%  compared to G3 (poorly‐differentiated) and significantly up‐regulated in G1 compared to G2. In the GSE23400 dataset, it was significantly down‐regulated in ESCC tissues compared to matched mucosa tissues. In addition, TIAF1 could discriminate between ESCC patients with well‐differentiated ESCC from those with poorly‐differentiated ESCC. The results of the present study indicate that TIAF1 might function as a tumor suppressor in the development of ESCC and might also be a potential biomarker for ESCC diagnosis. TIAF1 contributes to the progress of Alzheimer's disease and tumor GOLGA7B and GOLGA1 in ESCC patients with G3 (poorly‐differentiated) were significantly lower than those with G2 (moderately‐differentiated) and G1 (well‐differentiated). GOLGA7B locates at chromosome 10 and encodes GOLGA7B. GOLGA1 locates at chromosome 8 and encodes golgin A1. The exact roles of GOLGA7B and GOLGA1 remain unknown. GOLGAl is reported to be stably expressed in 17 tissues samples, including breast, esophagus, lung, pancreases, colon, rectum and duodenum GOLGA2 encodes golgin A, another member of golgin family, which is required for efficient tracking in the secretory pathway at the Golgi complex In vivo mice model experiments show that down‐regulation of GOLGA2 suppresses lung cancer tumorigenesis by inhibiting angiogenesis and cell invasion GOLGA7B and GOLGA1 were identified as novel genes associated with the tumor grade of ESCC. The biological roles of GOLGA7B and GOLGA1 in ESCC are unknown. Further investigation is necessary to clarify the mechanism of GOLGA7B and GOLGA1 with respect to the initiation and development of ESCC through in vitro and in vivo experiments.In the present study, the expression levels of ARNT2, PPARG, PPARD, FGF11, JUP, CDKN1A, bradykinin receptor B2 (BDKRB2) and LAMB3, overlapped from the 56 genes mentioned above, and 702 differentially expressed genes between the G3 and G1 groups and the top 50 genes negatively/positively correlated with tumor grade were obtained.A total of 56 genes associated with the tumor grade of ESCC was significantly enriched in pathways in cancer Table . Eight kLAMB3 encodes laminin subunit α3 and belongs to laminin family of extracellular matrix glycoproteins (secreted molecules) that have a heterotrimeric structure consisting of α, β and γ subunits. LAMB3, along with LAMA1, LAMA3 and LAMC2, was enriched in pathways involved in cancer. Figure LAMBA3 was significantly down‐regulated in ESCC with G3 (poorly‐differentiated) compared to those with G1 (well‐differentiated) and G2 (moderately‐differentiated). However, the expression status of LAMB3 in GSE23400 was in agreement with the previous studies. The expression level of LAMB3 is reported to be up‐regulated in ESCC compared to normal tissues and LABM3 expression is correlated with the depth of invasion and malignancy ARNT2 and PPARG showed positive correlations with the tumor grade of ESCC and were up‐regulated in G3 (poorly‐differentiated) compared to G1 (well‐differentiated). ARTN2, which encodes aryl hydrocarbon receptor nuclear translocator, is a transcriptional factor related to adaptive responses PPARG encodes peroxisome proliferator activated receptor γ, which is a member of PPAR subfamily of nuclear receptor. PPARG was significantly down‐regulated in ESCC tissues compared to matched mucosa tissues, for which the results were not compatible with our bioinformatics analyses showing that PPARG was significantly up‐regulated in ESCC with G3 (poorly‐differentiated) compared to those with G1 (well‐differentiated) and G2 (moderately‐differentiated). However, the expression status of PPARG in GSE23400 was in agreement with previous studies. PPARG is reported to function as an oncogene in ESCC and the activation of PPARG suppresses cell proliferation and induces cell apoptosis of esophageal cancer cells by inhibiting the TLR‐4 dependent mitogen‐activated protein kinase pathway CDKN1A, also known as CDKN1 and P21, encodes cyclin‐dependent kinase inhibitor 1A. It contributes to regulate cell‐cycle progression by inhibiting the activity of cyclin‐cyclin‐dependent kinase 2/4. In the present study, the expression level of CDKN1A was negatively correlated with the histological grade of ESCC. It was significantly down‐regulated in ESCC patients with G3 (poorly‐differentiated) compared to those patients with G1 (well‐differentiated). Decreased expression of nucleostemin suppresses cell proliferation of esophageal carcinoma by up‐regulating CDKN1A CDKN1A, GOLGA7B and TIAF1 was greater than 0.85, indicating that CDKN1A and GOLGA7B might be potential biomarkers for discriminating ESCC patients with G1 (well‐differentiated) from those with G3 (poorly‐differentiated).The dysregulated genes correlated with the tumor grade of ESCC were identified by bioinformatics analyses based on the TCGA database in the present study. To access the discriminatory ability of those dysregulated genes between ESCC patients with G1 and those with G2, the counts of candidate genes originating from 188 ESCC patients incorporated into our in silicon analysis in the TCGA database were used to depict the ROC curve. The ROC analyses showed that the AUC of GSE23400 dataset was used in our analyses. The expression level of TIAF1, LAMB3 and PPARG was statistically different between ESCC tissues and matched mucosa tissues; however, the expression levels of GOLGA7B, GOLGA1, ARNT2 and CDKN1A were not statistically different between ESCC and matched mucosa tissues. There is heterogeneity of the expression status of candidate genes between the TCGA and GSE23400 datasets. Two confounding factors might contribute to the the contradictory results of the expression status of candidate genes in ESCC between the these datasets. First, different sequencing platforms were used. The mRNA expression profiling of the GSE23400 dataset was based on an Affymetrix Human Genome U133A Array and an Affymetrix Human Genome U133B Array and the mRNA expression profiling of the TCGA dataset was based on RNA‐sequencing. Different sequencing platforms might lead to heterogeneity between the TCGA and GSE23400 datasets. Second, there were different procurement countries for the ESCC samples available in the respective datasets. The procurement countries of ESCC samples in the TCGA dataset included Netherlands, USA, Russia, Ukraine and Canada, whereas the ESCC samples in the GSE23400 dataset were obtained from patients who underwent surgery in China.To detect the expression levels of candidate genes in ESCC tissues, the in vivo and in vitro experiments. Second, the expression levels of candidate genes in ESCC patients compared to matched mucosa tissues and in ESCC patients with well‐, moderate‐ and poor differentiation need to be validated in a large sample size of ESCC patients in future studies. Third, the diagnostic value of CDKN1A, GOLGA7B and TIAF1 needs to be validated by use of a large cohort in clinical practice.The present study comprises a preliminary analysis for the identification of genes associated with the tumor grade of ESCC. There are limitations to our study. First, several candidate genes associated with the tumor grade of ESCC were identified, although the biological roles of corresponding genes with respect to cell differentiation, migration and adhesion of ESCC were not explored through CDKN1A, GOLGA7B and TIAF1 have potential diagnostic value for discriminating ESCC patients with G1 (well‐differentiated) from those with G3 (poorly‐differentiated).Taken together, the association between dysregulated genes and the tumor grade of ESCC was analyzed based on the metadata of patients retrieved from the TCGA database. In total, 1032 dysregulated genes associated with the tumor grade in ESCC were identified and these genes were significantly enriched in the phospholipase D signaling pathway, as well as in pathways involved in cancer and small cell lung cancer. CL designed the experiment. CL and JX analyzed the data. JX and CL collected patient information. JX and CL drafted the manuscript.Table S1. The full list of genes associated with the tumor grade in ESCC.Click here for additional data file.Table S2. The identified differentially expressed genes in G1 versus G2, G2 versus G3 and G3 versus G1.Click here for additional data file.


