Estrogen regulation of uterine genes in vivo detected by complementary DNA array.

PubMed

Andrade, P M; Silva, I D C G; Borra, R C; de Lima, G R; Baracat, E C

2002-05-01

In the present study, our aim was to identify differentially expressed genes involved in estrogen actions at the endometrium level in rats. Thirty adult rats were ovariectomized four days prior to drug administration for 48 days. Rats were divided in 2 groups: I, control and II, conjugated equine estrogens (CCE). Total RNA was isolated from uterus, and differential expression was analyzed by array technology and RT-PCR. A total of 32 candidate genes were shown to be upregulated or downregulated in groups I or II. Among them, differential expression was already confirmed by RT-PCR for IGFBP5, S12, c-kit, and VEGF, genes whose expression was up regulated during CCE therapy, and casein kinase II and serine kinase expression was the same level in both groups. We have demonstrated that cDNA array represents a powerful approach to identify key molecules in the estrogens therapy. A number of the candidates reported here should provide new markers that may contribute to the detection of target estrogen receptor. This information may also aid the development of new approaches to therapeutic intervention.

MicroRNA-203 Induces Apoptosis by Targeting Bmi-1 in YD-38 Oral Cancer Cells.

PubMed

Kim, Jae-Sung; Choi, Dae Woo; Kim, Chun Sung; Yu, Sun-Kyoung; Kim, Heung-Joong; Go, Dae-San; Lee, Seul Ah; Moon, Sung Min; Kim, Su Gwan; Chun, Hong Sung; Kim, Jeongsun; Kim, Jong-Keun; Kim, DO Kyung

2018-06-01

MicroRNAs (miRNAs) are closely associated with a number of cellular processes, including cell development, differentiation, proliferation, carcinogenesis, and apoptosis. The aim of the present study was to elucidate the molecular mechanisms underlying the tumor suppressor activity of miRNA-203 (miR-203) in YD-38 human oral cancer cells. Polymerase chain reaction analysis, MTT assay, DNA fragmentation assay, fluorescence-activated cell-sorting analysis, gene array, immunoblotting, and luciferase assay were carried out in YD-38 cells. miR-203 expression was significantly down-regulated in YD-38 cells compared to expression levels in normal human oral keratinocytes. miR-203 decreased the viability of YD-38 cells in a time- and dose-dependent manner. In addition, over-expression of miR-203 significantly increased not only DNA segmentation, but also the apoptotic population of YD-38 cells. These results indicate that miR-203 overexpression induces apoptosis in YD-38 cells. Target gene array analysis revealed that the expression of the polycomb complex protein gene Bmi-1, a representative oncogene, was significantly down-regulated by miR-203 in YD-38 cells. Moreover, both mRNA and protein levels of Bmi-1 were significantly reduced in YD-38 cells transfected with miR-203. These results indicate that Bmi-1 is a target gene of miR-203. A luciferase reporter assay confirmed that miR-203 suppressed Bmi-1 expression by directly targeting the 3'-untranslated region. miR-203 induces apoptosis in YD-38 cells by directly targeting Bmi-1, which suggests its possible application as an anti-cancer therapeutic. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Expression of Biglycan in First Trimester Chorionic Villous Sampling Placental Samples and Altered Function in Telomerase-Immortalized Microvascular Endothelial Cells.

PubMed

Chui, Amy; Gunatillake, Tilini; Brennecke, Shaun P; Ignjatovic, Vera; Monagle, Paul T; Whitelock, John M; van Zanten, Dagmar E; Eijsink, Jasper; Wang, Yao; Deane, James; Borg, Anthony J; Stevenson, Janet; Erwich, Jan Jaap; Said, Joanne M; Murthi, Padma

2017-06-01

Biglycan (BGN) has reduced expression in placentae from pregnancies complicated by fetal growth restriction (FGR). We used first trimester placental samples from pregnancies with later small for gestational age (SGA) infants as a surrogate for FGR. The functional consequences of reduced BGN and the downstream targets of BGN were determined. Furthermore, the expression of targets was validated in primary placental endothelial cells isolated from FGR or control pregnancies. APPROACH AND RESULTS: BGN expression was determined using real-time polymerase chain reaction in placental tissues collected during chorionic villous sampling performed at 10 to 12 weeks' gestation from pregnancies that had known clinical outcomes, including SGA. Short-interference RNA reduced BGN expression in telomerase-immortalized microvascular endothelial cells, and the effect on proliferation, angiogenesis, and thrombin generation was determined. An angiogenesis array identified downstream targets of BGN, and their expression in control and FGR primary placental endothelial cells was validated using real-time polymerase chain reaction. Reduced BGN expression was observed in SGA placental tissues. BGN reduction decreased network formation of telomerase-immortalized microvascular endothelial cells but did not affect thrombin generation or cellular proliferation. The array identified target genes, which were further validated: angiopoetin 4 ( ANGPT4 ), platelet-derived growth factor receptor α ( PDGFRA ), tumor necrosis factor superfamily member 15 ( TNFSF15 ), angiogenin ( ANG ), serpin family C member 1 ( SERPIN1 ), angiopoietin 2 ( ANGPT2 ), and CXC motif chemokine 12 ( CXCL12 ) in telomerase-immortalized microvascular endothelial cells and primary placental endothelial cells obtained from control and FGR pregnancies. This study reports a temporal relationship between altered placental BGN expression and subsequent development of SGA. Reduction of BGN in vascular endothelial cells leads to disrupted network formation and alterations in the expression of genes involved in angiogenesis. Therefore, differential expression of these may contribute to aberrant angiogenesis in SGA pregnancies. © 2017 American Heart Association, Inc.

Digital analysis of the expression levels of multiple colorectal cancer-related genes by multiplexed digital-PCR coupled with hydrogel bead-array.

PubMed

Qi, Zongtai; Ma, Yinjiao; Deng, Lili; Wu, Haiping; Zhou, Guohua; Kajiyama, Tomoharu; Kambara, Hideki

2011-06-07

To digitally analyze expression levels of multiple genes in one reaction, we proposed a method termed as 'MDHB' (Multiplexed Digital-PCR coupled with Hydrogel Bead-array). The template for bead-based emulsion PCR (emPCR) was prepared by reverse transcription using sequence-tagged primers. The beads recovered from emPCR were immobilized with hydrogel to form a single-bead layer on a chip, and then decoded by gene-specific probe hybridization and Cy3-dUTP based primer extension reaction. The specificity of probe hybridization was improved by using electrophoresis to remove mismatched probes on the bead's surface. The number of positive beads reflects the abundance of expressed genes; the expression levels of target genes were normalized to a housekeeping gene and expressed as the number ratio of green beads to red beads. The discrimination limit of MDHB is 0.1% (i.e., one target molecule from 1000 background molecules), and the sensitivity of the method is below 100 cells when using the β-actin gene as the detection target. We have successfully employed MDHB to detect the relative expression levels of four colorectal cancer (CRC)-related genes (c-myc, COX-2, MMP7, and DPEP1) in 8 tissue samples and 9 stool samples from CRC patients, giving the detection rates of 100% and 77%, respectively. The results suggest that MDHB could be a potential tool for early non-invasive diagnosis of CRC.

Differentially expressed microRNAs in the corpus cavernosum from a murine model with type 2 diabetes mellitus-associated erectile dysfunction.

PubMed

Pan, Feng; You, Jinwei; Liu, Yuan; Qiu, Xuefeng; Yu, Wen; Ma, Jiehua; Pan, Lianjun; Zhang, Aixia; Zhang, Qipeng

2016-12-01

To better understand the molecular aetiology of type 2 diabetes mellitus-associated erectile dysfunction (T2DMED) and to provide candidates for further study of its diagnosis and treatment, this study was designed to investigate differentially expressed microRNAs (miRNAs) in the corpus cavernosum (CC) of mice with T2DMED using GeneChip array techniques (Affymetrix miRNA 4.0 Array) and to predict target genes and signalling pathways regulated by these miRNAs based on bioinformatic analysis using TargetScan, the DAIAN web platform and DAVID. In the initial screening, 21 miRNAs appeared distinctly expressed in the T2DMED group (fold change ≥3, p ≤ 0.01). Among them, the differential expression of miR-18a, miR-206, miR-122, and miR-133 were confirmed by qRT-PCR (p < 0.05 and FDR <5 %). According to bioinformatic analysis, the four miRNAs were speculated to play potential roles in the mechanisms of T2DMED via regulating 28 different genes and several pathways, including apoptosis, fibrosis, eNOS/cGMP/PKG, and vascular smooth muscle contraction processes, which mainly focused on influencing the functions of the endothelium and smooth muscle in the CC. IGF-1, as one of the target genes, was verified to decrease in the CCs of T2DMED animals via ELISA and was confirmed as the target of miR-18a or miR-206 via luciferase assay. Finally, these four miRNAs deserve further confirmation as biomarkers of T2DMED in larger studies. Additionally, miR-18a and/or miR-206 may provide new preventive/therapeutic targets for ED management by targeting IGF-1.

Identification of potential target genes of ROR-alpha in THP1 and HUVEC cell lines.

PubMed

Gulec, Cagri; Coban, Neslihan; Ozsait-Selcuk, Bilge; Sirma-Ekmekci, Sema; Yildirim, Ozlem; Erginel-Unaltuna, Nihan

2017-04-01

ROR-alpha is a nuclear receptor, activity of which can be modulated by natural or synthetic ligands. Due to its possible involvement in, and potential therapeutic target for atherosclerosis, we aimed to identify ROR-alpha target genes in monocytic and endothelial cell lines. We performed chromatin immunoprecipitation (ChIP) followed by tiling array (ChIP-on-chip) for ROR-alpha in monocytic cell line THP1 and endothelial cell line HUVEC. Following bioinformatic analysis of the array data, we tested four candidate genes in terms of dependence of their expression level on ligand-mediated ROR-alpha activity, and two of them in terms of promoter occupancy by ROR-alpha. Bioinformatic analyses of ChIP-on-chip data suggested that ROR-alpha binds to genomic regions near the transcription start site (TSS) of more than 3000 genes in THP1 and HUVEC. Potential ROR-alpha target genes in both cell types seem to be involved mainly in membrane receptor activity, signal transduction and ion transport. While SPP1 and IKBKA were shown to be direct target genes of ROR-alpha in THP1 monocytes, inflammation related gene HMOX1 and heat shock protein gene HSPA8 were shown to be potential target genes of ROR-alpha. Our results suggest that ROR-alpha may regulate signaling receptor activity, and transmembrane transport activity through its potential target genes. ROR-alpha seems also to play role in cellular sensitivity to environmental substances like arsenite and chloroprene. Although, the expression analyses have shown that synthetic ROR-alpha ligands can modulate some of potential ROR-alpha target genes, functional significance of ligand-dependent modulation of gene expression needs to be confirmed with further analyses. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparison of gene expression responses to hypoxia in viviparous (Xiphophorus) and oviparous (Oryzias) fishes using a medaka microarray.

PubMed

Boswell, Mikki G; Wells, Melissa C; Kirk, Lyndsey M; Ju, Zhenlin; Zhang, Ziping; Booth, Rachell E; Walter, Ronald B

2009-03-01

Gene expression profiling using DNA microarray technology is a useful tool for assessing gene transcript level responses after an organism is exposed to environmental stress. Herein, we detail results from studies using an 8 k medaka (Oryzias latipes) microarray to assess modulated gene expression patterns upon hypoxia exposure of the live-bearing aquaria fish, Xiphophorus maculatus. To assess the reproducibility and reliability of using the medaka array in cross-genus hybridization, a two-factor ANOVA analysis of gene expression was employed. The data show the tissue source of the RNA used for array hybridization contributed more to the observed response of modulated gene targets than did the species source of the RNA. In addition, hierarchical clustering via heat map analyses of groupings of tissues and species (Xiphophorus and medaka) suggests that hypoxia induced similar responses in the same tissues from these two diverse aquatic model organisms. Our Xiphophorus results indicate 206 brain, 37 liver, and 925 gill gene targets exhibit hypoxia induced expression changes. Analysis of the Xiphophorus data to determine those features exhibiting a significant (p<0.05)+/-3 fold change produced only two gene targets within brain tissue and 80 features within gill tissue. Of these 82 characterized features, 39 were identified via homology searching (cut-off E-value of 1 x 10(-5)) and placed into one or more biological process gene ontology groups. Among these 39 genes, metabolic energy changes and manipulation was the most affected biological pathway (13 genes).

Role of miR-1 expression in clear cell renal cell carcinoma (ccRCC): A bioinformatics study based on GEO, ArrayExpress microarrays and TCGA database.

PubMed

Yan, Hai-Biao; Huang, Jia-Cheng; Chen, You-Rong; Yao, Jian-Ni; Cen, Wei-Ning; Li, Jia-Yi; Jiang, Yi-Fan; Chen, Gang; Li, Sheng-Hua

2018-02-01

To investigate the clinical value and potential molecular mechanisms of miR-1 in clear cell renal cell carcinoma (ccRCC). We searched the Gene Expression Omnibus (GEO), ArrayExpress, several online publication databases and the Cancer Genome Atlas (TCGA). Continuous variable meta-analysis and diagnostic meta-analysis were conducted, both in Stata 14, to show the expression of miR-1 in ccRCC. Furthermore, we acquired the potential targets of miR-1 from datasets that transfected miR-1 into ccRCC cells, online prediction databases, differentially expressed genes from TCGA and literature. Subsequently bioinformatics analysis based on aforementioned selected target genes was conducted. The combined effect was -0.92 with the 95% confidence interval (CI) of -1.08 to -0.77 based on fixed effect model (I 2  = 81.3%, P < 0.001). No publication bias was found in our investigation. Sensitivity analysis showed that GSE47582 and 2 TCGA studies might cause heterogeneity. After eliminating them, the combined effect was -0.47 (95%CI: -0.78, -0.16) with I 2  = 18.3%. As for the diagnostic meta-analysis, the combined sensitivity and specificity were 0.90 (95%CI: 0.61, 0.98) and 0.63 (95%CI: 0.39, 0.82). The area under the curve (AUC) in the summarized receiver operating characteristic (SROC) curve was 0.83 (95%CI: 0.80, 0.86). No publication bias was found (P = 0.15). We finally got 67 genes which were defined the promising target genes of miR-1 in ccRCC. The most three significant KEGG pathways based on the aforementioned genes were Complement and coagulation cascades, ECM-receptor interaction and Focal adhesion. The downregulation of miR-1 might play an important role in ccRCC by targeting its target genes. Copyright © 2017 Elsevier GmbH. All rights reserved.

Expression Profiling Smackdown: Human Transcriptome Array HTA 2.0 vs. RNA-Seq

PubMed Central

Palermo, Meghann; Driscoll, Heather; Tighe, Scott; Dragon, Julie; Bond, Jeff; Shukla, Arti; Vangala, Mahesh; Vincent, James; Hunter, Tim

2014-01-01

The advent of both microarray and massively parallel sequencing have revolutionized high-throughput analysis of the human transcriptome. Due to limitations in microarray technology, detecting and quantifying coding transcript isoforms, in addition to non-coding transcripts, has been challenging. As a result, RNA-Seq has been the preferred method for characterizing the full human transcriptome, until now. A new high-resolution array from Affymetrix, GeneChip Human Transcriptome Array 2.0 (HTA 2.0), has been designed to interrogate all transcript isoforms in the human transcriptome with >6 million probes targeting coding transcripts, exon-exon splice junctions, and non-coding transcripts. Here we compare expression results from GeneChip HTA 2.0 and RNA-Seq data using identical RNA extractions from three samples each of healthy human mesothelial cells in culture, LP9-C1, and healthy mesothelial cells treated with asbestos, LP9-A1. For GeneChip HTA 2.0 sample preparation, we chose to compare two target preparation methods, NuGEN Ovation Pico WTA V2 with the Encore Biotin Module versus Affymetrix's GeneChip WT PLUS with the WT Terminal Labeling Kit, on identical RNA extractions from both untreated and treated samples. These same RNA extractions were used for the RNA-Seq library preparation. All analyses were performed in Partek Genomics Suite 6.6. Expression profiles for control and asbestos-treated mesothelial cells prepared with NuGEN versus Affymetrix target preparation methods (GeneChip HTA 2.0) are compared to each other as well as to RNA-Seq results.

Multiplexing of miniaturized planar antibody arrays for serum protein profiling--a biomarker discovery in SLE nephritis.

PubMed

Petersson, Linn; Dexlin-Mellby, Linda; Bengtsson, Anders A; Sturfelt, Gunnar; Borrebaeck, Carl A K; Wingren, Christer

2014-06-07

In the quest to decipher disease-associated biomarkers, miniaturized and multiplexed antibody arrays may play a central role in generating protein expression profiles, or protein maps, of crude serum samples. In this conceptual study, we explored a novel, 4-times larger pen design, enabling us to, in a unique manner, simultaneously print 48 different reagents (antibodies) as individual 78.5 μm(2) (10 μm in diameter) sized spots at a density of 38,000 spots cm(-2) using dip-pen nanolithography technology. The antibody array set-up was interfaced with a high-resolution fluorescent-based scanner for sensitive sensing. The performance and applicability of this novel 48-plex recombinant antibody array platform design was demonstrated in a first clinical application targeting SLE nephritis, a severe chronic autoimmune connective tissue disorder, as the model disease. To this end, crude, directly biotinylated serum samples were targeted. The results showed that the miniaturized and multiplexed array platform displayed adequate performance, and that SLE-associated serum biomarker panels reflecting the disease process could be deciphered, outlining the use of miniaturized antibody arrays for disease proteomics and biomarker discovery.

Construction and application of a bovine immune-endocrine cDNA microarray.

PubMed

Tao, Wenjing; Mallard, Bonnie; Karrow, Niel; Bridle, Byram

2004-09-01

A variety of commercial DNA arrays specific for humans and rodents are widely available; however, microarrays containing well-characterized genes to study pathway-specific gene expression are not as accessible for domestic animals, such as cattle, sheep and pigs. Therefore, a small-scale application-targeted bovine immune-endocrine cDNA array was developed to evaluate genetic pathways involved in the immune-endocrine axis of cattle during periods of altered homeostasis provoked by physiological or environmental stressors, such as infection, vaccination or disease. For this purpose, 167 cDNA sequences corresponding to immune, endocrine and inflammatory response genes were collected and categorized. Positive controls included 5 housekeeping genes (glyceraldehydes-3-phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase, ribosomal protein L19, beta-actin, beta2-microglobulin) and bovine genomic DNA. Negative controls were a bacterial gene (Rhodococcus equi 17-kDa virulence-associated protein) and a partial sequence of the plasmid pACYC177. In addition, RNA extracted from un-stimulated, as well as superantigen (Staphylococcus aureus enterotoxin-A, S. aureus Cowan Pansorbin Cells) and mitogen-stimulated (LPS, ConA) bovine blood leukocytes was mixed, reverse transcribed and PCR amplified using gene-specific primers. The endocrine-associated genes were amplified from cDNA derived from un-stimulated bovine hypothalamus, pituitary, adrenal and thyroid gland tissues. The array was constructed in 4 repeating grids of 180 duplicated spots by coupling the PCR amplified 213-630 bp gene fragments onto poly-l-lysine coated glass slides. The bovine immune-endocrine arrays were standardized and preliminary gene expression profiles generated using Cy3 and Cy5 labelled cDNA from un-stimulated and ConA (5 microg/ml) stimulated PBMC of 4 healthy Holstein cows (2-4 replicate arrays/cow) in a time course study. Mononuclear cell-derived cytokine and chemokine (IL-2, IL-1alpha, TNFalpha, IFN-gamma, TGFbeta-1, MCP-1, MCP-2 and MIP-3alpha) mRNA exhibited a repeatable and consistently low expression in un-stimulated cells and at least a two-fold increased expression following 6 and 24 h ConA stimulation as compared to 0 h un-stimulated controls. In contrast, expression of antigen presenting molecules, MHC-DR, MHC-DQ and MHC-DY, were consistently at least two-fold lower following 6 and 24 h ConA stimulation. The only endocrine gene with differential expression following ConA stimulation was prolactin. Additionally, due to the high level of genetic homology between ovine, swine and bovine genes, RNA similarly acquired from sheep and pigs was evaluated and similar gene expression patterns were noted. These data demonstrate that this application-targeted array containing a set of well characterized genes can be used to determine the relative gene expression corresponding to immune-endocrine responses of cattle and related species, sheep and pigs.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gulec, Cagri, E-mail: cagri.gulec@gmail.com; Coban, Neslihan, E-mail: neslic@istanbul.edu.tr; Ozsait-Selcuk, Bilge, E-mail: ozsaitb@istanbul.edu.tr

ROR-alpha is a nuclear receptor, activity of which can be modulated by natural or synthetic ligands. Due to its possible involvement in, and potential therapeutic target for atherosclerosis, we aimed to identify ROR-alpha target genes in monocytic and endothelial cell lines. We performed chromatin immunoprecipitation (ChIP) followed by tiling array (ChIP-on-chip) for ROR-alpha in monocytic cell line THP1 and endothelial cell line HUVEC. Following bioinformatic analysis of the array data, we tested four candidate genes in terms of dependence of their expression level on ligand-mediated ROR-alpha activity, and two of them in terms of promoter occupancy by ROR-alpha. Bioinformatic analysesmore » of ChIP-on-chip data suggested that ROR-alpha binds to genomic regions near the transcription start site (TSS) of more than 3000 genes in THP1 and HUVEC. Potential ROR-alpha target genes in both cell types seem to be involved mainly in membrane receptor activity, signal transduction and ion transport. While SPP1 and IKBKA were shown to be direct target genes of ROR-alpha in THP1 monocytes, inflammation related gene HMOX1 and heat shock protein gene HSPA8 were shown to be potential target genes of ROR-alpha. Our results suggest that ROR-alpha may regulate signaling receptor activity, and transmembrane transport activity through its potential target genes. ROR-alpha seems also to play role in cellular sensitivity to environmental substances like arsenite and chloroprene. Although, the expression analyses have shown that synthetic ROR-alpha ligands can modulate some of potential ROR-alpha target genes, functional significance of ligand-dependent modulation of gene expression needs to be confirmed with further analyses.« less

Performance Analysis for Joint Target Parameter Estimation in UMTS-Based Passive Multistatic Radar with Antenna Arrays Using Modified Cramér-Rao Lower Bounds.

PubMed

Shi, Chenguang; Wang, Fei; Salous, Sana; Zhou, Jianjiang

2017-10-18

In this study, the modified Cramér-Rao lower bounds (MCRLBs) on the joint estimation of target position and velocity is investigated for a universal mobile telecommunication system (UMTS)-based passive multistatic radar system with antenna arrays. First, we analyze the log-likelihood redfunction of the received signal for a complex Gaussian extended target. Then, due to the non-deterministic transmitted data symbols, the analytically closed-form expressions of the MCRLBs on the Cartesian coordinates of target position and velocity are derived for a multistatic radar system with N t UMTS-based transmit station of L t antenna elements and N r receive stations of L r antenna elements. With the aid of numerical simulations, it is shown that increasing the number of receiving elements in each receive station can reduce the estimation errors. In addition, it is demonstrated that the MCRLB is not only a function of signal-to-noise ratio (SNR), the number of receiving antenna elements and the properties of the transmitted UMTS signals, but also a function of the relative geometric configuration between the target and the multistatic radar system.The analytical expressions for MCRLB will open up a new dimension for passive multistatic radar system by aiding the optimal placement of receive stations to improve the target parameter estimation performance.

Performance Analysis for Joint Target Parameter Estimation in UMTS-Based Passive Multistatic Radar with Antenna Arrays Using Modified Cramér-Rao Lower Bounds

PubMed Central

Wang, Fei; Salous, Sana; Zhou, Jianjiang

2017-01-01

In this study, the modified Cramér-Rao lower bounds (MCRLBs) on the joint estimation of target position and velocity is investigated for a universal mobile telecommunication system (UMTS)-based passive multistatic radar system with antenna arrays. First, we analyze the log-likelihood redfunction of the received signal for a complex Gaussian extended target. Then, due to the non-deterministic transmitted data symbols, the analytically closed-form expressions of the MCRLBs on the Cartesian coordinates of target position and velocity are derived for a multistatic radar system with Nt UMTS-based transmit station of Lt antenna elements and Nr receive stations of Lr antenna elements. With the aid of numerical simulations, it is shown that increasing the number of receiving elements in each receive station can reduce the estimation errors. In addition, it is demonstrated that the MCRLB is not only a function of signal-to-noise ratio (SNR), the number of receiving antenna elements and the properties of the transmitted UMTS signals, but also a function of the relative geometric configuration between the target and the multistatic radar system.The analytical expressions for MCRLB will open up a new dimension for passive multistatic radar system by aiding the optimal placement of receive stations to improve the target parameter estimation performance. PMID:29057805

Genome-wide analysis of endogenously expressed ZEB2 binding sites reveals inverse correlations between ZEB2 and GalNAc-transferase GALNT3 in human tumors.

PubMed

Balcik-Ercin, Pelin; Cetin, Metin; Yalim-Camci, Irem; Odabas, Gorkem; Tokay, Nurettin; Sayan, A Emre; Yagci, Tamer

2018-03-07

ZEB2 is a transcriptional repressor that regulates epithelial-to-mesenchymal transition (EMT) through binding to bipartite E-box motifs in gene regulatory regions. Despite the abundant presence of E-boxes within the human genome and the multiplicity of pathophysiological processes regulated during ZEB2-induced EMT, only a small fraction of ZEB2 targets has been identified so far. Hence, we explored genome-wide ZEB2 binding by chromatin immunoprecipitation-sequencing (ChIP-seq) under endogenous ZEB2 expression conditions. For ChIP-Seq we used an anti-ZEB2 monoclonal antibody, clone 6E5, in SNU398 hepatocellular carcinoma cells exhibiting a high endogenous ZEB2 expression. The ChIP-Seq targets were validated using ChIP-qPCR, whereas ZEB2-dependent expression of target genes was assessed by RT-qPCR and Western blotting in shRNA-mediated ZEB2 silenced SNU398 cells and doxycycline-induced ZEB2 overexpressing colorectal carcinoma DLD1 cells. Changes in target gene expression were also assessed using primary human tumor cDNA arrays in conjunction with RT-qPCR. Additional differential expression and correlation analyses were performed using expO and Human Protein Atlas datasets. Over 500 ChIP-Seq positive genes were annotated, and intervals related to these genes were found to include the ZEB2 binding motif CACCTG according to TOMTOM motif analysis in the MEME Suite database. Assessment of ZEB2-dependent expression of target genes in ZEB2-silenced SNU398 cells and ZEB2-induced DLD1 cells revealed that the GALNT3 gene serves as a ZEB2 target with the highest, but inversely correlated, expression level. Remarkably, GALNT3 also exhibited the highest enrichment in the ChIP-qPCR validation assays. Through the analyses of primary tumor cDNA arrays and expO datasets a significant differential expression and a significant inverse correlation between ZEB2 and GALNT3 expression were detected in most of the tumors. We also explored ZEB2 and GALNT3 protein expression using the Human Protein Atlas dataset and, again, observed an inverse correlation in all analyzed tumor types, except malignant melanoma. In contrast to a generally negative or weak ZEB2 expression, we found that most tumor tissues exhibited a strong or moderate GALNT3 expression. Our observation that ZEB2 negatively regulates a GalNAc-transferase (GALNT3) that is involved in O-glycosylation adds another layer of complexity to the role of ZEB2 in cancer progression and metastasis. Proteins glycosylated by GALNT3 may be exploited as novel diagnostics and/or therapeutic targets.

Fusion genes in solid tumors: an emerging target for cancer diagnosis and treatment.

PubMed

Parker, Brittany C; Zhang, Wei

2013-11-01

Studies over the past decades have uncovered fusion genes, a class of oncogenes that provide immense diagnostic and therapeutic advantages because of their tumor-specific expression. Originally associated with hemotologic cancers, fusion genes have recently been discovered in a wide array of solid tumors, including sarcomas, carcinomas, and tumors of the central nervous system. Fusion genes are attractive as both therapeutic targets and diagnostic tools due to their inherent expression in tumor tissue alone. Therefore, the discovery and elucidation of fusion genes in various cancer types may provide more effective therapies in the future for cancer patients.

Mobilization of Neural Precursors in the Circulating Blood of Patients with Multiple Sclerosis

DTIC Science & Technology

2013-09-01

Bongarzone ER. Expression of sonic hedgehog targeted genes in peripheral blood mononuclear cells of patients with multiple sclerosis. Society for...Print Program#/Poster#: 322.13 Presentation Title: Expression of sonic  hedgehog  targeted genes in peripheral blood mononuclear cells of patients with...analyses. Gene array hybridization showed up­ regulation of various components of the Sonic  hedgehog  (Shh) pathway including, Olig1 and Olig2. Taken

Organ-specific gene expression: the bHLH protein Sage provides tissue specificity to Drosophila FoxA.

PubMed

Fox, Rebecca M; Vaishnavi, Aria; Maruyama, Rika; Andrew, Deborah J

2013-05-01

FoxA transcription factors play major roles in organ-specific gene expression, regulating, for example, glucagon expression in the pancreas, GLUT2 expression in the liver, and tyrosine hydroxylase expression in dopaminergic neurons. Organ-specific gene regulation by FoxA proteins is achieved through cooperative regulation with a broad array of transcription factors with more limited expression domains. Fork head (Fkh), the sole Drosophila FoxA family member, is required for the development of multiple distinct organs, yet little is known regarding how Fkh regulates tissue-specific gene expression. Here, we characterize Sage, a bHLH transcription factor expressed exclusively in the Drosophila salivary gland (SG). We show that Sage is required for late SG survival and normal tube morphology. We find that many Sage targets, identified by microarray analysis, encode SG-specific secreted cargo, transmembrane proteins, and the enzymes that modify these proteins. We show that both Sage and Fkh are required for the expression of Sage target genes, and that co-expression of Sage and Fkh is sufficient to drive target gene expression in multiple cell types. Sage and Fkh drive expression of the bZip transcription factor Senseless (Sens), which boosts expression of Sage-Fkh targets, and Sage, Fkh and Sens colocalize on SG chromosomes. Importantly, expression of Sage-Fkh target genes appears to simply add to the tissue-specific gene expression programs already established in other cell types, and Sage and Fkh cannot alter the fate of most embryonic cell types even when expressed early and continuously.

Organ-specific gene expression: the bHLH protein Sage provides tissue specificity to Drosophila FoxA

PubMed Central

Fox, Rebecca M.; Vaishnavi, Aria; Maruyama, Rika; Andrew, Deborah J.

2013-01-01

FoxA transcription factors play major roles in organ-specific gene expression, regulating, for example, glucagon expression in the pancreas, GLUT2 expression in the liver, and tyrosine hydroxylase expression in dopaminergic neurons. Organ-specific gene regulation by FoxA proteins is achieved through cooperative regulation with a broad array of transcription factors with more limited expression domains. Fork head (Fkh), the sole Drosophila FoxA family member, is required for the development of multiple distinct organs, yet little is known regarding how Fkh regulates tissue-specific gene expression. Here, we characterize Sage, a bHLH transcription factor expressed exclusively in the Drosophila salivary gland (SG). We show that Sage is required for late SG survival and normal tube morphology. We find that many Sage targets, identified by microarray analysis, encode SG-specific secreted cargo, transmembrane proteins, and the enzymes that modify these proteins. We show that both Sage and Fkh are required for the expression of Sage target genes, and that co-expression of Sage and Fkh is sufficient to drive target gene expression in multiple cell types. Sage and Fkh drive expression of the bZip transcription factor Senseless (Sens), which boosts expression of Sage-Fkh targets, and Sage, Fkh and Sens colocalize on SG chromosomes. Importantly, expression of Sage-Fkh target genes appears to simply add to the tissue-specific gene expression programs already established in other cell types, and Sage and Fkh cannot alter the fate of most embryonic cell types even when expressed early and continuously. PMID:23578928

Arrayed antibody library technology for therapeutic biologic discovery.

PubMed

Bentley, Cornelia A; Bazirgan, Omar A; Graziano, James J; Holmes, Evan M; Smider, Vaughn V

2013-03-15

Traditional immunization and display antibody discovery methods rely on competitive selection amongst a pool of antibodies to identify a lead. While this approach has led to many successful therapeutic antibodies, targets have been limited to proteins which are easily purified. In addition, selection driven discovery has produced a narrow range of antibody functionalities focused on high affinity antagonism. We review the current progress in developing arrayed protein libraries for screening-based, rather than selection-based, discovery. These single molecule per microtiter well libraries have been screened in multiplex formats against both purified antigens and directly against targets expressed on the cell surface. This facilitates the discovery of antibodies against therapeutically interesting targets (GPCRs, ion channels, and other multispanning membrane proteins) and epitopes that have been considered poorly accessible to conventional discovery methods. Copyright © 2013. Published by Elsevier Inc.

Understanding the dynamics of superparamagnetic particles under the influence of high field gradient arrays

NASA Astrophysics Data System (ADS)

Barnsley, Lester C.; Carugo, Dario; Aron, Miles; Stride, Eleanor

2017-03-01

The aim of this study was to characterize the behaviour of superparamagnetic particles in magnetic drug targeting (MDT) schemes. A 3-dimensional mathematical model was developed, based on the analytical derivation of the trajectory of a magnetized particle suspended inside a fluid channel carrying laminar flow and in the vicinity of an external source of magnetic force. Semi-analytical expressions to quantify the proportion of captured particles, and their relative accumulation (concentration) as a function of distance along the wall of the channel were also derived. These were expressed in terms of a non-dimensional ratio of the relevant physical and physiological parameters corresponding to a given MDT protocol. The ability of the analytical model to assess magnetic targeting schemes was tested against numerical simulations of particle trajectories. The semi-analytical expressions were found to provide good first-order approximations for the performance of MDT systems in which the magnetic force is relatively constant over a large spatial range. The numerical model was then used to test the suitability of a range of different designs of permanent magnet assemblies for MDT. The results indicated that magnetic arrays that emit a strong magnetic force that varies rapidly over a confined spatial range are the most suitable for concentrating magnetic particles in a localized region. By comparison, commonly used magnet geometries such as button magnets and linear Halbach arrays result in distributions of accumulated particles that are less efficient for delivery. The trajectories predicted by the numerical model were verified experimentally by acoustically focusing magnetic microbeads flowing in a glass capillary channel, and optically tracking their path past a high field gradient Halbach array.

Extract of Stellerachamaejasme L(ESC) inhibits growth and metastasis of human hepatocellular carcinoma via regulating microRNA expression.

PubMed

Liu, Xiaoni; Wang, Shuang; Xu, Jianji; Kou, Buxin; Chen, Dexi; Wang, Yajie; Zhu, Xiaoxin

2018-03-20

MicroRNAs(miRNAs)are involved in the initiation and progression of hepatocellular carcinoma. ESC, an extract of Stellerachamaejasme L, had been confirmed as a potential anti-tumor extract of Traditional Chinese Medicine. In light of the important role of miRNAs in hepatocellular carcinoma, we questioned whether the inhibitory effects of ESC on hepatocellular carcinoma (HCC) were associated with miRNAs. The proliferation inhibition of ESC on HCC cells was measured with MTT assay. The migration inhibition of ESC on HCC cells was measured with transwell assay. The influences of ESC on growth and metastasis inhibition were evaluated with xenograft tumor model of HCC. Protein expressions were measured with western blot and immunofluorescence methods and miRNA profiles were detected with miRNA array. Differential miRNA and target mRNAs were verified with real-time PCR. The results showed that ESC could inhibit proliferation and epithelial mesenchymal transition (EMT) in HCC cells in vitro and tumor growth and metastasis in xenograft models in vivo. miRNA array results showed that 69 differential miRNAs in total of 429 ones were obtained in MHCC97H cells treated by ESC. hsa-miR-107, hsa-miR-638, hsa-miR-106b-5p were selected to be validated with real-time PCR method in HepG2 and MHCC97H cells. Expressions of hsa-miR-107 and hsa-miR-638 increased obviously in HCC cells treated by ESC. Target genes of three miRNAs were also validated with real-time PCR. Interestingly, only target genes of hsa-miR-107 changed greatly. ESC downregulated the MCL1, SALL4 and BCL2 gene expressions significantly but did not influence the expression of CACNA2D1. The findings suggested ESC regressed growth and metastasis of human hepatocellular carcinoma via regulating microRNAs expression and their corresponding target genes.

Aging and calorie restriction regulate the expression of miR-125a-5p and its target genes Stat3, Casp2 and Stard13.

PubMed

Makwana, Kuldeep; Patel, Sonal Arvind; Velingkaar, Nikkhil; Ebron, Jey Sabith; Shukla, Girish C; Kondratov, Roman V Kondratov V

2017-07-31

Calorie restriction (CR) is a dietary intervention known to delay aging. In order, to understand molecular mechanisms of CR, we analyzed the expression of 983 MicroRNAs (miRNAs) in the liver of female mice after 2 years of 30% CR using micro-array. 16 miRNAs demonstrated significant changes in their expression upon CR in comparison with age-matched control. mmu-miR-125a-5p (miR-125a-5p) was significantly upregulated upon CR, and in agreement with this, the expression of mRNAs for its three predicted target genes: Stat3, Casp2, and Stard13 was significantly downregulated in the liver of CR animals. The expression of precursor miRNA for miR-125a-5p was also upregulated upon CR, which suggests its regulation at the level of transcription. Upon aging miR-125a-5p expression was downregulated while the expression of its target genes was upregulated. Thus, CR prevented age-associated changes in the expression of miR-125a-5p and its targets. We propose that miR-125a-5p dependent downregulation of Stat3, Casp2, and Stard13 contributes to the calorie restriction-mediated delay of aging.

Molecular Targeted Therapies of Childhood Choroid Plexus Carcinoma

DTIC Science & Technology

2012-10-01

Microarray intensities were analyzed in PGS, using the benign human choroid plexus papilloma (CPP) samples as an expression baseline reference...identify candidate drug targets of CPC. Task 1: Generation of additional human and mouse CPC genomic profiles (timeframe: months 1-5). The goal...of these studies is to expand our number of genomic profiles (DNA and mRNA arrays) of both human and mouse CPCs to provide a comprehensive dataset

Integrated analysis of copy number alteration and RNA expression profiles of cancer using a high-resolution whole-genome oligonucleotide array.

PubMed

Jung, Seung-Hyun; Shin, Seung-Hun; Yim, Seon-Hee; Choi, Hye-Sun; Lee, Sug-Hyung; Chung, Yeun-Jun

2009-07-31

Recently, microarray-based comparative genomic hybridization (array-CGH) has emerged as a very efficient technology with higher resolution for the genome-wide identification of copy number alterations (CNA). Although CNAs are thought to affect gene expression, there is no platform currently available for the integrated CNA-expression analysis. To achieve high-resolution copy number analysis integrated with expression profiles, we established human 30k oligoarray-based genome-wide copy number analysis system and explored the applicability of this system for integrated genome and transcriptome analysis using MDA-MB-231 cell line. We compared the CNAs detected by the oligoarray with those detected by the 3k BAC array for validation. The oligoarray identified the single copy difference more accurately and sensitively than the BAC array. Seventeen CNAs detected by both platforms in MDA-MB-231 such as gains of 5p15.33-13.1, 8q11.22-8q21.13, 17p11.2, and losses of 1p32.3, 8p23.3-8p11.21, and 9p21 were consistently identified in previous studies on breast cancer. There were 122 other small CNAs (mean size 1.79 mb) that were detected by oligoarray only, not by BAC-array. We performed genomic qPCR targeting 7 CNA regions, detected by oligoarray only, and one non-CNA region to validate the oligoarray CNA detection. All qPCR results were consistent with the oligoarray-CGH results. When we explored the possibility of combined interpretation of both DNA copy number and RNA expression profiles, mean DNA copy number and RNA expression levels showed a significant correlation. In conclusion, this 30k oligoarray-CGH system can be a reasonable choice for analyzing whole genome CNAs and RNA expression profiles at a lower cost.

Genomewide Analysis of Aryl Hydrocarbon Receptor Binding Targets Reveals an Extensive Array of Gene Clusters that Control Morphogenetic and Developmental Programs

PubMed Central

Sartor, Maureen A.; Schnekenburger, Michael; Marlowe, Jennifer L.; Reichard, John F.; Wang, Ying; Fan, Yunxia; Ma, Ci; Karyala, Saikumar; Halbleib, Danielle; Liu, Xiangdong; Medvedovic, Mario; Puga, Alvaro

2009-01-01

Background The vertebrate aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates cellular responses to environmental polycyclic and halogenated compounds. The naive receptor is believed to reside in an inactive cytosolic complex that translocates to the nucleus and induces transcription of xenobiotic detoxification genes after activation by ligand. Objectives We conducted an integrative genomewide analysis of AHR gene targets in mouse hepatoma cells and determined whether AHR regulatory functions may take place in the absence of an exogenous ligand. Methods The network of AHR-binding targets in the mouse genome was mapped through a multipronged approach involving chromatin immunoprecipitation/chip and global gene expression signatures. The findings were integrated into a prior functional knowledge base from Gene Ontology, interaction networks, Kyoto Encyclopedia of Genes and Genomes pathways, sequence motif analysis, and literature molecular concepts. Results We found the naive receptor in unstimulated cells bound to an extensive array of gene clusters with functions in regulation of gene expression, differentiation, and pattern specification, connecting multiple morphogenetic and developmental programs. Activation by the ligand displaced the receptor from some of these targets toward sites in the promoters of xenobiotic metabolism genes. Conclusions The vertebrate AHR appears to possess unsuspected regulatory functions that may be potential targets of environmental injury. PMID:19654925

MicroRNA signature of the human developing pancreas.

PubMed

Rosero, Samuel; Bravo-Egana, Valia; Jiang, Zhijie; Khuri, Sawsan; Tsinoremas, Nicholas; Klein, Dagmar; Sabates, Eduardo; Correa-Medina, Mayrin; Ricordi, Camillo; Domínguez-Bendala, Juan; Diez, Juan; Pastori, Ricardo L

2010-09-22

MicroRNAs are non-coding RNAs that regulate gene expression including differentiation and development by either inhibiting translation or inducing target degradation. The aim of this study is to determine the microRNA expression signature during human pancreatic development and to identify potential microRNA gene targets calculating correlations between the signature microRNAs and their corresponding mRNA targets, predicted by bioinformatics, in genome-wide RNA microarray study. The microRNA signature of human fetal pancreatic samples 10-22 weeks of gestational age (wga), was obtained by PCR-based high throughput screening with Taqman Low Density Arrays. This method led to identification of 212 microRNAs. The microRNAs were classified in 3 groups: Group number I contains 4 microRNAs with the increasing profile; II, 35 microRNAs with decreasing profile and III with 173 microRNAs, which remain unchanged. We calculated Pearson correlations between the expression profile of microRNAs and target mRNAs, predicted by TargetScan 5.1 and miRBase algorithms, using genome-wide mRNA expression data. Group I correlated with the decreasing expression of 142 target mRNAs and Group II with the increasing expression of 876 target mRNAs. Most microRNAs correlate with multiple targets, just as mRNAs are targeted by multiple microRNAs. Among the identified targets are the genes and transcription factors known to play an essential role in pancreatic development. We have determined specific groups of microRNAs in human fetal pancreas that change the degree of their expression throughout the development. A negative correlative analysis suggests an intertwined network of microRNAs and mRNAs collaborating with each other. This study provides information leading to potential two-way level of combinatorial control regulating gene expression through microRNAs targeting multiple mRNAs and, conversely, target mRNAs regulated in parallel by other microRNAs as well. This study may further the understanding of gene expression regulation in the human developing pancreas.

MicroRNA signature of the human developing pancreas

PubMed Central

2010-01-01

Background MicroRNAs are non-coding RNAs that regulate gene expression including differentiation and development by either inhibiting translation or inducing target degradation. The aim of this study is to determine the microRNA expression signature during human pancreatic development and to identify potential microRNA gene targets calculating correlations between the signature microRNAs and their corresponding mRNA targets, predicted by bioinformatics, in genome-wide RNA microarray study. Results The microRNA signature of human fetal pancreatic samples 10-22 weeks of gestational age (wga), was obtained by PCR-based high throughput screening with Taqman Low Density Arrays. This method led to identification of 212 microRNAs. The microRNAs were classified in 3 groups: Group number I contains 4 microRNAs with the increasing profile; II, 35 microRNAs with decreasing profile and III with 173 microRNAs, which remain unchanged. We calculated Pearson correlations between the expression profile of microRNAs and target mRNAs, predicted by TargetScan 5.1 and miRBase altgorithms, using genome-wide mRNA expression data. Group I correlated with the decreasing expression of 142 target mRNAs and Group II with the increasing expression of 876 target mRNAs. Most microRNAs correlate with multiple targets, just as mRNAs are targeted by multiple microRNAs. Among the identified targets are the genes and transcription factors known to play an essential role in pancreatic development. Conclusions We have determined specific groups of microRNAs in human fetal pancreas that change the degree of their expression throughout the development. A negative correlative analysis suggests an intertwined network of microRNAs and mRNAs collaborating with each other. This study provides information leading to potential two-way level of combinatorial control regulating gene expression through microRNAs targeting multiple mRNAs and, conversely, target mRNAs regulated in parallel by other microRNAs as well. This study may further the understanding of gene expression regulation in the human developing pancreas. PMID:20860821

Inflammation-Mediated Regulation of MicroRNA Expression in Transplanted Pancreatic Islets

PubMed Central

Bravo-Egana, Valia; Rosero, Samuel; Klein, Dagmar; Jiang, Zhijie; Vargas, Nancy; Tsinoremas, Nicholas; Doni, Marco; Podetta, Michele; Ricordi, Camillo; Molano, R. Damaris; Pileggi, Antonello; Pastori, Ricardo L.

2012-01-01

Nonspecific inflammation in the transplant microenvironment results in β-cell dysfunction and death influencing negatively graft outcome. MicroRNA (miRNA) expression and gene target regulation in transplanted islets are not yet well characterized. We evaluated the impact of inflammation on miRNA expression in transplanted rat islets. Islets exposed in vitro to proinflammatory cytokines and explanted syngeneic islet grafts were evaluated by miRNA arrays. A subset of 26 islet miRNAs was affected by inflammation both in vivo and in vitro. Induction of miRNAs was dependent on NF-κB, a pathway linked with cytokine-mediated islet cell death. RT-PCR confirmed expression of 8 miRNAs. The association between these miRNAs and mRNA target-predicting algorithms in genome-wide RNA studies of β-cell inflammation identified 238 potential miRNA gene targets. Several genes were ontologically associated with regulation of insulin signaling and secretion, diabetes, and islet physiology. One of the most activated miRNAs was miR-21. Overexpression of miR-21 in insulin-secreting MIN6 cells downregulated endogenous expression of the tumor suppressor Pdcd4 and of Pclo, a Ca2+ sensor protein involved in insulin secretion. Bioinformatics identified both as potential targets. The integrated analysis of miRNA and mRNA expression profiles revealed potential targets that may identify molecular targets for therapeutic interventions. PMID:22655170

Multiplexed chemiluminescent assays in ArrayPlates for high-throughput measurement of gene expression

NASA Astrophysics Data System (ADS)

Martel, Ralph R.; Rounseville, Matthew P.; Botros, Ihab W.; Seligmann, Bruce E.

2002-06-01

Multiplexed Molecular Profiling (MMP) assays for drug discovery are performed in ArrayPlates. ArrayPlates are 96- well microtiter plates that contain a 16-element array at the bottom of each well. Each element within an array measures one analyte in a sample. A CCD imager records the quantitative chemiluminescent readout of all 1,536 elements in a 96-well plate simultaneously. Since array elements are reagent modifiable by the end-user, ArrayPlates can be adapted to a broad range of nucleic acid- and protein-based assays. Such multiplexed assays are rapidly established, flexible, robust, automation-friendly and cost-effective. Nucleic acid assays in ArrayPlates can detect DNA and RNA, including SNPs and ESTs. A multiplexed mRNA assay to measure the expression of 16 genes is described. The assay combines a homogeneous nuclease protection assay with subsequent probe immobilization to the array by means of a sandwich hybridization followed with chemiluminescent detection. This assay was used to examine cells grown and treated in microplates and avoided cloning, transfection, RNA insolation, reverse transcription, amplification and fluorochrome labeling. Standard deviations for the measurement of 16 genes ranged from 3 percent to 13 percent in samples of 30,000 cells. Such ArrayPlates transcription assays are useful in drug discovery and development for target validation, screening, lead optimization, metabolism and toxicity profiling. Chemiluminescent detection provides ArrayPlates assays with high signal-to-noise readout and simplifies imager requirements. Imaging a 2D surface that contains arrays simplifies lens requirements relative to imaging columns of liquid in microtiter plate wells. The Omix imager for ArrayPlates is described.

Three-dimensional ultrasound molecular imaging of angiogenesis in colon cancer using a clinical matrix array ultrasound transducer.

PubMed

Wang, Huaijun; Kaneko, Osamu F; Tian, Lu; Hristov, Dimitre; Willmann, Jürgen K

2015-05-01

We sought to assess the feasibility and reproducibility of 3-dimensional ultrasound molecular imaging (USMI) of vascular endothelial growth factor receptor 2 (VEGFR2) expression in tumor angiogenesis using a clinical matrix array transducer and a clinical grade VEGFR2-targeted contrast agent in a murine model of human colon cancer. Animal studies were approved by the Institutional Administrative Panel on Laboratory Animal Care. Mice with human colon cancer xenografts (n = 33) were imaged with a clinical ultrasound system and transducer (Philips iU22; X6-1) after intravenous injection of either clinical grade VEGFR2-targeted microbubbles or nontargeted control microbubbles. Nineteen mice were scanned twice to assess imaging reproducibility. Fourteen mice were scanned both before and 24 hours after treatment with either bevacizumab (n = 7) or saline only (n = 7). Three-dimensional USMI data sets were retrospectively reconstructed into multiple consecutive 1-mm-thick USMI data sets to simulate 2-dimensional imaging. Vascular VEGFR2 expression was assessed ex vivo using immunofluorescence. Three-dimensional USMI was highly reproducible using both VEGFR2-targeted microbubbles and nontargeted control microbubbles (intraclass correlation coefficient, 0.83). The VEGFR2-targeted USMI signal significantly (P = 0.02) decreased by 57% after antiangiogenic treatment compared with the control group, which correlated well with ex vivo VEGFR2 expression on immunofluorescence (ρ = 0.93, P = 0.003). If only central 1-mm tumor planes were analyzed to assess antiangiogenic treatment response, the USMI signal change was significantly (P = 0.006) overestimated by an average of 27% (range, 2%-73%) compared with 3-dimensional USMI. Three-dimensional USMI is feasible and highly reproducible and allows accurate assessment and monitoring of VEGFR2 expression in tumor angiogenesis in a murine model of human colon cancer.

Lectin array and glycogene expression analyses of ovarian cancer cell line A2780 and its cisplatin-resistant derivate cell line A2780-cp.

PubMed

Zhao, Ran; Qin, Wenjun; Qin, Ruihuan; Han, Jing; Li, Can; Wang, Yisheng; Xu, Congjian

2017-01-01

Ovarian cancer is one of the most lethal gynecological malignancies, in which platinum resistance is a common cause of its relapse and death. Glycosylation has been reported to be involved in drug resistance, and glycomic analyses of ovarian cancer may improve our understanding of the mechanisms underlying cancer cell drug resistance and provide potential biomarkers and therapeutic targets. The serous ovarian cancer cell line A2780 and its platinum-resistant counterpart A2780-cp were used in this study. We performed a lectin array analysis to compare the glycosylation patterns of the two cell lines, a gene expression array was employed to probe the differences in glycogenes. Furthermore, the results were verified by lectin blots. A2780-cp cell exhibited stronger intensities of Lens culinaris (LCA) Canavalia ensiformis (ConA), and Lycopersicon esculentum (LEL) and weaker intensities of Sambucus nigra (SNA) lectins. The gene expression array analysis revealed increased expression of Fut8, B3gnt4, B3gnt5, B4galt2 and decreased expression of Fut1 and ST6GalNAc 6 expression were evident in the A2780-cp cells. The lectin blot confirmed the differences in LCA, ConA, SNA and LEL between the A2780 and A2780-cp cells. The combination of the lectin and gene expression analyses showed that the levels of core fucosylation and poly-LacNAc were increased in the A2780-cp cells and the levels of Fuc α1-2(gal β1-4) GlcNAc and α2-6-linked sialic structures were decreased in the A2780-cp cells. These glycans represent potential biomarkers and might be involved in the mechanism of drug resistance in ovarian cancer.

Engineering customized TALE nucleases (TALENs) and TALE transcription factors by fast ligation-based automatable solid-phase high-throughput (FLASH) assembly.

PubMed

Reyon, Deepak; Maeder, Morgan L; Khayter, Cyd; Tsai, Shengdar Q; Foley, Jonathan E; Sander, Jeffry D; Joung, J Keith

2013-07-01

Customized DNA-binding domains made using transcription activator-like effector (TALE) repeats are rapidly growing in importance as widely applicable research tools. TALE nucleases (TALENs), composed of an engineered array of TALE repeats fused to the FokI nuclease domain, have been used successfully for directed genome editing in various organisms and cell types. TALE transcription factors (TALE-TFs), consisting of engineered TALE repeat arrays linked to a transcriptional regulatory domain, have been used to up- or downregulate expression of endogenous genes in human cells and plants. This unit describes a detailed protocol for the recently described fast ligation-based automatable solid-phase high-throughput (FLASH) assembly method. FLASH enables automated high-throughput construction of engineered TALE repeats using an automated liquid handling robot or manually using a multichannel pipet. Using the automated approach, a single researcher can construct up to 96 DNA fragments encoding TALE repeat arrays of various lengths in a single day, and then clone these to construct sequence-verified TALEN or TALE-TF expression plasmids in a week or less. Plasmids required for FLASH are available by request from the Joung lab (http://eGenome.org). This unit also describes improvements to the Zinc Finger and TALE Targeter (ZiFiT Targeter) web server (http://ZiFiT.partners.org) that facilitate the design and construction of FLASH TALE repeat arrays in high throughput. © 2013 by John Wiley & Sons, Inc.

Engineering Customized TALE Nucleases (TALENs) and TALE Transcription Factors by Fast Ligation-based Automatable Solid-phase High-throughput (FLASH) Assembly

PubMed Central

Reyon, Deepak; Maeder, Morgan L.; Khayter, Cyd; Tsai, Shengdar Q.; Foley, Jonathan E.; Sander, Jeffry D.; Joung, J. Keith

2013-01-01

Customized DNA-binding domains made using Transcription Activator-Like Effector (TALE) repeats are rapidly growing in importance as widely applicable research tools. TALE nucleases (TALENs), composed of an engineered array of TALE repeats fused to the FokI nuclease domain, have been used successfully for directed genome editing in multiple different organisms and cell types. TALE transcription factors (TALE-TFs), consisting of engineered TALE repeat arrays linked to a transcriptional regulatory domain, have been used to up- or down-regulate expression of endogenous genes in human cells and plants. Here we describe a detailed protocol for practicing the recently described Fast Ligation-based Automatable Solid-phase High-throughput (FLASH) assembly method. FLASH enables automated high-throughput construction of engineered TALE repeats using an automated liquid handling robot or manually using a multi-channel pipet. With the automated version of FLASH, a single researcher can construct up to 96 DNA fragments encoding various length TALE repeat arrays in one day and then clone these to construct sequence-verified TALEN or TALE-TF expression plasmids in one week or less. Plas-mids required to practice FLASH are available by request from the Joung Lab (http://www.jounglab.org/). We also describe here improvements to the Zinc Finger and TALE Targeter (ZiFiT Targeter) webserver (http://ZiFiTBeta.partners.org) that facilitate the design and construction of FLASH TALE repeat arrays in high-throughput. PMID:23821439

Correction of Spatial Bias in Oligonucleotide Array Data

PubMed Central

Lemieux, Sébastien

2013-01-01

Background. Oligonucleotide microarrays allow for high-throughput gene expression profiling assays. The technology relies on the fundamental assumption that observed hybridization signal intensities (HSIs) for each intended target, on average, correlate with their target's true concentration in the sample. However, systematic, nonbiological variation from several sources undermines this hypothesis. Background hybridization signal has been previously identified as one such important source, one manifestation of which appears in the form of spatial autocorrelation. Results. We propose an algorithm, pyn, for the elimination of spatial autocorrelation in HSIs, exploiting the duality of desirable mutual information shared by probes in a common probe set and undesirable mutual information shared by spatially proximate probes. We show that this correction procedure reduces spatial autocorrelation in HSIs; increases HSI reproducibility across replicate arrays; increases differentially expressed gene detection power; and performs better than previously published methods. Conclusions. The proposed algorithm increases both precision and accuracy, while requiring virtually no changes to users' current analysis pipelines: the correction consists merely of a transformation of raw HSIs (e.g., CEL files for Affymetrix arrays). A free, open-source implementation is provided as an R package, compatible with standard Bioconductor tools. The approach may also be tailored to other platform types and other sources of bias. PMID:23573083

A composite transcriptional signature differentiates responses towards closely related herbicides in Arabidopsis thaliana and brassica napus

EPA Science Inventory

In this study, genome-wide expression profiling based on Affymetrix ATH1 arrays was used to identify discriminating responses of Arabidopsis thaliana to five herbicides, which contain active ingredients targeting two different branches of amino acid biosynthesis. One herbicide co...

COMPARISON OF PM-INDUCED GENE EXPRESSION PROFILES BETWEEN BRONCHIAL EPITHELIAL CELLS AND NASAL EPITHELIAL CELLS IN HUMAN

EPA Science Inventory

Epidemiologic studies have linked exposures to particulate matter (PM) and increased pulmonary mortality and morbidity. Bronchial epithelial cells (BEC) are the primary target of PM. PM exposure induces a wide array of biological responses in BEC. Primary human BEC, however, need...

TARGET Research Goals

Cancer.gov

TARGET researchers use various sequencing and array-based methods to examine the genomes, transcriptomes, and for some diseases epigenomes of select childhood cancers. This “multi-omic” approach generates a comprehensive profile of molecular alterations for each cancer type. Alterations are changes in DNA or RNA, such as rearrangements in chromosome structure or variations in gene expression, respectively. Through computational analyses and assays to validate biological function, TARGET researchers predict which alterations disrupt the function of a gene or pathway and promote cancer growth, progression, and/or survival. Researchers identify candidate therapeutic targets and/or prognostic markers from the cancer-associated alterations.

Mapping and characterization of the amplicon near APOA2 in 1q23 in human sarcomas by FISH and array CGH.

PubMed

Kresse, Stine H; Berner, Jeanne-Marie; Meza-Zepeda, Leonardo A; Gregory, Simon G; Kuo, Wen-Lin; Gray, Joe W; Forus, Anne; Myklebost, Ola

2005-11-07

Amplification of the q21-q23 region on chromosome 1 is frequently found in sarcomas and a variety of other solid tumours. Previous analyses of sarcomas have indicated the presence of at least two separate amplicons within this region, one located in 1q21 and one located near the apolipoprotein A-II (APOA2) gene in 1q23. In this study we have mapped and characterized the amplicon in 1q23 in more detail. We have used fluorescence in situ hybridisation (FISH) and microarray-based comparative genomic hybridisation (array CGH) to map and define the borders of the amplicon in 10 sarcomas. A subregion of approximately 800 kb was identified as the core of the amplicon. The amplification patterns of nine possible candidate target genes located to this subregion were determined by Southern blot analysis. The genes activating transcription factor 6 (ATF6) and dual specificity phosphatase 12 (DUSP12) showed the highest level of amplification, and they were also shown to be over-expressed by quantitative real-time reverse transcription PCR (RT-PCR). In general, the level of expression reflected the level of amplification in the different tumours. DUSP12 was expressed significantly higher than ATF6 in a subset of the tumours. In addition, two genes known to be transcriptionally activated by ATF6, glucose-regulated protein 78 kDa and -94 kDa (GRP78 and GRP94), were shown to be over-expressed in the tumours that showed over-expression of ATF6. ATF6 and DUSP12 seem to be the most likely candidate target genes for the 1q23 amplification in sarcomas. Both genes have possible roles in promoting cell growth, which makes them interesting candidate targets.

Inhibiting MicroRNA-503 and MicroRNA-181d with Losartan Ameliorates Diabetic Nephropathy in KKAy Mice.

PubMed

Zhu, XinWang; Zhang, CongXiao; Fan, QiuLing; Liu, XiaoDan; Yang, Gang; Jiang, Yi; Wang, LiNing

2016-10-22

BACKGROUND Diabetic nephropathy (DN) is the most lethal diabetic microvascular complication; it is a major cause of renal failure, and an increasingly globally prominent healthcare problem. MATERIAL AND METHODS To identify susceptible microRNAs for the pathogenesis of DN and the targets of losartan treatment, microRNA arrays were employed to survey the glomerular microRNA expression profiles of KKAy mice treated with or without losartan. KKAy mice were assigned to either a losartan-treated group or a non-treatment group, with C57BL/6 mice used as a normal control. Twelve weeks after treatment, glomeruli from the mice were isolated. MicroRNA expression profiles were analyzed using microRNA arrays. Real-time PCR was used to confirm the results. RESULTS Losartan treatment improved albuminuria and the pathological lesions of KKAy mice. The expression of 10 microRNAs was higher, and the expression of 12 microRNAs was lower in the glomeruli of the KKAy untreated mice than that of the CL57BL/6 mice. The expression of 4 microRNAs was down-regulated in the glomeruli of the KKAy losartan-treated mice compared to that of the untreated mice. The expression of miRNA-503 and miRNA-181d was apparently higher in the glomeruli of the KKAy untreated mice, and was inhibited by losartan treatment. CONCLUSIONS The over-expression of miR-503 and miR-181d in glomeruli of KKAy mice may be responsible for the pathogenesis of DN and are potential therapeutic targets for DN.

miR-340 alleviates chemoresistance of osteosarcoma cells by targeting ZEB1.

PubMed

Yan, Haibin; Zhang, Bingyun; Fang, Chongbin; Chen, Liqiu

2018-06-01

Chemoresistance during treatment of osteosarcoma (OS) is attracting more and more attention as the main clinical obstacle. The purpose of this study was to elucidate the role of miR-340 in chemoresistance of OS. Plasmid construction and transfection, miRNA arrays, PCR analyses, and western blot analysis, as well as MTT, apoptosis, and luciferase assays were carried out in MG-63 cells and MG-63/cisplatin (DDP)-resistant cells. The results showed that miR-340 was downregulated in OS tissues and drug-resistant OS cells. Moreover, a negative correlation was observed between miR-340 and ZEB1 expression in OS tissues. Forced expression of miR-340 in drug-resistant OS cells significantly reduced multidrug resistance-1 and P-gp expression. Overexpression of miR-340 enhanced sensitivity to DDP by inhibiting viability and promoting apoptosis. The luciferase assay and western blot analysis identified ZEB1 as a direct target of miR-340, and miR-340 negatively regulated ZEB1 expression. Ectopic expression of ZEB1 reversed the effects of miR-340 on P-gp expression, cell viability, and apoptosis. miR-340 alleviated chemoresistance of OS cells by targeting ZEB1. Our results indicate that targeting miR-340 may be a potential therapeutic approach to treat drug-resistant OS.

A New Cell Separation Method Based on Antibody-Immobilized Nanoneedle Arrays for the Detection of Intracellular Markers.

PubMed

Kawamura, Ryuzo; Miyazaki, Minami; Shimizu, Keita; Matsumoto, Yuta; Silberberg, Yaron R; Sathuluri, Ramachandra Rao; Iijima, Masumi; Kuroda, Shun'ichi; Iwata, Futoshi; Kobayashi, Takeshi; Nakamura, Chikashi

2017-11-08

Focusing on intracellular targets, we propose a new cell separation technique based on a nanoneedle array (NNA) device, which allows simultaneous insertion of multiple needles into multiple cells. The device is designed to target and lift ("fish") individual cells from a mixed population of cells on a substrate using an antibody-functionalized NNA. The mechanics underlying this approach were validated by force analysis using an atomic force microscope. Accurate high-throughput separation was achieved using one-to-one contacts between the nanoneedles and the cells by preparing a single-cell array in which the positions of the cells were aligned with 10,000 nanoneedles in the NNA. Cell-type-specific separation was realized by controlling the adhesion force so that the cells could be detached in cell-type-independent manner. Separation of nestin-expressing neural stem cells (NSCs) derived from human induced pluripotent stem cells (hiPSCs) was demonstrated using the proposed technology, and successful differentiation to neuronal cells was confirmed.

The chemiluminescence based Ziplex automated workstation focus array reproduces ovarian cancer Affymetrix GeneChip expression profiles.

PubMed

Quinn, Michael C J; Wilson, Daniel J; Young, Fiona; Dempsey, Adam A; Arcand, Suzanna L; Birch, Ashley H; Wojnarowicz, Paulina M; Provencher, Diane; Mes-Masson, Anne-Marie; Englert, David; Tonin, Patricia N

2009-07-06

As gene expression signatures may serve as biomarkers, there is a need to develop technologies based on mRNA expression patterns that are adaptable for translational research. Xceed Molecular has recently developed a Ziplex technology, that can assay for gene expression of a discrete number of genes as a focused array. The present study has evaluated the reproducibility of the Ziplex system as applied to ovarian cancer research of genes shown to exhibit distinct expression profiles initially assessed by Affymetrix GeneChip analyses. The new chemiluminescence-based Ziplex gene expression array technology was evaluated for the expression of 93 genes selected based on their Affymetrix GeneChip profiles as applied to ovarian cancer research. Probe design was based on the Affymetrix target sequence that favors the 3' UTR of transcripts in order to maximize reproducibility across platforms. Gene expression analysis was performed using the Ziplex Automated Workstation. Statistical analyses were performed to evaluate reproducibility of both the magnitude of expression and differences between normal and tumor samples by correlation analyses, fold change differences and statistical significance testing. Expressions of 82 of 93 (88.2%) genes were highly correlated (p < 0.01) in a comparison of the two platforms. Overall, 75 of 93 (80.6%) genes exhibited consistent results in normal versus tumor tissue comparisons for both platforms (p < 0.001). The fold change differences were concordant for 87 of 93 (94%) genes, where there was agreement between the platforms regarding statistical significance for 71 (76%) of 87 genes. There was a strong agreement between the two platforms as shown by comparisons of log2 fold differences of gene expression between tumor versus normal samples (R = 0.93) and by Bland-Altman analysis, where greater than 90% of expression values fell within the 95% limits of agreement. Overall concordance of gene expression patterns based on correlations, statistical significance between tumor and normal ovary data, and fold changes was consistent between the Ziplex and Affymetrix platforms. The reproducibility and ease-of-use of the technology suggests that the Ziplex array is a suitable platform for translational research.

Quantitative Magnetic Separation of Particles and Cells using Gradient Magnetic Ratcheting

PubMed Central

Murray, Coleman; Pao, Edward; Tseng, Peter; Aftab, Shayan; Kulkarni, Rajan; Rettig, Matthew; Di Carlo, Dino

2016-01-01

Extraction of rare target cells from biosamples is enabling for life science research. Traditional rare cell separation techniques, such as magnetic activated cell sorting (MACS), are robust but perform coarse, qualitative separations based on surface antigen expression. We report a quantitative magnetic separation technology using high-force magnetic ratcheting over arrays of magnetically soft micro-pillars with gradient spacing, and use the system to separate and concentrate magnetic beads based on iron oxide content (IOC) and cells based on surface expression. The system consists of a microchip of permalloy micro-pillar arrays with increasing lateral pitch and a mechatronic device to generate a cycling magnetic-field. Particles with higher IOC separate and equilibrate along the miro-pillar array at larger pitches. We develop a semi-analytical model that predicts behavior for particles and cells. Using the system, LNCaP cells were separated based on the bound quantity of 1μm anti-EpCAM particles as a metric for expression. The ratcheting cytometry system was able to resolve a ±13 bound particle differential, successfully distinguishing LNCaP from PC3 populations based on EpCAM expression, correlating with flow cytometry analysis. As a proof of concept, EpCAM-labeled cells from patient blood were isolated with 74% purity, demonstrating potential towards a quantitative magnetic separation instrument. PMID:26890496

Use of planar array electrophysiology for the development of robust ion channel cell lines.

PubMed

Clare, Jeffrey J; Chen, Mao Xiang; Downie, David L; Trezise, Derek J; Powell, Andrew J

2009-01-01

The tractability of ion channels as drug targets has been significantly improved by the advent of planar array electrophysiology platforms which have dramatically increased the capacity for electrophysiological profiling of lead series compounds. However, the data quality and through-put obtained with these platforms is critically dependent on the robustness of the expression reagent being used. The generation of high quality, recombinant cell lines is therefore a key step in the early phase of ion channel drug discovery and this can present significant challenges due to the diversity and organisational complexity of many channel types. This article focuses on several complex and difficult to express ion channels and illustrates how improved stable cell lines can be obtained by integration of planar array electrophysiology systems into the cell line generation process per se. By embedding this approach at multiple stages (e.g., during development of the expression strategy, during screening and validation of clonal lines, and during characterisation of the final cell line), the cycle time and success rate in obtaining robust expression of complex multi-subunit channels can be significantly improved. We also review how recent advances in this technology (e.g., population patch clamp) have further widened the versatility and applicability of this approach.

