Glioblastoma (GBM) is both the most common and the most lethal primary brain tumor. It is thought that GBM stem cells (GSCs) are critically important in resistance to therapy. Therefore, there is a strong rationale to target these cells in order to develop new molecular therapies.PBK/TOPK, CENPA, KIF15, DEPDC1, CDC6, DLG7/DLGAP5/HURP, KIF18A, EZH2, HMMR/RHAMM/CD168, NOL4, MPP6, MDM1, RAPGEF4, RHBDD1, FNDC3B, FILIP1L, MCC, ATXN7L4/ATXN7L1, P2RY5/LPAR6 and FAM118A) that were consistently expressed in GSC cultures and consistently not expressed in NSC cultures. The expression of these genes was confirmed in clinical samples (TCGA and REMBRANDT). The first nine genes were highly co-expressed in all GBM subtypes and were part of the same protein-protein interaction network. Furthermore, their combined up-regulation correlated negatively with patient survival in the mesenchymal GBM subtype. Using targeted proteomics and the COGNOSCENTE database we linked these genes to GBM signalling pathways.To identify molecular targets in GSCs, we compared gene expression in GSCs to that in neural stem cells (NSCs) from the adult human brain, using microarrays. Bioinformatic filtering identified 20 genes (PBK, CENPA, KIF15, DEPDC1, CDC6, DLG7, KIF18A, EZH2 and HMMR should be further explored as targets for treatment of GBM.Nine genes:  Glioblastoma (GBM) is the most frequent primary brain tumor. Patient prognosis is poor because tumor cells infiltrating brain tissue surrounding the tumor elude surgery, and adjuvant treatment with irradiation and chemotherapy has only a moderate effect on these remaining cells. As a result, median survival is less than one year , althougUsing methods developed to investigate neural stem cells (NSCs) from the adult human brain –6, we anThe non-cancerous cell type that most closely resembles the GSC is the NSC. NSCs can differentiate into astrocytes, oligodendrocytes and fully functional neurons , 4, and PBK, CENPA, KIF15, DEPDC1, CDC6, DLG7, KIF18A, EZH2 and HMMR) in GSCs and GBM tissues was confirmed with all experimental and bioinformatic methods. They were highly co-expressed in all GBM subtypes and their combined up-regulation correlated with poor patient survival. Thus, there is a strong rationale to explore these nine genes as targets for treatment of GBM.To identify therapeutic targets in GSCs, we combined experimental techniques for analysis of gene and protein expression with public database mining. The increased expression of the selected candidate genes at RNA and protein levels was verified in GSC cultures from independent patient cohorts using qPCR, western blot and immunolabeling. Combining all our results, we found that the increased expression of nine genes  were significantly up-regulated while FAM118A and P2RY5 were down-regulated  coefficient between fold change values on microarrays and qPCR. Average correlation for all genes was r = 0.51, while the best correlation (r = 0.94) was observed for the following genes: CENPA, DLG7, PBK, MCC, MPPG, KIF18A and DEPDC1.To confirm the expression of the 20 selected genes, we used real-time quantitative reverse-transcription PCR (qPCR) analysis on independent sets of freshly isolated samples: seven new GSC and four new NSC cultures Figure ’. For ead Figure ’. We didAD1% medium) [Differences in culturing conditions are known to influence RNA and protein expression levels, as well as causing differentiation of GSCs . Before  medium) , and 3.  medium) . This laNES, GFAP and TUBB3 expressions in GSC, NSC and NFC cultures at RNA and protein levels using qPCR and western blot are also presented  were analyzed Figure . The avey Figure . In compy Figure . The PDTr = 0.88)  Figure  and 2I.AD1% medium, with PDT comparable to the fastest growing GSC cultures, did not express increased levels of these genes  and TCGA (http://cancergenome.nih.gov/). Analysis of the data from these databases confirmed the expression of all 20 genes in GBM tissues , the second eight genes , and the third two genes   were highly expressed in proneural and weakly expressed in mesenchymal tumors  were consistently up-regulated in mesenchymal tumors  Figure . While t) Figure , only th) Figure  and 3B. s Figure . NOL4 ans Figure . FNDC3B e Figure . FNDC3B,s Figure . Only NOectively . This isCENPA, DLG7, PBK, CDC6, KIF15, KIF18A, EZH2, DEPDC1, HMMR, FILIP1L and FNDC3B)  .mesenchymal  correlated with the least favorable survival  all gliomas irrespective of histological grade, and b) LGGs (predominantly grade II). The expression values for 51 probes representing 18 of our genes were significant and could predict survival (not shown). When considering glioma patients as one group irrespective of histological grade, the increased expression of 15 genes , we found good correlation (r = 0.64 + 021) between the RNA and the corresponding protein levels , we found negative RNA-protein correlation (r = −0.35 ± 0.27). The overall correlation for the entire set of 20 genes was r = 0.55 ± 0.41. The nine genes from gene cluster I exhibited very high RNA-protein correlation (r = 0.75 ± 0.12).To further explore the potential for therapeutic targeting, we analyzed the expression of the proteins encoded by the 20 selected genes using western blot, immunolabeling, targeted proteomics and bioinformatics. Western blot showed that 15 of the proteins encoded by the 20 selected genes were up-regulated in all tested GSC cultures: CENPA, DLG7, PBK, FILIPL1, DEPDC1, NOL4, CDC6, KIF15, MPP6, KIF18A, EZH2, HMMR, FAM118A, FNDC3B and MDM1 Figure . Two of 727), DLL3 and PDGFB  that contains information about biomolecular interactions based on experimental evidence. The interactions between molecules represented in this database included protein-protein, protein-DNA, protein-RNA and genetic interactions. In this analysis, we included proteins used in targeted proteomics  were functionally interconnected and built a protein interaction network , whose gene products interacted with a high number of proteins and qualify to be called high degree nodes or “hubs”. KIF15, HMMR and DLG7 were also highly interconnected within this network. EZH2 and CDC6 were connected via CDK2, CDK6, RBL2, and UBB. MCC interacted with DLG7 via APP and with HMMR via CSNK1E. HMMR was further connected with CENPA via ACTB. PBK was interconnected with CENPA, KIF15, CDC6, HMMR, DLG7 and EZH2  and bound 5 proteins  as well as DLL3 and NF1. Cyclin-dependent kinase 1 (CDK1) bound four proteins , while beta actin (ACTB) and retinoblastoma binding protein 4 (RBBP4) bound three proteins each. ACTB bound PBK, CENPA and HMMR while RBBP4 bound CDC6, EZH2 and PBK. Moreover, V-myc avian myelocytomatosis viral oncogene homolog (MYC) bound CDC6 and HIF1A and was functionally related to KIF18A and PBK inferred from genetic evidence  that bound 11 proteins encoded by the following genes: B Figure  as well e Figure . MDM1 anIncreased protein levels for a set of the selected genes were also confirmed in GSCs, NSCs and GBM tissues using immunolabeling Figure . InteresWe have performed a global analysis comparing GSC and NSC cultures and GBM tissues used in this work to iPS cells, neurons, iPS-derived neurons, astrocytes, fibroblasts, NSCs, NFCs, breast cancer cells (BCC), leucocytes, brain tissue, GBM, ESCs and many additional sets of GSCs from the GEO database. For visualization we used principal component analysis (PCA). This analysis showed that our sets of NSCs and GSCs clustered together with other NSCs and GSCs from the GEO database Figure . The firEZH2) showed reduced expression in astrocytes when compared to GSCs. Although the trend was similar for all eight genes, statistical significance was not reached in all tests . Eight genes  showed reduced expression in neurons when compared to GSCs. Also here the trend was similar for all eight genes although statistical significance was not reached in all tests. The expression of all genes, except CDC6, was relatively high in ESCs. The expression of DEPDC1 and HMMR was significantly higher in ESCs than in GSCs. The expression of HMMR and DLG7 was significantly higher in iPS cells than in GSCs.The GEO data sets were also used to compare expression of the nine potential target genes in GSCs, astrocytes, DA-neurons, iPS-derived neurons, iPS cells and ESCs . Eight gIt has been shown that GSCs are involved in GBM development and resistance to therapy –15. HencThe present study identified a set of 20 genes that does not overlap with the 30 genes from our previous study . In the Unlike many studies using brain tissue as a control, we used NSCs isolated from different areas of the adult human brain. Altogether we used 16 GSC and nine NSC cultures. To our knowledge, few reports have used such a high number of primary stem cell cultures to identify and verify candidate genes. While GSCs were propagated as spheres, the NSCs used for the validation experiments were cultured under three different sets of conditions, which reinforced the robustness of our findings. Both GSC cultures and the controls (NSC and NFC cultures) were properly matched with regard to state of differentiation and cell PDT Figure . AlthougPBK, CENPA, KIF15, DEPDC1, CDC6, DLG7, KIF18A, EZH2, and HMMR) whose expression was confirmed using all experimental and bioinformatics verification methods    were shown to be up-regulated in GBM [PBK, DLG7, and KIF18A as up-regulated in GBM and classified them as mitosis, DNA replication, and chromosome (MRC) organization genes [EZH2 and HMMR have been extensively studied in GBM and GSCs [EZH2 has been identified as one of the key players in GBM and its increased expression correlates with a poor clinical outcome [HMMR has recently been suggested as a potential therapeutic target in GSCs [HMMR is also involved in cell division [DLG7 also regulates KIF18A localization in the cell [Prior to the present study, six of the nine potential molecular targets  were all