Longevity assurance homolog 2 of yeast LAG1 (LASS2) has been reported to act as an important tumor suppressor in the development of human cancers. However, little is known about the prognostic value of LASS2 in hepatocellular carcinoma (HCC) . In the present study, we analyzed correlation between LASS2 and TGF-β1 levels, and evaluated their prognostic values in HCC patients. We first analyzed the expression of LASS2 and TGF-β1 in two independent cohorts  using immunohistochemistry (IHC). Kaplan-Meier survival and Cox regression analyses were executed to evaluate the prognosis of HCC. The results of IHC analysis revealed a positive correlation between the expression of LASS2 and TGF-β1. HCC Patients with low expression of LASS2 and TGF-β1 had shorter overall survival (OS) and time to recurrence (TTR) than patients with high expression of LASS2 and TGF-β1. Furthermore, combination of LASS2 and TGF-β1 was an independent and significant risk factor for OS and TTR. In conclusion, low expression of LASS2 and TGF-β1 contributes to the aggressiveness and poor prognosis of HCC, and may represent a novel prognostic biomarker for HCC patients. Hepatocellular carcinoma (HCC) is one of the most common malignant tumors, the third major cause of cancer-related death worldwide245Homo sapiens longevity assurance homolog 2 of yeast LAG1 (LASS2), which is also known as tumor metastasis suppressor gene (TMSG1) or ceramide synthase 2 (CerS2), is the gene identified from a human liver cDNA library by our laboratory13Transforming growth factor-β1 (TGF-β1) plays biphasic functions in tumor tumorigenesis, having a growth inhibitory effect at early stages, but at later stages enhancing the malignant conversion. The TGF-β1 signaling could cross-talk with multiple intracellular signaling in tumor cell. Generally, TGF-β1 signal is transduced from plasma membrane to the nucleus via serine/threonine kinase receptor type I (TβR-I) and type II (TβR-II) and their downstream effectors, smads. Moreover, TGF-β1 signal is also transduced via Smad-independent pathways through activation of mitogen-activated protein kinase (MAPK), nuclear factor kappa-B (NF-κB) and phosphatidylinositol 3-kinase (PI3K)161718202123182627We enrolled a test cohort consisting of 184 HCC patients and a validation cohort of 118 HCC patients, who had undergone curative resection between 2008 and 2013. Using immunohistochemical staining, we examined expression of LASS2 and TGF-β1 in the two independent cohorts, and analyzed their coexpression. The observed LASS2 staining pattern in HCC tissues was both plasma membranous and cytoplasmic. The level and extent of staining varied from weak focal to extensive strong expression. For TGF-β1 staining, TGF-β1 was primarily localized in the cytoplasm of tumor cells, which were producing TGF-β1 . Based oAmong the 184 HCC patients of test cohort, 64 (34.8%) and 100 (54.3%) cases had high expression of LASS2 and TGF-β1, respectively. We performed further analyses to determine the clinicopathological significance of LASS2 and TGF-β1 in HCC. The expression level of LASS2 was negatively correlated with the tumor size, tumor differentiation, and TNM stage . In addiWe determine the prognostic value of LASS2 and TGF-β1 in the test cohort of HCC patients. OS and TTR were evaluated using the Kaplan-Meier method. Our results showed that the median OS time was 46.5 (95% CI: 39.7–53.3) months for HCC patients with low expression of LASS2 and 58.4 (95% CI: 51.9–64.9) months for HCC patients with high expression of LASS2  and TTR , whereas high LASS2 high TGF-β1 group had the longest OS  and TTR  . CollectTo evaluate whether the expression levels of LASS2 and TGF-β1 were independent prognostic value for HCC patients in the test cohort, univariate and multivariate analyses using a Cox regression model were applied. As shown in We observed similar prognostic results of LASS2 and TGF-β1 in another independent cohort consisting of 114 HCC patients after surgery. Among the 114 HCC patients of validation cohort, 40 (35.1%) and 53 (46.5%) cases had high expression of LASS2 and TGF-β1, respectively. Kaplan–Meier analysis showed that low LASS2 group had the shorter OS  and TTR , whereas high LASS2 group had the longrer OS  and TTR  .Furthermore, we found that patients with low TGF-β1 expression also had shorter OS in vitro through interacting with vacuolar ATPase3213Hepatocellular carcinoma is one of the most aggressive cancers featured with high rate of metastasis and mortality. Although several molecular biomarkers have been reported to have clinical significance for predicting HCC prognosis2930Our previous study showed that knockout of LASS2 significantly upregulated the expression of TGF-β1 in DEN-induced liver tumourigenesis of miceTGF-β1 particularly achieves its tumor suppressive effect through the inhibition of proliferation and the induction of apoptosis or senescense in the early stage-tumors18In the present study, we provided the evidence that high level of LASS2/TGF-β1 expression predicted a favorable OS and TTR rate for HCC patients. The 1-, 3- and 5-year OS rates of HCC patients with high level of LASS2 or TGF-β1 expression were remarkably higher than those of HCC patients with low levels of LASS2 or TGF-β1 expression, respectively. Accordingly, the 1-, 3- and 5-year TTR rates of HCC patients with high level of LASS2 or TGF-β1 expression were significantly lower than those of HCC patients with low levels of LASS2 or TGF-β1 expression, respectively. As combination of multiple markers might yield more information for predicting clinical outcome of HCC patients, combination of LASS2 and TGF-β1 expression were therefore used as a predictor of clinical outcome. In both test cohort and validation cohort, HCC patients with both high expression of LASS2 and TGF-β1 had the most favorable OS and TTR rates, whereas those patients with both low expression of LASS2 and TGF-β1 had the poorest OS and TTR rates. Besides, we used univariate and multivariate analyses to further study the prognostic value of LASS2 and TGF-β1, the results showed that combination of LASS2 and TGF-β1 was significant and independent prognostic factor for HCC patients, but either LASS2 or TGF-β1 was not.In conclusion, our study demonstrated that high levels of LASS2 expression and TGF-β1 expression have a positive relationship with prognosis of HCC patients. The combination of LASS2 and TGF-β1 was more sensitive than either of them alone with respect to OS and TTR, and could be used as a new independent prognostic marker of HCC patients.This study involved two independent cohorts of patients with HCC: test cohort (184 HCC patients) and validation cohort (118 HCC patients), obtained from Eastern Hepatobiliary Surgery Hospital  between January 2003 and April 2006. For suspected cases with elevated serum AFP level (>20 ng/ml as positive), computed tomography (CT) and/or magnetic resonance imaging (MRI) were used to verify tumor recurrence. No patient received either radiotherapy or chemotherapy before the surgery, and no other cancers co-occurrence. The overall survival (OS) was defined as the length of time between the surgery and death or the last follow-up examination. The time to recurrence (TTR) was calculated from the date of tumor resection until the detection of tumor recurrence, death or the last observation. Tumor stage was defined according to the American Joint Committee on Cancer  TNM staging systemFor the use of clinical materials for research purposes, prior patients’ consents and approval were obtained from the Ethics Committee of Renji Hospital, Shanghai Jiao Tong University School of Medicine and EHBH of the Second Military Medical University. Written informed consent was obtained from all subjects for publication of this study. All experiments were performed in accordance with approved guidelines of Shanghai Jiao Tong University School of Medicine.HCC tumor tissues were resected from the patients, and then were fixed with 10% formalin and embedded in paraffin before prepared for tissue microarray. Briefly, 1-mm cores were taken from intratumoral tissue and serial sections (4-μm thick) were placed on slides coated with 3-aminopropyltriethoxysilane. The immunohistochemistry analysis was conducted as described previously2 analysis or two-tailed Student t test. Kaplan-Meier analysis was used to assess survival. Log-rank tests were used to compare survival of patients between subgroups. Multivariate analyses were performed by multivariate Cox proportional hazard regression model. Data were presented as mean ± SEM. Differences were considered to be statistically significant for p < 0.05.Differences among variables were assessed by χHow to cite this article: Ruan, H. et al. Co-expression of LASS2 and TGF-β1 predicts poor prognosis in hepatocellular carcinoma. Sci. Rep. 6, 32421; doi: 10.1038/srep32421 (2016).