Gene chips and arrays revealed: a primer on their power and their uses.

PubMed

Watson, S J; Akil, H

1999-03-01

This article provides an overview and general explanation of the rapidly developing area of gene chips and expression array technology. These are methods targeted at allowing the simultaneous study of thousands of genes or messenger RNAs under various physiological and pathological states. Their technical basis grows from the Human Genome Project. Both methods place DNA strands on glass computer chips (or microscope slides). Expression arrays start with complementary DNA (cDNA) clones derived from the EST data base, whereas Gene Chips synthesize oligonucleotides directly on the chip itself. Both are analyzed using image analysis systems, are capable of reading values from two different individuals at any one site, and can yield quantitative data for thousands of genes or mRNAs per slide. These methods promise to revolutionize molecular biology, cell biology, neuroscience and psychiatry. It is likely that this technology will radically open up our ability to study the actions and structure of the multiple genes involved in the complex genetics of brain disorders.

The PPARγ2 A/B-Domain Plays a Gene-Specific Role in Transactivation and Cofactor Recruitment

PubMed Central

Bugge, Anne; Grøntved, Lars; Aagaard, Mads M.; Borup, Rehannah; Mandrup, Susanne

2009-01-01

We have previously shown that adenoviral expression of peroxisome proliferator-activated receptors (PPARs) leads to rapid establishment of transcriptionally active complexes and activation of target gene expression within 5–8 h after transduction. Here we have used the adenoviral delivery system combined with expression array analysis to identify novel putative PPARγ target genes in murine fibroblasts and to determine the role of the A/B-domain in PPARγ-mediated transactivation of genomic target genes. Of the 257 genes found to be induced by PPARγ2 expression, only 25 displayed A/B-domain dependency, i.e. significantly reduced induction in the cells expressing the truncated PPARγ lacking the A/B-domain (PPARγCDE). Nine of the 25 A/B-domain-dependent genes were involved in lipid storage, and in line with this, triglyceride accumulation was considerably decreased in the cells expressing PPARγCDE compared with cells expressing full-length PPARγ2. Using chromatin immunoprecipitation, we demonstrate that PPARγ binding to genomic target sites and recruitment of the mediator component TRAP220/MED1/PBP/DRIP205 is not affected by the deletion of the A/B-domain. By contrast, the PPARγ-mediated cAMP response element-binding protein (CREB)-binding protein (CBP) and p300 recruitment to A/B-domain-dependent target genes is compromised by deletion of the A/B-domain. These results indicate that the A/B-domain of PPARγ2 is specifically involved in the recruitment or stabilization of CBP- and p300-containing cofactor complexes to a subset of target genes. PMID:19282365

Detection of Genes Regulated by Lmx1b During Limb Dorsalization

PubMed Central

Feenstra, Jennifer M.; Kanaya, Kohei; Pira, Charmaine U; Hoffman, Sarah E.; Eppey, Richard J.; Oberg, Kerby C.

2012-01-01

Lmx1b is a homeodomain transcription factor that regulates dorsal identity during limb development. Lmx1b knockout (KO) mice develop distal ventral-ventral limbs. Although induction of Lmx1b is linked to Wnt7a expression in the dorsal limb ectoderm, the downstream targets of Lmx1b that accomplish limb dorsalization are unknown. To identify genes targeted by Lmx1b, we compared gene arrays from Lmx1b KO and wildtype mouse limbs during limb dorsalization, i.e., 11.5, 12.5, and 13.5 days post coitum. We identified 54 target genes differentially expressed in all three stages. Several skeletal targets, including Emx2, Matrilin1 and Matrilin4, demonstrated a loss of scapular expression in the Lmx1b KO mice, supporting a role for Lmx1b in scapula development. Furthermore, the relative abundance of extracellular matrix-related soft tissue targets regulated by Lmx1b, such as collagens and proteoglycans, suggests a mechanism which includes changes in the extracellular matrix composition to accomplish limb dorsalization. Our study provides the most comprehensive characterization of genes regulated by Lmx1b during limb development to-date and provides targets for further investigation. PMID:22417325

Genomic biomarkers of phthalate-induced male reproductive developmental toxicity: A targeted rtPCR array approach for defining relative potency

EPA Science Inventory

Male rat fetuses exposed to certain phthalate esters (PEs) during sexual differentiation display reproductive tract malformations due to reductions in testosterone (T) production and the expression of steroidogenesis-and INSL3-related genes. In the current study} we used a 96well...

Evaluation of genomic biomarkers and relative potency of phthalate-induced male reproductive developmental toxicity using a targeted rtPCR array approach**

EPA Science Inventory

Exposure to certain phthalate esters (PEs) during sexual differentiation induces reproductive tract malformations in male rats due to reductions in fetal testicular testosterone (T) production and expression of steroidogenesis-and insl3-related genes. In the current study, we use...

Therapeutic targeting of sunitinib-induced AR phosphorylation in renal cell carcinoma.

PubMed

Adelaiye-Ogala, Remi; Damayanti, Nur P; Orillion, Ashley R; Arisa, Sreevani; Chintala, Sreenivasulu; Titus, Mark A; Kao, Chinghai; Pili, Roberto

2018-03-23

Androgen receptor (AR) plays a crucial role in the development and progression of prostate cancer. AR expression has also been reported in other solid tumors, including renal cell carcinoma (RCC), but its biological role here remains unclear. Through integrative analysis of a reverse phase protein array (RPPA), we discovered increased expression of AR in an RCC patient-derived xenograft model of acquired resistance to the receptor tyrosine kinase inhibitor (RTKi) sunitinib. AR expression was increased in RCC cell lines with either acquired or intrinsic sunitinib resistance in vitro. An AR signaling gene array profiler indicated elevated levels of AR target genes in sunitinib-resistant cells. Sunitinib-induced AR transcriptional activity was associated with increased phosphorylation of serine 81 (pS81) on AR. Additionally, AR overexpression resulted in acquired sunitinib resistance, and the AR antagonist enzalutamide-induced AR degradation and attenuated AR downstream activity in sunitinib-resistant cells, also indicated by decreased secretion of human kallikrein 2 (KLK2). Enzalutamide-induced AR degradation was rescued by either proteasome inhibition or by knockdown of the AR ubiquitin ligase speckle-type POZ protein (SPOP). In vivo treatment with enzalutamide and sunitinib demonstrated that this combination efficiently induced tumor regression in an RCC model following acquired sunitinib resistance. Overall, our results suggest the potential role of AR as a target for therapeutic interventions, in combination with RTKi, to overcome drug resistance in RCC. Copyright ©2018, American Association for Cancer Research.

Fabrication and characterization of gold nano-wires templated on virus-like arrays of tobacco mosaic virus coat proteins

NASA Astrophysics Data System (ADS)

Wnęk, M.; Górzny, M. Ł.; Ward, M. B.; Wälti, C.; Davies, A. G.; Brydson, R.; Evans, S. D.; Stockley, P. G.

2013-01-01

The rod-shaped plant virus tobacco mosaic virus (TMV) is widely used as a nano-fabrication template, and chimeric peptide expression on its major coat protein has extended its potential applications. Here we describe a simple bacterial expression system for production and rapid purification of recombinant chimeric TMV coat protein carrying C-terminal peptide tags. These proteins do not bind TMV RNA or form disks at pH 7. However, they retain the ability to self-assemble into virus-like arrays at acidic pH. C-terminal peptide tags in such arrays are exposed on the protein surface, allowing interaction with target species. We have utilized a C-terminal His-tag to create virus coat protein-templated nano-rods able to bind gold nanoparticles uniformly. These can be transformed into gold nano-wires by deposition of additional gold atoms from solution, followed by thermal annealing. The resistivity of a typical annealed wire created by this approach is significantly less than values reported for other nano-wires made using different bio-templates. This expression construct is therefore a useful additional tool for the creation of chimeric TMV-like nano-rods for bio-templating.

The Use of Protein-DNA, Chromatin Immunoprecipitation, and Transcriptome Arrays to Describe Transcriptional Circuits in the Dehydrated Male Rat Hypothalamus

PubMed Central

Qiu, Jing; Kleineidam, Anna; Gouraud, Sabine; Yao, Song Tieng; Greenwood, Mingkwan; Hoe, See Ziau; Hindmarch, Charles

2014-01-01

The supraoptic nucleus (SON) of the hypothalamus is responsible for maintaining osmotic stability in mammals through its elaboration of the antidiuretic hormone arginine vasopressin. Upon dehydration, the SON undergoes a function-related plasticity, which includes remodeling of morphology, electrical properties, and biosynthetic activity. This process occurs alongside alterations in steady state transcript levels, which might be mediated by changes in the activity of transcription factors. In order to identify which transcription factors might be involved in changing patterns of gene expression, an Affymetrix protein-DNA array analysis was carried out. Nuclear extracts of SON from dehydrated and control male rats were analyzed for binding to the 345 consensus DNA transcription factor binding sequences of the array. Statistical analysis revealed significant changes in binding to 26 consensus elements, of which EMSA confirmed increased binding to signal transducer and activator of transcription (Stat) 1/Stat3, cellular Myelocytomatosis virus-like cellular proto-oncogene (c-Myc)-Myc-associated factor X (Max), and pre-B cell leukemia transcription factor 1 sequences after dehydration. Focusing on c-Myc and Max, we used quantitative PCR to confirm previous transcriptomic analysis that had suggested an increase in c-Myc, but not Max, mRNA levels in the SON after dehydration, and we demonstrated c-Myc- and Max-like immunoreactivities in SON arginine vasopressin-expressing cells. Finally, by comparing new data obtained from Roche-NimbleGen chromatin immunoprecipitation arrays with previously published transcriptomic data, we have identified putative c-Myc target genes whose expression changes in the SON after dehydration. These include known c-Myc targets, such as the Slc7a5 gene, which encodes the L-type amino acid transporter 1, ribosomal protein L24, histone deactylase 2, and the Rat sarcoma proto-oncogene (Ras)-related nuclear GTPase. PMID:25144923

Multiple chimeric antigen receptors successfully target chondroitin sulfate proteoglycan 4 in several different cancer histologies and cancer stem cells

PubMed Central

2014-01-01

Background The development of immunotherapy has led to significant progress in the treatment of metastatic cancer, including the development of genetic engineering technologies that redirect lymphocytes to recognize and target a wide variety of tumor antigens. Chimeric antigen receptors (CARs) are hybrid proteins combining antibody recognition domains linked to T cell signaling elements. Clinical trials of CAR-transduced peripheral blood lymphocytes (PBL) have induced remission of both solid organ and hematologic malignancies. Chondroitin sulfate proteoglycan 4 (CSPG4) is a promising target antigen that is overexpressed in multiple cancer histologies including melanoma, triple-negative breast cancer, glioblastoma, mesothelioma and sarcoma. Methods CSPG4 expression in cancer cell lines was assayed using flow cytometry (FACS) and reverse-transcription PCR (RT-PCR). Immunohistochemistry was utilized to assay resected melanomas and normal human tissues (n = 30) for CSPG4 expression and a reverse-phase protein array comprising 94 normal tissue samples was also interrogated for CSPG4 expression. CARs were successfully constructed from multiple murine antibodies (225.28S, TP41.2, 149.53) using second generation (CD28.CD3ζ) signaling domains. CAR sequences were cloned into a gamma-retroviral vector with subsequent successful production of retroviral supernatant and PBL transduction. CAR efficacy was assayed by cytokine release and cytolysis following coculture with target cell lines. Additionally, glioblastoma stem cells were generated from resected human tumors, and CSPG4 expression was determined by RT-PCR and FACS. Results Immunohistochemistry demonstrated prominent CSPG4 expression in melanoma tumors, but failed to demonstrate expression in any of the 30 normal human tissues studied. Two of 94 normal tissue protein lysates were positive by protein array. CAR constructs demonstrated cytokine secretion and cytolytic function after co-culture with tumor cell lines from multiple different histologies, including melanoma, breast cancer, mesothelioma, glioblastoma and osteosarcoma. Furthermore, we report for the first time that CSPG4 is expressed on glioblastoma cancer stem cells (GSC) and demonstrate that anti-CSPG4 CAR-transduced T cells recognize and kill these GSC. Conclusions The functionality of multiple different CARs, with the widespread expression of CSPG4 on multiple malignancies, suggests that CSPG4 may be an attractive candidate tumor antigen for CAR-based immunotherapies using appropriate technology to limit possible off-tumor toxicity. PMID:25197555

MiR-422a as a Potential Cellular MicroRNA Biomarker for Postmenopausal Osteoporosis

PubMed Central

Cao, Zheng; Moore, Benjamin T.; Wang, Yang; Peng, Xian-Hao; Lappe, Joan M.; Recker, Robert R.; Xiao, Peng

2014-01-01

Background MicroRNAs (miRNAs) are a class of short non-coding RNA molecules that regulate gene expression by targeting mRNAs. Recently, miRNAs have been shown to play important roles in the etiology of various diseases. However, little is known about their roles in the development of osteoporosis. Circulating monocytes are osteoclast precursors that also produce various factors important for osteoclastogenesis. Previously, we have identified a potential biomarker miR-133a in circulating monocytes for postmenopausal osteoporosis. In this study, we aimed to further identify significant miRNA biomarkers in human circulating monocytes underlying postmenopausal osteoporosis. Methodology/Principal Findings We used ABI TaqMan miRNA array followed by qRT-PCR validation in human circulating monocytes from 10 high BMD and 10 low BMD postmenopausal Caucasian women to identify miRNA biomarkers. MiR-422a was up-regulated with marginal significance (P = 0.065) in the low compared with the high BMD group in the array analysis. However, a significant up-regulation of miR-422a was identified in the low BMD group by qRT-PCR analysis (P = 0.029). We also performed bioinformatic target gene analysis and found several potential target genes of miR-422a which are involved in osteoclastogenesis. Further qRT-PCR analyses of the target genes in the same study subjects showed that the expression of five of these genes (CBL, CD226, IGF1, PAG1, and TOB2) correlated negatively with miR-422a expression. Conclusions/Significance Our study suggests that miR-422a in human circulating monocytes (osteoclast precursors) is a potential miRNA biomarker underlying postmenopausal osteoporosis. PMID:24820117

MiR-422a as a potential cellular microRNA biomarker for postmenopausal osteoporosis.

PubMed

Cao, Zheng; Moore, Benjamin T; Wang, Yang; Peng, Xian-Hao; Lappe, Joan M; Recker, Robert R; Xiao, Peng

2014-01-01

MicroRNAs (miRNAs) are a class of short non-coding RNA molecules that regulate gene expression by targeting mRNAs. Recently, miRNAs have been shown to play important roles in the etiology of various diseases. However, little is known about their roles in the development of osteoporosis. Circulating monocytes are osteoclast precursors that also produce various factors important for osteoclastogenesis. Previously, we have identified a potential biomarker miR-133a in circulating monocytes for postmenopausal osteoporosis. In this study, we aimed to further identify significant miRNA biomarkers in human circulating monocytes underlying postmenopausal osteoporosis. We used ABI TaqMan miRNA array followed by qRT-PCR validation in human circulating monocytes from 10 high BMD and 10 low BMD postmenopausal Caucasian women to identify miRNA biomarkers. MiR-422a was up-regulated with marginal significance (P = 0.065) in the low compared with the high BMD group in the array analysis. However, a significant up-regulation of miR-422a was identified in the low BMD group by qRT-PCR analysis (P = 0.029). We also performed bioinformatic target gene analysis and found several potential target genes of miR-422a which are involved in osteoclastogenesis. Further qRT-PCR analyses of the target genes in the same study subjects showed that the expression of five of these genes (CBL, CD226, IGF1, PAG1, and TOB2) correlated negatively with miR-422a expression. Our study suggests that miR-422a in human circulating monocytes (osteoclast precursors) is a potential miRNA biomarker underlying postmenopausal osteoporosis.

At-TAX: a whole genome tiling array resource for developmental expression analysis and transcript identification in Arabidopsis thaliana

PubMed Central

Laubinger, Sascha; Zeller, Georg; Henz, Stefan R; Sachsenberg, Timo; Widmer, Christian K; Naouar, Naïra; Vuylsteke, Marnik; Schölkopf, Bernhard; Rätsch, Gunnar; Weigel, Detlef

2008-01-01

Gene expression maps for model organisms, including Arabidopsis thaliana, have typically been created using gene-centric expression arrays. Here, we describe a comprehensive expression atlas, Arabidopsis thaliana Tiling Array Express (At-TAX), which is based on whole-genome tiling arrays. We demonstrate that tiling arrays are accurate tools for gene expression analysis and identified more than 1,000 unannotated transcribed regions. Visualizations of gene expression estimates, transcribed regions, and tiling probe measurements are accessible online at the At-TAX homepage. PMID:18613972

University of Texas Southwestern Medical Center: High-Throughput siRNA Screening of a Non-Small Cell Lung Cancer (NSCLC) Cell Line Panel | Office of Cancer Genomics

Cancer.gov

The goal of this project is to use siRNA screens to identify NSCLC-selective siRNAs from two genome-wide libraries that will allow us to functionally define genetic dependencies of subtypes of NSCLC. Using bioinformatics tools, the CTD2 center at the University of Texas Southwestern Medical Center are discovering associations between this functional data (siRNAs) and NSCLC mutational status, methylation arrays, gene expression arrays, and copy number variation data that will help us identify new targets and enrollment biomarkers. 

University of Texas Southwestern Medical Center (UTSW): High-Throughput siRNA Screening of a Non-Small Cell Lung Cancer (NSCLC) Cell Line Panel | Office of Cancer Genomics

Cancer.gov

The goal of this project is to use siRNA screens to identify NSCLC-selective siRNAs from two genome-wide libraries that will allow us to functionally define genetic dependencies of subtypes of NSCLC. Using bioinformatics tools, the CTD2 center at the University of Texas Southwestern Medical Center are discovering associations between this functional data (siRNAs) and NSCLC mutational status, methylation arrays, gene expression arrays, and copy number variation data that will help us identify new targets and enrollment biomarkers. 

Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array

PubMed Central

Gardina, Paul J; Clark, Tyson A; Shimada, Brian; Staples, Michelle K; Yang, Qing; Veitch, James; Schweitzer, Anthony; Awad, Tarif; Sugnet, Charles; Dee, Suzanne; Davies, Christopher; Williams, Alan; Turpaz, Yaron

2006-01-01

Background Alternative splicing is a mechanism for increasing protein diversity by excluding or including exons during post-transcriptional processing. Alternatively spliced proteins are particularly relevant in oncology since they may contribute to the etiology of cancer, provide selective drug targets, or serve as a marker set for cancer diagnosis. While conventional identification of splice variants generally targets individual genes, we present here a new exon-centric array (GeneChip Human Exon 1.0 ST) that allows genome-wide identification of differential splice variation, and concurrently provides a flexible and inclusive analysis of gene expression. Results We analyzed 20 paired tumor-normal colon cancer samples using a microarray designed to detect over one million putative exons that can be virtually assembled into potential gene-level transcripts according to various levels of prior supporting evidence. Analysis of high confidence (empirically supported) transcripts identified 160 differentially expressed genes, with 42 genes occupying a network impacting cell proliferation and another twenty nine genes with unknown functions. A more speculative analysis, including transcripts based solely on computational prediction, produced another 160 differentially expressed genes, three-fourths of which have no previous annotation. We also present a comparison of gene signal estimations from the Exon 1.0 ST and the U133 Plus 2.0 arrays. Novel splicing events were predicted by experimental algorithms that compare the relative contribution of each exon to the cognate transcript intensity in each tissue. The resulting candidate splice variants were validated with RT-PCR. We found nine genes that were differentially spliced between colon tumors and normal colon tissues, several of which have not been previously implicated in cancer. Top scoring candidates from our analysis were also found to substantially overlap with EST-based bioinformatic predictions of alternative splicing in cancer. Conclusion Differential expression of high confidence transcripts correlated extremely well with known cancer genes and pathways, suggesting that the more speculative transcripts, largely based solely on computational prediction and mostly with no previous annotation, might be novel targets in colon cancer. Five of the identified splicing events affect mediators of cytoskeletal organization (ACTN1, VCL, CALD1, CTTN, TPM1), two affect extracellular matrix proteins (FN1, COL6A3) and another participates in integrin signaling (SLC3A2). Altogether they form a pattern of colon-cancer specific alterations that may particularly impact cell motility. PMID:17192196

Detection of Emotional Faces: Salient Physical Features Guide Effective Visual Search

ERIC Educational Resources Information Center

Calvo, Manuel G.; Nummenmaa, Lauri

2008-01-01

In this study, the authors investigated how salient visual features capture attention and facilitate detection of emotional facial expressions. In a visual search task, a target emotional face (happy, disgusted, fearful, angry, sad, or surprised) was presented in an array of neutral faces. Faster detection of happy and, to a lesser extent,…

Altered expression of four miRNA (miR-1238-3p, miR-202-3p, miR-630 and miR-766-3p) and their potential targets in peripheral blood from vitiligo patients.

PubMed

Shang, Zhiwei; Li, Hongwen

2017-10-01

Vitiligo is an acquired skin disease with pigmentary disorder. Autoimmune destruction of melanocytes is thought to be major factor in the etiology of vitiligo. miRNA-based regulators of gene expression have been reported to play crucial roles in autoimmune disease. Therefore, we attempt to profile the miRNA expressions and predict their potential targets, assessing the biological functions of differentially expressed miRNA. Total RNA was extracted from peripheral blood of vitiligo (experimental group, n = 5) and non-vitiligo (control group, n = 5) age-matched patients. Samples were hybridized to a miRNA array. Box, scatter and principal component analysis plots were performed, followed by unsupervised hierarchical clustering analysis to classify the samples. Quantitative reverse transcription polymerase chain reaction (RT-PCR) was conducted for validation of microarray data. Three different databases, TargetScan, PITA and microRNA.org, were used to predict the potential target genes. Gene ontology (GO) annotation and pathway analysis were performed to assess the potential functions of predicted genes of identified miRNA. A total of 100 (29 upregulated and 71 downregulated) miRNA were filtered by volcano plot analysis. Four miRNA were validated by quantitative RT-PCR as significantly downregulated in the vitiligo group. The functions of predicted target genes associated with differentially expressed miRNA were assessed by GO analysis, showing that the GO term with most significantly enriched target genes was axon guidance, and that the axon guidance pathway was most significantly correlated with these miRNA. In conclusion, we identified four downregulated miRNA in vitiligo and assessed the potential functions of target genes related to these differentially expressed miRNA. © 2017 Japanese Dermatological Association.

Harnessing Gene Conversion in Chicken B Cells to Create a Human Antibody Sequence Repertoire

PubMed Central

Schusser, Benjamin; Yi, Henry; Collarini, Ellen J.; Izquierdo, Shelley Mettler; Harriman, William D.; Etches, Robert J.; Leighton, Philip A.

2013-01-01

Transgenic chickens expressing human sequence antibodies would be a powerful tool to access human targets and epitopes that have been intractable in mammalian hosts because of tolerance to conserved proteins. To foster the development of the chicken platform, it is beneficial to validate transgene constructs using a rapid, cell culture-based method prior to generating fully transgenic birds. We describe a method for the expression of human immunoglobulin variable regions in the chicken DT40 B cell line and the further diversification of these genes by gene conversion. Chicken VL and VH loci were knocked out in DT40 cells and replaced with human VK and VH genes. To achieve gene conversion of human genes in chicken B cells, synthetic human pseudogene arrays were inserted upstream of the functional human VK and VH regions. Proper expression of chimeric IgM comprised of human variable regions and chicken constant regions is shown. Most importantly, sequencing of DT40 genetic variants confirmed that the human pseudogene arrays contributed to the generation of diversity through gene conversion at both the Igl and Igh loci. These data show that engineered pseudogene arrays produce a diverse pool of human antibody sequences in chicken B cells, and suggest that these constructs will express a functional repertoire of chimeric antibodies in transgenic chickens. PMID:24278246

Quantitative Magnetic Separation of Particles and Cells Using Gradient Magnetic Ratcheting.

PubMed

Murray, Coleman; Pao, Edward; Tseng, Peter; Aftab, Shayan; Kulkarni, Rajan; Rettig, Matthew; Di Carlo, Dino

2016-04-13

Extraction of rare target cells from biosamples is enabling for life science research. Traditional rare cell separation techniques, such as magnetic activated cell sorting, are robust but perform coarse, qualitative separations based on surface antigen expression. A quantitative magnetic separation technology is reported using high-force magnetic ratcheting over arrays of magnetically soft micropillars with gradient spacing, and the system is used to separate and concentrate magnetic beads based on iron oxide content (IOC) and cells based on surface expression. The system consists of a microchip of permalloy micropillar arrays with increasing lateral pitch and a mechatronic device to generate a cycling magnetic field. Particles with higher IOC separate and equilibrate along the miropillar array at larger pitches. A semi-analytical model is developed that predicts behavior for particles and cells. Using the system, LNCaP cells are separated based on the bound quantity of 1 μm anti-epithelial cell adhesion molecule (EpCAM) particles as a metric for expression. The ratcheting cytometry system is able to resolve a ±13 bound particle differential, successfully distinguishing LNCaP from PC3 populations based on EpCAM expression, correlating with flow cytometry analysis. As a proof-of-concept, EpCAM-labeled cells from patient blood are isolated with 74% purity, demonstrating potential toward a quantitative magnetic separation instrument. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

MicroRNA Expression Profiling of the Armed Forces Health Surveillance Branch Cohort for Identification of "Enviro-miRs" Associated With Deployment-Based Environmental Exposure.

PubMed

Dalgard, Clifton L; Polston, Keith F; Sukumar, Gauthaman; Mallon, Col Timothy M; Wilkerson, Matthew D; Pollard, Harvey B

2016-08-01

The aim of this study was to identify serum microRNA (miRNA) biomarkers that indicate deployment-associated exposures in service members at military installations with open burn pits. Another objective was to determine detection rates of miRNAs in Department of Defense Serum Repository (DoDSR) samples with a high-throughput methodology. Low-volume serum samples (n = 800) were profiled by miRNA-capture isolation, pre-amplification, and measurement by a quantitative PCR-based OpenArray platform. Normalized quantitative cycle values were used for differential expression analysis between groups. Assay specificity, dynamic range, reproducibility, and detection rates by OpenArray passed target desired specifications. Serum abundant miRNAs were consistently measured in study specimens. Four miRNAs were differentially expressed in the case deployment group subjects. miRNAs are suitable RNA species for biomarker discovery in the DoDSR serum specimens. Serum miRNAs are candidate biomarkers for deployment and environmental exposure in military service members.

Detection of genes regulated by Lmx1b during limb dorsalization.

PubMed

Feenstra, Jennifer M; Kanaya, Kohei; Pira, Charmaine U; Hoffman, Sarah E; Eppey, Richard J; Oberg, Kerby C

2012-05-01

Lmx1b is a homeodomain transcription factor that regulates dorsal identity during limb development. Lmx1b knockout (KO) mice develop distal ventral-ventral limbs. Although induction of Lmx1b is linked to Wnt7a expression in the dorsal limb ectoderm, the downstream targets of Lmx1b that accomplish limb dorsalization are unknown. To identify genes targeted by Lmx1b, we compared gene arrays from Lmx1b KO and wild type mouse limbs during limb dorsalization, i.e., 11.5, 12.5, and 13.5 days post coitum. We identified 54 target genes that were differentially expressed in all three stages. Several skeletal targets, including Emx2, Matrilin1 and Matrilin4, demonstrated a loss of scapular expression in the Lmx1b KO mice, supporting a role for Lmx1b in scapula development. Furthermore, the relative abundance of extracellular matrix-related soft tissue targets regulated by Lmx1b, such as collagens and proteoglycans, suggests a mechanism that includes changes in the extracellular matrix composition to accomplish limb dorsalization. Our study provides the most comprehensive characterization of genes regulated by Lmx1b during limb development to-date and provides targets for further investigation. © 2012 The Authors. Development, Growth & Differentiation © 2012 Japanese Society of Developmental Biologists.

Mapping and characterization of the amplicon near APOA2 in 1q23 in human sarcomas by FISH and array CGH

PubMed Central

Kresse, Stine H; Berner, Jeanne-Marie; Meza-Zepeda, Leonardo A; Gregory, Simon G; Kuo, Wen-Lin; Gray, Joe W; Forus, Anne; Myklebost, Ola

2005-01-01

Background Amplification of the q21-q23 region on chromosome 1 is frequently found in sarcomas and a variety of other solid tumours. Previous analyses of sarcomas have indicated the presence of at least two separate amplicons within this region, one located in 1q21 and one located near the apolipoprotein A-II (APOA2) gene in 1q23. In this study we have mapped and characterized the amplicon in 1q23 in more detail. Results We have used fluorescence in situ hybridisation (FISH) and microarray-based comparative genomic hybridisation (array CGH) to map and define the borders of the amplicon in 10 sarcomas. A subregion of approximately 800 kb was identified as the core of the amplicon. The amplification patterns of nine possible candidate target genes located to this subregion were determined by Southern blot analysis. The genes activating transcription factor 6 (ATF6) and dual specificity phosphatase 12 (DUSP12) showed the highest level of amplification, and they were also shown to be over-expressed by quantitative real-time reverse transcription PCR (RT-PCR). In general, the level of expression reflected the level of amplification in the different tumours. DUSP12 was expressed significantly higher than ATF6 in a subset of the tumours. In addition, two genes known to be transcriptionally activated by ATF6, glucose-regulated protein 78 kDa and -94 kDa (GRP78 and GRP94), were shown to be over-expressed in the tumours that showed over-expression of ATF6. Conclusion ATF6 and DUSP12 seem to be the most likely candidate target genes for the 1q23 amplification in sarcomas. Both genes have possible roles in promoting cell growth, which makes them interesting candidate targets. PMID:16274472

Transcriptional network inference from functional similarity and expression data: a global supervised approach.

PubMed

Ambroise, Jérôme; Robert, Annie; Macq, Benoit; Gala, Jean-Luc

2012-01-06

An important challenge in system biology is the inference of biological networks from postgenomic data. Among these biological networks, a gene transcriptional regulatory network focuses on interactions existing between transcription factors (TFs) and and their corresponding target genes. A large number of reverse engineering algorithms were proposed to infer such networks from gene expression profiles, but most current methods have relatively low predictive performances. In this paper, we introduce the novel TNIFSED method (Transcriptional Network Inference from Functional Similarity and Expression Data), that infers a transcriptional network from the integration of correlations and partial correlations of gene expression profiles and gene functional similarities through a supervised classifier. In the current work, TNIFSED was applied to predict the transcriptional network in Escherichia coli and in Saccharomyces cerevisiae, using datasets of 445 and 170 affymetrix arrays, respectively. Using the area under the curve of the receiver operating characteristics and the F-measure as indicators, we showed the predictive performance of TNIFSED to be better than unsupervised state-of-the-art methods. TNIFSED performed slightly worse than the supervised SIRENE algorithm for the target genes identification of the TF having a wide range of yet identified target genes but better for TF having only few identified target genes. Our results indicate that TNIFSED is complementary to the SIRENE algorithm, and particularly suitable to discover target genes of "orphan" TFs.

Gene targeting in mosquito cells: a demonstration of 'knockout' technology in extrachromosomal gene arrays

PubMed Central

Eggleston, Paul; Zhao, Yuguang

2001-01-01

Background Gene targeting would offer a number of advantages over current transposon-based strategies for insect transformation. These include freedom from both position effects associated with quasi-random integration and concerns over transgene instability mediated by endogenous transposases, independence from phylogenetic restrictions on transposon mobility and the ability to generate gene knockouts. Results We describe here our initial investigations of gene targeting in the mosquito. The target site was a hygromycin resistance gene, stably maintained as part of an extrachromosomal array. Using a promoter-trap strategy to enrich for targeted events, a neomycin resistance gene was integrated into the target site. This resulted in knockout of hygromycin resistance concurrent with the expression of high levels of neomycin resistance from the resident promoter. PCR amplification of the targeted site generated a product that was specific to the targeted cell line and consistent with precise integration of the neomycin resistance gene into the 5' end of the hygromycin resistance gene. Sequencing of the PCR product and Southern analysis of cellular DNA subsequently confirmed this molecular structure. Conclusions These experiments provide the first demonstration of gene targeting in mosquito tissue and show that mosquito cells possess the necessary machinery to bring about precise integration of exogenous sequences through homologous recombination. Further development of these procedures and their extension to chromosomally located targets hold much promise for the exploitation of gene targeting in a wide range of medically and economically important insect species. PMID:11513755

Methylselenol, a selenium metabolite, modulates p53 pathway and inhibits the growth of colon cancer xenografts in Balb/c mice.

PubMed

Zeng, Huawei; Cheng, Wen-Hsing; Johnson, Luann K

2013-05-01

It is has been hypothesized that methylselenol is a critical selenium metabolite for anticancer activity in vivo. In this study, we used a protein array which contained 112 different antibodies known to be involved in the p53 pathway to investigate the molecular targets of methylselenol in human HCT116 colon cancer cells. The array analysis indicated that methylselenol exposure changed the expression of 11 protein targets related to the regulation of cell cycle and apoptosis. Subsequently, we confirmed these proteins with the Western blotting approach, and found that methylselenol increased the expression of GADD 153 and p21 but reduced the level of c-Myc, E2F1 and Phos p38 MAP kinase. Similar to our previous report on human HCT116 colon cancer cells, methylselenol also inhibited cell growth and led to an increase in G1 and G2 fractions with a concomitant drop in S-phase in mouse colon cancer MC26 cells. When the MC26 cells were transplanted to their immune-competent Balb/c mice, methylselenol-treated MC26 cells had significantly less tumor growth potential than that of untreated MC26 cells. Taken together, our data suggest that methylselenol modulates the expression of key genes related to cell cycle and apoptosis and inhibits colon cancer cell proliferation and tumor growth. Copyright © 2013. Published by Elsevier Inc.

Naturally Inspired Peptide Leads: Alanine Scanning Reveals an Actin‐Targeting Thiazole Analogue of Bisebromoamide

PubMed Central

Johnston, Heather J.; Boys, Sarah K.; Makda, Ashraff; Carragher, Neil O.

2016-01-01

Abstract Systematic alanine scanning of the linear peptide bisebromoamide (BBA), isolated from a marine cyanobacterium, was enabled by solid‐phase peptide synthesis of thiazole analogues. The analogues have comparable cytotoxicity (nanomolar) to that of BBA, and cellular morphology assays indicated that they target the actin cytoskeleton. Pathway inhibition in human colon tumour (HCT116) cells was explored by reverse phase protein array (RPPA) analysis, which showed a dose‐dependent response in IRS‐1 expression. Alanine scanning reveals a structural dependence to the cytotoxicity, actin targeting and pathway inhibition, and allows a new readily synthesised lead to be proposed. PMID:27304907

Targeting the Regulatory Machinery of BIM for Cancer Therapy

PubMed Central

Harada, Hisashi; Grant, Steven

2013-01-01

BIM represents a BH3-only proapoptotic member of the BCL-2 family of apoptotic regulatory proteins. Recent evidence suggests that in addition to its involvement in normal homeostasis, BIM plays a critical role in tumor cell biology, including the regulation of tumorigenesis through activities as a tumor suppressor, tumor metastasis, and tumor cell survival. Consequently, BIM has become the focus of intense interest as a potential target for cancer chemotherapy. The control of BIM expression is complex, and involves multiple factors, including epigenetic events (i.e., promoter acetylation or methylation, miRNA), transcription factors, posttranscriptional regulation, and posttranslational modifications, most notably phosphorylation. Significantly, the expression of BIM by tumor cells has been shown to play an important role in determining the response of transformed cells to not only conventional cytotoxic agents, but also to a broad array of targeted agents that interrupt cell signaling and survival pathways. Furthermore, modifications in BIM expression may be exploited to improve the therapeutic activity and potentially the selectivity of such agents. It is likely that evolving insights into the factors that regulate BIM expression will ultimately lead to novel BIM-based therapeutic strategies in the future. PMID:22856430

MicroRNA-320 family is downregulated in colorectal adenoma and affects tumor proliferation by targeting CDK6.

PubMed

Tadano, Toshihiro; Kakuta, Yoichi; Hamada, Shin; Shimodaira, Yosuke; Kuroha, Masatake; Kawakami, Yoko; Kimura, Tomoya; Shiga, Hisashi; Endo, Katsuya; Masamune, Atsushi; Takahashi, Seiichi; Kinouchi, Yoshitaka; Shimosegawa, Tooru

2016-07-15

To investigate the microRNA (miRNA) expression during histological progression from colorectal normal mucosa through adenoma to carcinoma within a lesion. Using microarray, the sequential changes in miRNA expression profiles were compared in colonic lesions from matched samples; histologically, non-neoplastic mucosa, adenoma, and submucosal invasive carcinoma were microdissected from a tissue sample. Cell proliferation assay was performed to observe the effect of miRNA, and its target genes were predicted using bioinformatics approaches and the expression profile of SW480 transfected with the miRNA mimics. mRNA and protein levels of the target gene in colon cancer cell lines with a mimic control or miRNA mimics were measured using qRT-PCR and Western blotting. The expression levels of miRNA and target gene in colorectal tissue samples were also measured. Microarray analysis identified that the miR-320 family, including miR-320a, miR-320b, miR-320c, miR-320d and miR-320e, were differentially expressed in adenoma and submucosal invasive carcinoma. The miR-320 family, which inhibits cell proliferation, is frequently downregulated in colorectal adenoma and submucosal invasive carcinoma tissues. Seven genes including CDK6 were identified to be common in the results of gene expression array and bioinformatics analyses performed to find the target gene of the miR-320 family. We confirmed that mRNA and protein levels of CDK6 were significantly suppressed in colon cancer cell lines with miR-320 family mimics. CDK6 expression was found to increase from non-neoplastic mucosa through adenoma to submucosal invasive carcinoma tissues and showed an inverse correlation with miR-320 family expression. MiR-320 family affects colorectal tumor proliferation by targeting CDK6, plays important role in its growth, and is considered to be a biomarker for its early detection.

MicroRNA-320 family is downregulated in colorectal adenoma and affects tumor proliferation by targeting CDK6

PubMed Central

Tadano, Toshihiro; Kakuta, Yoichi; Hamada, Shin; Shimodaira, Yosuke; Kuroha, Masatake; Kawakami, Yoko; Kimura, Tomoya; Shiga, Hisashi; Endo, Katsuya; Masamune, Atsushi; Takahashi, Seiichi; Kinouchi, Yoshitaka; Shimosegawa, Tooru

2016-01-01

AIM: To investigate the microRNA (miRNA) expression during histological progression from colorectal normal mucosa through adenoma to carcinoma within a lesion. METHODS: Using microarray, the sequential changes in miRNA expression profiles were compared in colonic lesions from matched samples; histologically, non-neoplastic mucosa, adenoma, and submucosal invasive carcinoma were microdissected from a tissue sample. Cell proliferation assay was performed to observe the effect of miRNA, and its target genes were predicted using bioinformatics approaches and the expression profile of SW480 transfected with the miRNA mimics. mRNA and protein levels of the target gene in colon cancer cell lines with a mimic control or miRNA mimics were measured using qRT-PCR and Western blotting. The expression levels of miRNA and target gene in colorectal tissue samples were also measured. RESULTS: Microarray analysis identified that the miR-320 family, including miR-320a, miR-320b, miR-320c, miR-320d and miR-320e, were differentially expressed in adenoma and submucosal invasive carcinoma. The miR-320 family, which inhibits cell proliferation, is frequently downregulated in colorectal adenoma and submucosal invasive carcinoma tissues. Seven genes including CDK6 were identified to be common in the results of gene expression array and bioinformatics analyses performed to find the target gene of the miR-320 family. We confirmed that mRNA and protein levels of CDK6 were significantly suppressed in colon cancer cell lines with miR-320 family mimics. CDK6 expression was found to increase from non-neoplastic mucosa through adenoma to submucosal invasive carcinoma tissues and showed an inverse correlation with miR-320 family expression. CONCLUSION: MiR-320 family affects colorectal tumor proliferation by targeting CDK6, plays important role in its growth, and is considered to be a biomarker for its early detection. PMID:27559432

miR-935 suppresses gastric signet ring cell carcinoma tumorigenesis by targeting Notch1 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, Chao; Yu, Jianchun, E-mail: yu_jchpumch@163.com; Kang, Weiming

Gastric signet ring cell carcinoma (GSRCC) is a unique pathological type of gastric carcinoma that is extremely invasive and has a poor prognosis. Expression of microRNAs (miRNAs) has been closely linked to the carcinogenesis of gastric cancer and has been considered as a powerful prognostic marker. The function of miR-935 has never been reported in cancer before. We found, using microRNA array, that expression of miR-935 in GSRCC cell lines is lower than in non-GSRCC cell lines, and enhanced expression of miR-935 in GSRCC cell-lines inhibit cell proliferation, migration and invasion. We also identified Notch1 as a direct target ofmore » miR-935. Knockdown of Notch1 reduced proliferation, migration/invasion of GSRCC cells, and overexpression Notch1's activated form (Notch intracellular domain) could rescue miR-935's tumor suppressive effect on GSRCC. Expression of miR-935 was lower in gastric carcinoma tissue than in paired normal tissue samples, and lower in GSRCC than in non-GSRCC. Our results demonstrate the inverse correlation between the expression of miR-935 and Notch1 in gastric tissues. We conclude that miR-935 inhibits gastric carcinoma cell proliferation, migration and invasion by targeting Notch1, suggesting potential applications of the miR-935-Notch1 pathway in gastric cancer clinical diagnosis and therapeutics, especially in gastric signet ring cell carcinoma. - Highlights: • The expression of miR-935 is lower in GC tissue than in paired normal tissue. • The expression of miR-935 is lower in GSRCC tissue than in non-GSRCC. • Enhanced expression of miR-935 suppresses tumorigenesis of GSRCC. • Notch1 is a direct target of miR-935.« less

Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos

PubMed Central

Ochiai, Hiroshi; Sakamoto, Naoaki; Fujita, Kazumasa; Nishikawa, Masatoshi; Suzuki, Ken-ichi; Matsuura, Shinya; Miyamoto, Tatsuo; Sakuma, Tetsushi; Shibata, Tatsuo; Yamamoto, Takashi

2012-01-01

To understand complex biological systems, such as the development of multicellular organisms, it is important to characterize the gene expression dynamics. However, there is currently no universal technique for targeted insertion of reporter genes and quantitative imaging in multicellular model systems. Recently, genome editing using zinc-finger nucleases (ZFNs) has been reported in several models. ZFNs consist of a zinc-finger DNA-binding array with the nuclease domain of the restriction enzyme FokI and facilitate targeted transgene insertion. In this study, we successfully inserted a GFP reporter cassette into the HpEts1 gene locus of the sea urchin, Hemicentrotus pulcherrimus. We achieved this insertion by injecting eggs with a pair of ZFNs for HpEts1 with a targeting donor construct that contained ∼1-kb homology arms and a 2A-histone H2B–GFP cassette. We increased the efficiency of the ZFN-mediated targeted transgene insertion by in situ linearization of the targeting donor construct and cointroduction of an mRNA for a dominant-negative form of HpLig4, which encodes the H. pulcherrimus homolog of DNA ligase IV required for error-prone nonhomologous end joining. We measured the fluorescence intensity of GFP at the single-cell level in living embryos during development and found that there was variation in HpEts1 expression among the primary mesenchyme cells. These findings demonstrate the feasibility of ZFN-mediated targeted transgene insertion to enable quantification of the expression levels of endogenous genes during development in living sea urchin embryos. PMID:22711830

Regulator of G protein signaling 4 is a novel target of GATA-6 transcription factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yonggang; Li, Fang; Xiao, Xiao

GATA transcription factors regulate an array of genes important in cell proliferation and differentiation. Here we report the identification of regulator of G protein signaling 4 (RGS4) as a novel target for GATA-6 transcription factor. Although three sites (a, b, c) within the proximal region of rabbit RGS4 promoter for GATA transcription factors were predicted by bioinformatics analysis, only GATA-a site (16 bp from the core TATA box) is essential for RGS4 transcriptional regulation. RT-PCR analysis demonstrated that only GATA-6 was highly expressed in rabbit colonic smooth muscle cells but GATA-4/6 were expressed in cardiac myocytes and GATA-1/2/3 expressed inmore » blood cells. Adenovirus-mediated expression of GATA-6 but not GATA-1 significantly increased the constitutive and IL-1β-induced mRNA expression of the endogenous RGS4 in colonic smooth muscle cells. IL-1β stimulation induced GATA-6 nuclear translocation and increased GATA-6 binding to RGS4 promoter. These data suggest that GATA factor could affect G protein signaling through regulating RGS4 expression, and GATA signaling may develop as a future therapeutic target for RGS4-related diseases. - Highlights: • GATA-6 is highly expressed in colonic smooth muscle cells. • RGS4 is a novel target for GATA-6 transcription factor. • GATA-a response element is essential to regulate the core promoter of RGS4. • GATA-6 regulates IL-1β-induced RGS4 upregulation.« less

Immunotherapy targeting folate receptor induces cell death associated with autophagy in ovarian cancer

PubMed Central

Wen, Yunfei; Graybill, Whitney S.; Previs, Rebecca A.; Hu, Wei; Ivan, Cristina; Mangala, Lingegowda S.; Zand, Behrouz; Nick, Alpa M.; Jennings, Nicholas B.; Dalton, Heather J.; Sehgal, Vasudha; Ram, Prahlad; Lee, Ju-Seog; Vivas-Mejia, Pablo E.; Coleman, Robert L.; Sood, Anil K.

2014-01-01

Purpose Cancer cells are highly dependent on folate metabolism, making them susceptible to drugs that inhibit folate receptor activities. Targeting overexpressed folate receptor alpha (FRα) in cancer cells offers a therapeutic opportunity. We investigated the functional mechanisms of MORAB-003 (farletuzumab), a humanized monoclonal antibody against FRα, in ovarian cancer models. Experimental Design We first examined FRα expression in an array of human ovarian cancer cell lines and then assessed the in vivo effect of MORAB-003 on tumor growth and progression in several orthotopic mouse models of ovarian cancer derived from these cell lines. Molecular mechanisms of tumor cell death induced by MORAB-003 were investigated by cDNA and protein expression profiling analysis. Mechanistic studies were performed to determine the role of autophagy in MORAB-003–induced cell death. Results MORAB-003 significantly decreased tumor growth in the high-FRα IGROV1 and SKOV3ip1 models but not in the low-FRα A2780 model. MORAB-003 reduced proliferation but had no significant effect on apoptosis. Protein expression and cDNA microarray analyses showed that MORAB-003 regulated an array of autophagy-related genes. It also significantly increased expression of LC3 isoform II and enriched autophagic vacuolization. Blocking autophagy with hydroxychloroquine or bafilomycin A1 reversed the growth inhibition induced by MORAB-003. In add, alteration of FOLR1 gene copy number significantly correlated with shorter disease-free survival in patients with ovarian serous cystadenocarcinoma. Conclusions MORAB-003 displays prominent antitumor activity in ovarian cancer models expressing FRα at high levels. Blockade of folate receptor by MORAB-003 induced sustained autophagy and suppressed cell proliferation. PMID:25416196

A Targetable Molecular Chaperone Hsp27 Confers Aggressiveness in Hepatocellular Carcinoma.

PubMed

Zhang, Yurong; Tao, Xuemei; Jin, Guangzhi; Jin, Haojie; Wang, Ning; Hu, Fangyuan; Luo, Qin; Shu, Huiqun; Zhao, Fangyu; Yao, Ming; Fang, Jingyuan; Cong, Wenming; Qin, Wenxin; Wang, Cun

2016-01-01

Heat shock protein 27 (Hsp27) is an ATP-independent molecular chaperone and confers survival advantages and resistance to cancer cells under stress conditions. The effects and molecular mechanisms of Hsp27 in HCC invasion and metastasis are still unclear. In this study, hepatocellular carcinoma (HCC) tissue array (n = 167) was used to investigate the expression and prognostic relevance of Hsp27 in HCC patients. HCC patients with high expression of Hsp27 exhibited poor prognosis. Overexpression of Hsp27 led to the forced invasion of HCC cells, whereas silencing Hsp27 attenuated invasion and metastasis of HCC cells in vitro and in vivo. We revealed that Hsp27 activated Akt signaling, which in turn promoted MMP2 and ITGA7 expression and HCC metastasis. We further observed that targeting Hsp27 using OGX-427 obviously suppressed HCC metastasis in two metastatic models. These findings indicate that Hsp27 is a useful predictive factor for prognosis of HCC and it facilitates HCC metastasis through Akt signaling. Targeting Hsp27 with OGX-427 may represent an attractive therapeutic option for suppressing HCC metastasis.

A Targetable Molecular Chaperone Hsp27 Confers Aggressiveness in Hepatocellular Carcinoma

PubMed Central

Zhang, Yurong; Tao, Xuemei; Jin, Guangzhi; Jin, Haojie; Wang, Ning; Hu, Fangyuan; Luo, Qin; Shu, Huiqun; Zhao, Fangyu; Yao, Ming; Fang, Jingyuan; Cong, Wenming; Qin, Wenxin; Wang, Cun

2016-01-01

Heat shock protein 27 (Hsp27) is an ATP-independent molecular chaperone and confers survival advantages and resistance to cancer cells under stress conditions. The effects and molecular mechanisms of Hsp27 in HCC invasion and metastasis are still unclear. In this study, hepatocellular carcinoma (HCC) tissue array (n = 167) was used to investigate the expression and prognostic relevance of Hsp27 in HCC patients. HCC patients with high expression of Hsp27 exhibited poor prognosis. Overexpression of Hsp27 led to the forced invasion of HCC cells, whereas silencing Hsp27 attenuated invasion and metastasis of HCC cells in vitro and in vivo. We revealed that Hsp27 activated Akt signaling, which in turn promoted MMP2 and ITGA7 expression and HCC metastasis. We further observed that targeting Hsp27 using OGX-427 obviously suppressed HCC metastasis in two metastatic models. These findings indicate that Hsp27 is a useful predictive factor for prognosis of HCC and it facilitates HCC metastasis through Akt signaling. Targeting Hsp27 with OGX-427 may represent an attractive therapeutic option for suppressing HCC metastasis. PMID:26941848

A new age in functional genomics using CRISPR/Cas9 in arrayed library screening.

PubMed

Agrotis, Alexander; Ketteler, Robin

2015-01-01

CRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development. What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9) to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA. Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions. For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene. In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening. In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening.

Electro-gene transfer to skin using a noninvasive multielectrode array

PubMed Central

Guo, Siqi; Donate, Amy; Basu, Gaurav; Lundberg, Cathryn; Heller, Loree; Heller, Richard

2011-01-01

Because of its large surface area and easy access for both delivery and monitoring, the skin is an attractive target for gene therapy for cutaneous diseases, vaccinations and several metabolic disorders. The critical factors for DNA delivery to the skin by electroporation (EP) are effective expression levels and minimal or no tissue damage. Here, we evaluated the non-invasive multielectrode array (MEA) for gene electrotransfer. For these studies we utilized a guinea pig model, which has been shown to have a similar thickness and structure to human skin. Our results demonstrate significantly increased gene expression 2 to 3 logs above injection of plasmid DNA alone over 15 days. Furthermore, gene expression could be enhanced by increasing the size of the treatment area. Transgene expressing cells were observed exclusively in the epidermal layer of the skin. In contrast to caliper or plate electrodes, skin EP with the MEA greatly reduced muscle twitching and resulted in minimal and completely recoverable skin damage. These results suggest EP with the MEA can be an efficient and non-invasive skin delivery method with less adverse side effects than other EP delivery systems and promising clinical applications. PMID:21262290

MicroRNA-dependent regulation of transcription in non-small cell lung cancer.

PubMed

Molina-Pinelo, Sonia; Gutiérrez, Gabriel; Pastor, Maria Dolores; Hergueta, Marta; Moreno-Bueno, Gema; García-Carbonero, Rocío; Nogal, Ana; Suárez, Rocío; Salinas, Ana; Pozo-Rodríguez, Francisco; Lopez-Rios, Fernando; Agulló-Ortuño, Maria Teresa; Ferrer, Irene; Perpiñá, Asunción; Palacios, José; Carnero, Amancio; Paz-Ares, Luis

2014-01-01

Squamous cell lung cancer (SCC) and adenocarcinoma are the most common histological subtypes of non-small cell lung cancer (NSCLC), and have been traditionally managed in the clinic as a single entity. Increasing evidence, however, illustrates the biological diversity of these two histological subgroups of lung cancer, and supports the need to improve our understanding of the molecular basis beyond the different phenotypes if we aim to develop more specific and individualized targeted therapy. The purpose of this study was to identify microRNA (miRNA)-dependent transcriptional regulation differences between SCC and adenocarcinoma histological lung cancer subtypes. In this work, paired miRNA (667 miRNAs by TaqMan Low Density Arrays (TLDA)) and mRNA profiling (Whole Genome 44 K array G112A, Agilent) was performed in tumor samples of 44 NSCLC patients. Nine miRNAs and 56 mRNAs were found to be differentially expressed in SCC versus adenocarcinoma samples. Eleven of these 56 mRNA were predicted as targets of the miRNAs identified to be differently expressed in these two histological conditions. Of them, 6 miRNAs (miR-149, miR-205, miR-375, miR-378, miR-422a and miR-708) and 9 target genes (CEACAM6, CGN, CLDN3, ABCC3, MLPH, ACSL5, TMEM45B, MUC1) were validated by quantitative PCR in an independent cohort of 41 lung cancer patients. Furthermore, the inverse correlation between mRNAs and microRNAs expression was also validated. These results suggest miRNA-dependent transcriptional regulation differences play an important role in determining key hallmarks of NSCLC, and may provide new biomarkers for personalized treatment strategies.

MicroRNA-Dependent Regulation of Transcription in Non-Small Cell Lung Cancer

PubMed Central

Molina-Pinelo, Sonia; Gutiérrez, Gabriel; Pastor, Maria Dolores; Hergueta, Marta; Moreno-Bueno, Gema; García-Carbonero, Rocío; Nogal, Ana; Suárez, Rocío; Salinas, Ana; Pozo-Rodríguez, Francisco; Lopez-Rios, Fernando; Agulló-Ortuño, Maria Teresa; Ferrer, Irene; Perpiñá, Asunción; Palacios, José; Carnero, Amancio; Paz-Ares, Luis

2014-01-01

Squamous cell lung cancer (SCC) and adenocarcinoma are the most common histological subtypes of non-small cell lung cancer (NSCLC), and have been traditionally managed in the clinic as a single entity. Increasing evidence, however, illustrates the biological diversity of these two histological subgroups of lung cancer, and supports the need to improve our understanding of the molecular basis beyond the different phenotypes if we aim to develop more specific and individualized targeted therapy. The purpose of this study was to identify microRNA (miRNA)-dependent transcriptional regulation differences between SCC and adenocarcinoma histological lung cancer subtypes. In this work, paired miRNA (667 miRNAs by TaqMan Low Density Arrays (TLDA)) and mRNA profiling (Whole Genome 44 K array G112A, Agilent) was performed in tumor samples of 44 NSCLC patients. Nine miRNAs and 56 mRNAs were found to be differentially expressed in SCC versus adenocarcinoma samples. Eleven of these 56 mRNA were predicted as targets of the miRNAs identified to be differently expressed in these two histological conditions. Of them, 6 miRNAs (miR-149, miR-205, miR-375, miR-378, miR-422a and miR-708) and 9 target genes (CEACAM6, CGN, CLDN3, ABCC3, MLPH, ACSL5, TMEM45B, MUC1) were validated by quantitative PCR in an independent cohort of 41 lung cancer patients. Furthermore, the inverse correlation between mRNAs and microRNAs expression was also validated. These results suggest miRNA-dependent transcriptional regulation differences play an important role in determining key hallmarks of NSCLC, and may provide new biomarkers for personalized treatment strategies. PMID:24625834

Can an anger face also be scared? Malleability of facial expressions.

PubMed

Widen, Sherri C; Naab, Pamela

2012-10-01

Do people always interpret a facial expression as communicating a single emotion (e.g., the anger face as only angry) or is that interpretation malleable? The current study investigated preschoolers' (N = 60; 3-4 years) and adults' (N = 20) categorization of facial expressions. On each of five trials, participants selected from an array of 10 facial expressions (an open-mouthed, high arousal expression and a closed-mouthed, low arousal expression each for happiness, sadness, anger, fear, and disgust) all those that displayed the target emotion. Children's interpretation of facial expressions was malleable: 48% of children who selected the fear, anger, sadness, and disgust faces for the "correct" category also selected these same faces for another emotion category; 47% of adults did so for the sadness and disgust faces. The emotion children and adults attribute to facial expressions is influenced by the emotion category for which they are looking.

In situ synthesis of protein arrays.

PubMed

He, Mingyue; Stoevesandt, Oda; Taussig, Michael J

2008-02-01

In situ or on-chip protein array methods use cell free expression systems to produce proteins directly onto an immobilising surface from co-distributed or pre-arrayed DNA or RNA, enabling protein arrays to be created on demand. These methods address three issues in protein array technology: (i) efficient protein expression and availability, (ii) functional protein immobilisation and purification in a single step and (iii) protein on-chip stability over time. By simultaneously expressing and immobilising many proteins in parallel on the chip surface, the laborious and often costly processes of DNA cloning, expression and separate protein purification are avoided. Recently employed methods reviewed are PISA (protein in situ array) and NAPPA (nucleic acid programmable protein array) from DNA and puromycin-mediated immobilisation from mRNA.

Over-expression of the miRNA cluster at chromosome 14q32 in the alcoholic brain correlates with suppression of predicted target mRNA required for oligodendrocyte proliferation.

PubMed

Manzardo, A M; Gunewardena, S; Butler, M G

2013-09-10

We examined miRNA expression from RNA isolated from the frontal cortex (Broadman area 9) of 9 alcoholics (6 males, 3 females, mean age 48 years) and 9 matched controls using both the Affymetrix GeneChip miRNA 2.0 and Human Exon 1.0 ST Arrays to further characterize genetic influences in alcoholism and the effects of alcohol consumption on predicted target mRNA expression. A total of 12 human miRNAs were significantly up-regulated in alcohol dependent subjects (fold change≥1.5, false discovery rate (FDR)≤0.3; p<0.05) compared with controls including a cluster of 4 miRNAs (e.g., miR-377, miR-379) from the maternally expressed 14q32 chromosome region. The status of the up-regulated miRNAs was supported using the high-throughput method of exon microarrays showing decreased predicted mRNA gene target expression as anticipated from the same RNA aliquot. Predicted mRNA targets were involved in cellular adhesion (e.g., THBS2), tissue differentiation (e.g., CHN2), neuronal migration (e.g., NDE1), myelination (e.g., UGT8, CNP) and oligodendrocyte proliferation (e.g., ENPP2, SEMA4D1). Our data support an association of alcoholism with up-regulation of a cluster of miRNAs located in the genomic imprinted domain on chromosome 14q32 with their predicted gene targets involved with oligodendrocyte growth, differentiation and signaling. Copyright © 2013 Elsevier B.V. All rights reserved.

Method and infrastructure for cycle-reproducible simulation on large scale digital circuits on a coordinated set of field-programmable gate arrays (FPGAs)

DOEpatents

Asaad, Sameh W; Bellofatto, Ralph E; Brezzo, Bernard; Haymes, Charles L; Kapur, Mohit; Parker, Benjamin D; Roewer, Thomas; Tierno, Jose A

2014-01-28

A plurality of target field programmable gate arrays are interconnected in accordance with a connection topology and map portions of a target system. A control module is coupled to the plurality of target field programmable gate arrays. A balanced clock distribution network is configured to distribute a reference clock signal, and a balanced reset distribution network is coupled to the control module and configured to distribute a reset signal to the plurality of target field programmable gate arrays. The control module and the balanced reset distribution network are cooperatively configured to initiate and control a simulation of the target system with the plurality of target field programmable gate arrays. A plurality of local clock control state machines reside in the target field programmable gate arrays. The local clock state machines are configured to generate a set of synchronized free-running and stoppable clocks to maintain cycle-accurate and cycle-reproducible execution of the simulation of the target system. A method is also provided.

Seromic profiling of colorectal cancer patients with novel glycopeptide microarray.

PubMed

Pedersen, Johannes W; Blixt, Ola; Bennett, Eric P; Tarp, Mads A; Dar, Imran; Mandel, Ulla; Poulsen, Steen S; Pedersen, Anders E; Rasmussen, Susanne; Jess, Per; Clausen, Henrik; Wandall, Hans H

2011-04-15

Cancer-associated autoantibodies hold promise as sensitive biomarkers for early detection of cancer. Aberrant post-translational variants of proteins are likely to induce autoantibodies, and changes in O-linked glycosylation represent one of the most important cancer-associated post-translational modifications (PTMs). Short aberrant O-glycans on proteins may introduce novel glycopeptide epitopes that can elicit autoantibodies because of lack of tolerance. Technical barriers, however, have hampered detection of such glycopeptide-specific autoantibodies. Here, we have constructed an expanded glycopeptide array displaying a comprehensive library of glycopeptides and glycoproteins derived from a panel of human mucins (MUC1, MUC2, MUC4, MUC5AC, MUC6 and MUC7) known to have altered glycosylation and expression in cancer. Seromic profiling of patients with colorectal cancer identified cancer-associated autoantibodies to a set of aberrant glycopeptides derived from MUC1 and MUC4. The cumulative sensitivity of the array analysis was 79% with a specificity of 92%. The most prevalent of the identified autoantibody targets were validated as authentic cancer immunogens by showing expression of the epitopes in cancer using novel monoclonal antibodies. Our study provides evidence for the value of glycopeptides and other PTM-peptide arrays in diagnostic measures. Copyright © 2011 UICC.

Partition resampling and extrapolation averaging: approximation methods for quantifying gene expression in large numbers of short oligonucleotide arrays.

PubMed

Goldstein, Darlene R

2006-10-01

Studies of gene expression using high-density short oligonucleotide arrays have become a standard in a variety of biological contexts. Of the expression measures that have been proposed to quantify expression in these arrays, multi-chip-based measures have been shown to perform well. As gene expression studies increase in size, however, utilizing multi-chip expression measures is more challenging in terms of computing memory requirements and time. A strategic alternative to exact multi-chip quantification on a full large chip set is to approximate expression values based on subsets of chips. This paper introduces an extrapolation method, Extrapolation Averaging (EA), and a resampling method, Partition Resampling (PR), to approximate expression in large studies. An examination of properties indicates that subset-based methods can perform well compared with exact expression quantification. The focus is on short oligonucleotide chips, but the same ideas apply equally well to any array type for which expression is quantified using an entire set of arrays, rather than for only a single array at a time. Software implementing Partition Resampling and Extrapolation Averaging is under development as an R package for the BioConductor project.

Biological mechanism analysis of acute renal allograft rejection: integrated of mRNA and microRNA expression profiles.

PubMed

Huang, Shi-Ming; Zhao, Xia; Zhao, Xue-Mei; Wang, Xiao-Ying; Li, Shan-Shan; Zhu, Yu-Hui

2014-01-01

Renal transplantation is the preferred method for most patients with end-stage renal disease, however, acute renal allograft rejection is still a major risk factor for recipients leading to renal injury. To improve the early diagnosis and treatment of acute rejection, study on the molecular mechanism of it is urgent. MicroRNA (miRNA) expression profile and mRNA expression profile of acute renal allograft rejection and well-functioning allograft downloaded from ArrayExpress database were applied to identify differentially expressed (DE) miRNAs and DE mRNAs. DE miRNAs targets were predicted by combining five algorithm. By overlapping the DE mRNAs and DE miRNAs targets, common genes were obtained. Differentially co-expressed genes (DCGs) were identified by differential co-expression profile (DCp) and differential co-expression enrichment (DCe) methods in Differentially Co-expressed Genes and Links (DCGL) package. Then, co-expression network of DCGs and the cluster analysis were performed. Functional enrichment analysis for DCGs was undergone. A total of 1270 miRNA targets were predicted and 698 DE mRNAs were obtained. While overlapping miRNA targets and DE mRNAs, 59 common genes were gained. We obtained 103 DCGs and 5 transcription factors (TFs) based on regulatory impact factors (RIF), then built the regulation network of miRNA targets and DE mRNAs. By clustering the co-expression network, 5 modules were obtained. Thereinto, module 1 had the highest degree and module 2 showed the most number of DCGs and common genes. TF CEBPB and several common genes, such as RXRA, BASP1 and AKAP10, were mapped on the co-expression network. C1R showed the highest degree in the network. These genes might be associated with human acute renal allograft rejection. We conducted biological analysis on integration of DE mRNA and DE miRNA in acute renal allograft rejection, displayed gene expression patterns and screened out genes and TFs that may be related to acute renal allograft rejection.

Biological mechanism analysis of acute renal allograft rejection: integrated of mRNA and microRNA expression profiles

PubMed Central

Huang, Shi-Ming; Zhao, Xia; Zhao, Xue-Mei; Wang, Xiao-Ying; Li, Shan-Shan; Zhu, Yu-Hui

2014-01-01

Objectives: Renal transplantation is the preferred method for most patients with end-stage renal disease, however, acute renal allograft rejection is still a major risk factor for recipients leading to renal injury. To improve the early diagnosis and treatment of acute rejection, study on the molecular mechanism of it is urgent. Methods: MicroRNA (miRNA) expression profile and mRNA expression profile of acute renal allograft rejection and well-functioning allograft downloaded from ArrayExpress database were applied to identify differentially expressed (DE) miRNAs and DE mRNAs. DE miRNAs targets were predicted by combining five algorithm. By overlapping the DE mRNAs and DE miRNAs targets, common genes were obtained. Differentially co-expressed genes (DCGs) were identified by differential co-expression profile (DCp) and differential co-expression enrichment (DCe) methods in Differentially Co-expressed Genes and Links (DCGL) package. Then, co-expression network of DCGs and the cluster analysis were performed. Functional enrichment analysis for DCGs was undergone. Results: A total of 1270 miRNA targets were predicted and 698 DE mRNAs were obtained. While overlapping miRNA targets and DE mRNAs, 59 common genes were gained. We obtained 103 DCGs and 5 transcription factors (TFs) based on regulatory impact factors (RIF), then built the regulation network of miRNA targets and DE mRNAs. By clustering the co-expression network, 5 modules were obtained. Thereinto, module 1 had the highest degree and module 2 showed the most number of DCGs and common genes. TF CEBPB and several common genes, such as RXRA, BASP1 and AKAP10, were mapped on the co-expression network. C1R showed the highest degree in the network. These genes might be associated with human acute renal allograft rejection. Conclusions: We conducted biological analysis on integration of DE mRNA and DE miRNA in acute renal allograft rejection, displayed gene expression patterns and screened out genes and TFs that may be related to acute renal allograft rejection. PMID:25664019

Cortisol inhibits mTOR signaling in avascular necrosis of the femoral head.

PubMed

Liao, Yun; Su, Rui; Zhang, Ping; Yuan, Bo; Li, Ling

2017-10-18

ANFH is a major health problem, to which long lasting and definitive treatments are lacking. The aim of this study is to study RNA alterations attributed to cortisol-induced ANFH. Rat models were stratified into three groups: in vitro group (n = 20) for molecular biological assays, control group (n = 3), and ANFH group induced using lipopolysaccharide and dexamethasone (n = 3). Bone marrow-derived endothelial progenitor cells (BM-EPCs) were extracted from the rats. An RNA expression array was performed on BM-EPCs, and enriched genes were subject to pathway analysis. In vitro studies following findings of array results were also performed using the isolated BM-EPCs. Significant alterations in mammalian target of rapamycin (mTOR) and HIF signaling pathways were identified in BM-EPCs of ANFH. By applying cortisol and dexamethasone to BM-EPCs, significant changes in mTOR and HIF elements were identified. The alteration of HIF pathways appeared to be downstream of mTOR signaling. Glucocorticoid receptor (GR) expression was related to glucocorticoid-dependent mRNA expression of mTOR/HIF genes. mTOR-dependent angiogenesis but not anabolism was the target of GR in ANFH. Inhibition of mTOR signaling also induced apoptosis of BM-EPCs via CHOP-dependent DR5 induction in response to GR stimulation. Decreased mTOR signaling in response to GR stimulation leading to downregulated HIF pathway as well as increased apoptosis could be the pathophysiology.

Cooled particle accelerator target

DOEpatents

Degtiarenko, Pavel V.

2005-06-14

A novel particle beam target comprising: a rotating target disc mounted on a retainer and thermally coupled to a first array of spaced-apart parallel plate fins that extend radially inwardly from the retainer and mesh without physical contact with a second array of spaced-apart parallel plate fins that extend radially outwardly from and are thermally coupled to a cooling mechanism capable of removing heat from said second array of spaced-apart fins and located within the first array of spaced-apart parallel fins. Radiant thermal exchange between the two arrays of parallel plate fins provides removal of heat from the rotating disc. A method of cooling the rotating target is also described.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bonachea, Dan; Hargrove, P.