part of the same protein-protein interaction network , and Hedgehog and canonical Wnt signaling Figure . It has em cells , whereasem cells , 55. Ourem cells .PBK, CENPA, KIF15, DEPDC1, CDC6, DLG7, KIF18A, EZH2, and HMMR) in GSCs, but also in the range of independent methods used to verify the results. Gene knockdowns of EZH2 and HMMR already revealed that these two genes are essential for survival of GSCs and thus very promising new molecular targets for treatment of GBM [in vitro and attenuates tumor growth in vivo [We believe that the value of our study lies not only in the identification of nine important genes and potential therapeutic targets  at a density of 105 cells/ml and supplemented with EGF and bFGF twice a week. GSC cultures were characterized for the following stemness markers: CD133, SSEA-1/CD15, CD44, CD166 and A2B5. GSC cultures were orthotopically xenografted to confirm tumor initiation properties [et al., in revision, Mughal et al., in prep).Following informed consent, tumor samples classified as GBM according to the World Health Organization criteria were obtained from patients undergoing surgical treatment at Oslo University Hospital in accordance with the appropriate Institutional Review Boards . Normal operties , 58, 59 AD1% medium) [AD1% medium for one passage and were thereafter incubated on RN  in serum-free Neurobasal A medium (the same medium as for NFCs).NFCs  were cultured as spheres in serum-free Neurobasal A medium (Gibco) containing B27 (Gibco), 2 mM L-glutamine, 10 ng/ml bFGF, and 20 ng/ml EGF (both from R&D Systems). Adherent NSCs were cultured in a modified neurosphere medium containing 1% FBS, 10 ng/ml bFGF and 20 ng/ml TGFα ( medium) . NSCs on−ΔΔCT method and REST software [Total RNA was isolated using the RNeasy Mini Kit (Qiagen) and the concentration was determined with a Nanodrop spectrophotometer. For cDNA synthesis, experimental set up and oligonucleotide design, we used the procedure previously described . cDNA wasoftware . Each GSr) calculation  and centrifuged through a QIAshredder . A total of 20–40 μg of whole protein extract was mixed with the loading buffer  and loaded onto a 4–12% gradient Nu-PAGE gel (Life Technologies). Protein gels were blotted onto 0.45-μm PVDF membranes. The membranes were blocked with 5% skimmed milk in TBS/0.1% Tween 20  and probed with primary antibodies diluted in the same solution. The primary antibodies, obtained from Cell Signaling Technologies , were incubated in bovine serum albumin according to the recommended procedures. The secondary antibodies were HRP-conjugated anti-rabbit/mouse/goat/rat IgGs (1:10000). For a complete list of antibodies and the working concentrations, see GBM tissue samples were fixed in 4% PFA, cryoprotected in 20% sucrose and frozen in OCT . The blocks were cryo-sectioned and immunolabeling was performed as previously described , 63. CelSphere-forming ability was measured by plating 500 cells/well in 200 μl neurosphere medium in 96-well plates. After 10 days, the plates were scanned using GelCount  and the sphere colonies were quantified using GelCount software.We used the following formula: PDT = t log 2 / log Nt- log No where t = time period, Nt = number of cells at time t and No = initial number of cells.The initial selection of the 20 candidate genes was performed using excel-based analysis of 32875 transcripts from the array data set GSE31262 . By filthttp://rembrandt.nci.nih.gov were accessed on May the 15th 2012. The repository contained 249 microarrays of GBM grade IV patients and 66 microarrays of Astrocytoma grade II patients. The microarrays were from the Affymetrix Gene Chip Human Genome U133 Plus 2.0 Array platform. The R/Bioconductor package Robust Multiarray Average (RMA) [P-values from the logistic regression were corrected for multiple testing using the Benjamini-Hochberg procedure [REMBRANDT: Microarray data from the Repository for Molecular Brain Neoplasia Data  , 65 was https://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp) are based on tissue samples from 598 GBMs and 10 normal samples (TCGA: The gene expression data ( samples . The reshttps://tcga-data.nci.nih.gov/docs/publications/gbm_exp/ and processed using J-Express (Molmine) for hierarchical clustering. Patient survival was calculated using Prism 6 .For Figure http://vanburenlab.tamhsc.edu/cognoscente.html) that enables visualization of biomolecular interaction documented in the literature. The gene P2RY5 was not in the database while there were no interactions found for FILIP1L. Due to complexity of the network only the protein-protein interactions concerning the 20 selected proteins, BMI1 and HIF1A are presented (Figure For Figure d Figure . Some ofAD1% medium.For Figure


Gossypium raimondii through database searches and were classified into four different subfamilies based on phylogenetic analysis. All 19 membrane-bound FAD proteins shared three highly conserved histidine boxes, except for GrFAD2.1, which lost the third histidine box in the C-terminal region. In the G. raimondii genome, tandem duplication might have led to the increasing size of the FAD2 cluster in the Omega Desaturase subfamily, whereas segmental duplication appeared to be the dominant mechanism for the expansion of the Sphingolipid and Front-end Desaturase subfamilies. Gene expression analysis showed that seven membrane-bound FAD genes were significantly up-regulated and that five genes were greatly suppressed in G. raimondii leaves exposed to low temperature conditions.To produce unsaturated fatty acids, membrane-bound fatty acid desaturases (FADs) can be exploited to introduce double bonds into the acyl chains of fatty acids. In this study, 19 membrane-bound FAD genes were identified in  Both saturated and unsaturated fatty acids are major components of membrane phospholipids in plants as well as triacylglycerols in seeds. Unsaturated fatty acids usually contain one or more double bonds in their acyl chains. The number and position of double bonds in fatty acids profoundly influence their physical and physiological properties ,2. The dOsFAD8 has been reported to have a functional role in stress tolerance at low temperatures  ,29 G. ra/search)  and SMARerg.de/)  were useMultiple sequence alignments of full-length protein sequences were performed using Clustal X version 2.0  with defhttp://gsds1.cbi.pku.edu.cn/) [To illustrate exon-intron organization for an individual gene, the Gene Structure Display Server   was emplG. raimondii chromosomes [The location data of all membrane-bound FAD genes were acquired from the genome annotation document. The chromosome location image of membrane-bound FAD genes was generated using MapInspect software according to their starting positions on the omosomes ,36.G. raimondii was defined according to (1) the length of aligned sequence cover was > 80% of the longer gene, (2) the identity of the aligned regions was > 80%, and (3) only one duplication event was counted for tightly linked genes [Gene duplication of membrane-bound FAD genes in ed genes ,37,38. WG. raimondii were grown in a temperature-controlled chamber at 28°C with a photoperiod of 16 hours light and 8 hours dark. After ten days, the leaves, stems, roots, and cotyledons of some seedlings were sampled to analyze tissue-specific expression. To examine the expression patterns of membrane-bound FAD genes under low temperature stress, the plant leaves of the remaining seedlings treated at 10°C in the temperature-controlled chamber were harvested at 0, 3, 6, and 12 hours, which represented normal plants, slight stress, moderate stress, and severe stress, respectively. All collected samples were immediately frozen in liquid nitrogen and stored at -80°C. Three biological replicates were conducted per sample.All the plants of Premix Ex Taq  in the BioRad CFX96 Real-time PCR System following the manufacturer’s instructions. The cotton UBQ7 gene was used as an internal reference for all the qRT-PCR analyses. Each sample was performed in three biological replicates. The relative expression levels were calculated according to the 2-ΔΔCT method [Total RNA was extracted from all samples using the EASYspin Plus Total RNA Extraction Kit , and first-strand cDNAs were synthesized with the PrimeScript 1st Strand cDNA Synthesis Kit  according to the manufacturer’s protocols. For quantitative real-time RT-PCR (qRT-PCR) assay, gene-specific primers were designed for the membrane-bound FAD genes according to their CDSs . The qRTT method . The expT method .G. raimondii genome using the BlastP and tBlastN programs with the query sequences of Arabidopsis membrane-bound FAD genes. The retrieved sequences were submitted to the Pfam and SMART databases to confirm the presence of conserved domains (Pfam: PF00487). As a result, 19 non-redundant membrane-bound FAD genes were confirmed in G. raimondii. For comparative analysis, the membrane-bound FAD genes in rice (http://rice.plantbiology.msu.edu/index.shtml) following the same strategy. All identified FAD genes were named according to their orthology with reported counterparts in Arabidopsis. Detailed information about the 19 membrane-bound FAD genes in G. raimondii is provided in The candidate membrane-bound FAD genes were identified from the  in rice  were alsG. raimondii, Arabidopsis, and rice. In previous reports, membrane-bound desaturases from eukaryotic genomes were divided into four functional subfamilies: First Desaturase, Omega Desaturase, Front-end Desaturase, and Sphingolipid Desaturase [To evaluate the phylogenetic relationships of membrane-bound FAD genes in different species, an unrooted phylogenetic tree was constructed according to the alignments of full-length protein sequences of membrane-bound FADs in saturase . As showArabidopsis, but only one gene was found in G.raimondii, which was a homolog of AtADS3 .The First Desaturase subfamily included Δ7 desaturases