Avian influenza continues to circulate and remains a global health threat not least because of the associated high mortality. In this study antibody persistence, booster vaccine response and cross-clade immune response between two influenza A(H5N1) vaccines were compared. Participants aged over 18-years who had previously been immunized with a clade 1, A/Vietnam vaccine were re-immunized at 6-months with 7.5 μg of the homologous strain or at 22-months with a clade 2, alum-adjuvanted, A/Indonesia vaccine. Blood sampled at 6, 15 and 22-months after the primary course was used to assess antibody persistence. Antibody concentrations 6-months after primary immunisation with either A/Vietnam vaccine 30 μg alum-adjuvanted vaccine or 7.5 μg dose vaccine were lower than 21-days after the primary course and waned further with time. Re-immunization with the clade 2, 30 μg alum-adjuvanted vaccine confirmed cross-clade reactogenicity. Antibody cross-reactivity between A(H5N1) clades suggests that in principle a prime-boost vaccination strategy may provide both early protection at the start of a pandemic and improved antibody responses to specific vaccination once available.Trial Registration: ClinicalTrials.gov NCT00415129 The first influenza pandemic of the 21st century in 2009 was caused by a novel influenza A(H1N1) strain that was first recognized in Mexico  as adjuvant [The booster study, presented here, was conducted in 2 phases. A cohort of participants at one study site (n = 154) received a booster dose of the 7.5 μg (H5N1) clade 1 vaccine, 6 months after having received the primary immunization course with the 7.5 μg (H5N1) clade 1 vaccine, either without adjuvant or with 30 μg alum. At a later stage and in 2 of the 3 remaining sites, subjects who had been primed with two doses of the 30 μg alum-adjuvanted vaccine, were invited to participate at the booster study which implied that they received one booster immunization with a high dose (30 μg) (H5N1) clade 2 alum-adjuvanted A(H5N1) vaccine at month 22 after the primary immunization . FurtherIn order to assess antibody persistence all participants were invited to undergo a blood test at 6 months after starting the study. Thereafter, all participants who did not receive a booster vaccine at 6 months were invited to have a blood test to assess antibody persistence at 15 and 22 months.In one study site, parallel to the 22-month clade 2 booster immunization an additional 50 A(H5N1) vaccine-naïve adults, aged between 18 and 60 years, were recruited to receive a primary course of the same clade 2 vaccine that was used as booster. These participants received 2 doses of the vaccine given three weeks apart.Adults who had previously participated in the primary study were invited to take part in the follow-up and booster phase . The folThe study was approved by the institutional ethics review boards based in each participating Belgium institution  and the National Health Service Oxford ethics committee for the Oxford site., The study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice. Written informed consent was obtained from all participants before enrolment in this part of the study. The trial is registered with Clinicaltrials.gov: NCT00415129.Up to 30 ml of blood was sampled to assess antibody persistence at 6, 15 and 22-months in the different groups of participants as described above. For those who received booster immunizations at 6 months, blood was sampled prior to and 21 days after immunization. For those who received the 22-month booster immunization, blood was sampled just prior to booster immunization and at 7 and 21 days after immunization. The 7-day blood was taken to look for evidence of early response suggestive of immunological memory. Finally those immunized with a two-dose schedule of the clade 2 vaccine as a primary course had blood sampled prior to each immunization and 21 days after the second dose.3), propagated in embryonated eggs. The dose of Al(0H)3, used as the adjuvant, was 600 μg/dose (expressed as the content of Al3+). Both formulations were presented in ready-to-use multi-dose vials from which 0.5 ml were withdrawn and injected intramuscularly into the deltoid muscle using a standard syringe and 25 G, 25 mm needle.The vaccines were inactivated, split-virion preparations of vaccine strains; clade 1: A/Vietnam/1194/2004/NIBRG-14 (H5N1) (7.5 μg HA without adjuvant) or clade 2: A/Indonesia/5/05-RG2 (H5N1) (30 μg with Al(OH)After each immunization participants were monitored for 30 minutes for immediate reactions. Participants kept diaries in which they recorded any solicited (prelisted) local or systemic reactions that occurred up to 7 days after immunization. Specifically participants were asked to record injection site induration, injection site ecchymosis, temperature, malaise and shivering for 3 days after vaccination. Participants were also asked to record any other adverse event that occurred up to 21 days after immunization. Serious adverse events were collected throughout the study. Participants were followed up between 7 and 28 months (12 months for the group receiving the clade 1 A(H5N1)/Vietnam booster; 22 months for participants who took part in the primary phase of the study only; 28 months for participants who received the clade 2 A/Indonesia booster at 22 months and 7 months for 50 additional participants primed with the clade 2 A(H5N1N)/Indonesia.Serum samples were used for antibody titration against one of the vaccine strains (clade 1 or clade 2) using haemagglutination inhibition (HI) and seroneutralization (SN) assays.The HI assay reflects the ability of the specific anti-influenza virus antibodies to inhibit haemagglutination of erythrocytes by influenza virus as previously described . BrieflyThe SN assay is based on the ability of antibodies to inhibit the infection of Madin-Darby canine kidney (MDCK) cell culture, as previously described . BrieflyIn all assays samples were tested in duplicate and the titer analyzed was the geometric mean of the duplicates, expressed as the reciprocal of dilution. Samples without detectable antibody activity were assigned the titer of half the assay detection limit. These analyses were performed under blinded conditions at the Public Health England laboratories .www.clinicalstudydatarequest.com. If further assistance is required then the corresponding author is to be contacted.Sample size for the additional participants receiving a primary vaccination series of the clade 2 vaccine A(H5N1)/Indonesia, was arbitrarily set at 50 subjects. The sample size required to determine the booster response to clade 2 A(H5N1)/Indonesia was deemed to be lower than the initial cohort size, as only half the original study population were eligible to continue in the study, i.e. those who received the higher dose vaccine during the primary phase. Accordingly, participants at centre 4 were not invited to take part in the booster phase of the study. The minimal data sets underlying the findings are available to interested researchers who in the first instance may request access to anonymized patient-level data and clinical study documents at The study was descriptive and no formal statistical comparisons between groups have been made. The results are presented with 95% confidence intervals of point estimates calculated using normal approximation for quantitative data and exact binomial distribution (Clopper-Pearson method) for proportions. The population analyzed for all the timepoints presented here are the full analysis set. A per-protocol analysis was pre-designated for the A(H5N1) vaccine naïve group receiving the primary course with clade 2 vaccine, however there were no protocol deviations in this group therefore the per protocol and full analysis set were equivalent. For the groups that received either booster vaccination the full analysis set is defined as all those who received the booster immunization. The analysis included all data available at each timepoint.HI data are expressed as