GASNet is a language-independent, low-level networking layer that provides network-independent, high-performance communication primitives tailored for implementing parallel global address space SPMD languages and libraries such as UPC, UPC++, Co-Array Fortran, Legion, Chapel, and many others. The interface is primarily intended as a compilation target and for use by runtime library writers (as opposed to end users), and the primary goals are high performance, interface portability, and expressiveness. GASNet stands for "Global-Address Space Networking".

Molecular Targeted Therapies of Childhood Choroid Plexus Carcinoma

DTIC Science & Technology

2013-10-01

Microarray intensities were analyzed in PGS, using the benign human choroid plexus papilloma (CPP) samples as an expression baseline reference. This...additional human and mouse CPC genomic profiles (timeframe: months 1-5). The goal of these studies is to expand our number of genomic profiles (DNA and...mRNA arrays) of both human and mouse CPCs to provide a comprehensive dataset with which to identify key candidate oncogenes, tumor suppressor genes

Molecular Targeted Therapies of Childhood Choroid Plexus Carcinoma

DTIC Science & Technology

2011-10-01

were analyzed in PGS, using the benign human choroid plexus papilloma (CPP) samples as an expression baseline reference. This analysis highlights...Task 1: Generation of additional human and mouse CPC genomic profiles (timeframe: months 1-5). The goal of these studies is to expand our...number of genomic profiles (DNA and mRNA arrays) of both human and mouse CPCs to provide a comprehensive dataset with which to identify key candidate

Expression patterns of bark beetle cytochromes P450 during host colonization: Likely physiological functions and potential targets for pest management

Treesearch

Dezene P. W. Huber; Melissa Erickson; Christian Leutenegger; Joerg Bohlmann; Steven J. Seybold

2007-01-01

Cytochromes P450 family genes (P450s) are found in a diverse array of organisms ranging from bacteria to mammals to plants to arthropods. Although there are exceptions to this rule, organisms generally contain a fairly large number of P450 genes and pseudogenes in their genomes. For instance, among arthropods whose genomes are well characterized, the mosquito,

Evaluation of the X-Linked High-Grade Myopia Locus (MYP1) with Cone Dysfunction and Color Vision Deficiencies

PubMed Central

Metlapally, Ravikanth; Michaelides, Michel; Bulusu, Anuradha; Li, Yi-Ju; Schwartz, Marianne; Rosenberg, Thomas; Hunt, David M.; Moore, Anthony T.; Züchner, Stephan; Rickman, Catherine Bowes; Young, Terri L.

2014-01-01

Purpose X-linked high myopia with mild cone dysfunction and color vision defects has been mapped to chromosome Xq28 (MYP1 locus). CXorf2/TEX28 is a nested, intercalated gene within the red-green opsin cone pigment gene tandem array on Xq28. The authors investigated whether TEX28 gene alterations were associated with the Xq28-linked myopia phenotype. Genomic DNA from five pedigrees (with high myopia and either protanopia or deuteranopia) that mapped to Xq28 were screened for TEX28 copy number variations (CNVs) and sequence variants. Methods To examine for CNVs, ultra-high resolution array-comparative genomic hybridization (array-CGH) assays were performed comparing the subject genomic DNA with control samples (two pairs from two pedigrees). Opsin or TEX28 gene-targeted quantitative real-time gene expression assays (comparative CT method) were performed to validate the array-CGH findings. All exons of TEX28, including intron/exon boundaries, were amplified and sequenced using standard techniques. Results Array-CGH findings revealed predicted duplications in affected patient samples. Although only three copies of TEX28 were previously reported within the opsin array, quantitative real-time analysis of the TEX28 targeted assay of affected male or carrier female individuals in these pedigrees revealed either fewer (one) or more (four or five) copies than did related and control unaffected individuals. Sequence analysis of TEX28 did not reveal any variants associated with the disease status. Conclusions CNVs have been proposed to play a role in disease inheritance and susceptibility as they affect gene dosage. TEX28 gene CNVs appear to be associated with the MYP1 X-linked myopia phenotypes. PMID:19098318

MicroRNA-188-5p suppresses tumor cell proliferation and metastasis by directly targeting FGF5 in hepatocellular carcinoma.

PubMed

Fang, Feng; Chang, Rui-min; Yu, Lei; Lei, Xiong; Xiao, Shuai; Yang, Hao; Yang, Lian-Yue

2015-10-01

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. However, the detailed molecular mechanisms underlying HCC progression are still not completely clear. Given the crucial role of microRNAs (miRNAs) in cancer metastasis, we aimed to analyze the expression and function of a metastasis-associated miRNA named miR-188-5p in HCC. miRNA array analysis was performed to search for metastasis-associated miRNAs in HCC. miR-188-5p expressions in tumor tissues and adjacent non-tumorous liver tissues of HCC patients and cell lines were evaluated by real-time PCR. The protein expression levels were analyzed by Western blot and immunohistochemistry. Luciferase reporter assays was used to validate the target of miR-188-5p. The effect of miR-188-5p on HCC progression was studied in vitro and in vivo. miR-188-5p was significantly decreased in HCC and its expression levels were highly correlated with multiple nodules, microvascular invasion, overall and disease-free survival of HCC. Ectopic expression of miR-188-5p suppressed HCC cell proliferation and metastasis in vitro and in vivo. Fibroblast growth factor 5 (FGF5) was identified as a major target of miR-188-5p. Enforced expression of miR-188-5p inhibited the expression of FGF5 significantly and the restoration of FGF5 expression reversed the inhibitory effects of miR-188-5p on HCC cell proliferation and metastasis. These findings collectively demonstrate a tumor suppressor role of miR-188-5p in HCC progression via targeting FGF5, suggesting that miR-188-5p could serve as a potential prognostic biomarker and therapeutic target for HCC. Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Layer-by-layer cell membrane assembly

NASA Astrophysics Data System (ADS)

Matosevic, Sandro; Paegel, Brian M.

2013-11-01

Eukaryotic subcellular membrane systems, such as the nuclear envelope or endoplasmic reticulum, present a rich array of architecturally and compositionally complex supramolecular targets that are as yet inaccessible. Here we describe layer-by-layer phospholipid membrane assembly on microfluidic droplets, a route to structures with defined compositional asymmetry and lamellarity. Starting with phospholipid-stabilized water-in-oil droplets trapped in a static droplet array, lipid monolayer deposition proceeds as oil/water-phase boundaries pass over the droplets. Unilamellar vesicles assembled layer-by-layer support functional insertion both of purified and of in situ expressed membrane proteins. Synthesis and chemical probing of asymmetric unilamellar and double-bilayer vesicles demonstrate the programmability of both membrane lamellarity and lipid-leaflet composition during assembly. The immobilized vesicle arrays are a pragmatic experimental platform for biophysical studies of membranes and their associated proteins, particularly complexes that assemble and function in multilamellar contexts in vivo.

Diverse solid tumors expressing a restricted epitope of L1-CAM can be targeted by chimeric antigen receptor redirected T lymphocytes.

PubMed

Hong, Hao; Stastny, Michael; Brown, Christine; Chang, Wen-Chung; Ostberg, Julie R; Forman, Stephen J; Jensen, Michael C

2014-01-01

Adhesion molecule L1-CAM (CD171) was originally reported to be overexpressed on neuroblastoma and to play an important role during tumor progression. More recently, it has been shown to be overexpressed on many other solid tumors such as melanoma and carcinomas of the cervix, ovary, bladder, and others. Thus, there has been a growing interest in using this cell-surface molecule as a target for both antibody-based and cellular-based therapy-our group has previously examined the clinical utility of chimeric antigen receptor (CAR)-redirected cytolytic T cells that specifically target the CE7 epitope of L1-CAM on neuroblastoma patients. Here, we sought to determine whether this CE7 epitope is present on other recently identified L1-CAM tumors and whether it too can be targeted by CAR T cells. Our studies demonstrate that a diverse array of human tumor cell lines and primary solid tumors (ovarian, lung, and renal carcinoma, glioblastoma and neuroblastoma) do express the CE7 epitope and can efficiently stimulate CE7-specific CAR-redirected (CE7R) T-cell lytic activity and secretion of proinflamatory cytokines. L1-CAM was also detected on a limited number of normal tissues; however, L1-CAM expressed on normal human monocytes was not bound by the CE7 mAb nor was it targeted by CE7R T cells, suggesting that the CE7 epitope is more tumor restricted and not expressed on all L1-CAM tissues. Overall, the CE7 epitope of L1-CAM on a variety of tumors may be amenable to targeting by CE7R T cells, making it a promising target for adoptive immunotherapy.

Desensitization and Incomplete Recovery of Hepatic Target Genes After Chronic Thyroid Hormone Treatment and Withdrawal in Male Adult Mice

PubMed Central

Ohba, Kenji; Singh, Brijesh Kumar; Sinha, Rohit Anthony; Lesmana, Ronny; Liao, Xiao-Hui; Ghosh, Sujoy; Refetoff, Samuel

2016-01-01

Clinical symptoms may vary and not necessarily reflect serum thyroid hormone (TH) levels during acute and chronic hyperthyroidism as well as recovery from hyperthyroidism. We thus examined changes in hepatic gene expression and serum TH/TSH levels in adult male mice treated either with a single T3 (20 μg per 100 g body weight) injection (acute T3) or daily injections for 14 days (chronic T3) followed by 10 days of withdrawal. Gene expression arrays from livers harvested at these time points showed that among positively-regulated target genes, 320 were stimulated acutely and 429 chronically by T3. Surprisingly, only 69 of 680 genes (10.1%) were induced during both periods, suggesting desensitization of the majority of acutely stimulated target genes. About 90% of positively regulated target genes returned to baseline expression levels after 10 days of withdrawal; however, 67 of 680 (9.9%) did not return to baseline despite normalization of serum TH/TSH levels. Similar findings also were observed for negatively regulated target genes. Chromatin immunoprecipitation analysis of representative positively regulated target genes suggested that acetylation of H3K9/K14 was associated with acute stimulation, whereas trimethylation of H3K4 was associated with chronic stimulation. In an in vivo model of chronic intrahepatic hyperthyroidism since birth, adult male monocarboxylate transporter-8 knockout mice also demonstrated desensitization of most acutely stimulated target genes that were examined. In summary, we have identified transcriptional desensitization and incomplete recovery of gene expression during chronic hyperthyroidism and recovery. Our findings may be a potential reason for discordance between clinical symptoms and serum TH levels observed in these conditions. PMID:26866609

miRNA-135b Contributes to Triple Negative Breast Cancer Molecular Heterogeneity: Different Expression Profile in Basal-like Versus non-Basal-like Phenotypes.

PubMed

Uva, Paolo; Cossu-Rocca, Paolo; Loi, Federica; Pira, Giovanna; Murgia, Luciano; Orrù, Sandra; Floris, Matteo; Muroni, Maria Rosaria; Sanges, Francesca; Carru, Ciriaco; Angius, Andrea; De Miglio, Maria Rosaria

2018-01-01

The clinical and genetic heterogeneity of Triple Negative Breast Cancer (TNBC) and the lack of unambiguous molecular targets contribute to the inadequacy of current therapeutic options for these variants. MicroRNAs (miRNA) are a class of small highly conserved regulatory endogenous non-coding RNA, which can alter the expression of genes encoding proteins and may play a role in the dysregulation of cellular pathways. Our goal was to improve the knowledge of the molecular pathogenesis of TNBC subgroups analyzing the miRNA expression profile, and to identify new prognostic and predictive biomarkers. We conducted a human miRNome analysis by TaqMan Low Density Array comparing different TNBC subtypes, defined by immunohistochemical basal markers EGFR and CK5/6. RT-qPCR confirmed differential expression of microRNAs. To inspect the function of the selected targets we perform Gene Ontology and KEGG enrichment analysis. We identified a single miRNA signature given by miR-135b expression level, which was strictly related to TNBC with basal-like phenotype. miR-135b target analysis revealed a role in the TGF-beta, WNT and ERBB pathways. A significant positive correlation was identified between neoplastic proliferative index and miR-135b expression. These findings confirm the oncogenic roles of miR-135b in the pathogenesis of TNBC expressing basal markers. A potential negative prognostic role of miR-135b overexpression might be related to the positive correlation with high proliferative index. Our study implies potential clinical applications: miR-135b could be a potential therapeutic target in basal-like TNBCs.

miRNA-135b Contributes to Triple Negative Breast Cancer Molecular Heterogeneity: Different Expression Profile in Basal-like Versus non-Basal-like Phenotypes

PubMed Central

Uva, Paolo; Cossu-Rocca, Paolo; Loi, Federica; Pira, Giovanna; Murgia, Luciano; Orrù, Sandra; Floris, Matteo; Muroni, Maria Rosaria; Sanges, Francesca; Carru, Ciriaco; Angius, Andrea; De Miglio, Maria Rosaria

2018-01-01

The clinical and genetic heterogeneity of Triple Negative Breast Cancer (TNBC) and the lack of unambiguous molecular targets contribute to the inadequacy of current therapeutic options for these variants. MicroRNAs (miRNA) are a class of small highly conserved regulatory endogenous non-coding RNA, which can alter the expression of genes encoding proteins and may play a role in the dysregulation of cellular pathways. Our goal was to improve the knowledge of the molecular pathogenesis of TNBC subgroups analyzing the miRNA expression profile, and to identify new prognostic and predictive biomarkers. We conducted a human miRNome analysis by TaqMan Low Density Array comparing different TNBC subtypes, defined by immunohistochemical basal markers EGFR and CK5/6. RT-qPCR confirmed differential expression of microRNAs. To inspect the function of the selected targets we perform Gene Ontology and KEGG enrichment analysis. We identified a single miRNA signature given by miR-135b expression level, which was strictly related to TNBC with basal-like phenotype. miR-135b target analysis revealed a role in the TGF-beta, WNT and ERBB pathways. A significant positive correlation was identified between neoplastic proliferative index and miR-135b expression. These findings confirm the oncogenic roles of miR-135b in the pathogenesis of TNBC expressing basal markers. A potential negative prognostic role of miR-135b overexpression might be related to the positive correlation with high proliferative index. Our study implies potential clinical applications: miR-135b could be a potential therapeutic target in basal-like TNBCs. PMID:29725243

miR-1271 inhibits migration, invasion and epithelial-mesenchymal transition by targeting ZEB1 and TWIST1 in pancreatic cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Huaize; Wang, Han; Liu, Xiaoxiao

Pancreatic cancer (PC) remains one of the most lethal types of cancer in adults. The purpose of this study was to determine the role of miR-1271 in regulation of epithelial mesenchymal transition (EMT) and metastasis of pancreatic cancer cells. miR-1271 was identified to be significantly down-regulated in PC tissues by miRNA array. Also, an increase of EMT-regulators ZEB1 and TWIST1 expression level is accompanied by a decrease of miR-1271. We showed that expression of miR-1271 was significantly down-regulated in PC tissues as compared with that in normal tissues. In addition, our results showed that miR-1271 expression levels were decreased whilemore » ZEB1 and TWIST1 expression levels were increased in detected PC cell lines. Moreover, ectopic expression of miR-1271 suppressed and antagomiR-1271 promoted proliferation, migration, and invasion in SW1990 and PANC-1 cells. Bioinformatics coupled with luciferase and Western blot assays also revealed that miR-1271 inhibited expression of ZEB1 and TWIST1, which are master regulators of tumor metastasis. Our study first indicates that miR-1271 functions as a suppressor in regulating of pancreatic cancer EMT by targeting ZEB1 and TWIST1, and it promise as a therapeutic target and prognostic marker for metastatic pancreatic cancer. - Highlights: • miR-1271 is downregulated in pancreatic cancer tissues and cell lines. • miR-1271 regulates cell metastasis ability and EMT marker expression. . • miR-1271 directly targets ZEB1 and TWIST1. • ZEB1 and TWIST1 are functionally related to the effects of miR-1271.« less

miR-137 Targets p160 Steroid Receptor Coactivators SRC1, SRC2, and SRC3 and Inhibits Cell Proliferation

PubMed Central

Eedunuri, Vijay Kumar; Rajapakshe, Kimal; Fiskus, Warren; Geng, Chuandong; Chew, Sue Anne; Foley, Christopher; Shah, Shrijal S.; Shou, John; Mohamed, Junaith S.; O'Malley, Bert W.

2015-01-01

The p160 family of steroid receptor coactivators (SRCs) are pleiotropic transcription factor coactivators and “master regulators” of gene expression that promote cancer cell proliferation, survival, metabolism, migration, invasion, and metastasis. Cancers with high p160 SRC expression exhibit poor clinical outcomes and resistance to therapy, highlighting the SRCs as critical oncogenic drivers and, thus, therapeutic targets. microRNAs are important epigenetic regulators of protein expression. To examine the regulation of p160 SRCs by microRNAs, we used and combined 4 prediction algorithms to identify microRNAs that could target SRC1, SRC2, and SRC3 expression. For validation of these predictions, we assessed p160 SRC protein expression and cell viability after transfection of corresponding microRNA mimetics in breast cancer, uveal melanoma, and prostate cancer (PC) cell lines. Transfection of selected microRNA mimetics into breast cancer, uveal melanoma, and PC cells depleted SRC protein expression levels and exerted potent antiproliferative activity in these cell types. In particular, microRNA-137 (miR-137) depleted expression of SRC1, SRC2, and very potently, SRC3. The latter effect can be attributed to the presence of 3 miR-137 recognition sequences within the SRC3 3′-untranslated region. Using reverse phase protein array analysis, we identified a network of proteins, in addition to SRC3, that were modulated by miR-137 in PC cells. We also found that miR-137 and its host gene are epigenetically silenced in human cancer specimens and cell lines. These results support the development and testing of microRNA-based therapies (in particular based on restoring miR-137 levels) for targeting the oncogenic family of p160 SRCs in cancer. PMID:26066330

Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates demonstrate tumor specificity and anti-tumor activity.

PubMed

Prendergast, Jillian M; Galvao da Silva, Ana Paula; Eavarone, David A; Ghaderi, Darius; Zhang, Mai; Brady, Dane; Wicks, Joan; DeSander, Julie; Behrens, Jeff; Rueda, Bo R

Targeted therapeutics that can differentiate between normal and malignant tumor cells represent the ideal standard for the development of a successful anti-cancer strategy. The Sialyl-Thomsen-nouveau antigen (STn or Sialyl-Tn, also known as CD175s) is rarely seen in normal adult tissues, but it is abundantly expressed in many types of human epithelial cancers. We have identified novel antibodies that specifically target with high affinity the STn glycan independent of its carrier protein, affording the potential to recognize a wider array of cancer-specific sialylated proteins. A panel of murine monoclonal anti-STn therapeutic antibodies were generated and their binding specificity and efficacy were characterized in vitro and in in vivo murine cancer models. A subset of these antibodies were conjugated to monomethyl auristatin E (MMAE) to generate antibody-drug conjugates (ADCs). These ADCs demonstrated in vitro efficacy in STn-expressing cell lines and significant tumor growth inhibition in STn-expressing tumor xenograft cancer models with no evidence of overt toxicity.

Ciculating miRNA-21 as a Biomarker Predicts Polycystic Ovary Syndrome (PCOS) in Patients.

PubMed

Jiang, Liyan; Li, Wei; Wu, Minmin; Cao, Sifan

2015-01-01

Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism, hyperinsulinemia, and infertility. In PCOS, abnormal regulation of relevant genes is required for follicular development. By binding to the 3' untranslated region (3'URT), microRNAs (miRNAs) are widely involved in posttranscriptional gene regulation. However, few studies have been conducted on circulating miRNA expression in PCOS. This study aims to describe altered expression of circulating miR-21 in PCOS. The expression of serum miRNAs of PCOS patients were explored using the TaqMan Low Density Array followed by individual quantitative reverse transcription polymerase chain reaction assays. The protein level of LATS1 was determined using Western blot. To validate whether miR-21 targeted LATS1, the luciferase assay was applied. In comparison with normal subjects, the circulating level of miRNA-21 was significantly enhanced in PCOS patients. In PCOS patients, the expression levels of MST1/2, LATS1/2, TAZ were much lower than the control subjects. Luciferase reporter assay revealed that LATS1 was a downstream target of miR-21. In comparison with normal subjects, serum miR-21 is obviously increased in PCOS patients. Through targeting LATS1, miR-21 could prompt PCOS progression and could act as a novel non-invasive biomarker for diagnosis of PCOS.

Identification of downstream metastasis-associated target genes regulated by LSD1 in colon cancer cells.

PubMed

Chen, Jiang; Ding, Jie; Wang, Ziwei; Zhu, Jian; Wang, Xuejian; Du, Jiyi

2017-03-21

This study aims to identify downstream target genes regulated by lysine-specific demethylase 1 (LSD1) in colon cancer cells and investigate the molecular mechanisms of LSD1 influencing invasion and metastasis of colon cancer. We obtained the expression changes of downstream target genes regulated by small-interfering RNA-LSD1 and LSD1-overexpression via gene expression profiling in two human colon cancer cell lines. An Affymetrix Human Transcriptome Array 2.0 was used to identify differentially expressed genes (DEGs). We screened out LSD1-target gene associated with proliferation, metastasis, and invasion from DEGs via Gene Ontology and Pathway Studio. Subsequently, four key genes (CABYR, FOXF2, TLE4, and CDH1) were computationally predicted as metastasis-related LSD1-target genes. ChIp-PCR was applied after RT-PCR and Western blot validations to detect the occupancy of LSD1-target gene promoter-bound LSD1. A total of 3633 DEGs were significantly upregulated, and 4642 DEGs were downregulated in LSD1-silenced SW620 cells. A total of 4047 DEGs and 4240 DEGs were upregulated and downregulated in LSD1-overexpressed HT-29 cells, respectively. RT-PCR and Western blot validated the microarray analysis results. ChIP assay results demonstrated that LSD1 might be negative regulators for target genes CABYR and CDH1. The expression level of LSD1 is negatively correlated with mono- and dimethylation of histone H3 lysine4(H3K4) at LSD1- target gene promoter region. No significant mono-methylation and dimethylation of H3 lysine9 methylation was detected at the promoter region of CABYR and CDH1. LSD1- depletion contributed to the upregulation of CABYR and CDH1 through enhancing the dimethylation of H3K4 at the LSD1-target genes promoter. LSD1- overexpression mediated the downregulation of CABYR and CDH1expression through decreasing the mono- and dimethylation of H3K4 at LSD1-target gene promoter in colon cancer cells. CABYR and CDH1 might be potential LSD1-target genes in colon carcinogenesis.

Efficient generation and transportation of energetic electrons in a carbon nanotube array target

NASA Astrophysics Data System (ADS)

Ji, Yanling; Jiang, Gang; Wu, Weidong; Wang, Chaoyang; Gu, Yuqiu; Tang, Yongjian

2010-01-01

Laser-driven energetic electron propagation in a carbon nanotube-array target is investigated using two-dimensional particle-in-cell simulations. Energetic electrons are efficiently generated when the array is irradiated by a short intense laser pulse. Confined and guided transportation of energetic electrons in the array is achieved by exploiting strong transient electromagnetic fields created at the wall surfaces of nanotubes. The underlying mechanisms are discussed in detail. Our investigation shows that the laser energy can be transferred more effectively to the target electrons in the array than that of in the flat foil due to the hole structures in the array.

Chimeric antigen receptors with human scFvs preferentially induce T cell anti-tumor activity against tumors with high B7H6 expression.

PubMed

Gacerez, Albert T; Hua, Casey K; Ackerman, Margaret E; Sentman, Charles L

2018-05-01

B7H6 is emerging as a promising tumor antigen that is known to be expressed on a wide array of tumors and is reported to stimulate anti-tumor responses from the immune system. As such, B7H6 presents a good target for tumor-specific immunotherapies. B7H6-specific chimeric antigen receptors (CAR) based on a murine antibody showed successful targeting and elimination of tumors expressing B7H6. However, mouse single chain variable fragments (scFvs) have the potential to induce host anti-CAR responses that may limit efficacy, so human scFvs specific for B7H6 were selected by yeast surface display. In this study, we validate the functionality of these human scFvs when formatted into chimeric antigen receptors. The data indicate that T cells expressing these B7H6-specific human scFvs as CARs induced potent anti-tumor activity in vitro and in vivo against tumors expressing high amounts of B7H6. Importantly, these human scFv-based CARs are sensitive to changes in B7H6 expression which may potentially spare non-tumor cells that express B7H6 and provides the foundation for future clinical development.

The Epidermal Growth Factor Receptor Critically Regulates Endometrial Function during Early Pregnancy

PubMed Central

Large, Michael J.; Wetendorf, Margeaux; Lanz, Rainer B.; Hartig, Sean M.; Creighton, Chad J.; Mancini, Michael A.; Kovanci, Ertug; Lee, Kuo-Fen; Threadgill, David W.; Lydon, John P.; Jeong, Jae-Wook; DeMayo, Francesco J.

2014-01-01

Infertility and adverse gynecological outcomes such as preeclampsia and miscarriage represent significant female reproductive health concerns. The spatiotemporal expression of growth factors indicates that they play an important role in pregnancy. The goal of this study is to define the role of the ERBB family of growth factor receptors in endometrial function. Using conditional ablation in mice and siRNA in primary human endometrial stromal cells, we identified the epidermal growth factor receptor (Egfr) to be critical for endometrial function during early pregnancy. While ablation of Her2 or Erbb3 led to only a modest reduction in litter size, mice lacking Egfr expression are severely subfertile. Pregnancy demise occurred shortly after blastocyst implantation due to defects in decidualization including decreased proliferation, cell survival, differentiation and target gene expression. To place Egfr in a genetic regulatory hierarchy, transcriptome analyses was used to compare the gene signatures from mice with conditional ablation of Egfr, wingless-related MMTV integration site 4 (Wnt4) or boneless morphogenic protein 2 (Bmp2); revealing that not only are Bmp2 and Wnt4 key downstream effectors of Egfr, but they also regulate distinct physiological functions. In primary human endometrial stromal cells, marker gene expression, a novel high content image-based approach and phosphokinase array analysis were used to demonstrate that EGFR is a critical regulator of human decidualization. Furthermore, inhibition of EGFR signaling intermediaries WNK1 and AKT1S1, members identified in the kinase array and previously unreported to play a role in the endometrium, also attenuate decidualization. These results demonstrate that EGFR plays an integral role in establishing the cellular context necessary for successful pregnancy via the activation of intricate signaling and transcriptional networks, thereby providing valuable insight into potential therapeutic targets. PMID:24945252

Canine tumor cross-species genomics uncovers targets linked to osteosarcoma progression

PubMed Central

2009-01-01

Background Pulmonary metastasis continues to be the most common cause of death in osteosarcoma. Indeed, the 5-year survival for newly diagnosed osteosarcoma patients has not significantly changed in over 20 years. Further understanding of the mechanisms of metastasis and resistance for this aggressive pediatric cancer is necessary. Pet dogs naturally develop osteosarcoma providing a novel opportunity to model metastasis development and progression. Given the accelerated biology of canine osteosarcoma, we hypothesized that a direct comparison of canine and pediatric osteosarcoma expression profiles may help identify novel metastasis-associated tumor targets that have been missed through the study of the human cancer alone. Results Using parallel oligonucleotide array platforms, shared orthologues between species were identified and normalized. The osteosarcoma expression signatures could not distinguish the canine and human diseases by hierarchical clustering. Cross-species target mining identified two genes, interleukin-8 (IL-8) and solute carrier family 1 (glial high affinity glutamate transporter), member 3 (SLC1A3), which were uniformly expressed in dog but not in all pediatric osteosarcoma patient samples. Expression of these genes in an independent population of pediatric osteosarcoma patients was associated with poor outcome (p = 0.020 and p = 0.026, respectively). Validation of IL-8 and SLC1A3 protein expression in pediatric osteosarcoma tissues further supported the potential value of these novel targets. Ongoing evaluation will validate the biological significance of these targets and their associated pathways. Conclusions Collectively, these data support the strong similarities between human and canine osteosarcoma and underline the opportunities provided by a comparative oncology approach as a means to improve our understanding of cancer biology and therapies. PMID:20028558

Therapeutic Potential of Targeting PAK Signaling.

PubMed

Senapedis, William; Crochiere, Marsha; Baloglu, Erkan; Landesman, Yosef

2016-01-01

The therapeutic potential of targeting p21-Activated Kinases (PAK1 - 6) for the treatment of cancer has recently gained traction in the biotech industry. Many pharmaceutically-viable ATP competitive inhibitors have been through different stages of pre-clinical development with only a single compound evaluated in human trails (PF-3758309). The best studied functional roles of PAK proteins are control of cell adhesion and migration. PAK proteins are known downstream effectors of Ras signaling with PAK expression elevated in cancer (pancreatic, colon, breast, lung and other solid tumors). In addition altered PAK expression is a confirmed driver of this disease, especially in tumors harboring oncogenic Ras. However, there are very few examples of gain-of-function PAK mutations, as a majority of the cancer types have elevated PAK expression due to gene amplification or transcriptional modifications. There is a substantial number of known substrates affected by this aberrant PAK activity. One particular substrate, β-catenin, has garnered interest given its importance in both normal and cancer cell development. These data place PAK proteins between two major signaling pathways in cancer (Ras and β -catenin), making therapeutic targeting of PAKs an intriguing approach for the treatment of a broad array of oncological malignancies.

Novel Array-Based Target Identification for Synergistic Sensitization of Breast Cancer to Herceptin

DTIC Science & Technology

2010-05-01

Tatsuya Azum, Eileen Adamson, Ryan Alipio, Becky Pio, Frank Jones, Dan Mercola. Chip- on- chip analysis of mechanism of action of HER2 inhibition in...Munawar, Kutbuddin S. Doctor, Michael Birrer, Michael McClelland, Eileen Adamson, Dan Mercola. Egr1 regulates the coordinated expression of numerous...Kemal Korkmaz, Mashide Ohmichi, Eileen Adamson, Michael McClelland, Dan Mercola. Identification of genes bound and regulated by ATF2/c-Jun

MicroRNA-211-5p suppresses tumour cell proliferation, invasion, migration and metastasis in triple-negative breast cancer by directly targeting SETBP1.

PubMed

Chen, Liang-Liang; Zhang, Zhou-Jing; Yi, Zhan-Bo; Li, Jian-Jun

2017-06-27

Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancer in women globally. This subtype often has early and high recurrence rates, resulting in poor survival, partially due to lack of targeted therapies. To date, the detailed molecular mechanisms underlying TNBC progression are unclear. Given the crucial role of microRNAs (miRNAs) in cancer metastasis, we aimed to analyse the expression and function of a metastasis-associated miRNA named miR-211-5p in TNBC. MiRNA array analysis was performed to search for metastasis-associated miRNAs in TNBC. The miR-211-5p expression in tumour tissues, adjacent non-tumourous breast tissues of TNBC patients and cell lines were evaluated by real-time PCR. The protein expression levels were analysed by western blot, immunohistochemistry and in situ hybridisation. Luciferase reporter assays were employed to validate the target of miR-211-5p. The effect of miR-211-5p on TNBC progression was investigated in vitro and in vivo. MiR-211-5p was significantly downregulated in TNBC, and its expression level was associated with overall survival in TNBC. The expression of miR-211-5p suppressed TNBC cell proliferation, invasion, migration and metastasis in vitro and in vivo. Furthermore, SETBP1 was identified as a target of miR-211-5p. Through gain-of-function and loss-of-function studies, SETBP1 was shown to significantly affect colony and cell number in vitro. Enforced expression of miR-211-5p inhibited the expression of SETBP1 significantly and the restoration of SETBP1 expression reversed the inhibitory effects of miR-211-5p on TNBC cell proliferation and metastasis. These findings collectively demonstrate a tumour suppressor role of miR-211-5p in TNBC progression by targeting SETBP1, suggesting that miR-211-5p could serve as a potential prognostic biomarker and therapeutic target for TNBC.

Exon-Specific QTLs Skew the Inferred Distribution of Expression QTLs Detected Using Gene Expression Array Data

PubMed Central

Veyrieras, Jean-Baptiste; Gaffney, Daniel J.; Pickrell, Joseph K.; Gilad, Yoav; Stephens, Matthew; Pritchard, Jonathan K.

2012-01-01

Mapping of expression quantitative trait loci (eQTLs) is an important technique for studying how genetic variation affects gene regulation in natural populations. In a previous study using Illumina expression data from human lymphoblastoid cell lines, we reported that cis-eQTLs are especially enriched around transcription start sites (TSSs) and immediately upstream of transcription end sites (TESs). In this paper, we revisit the distribution of eQTLs using additional data from Affymetrix exon arrays and from RNA sequencing. We confirm that most eQTLs lie close to the target genes; that transcribed regions are generally enriched for eQTLs; that eQTLs are more abundant in exons than introns; and that the peak density of eQTLs occurs at the TSS. However, we find that the intriguing TES peak is greatly reduced or absent in the Affymetrix and RNA-seq data. Instead our data suggest that the TES peak observed in the Illumina data is mainly due to exon-specific QTLs that affect 3′ untranslated regions, where most of the Illumina probes are positioned. Nonetheless, we do observe an overall enrichment of eQTLs in exons versus introns in all three data sets, consistent with an important role for exonic sequences in gene regulation. PMID:22359548

Stress field forming of sector array transducers for vibro-acoustography.

PubMed

Silva, Glauber T; Chen, Shigao; Frery, Alejandro C; Greenleaf, James F; Fatemi, Mostafa

2005-11-01

This paper presents a study of the stress field forming of sector array transducers for vibro-acoustography applications. The system point-spread function (PSF) is given in terms of the dynamic radiation stress exerted on a point target by a dual ultrasound beam with slightly different frequencies. The radiation stress is calculated by assuming that the resulting ultrasound beam is a plane wave. The stress is proportional to the product of the velocity potential of each incident ultrasound beam. The beamforming and stress field forming of sector array transducers are analyzed through linear acoustics. An expression for the velocity potential produced by sector array transducers is derived. The vibro-acoustography PSF is evaluated numerically. A comparison between the PSF of a sector array and a confocal transducers is presented. The compared characteristics of the PSF are sidelobe levels, transverse, and in-depth spatial resolution. Indeed, one motivation to study sector transducers is the fact the depth-of-field of these transducers should be smaller than that of same size confocal transducers. An experimental setup was used to validate the theoretical PSF of sector array transducers. Results show that the measured PSF is in good agreement with the theoretical predications. Vibro-acoustography images of a breast-phantom by both transducers are presented and discussed.

USGS aerial resolution targets.

USGS Publications Warehouse

Salamonowicz, P.H.

1982-01-01

It is necessary to measure the achievable resolution of any airborne sensor that is to be used for metric purposes. Laboratory calibration facilities may be inadequate or inappropriate for determining the resolution of non-photographic sensors such as optical-mechanical scanners, television imaging tubes, and linear arrays. However, large target arrays imaged in the field can be used in testing such systems. The USGS has constructed an array of resolution targets in order to permit field testing of a variety of airborne sensing systems. The target array permits any interested organization with an airborne sensing system to accurately determine the operational resolution of its system. -from Author

Combining mouse mammary gland gene expression and comparative mapping for the identification of candidate genes for QTL of milk production traits in cattle

PubMed Central

Ron, Micha; Israeli, Galit; Seroussi, Eyal; Weller, Joel I; Gregg, Jeffrey P; Shani, Moshe; Medrano, Juan F

2007-01-01

Background Many studies have found segregating quantitative trait loci (QTL) for milk production traits in different dairy cattle populations. However, even for relatively large effects with a saturated marker map the confidence interval for QTL location by linkage analysis spans tens of map units, or hundreds of genes. Combining mapping and arraying has been suggested as an approach to identify candidate genes. Thus, gene expression analysis in the mammary gland of genes positioned in the confidence interval of the QTL can bridge the gap between fine mapping and quantitative trait nucleotide (QTN) determination. Results We hybridized Affymetrix microarray (MG-U74v2), containing 12,488 murine probes, with RNA derived from mammary gland of virgin, pregnant, lactating and involuting C57BL/6J mice in a total of nine biological replicates. We combined microarray data from two additional studies that used the same design in mice with a total of 75 biological replicates. The same filtering and normalization was applied to each microarray data using GeneSpring software. Analysis of variance identified 249 differentially expressed probe sets common to the three experiments along the four developmental stages of puberty, pregnancy, lactation and involution. 212 genes were assigned to their bovine map positions through comparative mapping, and thus form a list of candidate genes for previously identified QTLs for milk production traits. A total of 82 of the genes showed mammary gland-specific expression with at least 3-fold expression over the median representing all tissues tested in GeneAtlas. Conclusion This work presents a web tool for candidate genes for QTL (cgQTL) that allows navigation between the map of bovine milk production QTL, potential candidate genes and their level of expression in mammary gland arrays and in GeneAtlas. Three out of four confirmed genes that affect QTL in livestock (ABCG2, DGAT1, GDF8, IGF2) were over expressed in the target organ. Thus, cgQTL can be used to determine priority of candidate genes for QTN analysis based on differential expression in the target organ. PMID:17584498

EzArray: A web-based highly automated Affymetrix expression array data management and analysis system

PubMed Central

Zhu, Yuerong; Zhu, Yuelin; Xu, Wei

2008-01-01

Background Though microarray experiments are very popular in life science research, managing and analyzing microarray data are still challenging tasks for many biologists. Most microarray programs require users to have sophisticated knowledge of mathematics, statistics and computer skills for usage. With accumulating microarray data deposited in public databases, easy-to-use programs to re-analyze previously published microarray data are in high demand. Results EzArray is a web-based Affymetrix expression array data management and analysis system for researchers who need to organize microarray data efficiently and get data analyzed instantly. EzArray organizes microarray data into projects that can be analyzed online with predefined or custom procedures. EzArray performs data preprocessing and detection of differentially expressed genes with statistical methods. All analysis procedures are optimized and highly automated so that even novice users with limited pre-knowledge of microarray data analysis can complete initial analysis quickly. Since all input files, analysis parameters, and executed scripts can be downloaded, EzArray provides maximum reproducibility for each analysis. In addition, EzArray integrates with Gene Expression Omnibus (GEO) and allows instantaneous re-analysis of published array data. Conclusion EzArray is a novel Affymetrix expression array data analysis and sharing system. EzArray provides easy-to-use tools for re-analyzing published microarray data and will help both novice and experienced users perform initial analysis of their microarray data from the location of data storage. We believe EzArray will be a useful system for facilities with microarray services and laboratories with multiple members involved in microarray data analysis. EzArray is freely available from . PMID:18218103

Inhibition of IGF-1-Mediated Cellular Migration and Invasion by Migracin A in Ovarian Clear Cell Carcinoma Cells.

PubMed

Ukaji, Tamami; Lin, Yinzhi; Banno, Kouji; Okada, Shoshiro; Umezawa, Kazuo

2015-01-01

Previously we isolated migracin A from a Streptomyces culture filtrate as an inhibitor of cancer cell migration. In the present research, we found that migracin A inhibited migration and invasion of ovarian clear cell carcinoma ES-2 cells. In the course of our mechanistic study, migracin A was shown to enhance vasohibin-1 expression in an angiogenesis array. We also confirmed that it increased the mRNA expression of this protein. Moreover, overexpression of vasohibin-1 lowered the migration but not the invasion of ES-2 cells. Then, we looked for another target protein employing a motility array, and found that migracin A lowered the IGF-1 expression. Knockdown of IGF-1 by siRNA decreased the migration and invasion of ES-2 cells. Migracin A also decreased Akt phosphorylation involved in the downstream signaling. Crosstalk analysis indicated that overexpression of vasohibin-1 decreased the IGF-1 expression. On the other hand, it showed no direct anticancer activity in terms of the ES-2 growth in agar. Migracin A inhibited the migration and IGF-1 expression in not only ES-2 but also another ovarian clear cell carcinoma JHOC-5 cells. In addition, it also inhibited capillary tube formation of human umbilical vein endothelial cells. Since its cytotoxicity is very low, migracin A may be a candidate for an anti-metastasis agent not exhibiting prominent toxicity.

Small RNA-mediated regulation in bacteria: A growing palette of diverse mechanisms.

PubMed

Dutta, Tanmay; Srivastava, Shubhangi

2018-05-20

Small RNAs (sRNAs) in bacteria have evolved with diverse mechanisms to balance their target gene expression in response to changes in the environment. Accumulating studies on bacterial regulatory processes firmly established that sRNAs modulate their target gene expression generally at the posttranscriptional level. Identification of large number of sRNAs by advanced technologies, like deep sequencing, tilling microarray, indicates the existence of a plethora of distinctive sRNA-mediated regulatory mechanisms in bacteria. Types of the novel mechanisms are increasing with the discovery of new sRNAs. Complementary base pairing between sRNAs and target RNAs assisted by RNA chaperones like Hfq and ProQ, in many occasions, to regulate the cognate gene expression is prevalent in sRNA mechanisms. sRNAs, in most studied cases, can directly base pair with target mRNA to remodel its expression. Base pairing can happen either in the untranslated regions or in the coding regions of mRNA to activate/repress its translation. sRNAs also act as target mimic to titrate away different regulatory RNAs from its target. Other mechanism includes the sequestration of regulatory proteins, especially transcription factors, by sRNAs. Numerous sRNAs, following analogous mechanism, are widespread in bacteria, and thus, has drawn immense attention for the development of RNA-based technologies. Nevertheless, typical sRNA mechanisms are also discovered to be confined in some bacteria. Analysis of the sRNA mechanisms unravels their existence in both the single step processes and the complex regulatory networks with a global effect on cell physiology. This review deals with the diverse array of mechanisms, which sRNAs follow to maintain bacterial lifestyle. Copyright © 2018 Elsevier B.V. All rights reserved.

Deficiency in Aryl Hydrocarbon Receptor (AHR) Expression throughout Aging Alters Gene Expression Profiles in Murine Long-Term Hematopoietic Stem Cells

PubMed Central

Bennett, John A.; Singh, Kameshwar P.; Unnisa, Zeenath; Welle, Stephen L.; Gasiewicz, Thomas A.

2015-01-01

Dysregulation of hematopoietic stem cell (HSC) signaling can contribute to the development of diseases of the blood system. Lack of aryl hydrocarbon receptor (AhR) has been associated with alterations in gene expression related to HSC function and the subsequent development of a myeloproliferative disorder in aging female mice. We sorted the most primitive population of HSCs with the highest stem cell potential (Long-term, or LT-HSCs) from 18-month-old AhR-null-allele (AhR-KO) and WT mice and analyzed gene expression using microarray to determine alterations in gene expression and cell signaling networks in HSCs that could potentially contribute to the aging phenotype of AhR-KO mice. Comparisons with previous array data from 8-week old mice indicated that aging alone is sufficient to alter gene expression. In addition, a significant number of gene expression differences were observed in aged LT-HSCs that are dependent on both aging and lack of AhR. Pathway analysis of these genes revealed networks related to hematopoietic stem cell activity or function. qPCR was used to confirm the differential expression of a subset of these genes, focusing on genes that may represent novel AhR targets due to the presence of a putative AhR binding site in their upstream regulatory region. We verified differential expression of PDGF-D, Smo, Wdfy1, Zbtb37 and Zfp382. Pathway analysis of this subset of genes revealed overlap between cellular functions of the novel AhR targets and AhR itself. Lentiviral-mediated knockdown of AhR in lineage-negative hematopoietic cells was sufficient to induce changes in all five of the candidate AhR targets identified. Taken together, these data suggest a role for AhR in HSC functional regulation, and identify novel HSC AhR target genes that may contribute to the phenotypes observed in AhR-KO mice. PMID:26208102

Passive cavitation imaging with ultrasound arrays

PubMed Central

Salgaonkar, Vasant A.; Datta, Saurabh; Holland, Christy K.; Mast, T. Douglas

2009-01-01

A method is presented for passive imaging of cavitational acoustic emissions using an ultrasound array, with potential application in real-time monitoring of ultrasound ablation. To create such images, microbubble emissions were passively sensed by an imaging array and dynamically focused at multiple depths. In this paper, an analytic expression for a passive image is obtained by solving the Rayleigh–Sommerfield integral, under the Fresnel approximation, and passive images were simulated. A 192-element array was used to create passive images, in real time, from 520-kHz ultrasound scattered by a 1-mm steel wire. Azimuthal positions of this target were accurately estimated from the passive images. Next, stable and inertial cavitation was passively imaged in saline solution sonicated at 520 kHz. Bubble clusters formed in the saline samples were consistently located on both passive images and B-scans. Passive images were also created using broadband emissions from bovine liver sonicated at 2.2 MHz. Agreement was found between the images and source beam shape, indicating an ability to map therapeutic ultrasound beams in situ. The relation between these broadband emissions, sonication amplitude, and exposure conditions are discussed. PMID:20000921

Passive cavitation imaging with ultrasound arrays.

PubMed

Salgaonkar, Vasant A; Datta, Saurabh; Holland, Christy K; Mast, T Douglas

2009-12-01

A method is presented for passive imaging of cavitational acoustic emissions using an ultrasound array, with potential application in real-time monitoring of ultrasound ablation. To create such images, microbubble emissions were passively sensed by an imaging array and dynamically focused at multiple depths. In this paper, an analytic expression for a passive image is obtained by solving the Rayleigh-Sommerfield integral, under the Fresnel approximation, and passive images were simulated. A 192-element array was used to create passive images, in real time, from 520-kHz ultrasound scattered by a 1-mm steel wire. Azimuthal positions of this target were accurately estimated from the passive images. Next, stable and inertial cavitation was passively imaged in saline solution sonicated at 520 kHz. Bubble clusters formed in the saline samples were consistently located on both passive images and B-scans. Passive images were also created using broadband emissions from bovine liver sonicated at 2.2 MHz. Agreement was found between the images and source beam shape, indicating an ability to map therapeutic ultrasound beams in situ. The relation between these broadband emissions, sonication amplitude, and exposure conditions are discussed.

Light-Inducible Gene Regulation with Engineered Zinc Finger Proteins

PubMed Central

Polstein, Lauren R.; Gersbach, Charles A.

2014-01-01

The coupling of light-inducible protein-protein interactions with gene regulation systems has enabled the control of gene expression with light. In particular, heterodimer protein pairs from plants can be used to engineer a gene regulation system in mammalian cells that is reversible, repeatable, tunable, controllable in a spatiotemporal manner, and targetable to any DNA sequence. This system, Light-Inducible Transcription using Engineered Zinc finger proteins (LITEZ), is based on the blue light-induced interaction of GIGANTEA and the LOV domain of FKF1 that drives the localization of a transcriptional activator to the DNA-binding site of a highly customizable engineered zinc finger protein. This chapter provides methods for modifying LITEZ to target new DNA sequences, engineering a programmable LED array to illuminate cell cultures, and using the modified LITEZ system to achieve spatiotemporal control of transgene expression in mammalian cells. PMID:24718797

HES1, a target of Notch signaling, is elevated in canine osteosarcoma, but reduced in the most aggressive tumors.

PubMed

Dailey, Deanna D; Anfinsen, Kristin P; Pfaff, Liza E; Ehrhart, E J; Charles, J Brad; Bønsdorff, Tina B; Thamm, Douglas H; Powers, Barbara E; Jonasdottir, Thora J; Duval, Dawn L

2013-07-01

Hairy and enhancer of split 1 (HES1), a basic helix-loop-helix transcriptional repressor, is a downstream target of Notch signaling. Notch signaling and HES1 expression have been linked to growth and survival in a variety of human cancer types and have been associated with increased metastasis and invasiveness in human osteosarcoma cell lines. Osteosarcoma (OSA) is an aggressive cancer demonstrating both high metastatic rate and chemotherapeutic resistance. The current study examined expression of Notch signaling mediators in primary canine OSA tumors and canine and human osteosarcoma cell lines to assess their role in OSA development and progression. Reverse transcriptase - quantitative PCR (RT-qPCR) was utilized to quantify HES1, HEY1, NOTCH1 and NOTCH2 gene expression in matched tumor and normal metaphyseal bone samples taken from dogs treated for appendicular OSA at the Colorado State University Veterinary Teaching Hospital. Gene expression was also assessed in tumors from dogs with a disease free interval (DFI) of <100 days compared to those with a DFI > 300 days following treatment with surgical amputation followed by standard chemotherapy. Immunohistochemistry was performed to confirm expression of HES1. Data from RT-qPCR and immunohistochemical (IHC) experiments were analyzed using REST2009 software and survival analysis based on IHC expression employed the Kaplan-Meier method and log rank analysis. Unbiased clustered images were generated from gene array analysis data for Notch/HES1 associated genes. Gene array analysis of Notch/HES1 associated genes suggested alterations in the Notch signaling pathway may contribute to the development of canine OSA. HES1 mRNA expression was elevated in tumor samples relative to normal bone, but decreased in tumor samples from dogs with a DFI < 100 days relative to those with a DFI > 300 days. NOTCH2 and HEY1 mRNA expression was also elevated in tumors relative to normal bone, but was not differentially expressed between the DFI tumor groups. Survival analysis confirmed an association between decreased HES1 immunosignal and shorter DFI. Our findings suggest that activation of Notch signaling occurs and may contribute to the development of canine OSA. However, association of low HES1 expression and shorter DFI suggests that mechanisms that do not alter HES1 expression may drive the most aggressive tumors.

Experimental Demonstration of Adaptive Infrared Multispectral Imaging using Plasmonic Filter Array.

PubMed

Jang, Woo-Yong; Ku, Zahyun; Jeon, Jiyeon; Kim, Jun Oh; Lee, Sang Jun; Park, James; Noyola, Michael J; Urbas, Augustine

2016-10-10

In our previous theoretical study, we performed target detection using a plasmonic sensor array incorporating the data-processing technique termed "algorithmic spectrometry". We achieved the reconstruction of a target spectrum by extracting intensity at multiple wavelengths with high resolution from the image data obtained from the plasmonic array. The ultimate goal is to develop a full-scale focal plane array with a plasmonic opto-coupler in order to move towards the next generation of versatile infrared cameras. To this end, and as an intermediate step, this paper reports the experimental demonstration of adaptive multispectral imagery using fabricated plasmonic spectral filter arrays and proposed target detection scenarios. Each plasmonic filter was designed using periodic circular holes perforated through a gold layer, and an enhanced target detection strategy was proposed to refine the original spectrometry concept for spatial and spectral computation of the data measured from the plasmonic array. Both the spectrum of blackbody radiation and a metal ring object at multiple wavelengths were successfully reconstructed using the weighted superposition of plasmonic output images as specified in the proposed detection strategy. In addition, plasmonic filter arrays were theoretically tested on a target at extremely high temperature as a challenging scenario for the detection scheme.

MALDI-TOF mass spectrometry for quantitative gene expression analysis of acid responses in Staphylococcus aureus.

PubMed

Rode, Tone Mari; Berget, Ingunn; Langsrud, Solveig; Møretrø, Trond; Holck, Askild

2009-07-01

Microorganisms are constantly exposed to new and altered growth conditions, and respond by changing gene expression patterns. Several methods for studying gene expression exist. During the last decade, the analysis of microarrays has been one of the most common approaches applied for large scale gene expression studies. A relatively new method for gene expression analysis is MassARRAY, which combines real competitive-PCR and MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry. In contrast to microarray methods, MassARRAY technology is suitable for analysing a larger number of samples, though for a smaller set of genes. In this study we compare the results from MassARRAY with microarrays on gene expression responses of Staphylococcus aureus exposed to acid stress at pH 4.5. RNA isolated from the same stress experiments was analysed using both the MassARRAY and the microarray methods. The MassARRAY and microarray methods showed good correlation. Both MassARRAY and microarray estimated somewhat lower fold changes compared with quantitative real-time PCR (qRT-PCR). The results confirmed the up-regulation of the urease genes in acidic environments, and also indicated the importance of metal ion regulation. This study shows that the MassARRAY technology is suitable for gene expression analysis in prokaryotes, and has advantages when a set of genes is being analysed for an organism exposed to many different environmental conditions.

Target tracking and pointing for arrays of phase-locked lasers

NASA Astrophysics Data System (ADS)

Macasaet, Van P.; Hughes, Gary B.; Lubin, Philip; Madajian, Jonathan; Zhang, Qicheng; Griswold, Janelle; Kulkarni, Neeraj; Cohen, Alexander; Brashears, Travis

2016-09-01

Arrays of phase-locked lasers are envisioned for planetary defense and exploration systems. High-energy beams focused on a threatening asteroid evaporate surface material, creating a reactionary thrust that alters the asteroid's orbit. The same system could be used to probe an asteroid's composition, to search for unknown asteroids, and to propel interplanetary and interstellar spacecraft. Phased-array designs are capable of producing high beam intensity, and allow beam steering and beam profile manipulation. Modular designs allow ongoing addition of emitter elements to a growing array. This paper discusses pointing control for extensible laser arrays. Rough pointing is determined by spacecraft attitude control. Lateral movement of the laser emitter tips behind the optical elements provides intermediate pointing adjustment for individual array elements and beam steering. Precision beam steering and beam formation is accomplished by coordinated phase modulation across the array. Added cells are incorporated into the phase control scheme by precise alignment to local mechanical datums using fast, optical relative position sensors. Infrared target sensors are also positioned within the datum scheme, and provide information about the target vector relative to datum coordinates at each emitter. Multiple target sensors allow refined determination of the target normal plane, providing information to the phase controller for each emitter. As emitters and sensors are added, local position data allows accurate prediction of the relative global position of emitters across the array, providing additional constraints to the phase controllers. Mechanical design and associated phase control that is scalable for target distance and number of emitters is presented.

Quantitative Expression and Co-Localization of Wnt Signalling Related Proteins in Feline Squamous Cell Carcinoma

PubMed Central

Marote, Georgina; Abramo, Francesca; McKay, Jenny; Thomson, Calum; Beltran, Mariana; Millar, Michael; Priestnall, Simon; Dobson, Jane; Costantino-Casas, Fernando; Petrou, Terry; McGonnell, Imelda M.; Davies, Anthony J.; Weetman, Malcolm; Garden, Oliver A.; Masters, John R.; Thrasivoulou, Christopher; Ahmed, Aamir

2016-01-01

Feline oral squamous cell carcinoma (FOSCC) is an aggressive neoplasm in cats. Little is known about the possible molecular mechanisms that may be involved in the initiation, maintenance and progression of FOSCC. Wnt signalling is critical in development and disease, including many mammalian cancers. In this study, we have investigated the expression of Wnt signalling related proteins using quantitative immunohistochemical techniques on tissue arrays. We constructed tissue arrays with 58 individual replicate tissue samples. We tested for the expression of four key Wnt/ß-catenin transcription targets, namely Cyclin D1 (CCND1 or CD1), FRA1, c-Myc and MMP7. All antibodies showed cross reactivity in feline tissue except MMP7. Quantitative immunohistochemical analysis of single proteins (expressed as area fraction / amount of tissue for normal vs tumor, mean ± SE) showed that the expression of CD1 (3.9 ± 0.5 vs 12.2 ± 0.9), FRA1 (5.5 ± 0.6 vs 16.8 ± 1.1) and c-Myc (5.4 ± 0.5 vs 12.5 ± 0.9) was increased in FOSCC tissue by 2.3 to 3 fold compared to normal controls (p<0.0001). By using a multilabel, quantitative fluorophore technique we further investigated if the co-localization of these proteins (all transcription factors) with each other and in the nucleus (stained with 4',6-diamidino-2-phenylindole, DAPI) was altered in FOSCC compared to normal tissue. The global intersection coefficients, a measure of the proximity of two fluorophore labeled entities, showed that there was a significant change (p < 0.01) in the co-localization for all permutations (e.g. CD1/FRA1 etc), except for the nuclear localization of CD1. Our results show that putative targets of Wnt signalling transcription are up-regulated in FOSCC with alterations in the co-localization of these proteins and could serve as a useful marker for the disease. PMID:27559731

Insights into the Sigma-1 receptor chaperone’s cellular functions: a microarray report

PubMed Central

Tsai, Shang-Yi; Rothman, Richard Kyle; Su, Tsung-Ping

2013-01-01

We previously demonstrated that Sig-1Rs are critical regulators in neuronal morphogenesis and development via the regulation of oxidative stress and mitochondrial functions. In the present study, we sought to identify pathways and genes that are affected by Sig-1R. Gene expression profiles were examined in rat hippocampal neurons that had been cultured for18 days in vitro (DIV). The cells were transduced with AAV siRNA targeting Sig-1R on DIV 10 for 7 days, followed by gene expression analysis using a rat genome cDNA array. The gene array results indicated that Sig-1R knockdown hampered cellular functions including steroid biogenesis, protein ubiquitination, actin cytoskeleton network, and Nrf-2 mediated oxidative stress. Many of the cellular components important for actin polymerization and synapse plasticity, including F-actin capping protein and neurofilaments, were significantly changed in AAV-siSig-1R neurons. Further, cytochrome c was reduced in AAV-Sig-1R neurons whereas free-radical generating enzymes including cytochrome p450 and cytochrome b-245 were increased. The microarray results also suggest that Sig-1Rs may regulate genes that are involved in the pathogenesis of many CNS diseases including Alzheimer’s disease and Parkinson’s disease. These data further confirmed that Sig-1Rs play critical roles in the CNS and thus these findings may aid in future development of therapeutic treatments targeting neurodegenerative disorders. PMID:21905129

A Customized Quantitative PCR MicroRNA Panel Provides a Technically Robust Context for Studying Neurodegenerative Disease Biomarkers and Indicates a High Correlation Between Cerebrospinal Fluid and Choroid Plexus MicroRNA Expression.

PubMed

Wang, Wang-Xia; Fardo, David W; Jicha, Gregory A; Nelson, Peter T

2017-12-01

MicroRNA (miRNA) expression varies in association with different tissue types and in diseases. Having been found in body fluids including blood and cerebrospinal fluid (CSF), miRNAs constitute potential biomarkers. CSF miRNAs have been proposed as biomarkers for neurodegenerative diseases; however, there is a lack of consensus about the best candidate miRNA biomarkers and there has been variability in results from different research centers, perhaps due to technical factors. Here, we sought to optimize technical parameters for CSF miRNA studies. We examined different RNA isolation methods and performed miRNA expression profiling with TaqMan® miRNA Arrays. More specifically, we developed a customized CSF-miRNA low-density array (TLDA) panel that contains 47 targets: miRNAs shown previously to be relevant to neurodegenerative disease, miRNAs that are abundant in CSF, data normalizers, and controls for potential blood and tissue contamination. The advantages of using this CSF-miRNA TLDA panel include specificity, sensitivity, fast processing and data analysis, and cost effectiveness. We optimized technical parameters for this assay. Further, the TLDA panel can be tailored to other specific purposes. We tested whether the profile of miRNAs in the CSF resembled miRNAs isolated from brain tissue (hippocampus or cerebellum), blood, or the choroid plexus. We found that the CSF miRNA expression profile most closely resembles that of choroid plexus tissue, underscoring the potential importance of choroid plexus-derived signaling through CSF miRNAs. In summary, the TLDA miRNA array panel will enable evaluation and discovery of CSF miRNA biomarkers and can potentially be utilized in clinical diagnosis and disease stage monitoring.

High-density fiber optic biosensor arrays

NASA Astrophysics Data System (ADS)

Epstein, Jason R.; Walt, David R.

2002-02-01

Novel approaches are required to coordinate the immense amounts of information derived from diverse genomes. This concept has influenced the expanded role of high-throughput DNA detection and analysis in the biological sciences. A high-density fiber optic DNA biosensor was developed consisting of oligonucleotide-functionalized, 3.1 mm diameter microspheres deposited into the etched wells on the distal face of a 500 micrometers imaging fiber bundle. Imaging fiber bundles containing thousands of optical fibers, each associated with a unique oligonucleotide probe sequence, were the foundation for an optically connected, individually addressable DNA detection platform. Different oligonucleotide-functionalized microspheres were combined in a stock solution, and randomly dispersed into the etched wells. Microsphere positions were registered from optical dyes incorporated onto the microspheres. The distribution process provided an inherent redundancy that increases the signal-to-noise ratio as the square root of the number of sensors examined. The representative amount of each probe-type in the array was dependent on their initial stock solution concentration, and as other sequences of interest arise, new microsphere elements can be added to arrays without altering the existing detection capabilities. The oligonucleotide probe sequences hybridize to fluorescently-labeled, complementary DNA target solutions. Fiber optic DNA microarray research has included DNA-protein interaction profiles, microbial strain differentiation, non-labeled target interrogation with molecular beacons, and single cell-based assays. This biosensor array is proficient in DNA detection linked to specific disease states, single nucleotide polymorphism (SNP's) discrimination, and gene expression analysis. This array platform permits multiple detection formats, provides smaller feature sizes, and enables sensor design flexibility. High-density fiber optic microarray biosensors provide a fast, reversible format with the detection limit of a few hundred molecules.

ArrayExpress update--trends in database growth and links to data analysis tools.

PubMed

Rustici, Gabriella; Kolesnikov, Nikolay; Brandizi, Marco; Burdett, Tony; Dylag, Miroslaw; Emam, Ibrahim; Farne, Anna; Hastings, Emma; Ison, Jon; Keays, Maria; Kurbatova, Natalja; Malone, James; Mani, Roby; Mupo, Annalisa; Pedro Pereira, Rui; Pilicheva, Ekaterina; Rung, Johan; Sharma, Anjan; Tang, Y Amy; Ternent, Tobias; Tikhonov, Andrew; Welter, Danielle; Williams, Eleanor; Brazma, Alvis; Parkinson, Helen; Sarkans, Ugis

2013-01-01

The ArrayExpress Archive of Functional Genomics Data (http://www.ebi.ac.uk/arrayexpress) is one of three international functional genomics public data repositories, alongside the Gene Expression Omnibus at NCBI and the DDBJ Omics Archive, supporting peer-reviewed publications. It accepts data generated by sequencing or array-based technologies and currently contains data from almost a million assays, from over 30 000 experiments. The proportion of sequencing-based submissions has grown significantly over the last 2 years and has reached, in 2012, 15% of all new data. All data are available from ArrayExpress in MAGE-TAB format, which allows robust linking to data analysis and visualization tools, including Bioconductor and GenomeSpace. Additionally, R objects, for microarray data, and binary alignment format files, for sequencing data, have been generated for a significant proportion of ArrayExpress data.

An integrated molecular analysis of lung adenocarcinomas identifies potential therapeutic targets among TTF1-negative tumors including DNA repair proteins and Nrf2

PubMed Central

Cardnell, Robert J.G.; Behrens, Carmen; Diao, Lixia; Fan, YouHong; Tang, Ximing; Tong, Pan; John D., Minna; Mills, Gordon B.; Heymach, John V.; Wistuba, Ignacio I.; Wang, Jing; Byers., Lauren A.

2015-01-01

Purpose Thyroid transcription factor-1 (TTF1) immunohistochemistry (IHC) is used clinically to differentiate primary lung adenocarcinomas (LUAD) from squamous lung cancers and metastatic adenocarcinomas from other primary sites. However, a subset of LUAD (15-20%) does not express TTF1 and TTF1-negative patients have worse clinical outcomes. As there are no established targeted agents with activity in TTF1-negative LUAD, we performed an integrated molecular analysis to identify potential therapeutic targets. Experimental Design Using two clinical LUAD cohorts (274 tumors), one from our institution (PROSPECT) and the TCGA, we interrogated proteomic profiles (by reverse-phase protein array (RPPA)), gene expression, and mutational data. Drug response data from 74 cell lines were used to validate potential therapeutic agents. Results Strong correlations were observed between TTF1 IHC and TTF1 measurements by RPPA (Rho=0.57, p<0.001) and gene expression (NKX2-1, Rho=0.61, p<0.001). Established driver mutations (e.g. BRAF and EGFR) were associated with high TTF1 expression. In contrast, TTF1-negative LUAD had a higher frequency of inactivating KEAP1 mutations (p=0.001). Proteomic profiling identified increased expression of DNA repair proteins (e.g., Chk1 and the DNA repair score) and suppressed PI3K/MAPK signaling among TTF1-negative tumors, with differences in total proteins confirmed at the mRNA level. Cell line analysis showed drugs targeting DNA repair to be more active in TTF1-low cell lines. Conclusions Combined genomic and proteomic analyses demonstrated infrequent alteration of validated lung cancer targets (including the absence of BRAF mutations in TTF1-negative LUAD), but identified novel potential targets for TTF1-negative LUAD includingKEAP1/Nrf2 and DNA repair pathways. PMID:25878335

Genome editing in Kluyveromyces and Ogataea yeasts using a broad-host-range Cas9/gRNA co-expression plasmid.

PubMed

Juergens, Hannes; Varela, Javier A; Gorter de Vries, Arthur R; Perli, Thomas; Gast, Veronica J M; Gyurchev, Nikola Y; Rajkumar, Arun S; Mans, Robert; Pronk, Jack T; Morrissey, John P; Daran, Jean-Marc G

2018-05-01

While CRISPR-Cas9-mediated genome editing has transformed yeast research, current plasmids and cassettes for Cas9 and guide-RNA expression are species specific. CRISPR tools that function in multiple yeast species could contribute to the intensifying research on non-conventional yeasts. A plasmid carrying a pangenomic origin of replication and two constitutive expression cassettes for Cas9 and ribozyme-flanked gRNAs was constructed. Its functionality was tested by analyzing inactivation of the ADE2 gene in four yeast species. In two Kluyveromyces species, near-perfect targeting (≥96%) and homologous repair (HR) were observed in at least 24% of transformants. In two Ogataea species, Ade- mutants were not observed directly after transformation, but prolonged incubation of transformed cells resulted in targeting efficiencies of 9% to 63% mediated by non-homologous end joining (NHEJ). In an Ogataea parapolymorpha ku80 mutant, deletion of OpADE2 mediated by HR was achieved, albeit at low efficiencies (<1%). Furthermore the expression of a dual polycistronic gRNA array enabled simultaneous interruption of OpADE2 and OpYNR1 demonstrating flexibility of ribozyme-flanked gRNA design for multiplexing. While prevalence of NHEJ prevented HR-mediated editing in Ogataea, such targeted editing was possible in Kluyveromyces. This broad-host-range CRISPR/gRNA system may contribute to exploration of Cas9-mediated genome editing in other Saccharomycotina yeasts.

BeadArray Expression Analysis Using Bioconductor

PubMed Central

Ritchie, Matthew E.; Dunning, Mark J.; Smith, Mike L.; Shi, Wei; Lynch, Andy G.

2011-01-01

Illumina whole-genome expression BeadArrays are a popular choice in gene profiling studies. Aside from the vendor-provided software tools for analyzing BeadArray expression data (GenomeStudio/BeadStudio), there exists a comprehensive set of open-source analysis tools in the Bioconductor project, many of which have been tailored to exploit the unique properties of this platform. In this article, we explore a number of these software packages and demonstrate how to perform a complete analysis of BeadArray data in various formats. The key steps of importing data, performing quality assessments, preprocessing, and annotation in the common setting of assessing differential expression in designed experiments will be covered. PMID:22144879

Biodistribution mechanisms of therapeutic monoclonal antibodies in health and disease.

PubMed

Tabrizi, Mohammad; Bornstein, Gadi Gazit; Suria, Hamza

2010-03-01

The monoclonal antibody market continues to witness an impressive rate of growth and has become the leading source of expansion in the biologic segment within the pharmaceutical industry. Currently marketed monoclonal antibodies target a diverse array of antigens. These antigens are distributed in a variety of tissues such as tumors, lungs, synovial fluid, psoriatic plaques, and lymph nodes. As the concentration of drug at the proximity of the biological receptor determines the magnitude of the observed pharmacological responses, a significant consideration in effective therapeutic application of monoclonal antibodies is a thorough understanding of the processes that regulate antibody biodistribution. Monoclonal antibody distribution is affected by factors such as molecular weight, blood flow, tissue and tumor heterogeneity, structure and porosity, target antigen density, turnover rate, and the target antigen expression profile.

Downregulation of SS18-SSX1 expression in synovial sarcoma by small interfering RNA enhances the focal adhesion pathway and inhibits anchorage-independent growth in vitro and tumor growth in vivo.

PubMed

Takenaka, Satoshi; Naka, Norifumi; Araki, Nobuhito; Hashimoto, Nobuyuki; Ueda, Takafumi; Yoshioka, Kiyoko; Yoshikawa, Hideki; Itoh, Kazuyuki

2010-04-01

Synovial sarcoma (SS) is an aggressive soft-tissue malignancy characterized by a unique t(X;18) translocation resulting in expression of SS18-SSX fusion protein. In order to investigate the biological function of this fusion protein and to develop a novel therapeutic option, we examined downregulation of SS18-SSX1 expression by small interfering RNA targeting SS18-SSX1 in three human SS cell lines. Microarray analysis comparing SS18-SSX1-silenced cells with control cells in three SS cell lines showed that SS18-SSX1 mainly affected the focal adhesion pathway. In accord with the array data, silencing of SS18-SSX1 enhances adhesion to the extracellular matrix through the induction of expression of myosin light-chain kinase. Furthermore, the silencing of SS18-SSX1 inhibits anchorage-independent growth in vitro and systemic delivery of siRNA against SS18-SSX1 using a nanoparticle system inhibited tumor growth in a nude mouse xenograft model. Our results demonstrate that siRNA targeting of SS18-SSX1 has therapeutic potential for the treatment of SS.