and Δ9 desaturases, encoded by ADS genes, which generally introduced the first double bond into the saturated acyl chain . There wG. raimondii, Arabidopsis and rice had one FAD6 gene each, but the number of FAD2 genes was diverse, with an expanded number (up to five) in G. raimondii, which was greater than that in Arabidopsis (one) and rice (three). FAD3, FAD7, and FAD8 formed the other cluster, which contained three FAD genes from Arabidopsis, four from rice, and five from G. raimondii.The Omega Desaturase subfamily contained Δ12 desaturases and Δ15 desaturases, which introduce a double bond between an existing double bond and the acyl end . The Δ12G. raimondii, two SLD genes in Arabidopsis, and one SLD gene in rice.The Front-end Desaturase subfamily was comprised of sphingolipid Δ8 desaturases, which were encoded by SLD genes ,15. FiveArabidopsis, one gene from rice, and two genes from G. raimondii.The last group was the Sphingolipid Desaturase subfamily, which was represented by sphingolipid Δ4 desaturases ,47. The G. raimondii was greater than that in Arabidopsis and rice in three of four subfamilies  retrieval motif consisting of Φ-X-X-K/R/D/E-Φ (Φ are large hydrophobic amino acid residues such as F/Y/W/I/L/V) at the C-terminus . Expecteal motif . The mot5 domain . Throughterminus .G. raimondii -PCR analysis (data not shown), this gene was not included in the qRT-PCR analysis. As shown in GrFAD3.2, GrFAD8.1, and GrSLD5 were expressed at high levels in roots. GrFAD2.3, GrSLD3, GrSLD2, GrSLD1, GrFAD3.1, and GrDSD1 shared high expression levels in young stems. GrFAD5 and GrFAD6 displayed the highest transcript abundance in cotyledons. And GrFAD7 and GrFAD2.4 were predominantly expressed in leaves. These results demonstrated that the majority of these membrane-bound FAD genes exhibited tissue-specific expression patterns, which was consistent with membrane-bound FAD genes in Arabidopsis and soybean, which also showed specific spatial expression patterns [To investigate the expression profiles of membrane-bound FAD genes in different tissues of patterns .GrFAD2.3 showed preferential expression in the stem and cotyledon, but that in the root and leaf was limited. And GrFAD2.4, which was involved in a duplication event with GrFAD2.3, showed preferential expression in the stem and leaf.Furthermore, the tissue-specific expression patterns of the genes involved in duplication events were compared. Although the genes in all four duplicated gene pairs shared high sequence similarity and the same gene structure, the gene expression patterns were highly diverse. For instance, G. raimondii membrane-bound FAD genes in the leaves of 10-day-old seedlings under low temperature stress (10°C) were investigated in this study, and all the gene expression levels responsive to slight (3 hours), moderate (6 hours), and severe (12 hours) cold stresses were compared with those of normal plants as shown in GrFAD2.1. Out of the 18 membrane-bound FAD genes, seven genes, i.e., GrFAD8.1, GrFAD2.2, GrFAD8.2, GrSLD2, GrSLD4, GrDSD1, and GrSLD5, showed a marked increase in transcript level when treated with cold stress. Among them, the expression levels of GrFAD8.1 and GrSLD5 were higher at 12 hours after cold treatment, and GrSLD2 and GrSLD4 almost reached their highest levels at 6 hours after cold treatment, whereas GrFAD2.2, GrFAD8.2, and GrDSD1 were expressed highly at 3 hours of cold treatment. Additionally, the expression levels of 5 other genes, i.e., GrFAD2.4, GrFAD3.1, GrFAD3.2, GrFAD2.5, and GrDSD2, were slightly up-regulated in response to cold stress. In contrast, the five genes GrFAD5, GrFAD7, GrFAD2.3, GrSLD1, and GrSLD3 were significantly down-regulated after long periods of cold stress treatment, and one gene, GrFAD6, was suppressed slightly during treatment with cold stress. There were also some differences in expression patterns between duplicated genes under low temperature.The expression patterns of the G. raimondii. Undoubtedly, the 19 membrane-bound FAD genes identified in the diploid cotton will provide candidate genes for the gene engineering of fatty acid biosynthesis in cotton.FAD genes encode the enzymes that catalyze the desaturation of fatty acids, which affect the oxidative stability and nutritional value of seed storage oils . Many stArabidopsis. However, following this nomenclature, it was difficult to distinguish FAD7 and FAD8, due to their high degree of homology. It has been demonstrated that FAD7 was highly expressed at high temperatures [FAD8 increased at low temperatures [GrFAD7 and two isoforms of GrFAD8 (GrFAD8.1 and GrFAD8.2) were designated. GrFAD7 was suppressed at low temperature, whereas GrFAD8.1 was consistently induced under long duration cold exposure and GrFAD8.2 was rapidly induced at 3 hours under low temperatures.The FAD genes were named based on their orthologous genes in eratures ,62 and teratures ,62,63,64G. raimondii, except for GrFAD2.1, had the three highly conserved histidine boxes, which contained strongly conserved histidine residues. GrFAD2.1 contained the first and second histidine boxes in the N-terminal region, but lost the third histidine box. Moreover, the gene structure of GrFAD2.1 was also different from other FAD2 genes. By using five gene-specific primers, GrFAD2.1 was not found to be expressed in this study. These observations suggested that GrFAD2.1 might not be a functional gene. Considering the adjacent location of GrFAD2.1 to GrFAD2.2 on chromosome 13, it could be deduced that GrFAD2.1 might have undergone significant functional divergence or even have become a pseudogene after originating from an ancient tandem duplication event of the FAD2 gene in the G. raimondii genome.Analysis of protein structure showed that all of the 19 membrane-bound desaturases in G. raimondii was much greater than that in Arabidopsis and rice. Phylogenetic analysis of membrane-bound desaturases in G. raimondii, Arabidopsis and rice indicated that all subfamilies except for the First Desaturase subfamily contained more gene members in G. raimondii than in Arabidopsis and rice. The increased number of members of the G. raimondii membrane-bound FAD gene family belonging to the three subfamilies suggested that they might have undergone species-specific expansion during the process of evolution. In this study, the gene duplication events, including tandem and segmental duplications, were investigated to elucidate the expansion mechanism of the membrane-bound FAD gene family in G. raimondii. Four duplicated gene pairs, including eight genes out of the 19 membrane-bound FAD genes, were identified. Among them, one tandem duplicated gene pair, GrFAD2.3/GrFAD2.4, belonged to FAD2 cluster of the Omega Desaturase subfamily. A segmental duplicated gene pair, GrDSD1/GrDSD2, belonged to the Sphingolipid Desaturase subfamily. And the remaining two segmental duplicated gene pairs, GrSLD1/GrSLD2 and GrSLD4/GrSLD5, belonged to the Front-end Desaturase subfamily. These results showed that the tandem duplication might contribute to the increasing size of the FAD2 cluster and that the expansion of the Sphingolipid and Front-end Desaturase subfamilies was due to the segmental duplication.Gene duplications play a significant role in the expansion of gene families in the genome ,66. The Duplicated genes may experience three outcomes, i.e., non-functionalization , neo-functionalization (acquisition of novel functions), or sub-functionalization , during the process of evolution . Gene exG. raimondii membrane-bound FAD genes in leaves under low temperature were investigated at the whole family level. As a result, GrFAD8.1, GrFAD2.2, GrFAD8.2, GrSLD2, GrSLD4, GrDSD1 and GrSLD5 were found to be significantly up-regulated in response to cold stress, which suggested that these genes might be required to maintain appropriate levels of related unsaturated fatty acids in cotton plants under low temperature conditions. Conversely, GrFAD5, GrFAD7, GrFAD2.3, GrSLD1 and GrSLD3 were heavily down-regulated after long periods of cold stress treatment. In the previous study, two FAD2 genes, designated FAD2-3 and FAD2-4, were found to be induced in upland cotton (G. hirsutum) under cold stress [G. raimondii, of which GrFAD2.2 was induced under low temperature, GrFAD2.4 and GrFAD2.5 were slightly up-regulated in response to cold, and GrFAD2.3 was suppressed under low temperature. These results suggested that specific isoforms of FAD2 genes might play a vital role in cotton response to cold stress.Low temperature is one of the serious environmental stresses affecting cotton development and production. Previous studies revealed that FAD2 and FAD7/8 genes participated in cotton adaptation to cold stress ,25, and d stress . In thisArabidopsis, and deficiency of the genes resulted in enhanced sensitivity to prolonged low-temperature exposure [GrSLD2, GrSLD4, and GrSLD5 were highly expressed after long periods of cold treatment, whereas GrSLD1 and GrSLD3 were suppressed under low temperatures, suggesting that SLD genes are critical for cotton response to cold stress.SLD genes encoding the sphingolipid Δ8 desaturases have been well studied in exposure . Here, GS1 FigGrFAD2.1, which was excluded from the Omega Desaturase subfamily.The height of the letter designating the amino acid residue at each position represents the degree of conservation. The numbers on the x-axis represent the residue positions within the boxes. The y-axis represents the information content measured in bits. Note that all protein sequences in each subfamily were included in the analysis, with the exception of (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Soluble tumor necrosis factor receptors 1 (sTNFR1) and 2 (sTNFR2) have been associated to progression of renal failure, end stage renal disease and mortality in early stages of chronic kidney disease (CKD), mostly in the context of diabetic nephropathy. The