GMT’s in accordance with criteria based on those defined for the HI method by the committee for Medicinal Products for Human Use (CHMP) for seasonal influenza vaccines which are applied to pandemic vaccines in absence of specific alternative criteria. These criteria are: a seroprotection rate (% with HI titers ≥ 40 dil-1) of over 70%; a rate of seroconversion or significant titer increase  of over 40% and an increase in geometric mean titer ratio (GMTR) between post and pre vaccination titer by over 2.5. In adults over the age of 60 these criteria are modified to a seroprotection rate of over 60%, seroconversion rate or significant increase in titer of over 30% and a GMTR of over 2 . It is rAnalyses were performed by the Sanofi Pasteur Biostatistics Department, Marcy I’Etoile, France, using SAS software version 8.2 ) and were independently validated by the Oxford Vaccine Group, University of Oxford, using SAS version 9.3 (M.V.).At 6-months after the start of the primary phase of the study 587 out of the original 600 participants remained eligible to continue in the study . For theIn total 33 serious adverse events (SAEs) have been reported in this phase of the study, that is up to 42 days after the start of the study. SAEs that occurred in the primary phase are reported in the primary phase publication . There wUp to 21 days after the booster dose of the 7.5 μg clade 1 A(H5N1)/Vietnam vaccine the percentage of adults aged between 18 and 60 years reporting at least one solicited reaction was 50% in those primed with the high dose adjuvanted vaccine and 35.9% in those primed with the low dose non-adjuvanted vaccine. In adults aged over 60 years at least one solicited event was reported by 19.5% and 27.8% in the high dose and low dose priming groups respectively.Up to 21 days after booster immunization with the high-dose alum-adjuvanted clade 2 A/Indonesia vaccine 74.6% of adults aged between 18 and 60 reported at least one solicited injection site reaction and 42.4% at least one solicited systemic reaction whilst 36.8% and 33.3% of those aged over 60 reported at least one solicited injection site and systemic reaction, respectively.Most common reactions in both groups were pain at the injection site, headache and malaise which started within 3 days of vaccination. Unsolicited reactions that started after 21 days were considered not related to the vaccine. A break down of the type of reactions in the first 7 days after immunization is given in In the naïve group (18–60 years old) who received a primary course of the clade 2 A(H5N1)/Indonesia vaccine no SAEs were reported. Overall 72% of participants experienced a solicited reaction within 21 days of immunization. Injection site pain and headache were the most commonly reported reactions in the first 7 days after immunization .≥32) was 6.2% in the 18 to 60 years group and 14.4% in the over 60 years group (At 6 months after primary immunization with the 7.5 μg clade 1 A(H5N1)/Vietnam vaccine the percentage of individuals who had antibody levels above the threshold for seroprotection (defined as HI antibody titer rs group .In the group who had received the 30 μg adjuvanted clade 1 A(H5N1)/Vietnam vaccine, 6.2% of participants aged 18 to 60 and 18.9% of participants aged over 60 had HI titres > 32. Again these levels decreased with time .Booster response to 7.5 μg clade 1 A/Vietnam vaccine at 6 months .The response to this booster vaccine at 6 months after the primary immunisation as measured by HI antibody did not meet any of the CHMP criteria in either of the two age groups.Booster response to 30μg with adjuvant clade 2 A/Indonesia vaccine at 22 months .The booster response was tested against both clade 1 and 2 viruses. At 7 days after the booster immunization the GMTR was above the threshold required by the CHMP (fulfilling 1 out 3 requirements) in the 18 to 60 year group against both clade 1 and clade 2 strains but not in the over 60 age group.By 21 days after immunization in the 18 to 60 year age group 2 out of 3 CHMP criteria (seroconversion rate and GMTR) were fulfilled when tested against the clade 2 virus and a single criterion (GMTR) was met when tested against the clade 1 virus. In the over 60 year group the only CHMP criterion met was the GMTR for both of the viral strains tested.Primary Response to clade 2 vaccine .In the naïve group who received the clade 2 vaccine only, prior to immunization there was no evidence of prior immunity to the clade 2 A/Indonesia virus. Twenty-one days after a single dose of vaccine one CHMP criterion (GMTR over 2.5) was met. This did not change 21 days after the second immunization. The demographics of the booster and naïve group are given in the supplementary material.This study investigated the potential usefulness of a prime-boost vaccination strategy using two different influenza A(H5N1) strains to induce cross clade protection. The data show that a heterologous booster vaccine strain can induce cross-clade reactivity. Despite the fact that the primary vaccination schedule had induced antibody responses that met CHMP criteria in some groups, 6 months after the booster dose antibody levels had waned below the CHMP thresholds established for primary vaccination. It is important to stress here that no CHMP criteria exist for the duration of antibody persistence and the extent of cross-clade reactivity of influenza vaccines. It may not be indicated to appreciate the value of this prime-boost strategy using a possibly non-appropriate scoring system .Few reports are available on antibody persistence with influenza A(H5N1) vaccines exceding a 6 month period whereas a pandemic threat can persist for several years. Lin et al., (2009) reported that 6-months after immunization with low dose, alum-adjuvanted, whole virion A(H5N1) vaccine antibodies were only present in 4.8% to 20.8% of adults aged under 60 years and thus failed to meet any CHMP at this time point . In contIn the present study participants were followed for 22 months following the first immunization, which is the longest follow-up period reported. Six months after primary vaccination the magnitude of HI titres differed more by age group than by primary vaccination group . In the younger age group 6% of participants had HI titres detected at 6 months, irrespective of the type of vaccine administered. In the older age group 14.4% and 18.9% had detectable HI titres with the low- and high-dose vaccine, respectively. This trend continued for 22 months after immunization and may reflect the effect of prior priming via natural exposure or seasonal influenza vaccine in the older group. In fact even at baseline, prior to A(H5N1) immunization, 16% of the older age group had elevated HI titres; similar results have been found by other investigators .Vaccine-induced cross-clade immune response is defined as the ability to induce immune responses against pathogen strains that are genetically distinct from the strain(s) used to produce the vaccine . PracticThe main study limitation stemmed from the lower than expected antibody responses to the low dose vaccine described in the primary phase of the study  this meaIn conclusion antibody cross-reactivity between A(H5N1) clades suggests that in principle a prime-boost vaccination strategy may provide both early protection at the start of a pandemic and improved antibody responses to specific vaccination once available. Further criteria to assess this strategy are needed.S1 Fig(DOCX)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