Wongabel rhabdovirus accessory protein U3 targets the SWI/SNF chromatin remodeling complex.

PubMed

Joubert, D Albert; Rodriguez-Andres, Julio; Monaghan, Paul; Cummins, Michelle; McKinstry, William J; Paradkar, Prasad N; Moseley, Gregory W; Walker, Peter J

2015-01-15

Wongabel virus (WONV) is an arthropod-borne rhabdovirus that infects birds. It is one of the growing array of rhabdoviruses with complex genomes that encode multiple accessory proteins of unknown function. In addition to the five canonical rhabdovirus structural protein genes (N, P, M, G, and L), the 13.2-kb negative-sense single-stranded RNA (ssRNA) WONV genome contains five uncharacterized accessory genes, one overlapping the N gene (Nx or U4), three located between the P and M genes (U1 to U3), and a fifth one overlapping the G gene (Gx or U5). Here we show that WONV U3 is expressed during infection in insect and mammalian cells and is required for efficient viral replication. A yeast two-hybrid screen against a mosquito cell cDNA library identified that WONV U3 interacts with the 83-amino-acid (aa) C-terminal domain of SNF5, a component of the SWI/SNF chromatin remodeling complex. The interaction was confirmed by affinity chromatography, and nuclear colocalization was established by confocal microscopy. Gene expression studies showed that SNF5 transcripts are upregulated during infection of mosquito cells with WONV, as well as West Nile virus (Flaviviridae) and bovine ephemeral fever virus (Rhabdoviridae), and that SNF5 knockdown results in increased WONV replication. WONV U3 also inhibits SNF5-regulated expression of the cytokine gene CSF1. The data suggest that WONV U3 targets the SWI/SNF complex to block the host response to infection. The rhabdoviruses comprise a large family of RNA viruses infecting plants, vertebrates, and invertebrates. In addition to the major structural proteins (N, P, M, G, and L), many rhabdoviruses encode a diverse array of accessory proteins of largely unknown function. Understanding the role of these proteins may reveal much about host-pathogen interactions in infected cells. Here we examine accessory protein U3 of Wongabel virus, an arthropod-borne rhabdovirus that infects birds. We show that U3 enters the nucleus and interacts with SNF5, a component of the chromatin remodeling complex that is upregulated in response to infection and restricts viral replication. We also show that U3 inhibits SNF5-regulated expression of the cytokine colony-stimulating factor 1 (CSF1), suggesting that it targets the chromatin remodeling complex to block the host response to infection. This study appears to provide the first evidence of a virus targeting SNF5 to inhibit host gene expression. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Wongabel Rhabdovirus Accessory Protein U3 Targets the SWI/SNF Chromatin Remodeling Complex

PubMed Central

Joubert, D. Albert; Rodriguez-Andres, Julio; Monaghan, Paul; Cummins, Michelle; McKinstry, William J.; Paradkar, Prasad N.; Moseley, Gregory W.

2014-01-01

ABSTRACT Wongabel virus (WONV) is an arthropod-borne rhabdovirus that infects birds. It is one of the growing array of rhabdoviruses with complex genomes that encode multiple accessory proteins of unknown function. In addition to the five canonical rhabdovirus structural protein genes (N, P, M, G, and L), the 13.2-kb negative-sense single-stranded RNA (ssRNA) WONV genome contains five uncharacterized accessory genes, one overlapping the N gene (Nx or U4), three located between the P and M genes (U1 to U3), and a fifth one overlapping the G gene (Gx or U5). Here we show that WONV U3 is expressed during infection in insect and mammalian cells and is required for efficient viral replication. A yeast two-hybrid screen against a mosquito cell cDNA library identified that WONV U3 interacts with the 83-amino-acid (aa) C-terminal domain of SNF5, a component of the SWI/SNF chromatin remodeling complex. The interaction was confirmed by affinity chromatography, and nuclear colocalization was established by confocal microscopy. Gene expression studies showed that SNF5 transcripts are upregulated during infection of mosquito cells with WONV, as well as West Nile virus (Flaviviridae) and bovine ephemeral fever virus (Rhabdoviridae), and that SNF5 knockdown results in increased WONV replication. WONV U3 also inhibits SNF5-regulated expression of the cytokine gene CSF1. The data suggest that WONV U3 targets the SWI/SNF complex to block the host response to infection. IMPORTANCE The rhabdoviruses comprise a large family of RNA viruses infecting plants, vertebrates, and invertebrates. In addition to the major structural proteins (N, P, M, G, and L), many rhabdoviruses encode a diverse array of accessory proteins of largely unknown function. Understanding the role of these proteins may reveal much about host-pathogen interactions in infected cells. Here we examine accessory protein U3 of Wongabel virus, an arthropod-borne rhabdovirus that infects birds. We show that U3 enters the nucleus and interacts with SNF5, a component of the chromatin remodeling complex that is upregulated in response to infection and restricts viral replication. We also show that U3 inhibits SNF5-regulated expression of the cytokine colony-stimulating factor 1 (CSF1), suggesting that it targets the chromatin remodeling complex to block the host response to infection. This study appears to provide the first evidence of a virus targeting SNF5 to inhibit host gene expression. PMID:25392228

Experimental Demonstration of Adaptive Infrared Multispectral Imaging using Plasmonic Filter Array

PubMed Central

Jang, Woo-Yong; Ku, Zahyun; Jeon, Jiyeon; Kim, Jun Oh; Lee, Sang Jun; Park, James; Noyola, Michael J.; Urbas, Augustine

2016-01-01

In our previous theoretical study, we performed target detection using a plasmonic sensor array incorporating the data-processing technique termed “algorithmic spectrometry”. We achieved the reconstruction of a target spectrum by extracting intensity at multiple wavelengths with high resolution from the image data obtained from the plasmonic array. The ultimate goal is to develop a full-scale focal plane array with a plasmonic opto-coupler in order to move towards the next generation of versatile infrared cameras. To this end, and as an intermediate step, this paper reports the experimental demonstration of adaptive multispectral imagery using fabricated plasmonic spectral filter arrays and proposed target detection scenarios. Each plasmonic filter was designed using periodic circular holes perforated through a gold layer, and an enhanced target detection strategy was proposed to refine the original spectrometry concept for spatial and spectral computation of the data measured from the plasmonic array. Both the spectrum of blackbody radiation and a metal ring object at multiple wavelengths were successfully reconstructed using the weighted superposition of plasmonic output images as specified in the proposed detection strategy. In addition, plasmonic filter arrays were theoretically tested on a target at extremely high temperature as a challenging scenario for the detection scheme. PMID:27721506

A functional gene array for detection of bacterial virulence elements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jaing, C

2007-11-01

We report our development of the first of a series of microarrays designed to detect pathogens with known mechanisms of virulence and antibiotic resistance. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples. To validate our approach, we developed a first generation array targeting genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for microorganism detection and discrimination, measured the required target concentration, and assessedmore » tolerance for mismatches between probe and target sequences. Mismatch tolerance is a priority for this application, due to DNA sequence variability among members of gene families. Arrays were created using the NimbleGen Maskless Array Synthesizer at Lawrence Livermore National Laboratory. Purified genomic DNA from combinations of one or more of the four target organisms, pure cultures of four related organisms, and environmental aerosol samples with spiked-in genomic DNA were hybridized to the arrays. Based on the success of this prototype, we plan to design further arrays in this series, with the goal of detecting all known virulence and antibiotic resistance gene families in a greatly expanded set of organisms.« less

STAT3 Target Genes Relevant to Human Cancers

PubMed Central

Carpenter, Richard L.; Lo, Hui-Wen

2014-01-01

Since its discovery, the STAT3 transcription factor has been extensively studied for its function as a transcriptional regulator and its role as a mediator of development, normal physiology, and pathology of many diseases, including cancers. These efforts have uncovered an array of genes that can be positively and negatively regulated by STAT3, alone and in cooperation with other transcription factors. Through regulating gene expression, STAT3 has been demonstrated to play a pivotal role in many cellular processes including oncogenesis, tumor growth and progression, and stemness. Interestingly, recent studies suggest that STAT3 may behave as a tumor suppressor by activating expression of genes known to inhibit tumorigenesis. Additional evidence suggested that STAT3 may elicit opposing effects depending on cellular context and tumor types. These mixed results signify the need for a deeper understanding of STAT3, including its upstream regulators, parallel transcription co-regulators, and downstream target genes. To help facilitate fulfilling this unmet need, this review will be primarily focused on STAT3 downstream target genes that have been validated to associate with tumorigenesis and/or malignant biology of human cancers. PMID:24743777

Standard, Random, and Optimum Array conversions from Two-Pole resistance data

DOE PAGES

Rucker, D. F.; Glaser, Danney R.

2014-09-01

We present an array evaluation of standard and nonstandard arrays over a hydrogeological target. We develop the arrays by linearly combining data from the pole-pole (or 2-pole) array. The first test shows that reconstructed resistances for the standard Schlumberger and dipoledipole arrays are equivalent or superior to the measured arrays in terms of noise, especially at large geometric factors. The inverse models for the standard arrays also confirm what others have presented in terms of target resolvability, namely the dipole-dipole array has the highest resolution. In the second test, we reconstruct random electrode combinations from the 2-pole data segregated intomore » inner, outer, and overlapping dipoles. The resistance data and inverse models from these randomized arrays show those with inner dipoles to be superior in terms of noise and resolution and that overlapping dipoles can cause model instability and low resolution. Finally, we use the 2-pole data to create an optimized array that maximizes the model resolution matrix for a given electrode geometry. The optimized array produces the highest resolution and target detail. Thus, the tests demonstrate that high quality data and high model resolution can be achieved by acquiring field data from the pole-pole array.« less

Target guided synthesis using DNA nano-templates for selectively assembling a G-quadruplex binding c-MYC inhibitor

NASA Astrophysics Data System (ADS)

Panda, Deepanjan; Saha, Puja; Das, Tania; Dash, Jyotirmayee

2017-07-01

The development of small molecules is essential to modulate the cellular functions of biological targets in living system. Target Guided Synthesis (TGS) approaches have been used for the identification of potent small molecules for biological targets. We herein demonstrate an innovative example of TGS using DNA nano-templates that promote Huisgen cycloaddition from an array of azide and alkyne fragments. A G-quadruplex and a control duplex DNA nano-template have been prepared by assembling the DNA structures on gold-coated magnetic nanoparticles. The DNA nano-templates facilitate the regioselective formation of 1,4-substituted triazole products, which are easily isolated by magnetic decantation. The G-quadruplex nano-template can be easily recovered and reused for five reaction cycles. The major triazole product, generated by the G-quadruplex inhibits c-MYC expression by directly targeting the c-MYC promoter G-quadruplex. This work highlights that the nano-TGS approach may serve as a valuable strategy to generate target-selective ligands for drug discovery.

αB-crystallin: Portrait of a malignant chaperone as a cancer therapeutic target

PubMed Central

Malin, Dmitry; Petrovic, Vladimir; Strekalova, Elena; Sharma, Bhawna; Cryns, Vincent L.

2016-01-01

αB-crystallin is a widely expressed member of the small heat shock protein family that protects cells from stress by its dual function as a molecular chaperone to preserve proteostasis and as a cell death antagonist that negatively regulates components of the conserved apoptotic cell death machinery. Deregulated expression of αB-crystallin occurs in a broad array of solid tumors and has been linked to tumor progression and poor clinical outcomes. This review will focus on new insights into the molecular mechanisms by which oncogenes, oxidative stress, matrix detachment and other tumor microenvironmental stressors deregulate αB-crystallin expression. We will also review accumulating evidence pointing to an essential role for αB-crystallin in the multi-step metastatic cascade whereby tumor cells colonize distant organs by circumventing a multitude of barriers to cell migration and survival. Finally, we will evaluate emerging strategies to therapeutically target αB-crystallin and/or interacting proteins to selectively activate apoptosis and/or derail the metastatic cascade in an effort to improve outcomes for patients with metastatic disease. PMID:26820756

αB-crystallin: Portrait of a malignant chaperone as a cancer therapeutic target.

PubMed

Malin, Dmitry; Petrovic, Vladimir; Strekalova, Elena; Sharma, Bhawna; Cryns, Vincent L

2016-04-01

αB-crystallin is a widely expressed member of the small heat shock protein family that protects cells from stress by its dual function as a molecular chaperone to preserve proteostasis and as a cell death antagonist that negatively regulates components of the conserved apoptotic cell death machinery. Deregulated expression of αB-crystallin occurs in a broad array of solid tumors and has been linked to tumor progression and poor clinical outcomes. This review will focus on new insights into the molecular mechanisms by which oncogenes, oxidative stress, matrix detachment and other tumor microenvironmental stressors deregulate αB-crystallin expression. We will also review accumulating evidence pointing to an essential role for αB-crystallin in the multi-step metastatic cascade whereby tumor cells colonize distant organs by circumventing a multitude of barriers to cell migration and survival. Finally, we will evaluate emerging strategies to therapeutically target αB-crystallin and/or interacting proteins to selectively activate apoptosis and/or derail the metastatic cascade in an effort to improve outcomes for patients with metastatic disease. Copyright © 2016 Elsevier Inc. All rights reserved.

Detection of doublecortin domain-containing 2 (DCDC2), a new candidate tumor suppressor gene of hepatocellular carcinoma, by triple combination array analysis

PubMed Central

2013-01-01

Background To detect genes correlated with hepatocellular carcinoma (HCC), we developed a triple combination array consisting of methylation array, gene expression array and single nucleotide polymorphism (SNP) array analysis. Methods A surgical specimen obtained from a 68-year-old female HCC patient was analyzed by triple combination array, which identified doublecortin domain-containing 2 (DCDC2) as a candidate tumor suppressor gene of HCC. Subsequently, samples from 48 HCC patients were evaluated for their DCDC2 methylation and expression status using methylation specific PCR (MSP) and semi-quantitative reverse transcriptase (RT) PCR, respectively. Then, we investigated the relationship between clinicopathological factors and methylation status of DCDC2. Results DCDC2 was revealed to be hypermethylated (methylation value 0.846, range 0–1.0) in cancer tissue, compared with adjacent normal tissue (0.212) by methylation array in the 68-year-old female patient. Expression array showed decreased expression of DCDC2 in cancerous tissue. SNP array showed that the copy number of chromosome 6p22.1, in which DCDC2 resides, was normal. MSP revealed hypermethylation of the promoter region of DCDC2 in 41 of the tumor samples. DCDC2 expression was significantly decreased in the cases with methylation (P = 0.048). Furthermore, the methylated cases revealed worse prognosis for overall survival than unmethylated cases (P = 0.048). Conclusions The present study indicates that triple combination array is an effective method to detect novel genes related to HCC. We propose that DCDC2 is a tumor suppressor gene of HCC. PMID:24034596

Design and characterisation of a phased antenna array for intact breast hyperthermia.

PubMed

Curto, Sergio; Garcia-Miquel, Aleix; Suh, Minyoung; Vidal, Neus; Lopez-Villegas, Jose M; Prakash, Punit

2018-05-01

Currently available hyperthermia technology is not well suited to treating cancer malignancies in the intact breast. This study investigates a microwave applicator incorporating multiple patch antennas, with the goal of facilitating controllable power deposition profiles for treating lesions at diverse locations within the intact breast. A 3D-computational model was implemented to assess power deposition profiles with 915 MHz applicators incorporating a hemispheric groundplane and configurations of 2, 4, 8, 12, 16 and 20 antennas. Hemispheric breast models of 90 mm and 150 mm diameter were considered, where cuboid target volumes of 10 mm edge length (1 cm 3 ) and 30 mm edge length (27 cm 3 ) were positioned at the centre of the breast, and also located 15 mm from the chest wall. The average power absorption (αPA) ratio expressed as the ratio of the PA in the target volume and in the full breast was evaluated. A 4-antenna proof-of-concept array was fabricated and experimentally evaluated. Computational models identified an optimal inter-antenna spacing of 22.5° along the applicator circumference. Applicators with 8 and 12 antennas excited with constant phase presented the highest αPA at centrally located and deep-seated targets, respectively. Experimental measurements with a 4-antenna proof-of-concept array illustrated the potential for electrically steering power deposition profiles by adjusting the relative phase of the signal at antenna inputs. Computational models and experimental results suggest that the proposed applicator may have potential for delivering conformal thermal therapy in the intact breast.

Targeting PIM kinase enhances the activity of sunitinib in renal cell carcinoma.

PubMed

Mahalingam, D; Espitia, C M; Medina, E C; Esquivel, J A; Kelly, K R; Bearss, D; Choy, G; Taverna, P; Carew, J S; Giles, F J; Nawrocki, S T

2011-11-08

Upregulation of PIM kinase expression has been reported in many malignancies, suggesting that inhibition of PIM kinase activity may be an attractive therapeutic strategy. We hypothesised that inhibition of PIM kinase activity with SGI-1776, a novel small molecule inhibitor of PIM kinase activity, would reduce the viability of renal cell carcinoma (RCC) cells and enhance the activity of sunitinib. Immunoblotting, qRT-PCR, and gene expression arrays were carried out to identify genes modulated by SGI-1776 treatment. The anticancer activity of SGI-1776 and sunitinib was determined by viability and apoptosis assays and in tumour xenografts in vivo. Treatment with SGI-1776 led to a decrease in phosphorylated and total c-Myc levels, which resulted in the modulation of c-Myc target genes. SGI-1776 in combination with sunitinib induced a further reduction in c-Myc levels, which was associated with enhanced anticancer activity. siRNA-mediated knockdown of c-Myc demonstrated that its expression has a key role in regulating the sensitivity to the combination of SGI-1776 and sunitinib. Importantly, the combination significantly reduced tumour burden in two RCC xenograft models compared with single-agent therapy and was very well tolerated. These data indicate that targeting PIM kinase signalling is a promising treatment strategy for RCC. 2011 Cancer Research UK

Targeting PIM kinase enhances the activity of sunitinib in renal cell carcinoma

PubMed Central

Mahalingam, D; Espitia, C M; Medina, E C; Esquivel, J A; Kelly, K R; Bearss, D; Choy, G; Taverna, P; Carew, J S; Giles, F J; Nawrocki, S T

2011-01-01

Background: Upregulation of PIM kinase expression has been reported in many malignancies, suggesting that inhibition of PIM kinase activity may be an attractive therapeutic strategy. We hypothesised that inhibition of PIM kinase activity with SGI-1776, a novel small molecule inhibitor of PIM kinase activity, would reduce the viability of renal cell carcinoma (RCC) cells and enhance the activity of sunitinib. Methods: Immunoblotting, qRT–PCR, and gene expression arrays were carried out to identify genes modulated by SGI-1776 treatment. The anticancer activity of SGI-1776 and sunitinib was determined by viability and apoptosis assays and in tumour xenografts in vivo. Results: Treatment with SGI-1776 led to a decrease in phosphorylated and total c-Myc levels, which resulted in the modulation of c-Myc target genes. SGI-1776 in combination with sunitinib induced a further reduction in c-Myc levels, which was associated with enhanced anticancer activity. siRNA-mediated knockdown of c-Myc demonstrated that its expression has a key role in regulating the sensitivity to the combination of SGI-1776 and sunitinib. Importantly, the combination significantly reduced tumour burden in two RCC xenograft models compared with single-agent therapy and was very well tolerated. Conclusion: These data indicate that targeting PIM kinase signalling is a promising treatment strategy for RCC. PMID:22015557

Transcriptome profiling confirmed correlations between symptoms and transcriptional changes in RDV infected rice and revealed nucleolus as a possible target of RDV manipulation.

PubMed

Yang, Liang; Du, Zhenguo; Gao, Feng; Wu, Kangcheng; Xie, Lianhui; Li, Yi; Wu, Zujian; Wu, Jianguo

2014-05-06

Rice dwarf virus (RDV) is the causal agent of rice dwarf disease, which limits rice production in many areas of south East Asia. Transcriptional changes of rice in response to RDV infection have been characterized by Shimizu et al. and Satoh et al.. Both studies found induction of defense related genes and correlations between transcriptional changes and symptom development in RDV-infected rice. However, the same rice cultivar, namely Nipponbare belonging to the Japonic subspecies of rice was used in both studies. Gene expression changes of the indica subspecies of rice, namely Oryza sativa L. ssp. indica cv Yixiang2292 that show moderate resistance to RDV, in response to RDV infection were characterized using an Affymetrix Rice Genome Array. Differentially expressed genes (DEGs) were classified according to their Gene Ontology (GO) annotation. The effects of transient expression of Pns11 in Nicotiana benthaminana on the expression of nucleolar genes were studied using real-time PCR (RT-PCR). 856 genes involved in defense or other physiological processes were identified to be DEGs, most of which showed up-regulation. Ribosome- and nucleolus related genes were significantly enriched in the DEGs. Representative genes related to nucleolar function exhibited altered expression in N. benthaminana plants transiently expressing Pns11 of RDV. Induction of defense related genes is common for rice infected with RDV. There is a co-relation between symptom severity and transcriptional alteration in RDV infected rice. Besides ribosome, RDV may also target nucleolus to manipulate the translation machinery of rice. Given the tight links between nucleolus and ribosome, it is intriguing to speculate that RDV may enhance expression of ribosomal genes by targeting nucleolus through Pns11.

Genome-wide profiling of gene expression in the epididymis of alpha-chlorohydrin-induced infertile rats using an oligonucleotide microarray

PubMed Central

2010-01-01

Background As one of the chlorinated antifertility compounds, alpha-chlorohydrin (ACH) can inhibit glyceraldehyde-3-phosphate dehydrogenase (G3PDH) activity in epididymal sperm and affect sperm energy metabolism, maturation and fertilization, eventually leading to male infertility. Further studies demonstrated that the inhibitory effect of ACH on G3PDH is not only confined to epididymal sperm but also to the epididymis. Moreover, little investigation on gene expression changes in the epididymis after ACH treatment has been conducted. Therefore, gene expression studies may indicate new epididymal targets related to sperm maturation and fertility through the analysis of ACH-treated infertile animals. Methods Rats were treated with ACH for ten consecutive days, and then each male rat copulated with two female rats in proestrus. Then sperm maturation and other fertility parameters were analyzed. Furthermore, we identified epididymal-specific genes that are associated with fertility between control and ACH groups using an Affymetrix Rat 230 2.0 oligo-microarray. Finally, we performed RT-PCR analysis for several differentially expressed genes to validate the alteration in gene expression observed by oligonucleotide microarray. Results Among all the differentially expressed genes, we analyzed and screened the down-regulated genes associated with metabolism processes, which are considered the major targets of ACH action. Simultaneously, the genes that were up-regulated by chlorohydrin were detected. The genes that negatively regulate sperm maturation and fertility include apoptosis and immune-related genes and have not been reported previously. The overall results of PCR analysis for selected genes were consistent with the array data. Conclusions In this study, we have described the genome-wide profiles of gene expression in the epididymides of infertile rats induced by ACH, which could become potential epididymal specific targets for male contraception and infertility treatment. PMID:20409345

Genome-wide profiling of gene expression in the epididymis of alpha-chlorohydrin-induced infertile rats using an oligonucleotide microarray.

PubMed

Xie, Shuwu; Zhu, Yan; Ma, Li; Lu, Yingying; Zhou, Jieyun; Gui, Youlun; Cao, Lin

2010-04-22

As one of the chlorinated antifertility compounds, alpha-chlorohydrin (ACH) can inhibit glyceraldehyde-3-phosphate dehydrogenase (G3PDH) activity in epididymal sperm and affect sperm energy metabolism, maturation and fertilization, eventually leading to male infertility. Further studies demonstrated that the inhibitory effect of ACH on G3PDH is not only confined to epididymal sperm but also to the epididymis. Moreover, little investigation on gene expression changes in the epididymis after ACH treatment has been conducted. Therefore, gene expression studies may indicate new epididymal targets related to sperm maturation and fertility through the analysis of ACH-treated infertile animals. Rats were treated with ACH for ten consecutive days, and then each male rat copulated with two female rats in proestrus. Then sperm maturation and other fertility parameters were analyzed. Furthermore, we identified epididymal-specific genes that are associated with fertility between control and ACH groups using an Affymetrix Rat 230 2.0 oligo-microarray. Finally, we performed RT-PCR analysis for several differentially expressed genes to validate the alteration in gene expression observed by oligonucleotide microarray. Among all the differentially expressed genes, we analyzed and screened the down-regulated genes associated with metabolism processes, which are considered the major targets of ACH action. Simultaneously, the genes that were up-regulated by chlorohydrin were detected. The genes that negatively regulate sperm maturation and fertility include apoptosis and immune-related genes and have not been reported previously. The overall results of PCR analysis for selected genes were consistent with the array data. In this study, we have described the genome-wide profiles of gene expression in the epididymides of infertile rats induced by ACH, which could become potential epididymal specific targets for male contraception and infertility treatment.

CLDN1 expression in cervical cancer cells is related to tumor invasion and metastasis.

PubMed

Zhang, Wei-Na; Li, Wei; Wang, Xiao-Li; Hu, Zheng; Zhu, Da; Ding, Wen-Cheng; Liu, Dan; Li, Ke-Zhen; Ma, Ding; Wang, Hui

2016-12-27

Even though infection with human papillomaviruses (HPV) is very important, it is not the sole cause of cervical cancer. Because it is known that genetic variations that result from HPV infection are probably the most important causes of cervical cancer, we used human whole genome array comparative genomic hybridization to detect the copy number variations of genes in cervical squamous cell carcinoma. The results of the array were validated by PCR, FISH and immunohistochemistry. We find that the copy number and protein expression of claudin-1 (CLDN1) increase with the progression of cervical cancer. The strong positive staining of CLDN1 in the cervical lymph node metastasis group received a significantly higher score than the staining in the group with no lymph node metastasis of cervical cancer tissues. The overexpression of CLDN1 in SiHa cells can increase anti-apoptosis ability and promote invasive ability of these cells accompanied by a decrease in expression of the epithelial marker E-cadherin as well as an increase in the expression of the mesenchymal marker vimentin. CLDN1 induces the epithelial-mesenchymal transition (EMT) through its interaction with SNAI1. Furthermore, we demonstrate that CLDN1 overexpression has significant effects on the growth and metastasis of xenografted tumors in athymic mice. These data suggest that CLDN1 promotes invasion and metastasis in cervical cancer cells via the expression of EMT/invasion-related genes. Therefore, CLDN1 could be a potential therapeutic target for the treatment of cervical cancer.

An alternative method to amplify RNA without loss of signal conservation for expression analysis with a proteinase DNA microarray in the ArrayTube format.

PubMed

Schüler, Susann; Wenz, Ingrid; Wiederanders, B; Slickers, P; Ehricht, R

2006-06-12

Recent developments in DNA microarray technology led to a variety of open and closed devices and systems including high and low density microarrays for high-throughput screening applications as well as microarrays of lower density for specific diagnostic purposes. Beside predefined microarrays for specific applications manufacturers offer the production of custom-designed microarrays adapted to customers' wishes. Array based assays demand complex procedures including several steps for sample preparation (RNA extraction, amplification and sample labelling), hybridization and detection, thus leading to a high variability between several approaches and resulting in the necessity of extensive standardization and normalization procedures. In the present work a custom designed human proteinase DNA microarray of lower density in ArrayTube format was established. This highly economic open platform only requires standard laboratory equipment and allows the study of the molecular regulation of cell behaviour by proteinases. We established a procedure for sample preparation and hybridization and verified the array based gene expression profile by quantitative real-time PCR (QRT-PCR). Moreover, we compared the results with the well established Affymetrix microarray. By application of standard labelling procedures with e.g. Klenow fragment exo-, single primer amplification (SPA) or In Vitro Transcription (IVT) we noticed a loss of signal conservation for some genes. To overcome this problem we developed a protocol in accordance with the SPA protocol, in which we included target specific primers designed individually for each spotted oligomer. Here we present a complete array based assay in which only the specific transcripts of interest are amplified in parallel and in a linear manner. The array represents a proof of principle which can be adapted to other species as well. As the designed protocol for amplifying mRNA starts from as little as 100 ng total RNA, it presents an alternative method for detecting even low expressed genes by microarray experiments in a highly reproducible and sensitive manner. Preservation of signal integrity is demonstrated out by QRT-PCR measurements. The little amounts of total RNA necessary for the analyses make this method applicable for investigations with limited material as in clinical samples from, for example, organ or tumour biopsies. Those are arguments in favour of the high potential of our assay compared to established procedures for amplification within the field of diagnostic expression profiling. Nevertheless, the screening character of microarray data must be mentioned, and independent methods should verify the results.

Methods for implementing microbeam radiation therapy

DOEpatents

Dilmanian, F. Avraham; Morris, Gerard M.; Hainfeld, James F.

2007-03-20

A method of performing radiation therapy includes delivering a therapeutic dose such as X-ray only to a target (e.g., tumor) with continuous broad beam (or in-effect continuous) using arrays of parallel planes of radiation (microbeams/microplanar beams). Microbeams spare normal tissues, and when interlaced at a tumor, form a broad-beam for tumor ablation. Bidirectional interlaced microbeam radiation therapy (BIMRT) uses two orthogonal arrays with inter-beam spacing equal to beam thickness. Multidirectional interlaced MRT (MIMRT) includes irradiations of arrays from several angles, which interleave at the target. Contrast agents, such as tungsten and gold, are administered to preferentially increase the target dose relative to the dose in normal tissue. Lighter elements, such as iodine and gadolinium, are used as scattering agents in conjunction with non-interleaving geometries of array(s) (e.g., unidirectional or cross-fired (intersecting) to generate a broad beam effect only within the target by preferentially increasing the valley dose within the tumor.

Patients with the worst outcomes after paracetamol (acetaminophen)-induced liver failure have an early monocytopenia.

PubMed

Moore, J K; MacKinnon, A C; Man, T Y; Manning, J R; Forbes, S J; Simpson, K J

2017-02-01

Acute liver failure (ALF) is associated with significant morbidity and mortality. Studies have implicated the immune response, especially monocyte/macrophages as being important in dictating outcome. To investigate changes in the circulating monocytes and other immune cells serially in patients with ALF, relate these with cytokine concentrations, monocyte gene expression and patient outcome. In a prospective case-control study in the Scottish Liver Transplant Unit, Royal Infirmary Edinburgh, 35 consecutive patients admitted with paracetamol-induced liver failure (POD-ALF), 10 patients with non-paracetamol causes of ALF and 16 controls were recruited. The peripheral blood monocyte phenotype was analysed by flow cytometry, circulating cytokines quantified by protein array and monocyte gene expression array performed and related to outcome. On admission, patients with worst outcomes after POD-ALF had a significant monocytopenia, characterised by reduced classical and expanded intermediate monocyte population. This was associated with reduced circulating lymphocytes and natural killer cells, peripheral cytokine patterns suggestive of a 'cytokine storm' and increased concentrations of cytokines associated with monocyte egress from the bone marrow. Gene expression array did not differentiate patient outcome. At day 4, there was no significant difference in monocyte, lymphocyte or natural killer cells between survivors and the patients with adverse outcomes. Severe paracetamol liver failure is associated with profound changes in the peripheral blood compartment, particularly in monocytes, related with worse outcomes. This is not seen in patients with non-paracetamol-induced liver failure. Significant monocytopenia on admission may allow earlier clarification of prognosis, and it highlights a potential target for therapeutic intervention. © 2016 John Wiley & Sons Ltd.

Impact of S100A8 expression on kidney cancer progression and molecular docking studies for kidney cancer therapeutics.

PubMed

Mirza, Zeenat; Schulten, Hans-Juergen; Farsi, Hasan Ma; Al-Maghrabi, Jaudah A; Gari, Mamdooh A; Chaudhary, Adeel Ga; Abuzenadah, Adel M; Al-Qahtani, Mohammed H; Karim, Sajjad

2014-04-01

The proinflammatory protein S100A8, which is expressed in myeloid cells under physiological conditions, is strongly expressed in human cancer tissues. Its role in tumor cell differentiation and tumor progression is largely unclear and virtually unstudied in kidney cancer. In the present study, we investigated whether S100A8 could be a potential anticancer drug target and therapeutic biomarker for kidney cancer, and the underlying molecular mechanisms by exploiting its interaction profile with drugs. Microarray-based transcriptomics experiments using Affymetrix HuGene 1.0 ST arrays were applied to renal cell carcinoma specimens from Saudi patients for identification of significant genes associated with kidney cancer. In addition, we retrieved selected expression data from the National Center for Biotechnology Information Gene Expression Omnibus database for comparative analysis and confirmation of S100A8 expression. Ingenuity Pathway Analysis (IPA) was used to elucidate significant molecular networks and pathways associated with kidney cancer. The probable polar and non-polar interactions of possible S100A8 inhibitors (aspirin, celecoxib, dexamethasone and diclofenac) were examined by performing molecular docking and binding free energy calculations. Detailed analysis of bound structures and their binding free energies was carried out for S100A8, its known partner (S100A9), and S100A8-S100A9 complex (calprotectin). In our microarray experiments, we identified 1,335 significantly differentially expressed genes, including S100A8, in kidney cancer using a cut-off of p<0.05 and fold-change of 2. Functional analysis of kidney cancer-associated genes showed overexpression of genes involved in cell-cycle progression, DNA repair, cell death, tumor morphology and tissue development. Pathway analysis showed significant disruption of pathways of atherosclerosis signaling, liver X receptor/retinoid X receptor (LXR/RXR) activation, notch signaling, and interleukin-12 (IL-12) signaling. We identified S100A8 as a prospective biomarker for kidney cancer and in silico analysis showed that aspirin, celecoxib, dexamethasone and diclofenac binds to S100A8 and may inhibit downstream signaling in kidney cancer. The present study provides an initial overview of differentially expressed genes in kidney cancer of Saudi Arabian patients using whole-transcript, high-density expression arrays. Our analysis suggests distinct transcriptomic signatures, with significantly high levels of S100A8, and underlying molecular mechanisms contributing to kidney cancer progression. Our docking-based findings shed insight into S100A8 protein as an attractive anticancer target for therapeutic intervention in kidney cancer. To our knowledge, this is the first structure-based docking study for the selected protein targets using the chosen ligands.

Multiwaveguide implantable probe for light delivery to sets of distributed brain targets.

PubMed

Zorzos, Anthony N; Boyden, Edward S; Fonstad, Clifton G

2010-12-15

Optical fibers are commonly inserted into living tissues such as the brain in order to deliver light to deep targets for neuroscientific and neuroengineering applications such as optogenetics, in which light is used to activate or silence neurons expressing specific photosensitive proteins. However, an optical fiber is limited to delivering light to a single target within the three-dimensional structure of the brain. We here demonstrate a multiwaveguide probe capable of independently delivering light to multiple targets along the probe axis, thus enabling versatile optical control of sets of distributed brain targets. The 1.45-cm-long probe is microfabricated in the form of a 360-μm-wide array of 12 parallel silicon oxynitride (SiON) multimode waveguides clad with SiO(2) and coated with aluminum; probes of custom dimensions are easily created as well. The waveguide array accepts light from a set of sources at the input end and guides the light down each waveguide to an aluminum corner mirror that efficiently deflects light away from the probe axis. Light losses at each stage are small (input coupling loss, 0.4 ± 0.3 dB; bend loss, negligible; propagation loss, 3.1 ± 1 dB/cm using the outscattering method and 3.2 ± 0.4 dB/cm using the cutback method; corner mirror loss, 1.5 ± 0.4 dB); a waveguide coupled, for example, to a 5 mW source will deliver over 1.5 mW to a target at a depth of 1 cm.

Parallel gene analysis with allele-specific padlock probes and tag microarrays

PubMed Central

Banér, Johan; Isaksson, Anders; Waldenström, Erik; Jarvius, Jonas; Landegren, Ulf; Nilsson, Mats

2003-01-01

Parallel, highly specific analysis methods are required to take advantage of the extensive information about DNA sequence variation and of expressed sequences. We present a scalable laboratory technique suitable to analyze numerous target sequences in multiplexed assays. Sets of padlock probes were applied to analyze single nucleotide variation directly in total genomic DNA or cDNA for parallel genotyping or gene expression analysis. All reacted probes were then co-amplified and identified by hybridization to a standard tag oligonucleotide array. The technique was illustrated by analyzing normal and pathogenic variation within the Wilson disease-related ATP7B gene, both at the level of DNA and RNA, using allele-specific padlock probes. PMID:12930977

Characterization of transformation related genes in oral cancer cells.

PubMed

Chang, D D; Park, N H; Denny, C T; Nelson, S F; Pe, M

1998-04-16

A cDNA representational difference analysis (cDNA-RDA) and an arrayed filter technique were used to characterize transformation-related genes in oral cancer. From an initial comparison of normal oral epithelial cells and a human papilloma virus (HPV)-immortalized oral epithelial cell line, we obtained 384 differentially expressed gene fragments and arrayed them on a filter. Two hundred and twelve redundant clones were identified by three rounds of back hybridization. Sequence analysis of the remaining clones revealed 99 unique clones corresponding to 69 genes. The expression of these transformation related gene fragments in three nontumorigenic HPV-immortalized oral epithelial cell lines and three oral cancer cell lines were simultaneously monitored using a cDNA array hybridization. Although there was a considerable cell line-to-cell line variability in the expression of these clones, a reliable prediction of their expression could be made from the cDNA array hybridization. Our study demonstrates the utility of combining cDNA-RDA and arrayed filters in high-throughput gene expression difference analysis. The differentially expressed genes identified in this study should be informative in studying oral epithelial cell carcinogenesis.

Identification of transcription coactivator OCA-B-dependent genes involved in antigen-dependent B cell differentiation by cDNA array analyses.

PubMed

Kim, Unkyu; Siegel, Rachael; Ren, Xiaodi; Gunther, Cary S; Gaasterland, Terry; Roeder, Robert G

2003-07-22

The tissue-specific transcriptional coactivator OCA-B is required for antigen-dependent B cell differentiation events, including germinal center formation. However, the identity of OCA-B target genes involved in this process is unknown. This study has used large-scale cDNA arrays to monitor changes in gene expression patterns that accompany mature B cell differentiation. B cell receptor ligation alone induces many genes involved in B cell expansion, whereas B cell receptor and helper T cell costimulation induce genes associated with B cell effector function. OCA-B expression is induced by both B cell receptor ligation alone and helper T cell costimulation, suggesting that OCA-B is involved in B cell expansion as well as B cell function. Accordingly, several genes involved in cell proliferation and signaling, such as Lck, Kcnn4, Cdc37, cyclin D3, B4galt1, and Ms4a11, have been identified as OCA-B-dependent genes. Further studies on the roles played by these genes in B cells will contribute to an understanding of B cell differentiation.

Neurons with object-centered spatial selectivity in macaque SEF: do they represent locations or rules?

PubMed

Tremblay, Léon; Gettner, Sonya N; Olson, Carl R

2002-01-01

In macaque monkeys performing a task that requires eye movements to the leftmost or rightmost of two dots in a horizontal array, some neurons in the supplementary eye field (SEF) fire differentially according to which side of the array is the target regardless of the array's location on the screen. We refer to these neurons as exhibiting selectivity for object-centered location. This form of selectivity might arise from involvement of the neurons in either of two processes: representing the locations of targets or representing the rules by which targets are selected. To distinguish between these possibilities, we monitored neuronal activity in the SEF of two monkeys performing a task that required the selection of targets by either an object-centered spatial rule or a color rule. On each trial, a sample array consisting of two side-by-side dots appeared; then a cue flashed on one dot; then the display vanished and a delay ensued. Next a target array consisting of two side-by-side dots appeared at an unpredictable location and another delay ensued; finally the monkey had to make an eye movement to one of the target dots. On some trials, the monkey had to select the dot on the same side as the cue (right or left). On other trials, he had to select the target of the same color as the cue (red or green). Neuronal activity robustly encoded the object-centered locations first of the cue and then of the target regardless of the whether the monkey was following a rule based on object-centered location or color. Neuronal activity was at most weakly affected by the type of rule the monkey was following (object-centered-location or color) or by the color of the cue and target (red or green). On trials involving a color rule, neuronal activity was moderately enhanced when the cue and target appeared on opposite sides of their respective arrays. We conclude that the general function of SEF neurons selective for object-centered location is to represent where the cue and target are in their respective arrays rather than to represent the rule for target selection.

Colloidal silica films for high-capacity DNA arrays

NASA Astrophysics Data System (ADS)

Glazer, Marc Irving

The human genome project has greatly expanded the amount of genetic information available to researchers, but before this vast new source of data can be fully utilized, techniques for rapid, large-scale analysis of DNA and RNA must continue to develop. DNA arrays have emerged as a powerful new technology for analyzing genomic samples in a highly parallel format. The detection sensitivity of these arrays is dependent on the quantity and density of immobilized probe molecules. We have investigated substrates with a porous, "three-dimensional" surface layer as a means of increasing the surface area available for the synthesis of oligonucleotide probes, thereby increasing the number of available probes and the amount of detectable bound target. Porous colloidal silica films were created by two techniques. In the first approach, films were deposited by spin-coating silica colloid suspensions onto flat glass substrates, with the pores being formed by the natural voids between the solid particles (typically 23nm pores, 35% porosity). In the second approach, latex particles were co-deposited with the silica and then pyrolyzed, creating films with larger pores (36 nm), higher porosity (65%), and higher surface area. For 0.3 mum films, enhancements of eight to ten-fold and 12- to 14-fold were achieved with the pure silica films and the films "templated" with polymer latex, respectively. In gene expression assays for up to 7,000 genes using complex biological samples, the high-capacity films provided enhanced signals and performed equivalently or better than planar glass on all other functional measures, confirming that colloidal silica films are a promising platform for high-capacity DNA arrays. We have also investigated the kinetics of hybridization on planar glass and high-capacity substrates. Adsorption on planar arrays is similar to ideal Langmuir-type adsorption, although with an "overshoot" at high solution concentration. Hybridization on high-capacity films is controlled by traditional adsorption (ka) and desorption (kd) coefficients, as well as morphology factors and transient binding interactions between the target and probes. The strength of the transient probe/target binding interactions are on the order of 5--7 DNA base pairs, which suggests the formation of nucleation or other metastable complexes, rather than fully-zippered duplexes.

Carbon Nanotube Arrays for Intracellular Delivery and Biological Applications

NASA Astrophysics Data System (ADS)

Golshadi, Masoud

Introducing nucleic acids into mammalian cells is a crucial step to elucidate biochemical pathways, modify gene expression in immortalized cells, primary cells, and stem cells, and intoduces new approaches for clinical diagnostics and therapeutics. Current gene transfer technologies, including lipofection, electroporation, and viral delivery, have enabled break-through advances in basic and translational science to enable derivation and programming of embryonic stem cells, advanced gene editing using CRISPR (Clustered regularly interspaced short palindromic repeats), and development of targeted anti-tumor therapy using chimeric antigen receptors in T-cells (CAR-T). Despite these successes, current transfection technologies are time consuming and limited by the inefficient introduction of test molecules into large populations of target cells, and the cytotoxicity of the techniques. Moreover, many cell types cannot be consistently transfected by lipofection or electroporation (stem cells, T-cells) and viral delivery has limitations to the size of experimental DNA that can be packaged. In this dissertation, a novel coverslip-like platform consisting of an array of aligned hollow carbon nanotubes (CNTs) embedded in a sacrificial template is developed that enhances gene transfer capabilities, including high efficiency, low toxicity, in an expanded range of target cells, with the potential to transfer mixed combinations of protein and nucleic acids. The CNT array devices are fabricated by a scalable template-based manufacturing method using commercially available membranes, eliminating the need for nano-assembly. High efficient transfection has been demonstrated by delivering various cargos (nanoparticles, dye and plasmid DNA) into populations of cells, achieving 85% efficiency of plasmid DNA delivery into immortalized cells. Moreover, the CNT-mediated transfection of stem cells shows 3 times higher efficiency compared to current lipofection methods. Evaluating the cell-CNT interaction elucidates the importance of the geometrical properties of CNT arrays (CNT exposed length and surface morphology) on transfection efficiency. The results indicate that densely-packed and shortly-exposed CNT arrays with planar surface will enhance gene delivery using this new platform. This technology offers a significant increase in efficiency and cell viability, along with the ease of use compared to current standard methods, which demonstrates its potential to accelerate the development of new cell models to study intractable diseases, decoding the signaling pathways, and drug discovery.

Identification and validation of cetuximab resistance associated long noncoding RNA biomarkers in metastatic colorectal cancer.

PubMed

Peng, Ke; Liu, Ruiqi; Yu, Yiyi; Liang, Li; Yu, Shan; Xu, Xiaojing; Liu, Tianshu

2018-01-01

Cetuximab is one of the most widely used epidermal growth factor receptor (EGFR) inhibitors to treat patients with metastatic colorectal cancer (mCRC) harboring wild-type of RAS/RAF status. However, primary and acquired resistance to cetuximab is often found during target therapy. To gain insights into the functions of long non-coding RNA (lncRNA) in cetuximab resistance, we used a lncRNA-mining approach to distinguish lncRNA specific probes in Affymetrix HG-U133A 2.0 arrays. Then we performed lncRNA expression profiling in a cetuximab treated mCRC cohort from Gene Expression Ominus (GEO). The potential lncRNAs were further validated in acquired cetuximab resistant cell lines and clinical samples of our hospital. The functions and associated pathways of the prognostic lncRNA were predicted by GO and KEGG analyses. 249 lncRNA-specific probe sets (corresponding to 212 lncRNAs) were represented in Affymetrix HG-U133A 2.0 arrays. We found that 9 lncRNAs were differentially expressed between disease control group (DCG) and non-responders, and 5 of these 9 lncRNAs were significantly related with the progression-free survival (PFS) of the patients. Among those 5 lncRNAs, POU5F1P4 was also down-regulated in acquired cetuximab resistant cells, as well as in cetuximab resistant patients. Downregulation of POU5F1P4 decreased the sensitivity of colorectal cancer cells to cetuximab. Our findings indicate the potential roles of lncRNAs in cetuximab resistance, and may provide the useful information for discovery of new biomarkers and therapeutic targets. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

DSSTox chemical-index files for exposure-related experiments in ArrayExpress and Gene Expression Omnibus: enabling toxico-chemogenomics data linkages

EPA Science Inventory

The Distributed Structure-Searchable Toxicity (DSSTox) ARYEXP and GEOGSE files are newly published, structure-annotated files of the chemical-associated and chemical exposure-related summary experimental content contained in the ArrayExpress Repository and Gene Expression Omnibus...

High precision computing with charge domain devices and a pseudo-spectral method therefor

NASA Technical Reports Server (NTRS)

Barhen, Jacob (Inventor); Toomarian, Nikzad (Inventor); Fijany, Amir (Inventor); Zak, Michail (Inventor)

1997-01-01

The present invention enhances the bit resolution of a CCD/CID MVM processor by storing each bit of each matrix element as a separate CCD charge packet. The bits of each input vector are separately multiplied by each bit of each matrix element in massive parallelism and the resulting products are combined appropriately to synthesize the correct product. In another aspect of the invention, such arrays are employed in a pseudo-spectral method of the invention, in which partial differential equations are solved by expressing each derivative analytically as matrices, and the state function is updated at each computation cycle by multiplying it by the matrices. The matrices are treated as synaptic arrays of a neural network and the state function vector elements are treated as neurons. In a further aspect of the invention, moving target detection is performed by driving the soliton equation with a vector of detector outputs. The neural architecture consists of two synaptic arrays corresponding to the two differential terms of the soliton-equation and an adder connected to the output thereof and to the output of the detector array to drive the soliton equation.

A bio-inspired structural health monitoring system based on ambient vibration

NASA Astrophysics Data System (ADS)

Lin, Tzu-Kang; Kiremidjian, Anne; Lei, Chi-Yang

2010-11-01

A structural health monitoring (SHM) system based on naïve Bayesian (NB) damage classification and DNA-like expression data was developed in this research. Adapted from the deoxyribonucleic acid (DNA) array concept in molecular biology, the proposed structural health monitoring system is constructed utilizing a double-tier regression process to extract the expression array from the structural time history recorded during external excitations. The extracted array is symbolized as the various genes of the structure from the viewpoint of molecular biology and reflects the possible damage conditions prevalent in the structure. A scaled down, six-story steel building mounted on the shaking table of the National Center for Research on Earthquake Engineering (NCREE) was used as the benchmark. The structural response at different damage levels and locations under ambient vibration was collected to support the database for the proposed SHM system. To improve the precision of detection in practical applications, the system was enhanced by an optimization process using the likelihood selection method. The obtained array representing the DNA array of the health condition of the structure was first evaluated and ranked. A total of 12 groups of expression arrays were regenerated from a combination of four damage conditions. To keep the length of the array unchanged, the best 16 coefficients from every expression array were selected to form the optimized SHM system. Test results from the ambient vibrations showed that the detection accuracy of the structural damage could be greatly enhanced by the optimized expression array, when compared to the original system. Practical verification also demonstrated that a rapid and reliable result could be given by the final system within 1 min. The proposed system implements the idea of transplanting the DNA array concept from molecular biology into the field of SHM.

Naturally occurring anti-glycan antibodies binding to Globo H-expressing cells identify ovarian cancer patients.

PubMed

Pochechueva, Tatiana; Alam, Shahidul; Schötzau, Andreas; Chinarev, Alexander; Bovin, Nicolai V; Hacker, Neville F; Jacob, Francis; Heinzelmann-Schwarz, Viola

2017-02-10

Glycosphingolipids are important compounds of the plasma membrane of mammalian cells and a number of them have been associated with malignant transformation and progression, reinforcing tumour aggressiveness and metastasis. Here we investigated the levels of naturally occurring anti-glycan antibodies to Globo H in blood plasma obtained from high-grade serous ovarian cancer patients (SOC) and women without gynaecological malignancies (control) using suspension glycan array technology employing chemically synthesized glycans as antibody targets. We found that anti-human Globo H IgG antibodies were able to significantly discriminate SOC from controls (P < 0.05). A combination with the clinically used tumour marker CA125 increased the diagnostic performance (AUC 0.8711). We next compared suspension array with standard flow cytometry in plasma samples and found that the level of anti-Globo H antibodies highly correlated (r = 0.992). The incubation of plasma-derived anti-glycan antibodies with chemically synthesized (presented on fluorescence microspheres) and native Globo H (expressed on Globo H-positive cell lines) revealed strong reactivity of naturally occurring human anti-Globo H antibodies towards its antigen expressed on ovarian cancer cells. Our data demonstrate that human plasma-derived antibodies to Globo H as well as the presence of the antigen might be considered as therapeutic option in ovarian cancer.

THE USE OF GENE ARRAYS TO DETERMINE TEMPORAL GENE INDUCTION IN SHEEPSHEAD MINNOWS EXPOSED TO E2

EPA Science Inventory

Gene arrays provide a means to study differential gene expression in fish exposed to environmental estrogens by providing a "snapshot" of the genes expressed at a given time. Such array data may also uncover previously unknown biochemical pathways affected by estrogenic compounds...

CIDR

Science.gov Websites

Consortium Developed Arrays Infinium Human Drug Core Array The Illumina nfinium DrugDev Consortium array drug target discovery, validation and treatment response. Detailed Information on Array Infinium Human

Method and apparatus for coherent imaging of infrared energy

DOEpatents

Hutchinson, Donald P.

1998-01-01

A coherent camera system performs ranging, spectroscopy, and thermal imaging. Local oscillator radiation is combined with target scene radiation to enable heterodyne detection by the coherent camera's two-dimensional photodetector array. Versatility enables deployment of the system in either a passive mode (where no laser energy is actively transmitted toward the target scene) or an active mode (where a transmitting laser is used to actively illuminate the target scene). The two-dimensional photodetector array eliminates the need to mechanically scan the detector. Each element of the photodetector array produces an intermediate frequency signal that is amplified, filtered, and rectified by the coherent camera's integrated circuitry. By spectroscopic examination of the frequency components of each pixel of the detector array, a high-resolution, three-dimensional or holographic image of the target scene is produced for applications such as air pollution studies, atmospheric disturbance monitoring, and military weapons targeting.

Method and apparatus for coherent imaging of infrared energy

DOEpatents

Hutchinson, D.P.

1998-05-12

A coherent camera system performs ranging, spectroscopy, and thermal imaging. Local oscillator radiation is combined with target scene radiation to enable heterodyne detection by the coherent camera`s two-dimensional photodetector array. Versatility enables deployment of the system in either a passive mode (where no laser energy is actively transmitted toward the target scene) or an active mode (where a transmitting laser is used to actively illuminate the target scene). The two-dimensional photodetector array eliminates the need to mechanically scan the detector. Each element of the photodetector array produces an intermediate frequency signal that is amplified, filtered, and rectified by the coherent camera`s integrated circuitry. By spectroscopic examination of the frequency components of each pixel of the detector array, a high-resolution, three-dimensional or holographic image of the target scene is produced for applications such as air pollution studies, atmospheric disturbance monitoring, and military weapons targeting. 8 figs.

High-resolution Antibody Array Analysis of Childhood Acute Leukemia Cells*

PubMed Central

Kanderova, Veronika; Kuzilkova, Daniela; Stuchly, Jan; Vaskova, Martina; Brdicka, Tomas; Fiser, Karel; Hrusak, Ondrej; Lund-Johansen, Fridtjof

2016-01-01

Acute leukemia is a disease pathologically manifested at both genomic and proteomic levels. Molecular genetic technologies are currently widely used in clinical research. In contrast, sensitive and high-throughput proteomic techniques for performing protein analyses in patient samples are still lacking. Here, we used a technology based on size exclusion chromatography followed by immunoprecipitation of target proteins with an antibody bead array (Size Exclusion Chromatography-Microsphere-based Affinity Proteomics, SEC-MAP) to detect hundreds of proteins from a single sample. In addition, we developed semi-automatic bioinformatics tools to adapt this technology for high-content proteomic screening of pediatric acute leukemia patients. To confirm the utility of SEC-MAP in leukemia immunophenotyping, we tested 31 leukemia diagnostic markers in parallel by SEC-MAP and flow cytometry. We identified 28 antibodies suitable for both techniques. Eighteen of them provided excellent quantitative correlation between SEC-MAP and flow cytometry (p < 0.05). Next, SEC-MAP was applied to examine 57 diagnostic samples from patients with acute leukemia. In this assay, we used 632 different antibodies and detected 501 targets. Of those, 47 targets were differentially expressed between at least two of the three acute leukemia subgroups. The CD markers correlated with immunophenotypic categories as expected. From non-CD markers, we found DBN1, PAX5, or PTK2 overexpressed in B-cell precursor acute lymphoblastic leukemias, LAT, SH2D1A, or STAT5A overexpressed in T-cell acute lymphoblastic leukemias, and HCK, GLUD1, or SYK overexpressed in acute myeloid leukemias. In addition, OPAL1 overexpression corresponded to ETV6-RUNX1 chromosomal translocation. In summary, we demonstrated that SEC-MAP technology is a powerful tool for detecting hundreds of proteins in clinical samples obtained from pediatric acute leukemia patients. It provides information about protein size and reveals differences in protein expression between particular leukemia subgroups. Forty-seven of SEC-MAP identified targets were validated by other conventional method in this study. PMID:26785729

Stretched Lens Array Squarerigger (SLASR) Technology Maturation

NASA Technical Reports Server (NTRS)

O'Neill, Mark; McDanal, A.J.; Howell, Joe; Lollar, Louis; Carrington, Connie; Hoppe, David; Piszczor, Michael; Suszuki, Nantel; Eskenazi, Michael; Aiken, Dan;

An EG-VEGF-dependent decrease in homeobox gene NKX3.1 contributes to cytotrophoblast dysfunction: a possible mechanism in human fetal growth restriction.

PubMed

Murthi, P; Brouillet, S; Pratt, A; Borg, Aj; Kalionis, B; Goffin, F; Tsatsaris, V; Munaut, C; Feige, Jj; Benharouga, M; Fournier, T; Alfaidy, N

2015-07-21

Idiopathic fetal growth restriction (FGR) is frequently associated with placental insufficiency. Previous reports have provided evidence that EG-VEGF (endocrine gland derived-vascular endothelial growth factor), a placental secreted protein, is expressed during the first trimester of pregnancy, controls both trophoblast proliferation and invasion, and its increased expression is associated with human FGR. In this study, we hypothesise that EG-VEGF-dependent change in placental homeobox gene expressions contribute to trophoblast dysfunction in idiopathic FGR. The changes in EG-VEGF-dependent homeobox gene expressions were determined using a Homeobox gene cDNA array on placental explants of 8-12 weeks' gestation after stimulation with EG-VEGF in vitro for 24 hours. The Homeobox gene array identified a >5-fold increase in HOXA9, HOXC8, HOXC10, HOXD1, HOXD8, HOXD9 and HOXD11, while NKX 3.1 showed a >2 fold-decrease in mRNA expression compared to untreated controls. Homeobox gene NKX3.1 was selected as a candidate because it is a downstream target of EG-VEGF and its expression and functional role are largely unknown in control and idiopathic FGR-affected placentae. Real-time PCR and immunoblotting showed a significant decrease in NKX3.1 mRNA and protein levels, respectively, in placentae from FGR compared to control pregnancies. Gene inactivation in vitro using short-interference RNA specific for NKX3.1 demonstrated an increase in BeWo cell differentiation and a decrease in HTR8-SVneo proliferation. We conclude that the decreased expression of homeobox gene NKX3.1 down-stream of EG-VEGF may contribute to the trophoblast dysfunction associated with idiopathic FGR pregnancies.

Gene Expression Analysis of CL-20-induced Reversible Neurotoxicity Reveals GABAA Receptors as Potential Target in the Earthworm Eisenia fetida

PubMed Central

Gong, Ping; Guan, Xin; Pirooznia, Mehdi; Liang, Chun; Perkins, Edward J.

2012-01-01

The earthworm Eisenia fetida is one of the most used species in standardized soil ecotoxicity tests. Endpoints such as survival, growth and reproduction are eco-toxicologically relevant but provide little mechanistic insight into toxicity pathways, especially at the molecular level. Here we applied a toxicogenomic approach to investigate the mode of action underlying the reversible neurotoxicity of hexanitrohexaazaisowurtzitane (CL-20), a cyclic nitroamine explosives compound. We developed an E. fetida-specific shotgun microarray targeting 15119 unique E. fetida transcripts. Using this array we profiled gene expression in E. fetida in response to exposure to CL-20. Eighteen earthworms were exposed for 6 days to 0.2 μg/cm2 of CL-20 on filter paper, half of which were allowed to recover in a clean environment for 7 days. Nine vehicle control earthworms were sacrificed at day 6 and 13, separately. Electrophysiological measurements indicated that the conduction velocity of earthworm medial giant nerve fiber decreased significantly after 6-day exposure to CL-20, but was restored after 7 days of recovery. Total RNA was isolated from the four treatment groups including 6-day control, 6-day exposed, 13-day control and 13-day exposed (i.e. 6-day exposure followed by 7-day recovery), and was hybridized to the 15K shot-gun oligo array. Statistical and bioinformatic analyses suggest that CL-20 initiated neurotoxicity by non-competitively blocking the ligand-gated GABAA receptor ion channel, leading to altered expression of genes involved in GABAergic, cholinergic, and Agrin-MuSK pathways. In the recovery phase, expression of affected genes returned to normality, possibly as a result of autophagy and CL-20 dissociation/metabolism. This study provides significant insights into potential mechanisms of CL-20-induced neurotoxicity and the recovery of earthworms from transient neurotoxicity stress. PMID:22191394

A facial expression of pax: Assessing children's "recognition" of emotion from faces.

PubMed

Nelson, Nicole L; Russell, James A

2016-01-01

In a classic study, children were shown an array of facial expressions and asked to choose the person who expressed a specific emotion. Children were later asked to name the emotion in the face with any label they wanted. Subsequent research often relied on the same two tasks--choice from array and free labeling--to support the conclusion that children recognize basic emotions from facial expressions. Here five studies (N=120, 2- to 10-year-olds) showed that these two tasks produce illusory recognition; a novel nonsense facial expression was included in the array. Children "recognized" a nonsense emotion (pax or tolen) and two familiar emotions (fear and jealousy) from the same nonsense face. Children likely used a process of elimination; they paired the unknown facial expression with a label given in the choice-from-array task and, after just two trials, freely labeled the new facial expression with the new label. These data indicate that past studies using this method may have overestimated children's expression knowledge. Copyright © 2015 Elsevier Inc. All rights reserved.

Arrays of probes for positional sequencing by hybridization

DOEpatents

Cantor, Charles R [Boston, MA; Prezetakiewiczr, Marek [East Boston, MA; Smith, Cassandra L [Boston, MA; Sano, Takeshi [Waltham, MA

2008-01-15

This invention is directed to methods and reagents useful for sequencing nucleic acid targets utilizing sequencing by hybridization technology comprising probes, arrays of probes and methods whereby sequence information is obtained rapidly and efficiently in discrete packages. That information can be used for the detection, identification, purification and complete or partial sequencing of a particular target nucleic acid. When coupled with a ligation step, these methods can be performed under a single set of hybridization conditions. The invention also relates to the replication of probe arrays and methods for making and replicating arrays of probes which are useful for the large scale manufacture of diagnostic aids used to screen biological samples for specific target sequences. Arrays created using PCR technology may comprise probes with 5'- and/or 3'-overhangs.

Evaluation of plasma membrane calcium/calmodulin-dependent ATPase isoform 4 as a potential target for fertility control.

PubMed

Cartwright, Elizabeth J; Neyses, Ludwig

2010-01-01

The array of contraceptives currently available is clearly inadequate and does not meet consumer demands since it is estimated that up to a quarter of all pregnancies worldwide are unintended. There is, therefore, an overwhelming global need to develop new effective, safe, ideally non-hormonal contraceptives for both male and female use. The contraceptive field, unlike other areas such as cancer, has a dearth of new targets. We have addressed this issue and propose that isoform 4 of the plasma membrane calcium ATPase is a potentially exciting novel target for fertility control. The plasma membrane calcium ATPase is a ubiquitously expressed calcium pump whose primary function in the majority of cells is to extrude calcium to the extracellular milieu. Two isoforms of this gene family, PMCA1 and PMCA4, are expressed in spermatozoa, with PMCA4 being the predominant isoform. Although this gene is ubiquitously expressed, its function is highly tissue-specific. Genetic deletion of PMCA4, in PMCA4 knockout mice, led to 100% infertility specifically in the male mutant mice due to a selective defect in sperm motility. It is important to note that the gene deletion did not affect normal mating characteristics in these mice. This phenotype was mimicked in wild-type sperm treated with the non-specific PMCA inhibitor 5-(and 6-) carboxyeosin diacetate succinimidyl ester; a proof-of-principle that inhibition of PMCA4 has potential importance in the control of fertility. This review outlines the potential for PMCA4 to be a novel target for fertility control by acting to inhibit sperm motility. It will outline the characteristics that make this target drugable and will describe methodologies to identify and validate novel inhibitors of this target.

Subcellular Localization Screening of Colletotrichum higginsianum Effector Candidates Identifies Fungal Proteins Targeted to Plant Peroxisomes, Golgi Bodies, and Microtubules.

PubMed

Robin, Guillaume P; Kleemann, Jochen; Neumann, Ulla; Cabre, Lisa; Dallery, Jean-Félix; Lapalu, Nicolas; O'Connell, Richard J

2018-01-01

The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum , encodes a large inventory of putative secreted effector proteins that are sequentially expressed at different stages of plant infection, namely appressorium-mediated penetration, biotrophy and necrotrophy. However, the destinations to which these proteins are addressed inside plant cells are unknown. In the present study, we selected 61 putative effector genes that are highly induced in appressoria and/or biotrophic hyphae. We then used Agrobacterium -mediated transformation to transiently express them as N -terminal fusions with fluorescent proteins in cells of Nicotiana benthamiana for imaging by confocal microscopy. Plant compartments labeled by the fusion proteins in N. benthamiana were validated by co-localization with specific organelle markers, by transient expression of the proteins in the true host plant, Arabidopsis thaliana , and by transmission electron microscopy-immunogold labeling. Among those proteins for which specific subcellular localizations could be verified, nine were imported into plant nuclei, three were imported into the matrix of peroxisomes, three decorated cortical microtubule arrays and one labeled Golgi stacks. Two peroxisome-targeted proteins harbored canonical C -terminal tripeptide signals for peroxisome import via the PTS1 (peroxisomal targeting signal 1) pathway, and we showed that these signals are essential for their peroxisome localization. Our findings provide valuable information about which host processes are potentially manipulated by this pathogen, and also reveal plant peroxisomes, microtubules, and Golgi as novel targets for fungal effectors.

Antisense Oligonucleotides Used to Target the DUX4 mRNA as Therapeutic Approaches in FaciosScapuloHumeral Muscular Dystrophy (FSHD)

PubMed Central

Ansseau, Eugénie; Vanderplanck, Céline; Wauters, Armelle; Harper, Scott Q.; Coppée, Frédérique; Belayew, Alexandra

2017-01-01

FacioScapuloHumeral muscular Dystrophy (FSHD) is one of the most prevalent hereditary myopathies and is generally characterized by progressive muscle atrophy affecting the face, scapular fixators; upper arms and distal lower legs. The FSHD locus maps to a macrosatellite D4Z4 repeat array on chromosome 4q35. Each D4Z4 unit contains a DUX4 gene; the most distal of which is flanked by a polyadenylation site on FSHD-permissive alleles, which allows for production of stable DUX4 mRNAs. In addition, an open chromatin structure is required for DUX4 gene transcription. FSHD thus results from a gain of function of the toxic DUX4 protein that normally is only expressed in germ line and stem cells. Therapeutic strategies are emerging that aim to decrease DUX4 expression or toxicity in FSHD muscle cells. We review here the heterogeneity of DUX4 mRNAs observed in muscle and stem cells; and the use of antisense oligonucleotides (AOs) targeting the DUX4 mRNA to interfere either with transcript cleavage/polyadenylation or intron splicing. We show in primary cultures that DUX4-targeted AOs suppress the atrophic FSHD myotube phenotype; but do not improve the disorganized FSHD myotube phenotype which could be caused by DUX4c over-expression. Thus; DUX4c might constitute another therapeutic target in FSHD. PMID:28273791

Single nucleotide polymorphisms at miR-146a/196a2 and their primary ovarian insufficiency-related target gene regulation in granulosa cells.

PubMed

Cho, Sung Hwan; An, Hui Jeong; Kim, Kyung Ah; Ko, Jung Jae; Kim, Ji Hyang; Kim, Young Ran; Ahn, Eun Hee; Rah, HyungChul; Lee, Woo Sik; Kim, Nam Keun

2017-01-01

MicroRNAs post-transcriptionally regulate gene expression in animals and plants. The aim of this study was to identify new target genes for microRNA polymorphisms (miR-146aC>G and miR-196a2T>C) in primary ovarian insufficiency (POI). We cloned and transfected miR-146aC>G and miR-196a2T>C into human granulosa cells and used microarrays and qPCR-arrays to examine the changes in the messenger RNA expression profile. We show miR-146aC>G and miR-196a2T>C change the mRNA expression patterns in granulosa cell. In each case, mRNAs were up or down-regulated after treatments with miR-146a C or G and miR-196a2 T or C. We found that miR-146a led to a significantly altered regulation of the mRNA levels of FOXO3, FOXL2 and CCND2 compared to controls. We also found that the polymorphisms of miR-146a led to a significantly altered regulation of CCND2 and FOXO3. Our results suggest that miR-146aC>G and miR-196a2T>C can regulate the levels of many of their target transcripts. In addition, specific target genes of miR-146aC>G polymorphisms may be involved in granulosa cell regulation.