predictive value of these markers in advanced stages of CKD irrespective of the specific causes of kidney disease has not yet been defined. In this study, the relationship between sTNFR1 and sTNFR2 and the risk for adverse cardiovascular events (CVE) and all-cause mortality was investigated in a population with CKD stage 4-5, not yet on dialysis, to minimize the confounding by renal function.In 131 patients, CKD stage 4-5, sTNFR1, sTNFR2 were analysed for their association to a composite endpoint of all-cause mortality or first non-fatal CVE by univariate and multivariate Cox proportional hazards models. In the multivariate models, age, gender, CRP, eGFR and significant comorbidities were included as covariates.During a median follow-up of 33 months, 40 events (30.5%) occurred of which 29 deaths (22.1%) and 11 (8.4%) first non-fatal CVE. In univariate analysis, the hazard ratios (HR) of sTNFR1 and sTNFR2 for negative outcome were 1.49 : 1.28-1.75) and 1.13 (95% CI: 1.06-1.20) respectively. After adjustment for clinical covariables  both sTNFRs remained independently associated to outcomes . A subanalysis of the non-diabetic patients in the study population confirmed these findings, especially for sTNFR1.sTNFR1 and sTNFR2 are independently associated to all-cause mortality or an increased risk for cardiovascular events in advanced CKD irrespective of the cause of kidney disease. Chronic kidney disease (CKD) has been linked to increased risk for cardiovascular disease and mortality independent of traditional cardiovascular risk factors . This haSoluble tumor necrosis factor receptor 1 (sTNFR1) and soluble tumor necrosis factor receptor 2 (sTNFR2) are the circulating forms of their membrane bound counterparts (mTNFR1 and mTNFR2) which are essential for tumor necrosis factor alpha (TNFα)-signalling via different pathways. Interaction between TNFα and both mTNFR leads to a pro-inflammatory stimulus via activation of nuclear factor kappa B (NF-κB) or activator protein 1 (AP-1), while only mTNFR1 contains a death domain through which signalling leads to apoptosis . The solIn contrast to TNFα, which failed to be associated to mortality or cardiovascular events in CKD , both ciIn the present study, we evaluated the predictive value of sTNFR1 and sTNFR2 for the risk for all-cause mortality or cardiovascular events in a population with advanced CKD . Evaluating outcome in advanced CKD minimizes the influence of eGFR on outcomes and on the concentration of the evaluated marker. This may allow to generate hypotheses on the contribution of other important mechanisms in the pathophysiology of CKD, such as inflammation, while reducing the impact of their intrinsic association to kidney function.This study was approved by the local Ethics Committee  and performed in accordance to the Declaration of Helsinki. Written informed consent was obtained from all participants.All non-transplanted CKD patients stage 4 and 5 not on dialysis, attending the Nephrology outpatient clinic and included in the biobank sample collection of the Nephrology Department of the Ghent University Hospital between January 2011 and August 2012, were included in this study (n = 131). Samples were processed immediately after collection and stored at—80°C. Outcomes were registered prospectively.2 (kg/m2), mean arterial pressure (MAP) as the sum of 1/3 of the systolic and 2/3 of the diastolic blood pressure and pulse pressure (PP) as the difference between systolic and diastolic blood pressure. Estimated glomerular filtration rate (eGFR) was calculated based on the creatinine-based CKD-EPI formula . Twenty-nine patients (22.1%) died (cardiovascular (n = 3), malignancy (n = 3), infection (n = 7), ESRD (n = 1), unknown (n = 15)) and 11 (8.4%) had a first non-fatal CVE (ACS (n = 1), PTCA (n = 4), CABG (n = 2), stroke (n = 4)), of which 5 patients died subsequently during follow-up. Seven events occurred after the start of RRT, which was initiated in 35 patients during follow-up. Baseline clinical characteristics and biochemical parameters of the entire population and those with and without event are presented in sTNFR1 and sTNFR2 were strongly correlated  . Linear In univariate Cox proportional hazards analysis, sTNFR1  and sTNFR2  were associated to adverse outcomes. Older age, a higher CRP, a lower albumin concentration, a history of cardiovascular disease and diabetes mellitus also showed an association . Kaplan In multivariate models, sTNFR1 and to a smaller extent sTNFR2 remained significantly associated to outcome. In the first model  after adWhen including also albumin in this multivariate model, sTNFR1  remained significantly associated to adverse outcome, while sTNFR2 was not. Albumin itself was not a significant covariate.In summary, after adjustment for significant clinical covariables, sTNFR1 and sTNFR2 remained independently associated to increased risk of death or cardiovascular events. This association was stronger for sTNFR1 than for sTNFR2 .Eighteen of the eighty patients without diabetes had an adverse event (22.5%). Patients, having an event, were older , had a higher CRP , had more often a history of malignancy  and were less likely to start RRT during FU (5.6% (n = 1) vs. 29% (n = 18), p < 0.05). There were no differences for the other clinical variables listed in In the subgroup of diabetes patients (n = 51), only sTNFR1 and not sTNFR2 concentrations were significantly higher in the patients reaching the composite endpoint  . In univThis study evaluated the value of sTNFR1 and sTNFR2 as biomarkers for their association to the composite endpoint of all-cause mortality or first non-fatal cardiovascular event in a cohort with advanced CKD irrespective of the underlying etiology. The main finding of this study is that sTNFR1 and sTNFR2 are associated to adverse outcome, even after adjustment for clinical covariables, such as age, gender, eGFR, CRP. The association with outcome was stronger for sTNFR1 than for sTNFR2. In the subgroups of patients without and with diabetes only sTNFR1 was after adjustment associated to outcome.2) minimized the impact of eGFR on outcome, which was confirmed by the lack of association to adverse outcomes  and also reported previously . sTNFR1 Constitutively, both sTNFRs are released from cell membranes by shedding ,25 or asThe limitations of the study are: the results cannot be generalized to all CKD stages, since we restricted the evaluation to CKD stage 4 and 5 intentionally to evaluate both receptors in a narrow range of eGFR, which also resulted in a rather small population. Furthermore, based on our results it is not yet possible to draw conclusions on the potential use or incremental value of these markers in clinical situations. Nevertheless, the study unravelled convincing correlations between sTNFRs and outcomes even after adjustment for confounders such as eGFR or diabetes. Hence our data underscore the relationship of TNFRs, with hard clinical outcomes irrespective of underlying kidney disease and the presence or absence of diabetes. The fact that especially sTNFR1 was consistently associated to different outcomes in advanced CKD as shown in this study as well as in community-based and specific high risk populations such as post-myocardial infarction, offers a strong argument in favour for its further evaluation in larger CKD cohorts.S1 TableData are presented as means ± standard deviation or medians with interquartile range between square brackets. For binary variables, frequencies with percentages between brackets are given. MACE: major adverse cardiovascular event, N = number of patients, M: male, BMI: body mass index, MAP: mean arterial pressure, PP: pulse pressure, HR: heart rate, eGFR: estimated glomerular filtration rate, CVD: history of cardiovascular disease, DM: diabetes mellitus, AHT: arterial hypertension, RRT: start of renal replacement therapy during follow-up, CRP: C-reactive protein, TNFα: tumor necrosis factor alpha, sTNFR1: soluble tumor necrosis factor receptor 1, sTNFR2: soluble tumor necrosis factor receptor 2.(DOC)Click here for additional data file.S2 TableHR: Hazard ratio, CI: confidence interval. In bold, variables with p-value < 0.05, included in the multivariate model. sTNFR1: soluble tumor necrosis factor receptor 1, sTNFR2: soluble tumor necrosis factor receptor 2, TNFα: tumor necrosis factor alpha, CRP: C-reactive protein, eGFR: estimated glomerular filtration rate, MAP: mean arterial pressure, PP: pulse pressure, BMI: body mass index, CVD: history of cardiovascular disease, DM: diabetes mellitus, AHT: arterial hypertension.(DOC)Click here for additional data file.S3 TableData are presented as means ± standard deviation or medians with interquartile range between square brackets. For binary variables, frequencies with percentages between brackets are given. MACE: major adverse cardiovascular event, N = number of patients, M: male, BMI: body mass index, MAP: mean arterial pressure, PP: pulse pressure, HR: heart rate, eGFR: estimated glomerular filtration rate, CVD: history of cardiovascular disease, DM: diabetes mellitus, AHT: arterial hypertension, RRT: start of renal replacement therapy during follow-up, CRP: C-reactive protein, TNFα: tumor necrosis factor alpha, sTNFR1: soluble tumor necrosis factor receptor 1, sTNFR2: soluble tumor necrosis factor receptor 2(DOC)Click here for additional data file.S4 TableHR: Hazard ratio, CI: confidence interval. In bold, variables with p-value < 0.05, included in the multivariate model. sTNFR1: soluble tumor necrosis factor receptor 1, sTNFR2: soluble tumor necrosis factor receptor 2, TNFα: tumor necrosis factor alpha, CRP: C-reactive protein, eGFR: estimated glomerular filtration rate, MAP: mean arterial pressure, PP: pulse pressure, BMI: body mass index, CVD: history of cardiovascular disease, DM: diabetes mellitus, AHT: arterial hypertension.(DOC)Click here for additional data file.


Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxins.The pathogenic bacteria  Clostridium (C.) difficile, C. perfringens and C. botulinum produce the binary protein toxins CDT [7), which bind to their cell surface receptors [The pathogenic clostridia xins CDT ,2,3,4, ixins CDT ,6,7,8 anxins CDT ,10,11, rxins CDT ,13. The eceptors ,15,16,17eceptors ,19,20. Ieceptors . In conteceptors .7-toxin complexes are internalized by receptor-mediated endocytosis [7 oligomer changes conformation and inserts into the endosomal membranes, forming trans-membrane channels, which allow the translocation of the unfolded A components from the endosomal lumen into the cytosol [The A components of the binary toxins bind to their respective cell-bound B components, and the ABocytosis ,24,25,26 cytosol ,29,30,31 cytosol ,35,36,37 cytosol ,43,44,45 cytosol ,50,51,52C. difficile infections (CDI). C. difficile causes severe enteric diseases in hospitalized patients treated with broad-spectrum antibiotics. The spectrum of CDI ranges from diarrhea to severe, potentially life-threatening pseudomembranous colitis due to the disturbed gut flora, which allows spore germination and the growth of C. difficile [C. difficile-associated diseases. These toxins inhibit signal transduction via these Rho GTPases, resulting in the destruction of the actin cytoskeleton, cell-rounding and loss of intestinal wall integrity [C. difficile strains produce CDT in addition to toxins A and B, and this most likely contributes to their hypervirulence and the increased morbidity/mortality of patients infected with these strains [The clostridial binary toxins are potent enterotoxins and cause severe diseases in humans and animals. From a medical point of view, CDT contributes to the severe forms of ifficile . Proteinntegrity ,55,56. H strains ,59,60,61C. difficile, the development of pharmacological inhibitors, acting directly on the toxins and protecting cells from intoxication, is an important goal. By performing high-throughput screening from a library of small molecules to find novel inhibitors against the anthrax lethal toxin [N-semicarbazone (EGA) as a potent inhibitor against multiple bacterial toxins and viruses, which require trafficking through acidic vesicles to enter the host cell cytosol [Because such hypervirulent strains are resistant to broad-spectrum antibiotics used to treat other bacterial infections and allow overgrowth of al toxin , Bradley cytosol . In part cytosol . More re cytosol .Prompted by these findings, we investigated the effect of EGA on the binary clostridial actin ADP-ribosylating toxins CDT, iota and C2, which also require trafficking through acidified endosomal vesicles in order to identify a novel pharmacological inhibitor against this toxin family.EGA protects cells from intoxication with the binary actin ADP-ribosylating toxins CDT, iota and C2. Vero cells were treated with each of the binary toxins in the presence or absence of EGA, and the intoxication process was monitored in terms of cell-rounding, which is a well-established, highly specific and sensitive endpoint to detect the uptake of the A components of these toxins into the host cell cytosol. Vero cells express the receptor for CDT and iota toxin and were therefore used as a model cell line in this study. As shown in C. difficile toxins A (TcdA) and B (TcdB)  . Like th+ as the co-substrate in the absence and presence of EGA, and the ADP-ribosylated, i.e., biotin-labelled, actin was analyzed by Western blotting . Complete® protease inhibitor was purchased from Roche . Biotinylated NAD+ was supplied by R & D Systems GmbH . Bafilomycin (Baf) A1 was obtained from Calbiochem , and 2-(4-bromobenzylidene)-N-hydrazinecarboxamide (EGA) was synthesized as described [® . CDTa and CDTb (from C. difficile strain 196) were expressed as recombinant His-tagged proteins in the Bacillus megaterium expression system and purified as described earlier [Cell culture media (MEM) and fetal calf serum were from Invitrogen . Cell culture materials were obtained from TPP . The protein molecular weight marker Page Ruler prestained Protein ladderescribed  and prov earlier . Ia and  earlier . The rec earlier ,43. Toxi earlier ,55.2 in MEM medium, containing 10% heat-inactivated fetal calf serum, 1 mM sodium-pyruvate, 2 mM l-glutamine, 0.1 mM non-essential amino acids and 1% penicillin-streptomycin. Cells were trypsinized and reseeded three times per week for at most 30 times.HeLa and African green monkey kidney (Vero) cells were cultivated at 37 °C and 5% COFor intoxication experiments, cells were seeded in culture dishes and pre-incubated with EGA or the vehicle DMSO in serum-free medium for 1 h at 37 °C. Control cells were incubated without inhibitor. Subsequently, CDT, iota toxin, C2, toxin A or toxin B was added, and cells were further incubated at 37 °C with toxin plus inhibitor. After the given incubation periods, the cells were visualized by using a Zeiss Axiovert 40CFI microscope  with a Jenoptik progress C10 CCD camera  to analyze the morphological changes caused by the toxins, and toxin-induced cell-rounding was taken as an indication of the intoxication process. Finally, cells were counted per picture (ImageJ software 1.45 (1.47v), NIH, Bethesda, MD, USA, 2013), and the amount of rounded cells was determined as a percent.For immunoblot analysis, equal amounts of protein were subjected to SDS-PAGE according to the method of Laemmli . Subsequ+ (10 µM) were added followed by an incubation of the samples for 10 min at 37 °C. Thereupon, the proteins were subjected to SDS-PAGE and, after blotting onto a nitrocellulose membrane, biotin-labelled  G-actin was detected using Western blotting.C2I, CDTa or Ia (100 ng in each case) was pre-incubated for 10 min at 37 °C with EGA (50 µM) or left untreated for the control. Subsequently, Vero lysate (20 µg of protein) and biotin-labelled NADi.e., bound C2IIa) was detected using a specific antibody against C2I and peroxidase-coupled secondary antibody.HeLa cells were pre-incubated in serum-free medium with EGA (50 µM) for 1 h at 37 °C or left untreated for the control. Afterwards, cells were kept at 4 °C. After 10 min, C2 was added followed by further incubation at 4 °C for 30 min to enable C2-binding to its cell surface receptor. Unbound C2 was subsequently removed by three washing steps with ice-cold PBS, and cells were scraped in 5-fold concentrated sample buffer containing 10% DTT . ProteinThe pH-dependent translocation of C2 across endosomal membranes was experimentally mimicked on the cytoplasmic membranes of intact cells as described earlier . Therefon = 3) as the means ± standard deviation (SD) with the Prism4 Software .All experiments were performed independently at least two times, and the results from representative experiments are shown in the figures. The quantification was performed by calculating the values , EGA emerges as an attractive lead compound to develop novel broad-spectrum inhibitors against a variety of bacterial protein toxins, including the binary actin ADP-ribosylating toxins from clostridia, because it efficiently protects cells without obvious cytotoxic effects.