To secure elimination, further monitoring is warranted to detect any use of tOPV or monovalent OPV type 2 (mOPV2).The Global Polio Eradication Initiative (GPEI) has made substantial progress since its launch in 1988; only 37 wild poliovirus type 1 (WPV1) cases were detected in 2016, the lowest annual count ever. Wild poliovirus type 3 has not been detected since November 2012, and wild poliovirus type 2 was officially declared eradicated in September 2015. This success is attributable to the wide use of live oral poliovirus vaccines (OPVs). Since 2001, numerous outbreaks were caused by the emergence of genetically divergent vaccine-derived polioviruses (VDPVs) whose genetic drift from the parental OPV strains indicates prolonged replication or circulation  comprises 146 World Health Organization (WHO)–accredited poliovirus laboratories in 92 countries located in the six WHO regions , and enterovirus surveillance . These surveillance systems ensure sensitive and timely detection of circulating polioviruses worldwide. Whereas AFP surveillance has been the standard surveillance system for poliovirus since the beginning of the GPEI, recently, existing environmental surveillance for poliovirus has been expanded  were achieved in all countries. From January to April 2016 , 46 countries were reporting PV2 detected by GPLN laboratories from specimens from persons with AFP or their contacts and sewage samples . From MaField investigations in response to detection of PV2 after the switch found breaches in OPV2 withdrawal with evidence of continued inadvertent use of tOPV in India (During the pre-switch period (January–April 2016), PV2 was detected through both AFP and environmental surveillance; after the switch, PV2 was detected primarily through environmental surveillance  Figure . In counTo provide evidence concerning the origin and significance of circulating PV2, on August 1, 2016, GPLN laboratories began to refer all PV2s detected from all sources for genetic sequencing. Isolation of Sabin-like poliovirus with zero or few nucleotide differences from Sabin2 by GPLN laboratories were instrumental in 1) identifying continued use of tOPV in some countries post-switch and in 2) confirmation of three post-switch cVDPV2 outbreaks caused by genetically related cVDPVs that began circulating before the switch.Virologic monitoring through AFP cases and sewage samples indicate that withdrawal of a live vaccine, OPV2, used in routine immunization programs and mass immunization campaigns, was successfully accomplished by the GPEI. Some evidence of limited use of tOPV after the global tOPV to bOPV switch was found; however, 1 year after OPV2 withdrawal, PV2 has been isolated only in the few areas where mOPV2 has been used in response to detection of cVDPV2 isolates. By expanding the preexisting surveillance network to include environmental surveillance for polioviruses during the last 5 years, GPLN successfully detected VDPV2 emergences and outbreaks to allow GPEI to respond in a timely manner. AFP and environmental surveillance with laboratory testing for poliovirus by GPLN will continue to play a long-term, critical role in ensuring polio eradication  to respond to this outbreak. Nigeria and Pakistan also have circulation of WPV1, and WPV1 circulation continues in Afghanistan.Reintroduction of live PV2-containing vaccine through the use of 19 mOPV2 immunization campaigns to interrupt VDPV2 transmission in six countries , from May 2016 to Mar 2017 has disrupted the goal of interrupting PV2 transmission globally after the switch. The GPEI has established a mOPV2 advisory group, which advises WHO about each use of mOPV2, after an in-depth review of risk assessments conducted after any VDPV2 event or outbreak detection. In countries where no type 2-containing vaccine has been used after the switch, only three countries  have reported VDPV2 detection since September 2016.Environmental surveillance for polioviruses detected the majority of PV2 from September 2016 to March 2017. Detection and sequencing of polioviruses isolated from sewage samples is difficult because these isolates often represent complex mixtures of viruses. Despite these challenges, further expansion of environmental surveillance is needed to maintain the high level of vigilance required to detect and respond to any type 2 poliovirus from all sources in the future, including breaches in containment in facilities retaining or still working with PV2 materials, including WPV2.PV2s were tracked in both human specimens and sewage samples using a newly designed molecular diagnostic assay and algorithm developed by CDC , which was rapidly and efficiently implemented in GPLN laboratories in 2016. PV2 detection and genetic sequencing has been essential for the following: 1) providing evidence of continued use of tOPV after the withdrawal of this vaccine in April 2016; 2) identifying and following up unusual patterns of PV2 detection or circulation that signal gaps in herd immunity against PV2; and 3) classifying VDPV2s as either circulating viruses (cVDPV2s) or originating from immunodeficient persons (iVDPV2s), or of ambiguous origin (aVDPV2s)  has made substantial progress since 1988; in 2016, only 37 wild poliovirus (WPV) type 1 (WPV1) cases were detected, the lowest number ever recorded. WPV type 2 has been eradicated, and WPV type 3 has not been detected since 2012. To reduce the risk for paralysis from infection with vaccine-derived polioviruses (VDPVs), in April 2016, all 155 oral poliovirus vaccine (OPV)–using countries switched from trivalent OPV (tOPV) to bivalent OPV (bOPV), containing vaccine virus types 1 and 3.After the withdrawal and destruction of tOPV, the GPEI devised mechanisms to monitor disappearance of type 2 polioviruses (PV2s) in human populations and the environment. Enhanced environmental surveillance and provision of clear guidance to the Global Polio Laboratory Network has allowed timely, accurate, and comprehensive detection of PV2 by examining approximately 208,000 stool specimens and sewage samples. Preceding the tOPV to bOPV switch (January–April 2016), 43 countries reported detection of PV2; during January–March 2017, the number of countries reporting PV2 had declined to seven.To prevent paralysis caused by VDPVs, elimination of vaccine viruses from the environment will be critical. Lessons learned from surveillance for PV2 after the global synchronized withdrawal of the PV2 component from vaccines have resulted in development of standardized procedures for investigation of PV2 detection in humans and the environment, and handling PV2 in diagnostic laboratories. These lessons will guide the elimination of OPV1 and OPV3 once eradication of polio has been certified.


In patients with ST elevation myocardial infarction (STEMI), myocardial tissue injury is not restricted to the territory supplied by the culprit artery but it affects also the remote myocardium supplied by unaffected arteries.(1) The aim was to investigate the T1 and T2 characteristics in infarcted and remote myocardium comparing it with normal reference standard.30 patients  with STEMI and successful revascularisation by percutaneous coronary intervention were included. Each subject underwent clinical CMR at 1.5 T with T2 and T1 mapping (MOLLI) pre and post contrast (equilibrium contrast technique for extracellular volume (ECV) quantification) within 48hours of presentation. The T2 and pre and post contrast T1 values were evaluated in each of the 16 AHA myocardial segments.Out of 480 myocardial segments, 143 were affected and the rest unaffected or remote. The mean native T1 and the mean ECV in the remote myocardium was higher than the reference standard (1054 ± 65 msec vs 950 ± 21 msec and 0.32 ± 0.06 vs 0.25 ± 0.04). (2) However, the mean native T1 and the mean ECV of the remote myocardium was significantly lower compared to infarcted myocardium . In addition the mean T2 of the remote myocardium was significantly lower compared to infarcted myocardium .This is the first study looking at the impact of STEMI on remote myocardium via non-invasive tissue characterisation by advanced CMR relaxometry technique. Our study highlights that the remote myocardium is also affected following STEMI when compared to normal. Albeit the myocardial tissue injury in the infarcted territory is significantly greater than remote myocardium. These findings may have significant future implications in the treatment of STEMI, including targeting the remote myocardium.