Single nucleotide polymorphisms at miR-146a/196a2 and their primary ovarian insufficiency-related target gene regulation in granulosa cells

PubMed Central

Cho, Sung Hwan; An, Hui Jeong; Kim, Kyung Ah; Ko, Jung Jae; Kim, Ji Hyang; Kim, Young Ran; Ahn, Eun Hee; Rah, HyungChul; Lee, Woo Sik

2017-01-01

MicroRNAs post-transcriptionally regulate gene expression in animals and plants. The aim of this study was to identify new target genes for microRNA polymorphisms (miR-146aC>G and miR-196a2T>C) in primary ovarian insufficiency (POI). We cloned and transfected miR-146aC>G and miR-196a2T>C into human granulosa cells and used microarrays and qPCR-arrays to examine the changes in the messenger RNA expression profile. We show miR-146aC>G and miR-196a2T>C change the mRNA expression patterns in granulosa cell. In each case, mRNAs were up or down-regulated after treatments with miR-146a C or G and miR-196a2 T or C. We found that miR-146a led to a significantly altered regulation of the mRNA levels of FOXO3, FOXL2 and CCND2 compared to controls. We also found that the polymorphisms of miR-146a led to a significantly altered regulation of CCND2 and FOXO3. Our results suggest that miR-146aC>G and miR-196a2T>C can regulate the levels of many of their target transcripts. In addition, specific target genes of miR-146aC>G polymorphisms may be involved in granulosa cell regulation. PMID:28841705

HES1, a target of Notch signaling, is elevated in canine osteosarcoma, but reduced in the most aggressive tumors

PubMed Central

2013-01-01

Background Hairy and enhancer of split 1 (HES1), a basic helix-loop-helix transcriptional repressor, is a downstream target of Notch signaling. Notch signaling and HES1 expression have been linked to growth and survival in a variety of human cancer types and have been associated with increased metastasis and invasiveness in human osteosarcoma cell lines. Osteosarcoma (OSA) is an aggressive cancer demonstrating both high metastatic rate and chemotherapeutic resistance. The current study examined expression of Notch signaling mediators in primary canine OSA tumors and canine and human osteosarcoma cell lines to assess their role in OSA development and progression. Results Reverse transcriptase - quantitative PCR (RT-qPCR) was utilized to quantify HES1, HEY1, NOTCH1 and NOTCH2 gene expression in matched tumor and normal metaphyseal bone samples taken from dogs treated for appendicular OSA at the Colorado State University Veterinary Teaching Hospital. Gene expression was also assessed in tumors from dogs with a disease free interval (DFI) of <100 days compared to those with a DFI > 300 days following treatment with surgical amputation followed by standard chemotherapy. Immunohistochemistry was performed to confirm expression of HES1. Data from RT-qPCR and immunohistochemical (IHC) experiments were analyzed using REST2009 software and survival analysis based on IHC expression employed the Kaplan-Meier method and log rank analysis. Unbiased clustered images were generated from gene array analysis data for Notch/HES1 associated genes. Gene array analysis of Notch/HES1 associated genes suggested alterations in the Notch signaling pathway may contribute to the development of canine OSA. HES1 mRNA expression was elevated in tumor samples relative to normal bone, but decreased in tumor samples from dogs with a DFI < 100 days relative to those with a DFI > 300 days. NOTCH2 and HEY1 mRNA expression was also elevated in tumors relative to normal bone, but was not differentially expressed between the DFI tumor groups. Survival analysis confirmed an association between decreased HES1 immunosignal and shorter DFI. Conclusions Our findings suggest that activation of Notch signaling occurs and may contribute to the development of canine OSA. However, association of low HES1 expression and shorter DFI suggests that mechanisms that do not alter HES1 expression may drive the most aggressive tumors. PMID:23816051

Epigenetic regulation of the expression of genes involved in steroid hormone biosynthesis and action

PubMed Central

Martinez-Arguelles, Daniel B.; Papadopoulos, Vassilios

2010-01-01

Steroid hormones participate in organ development, reproduction, body homeostasis, and stress responses. The steroid machinery is expressed in a development- and tissue-specific manner, with the expression of these factors being tightly regulated by an array of transcription factors (TFs). Epigenetics provides an additional layer of gene regulation through DNA methylation and histone tail modifications. Evidence of epigenetic regulation of key steroidogenic enzymes is increasing, though this does not seem to be a predominant regulatory pathway. Steroid hormones exert their action in target tissues through steroid nuclear receptors belonging to the NR3A and NR3C families. Nuclear receptor expression levels and post-translational modifications regulate their function and dictate their sensitivity to steroid ligands. Nuclear receptors and TFs are more likely to be epigenetically regulated than proteins involved in steroidogenesis and have secondary impact on the expression of these steroidogenic enzymes. Here we review evidence for epigenetic regulation of enzymes, transcription factors, and nuclear receptors related to steroid biogenesis and action. PMID:20156469

Adaptation and adaptation transfer characteristics of five different saccade types in the monkey

PubMed Central

Fuchs, Albert F.; Soetedjo, Robijanto

2015-01-01

Shifts in the direction of gaze are accomplished by different kinds of saccades, which are elicited under different circumstances. Saccade types include targeting saccades to simple jumping targets, delayed saccades to visible targets after a waiting period, memory-guided (MG) saccades to remembered target locations, scanning saccades to stationary target arrays, and express saccades after very short latencies. Studies of human cases and neurophysiological experiments in monkeys suggest that separate pathways, which converge on a common locus that provides the motor command, generate these different types of saccade. When behavioral manipulations in humans cause targeting saccades to have persistent dysmetrias as might occur naturally from growth, aging, and injury, they gradually adapt to reduce the dysmetria. Although results differ slightly between laboratories, this adaptation generalizes or transfers to all the other saccade types mentioned above. Also, when one of the other types of saccade undergoes adaptation, it often transfers to another saccade type. Similar adaptation and transfer experiments, which allow inferences to be drawn about the site(s) of adaptation for different saccade types, have yet to be done in monkeys. Here we show that simian targeting and MG saccades adapt more than express, scanning, and delayed saccades. Adaptation of targeting saccades transfers to all the other saccade types. However, the adaptation of MG saccades transfers only to delayed saccades. These data suggest that adaptation of simian targeting saccades occurs on the pathway common to all saccade types. In contrast, only the delayed saccade command passes through the adaptation site of the MG saccade. PMID:25855693

MicroRNA Expression Profiles as Biomarkers of Minor Salivary Gland Inflammation and Dysfunction in Sjögren's Syndrome

PubMed Central

Alevizos, Ilias; Alexander, Stefanie; Turner, R. James; Illei, Gabor G.

2013-01-01

Objective MicroRNA reflect physiologic and pathologic processes and may be used as biomarkers of concurrent pathophysiologic events in complex settings such as autoimmune diseases. We generated microRNA microarray profiles from the minor salivary glands of control subjects without Sjögren's syndrome (SS) and patients with SS who had low-grade or high-grade inflammation and impaired or normal saliva production, to identify microRNA patterns specific to salivary gland inflammation or dysfunction. Methods MicroRNA expression profiles were generated by Agilent microRNA arrays. We developed a novel method for data normalization by identifying housekeeping microRNA. MicroRNA profiles were compared by unsupervised mathematical methods to test how well they distinguish between control subjects and various subsets of patients with SS. Several bioinformatics methods were used to predict the messenger RNA targets of the differentially expressed microRNA. Results MicroRNA expression patterns accurately distinguished salivary glands from control subjects and patients with SS who had low-degree or high-degree inflammation. Using real-time quantitative polymerase chain reaction, we validated 2 microRNA as markers of inflammation in an independent cohort. Comparing microRNA from patients with preserved or low salivary flow identified a set of differentially expressed microRNA, most of which were up-regulated in the group with decreased salivary gland function, suggesting that the targets of microRNA may have a protective effect on epithelial cells. The predicted biologic targets of microRNA associated with inflammation or salivary gland dysfunction identified both overlapping and distinct biologic pathways and processes. Conclusion Distinct microRNA expression patterns are associated with salivary gland inflammation and dysfunction in patients with SS, and microRNA represent a novel group of potential biomarkers. PMID:21280008

Uncovering leaf rust responsive miRNAs in wheat (Triticum aestivum L.) using high-throughput sequencing and prediction of their targets through degradome analysis.

PubMed

Kumar, Dhananjay; Dutta, Summi; Singh, Dharmendra; Prabhu, Kumble Vinod; Kumar, Manish; Mukhopadhyay, Kunal

2017-01-01

Deep sequencing identified 497 conserved and 559 novel miRNAs in wheat, while degradome analysis revealed 701 targets genes. QRT-PCR demonstrated differential expression of miRNAs during stages of leaf rust progression. Bread wheat (Triticum aestivum L.) is an important cereal food crop feeding 30 % of the world population. Major threat to wheat production is the rust epidemics. This study was targeted towards identification and functional characterizations of micro(mi)RNAs and their target genes in wheat in response to leaf rust ingression. High-throughput sequencing was used for transcriptome-wide identification of miRNAs and their expression profiling in retort to leaf rust using mock and pathogen-inoculated resistant and susceptible near-isogenic wheat plants. A total of 1056 mature miRNAs were identified, of which 497 miRNAs were conserved and 559 miRNAs were novel. The pathogen-inoculated resistant plants manifested more miRNAs compared with the pathogen infected susceptible plants. The miRNA counts increased in susceptible isoline due to leaf rust, conversely, the counts decreased in the resistant isoline in response to pathogenesis illustrating precise spatial tuning of miRNAs during compatible and incompatible interaction. Stem-loop quantitative real-time PCR was used to profile 10 highly differentially expressed miRNAs obtained from high-throughput sequencing data. The spatio-temporal profiling validated the differential expression of miRNAs between the isolines as well as in retort to pathogen infection. Degradome analysis provided 701 predicted target genes associated with defense response, signal transduction, development, metabolism, and transcriptional regulation. The obtained results indicate that wheat isolines employ diverse arrays of miRNAs that modulate their target genes during compatible and incompatible interaction. Our findings contribute to increase knowledge on roles of microRNA in wheat-leaf rust interactions and could help in rust resistance breeding programs.

PCR-free Quantification of Multiple Splice Variants in Cancer Gene by Surface Enhanced Raman Spectroscopy

PubMed Central

Sun, Lan; Irudayaraj, Joseph

2009-01-01

We demonstrate a surface enhanced Raman spectroscopy (SERS) based array platform to monitor gene expression in cancer cells in a multiplex and quantitative format without amplification steps. A strategy comprising of DNA/RNA hybridization, S1 nuclease digestion, and alkaline hydrolysis was adopted to obtain DNA targets specific to two splice junction variants Δ(9, 10) and Δ(5) of the breast cancer susceptibility gene 1 (BRCA1) from MCF-7 and MDA-MB-231 breast cancer cell lines. These two targets were identified simultaneously and their absolute quantities were estimated by a SERS strategy utilizing the inherent plasmon-phonon Raman mode of gold nanoparticle probes as a self-referencing standard to correct for variability in surface enhancement. Results were then validated by reverse transcription PCR (RT-PCR). Our proposed methodology could be expanded to a higher level of multiplexing for quantitative gene expression analysis of any gene without any amplification steps. PMID:19780515

Screening unlabeled DNA targets with randomly ordered fiber-optic gene arrays.

PubMed

Steemers, F J; Ferguson, J A; Walt, D R

2000-01-01

We have developed a randomly ordered fiber-optic gene array for rapid, parallel detection of unlabeled DNA targets with surface immobilized molecular beacons (MB) that undergo a conformational change accompanied by a fluorescence change in the presence of a complementary DNA target. Microarrays are prepared by randomly distributing MB-functionalized 3-microm diameter microspheres in an array of wells etched in a 500-microm diameter optical imaging fiber. Using several MBs, each designed to recognize a different target, we demonstrate the selective detection of genomic cystic fibrosis related targets. Positional registration and fluorescence response monitoring of the microspheres was performed using an optical encoding scheme and an imaging fluorescence microscope system.

Statistical Analysis of Microarray Data with Replicated Spots: A Case Study with Synechococcus WH8102

PubMed Central

Thomas, E. V.; Phillippy, K. H.; Brahamsha, B.; Haaland, D. M.; Timlin, J. A.; Elbourne, L. D. H.; Palenik, B.; Paulsen, I. T.

2009-01-01

Until recently microarray experiments often involved relatively few arrays with only a single representation of each gene on each array. A complete genome microarray with multiple spots per gene (spread out spatially across the array) was developed in order to compare the gene expression of a marine cyanobacterium and a knockout mutant strain in a defined artificial seawater medium. Statistical methods were developed for analysis in the special situation of this case study where there is gene replication within an array and where relatively few arrays are used, which can be the case with current array technology. Due in part to the replication within an array, it was possible to detect very small changes in the levels of expression between the wild type and mutant strains. One interesting biological outcome of this experiment is the indication of the extent to which the phosphorus regulatory system of this cyanobacterium affects the expression of multiple genes beyond those strictly involved in phosphorus acquisition. PMID:19404483

Statistical Analysis of Microarray Data with Replicated Spots: A Case Study with Synechococcus WH8102

DOE PAGES

Thomas, E. V.; Phillippy, K. H.; Brahamsha, B.; ...

2009-01-01

Until recently microarray experiments often involved relatively few arrays with only a single representation of each gene on each array. A complete genome microarray with multiple spots per gene (spread out spatially across the array) was developed in order to compare the gene expression of a marine cyanobacterium and a knockout mutant strain in a defined artificial seawater medium. Statistical methods were developed for analysis in the special situation of this case study where there is gene replication within an array and where relatively few arrays are used, which can be the case with current array technology. Due in partmore » to the replication within an array, it was possible to detect very small changes in the levels of expression between the wild type and mutant strains. One interesting biological outcome of this experiment is the indication of the extent to which the phosphorus regulatory system of this cyanobacterium affects the expression of multiple genes beyond those strictly involved in phosphorus acquisition.« less

Sex-specific microRNA expression networks in an acute mouse model of ozone-induced lung inflammation.

PubMed

Fuentes, Nathalie; Roy, Arpan; Mishra, Vikas; Cabello, Noe; Silveyra, Patricia

2018-05-08

Sex differences in the incidence and prognosis of respiratory diseases have been reported. Studies have shown that women are at increased risk of adverse health outcomes from air pollution than men, but sex-specific immune gene expression patterns and regulatory networks have not been well studied in the lung. MicroRNAs (miRNAs) are environmentally sensitive posttranscriptional regulators of gene expression that may mediate the damaging effects of inhaled pollutants in the lung, by altering the expression of innate immunity molecules. Male and female mice of the C57BL/6 background were exposed to 2 ppm of ozone or filtered air (control) for 3 h. Female mice were also exposed at different stages of the estrous cycle. Following exposure, lungs were harvested and total RNA was extracted. We used PCR arrays to study sex differences in the expression of 84 miRNAs predicted to target inflammatory and immune genes. We identified differentially expressed miRNA signatures in the lungs of male vs. female exposed to ozone. In silico pathway analyses identified sex-specific biological networks affected by exposure to ozone that ranged from direct predicted gene targeting to complex interactions with multiple intermediates. We also identified differences in miRNA expression and predicted regulatory networks in females exposed to ozone at different estrous cycle stages. Our results indicate that both sex and hormonal status can influence lung miRNA expression in response to ozone exposure, indicating that sex-specific miRNA regulation of inflammatory gene expression could mediate differential pollution-induced health outcomes in men and women.

Transcript profiling of Wilms tumors reveals connections to kidney morphogenesis and expression patterns associated with anaplasia.

PubMed

Li, Wenliang; Kessler, Patricia; Williams, Bryan R G

2005-01-13

Anaplasia (unfavorable histology) is associated with therapy resistance and poor prognosis of Wilms tumor, but the molecular basis for this phenotype is unclear. Here, we used a cDNA array with 9240 clones relevant to cancer biology and/or kidney development to examine the expression profiles of 54 Wilms tumors, five normal kidneys and fetal kidney. By linking genes differentially expressed between fetal kidney and Wilms tumors to kidney morphogenesis, we found that genes expressed at a higher level in Wilms tumors tend to be expressed more in uninduced metanephrogenic mesenchyme or blastema than in their differentiated structures. Conversely, genes expressed at a lower level in Wilms tumors tend to be expressed less in uninduced metanephrogenic mesenchyme or blastema. We also identified 97 clones representing 76 Unigenes or unclustered ESTs that clearly separate anaplastic Wilms tumors from tumors with favorable histology. Genes in this set provide insight into the nature of the abnormal nuclear morphology of anaplastic tumors and may facilitate identification of molecular targets to improve their responsiveness to treatment.

MicroRNA-139-5p acts as a tumor suppressor by targeting ELTD1 and regulating cell cycle in glioblastoma multiforme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dai, Shouping; Wang, Xianjun; Li, Xiao

MicroRNA-139-5p was identified to be significantly down-regulated in glioblastoma multiform (GBM) by miRNA array. In this report we aimed to clarify its biological function, molecular mechanisms and direct target gene in GBM. Twelve patients with GBM were analyzed for the expression of miR-139-5p by quantitative RT-PCR. miR-139-5p overexpression was established by transfecting miR-139-5p-mimic into U87MG and T98G cells, and its effects on cell proliferation were studied using MTT assay and colony formation assays. We concluded that ectopic expression of miR-139-5p in GBM cell lines significantly suppressed cell proliferation and inducing apoptosis. Bioinformatics coupled with luciferase and western blot assays alsomore » revealed that miR-139-5p suppresses glioma cell proliferation by targeting ELTD1 and regulating cell cycle. - Highlights: • miR-139-5p is downregulated in GBM. • miR-139-5p regulates cell proliferation through inducing apoptosis. • miR-139-5p regulates glioblastoma tumorigenesis by targeting 3′UTR of ELTD1. • miR-139-5p is involved in cell cycle regulation.« less

Simple method for assembly of CRISPR synergistic activation mediator gRNA expression array.

PubMed

Vad-Nielsen, Johan; Nielsen, Anders Lade; Luo, Yonglun

2018-05-20

When studying complex interconnected regulatory networks, effective methods for simultaneously manipulating multiple genes expression are paramount. Previously, we have developed a simple method for generation of an all-in-one CRISPR gRNA expression array. We here present a Golden Gate Assembly-based system of synergistic activation mediator (SAM) compatible CRISPR/dCas9 gRNA expression array for the simultaneous activation of multiple genes. Using this system, we demonstrated the simultaneous activation of the transcription factors, TWIST, SNAIL, SLUG, and ZEB1 a human breast cancer cell line. Copyright © 2018 Elsevier B.V. All rights reserved.

Fiber-array based optogenetic prosthetic system for stimulation therapy

NASA Astrophysics Data System (ADS)

Gu, Ling; Cote, Chris; Tejeda, Hector; Mohanty, Samarendra

2012-02-01

Recent advent of optogenetics has enabled activation of genetically-targeted neuronal cells using low intensity blue light with high temporal precision. Since blue light is attenuated rapidly due to scattering and absorption in neural tissue, optogenetic treatment of neurological disorders may require stimulation of specific cell types in multiple regions of the brain. Further, restoration of certain neural functions (vision, and auditory etc) requires accurate spatio-temporal stimulation patterns rather than just precise temporal stimulation. In order to activate multiple regions of the central nervous system in 3D, here, we report development of an optogenetic prosthetic comprising of array of fibers coupled to independently-controllable LEDs. This design avoids direct contact of LEDs with the brain tissue and thus does not require electrical and heat isolation, which can non-specifically stimulate and damage the local brain regions. The intensity, frequency, and duty cycle of light pulses from each fiber in the array was controlled independently using an inhouse developed LabView based program interfaced with a microcontroller driving the individual LEDs. While the temporal profile of the light pulses was controlled by varying the current driving the LED, the beam profile emanating from each fiber tip could be sculpted by microfabrication of the fiber tip. The fiber array was used to stimulate neurons, expressing channelrhodopsin-2, in different locations within the brain or retina. Control of neural activity in the mice cortex, using the fiber-array based prosthetic, is evaluated from recordings made with multi-electrode array (MEA). We also report construction of a μLED array based prosthetic for spatio-temporal stimulation of cortex.

Processes for design, construction and utilisation of arrays of light-emitting diodes and light-emitting diode-coupled optical fibres for multi-site brain light delivery

PubMed Central

Bernstein, Jacob G.; Allen, Brian D.; Guerra, Alexander A.; Boyden, Edward S.

2016-01-01

Optogenetics enables light to be used to control the activity of genetically targeted cells in the living brain. Optical fibers can be used to deliver light to deep targets, and LEDs can be spatially arranged to enable patterned light delivery. In combination, arrays of LED-coupled optical fibers can enable patterned light delivery to deep targets in the brain. Here we describe the process flow for making LED arrays and LED-coupled optical fiber arrays, explaining key optical, electrical, thermal, and mechanical design principles to enable the manufacturing, assembly, and testing of such multi-site targetable optical devices. We also explore accessory strategies such as surgical automation approaches as well as innovations to enable low-noise concurrent electrophysiology. PMID:26798482

A microRNA profile of human CD8(+) regulatory T cells and characterization of the effects of microRNAs on Treg cell-associated genes.

PubMed

Jebbawi, Fadi; Fayyad-Kazan, Hussein; Merimi, Makram; Lewalle, Philippe; Verougstraete, Jean-Christophe; Leo, Oberdan; Romero, Pedro; Burny, Arsene; Badran, Bassam; Martiat, Philippe; Rouas, Redouane

2014-08-06

Recently, regulatory T (Treg) cells have gained interest in the fields of immunopathology, transplantation and oncoimmunology. Here, we investigated the microRNA expression profile of human natural CD8(+)CD25(+) Treg cells and the impact of microRNAs on molecules associated with immune regulation. We purified human natural CD8(+) Treg cells and assessed the expression of FOXP3 and CTLA-4 by flow cytometry. We have also tested the ex vivo suppressive capacity of these cells in mixed leukocyte reactions. Using TaqMan low-density arrays and microRNA qPCR for validation, we could identify a microRNA 'signature' for CD8(+)CD25(+)FOXP3(+)CTLA-4(+) natural Treg cells. We used the 'TargetScan' and 'miRBase' bioinformatics programs to identify potential target sites for these microRNAs in the 3'-UTR of important Treg cell-associated genes. The human CD8(+)CD25(+) natural Treg cell microRNA signature includes 10 differentially expressed microRNAs. We demonstrated an impact of this signature on Treg cell biology by showing specific regulation of FOXP3, CTLA-4 and GARP gene expression by microRNA using site-directed mutagenesis and a dual-luciferase reporter assay. Furthermore, we used microRNA transduction experiments to demonstrate that these microRNAs impacted their target genes in human primary Treg cells ex vivo. We are examining the biological relevance of this 'signature' by studying its impact on other important Treg cell-associated genes. These efforts could result in a better understanding of the regulation of Treg cell function and might reveal new targets for immunotherapy in immune disorders and cancer.

The HIV Nef protein modulates cellular and exosomal miRNA profiles in human monocytic cells.

PubMed

Aqil, Madeeha; Naqvi, Afsar Raza; Mallik, Saurav; Bandyopadhyay, Sanghamitra; Maulik, Ujjwal; Jameel, Shahid

2014-01-01

The HIV Nef protein is a multifunctional virulence factor that perturbs intracellular membranes and signalling and is secreted into exosomes. While Nef-containing exosomes have a distinct proteomic profile, no comprehensive analysis of their miRNA cargo has been carried out. Since Nef functions as a viral suppressor of RNA interference and disturbs the distribution of RNA-induced silencing complex proteins between cells and exosomes, we hypothesized that it might also affect the export of miRNAs into exosomes. Exosomes were purified from human monocytic U937 cells that stably expressed HIV-1 Nef. The RNA from cells and exosomes was profiled for 667 miRNAs using a Taqman Low Density Array. Selected miRNAs and their mRNA targets were validated by quantitative RT-PCR. Bioinformatics analyses were used to identify targets and predict pathways. Nef expression affected a significant fraction of miRNAs in U937 cells. Our analysis showed 47 miRNAs to be selectively secreted into Nef exosomes and 2 miRNAs to be selectively retained in Nef-expressing cells. The exosomal miRNAs were predicted to target several cellular genes in inflammatory cytokine and other pathways important for HIV pathogenesis, and an overwhelming majority had targets within the HIV genome. This is the first study to report miRnome analysis of HIV Nef expressing monocytes and exosomes. Our results demonstrate that Nef causes large-scale dysregulation of cellular miRNAs, including their secretion through exosomes. We suggest this to be a novel viral strategy to affect pathogenesis and to limit the effects of RNA interference on viral replication and persistence.

Analysis of differential gene expression by bead-based fiber-optic array in nonfunctioning pituitary adenomas.

PubMed

Jiang, Z; Gui, S; Zhang, Y

2011-05-01

Nonfunctioning pituitary adenomas (NFPAs) are relatively common, accounting for 30% of all pituitary adenomas; however, their pathogenesis remains enigmatic. To explore the possible pathogenesis of NFPAs, we used fiber-optic BeadArray to examine gene expression in 5 NFPAs compared with 3 normal pituitaries. 4 differentially expressed genes were chosen randomly for validation by reverse transcriptase-real time quantitative polymerase chain reaction (RT-qPCR). We then analyzed the differentially expressed gene profile with Kyoto Encyclopedia of Genes and Genomes (KEGG). The array analysis indentified significant increases in the expression of 1,402 genes and 383 expressed sequence tags (ESTs), and decreases in 1,697 genes and 113 ESTs in the NFPAs. Bioinformatic and pathway analysis showed that the genes HIGD1B, FAM5C, PMAIP1 and the pathway cell-cycle regulation may play an important role in tumorigenesis and progression of NFPAs. Our data suggest fiber-optic BeadArray combined with pathway analysis of differential gene expression profile appears to be a valid approach for investigating the pathogenesis of tumors. © Georg Thieme Verlag KG Stuttgart · New York.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Luo-Qiao; Zhang, Yue; Yan, Huan

miR-373 was reported to be elevated in several tumors; however, the role of miR-373 in cervical cancer has not been investigated. In this study we aimed to investigate the role of miR-373 in tumorigenicity of cervical cancer cells in vivo and in vitro. The expression of miR-373 was investigated using real-time reverse transcription-polymerase chain reaction assay in 45 cervical specimens and cervical cancer cell lines. The role of miR-373 in tumorigenicity of cervical cancer cells was assessed by cell proliferation, colony formation in vitro as well as tumor growth assays in vivo with the overexpression of miR-373 or gene silencing. The functional target genemore » of miR-373 in cervical cancer cells was identified using integrated bioinformatics analysis, gene expression arrays, and luciferase assay. We founded that the expression of miR-373 is upregulated in human cervical cancer tissues and cervical carcinoma cell lines when compared to the corresponding noncancerous tissues. Ectopic overexpression of miR-373 in human cervical cancer cells promoted cell growth in vitro and tumorigenicity in vivo, whereas silencing the expression of miR-373 decreased the rate of cell growth. YOD1 was identified as a direct and functional target of miR-373 in cervical cancer cells. Expression levels of miR-373 were inversely correlated with YOD1 levels in human cervical cancer tissues. RNAi-mediated knockdown of YOD1 phenocopied the proliferation-promoting effect of miR-373. Moreover, overexpression of YOD1 abrogated miR-373-induced proliferation of cervical cancer cells. These results demonstrate that miR-373 increases proliferation by directly targeting YOD1, a new potential therapeutic target in cervical cancer. - Highlights: • The expression of miR-373 is upregulated in human cervical cancer tissues. • miR-373 effects as oncogenic miRNA in cervical cancer in vitro and in vivo. • miR-373 increases proliferation of cervical cancer cells by directly targeting YOD1.« less

Cigarette Smoking Decreases Global MicroRNA Expression in Human Alveolar Macrophages

PubMed Central

Graff, Joel W.; Powers, Linda S.; Dickson, Anne M.; Kim, Jongkwang; Reisetter, Anna C.; Hassan, Ihab H.; Kremens, Karol; Gross, Thomas J.

2012-01-01

Human alveolar macrophages are critical components of the innate immune system. Cigarette smoking-induced changes in alveolar macrophage gene expression are linked to reduced resistance to pulmonary infections and to the development of emphysema/COPD. We hypothesized that microRNAs (miRNAs) could control, in part, the unique messenger RNA (mRNA) expression profiles found in alveolar macrophages of cigarette smokers. Activation of macrophages with different stimuli in vitro leads to a diverse range of M1 (inflammatory) and M2 (anti-inflammatory) polarized phenotypes that are thought to mimic activated macrophages in distinct tissue environments. Microarray mRNA data indicated that smoking promoted an “inverse” M1 mRNA expression program, defined by decreased expression of M1-induced transcripts and increased expression of M1-repressed transcripts with few changes in M2-regulated transcripts. RT-PCR arrays identified altered expression of many miRNAs in alveolar macrophages of smokers and a decrease in global miRNA abundance. Stratification of human subjects suggested that the magnitude of the global decrease in miRNA abundance was associated with smoking history. We found that many of the miRNAs with reduced expression in alveolar macrophages of smokers were predicted to target mRNAs upregulated in alveolar macrophages of smokers. For example, miR-452 is predicted to target the transcript encoding MMP12, an important effector of smoking-related diseases. Experimental antagonism of miR-452 in differentiated monocytic cells resulted in increased expression of MMP12. The comprehensive mRNA and miRNA expression profiles described here provide insight into gene expression regulation that may underlie the adverse effects cigarette smoking has on alveolar macrophages. PMID:22952876

MicroRNA Expression in Alpha and Beta Cells of Human Pancreatic Islets

PubMed Central

Vargas, Nancy; Rosero, Samuel; Piroso, Julieta; Ichii, Hirohito; Umland, Oliver; Zhijie, Jiang; Tsinoremas, Nicholas; Ricordi, Camillo; Inverardi, Luca; Domínguez-Bendala, Juan; Pastori, Ricardo L.

2013-01-01

microRNAs (miRNAs) play an important role in pancreatic development and adult β-cell physiology. Our hypothesis is based on the assumption that each islet cell type has a specific pattern of miRNA expression. We sought to determine the profile of miRNA expression in α-and β-cells, the main components of pancreatic islets, because this analysis may lead to a better understanding of islet gene regulatory pathways. Highly enriched (>98%) subsets of human α-and β-cells were obtained by flow cytometric sorting after intracellular staining with c-peptide and glucagon antibody. The method of sorting based on intracellular staining is possible because miRNAs are stable after fixation. MiRNA expression levels were determined by quantitative high throughput PCR-based miRNA array platform screening. Most of the miRNAs were preferentially expressed in β-cells. From the total of 667 miRNAs screened, the Significant Analysis of Microarray identified 141 miRNAs, of which only 7 were expressed more in α-cells (α-miRNAs) and 134 were expressed more in β-cells (β-miRNAs). Bioinformatic analysis identified potential targets of β-miRNAs analyzing the Beta Cell Gene Atlas, described in the T1Dbase, the web platform, supporting the type 1 diabetes (T1D) community. cMaf, a transcription factor regulating glucagon expression expressed selectively in α-cells (TFα) is targeted by β-miRNAs; miR-200c, miR-125b and miR-182. Min6 cells treated with inhibitors of these miRNAs show an increased expression of cMaf RNA. Conversely, over expression of miR-200c, miR-125b or miR-182 in the mouse alpha cell line αTC6 decreases the level of cMAF mRNA and protein. MiR-200c also inhibits the expression of Zfpm2, a TFα that inhibits the PI3K signaling pathway, at both RNA and protein levels. In conclusion, we identified miRNAs differentially expressed in pancreatic α- and β-cells and their potential transcription factor targets that could add new insights into different aspects of islet biology and pathophysiology. PMID:23383059

MicroRNA expression in alpha and beta cells of human pancreatic islets.

PubMed

Klein, Dagmar; Misawa, Ryosuke; Bravo-Egana, Valia; Vargas, Nancy; Rosero, Samuel; Piroso, Julieta; Ichii, Hirohito; Umland, Oliver; Zhijie, Jiang; Tsinoremas, Nicholas; Ricordi, Camillo; Inverardi, Luca; Domínguez-Bendala, Juan; Pastori, Ricardo L

2013-01-01

microRNAs (miRNAs) play an important role in pancreatic development and adult β-cell physiology. Our hypothesis is based on the assumption that each islet cell type has a specific pattern of miRNA expression. We sought to determine the profile of miRNA expression in α-and β-cells, the main components of pancreatic islets, because this analysis may lead to a better understanding of islet gene regulatory pathways. Highly enriched (>98%) subsets of human α-and β-cells were obtained by flow cytometric sorting after intracellular staining with c-peptide and glucagon antibody. The method of sorting based on intracellular staining is possible because miRNAs are stable after fixation. MiRNA expression levels were determined by quantitative high throughput PCR-based miRNA array platform screening. Most of the miRNAs were preferentially expressed in β-cells. From the total of 667 miRNAs screened, the Significant Analysis of Microarray identified 141 miRNAs, of which only 7 were expressed more in α-cells (α-miRNAs) and 134 were expressed more in β-cells (β-miRNAs). Bioinformatic analysis identified potential targets of β-miRNAs analyzing the Beta Cell Gene Atlas, described in the T1Dbase, the web platform, supporting the type 1 diabetes (T1D) community. cMaf, a transcription factor regulating glucagon expression expressed selectively in α-cells (TFα) is targeted by β-miRNAs; miR-200c, miR-125b and miR-182. Min6 cells treated with inhibitors of these miRNAs show an increased expression of cMaf RNA. Conversely, over expression of miR-200c, miR-125b or miR-182 in the mouse alpha cell line αTC6 decreases the level of cMAF mRNA and protein. MiR-200c also inhibits the expression of Zfpm2, a TFα that inhibits the PI3K signaling pathway, at both RNA and protein levels.In conclusion, we identified miRNAs differentially expressed in pancreatic α- and β-cells and their potential transcription factor targets that could add new insights into different aspects of islet biology and pathophysiology.

Testing implications of varying targets for Bordetella pertussis: comparison of the FilmArray Respiratory Panel and the Focus B. pertussis PCR assay

PubMed Central

Jerris, Robert C; Williams, Sally R; MacDonald, Heather J; Ingebrigtsen, Danielle R; Westblade, Lars F; Rogers, Beverly B

2015-01-01

Background The FilmArray Respiratory Panel (RP) detects multiple pathogens, including Bordetella pertussis. The multiplex PCR system is appropriate for a core laboratory or point of care due to ease of use. The purpose of this study is to compare the analytical sensitivity of the FilmArray RP, which targets the promoter region of the B. pertussis toxin gene, with the Focus real-time PCR assay, which targets the insertion sequence IS481. Methods Seventy-one specimens from patients aged 1 month to 18 years, which had tested positive for B. pertussis using the Focus assay, were analysed using the FilmArray RP. Results Forty-six specimens were positive for B. pertussis by both the Focus and the FilmArray RP assays. Twenty-five specimens were negative for B. pertussis using the FilmArray RP assay, but positive using the Focus assay. Conclusions The FilmArray RP assays will detect approximately 1/3 less cases of B. pertussis than the Focus assay. PMID:25742911

GeneChip{sup {trademark}} screening assay for cystic fibrosis mutations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cronn, M.T.; Miyada, C.G.; Fucini, R.V.

1994-09-01

GeneChip{sup {trademark}} assays are based on high density, carefully designed arrays of short oligonucleotide probes (13-16 bases) built directly on derivatized silica substrates. DNA target sequence analysis is achieved by hybridizing fluorescently labeled amplification products to these arrays. Fluorescent hybridization signals located within the probe array are translated into target sequence information using the known probe sequence at each array feature. The mutation screening assay for cystic fibrosis includes sets of oligonucleotide probes designed to detect numerous different mutations that have been described in 14 exons and one intron of the CFTR gene. Each mutation site is addressed by amore » sub-array of at least 40 probe sequences, half designed to detect the wild type gene sequence and half designed to detect the reported mutant sequence. Hybridization with homozygous mutant, homozygous wild type or heterozygous targets results in distinctive hybridization patterns within a sub-array, permitting specific discrimination of each mutation. The GeneChip probe arrays are very small (approximately 1 cm{sup 2}). There miniature size coupled with their high information content make GeneChip probe arrays a useful and practical means for providing CF mutation analysis in a clinical setting.« less

Reverse-phase protein array profiling of oropharyngeal cancer and significance of PIK3CA mutations in HPV-associated head and neck cancer.

PubMed

Sewell, Andrew; Brown, Brandee; Biktasova, Asel; Mills, Gordon B; Lu, Yiling; Tyson, Darren R; Issaeva, Natalia; Yarbrough, Wendell G

2014-05-01

Human papilloma virus (HPV)-associated (HPV+) oropharyngeal squamous cell carcinomas (OPSCC) have different molecular and biologic characteristics and clinical behavior compared with HPV-negative (HPV-) OPSCC. PIK3CA mutations are more common in HPV(+) OPSCC. To define molecular differences and tumor subsets, protein expression and phosphorylation were compared between HPV(+) and HPV(-) OPSCC and between tumors with and without PIK3CA mutations. Expression of 137 total and phosphorylated proteins was evaluated by reverse-phase protein array in 29 HPV(+) and 13 HPV(-) prospectively collected OPSCCs. Forty-seven OPSCCs were tested for hotspot-activating mutations in PIK3CA and AKT. Activation of PIK3CA downstream targets and sensitivity to pathway inhibitors were determined in HPV(+) head and neck cancer cells overexpressing wild-type or mutant PIK3CA. Analyses revealed 41 differentially expressed proteins between HPV(+) and HPV(-) OPSCC categorized into functional groups: DNA repair, cell cycle, apoptosis, phosphoinositide 3-kinase (PI3K)/AKT/mTOR, and receptor kinase pathways. All queried DNA repair proteins were significantly upregulated in HPV(+) samples. A total of 8 of 33 HPV(+) and 0 of 14 HPV(-) tumors contained activating PIK3CA mutations. Despite all activating PIK3CA mutations occurring in HPV(+) samples, HPV(+) tumors had lower mean levels of activated AKT and downstream AKT target phosphorylation. Ectopic expression of mutant PIK3CA in HPV(+) cells increased mTOR, but not AKT activity. HPV E6/E7 overexpression inhibited AKT phosphorylation in HPV-negative cells. Mutant PIK3CA overexpressing cells were more sensitive to a dual PI3K/mTOR inhibitor compared with an AKT inhibitor. Protein expression analyses suggest that HPV(+) and HPV(-) OPSCC differentially activate DNA repair, cell cycle, apoptosis, PI3K/AKT/mTOR, and receptor kinase pathways. PIK3CA mutations are more common in HPV(+) OPSCC and are associated with activation of mTOR, but not AKT. These data suggest that inhibitors for mTOR may have activity against HPV(+) PIK3CA mutant oropharyngeal cancers. ©2014 AACR.

Downregulation of MCT1 inhibits tumor growth, metastasis and enhances chemotherapeutic efficacy in osteosarcoma through regulation of the NF-κB pathway.

PubMed

Zhao, Zhiqiang; Wu, Man-Si; Zou, Changye; Tang, Qinglian; Lu, Jinchang; Liu, Dawei; Wu, Yuanzhong; Yin, Junqiang; Xie, Xianbiao; Shen, Jingnan; Kang, Tiebang; Wang, Jin

2014-01-01

Monocarboxylate transporter isoform 1 (MCT1) is an important member of the proton-linked MCT family and has been reported in an array of human cancer cell lines and primary human tumors. MCT1 expression is associated with developing a new therapeutic approach for cancer. In this study, we initially showed that MCT1 is expressed in a variety of human osteosarcoma cell lines. Moreover, we evaluated the therapeutic response of targeting MCT1 using shRNA or MCT1 inhibitor. Inhibiting MCT1 delayed tumor growth in vitro and in vivo, including in an orthotopic model of osteosarcoma. Targeting MCT1 greatly enhanced the sensitivity of human osteosarcoma cells to the chemotherapeutic drugs adriamycin (ADM). In addition, we observed that MCT1 knockdown significantly suppressed the metastatic activity of osteosarcoma, including wound healing, invasion and migration. Further mechanistic studies revealed that the antitumor effects of targeting MCT1 might be related to the NF-κB pathway. Immunochemistry assay showed that MCT1 was an independent positive prognostic marker in osteosarcoma patients. In conclusion, our data, for the first time, demonstrate that MCT1 inhibition has antitumor potential which is associated with the NF-κB pathway, and high MCT1 expression predicates poor overall survival in patients with osteosarcoma. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Functional Analysis With a Barcoder Yeast Gene Overexpression System

PubMed Central

Douglas, Alison C.; Smith, Andrew M.; Sharifpoor, Sara; Yan, Zhun; Durbic, Tanja; Heisler, Lawrence E.; Lee, Anna Y.; Ryan, Owen; Göttert, Hendrikje; Surendra, Anu; van Dyk, Dewald; Giaever, Guri; Boone, Charles; Nislow, Corey; Andrews, Brenda J.

2012-01-01

Systematic analysis of gene overexpression phenotypes provides an insight into gene function, enzyme targets, and biological pathways. Here, we describe a novel functional genomics platform that enables a highly parallel and systematic assessment of overexpression phenotypes in pooled cultures. First, we constructed a genome-level collection of ~5100 yeast barcoder strains, each of which carries a unique barcode, enabling pooled fitness assays with a barcode microarray or sequencing readout. Second, we constructed a yeast open reading frame (ORF) galactose-induced overexpression array by generating a genome-wide set of yeast transformants, each of which carries an individual plasmid-born and sequence-verified ORF derived from the Saccharomyces cerevisiae full-length EXpression-ready (FLEX) collection. We combined these collections genetically using synthetic genetic array methodology, generating ~5100 strains, each of which is barcoded and overexpresses a specific ORF, a set we termed “barFLEX.” Additional synthetic genetic array allows the barFLEX collection to be moved into different genetic backgrounds. As a proof-of-principle, we describe the properties of the barFLEX overexpression collection and its application in synthetic dosage lethality studies under different environmental conditions. PMID:23050238

PhylArray: phylogenetic probe design algorithm for microarray.

PubMed

Militon, Cécile; Rimour, Sébastien; Missaoui, Mohieddine; Biderre, Corinne; Barra, Vincent; Hill, David; Moné, Anne; Gagne, Geneviève; Meier, Harald; Peyretaillade, Eric; Peyret, Pierre

2007-10-01

Microbial diversity is still largely unknown in most environments, such as soils. In order to get access to this microbial 'black-box', the development of powerful tools such as microarrays are necessary. However, the reliability of this approach relies on probe efficiency, in particular sensitivity, specificity and explorative power, in order to obtain an image of the microbial communities that is close to reality. We propose a new probe design algorithm that is able to select microarray probes targeting SSU rRNA at any phylogenetic level. This original approach, implemented in a program called 'PhylArray', designs a combination of degenerate and non-degenerate probes for each target taxon. Comparative experimental evaluations indicate that probes designed with PhylArray yield a higher sensitivity and specificity than those designed by conventional approaches. Applying the combined PhyArray/GoArrays strategy helps to optimize the hybridization performance of short probes. Finally, hybridizations with environmental targets have shown that the use of the PhylArray strategy can draw attention to even previously unknown bacteria.

Colorectal cancer cell-derived exosomes containing miR-10b regulate fibroblast cells via the PI3K/Akt pathway.

PubMed

Dai, Guangyao; Yao, Xiaoguang; Zhang, Yubin; Gu, Jianbin; Geng, Yunfeng; Xue, Fei; Zhang, Jingcheng

2018-04-01

Cancer-associated fibroblasts (CAFs) contribute to the proliferation of colorectal cancer(CRC) cells. However, the mechanism by which CAFs develop in the tumor microenvironment remains unknown. Exosomes may be involved in activating CAFs. Using a miRNA expression profiling array, we determined the miRNA expression profile of secretory exosomes in CRC cells and then identified potential miRNAs with significant differential expression compared to normal cells via enrichment analysis. Predicted targets of candidate miRNAs were then assessed via bioinformatics analysis. Realtime qPCR, western blot, and cell cycle analyses were performed to evaluate the role of candidate exosomal miRNAs. Luciferase reporter assays were applied to confirm whether candidate exosomal miRNAs control target pathway expression. A CRC xenograft mouse model was constructed to evaluate tumor growth in vivo. Exosomes from CRC cells contained significantly higher levels of miR-10b than did exosomes from normal colorectal epithelial cells. Moreover, exosomes containing miR-10b were transferred to fibroblasts. Bioinformatics analysis identified PIK3CA, as a potential target of miR-10b. Luciferase reporter assays confirmed that miR-10b directly inhibited PIK3CA expression. Co-culturing fibroblasts with exosomes containing miR-10b significantly suppressed PIK3CA expression and decreased PI3K/Akt/mTOR pathway activity. Finally, exosomes containing miR-10b reduced fibroblast proliferation but promoted expression of TGF-β and SM α-actin, suggesting that exosomal miR-10b may activate fibroblasts to become CAFs that express myofibroblast markers. These activated fibroblasts were able to promote CRC growth in vitro and in vivo. CRC-derived exosomes actively promote disease progression by modulating surrounding stromal cells, which subsequently acquire features of CAFs. Copyright © 2018 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.

Active suppression after involuntary capture of attention.

PubMed

Sawaki, Risa; Luck, Steven J

2013-04-01

After attention has been involuntarily captured by a distractor, how is it reoriented toward a target? One possibility is that attention to the distractor passively fades over time, allowing the target to become attended. Another possibility is that the captured location is actively suppressed so that attention can be directed toward the target location. The present study investigated this issue with event-related potentials (ERPs), focusing on the N2pc component (a neural measure of attentional deployment) and the Pd component (a neural measure of attentional suppression). Observers identified a color-defined target in a search array, which was preceded by a task-irrelevant cue array. When the cue array contained an item that matched the target color, this item captured attention (as measured both behaviorally and with the N2pc component). This capture of attention was followed by active suppression (indexed by the Pd component), and this was then followed by a reorienting of attention toward the target in the search array (indexed by the N2pc component). These findings indicate that the involuntary capture of attention by a distractor is followed by an active suppression process that presumably facilitates the subsequent voluntary orienting of attention to the target.

Enhanced identification and biological validation of differential gene expression via Illumina whole-genome expression arrays through the use of the model-based background correction methodology

PubMed Central

Ding, Liang-Hao; Xie, Yang; Park, Seongmi; Xiao, Guanghua; Story, Michael D.

2008-01-01

Despite the tremendous growth of microarray usage in scientific studies, there is a lack of standards for background correction methodologies, especially in single-color microarray platforms. Traditional background subtraction methods often generate negative signals and thus cause large amounts of data loss. Hence, some researchers prefer to avoid background corrections, which typically result in the underestimation of differential expression. Here, by utilizing nonspecific negative control features integrated into Illumina whole genome expression arrays, we have developed a method of model-based background correction for BeadArrays (MBCB). We compared the MBCB with a method adapted from the Affymetrix robust multi-array analysis algorithm and with no background subtraction, using a mouse acute myeloid leukemia (AML) dataset. We demonstrated that differential expression ratios obtained by using the MBCB had the best correlation with quantitative RT–PCR. MBCB also achieved better sensitivity in detecting differentially expressed genes with biological significance. For example, we demonstrated that the differential regulation of Tnfr2, Ikk and NF-kappaB, the death receptor pathway, in the AML samples, could only be detected by using data after MBCB implementation. We conclude that MBCB is a robust background correction method that will lead to more precise determination of gene expression and better biological interpretation of Illumina BeadArray data. PMID:18450815

Arabidopsis gene expression patterns during spaceflight

NASA Astrophysics Data System (ADS)

Paul, A.-L.; Ferl, R. J.

The exposure of Arabidopsis thaliana (Arabidopsis) plants to spaceflight environments resulted in the differential expression of hundreds of genes. A 5 day mission on orbiter Columbia in 1999 (STS-93) carried transgenic Arabidopsis plants engineered with a transgene composed of the alcohol dehydrogenase (Adh) gene promoter linked to the β -Glucuronidase (GUS) reporter gene. The plants were used to evaluate the effects of spaceflight on two fronts. First, expression patterns visualized with the Adh/GUS transgene were used to address specifically the possibility that spaceflight induces a hypoxic stress response, and to assess whether any spaceflight response was similar to control terrestrial hypoxia-induced gene expression patterns. (Paul et al., Plant Physiol. 2001, 126:613). Second, genome-wide patterns of native gene expression were evaluated utilizing the Affymetrix ATH1 GeneChip? array of 8,000 Arabidopsis genes. As a control for the veracity of the array analyses, a selection of genes identified with the arrays was further characterized with quantitative Real-Time RT PCR (ABI - TaqmanTM). Comparison of the patterns of expression for arrays of hybridized with RNA isolated from plants exposed to spaceflight compared to the control arrays revealed hundreds of genes that were differentially expressed in response to spaceflight, yet most genes that are hallmarks of hypoxic stress were unaffected. These results will be discussed in light of current models for plant responses to the spaceflight environment, and with regard to potential future flight opportunities.

Dimensionality of Data Matrices with Applications to Gene Expression Profiles

ERIC Educational Resources Information Center

Feng, Xingdong

2009-01-01

Probe-level microarray data are usually stored in matrices. Take a given probe set (gene), for example, each row of the matrix corresponds to an array, and each column corresponds to a probe. Often, people summarize each array by the gene expression level. Is one number sufficient to summarize a whole probe set for a specific gene in an array?…

Skin Dendritic Cell Targeting via Microneedle Arrays Laden with Antigen-Encapsulated Poly-d,l-lactide-co-Glycolide Nanoparticles Induces Efficient Antitumor and Antiviral Immune Responses

PubMed Central

2013-01-01

The efficacious delivery of antigens to antigen-presenting cells (APCs), in particular, to dendritic cells (DCs), and their subsequent activation remains a significant challenge in the development of effective vaccines. This study highlights the potential of dissolving microneedle (MN) arrays laden with nanoencapsulated antigen to increase vaccine immunogenicity by targeting antigen specifically to contiguous DC networks within the skin. Following in situ uptake, skin-resident DCs were able to deliver antigen-encapsulated poly-d,l-lactide-co-glycolide (PGLA) nanoparticles to cutaneous draining lymph nodes where they subsequently induced significant expansion of antigen-specific T cells. Moreover, we show that antigen-encapsulated nanoparticle vaccination via microneedles generated robust antigen-specific cellular immune responses in mice. This approach provided complete protection in vivo against both the development of antigen-expressing B16 melanoma tumors and a murine model of para-influenza, through the activation of antigen-specific cytotoxic CD8+ T cells that resulted in efficient clearance of tumors and virus, respectively. In addition, we show promising findings that nanoencapsulation facilitates antigen retention into skin layers and provides antigen stability in microneedles. Therefore, the use of biodegradable polymeric nanoparticles for selective targeting of antigen to skin DC subsets through dissolvable MNs provides a promising technology for improved vaccination efficacy, compliance, and coverage. PMID:23373658

Short-range/Long-range Integrated Target (SLIT) for Video Guidance Sensor Rendezvous and Docking

NASA Technical Reports Server (NTRS)

Roe, Fred D. (Inventor); Bryan, Thomas C. (Inventor)

2009-01-01

A laser target reflector assembly for mounting upon spacecraft having a long-range reflector array formed from a plurality of unfiltered light reflectors embedded in an array pattern upon a hemispherical reflector disposed upon a mounting plate. The reflector assembly also includes a short-range reflector array positioned upon the mounting body proximate to the long-range reflector array. The short-range reflector array includes three filtered light reflectors positioned upon extensions from the mounting body. The three filtered light reflectors retro-reflect substantially all incident light rays that are transmissive by their monochromatic filters and received by the three filtered light reflectors. In one embodiment the short-range reflector array is embedded within the hemispherical reflector,

Visual and Vestibular Determinants of Perceived Eye-Level

NASA Technical Reports Server (NTRS)

Cohen, Malcolm Martin

2003-01-01

Both gravitational and optical sources of stimulation combine to determine the perceived elevations of visual targets. The ways in which these sources of stimulation combine with one another in operational aeronautical environments are critical for pilots to make accurate judgments of the relative altitudes of other aircraft and of their own altitude relative to the terrain. In a recent study, my colleagues and I required eighteen observers to set visual targets at their apparent horizon while they experienced various levels of G(sub z) in the human centrifuge at NASA-Ames Research Center. The targets were viewed in darkness and also against specific background optical arrays that were oriented at various angles with respect to the vertical; target settings were lowered as Gz was increased; this effect was reduced when the background optical array was visible. Also, target settings were displaced in the direction that the background optical array was pitched. Our results were attributed to the combined influences of otolith-oculomotor mechanisms that underlie the elevator illusion and visual-oculomotor mechanisms (optostatic responses) that underlie the perceptual effects of viewing pitched optical arrays that comprise the background. In this paper, I present a mathematical model that describes the independent and combined effects of G(sub z) intensity and the orientation and structure of background optical arrays; the model predicts quantitative deviations from normal accurate perceptions of target localization under a variety of conditions. Our earlier experimental results and the mathematical model are described in some detail, and the effects of viewing specific optical arrays under various gravitational-inertial conditions encountered in aeronautical environments are discussed.

Novel statistical framework to identify differentially expressed genes allowing transcriptomic background differences.

PubMed

Ling, Zhi-Qiang; Wang, Yi; Mukaisho, Kenichi; Hattori, Takanori; Tatsuta, Takeshi; Ge, Ming-Hua; Jin, Li; Mao, Wei-Min; Sugihara, Hiroyuki

2010-06-01

Tests of differentially expressed genes (DEGs) from microarray experiments are based on the null hypothesis that genes that are irrelevant to the phenotype/stimulus are expressed equally in the target and control samples. However, this strict hypothesis is not always true, as there can be several transcriptomic background differences between target and control samples, including different cell/tissue types, different cell cycle stages and different biological donors. These differences lead to increased false positives, which have little biological/medical significance. In this article, we propose a statistical framework to identify DEGs between target and control samples from expression microarray data allowing transcriptomic background differences between these samples by introducing a modified null hypothesis that the gene expression background difference is normally distributed. We use an iterative procedure to perform robust estimation of the null hypothesis and identify DEGs as outliers. We evaluated our method using our own triplicate microarray experiment, followed by validations with reverse transcription-polymerase chain reaction (RT-PCR) and on the MicroArray Quality Control dataset. The evaluations suggest that our technique (i) results in less false positive and false negative results, as measured by the degree of agreement with RT-PCR of the same samples, (ii) can be applied to different microarray platforms and results in better reproducibility as measured by the degree of DEG identification concordance both intra- and inter-platforms and (iii) can be applied efficiently with only a few microarray replicates. Based on these evaluations, we propose that this method not only identifies more reliable and biologically/medically significant DEG, but also reduces the power-cost tradeoff problem in the microarray field. Source code and binaries freely available for download at http://comonca.org.cn/fdca/resources/softwares/deg.zip.

PD-L1 Is a Therapeutic Target of the Bromodomain Inhibitor JQ1 and, Combined with HLA Class I, a Promising Prognostic Biomarker in Neuroblastoma.

PubMed

Melaiu, Ombretta; Mina, Marco; Chierici, Marco; Boldrini, Renata; Jurman, Giuseppe; Romania, Paolo; D'Alicandro, Valerio; Benedetti, Maria C; Castellano, Aurora; Liu, Tao; Furlanello, Cesare; Locatelli, Franco; Fruci, Doriana

2017-08-01

Purpose: This study sought to evaluate the expression of programmed cell death-ligand-1 (PD-L1) and HLA class I on neuroblastoma cells and programmed cell death-1 (PD-1) and lymphocyte activation gene 3 (LAG3) on tumor-infiltrating lymphocytes to better define patient risk stratification and understand whether this tumor may benefit from therapies targeting immune checkpoint molecules. Experimental Design: In situ IHC staining for PD-L1, HLA class I, PD-1, and LAG3 was assessed in 77 neuroblastoma specimens, previously characterized for tumor-infiltrating T-cell density and correlated with clinical outcome. Surface expression of PD-L1 was evaluated by flow cytometry and IHC in neuroblastoma cell lines and tumors genetically and/or pharmacologically inhibited for MYC and MYCN. A dataset of 477 human primary neuroblastomas from GEO and ArrayExpress databases was explored for PD-L1, MYC, and MYCN correlation. Results: Multivariate Cox regression analysis demonstrated that the combination of PD-L1 and HLA class I tumor cell density is a prognostic biomarker for predicting overall survival in neuroblastoma patients ( P = 0.0448). MYC and MYCN control the expression of PD-L1 in neuroblastoma cells both in vitro and in vivo Consistently, abundance of PD-L1 transcript correlates with MYC expression in primary neuroblastoma. Conclusions: The combination of PD-L1 and HLA class I represents a novel prognostic biomarker for neuroblastoma. Pharmacologic inhibition of MYCN and MYC may be exploited to target PD-L1 and restore an efficient antitumor immunity in high-risk neuroblastoma. Clin Cancer Res; 23(15); 4462-72. ©2017 AACR . ©2017 American Association for Cancer Research.

Inferring microRNA regulation of mRNA with partially ordered samples of paired expression data and exogenous prediction algorithms.

PubMed

Godsey, Brian; Heiser, Diane; Civin, Curt

2012-01-01

MicroRNAs (miRs) are known to play an important role in mRNA regulation, often by binding to complementary sequences in "target" mRNAs. Recently, several methods have been developed by which existing sequence-based target predictions can be combined with miR and mRNA expression data to infer true miR-mRNA targeting relationships. It has been shown that the combination of these two approaches gives more reliable results than either by itself. While a few such algorithms give excellent results, none fully addresses expression data sets with a natural ordering of the samples. If the samples in an experiment can be ordered or partially ordered by their expected similarity to one another, such as for time-series or studies of development processes, stages, or types, (e.g. cell type, disease, growth, aging), there are unique opportunities to infer miR-mRNA interactions that may be specific to the underlying processes, and existing methods do not exploit this. We propose an algorithm which specifically addresses [partially] ordered expression data and takes advantage of sample similarities based on the ordering structure. This is done within a Bayesian framework which specifies posterior distributions and therefore statistical significance for each model parameter and latent variable. We apply our model to a previously published expression data set of paired miR and mRNA arrays in five partially ordered conditions, with biological replicates, related to multiple myeloma, and we show how considering potential orderings can improve the inference of miR-mRNA interactions, as measured by existing knowledge about the involved transcripts.

MicroRNAs associated with the efficacy of photodynamic therapy in biliary tract cancer cell lines.

PubMed

Wagner, Andrej; Mayr, Christian; Bach, Doris; Illig, Romana; Plaetzer, Kristjan; Berr, Frieder; Pichler, Martin; Neureiter, Daniel; Kiesslich, Tobias

2014-11-05

Photodynamic therapy (PDT) is a palliative treatment option for unresectable hilar biliary tract cancer (BTC) showing a considerable benefit for survival and quality of life with few side effects. Currently, factors determining the cellular response of BTC cells towards PDT are unknown. Due to their multifaceted nature, microRNAs (miRs) are a promising analyte to investigate the cellular mechanisms following PDT. For two photosensitizers, Photofrin® and Foscan®, the phototoxicity was investigated in eight BTC cell lines. Each cell line (untreated) was profiled for expression of n=754 miRs using TaqMan® Array Human MicroRNA Cards. Statistical analysis and bioinformatic tools were used to identify miRs associated with PDT efficiency and their putative targets, respectively. Twenty miRs correlated significantly with either high or low PDT efficiency. PDT was particularly effective in cells with high levels of clustered miRs 25-93*-106b and (in case of miR-106b) a phenotype characterized by high expression of the mesenchymal marker vimentin and high proliferation (cyclinD1 and Ki67 expression). Insensitivity towards PDT was associated with high miR-200 family expression and (for miR-cluster 200a/b-429) expression of differentiation markers Ck19 and Ck8/18. Predicted and validated downstream targets indicate plausible involvement of miRs 20a*, 25, 93*, 130a, 141, 200a, 200c and 203 in response mechanisms to PDT, suggesting that targeting these miRs could improve susceptibility to PDT in insensitive cell lines. Taken together, the miRNome pattern may provide a novel tool for predicting the efficiency of PDT and-following appropriate functional verification-may subsequently allow for optimization of the PDT protocol.

MicroRNAs Associated with the Efficacy of Photodynamic Therapy in Biliary Tract Cancer Cell Lines

PubMed Central

Wagner, Andrej; Mayr, Christian; Bach, Doris; Illig, Romana; Plaetzer, Kristjan; Berr, Frieder; Pichler, Martin; Neureiter, Daniel; Kiesslich, Tobias

2014-01-01

Photodynamic therapy (PDT) is a palliative treatment option for unresectable hilar biliary tract cancer (BTC) showing a considerable benefit for survival and quality of life with few side effects. Currently, factors determining the cellular response of BTC cells towards PDT are unknown. Due to their multifaceted nature, microRNAs (miRs) are a promising analyte to investigate the cellular mechanisms following PDT. For two photosensitizers, Photofrin® and Foscan®, the phototoxicity was investigated in eight BTC cell lines. Each cell line (untreated) was profiled for expression of n = 754 miRs using TaqMan® Array Human MicroRNA Cards. Statistical analysis and bioinformatic tools were used to identify miRs associated with PDT efficiency and their putative targets, respectively. Twenty miRs correlated significantly with either high or low PDT efficiency. PDT was particularly effective in cells with high levels of clustered miRs 25-93*-106b and (in case of miR-106b) a phenotype characterized by high expression of the mesenchymal marker vimentin and high proliferation (cyclinD1 and Ki67 expression). Insensitivity towards PDT was associated with high miR-200 family expression and (for miR-cluster 200a/b-429) expression of differentiation markers Ck19 and Ck8/18. Predicted and validated downstream targets indicate plausible involvement of miRs 20a*, 25, 93*, 130a, 141, 200a, 200c and 203 in response mechanisms to PDT, suggesting that targeting these miRs could improve susceptibility to PDT in insensitive cell lines. Taken together, the miRNome pattern may provide a novel tool for predicting the efficiency of PDT and—following appropriate functional verification—may subsequently allow for optimization of the PDT protocol. PMID:25380521

MiRNA-362-3p induces cell cycle arrest through targeting of E2F1, USF2 and PTPN1 and is associated with recurrence of colorectal cancer.

PubMed

Christensen, Lise Lotte; Tobiasen, Heidi; Holm, Anja; Schepeler, Troels; Ostenfeld, Marie S; Thorsen, Kasper; Rasmussen, Mads H; Birkenkamp-Demtroeder, Karin; Sieber, Oliver M; Gibbs, Peter; Lubinski, Jan; Lamy, Philippe; Laurberg, Søren; Oster, Bodil; Hansen, Kristian Q; Hagemann-Madsen, Rikke; Byskov, Kristina; Ørntoft, Torben F; Andersen, Claus L

2013-07-01

Colorectal cancer (CRC) is one of the leading causes of cancer deaths in Western countries. A significant number of CRC patients undergoing curatively intended surgery subsequently develop recurrence and die from the disease. MicroRNAs (miRNAs) are aberrantly expressed in cancers and appear to have both diagnostic and prognostic significance. In this study, we identified novel miRNAs associated with recurrence of CRC, and their possible mechanism of action. TaqMan(®) Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosas and 46 microsatellite stable CRC tumors. Four miRNAs (miR-362-3p, miR-570, miR-148 a* and miR-944) were expressed at a higher level in tumors from patients with no recurrence (p<0.015), compared with tumors from patients with recurrence. A significant association with increased disease free survival was confirmed for miR-362-3p in a second independent cohort of 43 CRC patients, using single TaqMan(®) microRNA assays. In vitro functional analysis showed that over-expression of miR-362-3p in colon cancer cell lines reduced cell viability, and proliferation mainly due to cell cycle arrest. E2F1, USF2 and PTPN1 were identified as potential miR-362-3p targets by mRNA profiling of HCT116 cells over-expressing miR-362-3p. Subsequently, these genes were confirmed as direct targets by Luciferase reporter assays and their knockdown in vitro phenocopied the effects of miR-362-3p over-expression. We conclude that miR-362-3p may be a novel prognostic marker in CRC, and hypothesize that the positive effects of augmented miR-362-3p expression may in part be mediated through the targets E2F1, USF2 and PTPN1. Copyright © 2012 UICC.

A microRNA expression signature of the postprandial state in response to a high-saturated-fat challenge.

PubMed

Lopez, Sergio; Bermudez, Beatriz; Montserrat-de la Paz, Sergio; Abia, Rocio; Muriana, Francisco J G

2018-07-01

The postprandial hypertriglyceridemia is an important and largely silent disturbance involved in the genesis of numerous pathological conditions. Exaggerated and prolonged states of postprandial hypertriglyceridemia are frequently related to the ingestion of meals enriched in saturated fatty acids (SFAs). MicroRNAs are noncoding RNAs that function as gene regulators and play significant roles in both health and disease. However, differential miRNA expression between fasting and postprandial states has never been elucidated. Here, we studied the impact of a high-saturated-fat meal, mainly rich in palmitic acid, on the miRNA signature in peripheral blood mononuclear cells (PBMCs) of nine male healthy individuals in the postprandial period by using a two-step analysis: miRNA array and validation through quantitative real-time polymerase chain reaction. Compared with miRNA expression signature in PBMCs at fasting, 36 miRNAs were down-regulated and 43 miRNAs were up-regulated in PBMCs at postprandial hypertriglyceridemic peak. Six chromosomes (3, 7, 8, 12, 14 and 19) had nearly half (48.1%) of dysregulated miRNA-gene-containing regions. Down-regulated miR-300 and miR-369-3p and up-regulated miR-495-3p, miR-129-5p and miR-7-2-3p had the highest number of target genes. The differentially expressed miRNAs and their predicted target genes involved pathways in cancer, MAPK signaling pathway, endocytosis and axon guidance. Only down-regulated miRNAs notably targeted PI3K-Akt signaling pathways, whereas only up-regulated miRNAs targeted focal adhesion, Wnt signaling pathway, transcriptional misregulation in cancer and ubiquitin-mediated proteolysis. This is the first study of miRNA expression analysis of human PBMCs during postprandial hypertriglyceridemia and offers insight into new potential mechanisms by which dietary SFAs influence health or disease. Copyright © 2018 Elsevier Inc. All rights reserved.

Identification of the collagen type 1 alpha 1 gene (COL1A1) as a candidate survival-related factor associated with hepatocellular carcinoma

PubMed Central

2014-01-01

Background Hepatocellular carcinoma (HCC) is one of the major causes of cancer-related death especially among Asian and African populations. It is urgent that we identify carcinogenesis-related genes to establish an innovative treatment strategy for this disease. Methods Triple-combination array analysis was performed using one pair each of HCC and noncancerous liver samples from a 68-year-old woman. This analysis consists of expression array, single nucleotide polymorphism array and methylation array. The gene encoding collagen type 1 alpha 1 (COL1A1) was identified and verified using HCC cell lines and 48 tissues from patients with primary HCC. Results Expression array revealed that COL1A1 gene expression was markedly decreased in tumor tissues (log2 ratio –1.1). The single nucleotide polymorphism array showed no chromosomal deletion in the locus of COL1A1. Importantly, the methylation value in the tumor tissue was higher (0.557) than that of the adjacent liver tissue (0.008). We verified that expression of this gene was suppressed by promoter methylation. Reactivation of COL1A1 expression by 5-aza-2′-deoxycytidine treatment was seen in HCC cell lines, and sequence analysis identified methylated CpG sites in the COL1A1 promoter region. Among 48 pairs of surgical specimens, 13 (27.1%) showed decreased COL1A1 mRNA expression in tumor sites. Among these 13 cases, 10 had promoter methylation at the tumor site. The log-rank test indicated that mRNA down-regulated tumors were significantly correlated with a poor overall survival rate (P = 0.013). Conclusions Triple-combination array analysis successfully identified COL1A1 as a candidate survival-related gene in HCCs. Epigenetic down-regulation of COL1A1 mRNA expression might have a role as a prognostic biomarker of HCC. PMID:24552139

Mapping of transcription factor binding regions in mammalian cells by ChIP: Comparison of array- and sequencing-based technologies

PubMed Central

Euskirchen, Ghia M.; Rozowsky, Joel S.; Wei, Chia-Lin; Lee, Wah Heng; Zhang, Zhengdong D.; Hartman, Stephen; Emanuelsson, Olof; Stolc, Viktor; Weissman, Sherman; Gerstein, Mark B.; Ruan, Yijun; Snyder, Michael

2007-01-01

Recent progress in mapping transcription factor (TF) binding regions can largely be credited to chromatin immunoprecipitation (ChIP) technologies. We compared strategies for mapping TF binding regions in mammalian cells using two different ChIP schemes: ChIP with DNA microarray analysis (ChIP-chip) and ChIP with DNA sequencing (ChIP-PET). We first investigated parameters central to obtaining robust ChIP-chip data sets by analyzing STAT1 targets in the ENCODE regions of the human genome, and then compared ChIP-chip to ChIP-PET. We devised methods for scoring and comparing results among various tiling arrays and examined parameters such as DNA microarray format, oligonucleotide length, hybridization conditions, and the use of competitor Cot-1 DNA. The best performance was achieved with high-density oligonucleotide arrays, oligonucleotides ≥50 bases (b), the presence of competitor Cot-1 DNA and hybridizations conducted in microfluidics stations. When target identification was evaluated as a function of array number, 80%–86% of targets were identified with three or more arrays. Comparison of ChIP-chip with ChIP-PET revealed strong agreement for the highest ranked targets with less overlap for the low ranked targets. With advantages and disadvantages unique to each approach, we found that ChIP-chip and ChIP-PET are frequently complementary in their relative abilities to detect STAT1 targets for the lower ranked targets; each method detected validated targets that were missed by the other method. The most comprehensive list of STAT1 binding regions is obtained by merging results from ChIP-chip and ChIP-sequencing. Overall, this study provides information for robust identification, scoring, and validation of TF targets using ChIP-based technologies. PMID:17568005

VX680/MK-0457, a potent and selective Aurora kinase inhibitor, targets both tumor and endothelial cells in clear cell renal cell carcinoma

PubMed Central

Li, Yan; Zhang, Zhong-Fa; Chen, Jindong; Huang, Dan; Ding, Yan; Tan, Min-Han; Qian, Chao-Nan; Resau, James H; Kim, Hyung; Teh, Bin Tean

2010-01-01

Aurora kinases are key regulators of cell mitosis and have been implicated in the process of tumorigenesis. In recent years, the Aurora kinases have attracted much interest as promising targets for cancer treatment. Here we report on the roles of Aurora A and Aurora B kinases in clear cell renal cell carcinoma (ccRCC). Using genomewide expression array analysis of 174 patient samples of ccRCC, we found that expression levels of Aurora A and B were significantly elevated in ccRCC compared to normal kidney samples. High expression levels of Aurora A and Aurora B were significantly associated with advanced tumor stage and poor patient survival. Inhibition of Aurora kinase activity with the drug VX680 (also referred to as MK-0457) inhibited ccRCC cell growth in vitro and led to ccRCC cell accumulation in the G2/M phase and apoptosis. Growth of ccRCC xenograft tumors was also inhibited by VX680 treatment, accompanied by a reduction of tumor microvessel density. Analysis of endothelial cell lines demonstrated that VX680 inhibits endothelial cell growth with effects similar to that seen in ccRCC cells. Our findings suggest that VX680 inhibits the growth of ccRCC tumors by targeting the proliferation of both ccRCC tumor cells and tumor-associated endothelial cells. Aurora kinases and their downstream cell cycle proteins have an important role in ccRCC and may be potent prognostic markers and therapy targets for this disease. PMID:20589168

Expression of 16 Nitrogenase Proteins within the Plant Mitochondrial Matrix

PubMed Central

Allen, Robert S.; Tilbrook, Kimberley; Warden, Andrew C.; Campbell, Peter C.; Rolland, Vivien; Singh, Surinder P.; Wood, Craig C.