Head and neck cancers, which affect 650,000 people and cause 350,000 deaths per year, is the sixth leading cancer by cancer incidence and eighth by cancer-related death worldwide. Oral cancer is the most common type of head and neck cancer. More than 90% of oral cancers are oral and oropharyngeal squamous cell carcinoma (OSCC). The overall five-year survival rate of OSCC patients is approximately 63%, which is due to the low response rate to current therapeutic drugs. In this review we discuss the possibility of using caffeic acid phenethyl ester (CAPE) as an alternative treatment for oral cancer. CAPE is a strong antioxidant extracted from honeybee hive propolis. Recent studies indicate that CAPE treatment can effectively suppress the proliferation, survival, and metastasis of oral cancer cells. CAPE treatment inhibits Akt signaling, cell cycle regulatory proteins, NF-κB function, as well as activity of matrix metalloproteinase (MMPs), epidermal growth factor receptor (EGFR), and Cyclooxygenase-2 (COX-2). Therefore, CAPE treatment induces cell cycle arrest and apoptosis in oral cancer cells. According to the evidence that aberrations in the EGFR/phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling, NF-κB function, COX-2 activity, and MMPs activity are frequently found in oral cancers, and that the phosphorylation of Akt, EGFR, and COX-2 correlates to oral cancer patient survival and clinical progression, we believe that CAPE treatment will be useful for treatment of advanced oral cancer patients. Head and neck cancers include cancers evolved from the oral cavity, pharynx, larynx, paranasal sinuses and nasal cavity, and salivary glands. Head and neck cancers rank as the sixth most common cancer worldwide, affecting 650,000 people and causing 350,000 deaths per year ,2. Oral Propolis (bee glue) is produced by honeybees through mixing the secretions of their hypopharyngeal glands with the digested product of resins collected from leaves, flowers, and tree barks, which is used to build honeybee hives . PropoliCaffeic acid phenethyl ester (CAPE)  is one oThere are several types of oral cancers. The majority (>90%) of oral cancers are OSCC ,4,29. OSCaffeic acid phenethyl ester (CAPE)  is one o50 of 6.3 nM in J774 macrophages [The epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK), is the cell-surface receptor for members of the epidermal growth factor (EGF) family. Elevated gene expression of EGFR has been reported to be associated with poor prognosis in OSCC ,83. Trearophages . Treatmerophages . Accordi50 of 175 and 341 μM, respectively [50 of CAPE to suppress proliferation of TW2.6 cells was 72.1, 41.5, and 19.0 μM for 24, 48, and 96 h of treatment, respectively. Although the IC50 of CAPE seems to be relatively high as compared to other chemotherapy drugs, CAPE can be applied in the form of topical cream or mouth rinsing solution. We believe that daily oral administration of CAPE for a week or longer is possible to cause regression of oral cancer cells.TW2.6 is an OSCC cancer cell line established from the untreated primary squamous cell carcinoma of the buccal mucosa from a 48-year-old Taiwanese male patient with a betel quid chewing and tobacco smoking habit . TW2.6 cectively ,90,  is a chemotherapy agent widely used for treating advanced head and neck cancer. 5-fluorouracil suppresses cancer cells by misincorporating fluoronucleotides into RNA and DNA as well as by inhibiting the nucleotide synthetic enzyme thymidylate synthase . HoweverThe PIK3CA oncogene is located at chromosome 3q26. Chromosome locus 3q26 is frequently amplified in OSCCs ,95,96,97N-terminal kinases (JNKs) [The tumor microenvironment is the environment in which the cancer cell exists. The status of the surrounding blood vessels, immune cells, fibroblasts, stroma cells, hypoxia, signaling molecules, and the extracellular matrix (ECM) determine the conditions of the tumor microenvironment. CAPE treatment reduces the secretion of vascular endothelial growth factor (VEGF) through the inhibition of the ROS, PI3K and HIF-1α signaling pathways in human retinal pigment epithelial cells under hypoxic conditions . Cancer s (JNKs) . These dNuclear factor kappa-B (NF-κB) is an important cell-survival signaling protein and plays a key role in regulating the cellular response to stress and the immune response to infection . DysreguKip1 and other proteins [Kip [CCND1 gene and forms a complex with cyclin-dependent kinase 4 (CDK4) or CDK6. These complexes are essential for cell cycle G1/S transition [CCND1 gene is positively regulated by Rb [Kip1 protein likely contribute to the induction of G2/M cell cycle arrest and growth inhibition in TW2.6 oral cancer cells.CAPE treatment decreased G1 cell population, increased G2/M cell population, and induced apoptosis in TW2.6 cells . Skp2 isproteins ,130. Treins [Kip . Rb is ains [Kip . Reductiins [Kip . As the ins [Kip . Cyclin ansition . Cyclin ed by Rb . DeclineAccording to the above summaries in this review, there is strong evidence that CAPE treatment suppress proliferation, survival, metastasis, EGFR and COX-2 activity, PI3K-Akt signaling, and Skp2 in human oral cancer cells. We summarize the effects of CAPE treatment on different signaling proteins and the potential effect on cell survival, cell cycle, cell proliferation, and metastasis of oral cancer cells in


Bacopa monniera extract in different dimensions, especially as a nerve tonic and memory enhancer. Studies in animal model evidenced that Bacopa treatment can attenuate dementia and enhances memory. Further, they demonstrate that Bacopa primarily either acts via antioxidant mechanism  or alters different neurotransmitters , dopamine (DA), acetylcholine (ACh), γ-aminobutyric acid (GABA)) to execute the pharmacological effect. Among them, 5-HT has been shown to fine tune the neural plasticity, which is a substrate for memory formation. This review focuses on the studies which trace the effect of Bacopa treatment on serotonergic system and 5-HT mediated key molecular changes that are associated with memory formation.Over the last 50 years, laboratories around the world analyzed the pharmacological effect of  Bacopa monniera (L.) Wettst., which belongs to the family Scrophulariaceae, is an annual creeping plant found in wet, damp, and marshy areas. The leaves and stem of the plant are used for medicinal purposes traditionally [ B. monniera known as “Bhrami” has been classified under Medhya Rasayana and described in ancient ayurvedic medical encyclopedias, namely, Charaka Samhita, Sushrutha Samhita, and Astanga Hrdaya, as cure for mental disorders and loss of intellect and memory. It has been tested in different animal models to understand its effect on memory [ B. monniera extract in elderly persons to improve cognitive performance and memory [ Bacopa is a main constituent in the preparation of ayurvedic medicine prescribed for cognitive dysfunction. In addition, several research groups and pharmaceutical companies formulated Bacopa for clinical use in different countries including India, New Zealand, Australia, and United States of America. Earlier, many reviews have discussed pharmacological property of B. monniera in a broad perspective; however, no comprehensive article has yet shown its effect on molecular level. In this review, we summarize the in vivo experiments that suggest that B. monniera treatment enhances cognitive function by altering the molecular targets through serotonergic system.tionally . In the n memory , 3 and an memory –9. Thesed memory –15. In p Bacopa, which include alkaloids , saponins , sterols , d-mannitol, acid A, and betulinic acid [ B. monniera are triterpene saponins of the dammarane class, which have been named bacosides and bacopasaponins. There are two types of saponins, jujubogenin and pseudojujubogenin, which differ only in the nature of the sugar units in the glycosidic chain and the position of the olefinic side chain in the aglycone. These saponins are complex mixture of closely related structures, namely, bacosides A1 [3 [. Two new dammarane-type jujubogenin bisdesmosides, bacopasaponins E and F [ B. monniera is bacoside A (64.28%) and bacoside B (27.11%); the latter differs only in optical rotation. The bacoside A  derives from two triterpenoid saponins: pseudojujubogenin and jujubogenin on acid hydrolysis [ Bacopa which is termed as bacosides-enriched standardized extract of Bacopa (BESEB CDRI-08) that contains 55 ± 5% bacosides , and BESEB CDRI-08 is mentioned as BME in this paper.Series of biochemical studies identified different pharmacological compounds from ethanolic extracts ofnic acid –18. The sides A1  and A3 [es A1 [3  and bacoes A1 [3 –23. Two  E and F , pseudoj E and F , phenyle E and F , and bac E and F  have alsdrolysis –18, 28. Bacopa treatment has been reported to improve behavior of different laboratory animal models under variety of experimental conditions. Oral administration of BME improved spatial learning of rats and mice in Morris water maze [ Bacopa treatment increased the transfer latency and freezing response [ Bacopa treatment induced dendritic arborization of neurons in hippocampal and basolateral amygdala [ter maze , 29–31. ter maze , 33, Y-mter maze , 35, radter maze , 36, Barter maze , Hole boter maze . In additer maze . Similarresponse , 40–42, response  and exhiresponse , 44, 45.amygdala , 47, whiγ-aminobutyric acid (GABA) type A receptor [ω-nitro-l-arginine (L-NNA), a nitric oxide synthase inhibitor [ Bacopa treatment.Interestingly, several studies investigated the pharmacological effect of BME against different chemical compounds that induce anterograde/retrograde amnesia by targeting different neuronal system. These studies reported that BME effectively attenuated anterograde/retrograde amnesia induced by chemical compounds such as scopolamine, an acetylcholine receptor antagonist , 48, 49,nhibitor , alumininhibitor , autistinhibitor  were als B. monniera extract [We have learned from pioneering works about different active compounds in extract –18. As a extract  confirme extract –27, they extract , and its extract  effectivγ-aminobutyric acid (GABA) [The balanced functions of various neurotransmitters such as acetylcholine (ACh) , 40, serd (GABA) , and glud (GABA)  were alld (GABA) , and alsd (GABA)  estimated (GABA) , 40. On d (GABA)  may actid (GABA) , 59, whid (GABA) , 61, butd (GABA) . These p in silico analysis suggested that interaction of bacosides  with TPH2 possibly alters the activity of TPH2 that could be one of the mechanisms for increased 5-HT synthesis [Earlier studies demonstrated that increasing level of tryptophan hydroxylase (TPH) mRNA expression elevated TPH activity and 5-HT metabolism, which profoundly could influence the synaptic 5-HT activity , 64. Furynthesis .1A, 5-HT2A, 5-HT4, 5-HT5A, 5-HT6, and 5-HT7) after BME treatment