LPP locus. The lesions occurred in the foot by presenting one lump in the plantar soft tissue, and three lesions were detected in the calcaneus and in the navicular bone. All tumors showed the double immunophenotype of epithelial markers and S100 protein expression. No rearrangement of the EWSR1 and FUS loci was detected as reported in myoepitheliomas. However, molecular karyotyping detected an unbalanced rearrangement of the LPP locus, not involving the HMGA2 locus, which is the most frequent translocation partner observed in benign mesenchymal tumors such as lipomas  and pulmonary chondroid hamartoma.We report a case of multiple myoepithelioma with synchronous bone and soft tissue tumors, associated with a new genomic alteration of the  Myoepithelial tumors (METs) of soft tissue and bone are rare tumors of uncertain histogenesis. The first deep tumor was described in the retroperitoneum , followeHistologically, METs are made of a homogeneous population of myoepithelial cells. They could be considered as a part of a continuum with mixed tumors when ductal differentiation is present. They may harbor chondroid and bone differentiation as observed in classical mixed tumors. The diagnosis of MET requires the coexpression of both epithelial markers and S100 protein , 9. TheyEWSR1(22q12) gene fusions have been detected in half of METs arising in deep soft tissues and in up to 70% cases of intraosseous MET [EWSR1 are described: POU5F1(6p21.33) (16%), PBX1 (16%), PBX3, ZNF444, ATF1, KLF17, and NFATC2 [EWSR1-POU5F1 more often occurs in children and young adults while the EWSR1-PBX1 occurs in middle-aged adult patients [EWSR1-PBX1 rearrangement presents a bland sclerotic appearance or clear cell morphology with a diffuse EMA staining. However, none of the EWSR1-rearranged tumors show the presence of ductal or glandular differentiation or cartilage/bone matrix formation [FUS(16p11) gene has also been reported in rare cases of MET arising in deep soft tissue as well as in bone [KLF17 and POU5F1. These FUS-rearranged tumors also lack ductal differentiation [A different genetic pattern distinguishes METs arising in the skin from those in deep soft tissue and bone. eous MET . Severald NFATC2 –16. The patients , 14. Theormation . Rearran in bone , 17. Twontiation .PLAG1 (8q12.1) and HMGA2 (12q14.3) rearrangements are the most common genetic events in pleomorphic adenomas [PLAG1 is also found in MET [PLAG1 gene rearrangement [adenomas . PLAG1 id in MET , 20. In angement . All tumLPP unbalanced rearrangement without EWSR1 and FUS alterations in both soft tissue and bone lesions.We report an unusual observation in a 52-year-old man of a multifocal MET without obvious ductular differentiation and harboring a new A 52-year-old patient complained of pain and swelling of the foot. MRI and plain radiography demonstrated a main lesion in the calcaneus, two others in the navicular bone, and a last one in the plantar soft tissue . The maiThe tumor of the calcaneus measured 4.4 × 3.4 cm , and theImmunohistochemistry demonstrated an intense and diffuse staining of plasmacytoid cells for the broad-spectrum cytokeratin, S100 protein Figures . There wThese results were in favor the diagnosis of a MET. Based on the multiplicity of localizations, a transtibial amputation was decided by the local multidisciplinary committee. Although lesions were multiple and some pictures were suspicious for vascular emboli, the patient had no recurrence or distant metastasis 2 years later. The last follow-up detected small lung lesions, which remained stable and were considered as aspecific.Conventional karyotyping detected the same abnormal complex pseudodiploid clone in the soft tissue tumor as in the bone tumor .EWSR1 and FUS. FISH detected no rearrangement of those loci. Other differential diagnoses were excluded by FISH: extraskeletal myxoid chondrosarcoma (NR4A3) and alveolar soft part sarcoma (TFE3).FISH experiments were performed to look for rearrangement of genes known to be altered in MET arising in deep soft and bone tissues, LPP gene, probably the 8 first exons (Molecular karyotyping detected 2 main deletions on the long arm of chromosome 3 located at 3q22.1-3q26.2 (37Mb)  and 3q27st exons . Three oLPP locus  [KMT2A(11q23) in a secondary acute leukemia [BCL6(3q27) through a 3q27 interstitial deletion in a primary central nervous system lymphomas [Three other A1(6p21) , and in leukemia  and BCL6ymphomas .LPP remains unclear, while the role of the partner gene seems to be crucial. Several lines of evidence suggest that HMGA2 truncation occurring in the most common t fusion gene is implicated in lipomagenesis. However, the HMGA2-LPP fusion protein retains the transactivation functions of two LPP LIM domains which might contribute to the mesenchymal tumorigenesis by directly affecting transcriptional regulation processes [The oncogenic role of rocesses .LPP locus suggests that carboxy-terminal LIM transactivating domains may contribute to the chimeric protein with the aminoterminal part of an unidentified gene. Although not rearranged by FISH, we cannot exclude a cryptic insertion of the 5' part of HMGA2 within the LPP locus. The only candidate locus for a gene fusion with LPP may be TRPS1 (8q23.3) whose 3' part is deleted. TRPS1 is known to be associated with tumorigenesis, metastasis, and angiogenesis in several tumors, including osteosarcoma [LPP-partner gene by 3' RACE-PCR or RNASeq.In the present case, the suspected breakpoint within the osarcoma . UnfortuPLAG1 and HMGA2 have been described in pleomorphic adenomas (mixed tumors) of salivary gland [PLAG1 rearrangements were mainly identified in a subset of cutaneous and superficial soft tissue MET tumors, often displaying ductal structures and considered as mixed tumors [PLAG1 has not been tested because of the absence of ductular differentiation in the tumor.No ductal differentiation was present although membranous EMA staining was focally detected. The stroma was focally myxoid but without a mesenchymal cell population as observed in mixed tumors. Recurrent genetic abnormalities involving ry gland . PLAG1 rd tumors . PLAG1 hLPP (3q27-3q28) locus in synchronous tumors presenting epithelioid features. The partner gene remains to be characterized. Analysis of the LPP locus should be performed by FISH on MET without EWSR1 or FUS rearrangements and pleomorphic adenomas of salivary gland without PLAG1 and HMGA2 aberrations to define the recurrence and the tumor characteristics associated with this new alteration.We describe here a new rearrangement of the


HvPIP2;5 from Hordeum vulgare and investigated its physiological roles in heterologous expression systems, yeast and Arabidopsis, under high salt and high osmotic stress conditions. In yeast, the expression of HvPIP2;5 enhanced abiotic stress tolerance under high salt and high osmotic conditions. Arabidopsis plants overexpressing HvPIP2;5 also showed better stress tolerance in germination and root growth under high salt and high osmotic stresses than the wild type (WT). HvPIP2;5 overexpressing plants were able to survive and recover after a 3-week drought period unlike the control plants which wilted and died during stress treatment. Indeed, overexpression of HvPIP2;5 caused higher retention of chlorophylls and water under salt and osmotic stresses than did control. We also observed lower accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), an end-product of lipid peroxidation in HvPIP2;5 overexpressing plants than in WT. These results suggest that HvPIP2;5 overexpression brought about stress tolerance, at least in part, by reducing the secondary oxidative stress caused by salt and osmotic stresses. Consistent with these stress tolerant phenotypes, HvPIP2;5 overexpressing Arabidopsis lines showed higher expression and activities of ROS scavenging enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), and ascorbate peroxidase (APX) under salt and osmotic stresses than did WT. In addition, the proline biosynthesis genes, Δ1-Pyrroline-5-Carboxylate Synthase 1 and 2 (P5CS1 and P5CS2) were up-regulated in HvPIP2;5 overexpressing plants under salt and osmotic stresses, which coincided with increased levels of the osmoprotectant proline. Together, these results suggested that HvPIP2;5 overexpression enhanced stress tolerance to high salt and high osmotic stresses by increasing activities and/or expression of ROS scavenging enzymes and osmoprotectant biosynthetic genes.We characterized an aquaporin gene  These proteins facilitate the transport of water and small uncharged molecules across biological membranes  was reduced by antisense technology, the NtAQP1-downregulated tobacco plants showed reduced root hydraulic conductivity and wilting phenotypes under water stress  have also been reported, implying the complexity of PIP function in plants  is one of the most agronomically cultivated crops; it is more adaptable to drought, salinity and cold than other cereal crops  in yeast and Arabidopsis and characterized these lines to understand the functions of the barley PIP gene under high salt and high osmotic stress conditions.In this study, we overexpressed barley Hordeum vulgare cv. NP21) cDNA was prepared using superscriptTM III reverse transcriptase , and total RNA was extracted with TRIzol® Reagent . A 873 bp-length HvPIP2;5 coding sequence (GenBank Accession number: AB377270.1) was cloned into TA cloning vector pTOPO2.1  using gene specific primers  at the EcoRI site and named pYES2: HvPIP2;5. For plant transformation, HvPIP2;5 coding sequence was cloned into a standard plant binary vector pCAMBIA2301. The resulting overexpression construct was named pCAMBIA2301-35S:HvPIP2;5.Barley  and FY3 strain only were used in stress assays. The yeast strain FY3 was transformed with a pYES2 empty vector or pYES2:HvPIP2;5 recombinant