2017-01-01

The industrial production and use of nitrogenous fertilizer involves significant environmental and economic costs. Strategies to reduce fertilizer dependency are required to address the world's increasing demand for sustainable food, fibers, and biofuels. Biological nitrogen fixation, a process unique to diazatrophic bacteria, is catalyzed by the nitrogenase complex, and reconstituting this function in plant cells is an ambitious biotechnological strategy to reduce fertilizer use. Here we establish that the full array of biosynthetic and catalytic nitrogenase (Nif) proteins from the diazotroph Klebsiella pneumoniae can be individually expressed as mitochondrial targeting peptide (MTP)-Nif fusions in Nicotiana benthamiana. We show that these are correctly targeted to the plant mitochondrial matrix, a subcellular location with biochemical and genetic characteristics potentially supportive of nitrogenase function. Although Nif proteins B, D, E, F, H, J, K, M, N, Q, S, U, V, X, Y, and Z were all detectable by Western blot analysis, the NifD catalytic component was the least abundant. To address this problem, a translational fusion between NifD and NifK was designed based on the crystal structure of the nitrogenase MoFe protein heterodimer. This fusion protein enabled equimolar NifD:NifK stoichiometry and improved NifD expression levels in plants. Finally, four MTP-Nif fusion proteins (B, S, H, Y) were successfully co-expressed, demonstrating that multiple components of nitrogenase can be targeted to plant mitochondria. These results establish the feasibility of reconstituting the complete componentry for nitrogenase in plant cells, within an intracellular environment that could support the conversion of nitrogen gas into ammonia. PMID:28316608

Gene expression signatures differentiate ovarian/peritoneal serous carcinoma from breast carcinoma in effusions

PubMed Central

Davidson, Ben; Stavnes, Helene Tuft; Holth, Arild; Chen, Xu; Yang, Yanqin; Shih, Ie-Ming; Wang, Tian-Li

2011-01-01

Abstract Ovarian/primary peritoneal carcinoma and breast carcinoma are the gynaecological cancers that most frequently involve the serosal cavities. With the objective of improving on the limited diagnostic panel currently available for the differential diagnosis of these two malignancies, as well as to define tumour-specific biological targets, we compared their global gene expression patterns. Gene expression profiles of 10 serous ovarian/peritoneal and eight ductal breast carcinoma effusions were analysed using the HumanRef-8 BeadChip from Illumina. Differentially expressed candidate genes were validated using quantitative real-time PCR and immunohistochemistry. Unsupervised hierarchical clustering using all 54,675 genes in the array separated ovarian from breast carcinoma samples. We identified 288 unique probes that were significantly differentially expressed in the two cancers by greater than 3.5-fold, of which 81 and 207 were overexpressed in breast and ovarian/peritoneal carcinoma, respectively. SAM analysis identified 1078 differentially expressed probes with false discovery rate less than 0.05. Genes overexpressed in breast carcinoma included TFF1, TFF3, FOXA1, CA12, GATA3, SDC1, PITX1, TH, EHFD1, EFEMP1, TOB1 and KLF2. Genes overexpressed in ovarian/peritoneal carcinoma included SPON1, RBP1, MFGE8, TM4SF12, MMP7, KLK5/6/7, FOLR1/3, PAX8, APOL2 and NRCAM. The differential expression of 14 genes was validated by quantitative real-time PCR, and differences in 5 gene products were confirmed by immunohistochemistry. Expression profiling distinguishes ovarian/peritoneal carcinoma from breast carcinoma and identifies genes that are differentially expressed in these two tumour types. The molecular signatures unique to these cancers may facilitate their differential diagnosis and may provide a molecular basis for therapeutic target discovery. PMID:20132413

Flexible Integration of Both High Imaging Resolution and High Power Arrays for Ultrasound-Induced Thermal Strain Imaging (US-TSI)

PubMed Central

Stephens, Douglas N.; Mahmoud, Ahmed M.; Ding, Xuan; Lucero, Steven; Dutta, Debaditya; Yu, Francois T.H.; Chen, Xucai

2013-01-01

Ultrasound-induced thermal strain imaging (US-TSI) for carotid artery plaque detection requires both high imaging resolution (<100 μm) and sufficient US induced heating to elevate the tissue temperature (~1-3°C within 1-3 cardiac cycles) in order to produce a noticeable change in sound speed in the targeted tissues. Since the optimization of both imaging and heating in a monolithic array design is particularly expensive and inflexible, a new integrated approach is presented that utilizes independent ultrasound arrays to meet the requirements for this particular application. This work demonstrates a new approach in dual-array construction. A 3D printed manifold was built to support both a high resolution 20 MHz commercial imaging array and 6 custom heating elements operating in the 3.5-4 MHz range. For the application of US-TSI on carotid plaque characterization, the tissue target site is 20 to 30 mm deep, with a typical target volume of 2 mm (elevation) × 8 mm (azimuthal) × 5 mm (depth). The custom heating array performance was fully characterized for two design variants (flat and spherical apertures), and can easily deliver 30 W of total acoustic power to produce intensities greater than 15 W/cm2 in tissue target region. PMID:24297029

A novel FOXA1/ESR1 interacting pathway: A study of Oncomine™ breast cancer microarrays

PubMed Central

Chaudhary, Sanjib; Krishna, B. Madhu; Mishra, Sandip K.

2017-01-01

Forkhead box protein A1 (FOXA1) is essential for the growth and differentiation of breast epithelium, and has a favorable outcome in breast cancer (BC). Elevated FOXA1 expression in BC also facilitates hormone responsiveness in estrogen receptor (ESR)-positive BC. However, the interaction between these two pathways is not fully understood. FOXA1 and GATA binding protein 3 (GATA3) along with ESR1 expression are responsible for maintaining a luminal phenotype, thus suggesting the existence of a strong association between them. The present study utilized the Oncomine™ microarray database to identify FOXA1:ESR1 and FOXA1:ESR1:GATA3 co-expression co-regulated genes. Oncomine™ analysis revealed 115 and 79 overlapping genes clusters in FOXA1:ESR1 and FOXA1:ESR1:GATA3 microarrays, respectively. Five ESR1 direct target genes [trefoil factor 1 (TFF1/PS2), B-cell lymphoma 2 (BCL2), seven in absentia homolog 2 (SIAH2), cellular myeloblastosis viral oncogene homolog (CMYB) and progesterone receptor (PGR)] were detected in the co-expression clusters. To further investigate the role of FOXA1 in ESR1-positive cells, MCF7 cells were transfected with a FOXA1 expression plasmid, and it was observed that the direct target genes of ESR1 (PS2, BCL2, SIAH2 and PGR) were significantly regulated upon transfection. Analysis of one of these target genes, PS2, revealed the presence of two FOXA1 binding sites in the vicinity of the estrogen response element (ERE), which was confirmed by binding assays. Under estrogen stimulation, FOXA1 protein was recruited to the FOXA1 site and could also bind to the ERE site (although in minimal amounts) in the PS2 promoter. Co-transfection of FOXA1/ESR1 expression plasmids demonstrated a significantly regulation of the target genes identified in the FOXA1/ESR1 multi-arrays compared with only FOXA1 transfection, which was suggestive of a synergistic effect of ESR1 and FOXA1 on the target genes. In summary, the present study identified novel FOXA1, ESR1 and GATA3 co-expressed genes that may be involved in breast tumorigenesis. PMID:28789340

A novel FOXA1/ESR1 interacting pathway: A study of Oncomine™ breast cancer microarrays.

PubMed

Chaudhary, Sanjib; Krishna, B Madhu; Mishra, Sandip K

2017-08-01

Forkhead box protein A1 (FOXA1) is essential for the growth and differentiation of breast epithelium, and has a favorable outcome in breast cancer (BC). Elevated FOXA1 expression in BC also facilitates hormone responsiveness in estrogen receptor ( ESR )-positive BC. However, the interaction between these two pathways is not fully understood. FOXA1 and GATA binding protein 3 ( GATA3 ) along with ESR1 expression are responsible for maintaining a luminal phenotype, thus suggesting the existence of a strong association between them. The present study utilized the Oncomine™ microarray database to identify FOXA1:ESR1 and FOXA1:ESR1:GATA3 co-expression co-regulated genes. Oncomine™ analysis revealed 115 and 79 overlapping genes clusters in FOXA1:ESR1 and FOXA1:ESR1:GATA3 microarrays, respectively. Five ESR1 direct target genes [trefoil factor 1 ( TFF1/PS2 ), B-cell lymphoma 2 ( BCL2 ), seven in absentia homolog 2 ( SIAH2 ), cellular myeloblastosis viral oncogene homolog ( CMYB ) and progesterone receptor ( PGR )] were detected in the co-expression clusters. To further investigate the role of FOXA1 in ESR1-positive cells, MCF7 cells were transfected with a FOXA1 expression plasmid, and it was observed that the direct target genes of ESR1 ( PS2, BCL2, SIAH2 and PGR ) were significantly regulated upon transfection. Analysis of one of these target genes, PS2 , revealed the presence of two FOXA1 binding sites in the vicinity of the estrogen response element (ERE), which was confirmed by binding assays. Under estrogen stimulation, FOXA1 protein was recruited to the FOXA1 site and could also bind to the ERE site (although in minimal amounts) in the PS2 promoter. Co-transfection of FOXA1 / ESR1 expression plasmids demonstrated a significantly regulation of the target genes identified in the FOXA1 / ESR1 multi-arrays compared with only FOXA1 transfection, which was suggestive of a synergistic effect of ESR1 and FOXA1 on the target genes. In summary, the present study identified novel FOXA1 , ESR1 and GATA 3 co-expressed genes that may be involved in breast tumorigenesis.

Identification of miRNAs during mouse postnatal ovarian development and superovulation.

PubMed

Khan, Hamid Ali; Zhao, Yi; Wang, Li; Li, Qian; Du, Yu-Ai; Dan, Yi; Huo, Li-Jun

2015-07-08

MicroRNAs are small noncoding RNAs that play critical roles in regulation of gene expression in wide array of tissues including the ovary through sequence complementarity at post-transcriptional level. Tight regulation of multitude of genes involved in ovarian development and folliculogenesis could be regulated at transcription level by these miRNAs. Therefore, tissue specific miRNAs identification is considered a key step towards understanding the role of miRNAs in biological processes. To investigate the role of microRNAs during ovarian development and folliculogenesis we sequenced eight different libraries using Illumina deep sequencing technology. Different developmental stages were selected to explore miRNAs expression pattern at different stages of gonadal maturation with/without treatment of PMSG/hCG for superovulation. From massive sequencing reads, clean reads of 16-26 bp were selected for further analysis of differential expression analysis and novel microRNA annotation. Expression analysis of all miRNAs at different developmental stages showed that some miRNAs were present ubiquitously while others were differentially expressed at different stages. Among differentially expressed miRNAs we reported 61 miRNAs with a fold change of more than 2 at different developmental stages among all libraries. Among the up-regulated miRNAs, mmu-mir-1298 had the highest fold change with 4.025 while mmu-mir-150 was down-regulated more than 3 fold. Furthermore, we found 2659 target genes for 20 differentially expressed microRNAs using seven different target predictions programs (DIANA-mT, miRanda, miRDB, miRWalk, RNAhybrid, PICTAR5, TargetScan). Analysis of the predicted targets showed certain ovary specific genes targeted by single or multiple microRNAs. Furthermore, pathway annotation and Gene ontology showed involvement of these microRNAs in basic cellular process. These results suggest the presence of different miRNAs at different stages of ovarian development and superovulation. Potential role of these microRNAs was elucidated using bioinformatics tools in regulation of different pathways, biological functions and cellular components underlying ovarian development and superovulation. These results provide a framework for extended analysis of miRNAs and their roles during ovarian development and superovulation. Furthermore, this study provides a base for characterization of individual miRNAs to discover their role in ovarian development and female fertility.

miR Profiling Identifies Cyclin-Dependent Kinase 6 Downregulation as a Potential Mechanism of Acquired Cisplatin Resistance in Non-Small-Cell Lung Carcinoma.

PubMed

Bar, Jair; Gorn-Hondermann, Ivan; Moretto, Patricia; Perkins, Theodore J; Niknejad, Nima; Stewart, David J; Goss, Glenwood D; Dimitroulakos, Jim

2015-11-01

To identify the mechanisms of cisplatin resistance, global microRNA (miR) expression was tested. The expression of miR-145 was consistently higher in resistant cells. The expression of cyclin-dependent kinase 6 (CDK6), a potential target of miR-145, was lower in resistant cells, and inhibition of CDK4/6 protected cells from cisplatin. Cell cycle inhibition, currently being tested in clinical trials, might be antagonistic to cisplatin and other cytotoxic drugs. Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related death. Platinum-based chemotherapeutic drugs are the most active agents in treating advanced disease. Resistance to these drugs is common and multifactorial; insight into the molecular mechanisms involved will likely enhance efficacy. A set of NSCLC platinum-resistant sublines was created from the Calu6 cell line. Cell viability was quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Differentially expressed microRNAs (miRs) in these lines were identified using Affymetrix miR arrays. The potential genes targeted by these miRs were searched using the TargetScan algorithm. The expression levels of miRs and mRNA were tested using real-time polymerase chain reaction. miR-145 was reproducibly elevated in all the resistant sublines tested; however, modulation of miR-145 levels alone in these cells did not affect their response to cisplatin. A potential target of miR-145 is cyclin-dependent kinase 6 (CDK6), an important regulator of cell proliferation. The mRNA and protein levels of CDK6 were both downregulated in the resistant sublines. An inhibitor of CDK4/6 (PD0332991) protected parental NSCLC cells from cisplatin cytotoxicity. In the present study, we identified miRs differentially expressed in cisplatin-resistant cell lines, including miR-145. A predicted target of miR-145 is CDK6, and its expression was found to be downregulated in the resistant sublines, although not directly by miR-145. Inhibition of CDK6 antagonizes cisplatin-induced NSCLC cell cytotoxicity, suggesting that agents that inhibit CDK6 should be avoided during cisplatin therapy. Copyright © 2015 Elsevier Inc. All rights reserved.

Analysis of differential gene expression by bead-based fiber-optic array in growth-hormone-secreting pituitary adenomas.

PubMed

Jiang, Zhiquan; Gui, Songbo; Zhang, Yazhuo

2010-09-01

Growth-hormone-secreting pituitary adenomas (GHomas) account for approximately 20% of all pituitary neoplasms. However, the pathogenesis of GHomas remains to be elucidated. To explore the possible pathogenesis of GHomas, we used bead-based fiber-optic arrays to examine the gene expression in five GHomas and compared them to three healthy pituitaries. Four differentially expressed genes were chosen randomly for validation by quantitative real-time reverse transcription-polymerase chain reaction. We then performed pathway analysis on the identified differentially expressed genes using the Kyoto Encyclopedia of Genes and Genomes. Array analysis showed significant increases in the expression of 353 genes and 206 expressed sequence tags (ESTs) and decreases in 565 genes and 29 ESTs. Bioinformatic analysis showed that the genes HIGD1B, HOXB2, ANGPT2, HPGD and BTG2 may play an important role in the tumorigenesis and progression of GHomas. Pathway analysis showed that the wingless-type signaling pathway and extracellular-matrix receptor interactions may play a key role in the tumorigenesis and progression of GHomas. Our data suggested that there are numerous aberrantly expressed genes and pathways involved in the pathogenesis of GHomas. Bead-based fiber-optic arrays combined with pathway analysis of differentially expressed genes appear to be a valid method for investigating the pathogenesis of tumors.

Analysis of differential gene expression by bead-based fiber-optic array in growth-hormone-secreting pituitary adenomas

PubMed Central

JIANG, ZHIQUAN; GUI, SONGBO; ZHANG, YAZHUO

2010-01-01

Growth-hormone-secreting pituitary adenomas (GHomas) account for approximately 20% of all pituitary neoplasms. However, the pathogenesis of GHomas remains to be elucidated. To explore the possible pathogenesis of GHomas, we used bead-based fiber-optic arrays to examine the gene expression in five GHomas and compared them to three healthy pituitaries. Four differentially expressed genes were chosen randomly for validation by quantitative real-time reverse transcription-polymerase chain reaction. We then performed pathway analysis on the identified differentially expressed genes using the Kyoto Encyclopedia of Genes and Genomes. Array analysis showed significant increases in the expression of 353 genes and 206 expressed sequence tags (ESTs) and decreases in 565 genes and 29 ESTs. Bioinformatic analysis showed that the genes HIGD1B, HOXB2, ANGPT2, HPGD and BTG2 may play an important role in the tumorigenesis and progression of GHomas. Pathway analysis showed that the wingless-type signaling pathway and extracellular-matrix receptor interactions may play a key role in the tumorigenesis and progression of GHomas. Our data suggested that there are numerous aberrantly expressed genes and pathways involved in the pathogenesis of GHomas. Bead-based fiber-optic arrays combined with pathway analysis of differentially expressed genes appear to be a valid method for investigating the pathogenesis of tumors. PMID:22993617

MicroRNA-targeted therapeutics for lung cancer treatment.

PubMed

Xue, Jing; Yang, Jiali; Luo, Meihui; Cho, William C; Liu, Xiaoming

2017-02-01

Lung cancer is one of the leading causes of cancer-related mortality worldwide. MicroRNAs (miRNAs) are endogenous non-coding small RNAs that repress the expression of a broad array of target genes. Many efforts have been made to therapeutically target miRNAs in cancer treatments using miRNA mimics and miRNA antagonists. Areas covered: This article summarizes the recent findings with the role of miRNAs in lung cancer, and discusses the potential and challenges of developing miRNA-targeted therapeutics in this dreadful disease. Expert opinion: The development of miRNA-targeted therapeutics has become an important anti-cancer strategy. Results from both preclinical and clinical trials of microRNA replacement therapy have shown some promise in cancer treatment. However, some obstacles, including drug delivery, specificity, off-target effect, toxicity mediation, immunological activation and dosage determination should be addressed. Several delivery strategies have been employed, including naked oligonucleotides, liposomes, aptamer-conjugates, nanoparticles and viral vectors. However, delivery remains a main challenge in miRNA-targeting therapeutics. Furthermore, immune-related serious adverse events are also a concern, which indicates the complexity of miRNA-based therapy in clinical settings.

Digital detection of multiple minority mutants and expression levels of multiple colorectal cancer-related genes using digital-PCR coupled with bead-array.

PubMed

Huang, Huan; Li, Shuo; Sun, Lizhou; Zhou, Guohua

2015-01-01

To simultaneously analyze mutations and expression levels of multiple genes on one detection platform, we proposed a method termed "multiplex ligation-dependent probe amplification-digital amplification coupled with hydrogel bead-array" (MLPA-DABA) and applied it to diagnose colorectal cancer (CRC). CRC cells and tissues were sampled to extract nucleic acid, perform MLPA with sequence-tagged probes, perform digital emulsion polymerase chain reaction (PCR), and produce a hydrogel bead-array to immobilize beads and form a single bead layer on the array. After hybridization with fluorescent probes, the number of colored beads, which reflects the abundance of expressed genes and the mutation rate, was counted for diagnosis. Only red or green beads occurred on the chips in the mixed samples, indicating the success of single-molecule PCR. When a one-source sample was analyzed using mixed MLPA probes, beads of only one color occurred, suggesting the high specificity of the method in analyzing CRC mutation and gene expression. In gene expression analysis of a CRC tissue from one CRC patient, the mutant percentage was 3.1%, and the expression levels of CRC-related genes were much higher than those of normal tissue. The highly sensitive MLPA-DABA succeeds in the relative quantification of mutations and gene expressions of exfoliated cells in stool samples of CRC patients on the same chip platform. MLPA-DABA coupled with hydrogel bead-array is a promising method in the non-invasive diagnosis of CRC.

Drosophila CLOCK target gene characterization: implications for circadian tissue-specific gene expression

PubMed Central

Abruzzi, Katharine Compton; Rodriguez, Joseph; Menet, Jerome S.; Desrochers, Jennifer; Zadina, Abigail; Luo, Weifei; Tkachev, Sasha; Rosbash, Michael

2011-01-01

CLOCK (CLK) is a master transcriptional regulator of the circadian clock in Drosophila. To identify CLK direct target genes and address circadian transcriptional regulation in Drosophila, we performed chromatin immunoprecipitation (ChIP) tiling array assays (ChIP–chip) with a number of circadian proteins. CLK binding cycles on at least 800 sites with maximal binding in the early night. The CLK partner protein CYCLE (CYC) is on most of these sites. The CLK/CYC heterodimer is joined 4–6 h later by the transcriptional repressor PERIOD (PER), indicating that the majority of CLK targets are regulated similarly to core circadian genes. About 30% of target genes also show cycling RNA polymerase II (Pol II) binding. Many of these generate cycling RNAs despite not being documented in prior RNA cycling studies. This is due in part to different RNA isoforms and to fly head tissue heterogeneity. CLK has specific targets in different tissues, implying that important CLK partner proteins and/or mechanisms contribute to gene-specific and tissue-specific regulation. PMID:22085964

Development of multitissue microfluidic dynamic array for assessing changes in gene expression associated with channel catfish appetite, growth, metabolism, and intestinal health

USDA-ARS?s Scientific Manuscript database

Large-scale, gene expression methods allow for high throughput analysis of physiological pathways at a fraction of the cost of individual gene expression analysis. Systems, such as the Fluidigm quantitative PCR array described here, can provide powerful assessments of the effects of diet, environme...

Characterization of the bionano interface and mapping extrinsic interactions of the corona of nanomaterials

NASA Astrophysics Data System (ADS)

O'Connell, D. J.; Bombelli, F. Baldelli; Pitek, A. S.; Monopoli, M. P.; Cahill, D. J.; Dawson, K. A.

2015-09-01

Nanoparticles in physiological environments are known to selectively adsorb proteins and other biomolecules forming a tightly bound biomolecular `corona' on their surface. Where the exchange times of the proteins are sufficiently long, it is believed that the protein corona constitutes the particle identity in biological milieu. Here we show that proteins in the corona retain their functional characteristics and can specifically bind to cognate proteins on arrays of thousands of immobilised human proteins. The biological identity of the nanomaterial is seen to be specific to the blood plasma concentration in which they are exposed. We show that the resulting in situ nanoparticle interactome is dependent on the protein concentration in plasma, with the emergence of a small number of dominant protein-protein interactions. These interactions are those driven by proteins that are adsorbed onto the particle surface and whose binding epitopes are subsequently expressed or presented suitably on the particle surface. We suggest that, since specific tailored protein arrays for target systems and organs can be designed, their use may be an important element in an overall study of the biomolecular corona.Nanoparticles in physiological environments are known to selectively adsorb proteins and other biomolecules forming a tightly bound biomolecular `corona' on their surface. Where the exchange times of the proteins are sufficiently long, it is believed that the protein corona constitutes the particle identity in biological milieu. Here we show that proteins in the corona retain their functional characteristics and can specifically bind to cognate proteins on arrays of thousands of immobilised human proteins. The biological identity of the nanomaterial is seen to be specific to the blood plasma concentration in which they are exposed. We show that the resulting in situ nanoparticle interactome is dependent on the protein concentration in plasma, with the emergence of a small number of dominant protein-protein interactions. These interactions are those driven by proteins that are adsorbed onto the particle surface and whose binding epitopes are subsequently expressed or presented suitably on the particle surface. We suggest that, since specific tailored protein arrays for target systems and organs can be designed, their use may be an important element in an overall study of the biomolecular corona. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr01970b

Heterologous Array Analysis in Pinaceae: Hybridization of Pinus Taeda cDNA Arrays With cDNA From Needles and Embryogenic Cultures of P. Taeda, P. Sylvestris or Picea Abies

PubMed Central

van Zyl, Leonel; von Arnold, Sara; Bozhkov, Peter; Chen, Yongzhong; Egertsdotter, Ulrika; MacKay, John; Sederoff, Ronald R.; Shen, Jing; Zelena, Lyubov

2002-01-01

Hybridization of labelled cDNA from various cell types with high-density arrays of expressed sequence tags is a powerful technique for investigating gene expression. Few conifer cDNA libraries have been sequenced. Because of the high level of sequence conservation between Pinus and Picea we have investigated the use of arrays from one genus for studies of gene expression in the other. The partial cDNAs from 384 identifiable genes expressed in differentiating xylem of Pinus taeda were printed on nylon membranes in randomized replicates. These were hybridized with labelled cDNA from needles or embryogenic cultures of Pinus taeda, P. sylvestris and Picea abies, and with labelled cDNA from leaves of Nicotiana tabacum. The Spearman correlation of gene expression for pairs of conifer species was high for needles (r2 = 0.78 − 0.86), and somewhat lower for embryogenic cultures (r2 = 0.68 − 0.83). The correlation of gene expression for tobacco leaves and needles of each of the three conifer species was lower but sufficiently high (r2 = 0.52 − 0.63) to suggest that many partial gene sequences are conserved in angiosperms and gymnosperms. Heterologous probing was further used to identify tissue-specific gene expression over species boundaries. To evaluate the significance of differences in gene expression, conventional parametric tests were compared with permutation tests after four methods of normalization. Permutation tests after Z-normalization provide the highest degree of discrimination but may enhance the probability of type I errors. It is concluded that arrays of cDNA from loblolly pine are useful for studies of gene expression in other pines or spruces. PMID:18629264

miR-34a: Multiple Opposing Targets and One Destiny in Hepatocellular Carcinoma.

PubMed

Yacoub, Radwa Alaa; Fawzy, Injie Omar; Assal, Reem Amr; Hosny, Karim Adel; Zekri, Abdel-Rahman Nabawy; Esmat, Gamal; El Tayebi, Hend Mohamed; Abdelaziz, Ahmed Ihab

2016-12-28

Background and Aims: The role of miR-34a in hepatocellular carcinoma (HCC) is controversial and several unresolved issues remain, including its expression pattern and relevance to tumor etiology, tumor stage and prognosis, and finally, its impact on apoptosis. Methods: miR-34a expression was assessed in hepatitis C virus (HCV)-induced non-metastatic HCC tissues by RT-Q-PCR. Huh-7 cells were transfected with miR-34a mimics and the impact of miR-34a was examined on 84 pro-apoptotic/anti-apoptotic genes using PCR array; its net effect was tested on cell viability via MTT assay. Results: miR-34a expression was up-regulated in HCC tissues. Moreover, miR-34a induced a large set of pro-apoptotic/anti-apoptotic genes, with a net result of triggering apoptosis and repressing cell viability. Conclusions: HCC-related differential expression of miR-34a could be etiology-based or stage-specific, and low expression of miR-34a may predict poor prognosis. This study's findings also emphasize the role of miR-34a in apoptosis.

The role of top-down spatial attention in contingent attentional capture.

PubMed

Huang, Wanyi; Su, Yuling; Zhen, Yanfen; Qu, Zhe

2016-05-01

It is well known that attentional capture by an irrelevant salient item is contingent on top-down feature selection, but whether attentional capture may be modulated by top-down spatial attention remains unclear. Here, we combined behavioral and ERP measurements to investigate the contribution of top-down spatial attention to attentional capture under modified spatial cueing paradigms. Each target stimulus was preceded by a peripheral circular cue array containing a spatially uninformative color singleton cue. We varied target sets but kept the cue array unchanged among different experimental conditions. When participants' task was to search for a colored letter in the target array that shared the same peripheral locations with the cue array, attentional capture by the peripheral color cue was reflected by both a behavioral spatial cueing effect and a cue-elicited N2pc component. When target arrays were presented more centrally, both the behavioral and N2pc effects were attenuated but still significant. The attenuated cue-elicited N2pc was found even when participants focused their attention on the fixed central location to identify a colored letter among an RSVP letter stream. By contrast, when participants were asked to identify an outlined or larger target, neither the behavioral spatial cueing effect nor the cue-elicited N2pc was observed, regardless of whether the target and cue arrays shared same locations or not. These results add to the evidence that attentional capture by salient stimuli is contingent upon feature-based task sets, and further indicate that top-down spatial attention is important but may not be necessary for contingent attentional capture. © 2016 Society for Psychophysiological Research.

Target genes discovery through copy number alteration analysis in human hepatocellular carcinoma.

PubMed

Gu, De-Leung; Chen, Yen-Hsieh; Shih, Jou-Ho; Lin, Chi-Hung; Jou, Yuh-Shan; Chen, Chian-Feng

2013-12-21

High-throughput short-read sequencing of exomes and whole cancer genomes in multiple human hepatocellular carcinoma (HCC) cohorts confirmed previously identified frequently mutated somatic genes, such as TP53, CTNNB1 and AXIN1, and identified several novel genes with moderate mutation frequencies, including ARID1A, ARID2, MLL, MLL2, MLL3, MLL4, IRF2, ATM, CDKN2A, FGF19, PIK3CA, RPS6KA3, JAK1, KEAP1, NFE2L2, C16orf62, LEPR, RAC2, and IL6ST. Functional classification of these mutated genes suggested that alterations in pathways participating in chromatin remodeling, Wnt/β-catenin signaling, JAK/STAT signaling, and oxidative stress play critical roles in HCC tumorigenesis. Nevertheless, because there are few druggable genes used in HCC therapy, the identification of new therapeutic targets through integrated genomic approaches remains an important task. Because a large amount of HCC genomic data genotyped by high density single nucleotide polymorphism arrays is deposited in the public domain, copy number alteration (CNA) analyses of these arrays is a cost-effective way to reveal target genes through profiling of recurrent and overlapping amplicons, homozygous deletions and potentially unbalanced chromosomal translocations accumulated during HCC progression. Moreover, integration of CNAs with other high-throughput genomic data, such as aberrantly coding transcriptomes and non-coding gene expression in human HCC tissues and rodent HCC models, provides lines of evidence that can be used to facilitate the identification of novel HCC target genes with the potential of improving the survival of HCC patients.

Long range heliostat target using array of normal incidence pyranometers to evaluate a beam of solar radiation

DOEpatents

Ghanbari, Cheryl M; Ho, Clifford K; Kolb, Gregory J

2014-03-04

Various technologies described herein pertain to evaluating a beam reflected by a heliostat. A portable target that has an array of sensors mounted thereupon is configured to capture the beam reflected by the heliostat. The sensors in the array output measured values indicative of a characteristic of the beam reflected by the heliostat. Moreover, a computing device can generate and output data corresponding to the beam reflected by the heliostat based on the measured values indicative of the characteristic of the beam received from the sensors in the array.

VEGF controls lung Th2 inflammation via the miR-1-Mpl (myeloproliferative leukemia virus oncogene)-P-selectin axis.

PubMed

Takyar, Seyedtaghi; Vasavada, Hema; Zhang, Jian-ge; Ahangari, Farida; Niu, Naiqian; Liu, Qing; Lee, Chun Geun; Cohn, Lauren; Elias, Jack A

2013-09-23

Asthma, the prototypic Th2-mediated inflammatory disorder of the lung, is an emergent disease worldwide. Vascular endothelial growth factor (VEGF) is a critical regulator of pulmonary Th2 inflammation, but the underlying mechanism and the roles of microRNAs (miRNAs) in this process have not been defined. Here we show that lung-specific overexpression of VEGF decreases miR-1 expression in the lung, most prominently in the endothelium, and a similar down-regulation occurs in lung endothelium in Th2 inflammation models. Intranasal delivery of miR-1 inhibited inflammatory responses to ovalbumin, house dust mite, and IL-13 overexpression. Blocking VEGF inhibited Th2-mediated lung inflammation, and this was restored by antagonizing miR-1. Using mRNA arrays, Argonaute pull-down assays, luciferase expression assays, and mutational analysis, we identified Mpl as a direct target of miR-1 and showed that VEGF controls the expression of endothelial Mpl during Th2 inflammation via the regulation of miR-1. In vivo knockdown of Mpl inhibited Th2 inflammation and indirectly inhibited the expression of P-selectin in lung endothelium. These experiments define a novel VEGF-miR-1-Mpl-P-selectin effector pathway in lung Th2 inflammation and herald the utility of miR-1 and Mpl as potential therapeutic targets for asthma.

VEGF controls lung Th2 inflammation via the miR-1–Mpl (myeloproliferative leukemia virus oncogene)–P-selectin axis

PubMed Central

Vasavada, Hema; Zhang, Jian-ge; Ahangari, Farida; Niu, Naiqian; Liu, Qing; Lee, Chun Geun; Cohn, Lauren

2013-01-01

Asthma, the prototypic Th2-mediated inflammatory disorder of the lung, is an emergent disease worldwide. Vascular endothelial growth factor (VEGF) is a critical regulator of pulmonary Th2 inflammation, but the underlying mechanism and the roles of microRNAs (miRNAs) in this process have not been defined. Here we show that lung-specific overexpression of VEGF decreases miR-1 expression in the lung, most prominently in the endothelium, and a similar down-regulation occurs in lung endothelium in Th2 inflammation models. Intranasal delivery of miR-1 inhibited inflammatory responses to ovalbumin, house dust mite, and IL-13 overexpression. Blocking VEGF inhibited Th2-mediated lung inflammation, and this was restored by antagonizing miR-1. Using mRNA arrays, Argonaute pull-down assays, luciferase expression assays, and mutational analysis, we identified Mpl as a direct target of miR-1 and showed that VEGF controls the expression of endothelial Mpl during Th2 inflammation via the regulation of miR-1. In vivo knockdown of Mpl inhibited Th2 inflammation and indirectly inhibited the expression of P-selectin in lung endothelium. These experiments define a novel VEGF–miR-1–Mpl–P-selectin effector pathway in lung Th2 inflammation and herald the utility of miR-1 and Mpl as potential therapeutic targets for asthma. PMID:24043765

Detection and characterisation of multi-drug resistance protein 1 (MRP-1) in human mitochondria.

PubMed

Roundhill, E A; Burchill, S A

2012-03-13

Overexpression of plasma membrane multi-drug resistance protein 1 (MRP-1) can lead to multidrug resistance. In this study, we describe for the first time the expression of mitochondrial MRP-1 in untreated human normal and cancer cells and tissues. MRP-1 expression and subcellular localisation in normal and cancer cells and tissues was examined by differential centrifugation and western blotting, and immunofluorescence microscopy. Viable mitochondria were isolated and MRP-1 efflux activity measured using the calcein-AM functional assay. MRP-1 expression was increased using retroviral infection and specific overexpression confirmed by RNA array. Cell viability was determined by trypan blue exclusion and annexin V-propidium iodide labelling of cells. MRP-1 was detected in the mitochondria of cancer and normal cells and tissues. The efflux activity of mitochondrial MRP-1 was more efficient (55-64%) than that of plasma membrane MRP-1 (11-22%; P<0.001). Induced MRP-1 expression resulted in a preferential increase in mitochondrial MRP-1, suggesting selective targeting to this organelle. Treatment with a non-lethal concentration of doxorubicin (0.85 nM, 8 h) increased mitochondrial and plasma membrane MRP-1, increasing resistance to MRP-1 substrates. For the first time, we have identified MRP-1 with efflux activity in human mitochondria. Mitochondrial MRP-1 may be an exciting new therapeutic target where historically MRP-1 inhibitor strategies have limited clinical success.

The innate immune response in fetal lung mesenchymal cells targets VEGFR2 expression and activity.

PubMed

Medal, Rachel M; Im, Amanda M; Yamamoto, Yasutoshi; Lakhdari, Omar; Blackwell, Timothy S; Hoffman, Hal M; Sahoo, Debashis; Prince, Lawrence S

2017-06-01

In preterm infants, soluble inflammatory mediators target lung mesenchymal cells, disrupting airway and alveolar morphogenesis. However, how mesenchymal cells respond directly to microbial stimuli remains poorly characterized. Our objective was to measure the genome-wide innate immune response in fetal lung mesenchymal cells exposed to the bacterial endotoxin lipopolysaccharide (LPS). With the use of Affymetrix MoGene 1.0st arrays, we showed that LPS induced expression of unique innate immune transcripts heavily weighted toward CC and CXC family chemokines. The transcriptional response was different between cells from E11, E15, and E18 mouse lungs. In all cells tested, LPS inhibited expression of a small core group of genes including the VEGF receptor Vegfr2 Although best characterized in vascular endothelial populations, we demonstrated here that fetal mouse lung mesenchymal cells express Vegfr2 and respond to VEGF-A stimulation. In mesenchymal cells, VEGF-A increased cell migration, activated the ERK/AKT pathway, and promoted FOXO3A nuclear exclusion. With the use of an experimental coculture model of epithelial-mesenchymal interactions, we also showed that VEGFR2 inhibition prevented formation of three-dimensional structures. Both LPS and tyrosine kinase inhibition reduced three-dimensional structure formation. Our data suggest a novel mechanism for inflammation-mediated defects in lung development involving reduced VEGF signaling in lung mesenchyme. Copyright © 2017 the American Physiological Society.

Functional analysis of Discoidin domain receptor 2 mutation and expression in squamous cell lung cancer.

PubMed

Kobayashi-Watanabe, Naomi; Sato, Akemi; Watanabe, Tatsuro; Abe, Tomonori; Nakashima, Chiho; Sueoka, Eisaburo; Kimura, Shinya; Sueoka-Aragane, Naoko

2017-08-01

Discoidin domain receptor (DDR) 2 mutations have recently been reported to be candidate targets of molecular therapy in lung squamous cell carcinoma (SQCC). However, the status of DDR2 expression and mutations, as well as their precise roles in lung SQCC, have not been clarified. We here report DDR2 mutation and expression status in clinical samples and its role of lung SQCC. We investigated DDR2 expression and mutation status in 44 human clinical samples and 7 cell lines. Biological functions of DDR2 were assessed by in vitro cell invasion assay and animal model experiments. Endogenous DDR2 protein expression levels were high in one cell line, PC-1, and immunohistochemistry of lung cancer tissue array showed high levels of DDR2 protein in 29% of lung SQCC patients. A mutation (T681I) identified in lung SQCC and the cell line EBC-1 was detected among 44 primary lung SQCC samples and 7 lung SQCC cell lines. Although Forced expression of DDR2 and its mutant (T681I) led to induce SQCC cell invasion in vitro, only wild type DDR2 enhanced lung metastasis in an animal model. We also found that ectopic expression of DDR2 induced MMP-1 mRNA expression accompanied by phosphorylation of c-Jun after treatment with its ligand, collagen type I, but DDR2 with the T681I mutation did not, suggesting that T681I mutation is an inactivating mutation. Overexpression of DDR2 might contribute to tumor progression in lung SQCC. The overexpression of DDR2 could be potential molecular target of lung SQCC. Copyright © 2017 Elsevier B.V. All rights reserved.

Subarray-based FDA radar to counteract deceptive ECM signals

NASA Astrophysics Data System (ADS)

Abdalla, Ahmed; Wang, Wen-Qin; Yuan, Zhao; Mohamed, Suhad; Bin, Tang

2016-12-01

In recent years, the frequency diverse array (FDA) radar concept has attracted extensive attention, as it may benefit from a small frequency increment, compared to the carrier frequency across the array elements and thereby achieve an array factor that is a function of the angle, the time, and the range which is superior to the conventional phase array radar (PAR). However, limited effort on the subject of FDA in electronic countermeasure scenarios, especially in the presence of mainbeam deceptive jamming, has been published. Basic FDA is not desirable for anti-jamming applications, due to the range-angle coupling response of targets. In this paper, a novel method based on subarrayed FDA signal processing is proposed to counteract deceptive ECM signals. We divide the FDA array into multiple subarrays, each of which employs a distinct frequency increment. As a result, in the subarray-based FDA, the desired target can be distinguished at subarray level in joint range-angle-Doppler domain by utilizing the fact that the jammer generates false targets with the same ranges to each subarray without reparations. The performance assessment shows that the proposed solution is effective for deceptive ECM targets suppression. The effectiveness is verified by simulation results.

Target Tracking Using SePDAF under Ambiguous Angles for Distributed Array Radar.

PubMed

Long, Teng; Zhang, Honggang; Zeng, Tao; Chen, Xinliang; Liu, Quanhua; Zheng, Le

2016-09-09

Distributed array radar can improve radar detection capability and measurement accuracy. However, it will suffer cyclic ambiguity in its angle estimates according to the spatial Nyquist sampling theorem since the large sparse array is undersampling. Consequently, the state estimation accuracy and track validity probability degrades when the ambiguous angles are directly used for target tracking. This paper proposes a second probability data association filter (SePDAF)-based tracking method for distributed array radar. Firstly, the target motion model and radar measurement model is built. Secondly, the fusion result of each radar's estimation is employed to the extended Kalman filter (EKF) to finish the first filtering. Thirdly, taking this result as prior knowledge, and associating with the array-processed ambiguous angles, the SePDAF is applied to accomplish the second filtering, and then achieving a high accuracy and stable trajectory with relatively low computational complexity. Moreover, the azimuth filtering accuracy will be promoted dramatically and the position filtering accuracy will also improve. Finally, simulations illustrate the effectiveness of the proposed method.

Targeting Staphylococcus aureus Toxins: A Potential form of Anti-Virulence Therapy

PubMed Central

Kong, Cin; Neoh, Hui-min; Nathan, Sheila

2016-01-01

Staphylococcus aureus is an opportunistic pathogen and the leading cause of a wide range of severe clinical infections. The range of diseases reflects the diversity of virulence factors produced by this pathogen. To establish an infection in the host, S. aureus expresses an inclusive set of virulence factors such as toxins, enzymes, adhesins, and other surface proteins that allow the pathogen to survive under extreme conditions and are essential for the bacteria’s ability to spread through tissues. Expression and secretion of this array of toxins and enzymes are tightly controlled by a number of regulatory systems. S. aureus is also notorious for its ability to resist the arsenal of currently available antibiotics and dissemination of various multidrug-resistant S. aureus clones limits therapeutic options for a S. aureus infection. Recently, the development of anti-virulence therapeutics that neutralize S. aureus toxins or block the pathways that regulate toxin production has shown potential in thwarting the bacteria’s acquisition of antibiotic resistance. In this review, we provide insights into the regulation of S. aureus toxin production and potential anti-virulence strategies that target S. aureus toxins. PMID:26999200

Identification of testis-specific male contraceptive targets: insights from transcriptional profiling of the cycle of the rat seminiferous epithelium and purified testicular cells.

PubMed

Johnston, Daniel S; Jelinsky, Scott A; Zhi, Yu; Finger, Joshua N; Kopf, Gregory S; Wright, William W

2007-12-01

In an effort to identify novel targets for the development of nonhormonal male contraceptives, genome-wide transcriptional profiling of the rat testis was performed. Specifically, enzymatically purified spermatogonia plus early spermatocyctes, pachytene spermatocytes, round spermatids, and Sertoli cells was analyzed along with microdissected rat seminiferous tubules at stages I, II-III, IV-V, VI, VIIa,b, VIIc,d, VIII, IX- XI, XII, XIII-XIV of the cycle of the seminiferous epithelium using RAE 230_2.0 microarrays. The combined analysis of these studies identified 16,971 expressed probe sets on the array. How these expression data, combined with additional bioinformatic data analysis and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis, led to the identification of 58 genes that have 1000-fold higher expression transcriptionally in the testis when compared to over 20 other nonreproductive tissues is described. The products of these genes may play important roles in testicular and/or sperm function, and further investigation on their utility as nonhormonal contraceptive targets is warranted. Moreover, these microarray data have been used to expedite the identification of a mutation in RIKEN cDNA 2410004F06 gene as likely being responsible for spermatogenic failure in a line of infertile mice generated by N-ethyl-N-nitrosourea (ENU) mutagenesis. The microarray data and the qRT-PCR data described are available in the Mammalian Reproductive Genetics database (http://mrg.genetics.washington.edu/).

Integrated Analysis of Dysregulated miRNA-gene Expression in HMGA2-silenced Retinoblastoma Cells

PubMed Central

Venkatesan, Nalini; Deepa, PR; Vasudevan, Madavan; Khetan, Vikas; Reddy, Ashwin M; Krishnakumar, Subramanian

2014-01-01

Retinoblastoma (RB) is a primary childhood eye cancer. HMGA2 shows promise as a molecule for targeted therapy. The involvement of miRNAs in genome-level molecular dys-regulation in HMGA2-silenced RB cells is poorly understood. Through miRNA expression microarray profiling, and an integrated array analysis of the HMGA2-silenced RB cells, the dysregulated miRNAs and the miRNA-target relationships were modelled. Loop network analysis revealed a regulatory association between the transcription factor (SOX5) and the deregulated miRNAs (miR-29a, miR-9*, miR-9-3). Silencing of HMGA2 deregulated the vital oncomirs (miR-7, miR-331, miR-26a, miR-221, miR-17~92 and miR-106b∼25) in RB cells. From this list, the role of the miR-106b∼25 cluster was examined further for its expression in primary RB tumor tissues (n = 20). The regulatory targets of miR-106b∼25 cluster namely p21 (cyclin-dependent kinase inhibitor) and BIM (pro-apoptotic gene) were elevated, and apoptotic cell death was observed, in RB tumor cells treated with the specific antagomirs of the miR-106b∼25 cluster. Thus, suppression of miR-106b∼25 cluster controls RB tumor growth. Taken together, HMGA2 mediated anti-tumor effect present in RB is, in part, mediated through the miR-106b∼25 cluster. PMID:25232279

Micro-RNAs in regenerating lungs: an integrative systems biology analysis of murine influenza pneumonia.

PubMed

Tan, Kai Sen; Choi, Hyungwon; Jiang, Xiaoou; Yin, Lu; Seet, Ju Ee; Patzel, Volker; Engelward, Bevin P; Chow, Vincent T

2014-07-11

Tissue regeneration in the lungs is gaining increasing interest as a potential influenza management strategy. In this study, we explored the role of microRNAs, short non-coding RNAs involved in post-transcriptional regulation, during pulmonary regeneration after influenza infection. We profiled miRNA and mRNA expression levels following lung injury and tissue regeneration using a murine influenza pneumonia model. BALB/c mice were infected with a sub-lethal dose of influenza A/PR/8(H1N1) virus, and their lungs were harvested at 7 and 15 days post-infection to evaluate the expression of ~300 miRNAs along with ~36,000 genes using microarrays. A global network was constructed between differentially expressed miRNAs and their potential target genes with particular focus on the pulmonary repair and regeneration processes to elucidate the regulatory role of miRNAs in the lung repair pathways. The miRNA arrays revealed a global down-regulation of miRNAs. TargetScan analyses also revealed specific miRNAs highly involved in targeting relevant gene functions in repair such as miR-290 and miR-505 at 7 dpi; and let-7, miR-21 and miR-30 at 15 dpi. The significantly differentially regulated miRNAs are implicated in the activation or suppression of cellular proliferation and stem cell maintenance, which are required during the repair of the damaged lungs. These findings provide opportunities in the development of novel repair strategies in influenza-induced pulmonary injury.

Identification of anti-inflammatory fractions of Geranium wilfordii using tumor necrosis factor-alpha as a drug target on Herbochip® - an array-based high throughput screening platform.

PubMed

Huang, Min; Yao, Pei-Wun; Chang, Margaret Dah-Tysr; Ng, Sim-Kun; Yu, Chien-Hui; Zhang, Yun-Feng; Wen, Meng-Liang; Yang, Xiao-Yuan; Lai, Yiu-Kay

2015-05-12

Geranium wilfordii is one of the major species used as Herba Geranii (lao-guan-cao) in China, it is commonly used solely or in polyherbal formulations for treatment of joint pain resulted from rheumatoid arthritis (RA) and gout. This herb is used to validate a target-based drug screening platform called Herbochip® and evaluate anti-inflammatory effects of Geranium wilfordii ethanolic extract (GWE) using tumor necrosis factor-alpha (TNF-α) as a drug target together with subsequent in vitro and in vivo assays. A microarray-based drug screening platform was constructed by arraying HPLC fractions of herbal extracts onto a surface-activated polystyrene slide (Herbochip®). Using TNF-α as a molecular probe, fractions of 82 selected herbal extracts, including GWE, were then screened to identify plant extracts containing TNF-α-binding agents. Cytotoxicity of GWE and modulatory effects of GWE on TNF-α expression were evaluated by cell-based assays using TNF-α sensitive murine fibrosarcoma L929 cells as an in vitro model. The in vivo anti-inflammatory effects of GWE were further assessed by animal models including carrageenan-induced hind paw edema in rats and xylene-induced ear edema in mice, in comparison with aspirin. The hybridization data obtained by Herbochip® analysis showed unambiguous signals which confirmed TNF-α binding activity in 46 herbal extracts including GWE. In L929 cells GWE showed significant inhibitory effect on TNF-α expression with negligible cytotoxicity. GWE also significantly inhibited formation of carrageenan-induced hind paw edema and xylene-induced ear edema in animal models, indicating that it indeed possessed anti-inflammatory activity. We have thus validated effectiveness of the Herbochip® drug screening platform using TNF-α as a molecular target. Subsequent experiments on GWE lead us to conclude that the anti-RA activity of GWE can be attributed to inhibitory effect of GWE on the key inflammatory factor, TNF-α. Our results contribute towards validation of the traditional use of GWE in the treatment of RA and other inflammatory joint disorders.

Dynamic Analysis of Human Natural Killer Cell Response at Single-Cell Resolution in B-Cell Non-Hodgkin Lymphoma.

PubMed

Sarkar, Saheli; Sabhachandani, Pooja; Ravi, Dashnamoorthy; Potdar, Sayalee; Purvey, Sneha; Beheshti, Afshin; Evens, Andrew M; Konry, Tania

2017-01-01

Natural killer (NK) cells are phenotypically and functionally diverse lymphocytes that recognize and kill cancer cells. The susceptibility of target cancer cells to NK cell-mediated cytotoxicity depends on the strength and balance of regulatory (activating/inhibitory) ligands expressed on target cell surface. We performed gene expression arrays to determine patterns of NK cell ligands associated with B-cell non-Hodgkin lymphoma (b-NHL). Microarray analyses revealed significant upregulation of a multitude of NK-activating and costimulatory ligands across varied b-NHL cell lines and primary lymphoma cells, including ULBP1, CD72, CD48, and SLAMF6. To correlate genetic signatures with functional anti-lymphoma activity, we developed a dynamic and quantitative cytotoxicity assay in an integrated microfluidic droplet generation and docking array. Individual NK cells and target lymphoma cells were co-encapsulated in picoliter-volume droplets to facilitate monitoring of transient cellular interactions and NK cell effector outcomes at single-cell level. We identified significant variability in NK-lymphoma cell contact duration, frequency, and subsequent cytolysis. Death of lymphoma cells undergoing single contact with NK cells occurred faster than cells that made multiple short contacts. NK cells also killed target cells in droplets via contact-independent mechanisms that partially relied on calcium-dependent processes and perforin secretion, but not on cytokines (interferon-γ or tumor necrosis factor-α). We extended this technique to characterize functional heterogeneity in cytolysis of primary cells from b-NHL patients. Tumor cells from two diffuse large B-cell lymphoma patients showed similar contact durations with NK cells; primary Burkitt lymphoma cells made longer contacts and were lysed at later times. We also tested the cytotoxic efficacy of NK-92, a continuously growing NK cell line being investigated as an antitumor therapy, using our droplet-based bioassay. NK-92 cells were found to be more efficient in killing b-NHL cells compared with primary NK cells, requiring shorter contacts for faster killing activity. Taken together, our combined genetic and microfluidic analysis demonstrate b-NHL cell sensitivity to NK cell-based cytotoxicity, which was associated with significant heterogeneity in the dynamic interaction at single-cell level.

Analysis of Antisense Expression by Whole Genome Tiling Microarrays and siRNAs Suggests Mis-Annotation of Arabidopsis Orphan Protein-Coding Genes

PubMed Central

Richardson, Casey R.; Luo, Qing-Jun; Gontcharova, Viktoria; Jiang, Ying-Wen; Samanta, Manoj; Youn, Eunseog; Rock, Christopher D.

2010-01-01

Background MicroRNAs (miRNAs) and trans-acting small-interfering RNAs (tasi-RNAs) are small (20–22 nt long) RNAs (smRNAs) generated from hairpin secondary structures or antisense transcripts, respectively, that regulate gene expression by Watson-Crick pairing to a target mRNA and altering expression by mechanisms related to RNA interference. The high sequence homology of plant miRNAs to their targets has been the mainstay of miRNA prediction algorithms, which are limited in their predictive power for other kingdoms because miRNA complementarity is less conserved yet transitive processes (production of antisense smRNAs) are active in eukaryotes. We hypothesize that antisense transcription and associated smRNAs are biomarkers which can be computationally modeled for gene discovery. Principal Findings We explored rice (Oryza sativa) sense and antisense gene expression in publicly available whole genome tiling array transcriptome data and sequenced smRNA libraries (as well as C. elegans) and found evidence of transitivity of MIRNA genes similar to that found in Arabidopsis. Statistical analysis of antisense transcript abundances, presence of antisense ESTs, and association with smRNAs suggests several hundred Arabidopsis ‘orphan’ hypothetical genes are non-coding RNAs. Consistent with this hypothesis, we found novel Arabidopsis homologues of some MIRNA genes on the antisense strand of previously annotated protein-coding genes. A Support Vector Machine (SVM) was applied using thermodynamic energy of binding plus novel expression features of sense/antisense transcription topology and siRNA abundances to build a prediction model of miRNA targets. The SVM when trained on targets could predict the “ancient” (deeply conserved) class of validated Arabidopsis MIRNA genes with an accuracy of 84%, and 76% for “new” rapidly-evolving MIRNA genes. Conclusions Antisense and smRNA expression features and computational methods may identify novel MIRNA genes and other non-coding RNAs in plants and potentially other kingdoms, which can provide insight into antisense transcription, miRNA evolution, and post-transcriptional gene regulation. PMID:20520764

System and method for generating a deselect mapping for a focal plane array

DOEpatents

Bixler, Jay V; Brandt, Timothy G; Conger, James L; Lawson, Janice K

2013-05-21

A method for generating a deselect mapping for a focal plane array according to one embodiment includes gathering a data set for a focal plane array when exposed to light or radiation from a first known target; analyzing the data set for determining which pixels or subpixels of the focal plane array to add to a deselect mapping; adding the pixels or subpixels to the deselect mapping based on the analysis; and storing the deselect mapping. A method for gathering data using a focal plane array according to another embodiment includes deselecting pixels or subpixels based on a deselect mapping; gathering a data set using pixels or subpixels in a focal plane array that are not deselected upon exposure thereof to light or radiation from a target of interest; and outputting the data set.

Expression analysis of G Protein-Coupled Receptors in mouse macrophages.

PubMed

Lattin, Jane E; Schroder, Kate; Su, Andrew I; Walker, John R; Zhang, Jie; Wiltshire, Tim; Saijo, Kaoru; Glass, Christopher K; Hume, David A; Kellie, Stuart; Sweet, Matthew J

2008-04-29

Monocytes and macrophages express an extensive repertoire of G Protein-Coupled Receptors (GPCRs) that regulate inflammation and immunity. In this study we performed a systematic micro-array analysis of GPCR expression in primary mouse macrophages to identify family members that are either enriched in macrophages compared to a panel of other cell types, or are regulated by an inflammatory stimulus, the bacterial product lipopolysaccharide (LPS). Several members of the P2RY family had striking expression patterns in macrophages; P2ry6 mRNA was essentially expressed in a macrophage-specific fashion, whilst P2ry1 and P2ry5 mRNA levels were strongly down-regulated by LPS. Expression of several other GPCRs was either restricted to macrophages (e.g. Gpr84) or to both macrophages and neural tissues (e.g. P2ry12, Gpr85). The GPCR repertoire expressed by bone marrow-derived macrophages and thioglycollate-elicited peritoneal macrophages had some commonality, but there were also several GPCRs preferentially expressed by either cell population. The constitutive or regulated expression in macrophages of several GPCRs identified in this study has not previously been described. Future studies on such GPCRs and their agonists are likely to provide important insights into macrophage biology, as well as novel inflammatory pathways that could be future targets for drug discovery.

MicroRNA-127 Promotes Mesendoderm Differentiation of Mouse Embryonic Stem Cells by Targeting Left-Right Determination Factor 2.

PubMed

Ma, Haixia; Lin, Yu; Zhao, Zhen-Ao; Lu, Xukun; Yu, Yang; Zhang, Xiaoxin; Wang, Qiang; Li, Lei

2016-06-03

Specification of the three germ layers is a fundamental process and is essential for the establishment of organ rudiments. Multiple genetic and epigenetic factors regulate this dynamic process; however, the function of specific microRNAs in germ layer differentiation remains unknown. In this study, we established that microRNA-127 (miR-127) is related to germ layer specification via microRNA array analysis of isolated three germ layers of E7.5 mouse embryos and was verified through differentiation of mouse embryonic stem cells. miR-127 is highly expressed in endoderm and primitive streak. Overexpression of miR-127 increases and inhibition of miR-127 decreases the expression of mesendoderm markers. We further show that miR-127 promotes mesendoderm differentiation through the nodal pathway, a determinative signaling pathway in early embryogenesis. Using luciferase reporter assay, left-right determination factor 2 (Lefty2), an antagonist of nodal, is identified to be a novel target of miR-127. Furthermore, the role of miR-127 in mesendoderm differentiation is attenuated by Lefty2 overexpression. Altogether, our results indicate that miR-127 accelerates mesendoderm differentiation of mouse embryonic stem cells through nodal signaling by targeting Lefty2. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Development of Inhibitors Targeting Hypoxia-Inducible Factor 1 and 2 for Cancer Therapy

PubMed Central

Yu, Tianchi

2017-01-01

Hypoxia is frequently observed in solid tumors and also one of the major obstacles for effective cancer therapies. Cancer cells take advantage of their ability to adapt hypoxia to initiate a special transcriptional program that renders them more aggressive biological behaviors. Hypoxia-inducible factors (HIFs) are the key factors that control hypoxia-inducible pathways by regulating the expression of a vast array of genes involved in cancer progression and treatment resistance. HIFs, mainly HIF-1 and -2, have become potential targets for developing novel cancer therapeutics. This article reviews the updated information in tumor HIF pathways, particularly recent advances in the development of HIF inhibitors. These inhibitors interfere with mRNA expression, protein synthesis, protein degradation and dimerization, DNA binding and transcriptional activity of HIF-1 and -2, or both. Despite efforts in the past two decades, no agents directly inhibiting HIFs have been approved for treating cancer patients. By analyzing results of the published reports, we put the perspectives at the end of the article. The therapeutic efficacy of HIF inhibitors may be improved if more efforts are devoted on developing agents that are able to simultaneously target HIF-1 and -2, increasing the penetrating capacity of HIF inhibitors, and selecting suitable patient subpopulations for clinical trials. PMID:28332352

miRNA-148a serves as a prognostic factor and suppresses migration and invasion through Wnt1 in non-small cell lung cancer.

PubMed

Chen, Yong; Min, Lingfeng; Ren, Chuanli; Xu, Xingxiang; Yang, Jianqi; Sun, Xinchen; Wang, Tao; Wang, Fang; Sun, Changjiang; Zhang, Xizhi

2017-01-01

Lung cancer is the leading cause of cancer death in the world, and aberrant expression of miRNA is a common feature during the cancer initiation and development. Our previous study showed that levels of miRNA-148a assessed by quantitative real-time polymerase chain reaction (qRT-PCR) were a good prognosis factor for non-small cell lung cancer (NSCLC) patients. In this study, we used high-throughput formalin-fixed and paraffin-embedded (FFPE) lung cancer tissue arrays and in situ hybridization (ISH) to determine the clinical significances of miRNA-148a and aimed to find novel target of miRNA-148a in lung cancer. Our results showed that there were 86 of 159 patients with low miRNA-148a expression and miRNA-148a was significantly down-regulated in primary cancer tissues when compared with their adjacent normal lung tissues. Low expression of miRNA-148a was strongly associated with high tumor grade, lymph node (LN) metastasis and a higher risk of tumor-related death in NSCLC. Lentivirus mediated overexpression of miRNA-148a inhibited migration and invasion of A549 and H1299 lung cancer cells. Furthermore, we validated Wnt1 as a direct target of miRNA-148a. Our data showed that the Wnt1 expression was negatively correlated with the expression of miRNA-148a in both primary cancer tissues and their corresponding adjacent normal lung tissues. In addition, overexpression of miRNA-148a inhibited Wnt1 protein expression in cancer cells. And knocking down of Wnt-1 by siRNA had the similar effect of miRNA-148a overexpression on cell migration and invasion in lung cancer cells. In conclusion, our results suggest that miRNA-148a inhibited cell migration and invasion through targeting Wnt1 and this might provide a new insight into the molecular mechanisms of lung cancer metastasis.

The bromodomain protein inhibitor I-BET151 suppresses expression of inflammatory genes and matrix degrading enzymes in rheumatoid arthritis synovial fibroblasts.

PubMed

Klein, Kerstin; Kabala, Pawel A; Grabiec, Aleksander M; Gay, Renate E; Kolling, Christoph; Lin, Lih-Ling; Gay, Steffen; Tak, Paul P; Prinjha, Rab K; Ospelt, Caroline; Reedquist, Kris A

2016-02-01

To investigate the effects of BET bromodomain protein inhibition on inflammatory activation and functional properties of rheumatoid arthritis synovial fibroblasts (RASF). The expression of the BET bromodomain proteins BRD2, BRD3 and BRD4 was analysed in synovial tissue by immunohistochemistry. RASF were stimulated with tumour necrosis factor (TNF)-α, interleukin (IL)-1β and toll-like receptor (TLR) ligands (Pam3, pIC and lipopolysaccharide (LPS)) in the presence or absence of the BET inhibitor I-BET151, or siRNA targeting BRD2, BRD3 and BRD4. RASF expression of inflammatory mediators, including MMP1, MMP3, IL-6 and IL-8, was measured by q-PCR, q-PCR array and ELISA. Cellular viability, apoptosis, proliferation and chemoattractive properties of RASF were investigated using MTT, cell apoptosis ELISA, BrdU-based proliferation and transwell migration assays. BRD2, BRD3 and BRD4 proteins were detected in rheumatoid arthritis (RA) synovial tissue, expressed in both RASF and macrophages. I-BET151 suppressed cytokine and TLR ligand-induced secretion of MMP1, MMP3, IL-6 and IL-8, and mRNA expression of more than 70% of genes induced by TNF-α and IL-1β. Combined silencing of BRD2, BRD3 and BRD4 significantly reduced cytokine and TLR ligand-induced expression of a subset of gene products targeted by I-BET151, including MMP1, CXCL10 and CXCL11. I-BET151 treatment of RASF reduced RASF proliferation, and the chemotactic potential for peripheral blood leucocytes of RASF conditioned medium. Inhibition of BET family proteins suppresses the inflammatory, matrix-degrading, proliferative and chemoattractive properties of RASF and suggests a therapeutic potential in the targeting of epigenetic reader proteins in RA. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Essential role for cyclic-AMP responsive element binding protein 1 (CREB) in the survival of acute lymphoblastic leukemia.

PubMed

van der Sligte, Naomi E; Kampen, Kim R; ter Elst, Arja; Scherpen, Frank J G; Meeuwsen-de Boer, Tiny G J; Guryev, Victor; van Leeuwen, Frank N; Kornblau, Steven M; de Bont, Eveline S J M

2015-06-20

Acute lymphoblastic leukemia (ALL) relapse remains a leading cause of cancer related death in children, therefore, new therapeutic options are needed. Recently, we showed that a peptide derived from Cyclic-AMP Responsive Element Binding Protein (CREB) was highly phosphorylated in pediatric leukemias. In this study, we determined CREB phosphorylation and mRNA levels showing that CREB expression was significantly higher in ALL compared to normal bone marrow (phosphorylation: P < 0.0001, mRNA: P = 0.004). High CREB and phospho-CREB expression was correlated with a lower median overall survival in a cohort of 140 adult ALL patients. ShRNA mediated knockdown of CREB in ALL cell lines blocked leukemic cell growth by inducing cell cycle arrest and apoptosis. Gene expression array analysis showed downregulation of CREB target genes regulating cell proliferation and glucose metabolism and upregulation of apoptosis inducing genes. Similar to CREB knockdown, the CREB inhibitor KG-501 decreased leukemic cell viability and induced apoptosis in ALL cell lines, as well as primary T-ALL samples, with cases showing high phospho-CREB levels being more sensitive than those with lower phospho-CREB levels. Together, these in vitro findings support an important role for CREB in the survival of ALL cells and identify this transcription factor as a potential target for treatment.

GENE EXPRESSION PATTERNS ASSOCIATED WITH INFERTILITY IN HUMAN AND RODENT MODELS

EPA Science Inventory

Modern genomic technologies such as DNA arrays provide the means to investigate molecular interactions at an unprecedented level, and arrays have been used to carry out gene expression profiling as a means of identifying candidate genes involved in molecular mechanisms underlying...

Refinement of light-responsive transcript lists using rice oligonucleotide arrays: evaluation of gene-redundancy.

PubMed

Jung, Ki-Hong; Dardick, Christopher; Bartley, Laura E; Cao, Peijian; Phetsom, Jirapa; Canlas, Patrick; Seo, Young-Su; Shultz, Michael; Ouyang, Shu; Yuan, Qiaoping; Frank, Bryan C; Ly, Eugene; Zheng, Li; Jia, Yi; Hsia, An-Ping; An, Kyungsook; Chou, Hui-Hsien; Rocke, David; Lee, Geun Cheol; Schnable, Patrick S; An, Gynheung; Buell, C Robin; Ronald, Pamela C

2008-10-06

Studies of gene function are often hampered by gene-redundancy, especially in organisms with large genomes such as rice (Oryza sativa). We present an approach for using transcriptomics data to focus functional studies and address redundancy. To this end, we have constructed and validated an inexpensive and publicly available rice oligonucleotide near-whole genome array, called the rice NSF45K array. We generated expression profiles for light- vs. dark-grown rice leaf tissue and validated the biological significance of the data by analyzing sources of variation and confirming expression trends with reverse transcription polymerase chain reaction. We examined trends in the data by evaluating enrichment of gene ontology terms at multiple false discovery rate thresholds. To compare data generated with the NSF45K array with published results, we developed publicly available, web-based tools (www.ricearray.org). The Oligo and EST Anatomy Viewer enables visualization of EST-based expression profiling data for all genes on the array. The Rice Multi-platform Microarray Search Tool facilitates comparison of gene expression profiles across multiple rice microarray platforms. Finally, we incorporated gene expression and biochemical pathway data to reduce the number of candidate gene products putatively participating in the eight steps of the photorespiration pathway from 52 to 10, based on expression levels of putatively functionally redundant genes. We confirmed the efficacy of this method to cope with redundancy by correctly predicting participation in photorespiration of a gene with five paralogs. Applying these methods will accelerate rice functional genomics.