was examined. Notably, 5-HT3A receptor expression was increased compared to all other receptors. It is the only metabotropic receptor, and its expression could be stimulated by endogenous 5-HT which may facilitate the hippocampal-dependent task [3A in hippocampal-dependent learning could be tested by using 5-HT3 antagonist 1-(m-chlorophenyl)-biguanide (mCPBG), which effectively impairs the retention of the conditioned response [3 antagonist mCPBG has facilitated gaining insight into the BME induced 5-HT3A receptor mediated role in hippocampal-dependent learning and its regulation of other neurotransmitters. Interestingly, treatment of BME ameliorated the antagonistic effect of mCPBG. The combination of mCPBG and BME treatment recorded improvement in behavioural task accompanying the upregulation of 5-HT3A receptor. Considering the interaction of multiple neurotransmitters involved in learning and memory network [3 receptor in activation/inhibition of other neurotransmitter systems.Previously, it has been found that synaptically released 5-HT exerts its function through their diverse receptors . Activatent task , 74. Henresponse  in both response . The 5-H network –80, it c3A receptor might regulate serotonergic system and may interact with other neurotransmitters that are involved in learning and memory [3A is a heteroreceptor; its stimulation by means of mCPBG has been reported to enhance GABA and DA levels and inhibit the release of ACh [3 receptors in dopaminergic neuron could facilitate the release of DA [ mCPBG inhibits dopamine uptake by binding with dopamine transporter [The upregulated 5-HTd memory , 67, 81.e of ACh . The actse of DA , 83, andnsporter , therebynsporter  and othensporter , 85.3A receptor [3A receptor.A noteworthy point is that it did not alter the level of Glu. This suggests that glutamate neurons in the hippocampus may not colocalize with 5-HTreceptor . The obs3A receptor regulated synaptic plasticity through protein kinase and cAMP response element binding (CREB) protein signaling pathway. It is noteworthy to mention that treatment of BME increased the phosphorylation of ERK1/2 and provides a physiological and functional meaning for the observed different forms of memory [ m-CPBG treatment, the mCPBG mediated suppression of CREB phosphorylation was attenuated by BME, thus adding additional support to the effect of BME in regulation of PKA-CREB pathway.A pioneering study in 1976 described that serotonin stimulation increases the level of cyclic adenosine monophosphate (cAMP) by the adenyl cyclase in the neuronal cells . Subsequf memory . If the f memory –97. It sf memory  and theif memory –102. Pref memory  found th mCPBG showed upregulated Dicer, Ago2, and miR-124 [ Creb1 mRNA level [ Creb1 3′UTR and regulates the expression of CREB [Long-term memory formation requires synthesis of new proteins , 104, wh miR-124 . It has  miR-124 . Though NA level , recent  of CREB , 113. In of CREB , 114. Th of CREB –118. Theα and PP2A level in hippocampus, which appears to be responsible for observed BME mediated enhanced memory [Contrary to the protein kinases, protein phosphatases (PPs) act as dephosphorylating enzymes that dephosphorylate the molecules like CREB . PPs crid memory . This std memory , 128–130 in vitro and in vivo studies claimed that transcriptional induction of CREB occurred by pSer133, which requires histone acetylase (HAT)—CREB binding protein (CBP/p300) [Studies in memory highlighted chromatin alteration and epigenetic changes that are associated with CREB activation. Contribution of histone tail acetylation and deacetylation in chromatin are widely known to be involved in the formation of long-term memory and synaptic changes –133. HisBP/p300) , 137. P3BP/p300) –140. ManBP/p300) –143. TheAn earlier study reported significant enhancement of p300 level in hippocampus of BME treated groups, but not in control groups after training . These r2+ and conserved at least in vertebrates [2+/calmodulin dependent protein kinase II (CaMKII)) distributed densely. Acute phosphorylation and localization of PSD-95 and CaMKII is fundamental to synaptic function [Behavioural response to the stimuli is basic functional circuit formation between the neuronal cells. The molecular mechanism underlying the circuit (synaptic plasticity) is likely to provide insight to role of molecules/molecular complexes. The communications between the neuronal cells are initiated by the recruitment of adhesion molecules in pre-post synaptic neurons , 152. Sytebrates . This sytebrates . Anothertebrates . It is atebrates , 156. Thtebrates . BME trefunction . They arfunction , 159. Trfunction . The indfunction . Geneticfunction , 162. BMfunction , 164 invfunction . The levfunction , 168. Eafunction ; upregulfunction –172. The Bacopa extract has been known to improve cognitive function by modulating different neurotransmitters. However, this review focused on the studies which provide much attention to the serotonergic system, in which, starting from in silico approach to alternation in 5-HT levels, their receptors and associated signaling cascades known to be involved in synaptic plasticity and memory enhancement were discussed. These studies provide molecular evidence to possible mechanism of BME on serotonergic system and its associated pathway.Taken together, bacosides present in the


AbstractLignobryconmyersi is an endemic fish species from a few coastal rivers in northeastern Brazil. Based on molecular evidence, Lignobryconmyersi and genera Triportheus Cope, 1872, Agoniates Müller & Troschel, 1845, Clupeacharax Pearson, 1924 and Engraulisoma Castro, 1981 were placed in the family Triportheidae. In the present work, we report the first cytogenetic data for Lignobryconmyersi to test the hypothesis that Lignobrycon and Triportheus are closely related. Studied specimens presented 2n=52 with 28 metacentric (m), 18 submetacentric (sm) and six subtelocentric (st) chromosomes for males and 27 m, 19 sm and 6 st for females, characterizing a ZZ/ZW sex chromosome system. The Z chromosome corresponds to the largest chromosome in karyotype while the W is about 50% smaller than the Z and largely heterochromatic. Terminal nucleolus organizer regions, GC-rich sites and 18S rDNA signals were detected on pair 14. However, additional 18S rDNA sites were observed in the W chromosome. The 5S rDNA was mainly detected on long arms of pair 7. The apparent synapomorphic chromosomal traits of Triportheus and Lignobryconmyersi reinforce their close phylogenetic relationship, suggesting that the ZZ/ZW chromosome system in both genera has arisen before cladogenic events. Lignobryconmyersi Miranda-Ribeiro, 1956 is a small characin fish (about 11 cm in length) characterized by a compressed body with keeled coracoids, adapted to swim near the surface. The type-locality of Lignobryconmyersi is located in the Almada river basin, a costal drainage in Bahia , including the following genera of tetras or freshwater sardines: Agoniates Müller & Troschel, 1845, Clupeacharax Pearson, 1924, Engraulisoma Castro, 1981, Triportheus and Lignobrycon  on Z chromosomes  were collected in their type-locality in Braço (14°40'52"S/39°14'39"W) and Almada (14°39'35"S/39°13'24"W) Rivers, both belonging to the Almada River basin in the state of Bahia, northeastern Brazil  were detected by silver nitrate staining (3 (CMA3) and 4,6-diamidino-2-phenylindole (DAPI), respectively (Metaphase chromosomes were obtained from anterior kidney cells as described by -banding  and actistaining . The GC-ectively .in situ hybridization fluorescence (FISH) was performed to map simultaneously 18S and 5S rDNA on chromosomes of Lignobryconmyersi according to Moenkhausiasanctafilomenae Steindachner, 1907 as described by PageBreaklabeled with biotin-16-dUTP via nick translation using the BioNick Labeling System kit (Invitrogen) and signals were detected using avidin-fluorescein isothiocyanate (FITC) conjugate (Sigma). The 5S rDNA probe was obtained from DNA of the headstander Leporinuselongatus Valenciennes, 1850 (DAPI (0.2 mg/mL) in Vectashield Mounting Medium (Vector) and slides were stored in a dark chamber up to analysis.The es, 1850 , labeledTriportheus  with heteromorphic marks at terminal regions on long arms in both sexes  were coincident with Ag-NORs  in both sexes  revealed three trends in this genus, as follows: (1) species PageBreakwith WRL higher than 60%, (2) species with WRL ranging from 40 to 60%, and (3) species with WRL below 40%  or a technical artifact related to differences in condensation of chromosomes or biased measurements by each author.In turn, ish taxa , but rardae e.g. , therebys family . The locFISH with 18S rDNA probes showed that, similarly to other Triportheus species, Lignobryconmyersi also bears NORs on the W chromosome, even though they were inactive in studied samples (i.e. undetected by silver nitrate staining) . Putatively, during the evolutionary history of Lignobryconmyersi, the 18S rDNA sequences may have partially degenerated and thus inactivated  Fig. . This reated see  while reLignobryconmyersi and several species of Triportheus  in Lignobryconmyersi were interspersed to Ag-NORs only, as reported in some species of Triportheus , conspicuous CMA3+ signals were reported in both autosomal NORs and W chromosomes of other species like Triportheusnematurus  by Lignobryconmyersi (e.g. Contas River) can be useful to evaluate interpopulation differences or the existence of cryptic forms that should be prioritized for conservation.In conclusion, the cytogenetic results agree with morphological  and molePageBreak