vector by lithium acetate method .The For stress analysis, transformed yeast cells were propagated in SC-U medium containing 2% galactose for 12 h and cell density was adjusted to 1.0 of OD600 followed by serial dilutions. Yeast cell were spotted on YPD medium supplemented with PEG (4%) or NaCl (200 mM), respectively. Plates were maintained at 30°C and growth was monitored after 2 days.Agrobacterium tumefaciens GV3101 harboring pCAMBIA2301-35S:HvPIP2;5, Arabidopsis Columbia-0 plants were transformed via floral dipping method  media and allowed to germinate at 22°C and 60% relative humidity with a 16/8 h light-dark photo cycle after 3 day stratification at 4°C.For germination analysis, the seeds of WT and homozygote Final concentration of 100–200 mM NaCl  or 10–20% PEG (for high osmotic stress) was supplemented to the MS media for stress administration. Hereby salt stress or osmotic stress indicates high salt stress or high osmotic stress, respectively. Emergence of cotyledons was used as a germination criterium. The number of germinated seeds was expressed as a percentage of total number of seeds planted after 7 days. For root elongation analysis, vertically grown seedlings with 1–1.5 cm long root were transferred onto a MS vertical plate supplemented with or without stress agents (200 mM NaCl and 20% PEG). Root length was measured 15 days after transfer. Root length of seedlings under stress conditions was expressed as a percentage of their respective controls grown on normal MS medium. All stress analysis experiments were conducted three times and each contained 3 biological repeats. For drought test with soil-grown plants, 4 week old seedlings were subjected to drought stress by withholding water supply for 21 days and then re-watered. For salt and osmotic stress, 3 week old plants were irrigated with either half strength MS liquid (control) or half strength MS liquid supplemented either with 200 mM NaCl or 20% PEG to impose stress for 15 days at 2 day intervals and then harvested for analysis. The chlorophyll, proline, and malondialdehyde (MDA) content in control or stress treated plants were determined by the methods reported previously by Lichtenthaler , Bates eHvPIP2;5 OE lines were excised and treated either with 200 mM NaCl or 20% PEG or deionized water after measuring the fresh weight. After 24 h of incubation, the tissues were weighed again for their turgid weight and then dried completely to obtain dry weight. The root RWC was calculated by the following formula; RWC (%) = [(fresh weight − dry weight)/(turgid weight − dry weight)] × 100  staining and hydrogen peroxide by 3, 3'diaminobenzidine (DAB) staining was performed according to methods previously described by Rao and Davis  or half strength MS liquid supplemented either with 200 mM NaCl or 20% PEG to impose stress for 15 days at 2 day intervals and then harvested for analysis. Contents of superoxide and hydrogen peroxide were estimated by methods previously described by Elstner and Heupel  and SagiPlants were treated with water for control or 300 mM NaCl for 6 h or 20% PEG for 6 h, and total RNAs were extracted using RNeasy Plant mini Kit . cDNA was synthesized using RevertAid First Strand cDNA Synthesis Kit , and resulting cDNAs were used for semi-quantitative RT or qRT-PCR experiments for selected genes. Primer pairs used in experiments are shown in Supplementary Table P ≤ 0.05).All statistical comparisons between variances were determined by ANOVA  and least significant differences (LSD) between variants were calculated using Statistix 8.1 computation software. Statistically significant mean values were denoted as * (HvPIP) genes and examined their expressions under salt and osmotic stress conditions , one of the HvPIP2 genes that possesses abundant transcripts and demonstrates down-regulation of gene expression under salt and osmotic stresses . HvPIP2;5 protein shares 81% identity with Arabidopsis PIP2;5 protein . As shown in Supplementary Figure PIP2;5 orthologs . As expected, HvPIP2 proteins showed high degree of homology among them, and HvPIP2;5 was located in the same clade as HvPIP2;1 with 85% identity to HvPIP2;1 , while yeast cells with HvPIP2;5 displayed growth until 10−6 dilution under the same condition  lines were selected. PCR analysis confirmed the presence of the HvPIP2;5 gene in transgenic plants (data not shown) and semi quantitative RT-PCR for HvPIP2;5 expression analysis demonstrated that transgenic lines were overexpressing HvPIP2;5 gene  and 20% PEG (high osmotic stress) was first tested for two independent  Figures . Also, H Figures .HvPIP2;5 OE lines under salinity and osmotic stresses compared to WT. The relative root length of HvPIP2;5 OE lines was significantly higher in the presence of 200 mM NaCl (59–62% vs. 34%) or 20% PEG (47–50% vs. 28%) compared to that of WT  was measured from the root tissues of WT and C Figure . LikewisC Figure .HvPIP2;5 OE lines under high salt (200 mM NaCl) and high osmotic stress (20% PEG). Compared to WT, the HvPIP2;5 OE lines showed significantly weaker NBT staining and less 2O2 content revealed lower amounts of H2O2 in the OE lines than in WT after NaCl treatment  are generated in plants under drought, salt, and temperature stresses. Using nitro blue tetrazolium (NBT) staining for superoxide and diaminobenzidine (DAB) staining for hydrogen peroxide, we measured the levels of ROS in WT and  Figures . Similar Figures . HoweverHvPIP2;5 OE lines than in WT, which implicates lesser membrane damage in HvPIP2;5 OE lines  is an end product of lipid peroxidation of cell membrane lipids and a good indicator of oxidative damage  and superoxide dismutase (SOD) in WT and s Figure , the levs Figure . InteresHvPIP2;5 OE lines under 100 and 200 mM NaCl conditions. GR and APX are major enzymes for the ascorbate-glutathione cycle which is an important component of the ROS scavenging system in plants  and ascorbate peroxidase (APX) in s Figure . The acts Figure .HvPIP2;5 OE lines under salt and osmotic stresses is possibly related to enhanced activities of ROS scavenging enzymes.Taken together, these results indicated that reduction of oxidative stress in 1-Pyrroline-5-Carboxylate Synthase 1 and 2 (P5CS1 and P5CS2) in WT and HvPIP2;5 OE lines. Real time PCR analysis revealed that gene expression levels of P5CS1 and P5CS2 were higher in HvPIP2;5 OE lines than in WT under salt and osmotic stresses  in both yeast and Arabidopsis improved tolerance to high salt and high osmotic stresses   overexpression brought about reduced osmotic stress tolerance in Arabidopsis and tobacco (Jang et al., AtPIP1;2, rice RWC3, and tobacco NtAQP1 share approximately 80% sequence identity. Despite this, overexpression of AtPIP1;2 in tobacco has caused reduced stress tolerance (Aharon et al., RWC3 overexpression in rice and NtAQP1 overexpression in tomato have shown enhanced tolerance under drought and salt stress, respectively (Lian et al., pip2;2 mutants display defects in hydraulic conductivity despite the expression of a very close homolog AtPIP2;3 which shares >96% homology, demonstrating that close aquaporin homologs could not function redundantly even within the same plant (Javot et al., While HvPIP2;5 and AtPIP2;5 share high sequence homology, there are differences in gene regulation. AtPIP2;5 expression levels remain low in Arabidopsis and are only up-regulated by drought and cold (Jang et al., HvPIP2;5 is one of the highly expressed PIPs in barley which is down-regulated by osmotic stress (Katsuhara et al., PIP2;5 in Arabidopsis and barley, and may be attributed to contrasting stress responses in HvPIP2;5 and AtPIP2;5 overexpressors.Although, Another explanation for the contrasting results might lie in the difference in protein sequence between HvPIP2;5 and AtPIP2;5 proteins. Amino acid sequence differences are mainly found in the N-terminus which is expected to be exposed on the cytosol side (Walz et al., HvPIP2;5 overexpressing Arabidopsis, up-regulation of P5CS2, high levels of proline, and increased activities of SOD, CAT, GR, and APX with reduced levels of ROS under drought and salt stress conditions. Particularly, up-regulation of the key proline biosynthetic P5CS genes coincided with increased levels of osmoprotectant proline under salt stress (Figure HvPIP2;5 overexpressing plants seemed to result, at least in part, from increased expression of proline biosynthetic genes and elevated activities of ROS scavenging enzymes. Similar to our findings, the overexpression of wheat TaAQP7, one of the closest homologs of HvPIP2;5, in tobacco enhanced drought tolerance in correlation with decreased levels of MDA and H2O2 and increased activities of SOD and CAT enzymes (Zhou et al., HvPIP2;5 overexpression might sensitize transgenic plants, making the overexpressors react faster to osmotic stress signals and eventually induce enhanced stress defense. Thus, one might speculate that HvPIP2;5 aquaporins in transgenic plants might activate osmotic and salt stress sensing or upstream steps in signaling pathways to induce better stress-tolerance mechanism than WT under stress conditions. Consistent with our speculation, it has been suggested that aquaporins may be part of an osmotic stress signaling cascade (Maurel et al., We found, in s Figure . Prolines Figure . Thus, eHvPIP2;5 can improve tolerance to salt and osmotic stresses when overexpressed in yeast and Arabidopsis. Our results contrast with a previous AtPIP2;5 overexpression study where osmotic stress sensitive phenotypes in AtPIP2;5 overexpressing plants were reported (Jang et al., In conclusion, we have shown that HA, BhL, and SKP designed the experiments; HA, JPA performed the experiments; GR, LS, BhL, and SKP advised the research; HA, BhL, and SKP discussed the results and wrote the paper.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.