Novel β-catenin target genes identified in thalamic neurons encode modulators of neuronal excitability

PubMed Central

2012-01-01

Background LEF1/TCF transcription factors and their activator β-catenin are effectors of the canonical Wnt pathway. Although Wnt/β-catenin signaling has been implicated in neurodegenerative and psychiatric disorders, its possible role in the adult brain remains enigmatic. To address this issue, we sought to identify the genetic program activated by β-catenin in neurons. We recently showed that β-catenin accumulates specifically in thalamic neurons where it activates Cacna1g gene expression. In the present study, we combined bioinformatics and experimental approaches to find new β-catenin targets in the adult thalamus. Results We first selected the genes with at least two conserved LEF/TCF motifs within the regulatory elements. The resulting list of 428 putative LEF1/TCF targets was significantly enriched in known Wnt targets, validating our approach. Functional annotation of the presumed targets also revealed a group of 41 genes, heretofore not associated with Wnt pathway activity, that encode proteins involved in neuronal signal transmission. Using custom polymerase chain reaction arrays, we profiled the expression of these genes in the rat forebrain. We found that nine of the analyzed genes were highly expressed in the thalamus compared with the cortex and hippocampus. Removal of nuclear β-catenin from thalamic neurons in vitro by introducing its negative regulator Axin2 reduced the expression of six of the nine genes. Immunoprecipitation of chromatin from the brain tissues confirmed the interaction between β-catenin and some of the predicted LEF1/TCF motifs. The results of these experiments validated four genes as authentic and direct targets of β-catenin: Gabra3 for the receptor of GABA neurotransmitter, Calb2 for the Ca2+-binding protein calretinin, and the Cacna1g and Kcna6 genes for voltage-gated ion channels. Two other genes from the latter cluster, Cacna2d2 and Kcnh8, appeared to be regulated by β-catenin, although the binding of β-catenin to the regulatory sequences of these genes could not be confirmed. Conclusions In the thalamus, β-catenin regulates the expression of a novel group of genes that encode proteins involved in neuronal excitation. This implies that the transcriptional activity of β-catenin is necessary for the proper excitability of thalamic neurons, may influence activity in the thalamocortical circuit, and may contribute to thalamic pathologies. PMID:23157480

Lung Microtissue Array to Screen the Fibrogenic Potential of Carbon Nanotubes

PubMed Central

Chen, Zhaowei; Wang, Qixin; Asmani, Mohammadnabi; Li, Yan; Liu, Chang; Li, Changning; Lippmann, Julian M.; Wu, Yun; Zhao, Ruogang

2016-01-01

Due to their excellent physical and chemical characteristics, multi-wall carbon nanotubes (MWCNT) have the potential to be used in structural composites, conductive materials, sensors, drug delivery and medical imaging. However, because of their small-size and light-weight, the applications of MWCNT also raise health concerns. In vivo animal studies have shown that MWCNT cause biomechanical and genetic alterations in the lung tissue which lead to lung fibrosis. To screen the fibrogenic risk factor of specific types of MWCNT, we developed a human lung microtissue array device that allows real-time and in-situ readout of the biomechanical properties of the engineered lung microtissue upon MWCNT insult. We showed that the higher the MWCNT concentration, the more severe cytotoxicity was observed. More importantly, short type MWCNT at low concentration of 50 ng/ml stimulated microtissue formation and contraction force generation, and caused substantial increase in the fibrogenic marker miR-21 expression, indicating the high fibrogenic potential of this specific carbon nanotube type and concentration. The presented microtissue array system provides a powerful tool for high-throughput examination of the therapeutic and toxicological effects of target compounds in realistic tissue environment. PMID:27510174

Method for replicating an array of nucleic acid probes

DOEpatents

Cantor, Charles R.; Przetakiewicz, Marek; Smith, Cassandra L.; Sano, Takeshi

1998-01-01

The invention relates to the replication of probe arrays and methods for replicating arrays of probes which are useful for the large scale manufacture of diagnostic aids used to screen biological samples for specific target sequences. Arrays created using PCR technology may comprise probes with 5'- and/or 3'-overhangs.

Transglutaminase 2 expression in acute myeloid leukemia: Association with adhesion molecule expression and leukemic blast motility

PubMed Central

Meyer, Stefan; Ravandi-Kashani, Farhad; Borthakur, Gautam; Coombes, Kevin R.; Zhang, Nianxiang; Kornblau, Steven

2016-01-01

Acute myeloid leukemia (AML) is a heterogenous disease with differential oncogene association, outcome and treatment regimens. Treatment strategies for AML have improved outcome but despite increased molecular biological information AML is still associated with poor prognosis. Proteomic analysis on the effects of a range of leukemogenic oncogenes showed that the protein transglutaminase 2 (TG2) is expressed at greater levels as a consequence of oncogenic transformation. Further analysis of this observation was performed with 511 AML samples using reverse phase proteomic arrays, demonstrating that TG2 expression was higher at relapse than diagnosis in many cases. In addition elevated TG2 expression correlated with increased expression of numerous adhesion proteins and many apoptosis regulating proteins, two processes related to leukemogenesis. TG2 has previously been linked to drug resistance in cancer and given the negative correlation between TG2 levels and peripheral blasts observed increased TG2 levels may lead to the protection of the leukemic stem cell due to increased adhesion/reduced motility. TG2 may therefore form part of a network of proteins that define poor outcome in AML patients and potentially offer a target to sensitize AML stem cells to drug treatment. PMID:23576428

The drift chamber array at the external target facility in HIRFL-CSR

NASA Astrophysics Data System (ADS)

Sun, Y. Z.; Sun, Z. Y.; Wang, S. T.; Duan, L. M.; Sun, Y.; Yan, D.; Tang, S. W.; Yang, H. R.; Lu, C. G.; Ma, P.; Yu, Y. H.; Zhang, X. H.; Yue, K.; Fang, F.; Su, H.

2018-06-01

A drift chamber array at the External Target Facility in HIRFL-CSR has been constructed for three-dimensional particle tracking in high-energy radioactive ion beam experiments. The design, readout, track reconstruction program and calibration procedures for the detector are described. The drift chamber array was tested in a 311 AMeV 40Ar beam experiment. The detector performance based on the measurements of the beam test is presented. A spatial resolution of 230 μm is achieved.

Portable pathogen detection system

DOEpatents

Colston, Billy W.; Everett, Matthew; Milanovich, Fred P.; Brown, Steve B.; Vendateswaran, Kodumudi; Simon, Jonathan N.

2005-06-14

A portable pathogen detection system that accomplishes on-site multiplex detection of targets in biological samples. The system includes: microbead specific reagents, incubation/mixing chambers, a disposable microbead capture substrate, and an optical measurement and decoding arrangement. The basis of this system is a highly flexible Liquid Array that utilizes optically encoded microbeads as the templates for biological assays. Target biological samples are optically labeled and captured on the microbeads, which are in turn captured on an ordered array or disordered array disposable capture substrate and then optically read.

A Single Multiplex crRNA Array for FnCpf1-Mediated Human Genome Editing.

PubMed

Sun, Huihui; Li, Fanfan; Liu, Jie; Yang, Fayu; Zeng, Zhenhai; Lv, Xiujuan; Tu, Mengjun; Liu, Yeqing; Ge, Xianglian; Liu, Changbao; Zhao, Junzhao; Zhang, Zongduan; Qu, Jia; Song, Zongming; Gu, Feng

2018-06-15

Cpf1 has been harnessed as a tool for genome manipulation in various species because of its simplicity and high efficiency. Our recent study demonstrated that FnCpf1 could be utilized for human genome editing with notable advantages for target sequence selection due to the flexibility of the protospacer adjacent motif (PAM) sequence. Multiplex genome editing provides a powerful tool for targeting members of multigene families, dissecting gene networks, modeling multigenic disorders in vivo, and applying gene therapy. However, there are no reports at present that show FnCpf1-mediated multiplex genome editing via a single customized CRISPR RNA (crRNA) array. In the present study, we utilize a single customized crRNA array to simultaneously target multiple genes in human cells. In addition, we also demonstrate that a single customized crRNA array to target multiple sites in one gene could be achieved. Collectively, FnCpf1, a powerful genome-editing tool for multiple genomic targets, can be harnessed for effective manipulation of the human genome. Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Protein-protein interactions: an application of Tus-Ter mediated protein microarray system.

PubMed

Sitaraman, Kalavathy; Chatterjee, Deb K

2011-01-01

In this chapter, we present a novel, cost-effective microarray strategy that utilizes expression-ready plasmid DNAs to generate protein arrays on-demand and its use to validate protein-protein interactions. These expression plasmids were constructed in such a way so as to serve a dual purpose of synthesizing the protein of interest as well as capturing the synthesized protein. The microarray system is based on the high affinity binding of Escherichia coli "Tus" protein to "Ter," a 20 bp DNA sequence involved in the regulation of DNA replication. The protein expression is carried out in a cell-free protein synthesis system, with rabbit reticulocyte lysates, and the target proteins are detected either by labeled incorporated tag specific or by gene-specific antibodies. This microarray system has been successfully used for the detection of protein-protein interaction because both the target protein and the query protein can be transcribed and translated simultaneously in the microarray slides. The utility of this system for detecting protein-protein interaction is demonstrated by a few well-known examples: Jun/Fos, FRB/FKBP12, p53/MDM2, and CDK4/p16. In all these cases, the presence of protein complexes resulted in the localization of fluorophores at the specific sites of the immobilized target plasmids. Interestingly, during our interactions studies we also detected a previously unknown interaction between CDK2 and p16. Thus, this Tus-Ter based system of protein microarray can be used for the validation of known protein interactions as well as for identifying new protein-protein interactions. In addition, it can be used to examine and identify targets of nucleic acid-protein, ligand-receptor, enzyme-substrate, and drug-protein interactions.

KRC-327, a selective novel inhibitor of c-Met receptor tyrosine kinase with anticancer activity.

PubMed

Park, Byung Hee; Jung, Kyung Hee; Yun, Sun-Mi; Hong, Sang-Won; Ryu, Jae Wook; Jung, Heejung; Ha, Jae Du; Lee, Jongkook; Hong, Soon-Sun

2013-05-01

c-Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), have been reported to be involved in tumorigenesis and metastatic progression. We synthesized a novel triazolopyridazine derivative KRC-327 which selectively targets the c-Met. When we performed receptor tyrosine kinases (RTKs) array with 42 different phosphorylated-RTKs, KRC-327 strongly inhibited expression of activated c-Met in MKN-45 cancer cells. This was confirmed by immunofluorescence staining. Also, KRC-327 decreased the expression of Gab1, Akt, signal transducer and activator of transcription 3 (STAT3) and Erk, down-stream signals of c-Met. KRC-327 strongly suppressed the growth of c-Met over-expressed cancer cells (MKN-45, SNU-638, SNU-5), while not in c-Met absent cancer cell lines (MKN-1, SNU-1). Furthermore, KRC-327 effectively induced cell cycle arrest, especially G0/G1 arrest by increasing expression of p21, p27 and decreasing that of cyclin D1. In the ligand-induced functional studies, KRC-327 inhibited proliferation of HGF-stimulated BxPC-3 cells, the migration of HGF-stimulated AGS cancer cells, and suppressed colony formation in HGF-stimulated U-87MG cells. In xenograft animal models, KRC-327 significantly not only delayed tumor growth but also suppressed phosphorylation of c-Met and its signaling cascades as well as proliferation. Taken together, these results demonstrate that KRC-327 selectively targets c-Met, resulting in inhibition of cell growth and proliferation. Therefore, we suggest that KRC-327 may be a novel drug candidate with the therapeutic potential of targeting c-Met in human cancer. Crown Copyright © 2013. Published by Elsevier Ireland Ltd. All rights reserved.

Anti-chondroitin sulfate proteoglycan 4-specific antibodies modify the effects of vemurafenib on melanoma cells differentially in normoxia and hypoxia

PubMed Central

PUCCIARELLI, DANIELA; LENGGER, NINA; TAKACOVA, MARTINA; CSADEROVA, LUCIA; BARTOSOVA, MARIA; BREITENEDER, HEIMO; PASTOREKOVA, SILVIA; HAFNER, CHRISTINE

2015-01-01

Chondroitin sulfate proteoglycan 4 (CSPG4), a highly immunogenic melanoma tumor antigen, is a potential target for antibody-based immunotherapy. The mechanism by which CSPG4 affects melanoma progression is only partly understood, in particular the involvement of other receptor tyrosine kinases and the tumor microenvironment. We have previously reported on a mimotope-based vaccine against CSPG4 in a human melanoma xenograft model that resulted in reduction of tumor growth. Herein we describe the influence of hypoxia on the response to polyclonal anti-CSPG4-antibodies induced by this vaccine in combination with the BRAF inhibitor vemurafenib to enhance therapeutic efficacy by simultaneously targeting multiple signaling pathways. Melanoma cells were treated with polyclonal anti-CSPG4-antibodies and vemurafenib. Proliferation, migration and invasion were evaluated in a real-time setting in the impedance-based x-CELLigence® system. Western blotting and quantitative PCR arrays were used to determine protein and mRNA expression of hypoxia inducible factor 1α (HIF1α), carbonic anhydrase IX (CAIX) and signaling pathway proteins. A melanoma xenograft model was used to detect HIF1α and CAIX expression in vivo. Hypoxia enhanced the antiproliferative response to vemurafenib. The migration and invasion capacities of vemurafenib-treated melanoma cells were increased, in spite of vemurafenib-decreased expression of HIF1α and CAIX. Polyclonal anti-CSPG4-antibodies reduced the Transwell migration of vemurafenib-treated, BRAF V600E-mutant and CSPG4-expressing melanoma cells in hypoxia. This was associated with the downregulation of phosphorylated AKT, a kinase contributing to tumor cell migration. Our results highlight CSPG4 as a potential target for modulating treatment resistance to vemurafenib induced by the hypoxic microenvironment. PMID:25997619

Anti-chondroitin sulfate proteoglycan 4-specific antibodies modify the effects of vemurafenib on melanoma cells differentially in normoxia and hypoxia.

PubMed

Pucciarelli, Daniela; Lengger, Nina; Takacova, Martina; Csaderova, Lucia; Bartosova, Maria; Breiteneder, Heimo; Pastorekova, Silvia; Hafner, Christine

2015-07-01

Chondroitin sulfate proteoglycan 4 (CSPG4), a highly immunogenic melanoma tumor antigen, is a potential target for antibody-based immunotherapy. The mechanism by which CSPG4 affects melanoma progression is only partly understood, in particular the involvement of other receptor tyrosine kinases and the tumor microenvironment. We have previously reported on a mimotope-based vaccine against CSPG4 in a human melanoma xenograft model that resulted in reduction of tumor growth. Herein we describe the influence of hypoxia on the response to polyclonal anti-CSPG4-antibodies induced by this vaccine in combination with the BRAF inhibitor vemurafenib to enhance therapeutic efficacy by simultaneously targeting multiple signaling pathways. Melanoma cells were treated with polyclonal anti-CSPG4-antibodies and vemurafenib. Proliferation, migration and invasion were evaluated in a real-time setting in the impedance-based x-CELLigence® system. Western blotting and quantitative PCR arrays were used to determine protein and mRNA expression of hypoxia inducible factor 1α (HIF1α), carbonic anhydrase IX (CAIX) and signaling pathway proteins. A melanoma xenograft model was used to detect HIF1α and CAIX expression in vivo. Hypoxia enhanced the antiproliferative response to vemurafenib. The migration and invasion capacities of vemurafenib-treated melanoma cells were increased, in spite of vemurafenib-decreased expression of HIF1α and CAIX. Polyclonal anti-CSPG4-antibodies reduced the Transwell migration of vemurafenib-treated, BRAF V600E-mutant and CSPG4-expressing melanoma cells in hypoxia. This was associated with the downregulation of phosphorylated AKT, a kinase contributing to tumor cell migration. Our results highlight CSPG4 as a potential target for modulating treatment resistance to vemurafenib induced by the hypoxic microenvironment.

A proposed metric for assessing the measurement quality of individual microarrays

PubMed Central

Kim, Kyoungmi; Page, Grier P; Beasley, T Mark; Barnes, Stephen; Scheirer, Katherine E; Allison, David B

2006-01-01

Background High-density microarray technology is increasingly applied to study gene expression levels on a large scale. Microarray experiments rely on several critical steps that may introduce error and uncertainty in analyses. These steps include mRNA sample extraction, amplification and labeling, hybridization, and scanning. In some cases this may be manifested as systematic spatial variation on the surface of microarray in which expression measurements within an individual array may vary as a function of geographic position on the array surface. Results We hypothesized that an index of the degree of spatiality of gene expression measurements associated with their physical geographic locations on an array could indicate the summary of the physical reliability of the microarray. We introduced a novel way to formulate this index using a statistical analysis tool. Our approach regressed gene expression intensity measurements on a polynomial response surface of the microarray's Cartesian coordinates. We demonstrated this method using a fixed model and presented results from real and simulated datasets. Conclusion We demonstrated the potential of such a quantitative metric for assessing the reliability of individual arrays. Moreover, we showed that this procedure can be incorporated into laboratory practice as a means to set quality control specifications and as a tool to determine whether an array has sufficient quality to be retained in terms of spatial correlation of gene expression measurements. PMID:16430768

The MSX1 homeobox transcription factor is a downstream target of PHOX2B and activates the Delta-Notch pathway in neuroblastoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Revet, Ingrid; Huizenga, Gerda; Chan, Alvin

Neuroblastoma is an embryonal tumour of the peripheral sympathetic nervous system (SNS). One of the master regulator genes for peripheral SNS differentiation, the homeobox transcription factor PHOX2B, is mutated in familiar and sporadic neuroblastomas. Here we report that inducible expression of PHOX2B in the neuroblastoma cell line SJNB-8 down-regulates MSX1, a homeobox gene important for embryonic neural crest development. Inducible expression of MSX1 in SJNB-8 caused inhibition of both cell proliferation and colony formation in soft agar. Affymetrix micro-array and Northern blot analysis demonstrated that MSX1 strongly up-regulated the Delta-Notch pathway genes DLK1, NOTCH3, and HEY1. In addition, the proneuralmore » gene NEUROD1 was down-regulated. Western blot analysis showed that MSX1 induction caused cleavage of the NOTCH3 protein to its activated form, further confirming activation of the Delta-Notch pathway. These experiments describe for the first time regulation of the Delta-Notch pathway by MSX1, and connect these genes to the PHOX2B oncogene, indicative of a role in neuroblastoma biology. Affymetrix micro-array analysis of a neuroblastic tumour series consisting of neuroblastomas and the more benign ganglioneuromas showed that MSX1, NOTCH3 and HEY1 are more highly expressed in ganglioneuromas. This suggests a block in differentiation of these tumours at distinct developmental stages or lineages.« less

Cancer testis antigens and NY-BR-1 expression in primary breast cancer: prognostic and therapeutic implications.

PubMed

Balafoutas, Dimitrios; zur Hausen, Axel; Mayer, Sebastian; Hirschfeld, Marc; Jaeger, Markus; Denschlag, Dominik; Gitsch, Gerald; Jungbluth, Achim; Stickeler, Elmar

2013-06-03

Cancer-testis antigens (CTA) comprise a family of proteins, which are physiologically expressed in adult human tissues solely in testicular germ cells and occasionally placenta. However, CTA expression has been reported in various malignancies. CTAs have been identified by their ability to elicit autologous cellular and or serological immune responses, and are considered potential targets for cancer immunotherapy. The breast differentiation antigen NY-BR-1, expressed specifically in normal and malignant breast tissue, has also immunogenic properties. Here we evaluated the expression patterns of CTAs and NY-BR-1 in breast cancer in correlation to clinico-pathological parameters in order to determine their possible impact as prognostic factors. The reactivity pattern of various mAbs (6C1, MA454, M3H67, 57B, E978, GAGE #26 and NY-BR-1 #5) were assessed by immunohistochemistry in a tissue micro array series of 210 randomly selected primary invasive breast cancers in order to study the diversity of different CTAs (e.g. MAGE-A, NY-ESO-1, GAGE) and NY-BR-1. These expression data were correlated to clinico-pathological parameters and outcome data including disease-free and overall survival. Expression of at least one CTA was detectable in the cytoplasm of tumor cells in 37.2% of the cases. NY-BR-1 expression was found in 46.6% of tumors, respectively. Overall, CTA expression seemed to be linked to adverse prognosis and M3H67 immunoreactivity specifically was significantly correlated to shorter overall and disease-free survival (p=0.000 and 0.024, respectively). Our findings suggest that M3H67 immunoreactivity could serve as potential prognostic marker in primary breast cancer patients. The exclusive expression of CTAs in tumor tissues as well as the frequent expression of NY-BR-1 could define new targets for specific breast cancer therapies.

Laser radar range and detection performance for MEMS corner cube retroreflector arrays

NASA Astrophysics Data System (ADS)

Grasso, Robert J.; Odhner, Jefferson E.; Stewart, Hamilton; McDaniel, Robert V.

2004-12-01

BAE SYSTEMS reports on a program to characterize the performance of MEMS corner cube retroreflector arrays under laser illumination. These arrays have significant military and commercial application in the areas of: 1) target identification; 2) target tracking; 3) target location; 4) identification friend-or-foe (IFF); 5) parcel tracking, and; 6) search and rescue assistance. BAE SYSTEMS has theoretically determined the feasibility of these devices to learn if sufficient signal-to-noise performance exists to permit a cooperative laser radar sensor to be considered for device location and interrogation. Results indicate that modest power-apertures are required to achieve SNR performance consistent with high probability of detection and low false alarm rates.

Laser radar range and detection performance for MEMS corner cube retroreflector arrays

NASA Astrophysics Data System (ADS)

Grasso, Robert J.; Jost, Steven R.; Smith, M. J.; McDaniel, Robert V.

2004-01-01

BAE SYSTEMS reports on a program to characterize the performance of MEMS corner cube retroreflector arrays under laser illumination. These arrays have significant military and commercial application in the areas of: (1) target identification; (2) target tracking; (3) target location; (4) identification friend-or-foe (IFF); (5) parcel tracking, and; (6) search and rescue assistance. BAE SYSTEMS has theoretically determined the feasibility of these devices to learn if sufficient signal-to-noise performance exists to permit a cooperative laser radar sensor to be considered for device location and interrogation. Results indicate that modest power-apertures are required to achieve SNR performance consistent with high probability of detection and low false alarm rates.

In vivo phosphorylation of a peptide tag for protein purification.

PubMed

Goux, Marine; Fateh, Amina; Defontaine, Alain; Cinier, Mathieu; Tellier, Charles

2016-05-01

To design a new system for the in vivo phosphorylation of proteins in Escherichia coli using the co-expression of the α-subunit of casein kinase II (CKIIα) and a target protein, (Nanofitin) fused with a phosphorylatable tag. The level of the co-expressed CKIIα was controlled by the arabinose promoter and optimal phosphorylation was obtained with 2 % (w/v) arabinose as inductor. The effectiveness of the phosphorylation system was demonstrated by electrophoretic mobility shift assay (NUT-PAGE) and staining with a specific phosphoprotein-staining gel. The resulting phosphorylated tag was also used to purify the phosphoprotein by immobilized metal affinity chromatography, which relies on the specific interaction of phosphate moieties with Fe(III). The use of a single tag for both the purification and protein array anchoring provides a simple and straightforward system for protein analysis.

Minimalist Antibodies and Mimetics: An Update and Recent Applications.

PubMed

Bruce, Virginia J; Ta, Angeline N; McNaughton, Brian R

2016-10-17

The immune system utilizes antibodies to recognize foreign or disease-relevant receptors, initiating an immune response to destroy unwelcomed guests. Because researchers can evolve antibodies to bind virtually any target, it is perhaps unsurprising that these reagents, and their small-molecule conjugates, are used extensively in clinical and basic research environments. However, virtues of antibodies are countered by significant challenges. Foremost among these is the need for expression in mammalian cells (largely due to often necessary post-translational modifications). In response to these challenges, researchers have developed an array of minimalist antibodies and mimetics, which are smaller, more stable, simpler to express in Escherichia coli, and amendable to laboratory evolution and protein engineering. Here we describe these scaffolds and discuss recent applications of minimalist antibodies and mimetics. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

DNA Microarray Profiling Highlights Nrf2-Mediated Chemoprevention Targeted by Wasabi-Derived Isothiocyanates in HepG2 Cells.

PubMed

Trio, Phoebe Zapanta; Kawahara, Atsuyoshi; Tanigawa, Shunsuke; Sakao, Kozue; Hou, De-Xing

2017-01-01

6-MSITC and 6-MTITC are sulforaphane (SFN) analogs found in Japanese Wasabi. As we reported previously, Wasabi isothiocyanates (ITCs) are activators of Nrf2-antioxidant response element pathway, and also inhibitors of pro-inflammatory cyclooxygenase-2. This study is the first to assess the global changes in transcript levels by Wasabi ITCs, comparing with SFN, in HepG2 cells. We performed comparative gene expression profiling by treating HepG2 cells with ITCs, followed by DNA microarray analyses using HG-U133 plus 2.0 oligonucleotide array. Partial array data on selected gene products were confirmed by RT-PCR and Western blotting. Ingenuity Pathway Analysis (IPA) was used to identify functional subsets of genes and biologically significant network pathways. 6-MTITC showed the highest number of differentially altered (≥2 folds) gene expression, of which 114 genes were upregulated and 75 were downregulated. IPA revealed that Nrf2-mediated pathway, together with glutamate metabolism, is the common significantly modulated pathway across treatments. Interestingly, 6-MSITC exhibited the most potent effect toward Nrf2-mediated pathway. Our data suggest that 6-MSITC could exert chemopreventive role against cancer through its underlying antioxidant activity via the activation of Nrf2-mediated subsequent induction of cytoprotective genes.

Genome‐Wide MicroRNA and Gene Analysis of Mesenchymal Stem Cell Chondrogenesis Identifies an Essential Role and Multiple Targets for miR‐140‐5p

PubMed Central

Tselepi, Maria; Gómez, Rodolfo; Woods, Steven; Hui, Wang; Smith, Graham R.; Shanley, Daryl P.; Clark, Ian M.; Young, David A.

2015-01-01

Abstract microRNAs (miRNAs) are abundantly expressed in development where they are critical determinants of cell differentiation and phenotype. Accordingly miRNAs are essential for normal skeletal development and chondrogenesis in particular. However, the question of which miRNAs are specific to the chondrocyte phenotype has not been fully addressed. Using microarray analysis of miRNA expression during mesenchymal stem cell chondrogenic differentiation and detailed examination of the role of essential differentiation factors, such as SOX9, TGF‐β, and the cell condensation phase, we characterize the repertoire of specific miRNAs involved in chondrocyte development, highlighting in particular miR‐140 and miR‐455. Further with the use of mRNA microarray data we integrate miRNA expression and mRNA expression during chondrogenesis to underline the particular importance of miR‐140, especially the ‐5p strand. We provide a detailed identification and validation of direct targets of miR‐140‐5p in both chondrogenesis and adult chondrocytes with the use of microarray and 3′UTR analysis. This emphasizes the diverse array of targets and pathways regulated by miR‐140‐5p. We are also able to confirm previous experimentally identified targets but, additionally, identify a novel positive regulation of the Wnt signaling pathway by miR‐140‐5p. Wnt signaling has a complex role in chondrogenesis and skeletal development and these findings illustrate a previously unidentified role for miR‐140‐5p in regulation of Wnt signaling in these processes. Together these developments further highlight the role of miRNAs during chondrogenesis to improve our understanding of chondrocyte development and guide cartilage tissue engineering. Stem Cells 2015;33:3266–3280 PMID:26175215

miR-145-dependent targeting of junctional adhesion molecule A and modulation of fascin expression are associated with reduced breast cancer cell motility and invasiveness.

PubMed

Götte, M; Mohr, C; Koo, C-Y; Stock, C; Vaske, A-K; Viola, M; Ibrahim, S A; Peddibhotla, S; Teng, Y H-F; Low, J-Y; Ebnet, K; Kiesel, L; Yip, G W

2010-12-16

Micro RNAs are small non-coding RNAs, which regulate fundamental cellular and developmental processes at the transcriptional and translational level. In breast cancer, miR-145 expression is downregulated compared with healthy control tissue. As several predicted targets of miR-145 potentially regulate cell motility, we aimed at investigating a potential role for miR-145 in breast cancer cell motility and invasiveness. Assisted by Affymetrix array technology, we demonstrate that overexpression of miR-145 in MDA-MB-231, MCF-7, MDA-MB-468 and SK-BR-3 breast cancer cells and in Ishikawa endometrial carcinoma cells leads to a downregulation of the cell-cell adhesion protein JAM-A and of the actin bundling protein fascin. Moreover, podocalyxin and Serpin E1 mRNA levels were downregulated, and gamma-actin, transgelin and MYL9 were upregulated upon miR-145 overexpression. These miR-145-dependent expression changes drastically decreased cancer cell motility, as revealed by time-lapse video microscopy, scratch wound closure assays and matrigel invasion assays. Immunofluorescence microscopy demonstrated restructuring of the actin cytoskeleton and a change in cell morphology by miR-145 overexpression, resulting in a more cortical actin distribution, and reduced actin stress fiber and filopodia formation. Nuclear rotation was observed in 10% of the pre-miR-145 transfected MDA-MB-231 cells, accompanied by a reduction of perinuclear actin. Luciferase activation assays confirmed direct miR-145-dependent regulation of the 3'UTR of JAM-A, whereas siRNA-mediated knockdown of JAM-A expression resulted in decreased motility and invasiveness of MDA-MB-231 and MCF-7 breast cancer cells. Our data identify JAM-A and fascin as novel targets of miR-145, firmly establishing a role for miR-145 in modulating breast cancer cell motility. Our data provide a rationale for future miR-145-targeted approaches of antimetastatic cancer therapy.

MicroRNA-320 is Involved in the Regulation of Cardiac Ischemia/Reperfusion Injury by Targeting Hsp20

PubMed Central

Ren, Xiao-Ping; Wang, Xiaohong; Sartor, Maureen A.; Jones, Keith; Qian, Jiang; Nicolaou, Persoulla; Pritchard, Tracy J.; Fan, Guo-Chang

2009-01-01

Background Recent studies have identified critical roles for microRNAs (miRNAs) in a variety of cellular processes, including regulation of cardiomyocyte death. However, the signature of miRNA expression and possible roles of miRNA in the ischemic heart have been less well-studied. Methods and Results Here we performed miRNA arrays to detect the expression pattern of miRNAs in murine hearts subjected to ischemia/reperfusion (I/R) in vivo and ex vivo. Surprisingly, we found that only miR-320 expression was significantly decreased in the hearts upon I/R in vivo and ex vivo. This was further confirmed by Taqman RT-PCR. Gain-of-function and loss-of-function approaches were employed in cultured adult rat cardiomyocytes to investigate the functional roles of miR-320. Overexpression of miR-320 enhanced cardiomyocyte death and apoptosis, while knock-down was cytoprotective, upon simulated I/R. Furthermore, transgenic mice with cardiac-specific overexpression of miR-320 revealed an increased extent of apoptosis and infarction size in the hearts upon I/R in vivo and ex vivo, relative to the WT controls. Conversely, in vivo treatment with antagomir-320 reduced the infarction size, relative to the administration of mutant antagomir-320 and saline controls. Using Target-Scan software and proteomic analysis, we identified Hsp20, a known cardioprotective protein, as an important candidate target for miR-320. This was validated experimentally by utilizing a luciferase/GFP reporter activity assay and examining the expression of Hsp20 upon miR-320 overexpression and knockdown in cardiomyocytes. Conclusions Our data demonstrate that miR-320 is involved in the regulation of I/R-induced cardiac injury and dysfunction via antithetical regulation of Hsp20. Thus, miR-320 may constitute a new therapeutic target for ischemic heart diseases. PMID:19380620

Arctigenin protects against steatosis in WRL68 hepatocytes through activation of phosphoinositide 3-kinase/protein kinase B and AMP-activated protein kinase pathways.

PubMed

Chen, Kung-Yen; Lin, Jui-An; Yao, Han-Yun; Hsu, An-Chih; Tai, Yu-Ting; Chen, Jui-Tai; Hsieh, Mao-Chih; Shen, Tang-Long; Hsu, Ren-Yi; Wu, Hong-Tan; Wang, Guey Horng; Ho, Bing-Ying; Chen, Yu-Pei

2018-04-01

Arctigenin (ATG), a lignin extracted from Arctium lappa (L.), exerts antioxidant and anti-inflammatory effects. We hypothesized that ATG exerts a protective effect on hepatocytes by preventing nonalcoholic fatty liver disease (NAFLD) progression associated with lipid oxidation-associated lipotoxicity and inflammation. We established an in vitro NAFLD cell model by using normal WRL68 hepatocytes to investigate oleic acid (OA) accumulation and the potential bioactive role of ATG. The results revealed that ATG inhibited OA-induced lipid accumulation, lipid peroxidation, and inflammation in WRL68 hepatocytes, as determined using Oil Red O staining, thiobarbituric acid reactive substance assay, and inflammation antibody array assays. Quantitative RT-PCR analysis demonstrated that ATG significantly mitigated the expression of acetylcoenzyme A carboxylase 1 and sterol regulatory element-binding protein-1 and significantly increased the expression of carnitine palmitoyltransferase 1 and peroxisome proliferator-activated receptor alpha. The 40 targets of the Human Inflammation Antibody Array indicated that ATG significantly inhibited the elevation of the U937 lymphocyte chemoattractant, ICAM-1, IL-1β, IL-6, IL-6sR, IL-7, and IL-8. ATG could activate the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and AMP-activated protein kinase (AMPK) pathways and could increase the phosphorylation levels of Akt and AMPK to mediate cell survival, lipid metabolism, oxidation stress, and inflammation. Thus, we demonstrated that ATG could inhibit NAFLD progression associated with lipid oxidation-associated lipotoxicity and inflammation, and we provided insights into the underlying mechanisms and revealed potential targets to enable a thorough understanding of NAFLD progression. Copyright © 2018 Elsevier Inc. All rights reserved.

Investigational therapies up to Phase II which target PDGF receptors: potential anti-cancer therapeutics.

PubMed

Arrondeau, Jennifer; Huillard, Olivier; Tlemsani, Camille; Cessot, Anatole; Boudou-Rouquette, Pascaline; Blanchet, Benoit; Thomas-Schoemann, Audrey; Vidal, Michel; Tigaud, Jean-Marie; Durand, Jean-Philippe; Alexandre, Jerome; Goldwasser, Francois

2015-05-01

The platelet-derived growth factor receptor (PDGFR) pathway has important functions in cell growth and, by overexpression or mutation, could also be a driver for tumor development. Moreover, PDGFR is expressed in a tumoral microenvironment and could promote tumorigenesis. With these biological considerations, the PDGFR pathway could be an interesting target for therapeutics. Currently, there are many molecules under development that target the PDGFR pathway in different types of cancer. In this review, the authors report the different molecules under development, as well as those approved albeit briefly, which inhibit the PDGFR pathway. Furthermore, the authors summarize their specificities, their toxicities, and their development. Currently, most PDGFR kinase inhibitors are multikinase inhibitors and therefore do not simply target the PDGFR pathway. The development of more specific PDGFR inhibitors could improve drug efficacy. Moreover, selecting tumors harboring mutations or amplifications of PDGFR could improve outcomes associated with the use of these molecules. The authors believe that new technologies, such as kinome arrays or pharmacologic assays, could be of benefit to understanding resistance mechanisms and develop more selective PDGFR inhibitors.

Design and Use of Microphone Directional Arrays for Aeroacoustic Measurements

NASA Technical Reports Server (NTRS)

Humphreys, William M., Jr.; Brooks, Thomas F.; Hunter, William W., Jr.; Meadows, Kristine R.

1998-01-01

An overview of the development of two microphone directional arrays for aeroacoustic testing is presented. These arrays were specifically developed to measure airframe noise in the NASA Langley Quiet Flow Facility. A large aperture directional array using 35 flush-mounted microphones was constructed to obtain high resolution noise localization maps around airframe models. This array possesses a maximum diagonal aperture size of 34 inches. A unique logarithmic spiral layout design was chosen for the targeted frequency range of 2-30 kHz. Complementing the large array is a small aperture directional array, constructed to obtain spectra and directivity information from regions on the model. This array, possessing 33 microphones with a maximum diagonal aperture size of 7.76 inches, is easily moved about the model in elevation and azimuth. Custom microphone shading algorithms have been developed to provide a frequency- and position-invariant sensing area from 10-40 kHz with an overall targeted frequency range for the array of 5-60 kHz. Both arrays are employed in acoustic measurements of a 6 percent of full scale airframe model consisting of a main element NACA 632-215 wing section with a 30 percent chord half-span flap. Representative data obtained from these measurements is presented, along with details of the array calibration and data post-processing procedures.

Bottleneck limitations for microRNA-based therapeutics from bench to the bedside.

PubMed

Chen, Yan; Zhao, Hongliang; Tan, Zhijun; Zhang, Cuiping; Fu, Xiaobing

2015-03-01

MicroRNAs are endogenous non-coding small RNAs that repress expression of a broad array of target genes. Research into the role and underlying molecular events of microRNAs in disease processes and the potential of microRNAs as drug targets has expanded rapidly. Significant advances have been made in identifying the associations of microRNAs with cancers, viral infections, immune diseases, cardiovascular diseases, wound healing, biological development and other areas of medicine. However, because of intense competition and financial risks, there is a series of stringent criteria and conditions that must be met before microRNA-based therapeutics could be pursued as new drug candidates. In this review, we specifically emphasized the obstacles for bench-based microRNA to the bedside, including common barriers in basic research, application limitations while moving to the clinic at the aspects of vector delivery, off-target effects, toxicity mediation, immunological activation and dosage determination, which should be overcome before microRNA-based therapeutics take their place in the clinic.

IL-17 for therapy.

PubMed

Kurschus, Florian C; Moos, Sonja

2017-09-01

The cytokine IL-17 is now a target for an array of therapeutic monoclonal antibodies supposed to treat a variety of inflammatory diseases. The forerunner Secukinumab, an IL-17A neutralizing antibody, is meanwhile approved as first-line treatments for moderate-to-severe plaque psoriasis, and as second-line treatment for psoriatic arthritis and ankylosing spondylitis. Ixekizumab and Brodalumab, both also targeting the IL-17 pathway, were also recently approved by the FDA for plaque psoriasis. Using mice overexpressing IL-17A in a tissue of choice, we showed that the ectopic expression of this cytokine in keratinocytes resulted in a spontaneous and very strong form of psoriasis-like dermatitis. Interestingly, this model showed some typical comorbidities found in humans with psoriasis. In this review, we will discuss why IL-17 is a good target especially in psoriasis and what we learned from mouse models about its functions in pathological situations. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Method for replicating an array of nucleic acid probes

DOEpatents

Cantor, C.R.; Przetakiewicz, M.; Smith, C.L.; Sano, T.

1998-08-18

The invention relates to the replication of probe arrays and methods for replicating arrays of probes which are useful for the large scale manufacture of diagnostic aids used to screen biological samples for specific target sequences. Arrays created using PCR technology may comprise probes with 5{prime}- and/or 3{prime}-overhangs. 16 figs.

Gene expression of cell surface antigens in the early phase of murine influenza pneumonia determined by a cDNA expression array technique.

PubMed

Sakai, Shinya; Mantani, Naoki; Kogure, Toshiaki; Ochiai, Hiroshi; Shimada, Yutaka; Terasawa, Katsutoshi

2002-12-01

Influenza virus is a worldwide health problem with significant economic consequences. To study the gene expression pattern induced by influenza virus infection, it is useful to reveal the pathogenesis of influenza virus infection; but this has not been well examined, especially in vivo study. To assess the influence of influenza virus infection on gene expression in mice, mRNA levels in the lung and tracheal tissue 48 h after infection were investigated by cDNA array analysis. Four-week-old outbred, specific pathogen free strain, ICR female mice were infected by intra-nasal inoculation of a virus solution under ether anesthesia. The mice were sacrificed 48 h after infection and the tracheas and lungs were removed. To determine gene expression, the membrane-based microtechnique with an Atlas cDNA expression array (mouse 1.2 array II) was performed in accordance with the manual provided. We focused on the expression of 46 mRNAs for cell surface antigens. Of these 46 mRNAs that we examined, four (CD1d2 antigen, CD39 antigen-like 1, CD39 antigen-like 3, CD68 antigen) were up-regulated and one (CD36 antigen) was down-regulated. Although further studies are required, these data suggest that these molecules play an important role in influenza virus infection, especially the phase before specific immunity.

Closed-Form Evaluation of Mutual Coupling in a Planar Array of Circular Apertures

NASA Technical Reports Server (NTRS)

Bailey, M. C.

1996-01-01

The integral expression for the mutual admittance between circular apertures in a planar array is evaluated in closed form. Very good accuracy is realized when compared with values that were obtained by numerical integration. Utilization of this closed-form expression, for all element pairs that are separated by more than one element spacing, yields extremely accurate results and significantly reduces the computation time that is required to analyze the performance of a large electronically scanning antenna array.

Selective functionalization of carbon nanotubes based upon distance traveled

NASA Technical Reports Server (NTRS)

Khare, Bishun N. (Inventor); Meyyappan, Meyya (Inventor)

2010-01-01

Method and system for functionalizing a collection of carbon nanotubes (CNTs). A selected precursor gas (e.g., H.sub.2 or NH.sub.3 or NF.sub.3 or F.sub.2 or CF.sub.4 or C.sub.nH.sub.m) is irradiated to provide a cold plasma of selected target particles, such as atomic H or F, in a first chamber. The target particles are directed toward an array of CNTs located in a second chamber while suppressing transport of ultraviolet radiation to the second chamber. A CNT array is functionalized with the target particles, at or below room temperature, to a point of saturation, in an exposure time interval no longer than about 30 sec. The predominant species that are deposited on the CNT array vary with the distance d measured along a path from the precursor gas to the CNT array; two or three different predominant species can be deposited on a CNT array for distances d=d1 and d=d2>d1 and d=d3>d2.

Target Tracking Using SePDAF under Ambiguous Angles for Distributed Array Radar

PubMed Central

Long, Teng; Zhang, Honggang; Zeng, Tao; Chen, Xinliang; Liu, Quanhua; Zheng, Le

2016-01-01

Distributed array radar can improve radar detection capability and measurement accuracy. However, it will suffer cyclic ambiguity in its angle estimates according to the spatial Nyquist sampling theorem since the large sparse array is undersampling. Consequently, the state estimation accuracy and track validity probability degrades when the ambiguous angles are directly used for target tracking. This paper proposes a second probability data association filter (SePDAF)-based tracking method for distributed array radar. Firstly, the target motion model and radar measurement model is built. Secondly, the fusion result of each radar’s estimation is employed to the extended Kalman filter (EKF) to finish the first filtering. Thirdly, taking this result as prior knowledge, and associating with the array-processed ambiguous angles, the SePDAF is applied to accomplish the second filtering, and then achieving a high accuracy and stable trajectory with relatively low computational complexity. Moreover, the azimuth filtering accuracy will be promoted dramatically and the position filtering accuracy will also improve. Finally, simulations illustrate the effectiveness of the proposed method. PMID:27618058

Modulation of intestinal gene expression by dietary zinc status: Effectiveness of cDNA arrays for expression profiling of a single nutrient deficiency

PubMed Central

Blanchard, Raymond K.; Moore, J. Bernadette; Green, Calvert L.; Cousins, Robert J.

2001-01-01

Mammalian nutritional status affects the homeostatic balance of multiple physiological processes and their associated gene expression. Although DNA array analysis can monitor large numbers of genes, there are no reports of expression profiling of a micronutrient deficiency in an intact animal system. In this report, we have tested the feasibility of using cDNA arrays to compare the global changes in expression of genes of known function that occur in the early stages of rodent zinc deficiency. The gene-modulating effects of this deficiency were demonstrated by real-time quantitative PCR measurements of altered mRNA levels for metallothionein 1, zinc transporter 2, and uroguanylin, all of which have been previously documented as zinc-regulated genes. As a result of the low level of inherent noise within this model system and application of a recently reported statistical tool for statistical analysis of microarrays [Tusher, V.G., Tibshirani, R. & Chu, G. (2001) Proc. Natl. Acad. Sci. USA 98, 5116–5121], we demonstrate the ability to reproducibly identify the modest changes in mRNA abundance produced by this single micronutrient deficiency. Among the genes identified by this array profile are intestinal genes that influence signaling pathways, growth, transcription, redox, and energy utilization. Additionally, the influence of dietary zinc supply on the expression of some of these genes was confirmed by real-time quantitative PCR. Overall, these data support the effectiveness of cDNA array expression profiling to investigate the pleiotropic effects of specific nutrients and may provide an approach to establishing markers for assessment of nutritional status. PMID:11717422

Detection of siRNA Mediated Target mRNA Cleavage Activities in Human Cells by a Novel Stem-Loop Array RT-PCR Analysis

DTIC Science & Technology

2016-09-07

sequences of the target mRNA, and a double stranded stem at the 5′ end that forms a stem -loop to function as a forceps to stabilize the secondary...E-mjournal homepage: www.elsevier.com/locate/bbrepDetection of siRNA-mediated target mRNA cleavage activities in human cells by a novel stem -loop...challenges for the accurate and efficient detection and verification of cleavage sites on target mRNAs. Here we used a sensitive stem -loop array reverse

MicroRNAs in the pathobiology of atherosclerosis

PubMed Central

Laffont, Benoit; Rayner, Katey J

2017-01-01

MicroRNAs are short non-coding RNAs, expressed in humans and involved in sequence-specific post-transcriptional regulation of gene expression. They have emerged as key players in a wide array of biological processes, and changes in their expression and/or function have been associated with plethora of human diseases. Atherosclerosis and its related clinical complications, such as myocardial infarction or stroke, represent the leading cause of death in the western world. Accumulating experimental evidence has revealed a key role for microRNAs in regulating cellular and molecular processes related to atherosclerosis development, ranging from risk factors, to plaque initiation and progression, up to atherosclerotic plaque rupture. In this review, we will focus on how microRNAs can influence atherosclerosis biology, as well as the potential clinical applications of microRNAs which are being developed as both targets and therapeutics for a growing industry hoping to harness the power of RNA-guided gene regulation to fight disease and infection. PMID:28232017

ATF3 plays a protective role against toxicity by N-terminal fragment of mutant huntingtin in stable PC12 cell line

PubMed Central

Liang, Yideng; Jiang, Haibing; Ratovitski, Tamara; Jie, Chunfa; Nakamura, Masayuki; Hirschhorn, Ricky R.; Wang, Xiaofang; Smith, Wanli W.; Hai, Tsonwin; Poirier, Michelle A.; Ross, Christopher A.

2009-01-01

Huntington's disease is a progressive neurodegenerative disorder caused by a polyglutamine expansion near the N-terminus of huntingtin. The mechanisms of polyglutamine neurotoxicity, and cellular responses are not fully understood. We have studied gene expression profiles by cDNA array using an inducible PC12 cell model expressing an N-terminal huntingtin fragment with expanded polyglutamine (Htt-N63-148Q). Mutant huntingtin Htt-N63 induced cell death and increased the mRNA and protein levels of activating transcription factor 3 (ATF3). Mutant Htt-N63 also significantly enhanced ATF3 transcriptional activity by a promoter-based reporter assay. Overexpression of ATF3 protects against mutant Htt-N63 toxicity and knocking down ATF3 expression reduced Htt-N63 toxicity in a stable PC12 cell line. These results indicated that ATF3 plays a critical role in toxicity induced by mutant Htt-N63 and may lead to a useful therapeutic target. PMID:19559011

You may look unhappy unless you smile: the distinctiveness of a smiling face against faces without an explicit smile.

PubMed

Park, Hyung-Bum; Han, Ji-Eun; Hyun, Joo-Seok

2015-05-01

An expressionless face is often perceived as rude whereas a smiling face is considered as hospitable. Repetitive exposure to such perceptions may have developed stereotype of categorizing an expressionless face as expressing negative emotion. To test this idea, we displayed a search array where the target was an expressionless face and the distractors were either smiling or frowning faces. We manipulated set size. Search reaction times were delayed with frowning distractors. Delays became more evident as the set size increased. We also devised a short-term comparison task where participants compared two sequential sets of expressionless, smiling, and frowning faces. Detection of an expression change across the sets was highly inaccurate when the change was made between frowning and expressionless face. These results indicate that subjects were confused with expressed emotions on frowning and expressionless faces, suggesting that it is difficult to distinguish expressionless face from frowning faces. Copyright © 2015 Elsevier B.V. All rights reserved.

Assessment of a novel multi-array normalization method based on spike-in control probes suitable for microRNA datasets with global decreases in expression.

PubMed

Sewer, Alain; Gubian, Sylvain; Kogel, Ulrike; Veljkovic, Emilija; Han, Wanjiang; Hengstermann, Arnd; Peitsch, Manuel C; Hoeng, Julia

2014-05-17

High-quality expression data are required to investigate the biological effects of microRNAs (miRNAs). The goal of this study was, first, to assess the quality of miRNA expression data based on microarray technologies and, second, to consolidate it by applying a novel normalization method. Indeed, because of significant differences in platform designs, miRNA raw data cannot be normalized blindly with standard methods developed for gene expression. This fundamental observation motivated the development of a novel multi-array normalization method based on controllable assumptions, which uses the spike-in control probes to adjust the measured intensities across arrays. Raw expression data were obtained with the Exiqon dual-channel miRCURY LNA™ platform in the "common reference design" and processed as "pseudo-single-channel". They were used to apply several quality metrics based on the coefficient of variation and to test the novel spike-in controls based normalization method. Most of the considerations presented here could be applied to raw data obtained with other platforms. To assess the normalization method, it was compared with 13 other available approaches from both data quality and biological outcome perspectives. The results showed that the novel multi-array normalization method reduced the data variability in the most consistent way. Further, the reliability of the obtained differential expression values was confirmed based on a quantitative reverse transcription-polymerase chain reaction experiment performed for a subset of miRNAs. The results reported here support the applicability of the novel normalization method, in particular to datasets that display global decreases in miRNA expression similarly to the cigarette smoke-exposed mouse lung dataset considered in this study. Quality metrics to assess between-array variability were used to confirm that the novel spike-in controls based normalization method provided high-quality miRNA expression data suitable for reliable downstream analysis. The multi-array miRNA raw data normalization method was implemented in an R software package called ExiMiR and deposited in the Bioconductor repository.

Assessment of a novel multi-array normalization method based on spike-in control probes suitable for microRNA datasets with global decreases in expression

PubMed Central

2014-01-01

Background High-quality expression data are required to investigate the biological effects of microRNAs (miRNAs). The goal of this study was, first, to assess the quality of miRNA expression data based on microarray technologies and, second, to consolidate it by applying a novel normalization method. Indeed, because of significant differences in platform designs, miRNA raw data cannot be normalized blindly with standard methods developed for gene expression. This fundamental observation motivated the development of a novel multi-array normalization method based on controllable assumptions, which uses the spike-in control probes to adjust the measured intensities across arrays. Results Raw expression data were obtained with the Exiqon dual-channel miRCURY LNA™ platform in the “common reference design” and processed as “pseudo-single-channel”. They were used to apply several quality metrics based on the coefficient of variation and to test the novel spike-in controls based normalization method. Most of the considerations presented here could be applied to raw data obtained with other platforms. To assess the normalization method, it was compared with 13 other available approaches from both data quality and biological outcome perspectives. The results showed that the novel multi-array normalization method reduced the data variability in the most consistent way. Further, the reliability of the obtained differential expression values was confirmed based on a quantitative reverse transcription–polymerase chain reaction experiment performed for a subset of miRNAs. The results reported here support the applicability of the novel normalization method, in particular to datasets that display global decreases in miRNA expression similarly to the cigarette smoke-exposed mouse lung dataset considered in this study. Conclusions Quality metrics to assess between-array variability were used to confirm that the novel spike-in controls based normalization method provided high-quality miRNA expression data suitable for reliable downstream analysis. The multi-array miRNA raw data normalization method was implemented in an R software package called ExiMiR and deposited in the Bioconductor repository. PMID:24886675

A revised design for microarray experiments to account for experimental noise and uncertainty of probe response.

PubMed

Pozhitkov, Alex E; Noble, Peter A; Bryk, Jarosław; Tautz, Diethard

2014-01-01

Although microarrays are analysis tools in biomedical research, they are known to yield noisy output that usually requires experimental confirmation. To tackle this problem, many studies have developed rules for optimizing probe design and devised complex statistical tools to analyze the output. However, less emphasis has been placed on systematically identifying the noise component as part of the experimental procedure. One source of noise is the variance in probe binding, which can be assessed by replicating array probes. The second source is poor probe performance, which can be assessed by calibrating the array based on a dilution series of target molecules. Using model experiments for copy number variation and gene expression measurements, we investigate here a revised design for microarray experiments that addresses both of these sources of variance. Two custom arrays were used to evaluate the revised design: one based on 25 mer probes from an Affymetrix design and the other based on 60 mer probes from an Agilent design. To assess experimental variance in probe binding, all probes were replicated ten times. To assess probe performance, the probes were calibrated using a dilution series of target molecules and the signal response was fitted to an adsorption model. We found that significant variance of the signal could be controlled by averaging across probes and removing probes that are nonresponsive or poorly responsive in the calibration experiment. Taking this into account, one can obtain a more reliable signal with the added option of obtaining absolute rather than relative measurements. The assessment of technical variance within the experiments, combined with the calibration of probes allows to remove poorly responding probes and yields more reliable signals for the remaining ones. Once an array is properly calibrated, absolute quantification of signals becomes straight forward, alleviating the need for normalization and reference hybridizations.

Slow down to stay alive: HER4 protects against cellular stress and confers chemoresistance in neuroblastoma.

PubMed

Hua, Yingqi; Gorshkov, Kirill; Yang, Yanwen; Wang, Wenyi; Zhang, Nianxiang; Hughes, Dennis P M

2012-10-15

Neuroblastoma (NBL) is a common pediatric solid tumor, and outcomes for patients with advanced neuroblastoma remain poor despite extremely aggressive treatment. Chemotherapy resistance at relapse contributes heavily to treatment failure. The poor survival of patients with high-risk NBL prompted this investigation into novel treatment options with the objective of gaining a better understanding of resistance mechanisms. On the basis of previous work and on data from publicly available studies, the authors hypothesized that human epidermal growth factor receptor 4 (Her4) contributes to resistance. Her4 expression was reduced with small-hairpin RNA (shRNA) to over express intracellular HER4, and the authors tested its impact on tumor cell survival under various culture conditions. The resulting changes in gene expression after HER4 knockdown were measured by using a messenger RNA (mRNA) array. HER4 expression was up-regulated in tumor spheres compared with the expression in monolayer culture. With HER4 knockdown, NBL cells became less resistant to anoikis and serum starvation. Moreover, HER4 knockdown increased the chemosensitivity of NBL cells to cisplatin, doxorubicin, etoposide, and activated ifosfamide. In mRNA array analysis, HER4 knockdown predominately altered genes related to cell cycle regulation. In NBL spheres compared with monolayers, cell proliferation was decreased, and cyclin D expression was reduced. HER4 knockdown reversed cyclin D suppression. Overexpressed intracellular HER4 slowed the cell cycle and induced chemoresistance. The current results indicated that HER4 protects NBL cells from multiple exogenous apoptotic stimuli, including anoikis, nutrient deficiency, and cytotoxic chemotherapy. The intracellular fragment of HER4 was sufficient to confer this phenotype. HER4 functions as a cell cycle suppressor, maintaining resistance to cellular stress. The current findings indicate that HER4 overexpression may be associated with refractory disease, and HER4 may be an important therapeutic target. Copyright © 2012 American Cancer Society.

Uniform and nonuniform V-shaped planar arrays for 2-D direction-of-arrival estimation

NASA Astrophysics Data System (ADS)

Filik, T.; Tuncer, T. E.

2009-10-01

In this paper, isotropic and directional uniform and nonuniform V-shaped arrays are considered for azimuth and elevation direction-of-arrival (DOA) angle estimation simultaneously. It is shown that the uniform isotropic V-shaped arrays (UI V arrays) have no angle coupling between the azimuth and elevation DOA. The design of the UI V arrays is investigated, and closed form expressions are presented for the parameters of the UI V arrays and nonuniform V arrays. These expressions allow one to find the isotropic V angle for different array types. The DOA performance of the UI V array is compared with the uniform circular array (UCA) for correlated signals and in case of mutual coupling between array elements. The modeling error for the sensor positions is also investigated. It is shown that V array and circular array have similar robustness for the position errors while the performance of UI V array is better than the UCA for correlated source signals and when there is mutual coupling. Nonuniform V-shaped isotropic arrays are investigated which allow good DOA performance with limited number of sensors. Furthermore, a new design method for the directional V-shaped arrays is proposed. This method is based on the Cramer-Rao Bound for joint estimation where the angle coupling effect between the azimuth and elevation DOA angles is taken into account. The design method finds an optimum angle between the linear subarrays of the V array. The proposed method can be used to obtain directional arrays with significantly better DOA performance.

miR-151-5p, targeting chromatin remodeler SMARCA5, as a marker for the BRCAness phenotype

PubMed Central

Tommasi, Stefania; Pinto, Rosamaria; Danza, Katia; Pilato, Brunella; Palumbo, Orazio; Micale, Lucia; Summa, Simona De

2016-01-01

In recent years, the assessment of biomarkers useful for “precision medicine” has been a hot topic in research. The involvement of microRNAs in the pathogenesis of breast cancer has been highly investigated with the aim of being able to molecularly stratify this highly heterogeneous disease. Our aim was to identify microRNAs targeting DNA repair machinery, through Affymetrix GeneChip miRNA Arrays, in a cohort of BRCA-related and sporadic breast cancers. Moreover, we analyzed microRNA expression taking into account our previous results on the expression of PARP1, because of its importance in targeted therapy. miR-361-5p and miR-151-5p were found to be overexpressed in PARP1-upregulating BRCA-germline mutated and sporadic breast tumors. Pathway enrichment analysis was performed to identify potential target genes to be analyzed in the validation step in an independent cohort. Our results confirmed the overexpression of miR-151-5p and, interestingly, its role in the targeting of SMARCA5, a chromatin remodeler. This result was also confirmed in vitro, both through luciferase assay and by analyzing endogenous levels of SMARCA5 in MCF-7 cell lines using miR-151-5p mimic and inhibitor. In conclusion, our data showed the possibility of considering the overexpression of PARP1 and miR-151-5p as biomarkers useful to correctly treat sporadic breast cancers, which eventually could be considered as BRCAness tumors, with PARP-inhibitors. PMID:27385001

CRISPR-Cas systems target a diverse collection of invasive mobile genetic elements in human microbiomes

PubMed Central

2013-01-01

Background Bacteria and archaea develop immunity against invading genomes by incorporating pieces of the invaders' sequences, called spacers, into a clustered regularly interspaced short palindromic repeats (CRISPR) locus between repeats, forming arrays of repeat-spacer units. When spacers are expressed, they direct CRISPR-associated (Cas) proteins to silence complementary invading DNA. In order to characterize the invaders of human microbiomes, we use spacers from CRISPR arrays that we had previously assembled from shotgun metagenomic datasets, and identify contigs that contain these spacers' targets. Results We discover 95,000 contigs that are putative invasive mobile genetic elements, some targeted by hundreds of CRISPR spacers. We find that oral sites in healthy human populations have a much greater variety of mobile genetic elements than stool samples. Mobile genetic elements carry genes encoding diverse functions: only 7% of the mobile genetic elements are similar to known phages or plasmids, although a much greater proportion contain phage- or plasmid-related genes. A small number of contigs share similarity with known integrative and conjugative elements, providing the first examples of CRISPR defenses against this class of element. We provide detailed analyses of a few large mobile genetic elements of various types, and a relative abundance analysis of mobile genetic elements and putative hosts, exploring the dynamic activities of mobile genetic elements in human microbiomes. A joint analysis of mobile genetic elements and CRISPRs shows that protospacer-adjacent motifs drive their interaction network; however, some CRISPR-Cas systems target mobile genetic elements lacking motifs. Conclusions We identify a large collection of invasive mobile genetic elements in human microbiomes, an important resource for further study of the interaction between the CRISPR-Cas immune system and invaders. PMID:23628424

Genomic Alteration in Head and Neck Squamous Cell Carcinoma (HNSCC) Cell Lines Inferred from Karyotyping, Molecular Cytogenetics, and Array Comparative Genomic Hybridization

PubMed Central

Rerkarmnuaychoke, Budsaba; Suntronpong, Aorarat; Fu, Beiyuan; Bodhisuwan, Winai; Peyachoknagul, Surin; Yang, Fengtang; Koontongkaew, Sittichai; Srikulnath, Kornsorn

2016-01-01

Genomic alteration in head and neck squamous cell carcinoma (HNSCC) was studied in two cell line pairs (HN30-HN31 and HN4-HN12) using conventional C-banding, multiplex fluorescence in situ hybridization (M-FISH), and array comparative genomic hybridization (array CGH). HN30 and HN4 were derived from primary lesions in the pharynx and base of tongue, respectively, and HN31 and HN12 were derived from lymph-node metastatic lesions belonging to the same patients. Gain of chromosome 1, 7, and 11 were shared in almost all cell lines. Hierarchical clustering revealed that HN31 was closely related to HN4, which shared eight chromosome alteration cases. Large C-positive heterochromatins were found in the centromeric region of chromosome 9 in HN31 and HN4, which suggests complex structural amplification of the repetitive sequence. Array CGH revealed amplification of 7p22.3p11.2, 8q11.23q12.1, and 14q32.33 in all cell lines involved with tumorigenesis and inflammation genes. The amplification of 2p21 (SIX3), 11p15.5 (H19), and 11q21q22.3 (MAML2, PGR, TRPC6, and MMP family) regions, and deletion of 9p23 (PTPRD) and 16q23.1 (WWOX) regions were identified in HN31 and HN12. Interestingly, partial loss of PTPRD (9p23) and WWOX (16q23.1) genes was identified in HN31 and HN12, and the level of gene expression tended to be the down-regulation of PTPRD, with no detectable expression of the WWOX gene. This suggests that the scarcity of PTPRD and WWOX genes might have played an important role in progression of HNSCC, and could be considered as a target for cancer therapy or a biomarker in molecular pathology. PMID:27501229

Effects of topography on the functional development of human neural progenitor cells.

PubMed

Wu, Ze-Zhi; Kisaalita, William S; Wang, Lina; Zachman, Angela L; Zhao, Yiping; Hasneen, Kowser; Machacek, Dave; Stice, Steven L

2010-07-01

We have fabricated a topographical substrate with a packed polystyrene bead array for the development of cell-based assay systems targeting voltage-gated calcium channels (VGCCs). Human neural progenitor cells (H945RB.3) cultured on both flat and topographical substrates were analyzed in terms of morphological spreading, neuronal commitment, resting membrane potential (V(m)) establishment and VGCC function development. We found, by SEM imaging, that arrayed substrates, formed with both sub-micrometer (of 0.51 microm in mean diameter) and micrometer (of 1.98 microm in mean diameter) beads, were capable of promoting the spreading of the progenitor cells as compared with the flat polystyrene surfaces. With the micrometer beads, it was found that arrayed substrates facilitated the neural progenitor cells' maintenance of less negative V(m) values upon differentiation with bFGF starvation, which favored predominant neuronal commitment. Almost all the progenitor cells were responsive to 50 mM K(+) depolarization with an increase in [Ca(2+)](i) either before or upon differentiation, suggesting the expression of functional VGCCs. Compared to the flat polystyrene surfaces, microbead arrayed substrates facilitated the development of higher VGCC responsiveness by the progenitor cells upon differentiation. The enhancement of both VGCC responsiveness and cell spreading by arrays of micrometer beads was most significant on day 14 into differentiation, which was the latest time point of measurement in this study. This study thus rationalized the possibility for future substrate topography engineering to manipulate ion channel function and to meet the challenge of low VGCC responsiveness found in early drug discovery.

Development of a Digital Microarray with Interferometric Reflectance Imaging

NASA Astrophysics Data System (ADS)

Sevenler, Derin

This dissertation describes a new type of molecular assay for nucleic acids and proteins. We call this technique a digital microarray since it is conceptually similar to conventional fluorescence microarrays, yet it performs enumerative ('digital') counting of the number captured molecules. Digital microarrays are approximately 10,000-fold more sensitive than fluorescence microarrays, yet maintain all of the strengths of the platform including low cost and high multiplexing (i.e., many different tests on the same sample simultaneously). Digital microarrays use gold nanorods to label the captured target molecules. Each gold nanorod on the array is individually detected based on its light scattering, with an interferometric microscopy technique called SP-IRIS. Our optimized high-throughput version of SP-IRIS is able to scan a typical array of 500 spots in less than 10 minutes. Digital DNA microarrays may have utility in applications where sequencing is prohibitively expensive or slow. As an example, we describe a digital microarray assay for gene expression markers of bacterial drug resistance.

Clustering and Network Analysis of Reverse Phase Protein Array Data.

PubMed

Byron, Adam

2017-01-01

Molecular profiling of proteins and phosphoproteins using a reverse phase protein array (RPPA) platform, with a panel of target-specific antibodies, enables the parallel, quantitative proteomic analysis of many biological samples in a microarray format. Hence, RPPA analysis can generate a high volume of multidimensional data that must be effectively interrogated and interpreted. A range of computational techniques for data mining can be applied to detect and explore data structure and to form functional predictions from large datasets. Here, two approaches for the computational analysis of RPPA data are detailed: the identification of similar patterns of protein expression by hierarchical cluster analysis and the modeling of protein interactions and signaling relationships by network analysis. The protocols use freely available, cross-platform software, are easy to implement, and do not require any programming expertise. Serving as data-driven starting points for further in-depth analysis, validation, and biological experimentation, these and related bioinformatic approaches can accelerate the functional interpretation of RPPA data.

HIF-2α dictates the susceptibility of pancreatic cancer cells to TRAIL by regulating survivin expression

PubMed Central

Harashima, Nanae; Takenaga, Keizo; Akimoto, Miho; Harada, Mamoru

2017-01-01

Cancer cells develop resistance to therapy by adapting to hypoxic microenvironments, and hypoxia-inducible factors (HIFs) play crucial roles in this process. We investigated the roles of HIF-1α and HIF-2α in cancer cell death induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) using human pancreatic cancer cell lines. siRNA-mediated knockdown of HIF-2α, but not HIF-1α, increased susceptibility of two pancreatic cancer cell lines, Panc-1 and AsPC-1, to TRAIL in vitro under normoxic and hypoxic conditions. The enhanced sensitivity to TRAIL was also observed in vivo. This in vitro increased TRAIL sensitivity was observed in other three pancreatic cancer cell lines. An array assay of apoptosis-related proteins showed that knockdown of HIF-2α decreased survivin expression. Additionally, survivin promoter activity was decreased in HIF-2α knockdown Panc-1 cells and HIF-2α bound to the hypoxia-responsive element in the survivin promoter region. Conversely, forced expression of the survivin gene in HIF-2α shRNA-expressing Panc-1 cells increased resistance to TRAIL. In a xenograft mouse model, the survivin suppressant YM155 sensitized Panc-1 cells to TRAIL. Collectively, our results indicate that HIF-2α dictates the susceptibility of human pancreatic cancer cell lines, Panc-1 and AsPC-1, to TRAIL by regulating survivin expression transcriptionally, and that survivin could be a promising target to augment the therapeutic efficacy of death receptor-targeting anti-cancer therapy. PMID:28476028

microRNA Profiling of Amniotic Fluid: Evidence of Synergy of microRNAs in Fetal Development.

PubMed

Sun, Tingting; Li, Weiyun; Li, Tianpeng; Ling, Shucai

2016-01-01

Amniotic fluid (AF) continuously exchanges molecules with the fetus, playing critical roles in fetal development especially via its complex components. Among these components, microRNAs are thought to be transferred between cells loaded in microvesicles. However, the functions of AF microRNAs remain unknown. To date, few studies have examined microRNAs in amniotic fluid. In this study, we employed miRCURY Locked Nucleotide Acid arrays to profile the dynamic expression of microRNAs in AF from mice on embryonic days E13, E15, and E17. At these times, 233 microRNAs were differentially expressed (p< 0.01), accounting for 23% of the total Mus musculus microRNAs. These differentially-expressed microRNAs were divided into two distinct groups based on their expression patterns. Gene ontology analysis showed that the intersectional target genes of these differentially-expressed microRNAs were mainly distributed in synapse, synaptosome, cell projection, and cytoskeleton. Pathway analysis revealed that the target genes of the two groups of microRNAs were synergistically enriched in axon guidance, focal adhesion, and MAPK signaling pathways. MicroRNA-mRNA network analysis and gene- mapping showed that these microRNAs synergistically regulated cell motility, cell proliferation and differentiation, and especially the axon guidance process. Cancer pathways associated with growth and proliferation were also enriched in AF. Taken together, the results of this study are the first to show the functions of microRNAs in AF during fetal development, providing novel insights into interpreting the roles of AF microRNAs in fetal development.

Retargeting of adenovirus vectors through genetic fusion of a single-chain or single-domain antibody to capsid protein IX.

PubMed

Poulin, Kathy L; Lanthier, Robert M; Smith, Adam C; Christou, Carin; Risco Quiroz, Milagros; Powell, Karen L; O'Meara, Ryan W; Kothary, Rashmi; Lorimer, Ian A; Parks, Robin J

2010-10-01

Adenovirus (Ad) vectors are the most commonly used system for gene therapy applications, due in part to their ability to infect a wide array of cell types and tissues. However, many therapies would benefit from the ability to target the Ad vector only to specific cells, such as tumor cells for cancer gene therapy. In this study, we investigated the utility of capsid protein IX (pIX) as a platform for the presentation of single-chain variable-fragment antibodies (scFv) and single-domain antibodies (sdAb) for virus retargeting. We show that scFv can be displayed on the capsid through genetic fusion to native pIX but that these molecules fail to retarget the virus, due to improper folding of the scFv. Redirecting expression of the fusion protein to the endoplasmic reticulum (ER) results in correct folding of the scFv and allows it to recognize its epitope; however, ER-targeted pIX-scFv was incorporated into the Ad capsid at a very low level which was not sufficient to retarget virus infection. In contrast, a pIX-sdAb construct was efficiently incorporated into the Ad capsid and enhanced virus infection of cells expressing the targeted receptor. Taken together, our data indicate that pIX is an effective platform for presentation of large targeting polypeptides on the surface of the virus capsid, but the nature of the ligand can significantly affect its association with virions.

MicroRNAs Associated with Epithelial-Mesenchymal Transition Can Be Targeted to Inhibit Peritoneal Dissemination of Human Scirrhous Gastric Cancers.