Notch, IL-1 and leptin are known pro-angiogenic factors linked to breast cancer development, tumor aggressiveness and poor prognosis. A complex crosstalk between these molecules (NILCO) has been reported in breast cancer cell lines. However, whether NILCO biomarkers are differentially expressed in estrogen responsive (ER+), unresponsive (ER-) and triple negative (TNBC) breast cancer tissues is unknown.Expression levels of nine NILCO and targets  were examined via immunohistochemistry in breast cancer tissue microarrays from Chinese patients  and non-malignant breast tissue  using a semi-quantitative analysis of intensity staining, HSCORE.Categorical expression of NILCO and targets (+ or -) was similar among all cancer tissues. However, TNBC showed differential localization pattern of NILCO. TNBC showed fewer nuclei and cytoplasms positive for Notch4 and JAG1, but more cytoplasms positive for leptin. In addition, fewer TNBC stromas were positive for Notch1 and Notch4, but 100% of TNBC stromas were positive for VEGFR2. Moreover, TNBC had lower DLL4 and IL-1R tI expression. TNBC and ER- showed higher expression of EGFR, but lower expression of AR. Leptin and OB-R were detected in more than 61% of samples. Leptin positively correlated to OB-R, JAG1, VEGF, and marginally to IL-1R tI. Notch1 positively correlated to IL-1R tI. EGFR and Ki67 were positively associated to Notch1, but no associations of NILCO and targets with p53 were found.Present data suggest that NILCO components are differentially expressed in breast cancer. TNBC showed distinctive patterns for NILCO expression and localization. The complex crosstalk between leptin, IL-1 and Notch could differentially drive breast cancer growth and angiogenesis. Furthermore, the analysis of NILCO and targets using Pathway Studio9 software (Ariadine Genomics) showed multiple molecular relationships that suggest NILCO has potential prognostic biomarker value in breast cancer. Breast cancer is a heterogeneous disease with four major genetic-signature subtypes . Howeveri.e., insulin, insulin-like growth factor-I (IGF-I) and adipokines]  ,4. This pokines] .Notch signaling is a hallmark of breast cancer ,7. The rNotch signaling is a key mediator of proliferation, survival, and possibly malignant invasion of TNBC. These data suggest that TNBC is heavily dependent on Notch signaling . In linei.e., insulin and IGF-1, and adipokines) [Recent data indicate that breast cancer development is likely related to lifestyle and the result of being overweight. Obesity is associated with more than 100,000 incidents of cancer in the United States every year, particularly cancers of the breast, colon, and endometrium. The specific molecular mechanisms involved in the development of obesity-related breast cancers are unknown. However, the general picture suggests that obesity-related breast cancer is the consequence of multi-factorial causes ,15. Sevepokines) . Leptin,pokines) -19, and pokines) . Leptin pokines) -24.We have previously unveiled a complex crosstalk between Notch, IL-1 and leptin (NILCO) in breast cancer cell lines, which could be essential for leptin-induced proliferation, inflammation and angiogenesis . Moreovein silico via Pathway Studio9 software  [We propose that specific associations between NILCO biomarkers occur in breast cancer, which may differ in TNBC. To this end, we examined the expression and cellular localization of NILCO, and targets, via immunohistochemistry in a commercial array of breast cancer biopsies from Chinese patients. Data were also analyzed ics, MD) . Data anPolyclonal antibodies for Notch4, OB-R amino terminus, DLL4, IL-1 R tI, VEGF, VEGFR2, Jagged1 (JAG1) and leptin were obtained from Santa Cruz Biotechnology, Inc. . Polyclonal anti-Notch2 and -Notch3 were from Abcam Inc. . Monoclonal anti-Notch1 antibody and other chemicals were purchased from Sigma-Aldrich . Vectastin ABC-APK and Vectamount were obtained from Vector Laboratories . Hematoxilyn was purchased from Dako Corporation, Carpinteria, CA., and p53 and Ki67 expression data. However, no information on body weight of patients was available. Each slide contained 150 cores, including 75 cases in duplicate of normal/hyperplastic specimens (n=3), fibroadenomas (n=2), ductal carcinoma in situ , Paget’s disease (n=1) and invasive carcinomas  showing diverse levels of PR and HER2 expression. The studies were focused on non-malignant (n=5) and invasive carcinoma samples .Breast cancer tissue arrays from female Chinese were obtained from Pantomics, Inc. . Biopsies features included age, grading, TNM staging, and receptor status of estrogen (ER), androgen (AR), progesterone (PR), epidermal growth factor receptors (ErbB1/EGFR/HER1 and ErbB2/HER2)IHC staining was performed on 12 separate microarray slides. The following specific antibodies were used to analyze nine antigens: anti-Notch1, Notch4, DLL4, JAG1, leptin, leptin receptor (OB-R), VEGF, VEGFR2 (FLK-1) and IL-1R tI. Staining patterns of 1206 tissue samples were evaluated by two independent observers in a blind manner. Three slides were used for negative controls (no primary antibody) incubated with secondary antibodies .Staining intensity of cells in tissue arrays was evaluated as negative or positive in three different bright fields (≥100 cells/field). Semi-quantitative HSCORE was calculated for each antigen using the following equation: HSCORE = ∑ pi(i +1), where “i” was the intensity with a value of 0, 1, 2, or 3  and “pi” was the percentage of stained cells for each intensity ,26.Pathway Studio9 software  was used to evaluate NILCO and its targets’ interactions in breast cancer tissue arrays. HSCORE of antigens showing significantly relationships in breast cancer were imported into the pathway software and analyzed.+, ER-and TNBC). Predictors were defined for A_HSCORE of nine antigens  and dichotomized A_HSCORE . Covariate analyses were performed for p53, EGFR, Ki67, AR, grade, stage and age. The patients’ clinicopathological and histology characteristics, and the categorical and continuous A_HSCOREs were summarized for breast cancer and non-malignant disease patients. Univariate association between categorical HSCOREs and continuous A_HSCOREs for ER, PR, HER2, EGFR, AR, Ki67 and p53 expressions and grade and stage were compared by Chi-square or Fisher’s exact test. ANOVA  was used to analyze age and Notch1 expression. Kruskal-Wallis test was also used to analyze continuous HSCOREs. All analyses were done using SAS 9.3  with a significance level of 0.05.HSCOREs for each antigen were determined twice, averaged, named A_HSCORE and used in the analyses. Pearson or Spearman correlation coefficients were used to compare the concordance between results from duplicate breast tissue samples and pairwise correlation between A_HSCOREs from the nine antigens analyzed in the microarray. The outcome was defined for three types of breast cancers according to the expression of ER, PR and HER2 . Age, grade, stage, Ki67, and p53 expression statuses were well-balanced among breast cancer tissues in the three groups . However, ER, PR, HER2, EGFR and AR statuses were significantly different among them.The clinicopathological and histological characteristics of non-malignant and breast cancer patients are summarized in Table The majority of TNBC were grade III and stage III invasive ductal carcinomas. ER+ and ER- breast cancers were mainly invasive ductal carcinomas grade III and stage II  of Notch1, Notch4, JAG1, OB-R, VEGF and, VEGFR2 were not significantly different among breast cancers irrespective of their expression of ER, HER2 and PR. Notch1, DLL-4 and, VEGF were detected in most malignant tissues. Leptin, OB-R, Notch4, JAG1 and, IL-1R tI were detected in more than 60% of breast cancer . In contrast, approximately 30% of these breast cancers showed immunoreactivity for VEGFR2. Leptin, OB-R, Notch1 and VEGFR2 were significantly lower in non-malignant tissues.Remarkably, cellular localization of NILCO and targets was significantly different in TNBC compared to ER- and ER+ breast cancer Table . A lowerEvaluation of A_HSCOREs showed diverse grade of expression of NILCO and targets in ER+, ER- and, TNBC Table . A_HSCORExpression of DLL4 and IL-1R tI were significantly different among the three breast cancer types  and target molecules (VEGF and VEGFR2) were co-expressed in breast cancer tissues, irrespective of ER, PR and, HER2 statuses. Remarkably, TNBC shows a differential pattern of expression and cellular localization of NILCO. TNBC showed lower protein levels of IL-1R tI and DLL4, and fewer nuclei and cytoplasms were positive for Notch4 and JAG1. In contrast, more TNBC cytoplasms were positive for leptin. Moreover, TNBC stromas showed distinctive patterns of Notch1 and VEGFR2 immnoreactivities. Notch1 and Notch4 expression were lower, but VEGFR2 expression was higher in stromas from TNBC compared with ER- and ER+ breast cancer stromas.Notch1 was found in the majority of breast cancer tissues evaluated. This data was in agreement with a previous report that showed 100% of Notch1 expression in TNBC . Notch4 Present data further confirm previous findings showing that TNBC cells in culture MDA-MB231) secreted more leptin (approximately four-fold) than ER+ breast cancer cells (MCF-7) . Notch-i31 secretNotch1 expression was also associated with the cell proliferation marker, Ki67. This marker was detected in approximately 60% of breast cancer independently of the expression of hormone receptors. It was previously reported that Ki67 is found in 90% of TNBC  and its in vitro[in vitro[in vitro[in vivo may not always be p53 dependent.The transformation of normal breast epithelial cells by increased Notch signaling was previously linked to the repression of apoptosis in vitro. Notch N[in vitro. Additio[in vitro. HoweverTNBC and ER- breast cancers are not responsive to steroid hormones, but are highly aggressive tumors that respond to several other growth factor-related signals . TNBC frObesity could affect breast carcinogenesis by autocrine and paracrine actions mediated by two major adipokines: leptin and adiponectin . Obese bFor the first time, we are reporting on a comprehensive data analysis on protein levels of NILCO and targets in three major groups of breast cancer: TNBC, ER-, and ER+. TNBC showed distinctive patterns of expression and localization of NILCO, which suggests these molecules may be useful as markers for disease progression and aggressiveness. It is known that inhibition of Notch  and leptOB-R: Leptin receptor; IL-1R tI: Interleukin 1 receptor type I; NILCO: Notch interleukin 1 leptin crosstalk outcome; EGFR: Epidermal growth factor receptor; NANOG: A homeobox protein and transcription factor; SOX2: Sex determining region Y-box 2 protein; OCT4: Octamer-binding transcription factor 4; IGF-1R: Insulin like growth factor receptor 1; ER: Estrogen receptor; AR: Androgen receptor; PR: Progesterone receptor; HER2: Epidermal growth factor receptor 2; DLL4: Delta like notch ligand 4; JAG1: Jagged 1 protein (a Notch ligand); VEGF: Vascular endothelial growth factor; VEGFR2: Vascular endothelial growth factor receptor 2 or KDR; TNBC: Triple negative breast cancer cells; HSCORE: A semi-quantitative analysis of intensity staining.The authors declare that they have no competing interests.in silico analysis of NILCO relationships using Pathway Studio9 Program and contributed to writing and editing the manuscript. KW, CG and TD developed immunohistochemistry assays and HSCORE determinations of NILCO, and targets. GO, performed immunohistochemical and pathological analyses of tissue samples. SK and YL performed the statistical analysis and interpretation of data. All authors read and approved the final manuscript.RRGP designed and wrote up the current study. LSC was involved in the study design, immunohistochemistry assays and HSCORE determinations of NILCO and targets. GN developed the The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/249/prepubList of references reporting relationships between NILCO and its targets in breast cancer.Click here for file