This study investigated the potential of a newly proposed scattering foil free (SFF) electron beam scanning technique for the treatment of skin cancer on the irregular patient surfaces using Monte Carlo (MC) simulation. After benchmarking of the MC simulations, we removed the scattering foil to generate SFF electron beams. Cylindrical and spherical phantoms with 1 cm boluses were generated and the target volume was defined from the surface to 5 mm depth. The SFF scanning technique with 6 MeV electrons was simulated using those phantoms. For comparison, volumetric modulated arc therapy (VMAT) plans were also generated with two full arcs and 6 MV photon beams. When the scanning resolution resulted in a larger separation between beams than the field size, the plan qualities were worsened. In the cylindrical phantom with a radius of 10 cm, the conformity indices, homogeneity indices and body mean doses of the SFF plans (scanning resolution = 1°) vs. VMAT plans were 1.04 vs. 1.54, 1.10 vs. 1.12 and 5 Gy vs. 14 Gy, respectively. Those of the spherical phantom were 1.04 vs. 1.83, 1.08 vs. 1.09 and 7 Gy vs. 26 Gy, respectively. The proposed SFF plans showed superior dose distributions compared to the VMAT plans. Advanced radiotherapy techniques such as intensity modulated radiation therapy (IMRT) as well as volumetric modulated arc therapy (VMAT) enable conformal delivery of a prescription dose to deeply seated target volumes while sparing dose to normal tissues –4. Howevet al. suggested using scattering foil free (SFF) electron beams for MERT ., 13.et a× 10 cm2 . Further2 generally resulted in a slightly better plan quality than did the beams with a field size of 1 × 1 cm2. For both cylindrical and spherical phantoms, target conformity as well as body mean dose with the field size of 4 × 4 cm2 were better than those with the field size of 1 × 1 cm2 when the scanning angle was equal to or less than 10° for all phantom sizes. Because fine resolution scanning takes a long beam delivery time, the scanning angle of 10° with a field size of 4 × 4 cm2 seems optimal considering both plan quality as well as treatment time.We tried to find optimal parameters for the SFF scanning technique. In the results, the plan quality was good when the scanning resolution was small enough to result in full overlap between the scanning beams. At the very least, the scanning intervals should be smaller than the field size of the scanning beam to ensure good plan quality. If the scanning resolution was larger than the field size of the scanning beam, drastic degradation of the plan quality was observed. If the scanning resolution was fine enough for full overlap between beams, the field size of 4 × 4 cmTreatment time for the proposed technique was not calculated in this study. The SFF scanning technique synchronized with gantry rotation and couch movement in order to deliver electron beams perpendicular to the patient surface while maintaining the SSD might result in long treatment times. However, we hypothesize that the increased beam output of the SFF beam may partially counteract the increased treatment time. Although the treatment time of the proposed technique is potentially long, the SFF scanning technique provides value due to its considerable dosimetric advantages in comparison with other radiotherapy techniques for the treatment of extensively developed skin cancer on irregular patient surfaces.In this study, we used virtual cylindrical and spherical phantoms with radii from 5 cm to 15 cm because the average radius of a 2-months-old female’s head is 5.9 cm and the average radius of Caucasian adult male’s head is 10.08 cm . As show2, beam output increased by 21 times and bremsstrahlung contamination decreased by 7 times compared to the 6 MeV electron beam with scattering foil. With MC simulations, we demonstrated that the SFF scanning technique with 6 MeV electrons can reduce mean dose to normal tissue by 260% in a spherical phantom that is comparable to the average size of a Caucasian adult male’s head (10 cm radius) while keeping same target coverage, as compared to the VMAT technique. The target conformity and homogeneity of the SFF scanning technique were also better than those of VMAT.By removing scattering foil in the treatment head of 6 MeV electron beam with a field size of 2 × 2 cmS1 TableThe benchmarking results of the simulated 6 MeV electron beam are shown.(XLSX)Click here for additional data file.S2 TableThe 6 MeV electron beams with a field size of 1 cm by 1 cm, which were collimated by the multi-leaf collimators are shown with and without scattering foil.(XLSX)Click here for additional data file.S3 TableThe 6 MeV electron beams with a field size of 2 cm by 2 cm, which were collimated by the multi-leaf collimators are shown with and without scattering foil.(XLSX)Click here for additional data file.S4 TableThe 6 MeV electron beams with a field size of 4 cm by 4 cm, which were collimated by the multi-leaf collimators are shown with and without scattering foil.(XLSX)Click here for additional data file.S5 TableThe 6 MeV electron beams with a field size of 10 cm by 10 cm, which were collimated by the multi-leaf collimators are shown with and without scattering foil.(XLSX)Click here for additional data file.


A previous multi-locus lineage (MLL) analysis of SSR-microsatellite data of old olive trees in the southeast Mediterranean area had shown the predominance of the Souri cultivar (MLL1) among grafted trees. The MLL analysis had also identified an MLL (MLL7) that was more common among rootstocks than other MLLs. We here present a comparison of the MLL combinations MLL1 (scion)/MLL7 (rootstock) and MLL1/MLL1 in order to investigate the possible influence of rootstock on scion phenotype.A linear regression analysis demonstrated that the abundance of MLL1/MLL7 trees decreases and of MLL1/MLL1 trees increases along a gradient of increasing aridity. Hypothesizing that grafting on MLL7 provides an advantage under certain conditions, Akaike information criterion (AIC) model selection procedure was used to assess the influence of different environmental conditions on phenotypic characteristics of the fruits and oil of the two MLL combinations. The most parsimonious models indicated differential influences of environmental conditions on parameters of olive oil quality in trees belonging to the MLL1/MLL7 and MLL1/MLL1 combinations, but a similar influence on fruit characteristics and oil content. These results suggest that in certain environments grafting of the local Souri cultivar on MLL7 rootstocks and the MLL1/MLL1 combination result in improved oil quality. The decreasing number of MLL1/MLL7 trees along an aridity gradient suggests that use of this genotype combination in arid sites was not favoured because of sensitivity of MLL7 to drought.Our results thus suggest that MLL1/MLL7 and MLL1/MLL1 combinations were selected by growers in traditional rain-fed cultivation under Mediterranean climate conditions in the southeast Mediterranean area.The online version of this article (doi:10.1186/s12898-017-0114-3) contains supplementary material, which is available to authorized users. Map3 content (2.0–57.7%)  Table . It has ne soils . We thusne soils . Support3 described above, that some environmental parameters have a differential influence on oil quality in GG1 vs. GG2 trees. The decreasing abundance of trees belonging to GG1 (i.e. common MLL1 grafted on MLL7) along an aridity gradient  on the MLL7 rootstock was governed by two opposing forces. On the one hand, growers in the past may have chosen to graft the common cultivar (MLL1) on MLL7 in order to enhance crop performance under certain environments. As MLL7 was used for grafting much more commonly than other MLLs , 27. As