PubMed

Takei, Yoshifumi; Shen, Guodong; Morita-Kondo, Ayami; Hara, Toshifumi; Mihara, Keichiro; Yanagihara, Kazuyoshi

2018-05-30

Scirrhous gastric cancers grow rapidly, and frequently invade the peritoneum. Such peritoneal dissemination properties markedly reduce patient survival. Thus, an effective means for inhibiting peritoneal dissemination is urgently required. We previously established a cell line, HSC-58, from a scirrhous gastric cancer patient, and further successfully isolated a metastatic line, 58As9, in nude mice upon orthotopic inoculation. Using the lines, we examined the mechanism underlying peritoneal dissemination from the viewpoint of microRNA (miRNA) expression. miRNA array and qRT-PCR analysis showed that the expressions of epithelial-mesenchymal transition (EMT)-associated miRNAs such as miR-200c and miR-141 were significantly low in 58As9. Using 58As9 with stably overexpressing miR-200c, miR-141, or both, together with a luciferase reporter assay, we found that miR-200c targeted zinc finger E-box-binding homeobox 1 (ZEB1) and miR-141 targeted ZEB2. The overexpressed lines reversed the EMT status from mesenchymal to epithelial in 58As9, and significantly reduced the invasion activity and peritoneal dissemination for a significant prolongation of survival in the orthotopic tumor models in nude mice. EMT-associated miRNAs such as miR-200c and miR-141 and their target genes ZEB1/ZEB2 have good potential for antiperitoneal dissemination therapy in patients with scirrhous gastric cancers. © 2018 S. Karger AG, Basel.

Microarray Analysis Gene Expression Profiles in Laryngeal Muscle After Recurrent Laryngeal Nerve Injury.

PubMed

Bijangi-Vishehsaraei, Khadijeh; Blum, Kevin; Zhang, Hongji; Safa, Ahmad R; Halum, Stacey L

2016-03-01

The pathophysiology of recurrent laryngeal nerve (RLN) transection injury is rare in that it is characteristically followed by a high degree of spontaneous reinnervation, with reinnervation of the laryngeal adductor complex (AC) preceding that of the abducting posterior cricoarytenoid (PCA) muscle. Here, we aim to elucidate the differentially expressed myogenic factors following RLN injury that may be at least partially responsible for the spontaneous reinnervation. F344 male rats underwent RLN injury (n = 12) or sham surgery (n = 12). One week after RLN injury, larynges were harvested following euthanasia. The mRNA was extracted from PCA and AC muscles bilaterally, and microarray analysis was performed using a full rat genome array. Microarray analysis of denervated AC and PCA muscles demonstrated dramatic differences in gene expression profiles, with 205 individual probes that were differentially expressed between the denervated AC and PCA muscles and only 14 genes with similar expression patterns. The differential expression patterns of the AC and PCA suggest different mechanisms of reinnervation. The PCA showed the gene patterns of Wallerian degeneration, while the AC expressed the gene patterns of reinnervation by adjacent axonal sprouting. This finding may reveal important therapeutic targets applicable to RLN and other peripheral nerve injuries. © The Author(s) 2015.

Dynamically re-configurable CMOS imagers for an active vision system

NASA Technical Reports Server (NTRS)

Yang, Guang (Inventor); Pain, Bedabrata (Inventor)

2005-01-01

A vision system is disclosed. The system includes a pixel array, at least one multi-resolution window operation circuit, and a pixel averaging circuit. The pixel array has an array of pixels configured to receive light signals from an image having at least one tracking target. The multi-resolution window operation circuits are configured to process the image. Each of the multi-resolution window operation circuits processes each tracking target within a particular multi-resolution window. The pixel averaging circuit is configured to sample and average pixels within the particular multi-resolution window.

Defining Genomic Changes in Triple-Negative Breast Cancer in Women of African Descent

DTIC Science & Technology

2012-06-01

Triple negative breast cancer • Ethnic disparities • Breast cancer amongst African Americans and Africans • Gene expression profiling • Array... negative cases seen in both African and African - American breast cancer cases. Gene Expression Array Studies The 31 triple negative Kijabe... African - American Adjacent Normal Breast Tissue PI: Pegram &

THE USE OF GENE ARRAYS TO MEASURE CHANGES IN GENE EXPRESSION PATTERNS IN FISH EXPOSED TO COMPOUNDS THAT MIMIC ESTROGEN

EPA Science Inventory

We have developed estrogen-sensitive gene arrays to measure changes in gene expression in sheepshead minnows and largemouth bass exposed to anthropogenic chemicals that mimic estrogen. The in vivo exposures, which realize the full physiological response in fish, result in changes...

DEVELOPMENT OF A 950-GENE DNA ARRAY FOR EXAMINING GENE EXPRESSION PATTERNS IN MOUSE TESTIS

EPA Science Inventory

Development of a 950-gene DNA array for examining gene expression patterns in mouse testis.

Rockett JC, Christopher Luft J, Brian Garges J, Krawetz SA, Hughes MR, Hee Kirn K, Oudes AJ, Dix DJ.

Reproductive Toxicology Division, National Health and Environmental Effec...

Single step generation of protein arrays from DNA by cell-free expression and in situ immobilisation (PISA method).

PubMed

He, M; Taussig, M J

2001-08-01

We describe a format for production of protein arrays termed 'protein in situ array' (PISA). A PISA is rapidly generated in one step directly from PCR-generated DNA fragments by cell-free protein expression and in situ immobilisation at a surface. The template for expression is DNA encoding individual proteins or domains, which is produced by PCR using primers designed from information in DNA databases. Coupled transcription and translation is carried out on a surface to which the tagged protein adheres as soon as it is synthesised. Because proteins generated by cell-free synthesis are usually soluble and functional, this method can overcome problems of insolubility or degradation associated with bacterial expression of recombinant proteins. Moreover, the use of PCR-generated DNA enables rapid production of proteins or domains based on genome information alone and will be particularly useful where cloned material is not available. Here we show that human single-chain antibody fragments (three domain, V(H)/K form) and an enzyme (luciferase) can be functionally arrayed by the PISA method.

Detection and characterisation of multi-drug resistance protein 1 (MRP-1) in human mitochondria

PubMed Central

Roundhill, E A; Burchill, S A

2012-01-01

Background: Overexpression of plasma membrane multi-drug resistance protein 1 (MRP-1) can lead to multidrug resistance. In this study, we describe for the first time the expression of mitochondrial MRP-1 in untreated human normal and cancer cells and tissues. Methods: MRP-1 expression and subcellular localisation in normal and cancer cells and tissues was examined by differential centrifugation and western blotting, and immunofluorescence microscopy. Viable mitochondria were isolated and MRP-1 efflux activity measured using the calcein-AM functional assay. MRP-1 expression was increased using retroviral infection and specific overexpression confirmed by RNA array. Cell viability was determined by trypan blue exclusion and annexin V-propidium iodide labelling of cells. Results: MRP-1 was detected in the mitochondria of cancer and normal cells and tissues. The efflux activity of mitochondrial MRP-1 was more efficient (55–64%) than that of plasma membrane MRP-1 (11–22% P<0.001). Induced MRP-1 expression resulted in a preferential increase in mitochondrial MRP-1, suggesting selective targeting to this organelle. Treatment with a non-lethal concentration of doxorubicin (0.85 n, 8 h) increased mitochondrial and plasma membrane MRP-1, increasing resistance to MRP-1 substrates. For the first time, we have identified MRP-1 with efflux activity in human mitochondria. Conclusion: Mitochondrial MRP-1 may be an exciting new therapeutic target where historically MRP-1 inhibitor strategies have limited clinical success. PMID:22353810

Profiling dendritic cell subsets in head and neck squamous cell tonsillar cancer and benign tonsils.

PubMed

Abolhalaj, Milad; Askmyr, David; Sakellariou, Christina Alexandra; Lundberg, Kristina; Greiff, Lennart; Lindstedt, Malin

2018-05-23

Dendritic cells (DCs) have a key role in orchestrating immune responses and are considered important targets for immunotherapy against cancer. In order to develop effective cancer vaccines, detailed knowledge of the micromilieu in cancer lesions is warranted. In this study, flow cytometry and human transcriptome arrays were used to characterize subsets of DCs in head and neck squamous cell tonsillar cancer and compare them to their counterparts in benign tonsils to evaluate subset-selective biomarkers associated with tonsillar cancer. We describe, for the first time, four subsets of DCs in tonsillar cancer: CD123 + plasmacytoid DCs (pDC), CD1c + , CD141 + , and CD1c - CD141 - myeloid DCs (mDC). An increased frequency of DCs and an elevated mDC/pDC ratio were shown in malignant compared to benign tonsillar tissue. The microarray data demonstrates characteristics specific for tonsil cancer DC subsets, including expression of immunosuppressive molecules and lower expression levels of genes involved in development of effector immune responses in DCs in malignant tonsillar tissue, compared to their counterparts in benign tonsillar tissue. Finally, we present target candidates selectively expressed by different DC subsets in malignant tonsils and confirm expression of CD206/MRC1 and CD207/Langerin on CD1c + DCs at protein level. This study descibes DC characteristics in the context of head and neck cancer and add valuable steps towards future DC-based therapies against tonsillar cancer.

PGK1, a glucose metabolism enzyme, may play an important role in rheumatoid arthritis.

PubMed

Zhao, Yan; Yan, Xinfeng; Li, Xia; Zheng, Yabing; Li, Shufeng; Chang, Xiaotian

2016-10-01

Some studies have indicated that glucose metabolism plays an important role in the pathogenesis of rheumatoid arthritis (RA). This study aimed to find the novel genes affecting glucose metabolism in RA. Synovial tissues of collagen-induced arthritis (CIA) were analyzed with Rat Glucose Metabolism RT(2) Profiler™ PCR Array to screen those genes with special expressions in glucose metabolism. Real-time PCR, western blotting, and ELISA were used to confirm the result in synovial tissues and blood of human RA. Culture synovial fibroblast cells (RASF) was treated with siRNA to suppress expressions of the target genes. CCK-8 cell proliferation assay and two-compartment transwell system were performed to examine cell proliferation and cell migration of the treated RASF. Both PCR array and real-time PCR detected the up-regulation of ENO1, HK2, and PGK1 and the down-regulation of PCK1 and PDK4 in synovial tissues of CIA rats. Real-time PCR and western blotting detected the increased expression of ENO1 and PGK1 in RA synovial tissues. ELISA detected a high level of PGK1 in the blood of RA patients. Decreased cell proliferation and cell migration capabilities were significantly detected in RASF following treatment of anti-PGK1 siRNA. IL-1β and IFN-γ rather than TNF-α and IL-1α levels were significantly declined in supernatants of the treated RASF. PGK1, a glycolytic enzyme catalyzing the conversion of 3-phosphoglycerate into 2-phosphoglycerate, has increased expression in synovial tissues and blood of RA, which may be involved in pro-inflammation and synovial hyperplasia of the disease.

Experimental Demonstration of Adaptive Infrared Multispectral Imaging Using Plasmonic Filter Array (Postprint)

DTIC Science & Technology

2016-10-10

AFRL-RX-WP-JA-2017-0189 EXPERIMENTAL DEMONSTRATION OF ADAPTIVE INFRARED MULTISPECTRAL IMAGING USING PLASMONIC FILTER ARRAY...March 2016 – 23 May 2016 4. TITLE AND SUBTITLE EXPERIMENTAL DEMONSTRATION OF ADAPTIVE INFRARED MULTISPECTRAL IMAGING USING PLASMONIC FILTER ARRAY...experimental demonstration of adaptive multispectral imagery using fabricated plasmonic spectral filter arrays and proposed target detection scenarios

Calling Chromosome Alterations, DNA Methylation Statuses, and Mutations in Tumors by Simple Targeted Next-Generation Sequencing: A Solution for Transferring Integrated Pangenomic Studies into Routine Practice?

PubMed

Garinet, Simon; Néou, Mario; de La Villéon, Bruno; Faillot, Simon; Sakat, Julien; Da Fonseca, Juliana P; Jouinot, Anne; Le Tourneau, Christophe; Kamal, Maud; Luscap-Rondof, Windy; Boeva, Valentina; Gaujoux, Sebastien; Vidaud, Michel; Pasmant, Eric; Letourneur, Franck; Bertherat, Jérôme; Assié, Guillaume

2017-09-01

Pangenomic studies identified distinct molecular classes for many cancers, with major clinical applications. However, routine use requires cost-effective assays. We assessed whether targeted next-generation sequencing (NGS) could call chromosomal alterations and DNA methylation status. A training set of 77 tumors and a validation set of 449 (43 tumor types) were analyzed by targeted NGS and single-nucleotide polymorphism (SNP) arrays. Thirty-two tumors were analyzed by NGS after bisulfite conversion, and compared to methylation array or methylation-specific multiplex ligation-dependent probe amplification. Considering allelic ratios, correlation was strong between targeted NGS and SNP arrays (r = 0.88). In contrast, considering DNA copy number, for variations of one DNA copy, correlation was weaker between read counts and SNP array (r = 0.49). Thus, we generated TARGOMICs, optimized for detecting chromosome alterations by combining allelic ratios and read counts generated by targeted NGS. Sensitivity for calling normal, lost, and gained chromosomes was 89%, 72%, and 31%, respectively. Specificity was 81%, 93%, and 98%, respectively. These results were confirmed in the validation set. Finally, TARGOMICs could efficiently align and compute proportions of methylated cytosines from bisulfite-converted DNA from targeted NGS. In conclusion, beyond calling mutations, targeted NGS efficiently calls chromosome alterations and methylation status in tumors. A single run and minor design/protocol adaptations are sufficient. Optimizing targeted NGS should expand translation of genomics to clinical routine. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

DNA detection on ultrahigh-density optical fiber-based nanoarrays.

PubMed

Tam, Jenny M; Song, Linan; Walt, David R

2009-04-15

Nanoarrays for DNA detection were fabricated on etched nanofiber bundles based on recently developed techniques for microscale arrays. Two different-sized nanoarrays were created: one with 700 nm feature sizes and a 1 microm center-to-center pitch (approximately 1x10(6) array elements/mm(2)) and one with 300 nm feature sizes and a 500 nm center-to-center pitch (4.6x10(6) array elements/mm(2)). A random, multiplexed array composed of oligonucleotide-functionalized nanospheres was constructed and used for parallel detection and analysis of fluorescently labeled DNA targets. We have used these arrays to detect a variety of target sequences including Bacillus thuringiensis kurstaki and vaccina virus sequences, two potential biowarfare agents, as well as interleukin-2 sequences, an immune system modulator that has been used for the diagnosis of HIV.

Receptor-Mediated Uptake and Intracellular Sorting of Multivalent Lipid Nanoparticles Against the Epidermal Growth Factor Receptor (EGFR) and the Human EGFR 2 (HER2)

NASA Astrophysics Data System (ADS)

Tran, David Tu

In the area of receptor-targeted lipid nanoparticles for drug delivery, efficiency has been mainly focused on cell-specificity, endocytosis, and subsequently effects on bioactivity such as cell growth inhibition. Aspects of targeted liposomal uptake and intracellular sorting are not well defined. This dissertation assessed a series of ligands as targeted functional groups against HER2 and EGFR for liposomal drug delivery. Receptor-mediated uptake, both mono-targeted and dual-targeted to multiple receptors of different ligand valence, and the intracellular sorting of lipid nanoparticles were investigated to improve the delivery of drugs to cancer cells. Lipid nanoparticles were functionalized through a new sequential micelle transfer---conjugation method, while the micelle transfer method was extended to growth factors. Through a combination of both techniques, anti-HER2 and anti-EGFR dual-targeted immunoliposomes with different combinations of ligand valence were developed for comparative studies. With the array of lipid nanoparticles, the uptake and cytotoxicity of lipid nanoparticles in relationship to ligand valence, both mono-targeting and dual-targeting, were evaluated on a small panel of breast cancer cell lines that express HER2 and EGFR of varying levels. Comparable uptake ratios of ligand to expressed receptor and apparent cooperativity were observed. For cell lines that express both receptors, additive dose-uptake effects were also observed with dual-targeted immunoliposomes, which translated to marginal improvements in cell growth inhibition with doxorubicin delivery. Colocalization analysis revealed that ligand-conjugated lipid nanoparticles settle to endosomal compartments similar to their attached ligands. Pathway transregulation and pathway saturation were also observed to affect trafficking. In the end, liposomes routed to the recycling endosomes were never observed to traffic beyond the endosomes nor to be exocytose like recycled ligands. Based on the experimental data, models were developed to help interpret and predict the binding and trafficking of lipid nanoparticles. The crosslink multivalent binding model of lipid nanoparticles to monovalent receptors was able to predict ligand valence for optimum binding, cell association concentrations, offer explanations to the antagonistic effects observed from high ligand valence, and predict the binding limitations of both ligand valence and ligand affinity. Hopefully, the models will serve as valuable tools for future optimizations in targeted liposomal drug delivery.

Radar Resource Management in a Dense Target Environment

DTIC Science & Technology

2014-03-01

problem faced by networked MFRs . While relaxing our assumptions concerning information gain presents numerous challenges worth exploring, future research...linear programming MFR multifunction phased array radar MILP mixed integer linear programming NATO North Atlantic Treaty Organization PDF probability...1: INTRODUCTION Multifunction phased array radars ( MFRs ) are capable of performing various tasks in rapid succession. The performance of target search

Choroid plexus epithelial cells express the adhesion protein P-cadherin at cell-cell contacts and syntaxin-4 in the luminal membrane domain.

PubMed

Christensen, Inga Baasch; Mogensen, Esben Nees; Damkier, Helle Hasager; Praetorius, Jeppe

2018-05-01

The choroid plexus epithelial cells (CPECs) belong to a small group of polarized cells, where the Na + -K + -ATPase is expressed in the luminal membrane. The basic polarity of the cells is, therefore, still debated. We investigated the subcellular distribution of an array of proteins known to play fundamental roles either in establishing and maintaining basic cell polarity or in the polarized delivery and recycling of plasma membrane proteins. Immunofluorescence histochemical analysis was applied to determine the subcellular localization of apical and basolateral membrane determinants. Mass spectrometry analysis of CPECs isolated by fluorescence-activated cell sorting was applied to determine the expression of specific forms of the proteins. CPECs mainly express the cell-adhesive P-cadherin, which is localized to the lateral membranes. Proteins belonging to the Crumbs and partitioning defective (Par) protein complexes were all localized to the luminal membrane domain. Par-1 and the Scribble complex were localized to the basolateral membrane domain. Lethal(2) giant larvae homolog 2 (Lgl2) labeling was preferentially observed in the luminal membrane domain. Phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) was immunolocalized to the basolateral membrane domain, while phosphatidylinositol 4,5-bisphosphate (PIP 2 ) staining was most prominent in the luminal membrane domain along with the PIP 3 phosphatase, Pten. The apical target-SNARE syntaxin-3 and the basolateral target-SNARE syntaxin-4 were both localized to the apical membrane domain in CPECs, which lack cellular expression of the clathrin adaptor protein AP-1B for basolateral protein recycling. In conclusion, the CPECs are conventionally polarized, but express P-cadherin at cell-cell contacts, and Lgl2 and syntaxin-4 in the luminal plasma membrane domain.

Downregulation of miR-15a due to LMP1 promotes cell proliferation and predicts poor prognosis in nasal NK/T-cell lymphoma.

PubMed

Komabayashi, Yuki; Kishibe, Kan; Nagato, Toshihiro; Ueda, Seigo; Takahara, Miki; Harabuchi, Yasuaki

2014-01-01

Nasal NK/T-cell lymphoma (NNKTL) is an Epstein-Barr virus (EBV)-associated malignancy and has distinct clinical and histological features. However, its genetic features are hitherto unclear. MicroRNAs (miRNAs) play a crucial role in the pathogenesis of several malignancies via regulating gene expression. In this study, we investigated whether the specific microRNAs were related to the tumor behaviors in NNKTL. MiRNA array and Quantitative RT-PCR analyses revealed that miR-15a was expressed at a much lower level in NNKTL cells (SNK-1, SNK-6, and SNT-8) than in normal peripheral NK cells and EBV-negative NK cell line KHYG-1. Quantitative PCR and western blot analyses showed that the expression of MYB and cyclin D1, which are validated targets of miR-15a, was higher in NNKTL cells. Transfection of NNKTL cells (SNK-6 and SNT-8) with a miR-15a precursor decreased MYB and cyclin D1 levels, thereby blocking G1/S transition and cell proliferation. Knockdown of EBV-encoded latent membrane protein 1 (LMP1) significantly increased miR-15a expression in SNK-6 cells. In NNKTL tissues, we found that reduced miR-15a expression, which correlated with MYB and cyclin D1 expression, was associated with poor prognosis of NNKTL patients. These data suggest that downregulation of miR-15a, possibly due to LMP1, implicates in the pathogenesis of NNKTL by inducing cell proliferation via MYB and cyclin D1. Thus, miR-15a could be a potential target for antitumor therapy and a prognostic predictor for NNKTL. Copyright © 2013 Wiley Periodicals, Inc.

Target detection and localization in shallow water: an experimental demonstration of the acoustic barrier problem at the laboratory scale.

PubMed

Marandet, Christian; Roux, Philippe; Nicolas, Barbara; Mars, Jérôme

2011-01-01

This study demonstrates experimentally at the laboratory scale the detection and localization of a wavelength-sized target in a shallow ultrasonic waveguide between two source-receiver arrays at 3 MHz. In the framework of the acoustic barrier problem, at the 1/1000 scale, the waveguide represents a 1.1-km-long, 52-m-deep ocean acoustic channel in the kilohertz frequency range. The two coplanar arrays record in the time-domain the transfer matrix of the waveguide between each pair of source-receiver transducers. Invoking the reciprocity principle, a time-domain double-beamforming algorithm is simultaneously performed on the source and receiver arrays. This array processing projects the multireverberated acoustic echoes into an equivalent set of eigenrays, which are defined by their launch and arrival angles. Comparison is made between the intensity of each eigenray without and with a target for detection in the waveguide. Localization is performed through tomography inversion of the acoustic impedance of the target, using all of the eigenrays extracted from double beamforming. The use of the diffraction-based sensitivity kernel for each eigenray provides both the localization and the signature of the target. Experimental results are shown in the presence of surface waves, and methodological issues are discussed for detection and localization.

Normal gene expression in male F344 rat nasal transitional and respiratory epithelium.

PubMed

Hester, Susan D; Benavides, Gina B; Sartor, Maureen; Yoon, Lawrence; Wolf, Douglas C; Morgan, Kevin T

2002-02-20

The nasal epithelium is an important target site for chemically-induced toxicity and carcinogenicity in rodents. Gene expression profiles were determined in order to provide normal baseline data for nasal transitional/respiratory epithelium from healthy rats. Cells lining the rat nasal passages were collected and gene expression analysis was performed using Clontech cDNA Rat Atlas 1.2 arrays (1185 genes). The percentages of genes within specific average expression ranges were 4.2% at 45,000-1000, 14.8% at 1000-200, 25.0% at 200-68, and 56.0% below 68. Nine out of a subset of ten genes were confirmed for relative signal intensity using quantitative real-time RT-PCR. The most highly expressed genes included those involved in phase I (e.g. cytochrome P450s) and phase II (e.g. glutathione S-transferases) xenobiotic metabolism, bioenergetics (e.g. cytochrome oxidase), osmotic balance (e.g. Na(+)/K(+) ATPase) and epithelial ionic homeostasis (e.g. ion channels). Such baseline data will contribute to further understanding the normal physiology of these cells and facilitate the interpretation of responses by the nasal epithelial cells to xenobiotic treatment or disease.

3D target array for pulsed multi-sourced radiography

DOEpatents

Le Galloudec, Nathalie Joelle

2016-02-23

The various technologies presented herein relate to the generation of x-rays and other charged particles. A plurality of disparate source materials can be combined on an array to facilitate fabrication of co-located mixed tips (point sources) which can be utilized to form a polychromatic cloud, e.g., a plurality of x-rays having a range of energies and or wavelengths, etc. The tips can be formed such that the x-rays are emitted in a direction different to other charged particles to facilitate clean x-ray sourcing. Particles, such as protons, can be directionally emitted to facilitate generation of neutrons at a secondary target. The various particles can be generated by interaction of a laser irradiating the array of tips. The tips can be incorporated into a plurality of 3D conical targets, the conical target sidewall(s) can be utilized to microfocus a portion of a laser beam onto the tip material.

miR-4725-3p targeting Stim1 signaling is involved in xanthohumol inhibition of glioma cell invasion.

PubMed

Ho, Kuo-Hao; Chang, Cheng-Kuei; Chen, Peng-Hsu; Wang, Yu-Jia; Chang, Wei-Chiao; Chen, Ku-Chung

2018-05-10

Glioblastoma multiforme (GBM) is the most common brain tumor in adults. Due to its highly invasive nature, it is not easy to treat, resulting in high mortality rates. Stromal interacting molecule 1 (Stim1) plays important roles in regulating store-operated Ca 2+ entry (SOCE), and controls invasion by cancer cells. However, the mechanisms and functions of Stim1 in glioma progression are still unclear. In this study, we investigated the effects of targeting Stim1 expression on glioma cell invasion. By analyzing profiles of GBM patients from RNA-sequencing data in The Cancer Genome Atlas (TCGA), higher expression levels of STIM1 were correlated with the poor survival. Furthermore, signaling pathways associated with tumor malignancy, including the epithelial-to-mesenchymal transition (EMT), were activated in patients with high STIM1 expression according to gene set enrichment analyses. Higher Stim1 levels were found in glioma cells compared to human astrocytes, and these higher levels enhanced glioma cell invasion. Xanthohumol (XN), a prenylated flavonoid extracted from the hop plant Humulus lupulus L. (Cannabaceae), significantly reduced cell invasion through inhibiting Stim1 expression. From an micro(mi)RNA array analysis, miR-4725-3p was upregulated by XN treatment. Overexpression of miR-4725-3p inhibited glioma cell invasion via directly targeting the 3'-untranslated region of STIM1. The extracellular signal-regulated kinase/c-Fos pathway was also validated to participate in XN-upregulated miR-4725-3p expression according to promoter and chromatin immunoprecipitation assays. These results emphasize that miR-4725-3p-inhibited STIM1 signaling is involved in XN-attenuated glioma cell invasion. These findings may provide insights into novel therapeutic strategies for future glioblastoma therapy and drug development. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Downregulation of miR-199b is associated with distant metastasis in colorectal cancer via activation of SIRT1 and inhibition of CREB/KISS1 signaling.

PubMed

Shen, Zhan-Long; Wang, Bo; Jiang, Ke-Wei; Ye, Chun-Xiang; Cheng, Cheng; Yan, Yi-Chao; Zhang, Ji-Zhun; Yang, Yang; Gao, Zhi-Dong; Ye, Ying-Jiang; Wang, Shan

2016-06-07

The progression of distant metastasis cascade is a multistep and complicated process, frequently leading to a poor prognosis in cancer patients. Recently, growing evidence has indicated that deregulation of microRNAs (miRNAs) contributes to tumorigenesis and tumor progression in colorectal cancer (CRC). In the present study, by comparing the miRNA expression profiles of CRC tissues and corresponding hepatic metastasis tissues, we established the downregulation of miR-199b in CRC metastasis tissues. The decrease in miR-199b expression was significantly correlated to late TNM stage and distant metastasis. Moreover, Kaplan-Meier curves showed that CRC patients with high expression level of miR-199b had a longer median survival. Functional assays results indicated that the restoration of miR-199b considerably reduced cell invasion and migration in vitro and in vivo, and increased the sensitivity to 5-FU and oxaliplatin. Further dual-luciferase reporter gene assays revealed that SIRT1 was the direct target of miR-199b in CRC. The expression of miR-199b was inversely correlated with SIRT1 in CRC specimens. SIRT1 knockdown produced effects on biological behavior that were similar to those of miR-199b overexpression. Furthermore, through Human Tumor Metastasis PCR Array we discovered KISS1 was one of the downstream targets of SIRT1. Silencing of SIRT1 upregulated KISS1 expression by enhancing the acetylation of the transcription factor CREB. The latter was further activated via binding to the promoter of KISS1 to induce transcription. Thus, we concluded that miR-199b regulates SIRT1/CREB/KISS1 signaling pathway and might serve as a prognosis marker or a novel therapeutic target for patients with CRC.

Probing the function of neuronal populations: combining micromirror-based optogenetic photostimulation with voltage-sensitive dye imaging

PubMed Central

Tsuda, Sachiko; Kee, Michelle Z.L.; Cunha, Catarina; Kim, Jinsook; Yan, Ping; Loew, Leslie M.; Augustine, George J.

2013-01-01

Recent advances in our understanding of brain function have come from using light to either control or image neuronal activity. Here we describe an approach that combines both techniques: a micromirror array is used to photostimulate populations of presynaptic neurons expressing channelrhodopsin-2, while a red-shifted voltage-sensitive dye allows optical detection of resulting postsynaptic activity. Such technology allowed us to control the activity of cerebellar interneurons while simultaneously recording inhibitory responses in multiple Purkinje neurons, their postsynaptic targets. This approach should substantially accelerate our understanding of information processing by populations of neurons within brain circuits. PMID:23254260

Pupil geometry and pupil re-imaging in telescope arrays

NASA Technical Reports Server (NTRS)

Traub, Wesley A.

1990-01-01

This paper considers the issues of lateral and longitudinal pupil geometry in ground-based telescope arrays, such as IOTA. In particular, it is considered whether or not pupil re-imaging is required before beam combination. By considering the paths of rays through the system, an expression is derived for the optical path errors in the combined wavefront as a function of array dimensions, telescope magnification factor, viewing angle, and field-of-view. By examining this expression for the two cases of pupil-plane and image-plane combination, operational limits can be found for any array. As a particular example, it is shown that for IOTA no pupil re-imaging optics will be needed.

A Dual-Layer Transducer Array for 3-D Rectilinear Imaging

PubMed Central

Yen, Jesse T.; Seo, Chi Hyung; Awad, Samer I.; Jeong, Jong S.

2010-01-01

2-D arrays for 3-D rectilinear imaging require very large element counts (16,000–65,000). The difficulties in fabricating and interconnecting 2-D arrays with a large number of elements (>5,000) have limited the development of suitable transducers for 3-D rectilinear imaging. In this paper, we propose an alternative solution to this problem by using a dual-layer transducer array design. This design consists of two perpendicular 1-D arrays for clinical 3-D imaging of targets near the transducer. These targets include the breast, carotid artery, and musculoskeletal system. This transducer design reduces the fabrication complexity and the channel count making 3-D rectilinear imaging more realizable. With this design, an effective N × N 2-D array can be developed using only N transmitters and N receivers. This benefit becomes very significant when N becomes greater than 128, for example. To demonstrate feasibility, we constructed a 4 × 4 cm prototype dual-layer array. The transmit array uses diced PZT-5H elements, and the receive array is a single sheet of undiced P[VDF-TrFE] copolymer. The receive elements are defined by the copper traces on the flexible interconnect circuit. The measured −6 dB fractional bandwidth was 80% with a center frequency of 4.8 MHz. At 5 MHz, the nearest neighbor crosstalk of the PZT array and PVDF array was −30.4 ± 3.1 dB and −28.8 ± 3.7 dB respectively. This dual-layer transducer was interfaced with an Ultrasonix Sonix RP system, and a synthetic aperture 3-D data set was acquired. We then performed off-line 3-D beamforming to obtain volumes of nylon wire targets. The theoretical lateral beamwidth was 0.52 mm compared to measured beamwidths of 0.65 mm and 0.67 mm in azimuth and elevation respectively. 3-D images of an 8 mm diameter anechoic cyst phantom were also acquired. PMID:19213647

Selection of novel reference genes for use in the human central nervous system: a BrainNet Europe Study.

PubMed

Durrenberger, Pascal F; Fernando, Francisca S; Magliozzi, Roberta; Kashefi, Samira N; Bonnert, Timothy P; Ferrer, Isidro; Seilhean, Danielle; Nait-Oumesmar, Brahim; Schmitt, Andrea; Gebicke-Haerter, Peter J; Falkai, Peter; Grünblatt, Edna; Palkovits, Miklos; Parchi, Piero; Capellari, Sabina; Arzberger, Thomas; Kretzschmar, Hans; Roncaroli, Federico; Dexter, David T; Reynolds, Richard

2012-12-01

The use of an appropriate reference gene to ensure accurate normalisation is crucial for the correct quantification of gene expression using qPCR assays and RNA arrays. The main criterion for a gene to qualify as a reference gene is a stable expression across various cell types and experimental settings. Several reference genes are commonly in use but more and more evidence reveals variations in their expression due to the presence of on-going neuropathological disease processes, raising doubts concerning their use. We conducted an analysis of genome-wide changes of gene expression in the human central nervous system (CNS) covering several neurological disorders and regions, including the spinal cord, and were able to identify a number of novel stable reference genes. We tested the stability of expression of eight novel (ATP5E, AARS, GAPVD1, CSNK2B, XPNPEP1, OSBP, NAT5 and DCTN2) and four more commonly used (BECN1, GAPDH, QARS and TUBB) reference genes in a smaller cohort using RT-qPCR. The most stable genes out of the 12 reference genes were tested as normaliser to validate increased levels of a target gene in CNS disease. We found that in human post-mortem tissue the novel reference genes, XPNPEP1 and AARS, were efficient in replicating microarray target gene expression levels and that XPNPEP1 was more efficient as a normaliser than BECN1, which has been shown to change in expression as a consequence of neuronal cell loss. We provide herein one more suitable novel reference gene, XPNPEP1, with no current neuroinflammatory or neurodegenerative associations that can be used for gene quantitative gene expression studies with human CNS post-mortem tissue and also suggest a list of potential other candidates. These data also emphasise the importance of organ/tissue-specific stably expressed genes as reference genes for RNA studies.

Expression analysis of G Protein-Coupled Receptors in mouse macrophages

PubMed Central

Lattin, Jane E; Schroder, Kate; Su, Andrew I; Walker, John R; Zhang, Jie; Wiltshire, Tim; Saijo, Kaoru; Glass, Christopher K; Hume, David A; Kellie, Stuart; Sweet, Matthew J

2008-01-01

Background Monocytes and macrophages express an extensive repertoire of G Protein-Coupled Receptors (GPCRs) that regulate inflammation and immunity. In this study we performed a systematic micro-array analysis of GPCR expression in primary mouse macrophages to identify family members that are either enriched in macrophages compared to a panel of other cell types, or are regulated by an inflammatory stimulus, the bacterial product lipopolysaccharide (LPS). Results Several members of the P2RY family had striking expression patterns in macrophages; P2ry6 mRNA was essentially expressed in a macrophage-specific fashion, whilst P2ry1 and P2ry5 mRNA levels were strongly down-regulated by LPS. Expression of several other GPCRs was either restricted to macrophages (e.g. Gpr84) or to both macrophages and neural tissues (e.g. P2ry12, Gpr85). The GPCR repertoire expressed by bone marrow-derived macrophages and thioglycollate-elicited peritoneal macrophages had some commonality, but there were also several GPCRs preferentially expressed by either cell population. Conclusion The constitutive or regulated expression in macrophages of several GPCRs identified in this study has not previously been described. Future studies on such GPCRs and their agonists are likely to provide important insights into macrophage biology, as well as novel inflammatory pathways that could be future targets for drug discovery. PMID:18442421

Stress amplifies sex differences in primate prefrontal profiles of gene expression.

PubMed

Lee, Alex G; Hagenauer, Megan; Absher, Devin; Morrison, Kathleen E; Bale, Tracy L; Myers, Richard M; Watson, Stanley J; Akil, Huda; Schatzberg, Alan F; Lyons, David M

2017-11-02

Stress is a recognized risk factor for mood and anxiety disorders that occur more often in women than men. Prefrontal brain regions mediate stress coping, cognitive control, and emotion. Here, we investigate sex differences and stress effects on prefrontal cortical profiles of gene expression in squirrel monkey adults. Dorsolateral, ventrolateral, and ventromedial prefrontal cortical regions from 18 females and 12 males were collected after stress or no-stress treatment conditions. Gene expression profiles were acquired using HumanHT-12v4.0 Expression BeadChip arrays adapted for squirrel monkeys. Extensive variation between prefrontal cortical regions was discerned in the expression of numerous autosomal and sex chromosome genes. Robust sex differences were also identified across prefrontal cortical regions in the expression of mostly autosomal genes. Genes with increased expression in females compared to males were overrepresented in mitogen-activated protein kinase and neurotrophin signaling pathways. Many fewer genes with increased expression in males compared to females were discerned, and no molecular pathways were identified. Effect sizes for sex differences were greater in stress compared to no-stress conditions for ventromedial and ventrolateral prefrontal cortical regions but not dorsolateral prefrontal cortex. Stress amplifies sex differences in gene expression profiles for prefrontal cortical regions involved in stress coping and emotion regulation. Results suggest molecular targets for new treatments of stress disorders in human mental health.

miRNA expression profiling in formalin-fixed paraffin-embedded endometriosis and ovarian cancer samples

PubMed Central

Braicu, Ovidiu-Leonard; Budisan, Liviuta; Buiga, Rares; Jurj, Ancuta; Achimas-Cadariu, Patriciu; Pop, Laura Ancuta; Braicu, Cornelia; Irimie, Alexandru; Berindan-Neagoe, Ioana

2017-01-01

Endometriosis is an inflammatory pathology associated with a negative effect on life quality. Recently, this pathology was connected to ovarian cancer, in particular with endometrioid ovarian cancer. microRNAs (miRNAs) are a class of RNA transcripts ~19–22 nucleotides in length, the altered miRNA pattern being connected to pathological status. miRNAs are highly stable transcripts, and these can be assessed from formalin-fixed paraffin-embedded (FFPE) samples leading to the identification of miRNAs that could be developed as diagnostic and prognostic biomarkers, in particular those involved in malignant transformation. The aim of our study was to evaluate miRNA expression pattern in FFPE samples from endometriosis and ovarian cancer patients using PCR-array technology and also to compare the differential expression pattern in ovarian cancer versus endometriosis. For the PCR-array study, we have used nine macrodissected FFPE samples from endometriosis tissue, eight samples of ovarian cancers and five normal ovarian tissues. Quantitative real-time PCR (qRT-PCR) was used for data validation in a new patient cohort of 17 normal samples, 33 endometriosis samples and 28 ovarian cancer macrodissected FFPE samples. Considering 1.5-fold expression difference as a cut-off level and a P-value <0.05, we have identified four miRNAs being overexpressed in endometrial tissue, while in ovarian cancer 15 were differentially expressed (nine overexpressed and six downregulated). The expression level was confirmed by qRT-PCR for miR-93, miR-141, miR-155, miR-429, miR-200c, miR-205 and miR-492. Using the interpretative program Ingenuity Pathway Analysis revealed several deregulated pathways due to abnormal miRNA expression in endometriosis and ovarian cancer, which in turn is responsible for pathogenesis; this differential expression of miRNAs can be exploited as a therapeutic target. A higher number of altered miRNAs were detected in endometriosis versus ovarian cancer tissue, most of them being linked with epithelial-to-mesenchymal transition. PMID:28894379

Phased-array radar for airborne systems

NASA Astrophysics Data System (ADS)

Tahim, Raghbir S.; Foshee, James J.; Chang, Kai

2003-09-01

Phased array antenna systems, which support high pulse rates and high transmit power, are well suited for radar and large-scale surveillance. Sensors and communication systems can function as the eyes and ears for ballistic missile defense applications, providing early warning of attack, target detection and identification, target tracking, and countermeasure decision. In such applications, active array radar systems that contain solid-state transmitter sources and low-noise preamplifiers for transmission and reception are preferred over the conventional radar antennas, because the phased array radar offers the advantages of power management and efficiency, reliability, signal reception, beam steering target detection. The current phased array radar designs are very large, complex and expensive and less efficient because of high RF losses in the phase control circuits used for beam scan. Several thousands of phase shifters and drivers may be required for a single system thus making the system very complex and expensive. This paper describes the phased array radar system based on high power T/R modules, wide-band radiating planar antenna elements and very low loss wide-band phase control circuits (requiring reduced power levels) for beam scan. The phase shifter design is based on micro-strip feed lines perturbed by the proximity of voltage controlled piezoelectric transducer (PET). Measured results have shown an added insertion loss of less than 1 dB for a phase shift of 450 degrees from 2 to 20 GHz. The new wideband phased array radar design provides significant reduction in size cost and weight. Compared to the conventional phased array systems, the cost saving is more than 15 to 1.

Evidence for Alteration of Gene Regulatory Networks through MicroRNAs of the HIV-infected brain: novel analysis of retrospective cases.

PubMed

Tatro, Erick T; Scott, Erick R; Nguyen, Timothy B; Salaria, Shahid; Banerjee, Sugato; Moore, David J; Masliah, Eliezer; Achim, Cristian L; Everall, Ian P

2010-04-26

HIV infection disturbs the central nervous system (CNS) through inflammation and glial activation. Evidence suggests roles for microRNA (miRNA) in host defense and neuronal homeostasis, though little is known about miRNAs' role in HIV CNS infection. MiRNAs are non-coding RNAs that regulate gene translation through post-transcriptional mechanisms. Messenger-RNA profiling alone is insufficient to elucidate the dynamic dance of molecular expression of the genome. We sought to clarify RNA alterations in the frontal cortex (FC) of HIV-infected individuals and those concurrently infected and diagnosed with major depressive disorder (MDD). This report is the first published study of large-scale miRNA profiling from human HIV-infected FC. The goals of this study were to: 1. Identify changes in miRNA expression that occurred in the frontal cortex (FC) of HIV individuals, 2. Determine whether miRNA expression profiles of the FC could differentiate HIV from HIV/MDD, and 3. Adapt a method to meaningfully integrate gene expression data and miRNA expression data in clinical samples. We isolated RNA from the FC (n = 3) of three separate groups (uninfected controls, HIV, and HIV/MDD) and then pooled the RNA within each group for use in large-scale miRNA profiling. RNA from HIV and HIV/MDD patients (n = 4 per group) were also used for non-pooled mRNA analysis on Affymetrix U133 Plus 2.0 arrays. We then utilized a method for integrating the two datasets in a Target Bias Analysis. We found miRNAs of three types: A) Those with many dysregulated mRNA targets of less stringent statistical significance, B) Fewer dysregulated target-genes of highly stringent statistical significance, and C) unclear bias. In HIV/MDD, more miRNAs were downregulated than in HIV alone. Specific miRNA families at targeted chromosomal loci were dysregulated. The dysregulated miRNAs clustered on Chromosomes 14, 17, 19, and X. A small subset of dysregulated genes had many 3' untranslated region (3'UTR) target-sites for dysregulated miRNAs. We provide evidence that certain miRNAs serve as key elements in gene regulatory networks in HIV-infected FC and may be implicated in neurobehavioral disorder. Finally, our data indicates that some genes may serve as hubs of miRNA activity.

Comprehensive Survey on Improved Focality and Penetration Depth of Transcranial Magnetic Stimulation Employing Multi-Coil Arrays.

PubMed

Wei, Xile; Li, Yao; Lu, Meili; Wang, Jiang; Yi, Guosheng

2017-11-14

Multi-coil arrays applied in transcranial magnetic stimulation (TMS) are proposed to accurately stimulate brain tissues and modulate neural activities by an induced electric field (EF). Composed of numerous independently driven coils, a multi-coil array has alternative energizing strategies to evoke EFs targeting at different cerebral regions. To improve the locating resolution and the stimulating focality, we need to fully understand the variation properties of induced EFs and the quantitative control method of the spatial arrangement of activating coils, both of which unfortunately are still unclear. In this paper, a comprehensive analysis of EF properties was performed based on multi-coil arrays. Four types of planar multi-coil arrays were used to study the relationship between the spatial distribution of EFs and the structure of stimuli coils. By changing coil-driven strategies in a basic 16-coil array, we find that an EF induced by compactly distributed coils decays faster than that induced by dispersedly distributed coils, but the former has an advantage over the latter in terms of the activated brain volume. Simulation results also indicate that the attenuation rate of an EF induced by the 36-coil dense array is 3 times and 1.5 times greater than those induced by the 9-coil array and the 16-coil array, respectively. The EF evoked by the 36-coil dispense array has the slowest decay rate. This result demonstrates that larger multi-coil arrays, compared to smaller ones, activate deeper brain tissues at the expense of decreased focality. A further study on activating a specific field of a prescribed shape and size was conducted based on EF variation. Accurate target location was achieved with a 64-coil array 18 mm in diameter. A comparison between the figure-8 coil, the planar array, and the cap-formed array was made and demonstrates an improvement of multi-coil configurations in the penetration depth and the focality. These findings suggest that there is a tradeoff between attenuation rate and focality in the application of multi-coil arrays. Coil-energizing strategies and array dimensions should be based on an adequate evaluation of these two important demands and the topological structure of target tissues.

Micropallet arrays for the capture, isolation and culture of circulating tumor cells from whole blood of mice engrafted with primary human pancreatic adenocarcinoma.

PubMed

Gach, Philip C; Attayek, Peter J; Whittlesey, Rebecca L; Yeh, Jen Jen; Allbritton, Nancy L

2014-04-15

Circulating tumor cells (CTCs) are important biomarkers of cancer progression and metastatic potential. The rarity of CTCs in peripheral blood has driven the development of technologies to isolate these tumor cells with high specificity; however, there are limited techniques available for isolating target CTCs following enumeration. A strategy is described to capture and isolate viable tumor cells from whole blood using an array of releasable microstructures termed micropallets. Specific capture of nucleated cells or cells expressing epithelial cell adhesion molecules (EpCAM) was achieved by functionalizing micropallet surfaces with either fibronectin, Matrigel or anti-EpCAM antibody. Surface grafting of poly(acrylic acid) followed by covalent binding of protein A/G enabled efficient capture of EpCAM antibody on the micropallet surface. MCF-7 cells, a human breast adenocarcinoma, were retained on the array surface with 90±8% efficiency when using an anti-EpCAM-coated array. To demonstrate the efficiency of tumor cell retention on micropallet arrays in the presence of blood, MCF-7 cells were mixed into whole blood and added to small arrays (71 mm(2)) coated with fibronectin, Matrigel or anti-EpCAM. These approaches achieved MCF-7 cell capture from ≤10 µL of whole blood with efficiencies greater than 85%. Furthermore, MCF-7 cells intermixed with 1 mL blood and loaded onto large arrays (7171 mm(2)) were captured with high efficiencies (≥97%), could be isolated from the array by a laser-based approach and were demonstrated to yield a high rate of colony formation (≥85%) after removal from the array. Clinical utility of this technology was shown through the capture, isolation and successful culture of CTCs from the blood of mice engrafted with primary human pancreatic tumors. Direct capture and isolation of living tumor cells from blood followed by analysis or culture will be a valuable tool for cancer cell characterization. © 2013 Elsevier B.V. All rights reserved.

Design and coverage of high throughput genotyping arrays optimized for individuals of East Asian, African American, and Latino race/ethnicity using imputation and a novel hybrid SNP selection algorithm.

PubMed

Hoffmann, Thomas J; Zhan, Yiping; Kvale, Mark N; Hesselson, Stephanie E; Gollub, Jeremy; Iribarren, Carlos; Lu, Yontao; Mei, Gangwu; Purdy, Matthew M; Quesenberry, Charles; Rowell, Sarah; Shapero, Michael H; Smethurst, David; Somkin, Carol P; Van den Eeden, Stephen K; Walter, Larry; Webster, Teresa; Whitmer, Rachel A; Finn, Andrea; Schaefer, Catherine; Kwok, Pui-Yan; Risch, Neil

2011-12-01

Four custom Axiom genotyping arrays were designed for a genome-wide association (GWA) study of 100,000 participants from the Kaiser Permanente Research Program on Genes, Environment and Health. The array optimized for individuals of European race/ethnicity was previously described. Here we detail the development of three additional microarrays optimized for individuals of East Asian, African American, and Latino race/ethnicity. For these arrays, we decreased redundancy of high-performing SNPs to increase SNP capacity. The East Asian array was designed using greedy pairwise SNP selection. However, removing SNPs from the target set based on imputation coverage is more efficient than pairwise tagging. Therefore, we developed a novel hybrid SNP selection method for the African American and Latino arrays utilizing rounds of greedy pairwise SNP selection, followed by removal from the target set of SNPs covered by imputation. The arrays provide excellent genome-wide coverage and are valuable additions for large-scale GWA studies. Copyright © 2011 Elsevier Inc. All rights reserved.

Spatial analysis of biomineralization associated gene expression from the mantle organ of the pearl oyster Pinctada maxima

PubMed Central

2011-01-01

Background Biomineralization is a process encompassing all mineral containing tissues produced within an organism. One of the most dynamic examples of this process is the formation of the mollusk shell, comprising a variety of crystal phases and microstructures. The organic component incorporated within the shell is said to dictate this architecture. However general understanding of how this process is achieved remains ambiguous. The mantle is a conserved organ involved in shell formation throughout molluscs. Specifically the mantle is thought to be responsible for secreting the protein component of the shell. This study employs molecular approaches to determine the spatial expression of genes within the mantle tissue to further the elucidation of the shell biomineralization. Results A microarray platform was custom generated (PmaxArray 1.0) from the pearl oyster Pinctada maxima. PmaxArray 1.0 consists of 4992 expressed sequence tags (ESTs) originating from mantle tissue. This microarray was used to analyze the spatial expression of ESTs throughout the mantle organ. The mantle was dissected into five discrete regions and analyzed for differential gene expression with PmaxArray 1.0. Over 2000 ESTs were determined to be differentially expressed among the tissue sections, identifying five major expression regions. In situ hybridization validated and further localized the expression for a subset of these ESTs. Comparative sequence similarity analysis of these ESTs revealed a number of the transcripts were novel while others showed significant sequence similarities to previously characterized shell related genes. Conclusions This investigation has mapped the spatial distribution for over 2000 ESTs present on PmaxArray 1.0 with reference to specific locations of the mantle. Expression profile clusters have indicated at least five unique functioning zones in the mantle. Three of these zones are likely involved in shell related activities including formation of nacre, periostracum and calcitic prismatic microstructure. A number of novel and known transcripts have been identified from these clusters. The development of PmaxArray 1.0, and the spatial map of its ESTs expression in the mantle has begun characterizing the molecular mechanisms linking the organics and inorganics of the molluscan shell. PMID:21936921

Coherent beam combining of collimated fiber array based on target-in-the-loop technique

NASA Astrophysics Data System (ADS)

Li, Xinyang; Geng, Chao; Zhang, Xiaojun; Rao, Changhui

2011-11-01

Coherent beam combining (CBC) of fiber array is a promising way to generate high power and high quality laser beams. Target-in-the-loop (TIL) technique might be an effective way to ensure atmosphere propagation compensation without wavefront sensors. In this paper, we present very recent research work about CBC of collimated fiber array using TIL technique at the Key Lab on Adaptive Optics (KLAO), CAS. A novel Adaptive Fiber Optics Collimator (AFOC) composed of phase-locking module and tip/tilt control module was developed. CBC experimental setup of three-element fiber array was established. Feedback control is realized using stochastic parallel gradient descent (SPGD) algorithm. The CBC based on TIL with piston and tip/tilt correction simultaneously is demonstrated. And the beam pointing to locate or sweep position of combined spot on target was achieved through TIL technique too. The goal of our work is achieve multi-element CBC for long-distance transmission in atmosphere.

Nonlinear Blind Compensation for Array Signal Processing Application

PubMed Central

Ma, Hong; Jin, Jiang; Zhang, Hua

2018-01-01

Recently, nonlinear blind compensation technique has attracted growing attention in array signal processing application. However, due to the nonlinear distortion stemming from array receiver which consists of multi-channel radio frequency (RF) front-ends, it is too difficult to estimate the parameters of array signal accurately. A novel nonlinear blind compensation algorithm aims at the nonlinearity mitigation of array receiver and its spurious-free dynamic range (SFDR) improvement, which will be more precise to estimate the parameters of target signals such as their two-dimensional directions of arrival (2-D DOAs). Herein, the suggested method is designed as follows: the nonlinear model parameters of any channel of RF front-end are extracted to synchronously compensate the nonlinear distortion of the entire receiver. Furthermore, a verification experiment on the array signal from a uniform circular array (UCA) is adopted to testify the validity of our approach. The real-world experimental results show that the SFDR of the receiver is enhanced, leading to a significant improvement of the 2-D DOAs estimation performance for weak target signals. And these results demonstrate that our nonlinear blind compensation algorithm is effective to estimate the parameters of weak array signal in concomitance with strong jammers. PMID:29690571

DNA ARRAYS TO MONITOR GENE EXPRESSION IN RAT BLOOD AND UTERUS FOLLOWING 17-BETA-ESTRADIOL EXPOSURE: BIOMONITORING ENVIRONMENTAL EFFECTS USING SURROGATE TISSUES

EPA Science Inventory

DNA arrays to monitor gene expression in rat blood and uterus following 17-b-estradiol exposure - biomonitoring environmental effects using surrogate tissues
John C. Rockett, Robert J. Kavlock, Christy R. Lambright, Louise G. Parks, Judith E. Schmid, Vickie S. Wilson, Carmen W...

ANALYSIS OF CHANGES IN GENE EXPRESSION PATTERNS IN FISH EXPOSED TO NATURAL PHARMACEUTICAL AND ENVIRONMENTAL ESTROGENS USING GENE ARRAYS.

EPA Science Inventory

Denslow, N.D., P. Larkin, T.L. Sabo-Attwood, J. Kocerha, K.J. Kroll, M.J. Hemmer and L.C. Folmar. 2004. Analysis of Changes in Gene Expression Patterns in Fish Exposed to Natural, Pharmaceutical and Environmental Estrogens Using Gene Arrays (Abstract). Mar. Environ. Res. 58(2-5):...

Profiling differential gene expression of corals along a transect of waters adjacent to the Bermuda municipal dump.

PubMed

Morgan, Michael B; Edge, Sara E; Snell, Terry W

2005-01-01

A coral cDNA array containing 32 genes was used to examine the gene expression profiles of coral populations located at four sites that varied with distance from a semi-submerged municipal dump in Castle Harbour, Bermuda (previously identified as a point source of anthropogenic stressors). Genes on the array represent transcripts induced under controlled laboratory conditions to a variety of stressors both natural (temperature, sediment, salinity, darkness) and xenobiotic (heavy metals, pesticides, PAH) in origin. The gene expression profiles produced revealed information about the types of stressors. Consistent with other studies undertaken in Castle Harbour, the coral cDNA array detected responses to heavy metals, sedimentation, as well as oxidative stress.

Recognition Imaging of Acetylated Chromatin Using a DNA Aptamer

PubMed Central

Lin, Liyun; Fu, Qiang; Williams, Berea A.R.; Azzaz, Abdelhamid M.; Shogren-Knaak, Michael A.; Chaput, John C.; Lindsay, Stuart

2009-01-01

Histone acetylation plays an important role in the regulation of gene expression. A DNA aptamer generated by in vitro selection to be highly specific for histone H4 protein acetylated at lysine 16 was used as a recognition element for atomic force microscopy-based recognition imaging of synthetic nucleosomal arrays with precisely controlled acetylation. The aptamer proved to be reasonably specific at recognizing acetylated histones, with recognition efficiencies of 60% on-target and 12% off-target. Though this selectivity is much poorer than the >2000:1 equilibrium specificity of the aptamer, it is a large improvement on the performance of a ChIP-quality antibody, which is not selective at all in this application, and it should permit high-fidelity recognition with repeated imaging. The ability to image the precise location of posttranslational modifications may permit nanometer-scale investigation of their effect on chromatin structure. PMID:19751687

AKT in cancer: new molecular insights and advances in drug development

PubMed Central

Mundi, Prabhjot S.; Sachdev, Jasgit; McCourt, Carolyn

2016-01-01

The phosphatidylinositol‐3 kinase (PI3K)–AKT pathway is one of the most commonly dysregulated pathways in all of cancer, with somatic mutations, copy number alterations, aberrant epigenetic regulation and increased expression in a number of cancers. The carefully maintained homeostatic balance of cell division and growth on one hand, and programmed cell death on the other, is universally disturbed in tumorigenesis, and downstream effectors of the PI3K–AKT pathway play an important role in this disturbance. With a wide array of downstream effectors involved in cell survival and proliferation, the well‐characterized direct interactions of AKT make it a highly attractive yet elusive target for cancer therapy. Here, we review the salient features of this pathway, evidence of its role in promoting tumorigenesis and recent progress in the development of therapeutic agents that target AKT. PMID:27232857

Antipsychotics activate the TGFβ pathway effector SMAD3

PubMed Central

Cohen, T.; Sundaresh, S.; Levine, F.

2014-01-01

Although effective in treating an array of neurological disorders, antipsychotics are associated with deleterious metabolic side effects. Through high-throughput screening, we previously identified phenothiazine antipsychotics as modulators of the human insulin promoter. Here, we extended our initial finding to structurally diverse typical and atypical antipsychotics. We then identified the TGFβ pathway as being involved in the effect of antipsychotics on the insulin promoter, finding that antipsychotics activated SMAD3, a downstream effector of the TGFβ pathway, through a receptor distinct from the TGFβ receptor family and known neurotransmitter receptor targets of antipsychotics. Of note, antipsychotics that do not cause metabolic side effects did not activate SMAD3. In vivo relevance was demonstrated by reanalysis of gene expression data from human brains treated with antipsychotics, which showed altered expression of SMAD3 responsive genes. This work raises the possibility that antipsychotics could be designed that retain beneficial CNS activity while lacking deleterious metabolic side effects. PMID:22290122

Involvement of MicroRNAs in Lung Cancer Biology and Therapy

PubMed Central

Liu, Xi; Sempere, Lorenzo F.; Guo, Yongli; Korc, Murray; Kauppinen, Sakari; Freemantle, Sarah J.; Dmitrovsky, Ethan

2011-01-01

MicroRNAs (miRNAs) are a class of small RNAs that regulate gene expression. Expression profiles of specific miRNAs have improved cancer diagnosis and classification and even provided prognostic information in many human cancers, including lung cancer. Tumor suppressive and oncogenic miRNAs were uncovered in lung carcinogenesis. The biological functions of these miRNAs in lung cancer were recently validated in well characterized cellular, murine transgenic as well as transplantable lung cancer models and in human paired normal-malignant lung tissue banks and tissue arrays. Tumor suppressive and oncogenic miRNAs that were identified in lung cancer will be reviewed here. Emphasis is placed on highlighting those functionally validated miRNAs that are not only biomarkers of lung carcinogenesis, but also candidate pharmacologic targets. How these miRNA findings advance an understanding of lung cancer biology and could improve lung cancer therapy are discussed in this article. PMID:21420030

A Label-Free Fluorescent Array Sensor Utilizing Liposome Encapsulating Calcein for Discriminating Target Proteins by Principal Component Analysis

PubMed Central

Imamura, Ryota; Murata, Naoki; Shimanouchi, Toshinori; Yamashita, Kaoru; Fukuzawa, Masayuki; Noda, Minoru

2017-01-01

A new fluorescent arrayed biosensor has been developed to discriminate species and concentrations of target proteins by using plural different phospholipid liposome species encapsulating fluorescent molecules, utilizing differences in permeation of the fluorescent molecules through the membrane to modulate liposome-target protein interactions. This approach proposes a basically new label-free fluorescent sensor, compared with the common technique of developed fluorescent array sensors with labeling. We have confirmed a high output intensity of fluorescence emission related to characteristics of the fluorescent molecules dependent on their concentrations when they leak from inside the liposomes through the perturbed lipid membrane. After taking an array image of the fluorescence emission from the sensor using a CMOS imager, the output intensities of the fluorescence were analyzed by a principal component analysis (PCA) statistical method. It is found from PCA plots that different protein species with several concentrations were successfully discriminated by using the different lipid membranes with high cumulative contribution ratio. We also confirmed that the accuracy of the discrimination by the array sensor with a single shot is higher than that of a single sensor with multiple shots. PMID:28714873

A Label-Free Fluorescent Array Sensor Utilizing Liposome Encapsulating Calcein for Discriminating Target Proteins by Principal Component Analysis.

PubMed

Imamura, Ryota; Murata, Naoki; Shimanouchi, Toshinori; Yamashita, Kaoru; Fukuzawa, Masayuki; Noda, Minoru

2017-07-15

A new fluorescent arrayed biosensor has been developed to discriminate species and concentrations of target proteins by using plural different phospholipid liposome species encapsulating fluorescent molecules, utilizing differences in permeation of the fluorescent molecules through the membrane to modulate liposome-target protein interactions. This approach proposes a basically new label-free fluorescent sensor, compared with the common technique of developed fluorescent array sensors with labeling. We have confirmed a high output intensity of fluorescence emission related to characteristics of the fluorescent molecules dependent on their concentrations when they leak from inside the liposomes through the perturbed lipid membrane. After taking an array image of the fluorescence emission from the sensor using a CMOS imager, the output intensities of the fluorescence were analyzed by a principal component analysis (PCA) statistical method. It is found from PCA plots that different protein species with several concentrations were successfully discriminated by using the different lipid membranes with high cumulative contribution ratio. We also confirmed that the accuracy of the discrimination by the array sensor with a single shot is higher than that of a single sensor with multiple shots.

Station-Keeping Requirements for Astronomical Imaging with Constellations of Free-Flying Collectors

NASA Technical Reports Server (NTRS)

Allen, Ronald J.

2004-01-01

The requirements on station-keeping for constellations of free-flying collectors coupled as (future) imaging arrays in space for astrophysics applications are discussed. The typical knowledge precision required in the plane of the array depends on the angular size of the targets of interest; it is generally at a level of tens of centimeters for typical stellar targets, becoming of order centimeters only for the widest attainable fields of view. In the "piston" direction, perpendicular to the array, the typical knowledge precision required depends on the bandwidth of the signal, and is at a level of tens of wavelengths for narrow approx. 1% signal bands, becoming of order one wavelength only for the broadest bandwidths expected to be useful. The significance of this result is that, at this level of precision, it may be possible to provide the necessary knowledge of array geometry without the use of signal photons, thereby allowing observations of faint targets. "Closure-phase" imaging is a technique which has been very successfully applied to surmount instabilities owing to equipment and to the atmosphere, and which appears to be directly applicable to space imaging arrays where station-keeping drifts play the same role as (slow) atmospheric and equipment instabilities.

Assessing differential gene expression with small sample sizes in oligonucleotide arrays using a mean-variance model.

PubMed

Hu, Jianhua; Wright, Fred A

2007-03-01

The identification of the genes that are differentially expressed in two-sample microarray experiments remains a difficult problem when the number of arrays is very small. We discuss the implications of using ordinary t-statistics and examine other commonly used variants. For oligonucleotide arrays with multiple probes per gene, we introduce a simple model relating the mean and variance of expression, possibly with gene-specific random effects. Parameter estimates from the model have natural shrinkage properties that guard against inappropriately small variance estimates, and the model is used to obtain a differential expression statistic. A limiting value to the positive false discovery rate (pFDR) for ordinary t-tests provides motivation for our use of the data structure to improve variance estimates. Our approach performs well compared to other proposed approaches in terms of the false discovery rate.

A model of binding on DNA microarrays: understanding the combined effect of probe synthesis failure, cross-hybridization, DNA fragmentation and other experimental details of affymetrix arrays

PubMed Central

2012-01-01

Background DNA microarrays are used both for research and for diagnostics. In research, Affymetrix arrays are commonly used for genome wide association studies, resequencing, and for gene expression analysis. These arrays provide large amounts of data. This data is analyzed using statistical methods that quite often discard a large portion of the information. Most of the information that is lost comes from probes that systematically fail across chips and from batch effects. The aim of this study was to develop a comprehensive model for hybridization that predicts probe intensities for Affymetrix arrays and that could provide a basis for improved microarray analysis and probe development. The first part of the model calculates probe binding affinities to all the possible targets in the hybridization solution using the Langmuir isotherm. In the second part of the model we integrate details that are specific to each experiment and contribute to the differences between hybridization in solution and on the microarray. These details include fragmentation, wash stringency, temperature, salt concentration, and scanner settings. Furthermore, the model fits probe synthesis efficiency and target concentration parameters directly to the data. All the parameters used in the model have a well-established physical origin. Results For the 302 chips that were analyzed the mean correlation between expected and observed probe intensities was 0.701 with a range of 0.88 to 0.55. All available chips were included in the analysis regardless of the data quality. Our results show that batch effects arise from differences in probe synthesis, scanner settings, wash strength, and target fragmentation. We also show that probe synthesis efficiencies for different nucleotides are not uniform. Conclusions To date this is the most complete model for binding on microarrays. This is the first model that includes both probe synthesis efficiency and hybridization kinetics/cross-hybridization. These two factors are sequence dependent and have a large impact on probe intensity. The results presented here provide novel insight into the effect of probe synthesis errors on Affymetrix microarrays; furthermore, the algorithms developed in this work provide useful tools for the analysis of cross-hybridization, probe synthesis efficiency, fragmentation, wash stringency, temperature, and salt concentration on microarray intensities. PMID:23270536

The COP9 Signalosome Converts Temporal Hormone Signaling to Spatial Restriction on Neural Competence

PubMed Central

Huang, Yi-Chun; Lu, Yu-Nung; Wu, June-Tai; Chien, Cheng-Ting; Pi, Haiwei

2014-01-01

During development, neural competence is conferred and maintained by integrating spatial and temporal regulations. The Drosophila sensory bristles that detect mechanical and chemical stimulations are arranged in stereotypical positions. The anterior wing margin (AWM) is arrayed with neuron-innervated sensory bristles, while posterior wing margin (PWM) bristles are non-innervated. We found that the COP9 signalosome (CSN) suppresses the neural competence of non-innervated bristles at the PWM. In CSN mutants, PWM bristles are transformed into neuron-innervated, which is attributed to sustained expression of the neural-determining factor Senseless (Sens). The CSN suppresses Sens through repression of the ecdysone signaling target gene broad (br) that encodes the BR-Z1 transcription factor to activate sens expression. Strikingly, CSN suppression of BR-Z1 is initiated at the prepupa-to-pupa transition, leading to Sens downregulation, and termination of the neural competence of PWM bristles. The role of ecdysone signaling to repress br after the prepupa-to-pupa transition is distinct from its conventional role in activation, and requires CSN deneddylating activity and multiple cullins, the major substrates of deneddylation. Several CSN subunits physically associate with ecdysone receptors to represses br at the transcriptional level. We propose a model in which nuclear hormone receptors cooperate with the deneddylation machinery to temporally shutdown downstream target gene expression, conferring a spatial restriction on neural competence at the PWM. PMID:25393278

Enhancing radiosensitization in EphB4 receptor-expressing Head and Neck Squamous Cell Carcinomas

PubMed Central

Bhatia, Shilpa; Hirsch, Kellen; Sharma, Jaspreet; Oweida, Ayman; Griego, Anastacia; Keysar, Stephen; Jimeno, Antonio; Raben, David; Krasnoperov, Valery; Gill, Parkash S.; Pasquale, Elena B.; Wang, Xiao-Jing; Karam, Sana D.

2016-01-01

Members of the Eph family of receptor tyrosine kinases have been implicated in a wide array of human cancers. The EphB4 receptor is ubiquitously expressed in head and neck squamous cell carcinoma (HNSCC) and has been shown to impart tumorigenic and invasive characteristics to these cancers. In this study, we investigated whether EphB4 receptor targeting can enhance the radiosensitization of HNSCC. Our data show that EphB4 is expressed at high to moderate levels in HNSCC cell lines and patient-derived xenograft (PDX) tumors. We observed decreased survival fractions in HNSCC cells following EphB4 knockdown in clonogenic assays. An enhanced G2 cell cycle arrest with activation of DNA damage response pathway and increased apoptosis was evident in HNSCC cells following combined EphB4 downregulation and radiation compared to EphB4 knockdown and radiation alone. Data using HNSCC PDX models showed significant reduction in tumor volume and enhanced delay in tumor regrowth following sEphB4-HSA administration with radiation compared to single agent treatment. sEphB4-HSA is a protein known to block the interaction between the EphB4 receptor and its ephrin-B2 ligand. Overall, our findings emphasize the therapeutic relevance of EphB4 targeting as a radiosensitizer that can be exploited for the treatment of human head and neck carcinomas. PMID:27941840

Dose-response relationships in gene expression profiles in rainbow trout, Oncorhyncus mykiss, exposed to ethynylestradiol.

PubMed

Hook, Sharon E; Skillman, Ann D; Small, Jack A; Schultz, Irvin R

2006-07-01

Determining how gene expression profiles change with toxicant dose will improve the utility of arrays in identifying biomarkers and modes of toxic action. Isogenic rainbow trout, Oncorhyncus mykiss,were exposed to 10, 50 or 100 ng/L ethynylestradiol (a xeno-estrogen) for 7 days. Following exposure hepatic RNA was extracted. Fluorescently labeled cDNA were generated and hybridized against a commercially available Atlantic Salmon/Trout array (GRASP project, University of Victoria) spotted with 16,000 cDNAs. Transcript expression in treated vs control fish was analyzed via Genespring (Silicon Genetics) to identify genes with altered expression, as well as to determine gene clustering patterns that can be used as "expression signatures". Array results were confirmed via qRT PCR. Our analysis indicates that gene expression profiles varied somewhat with dose. Established biomarkers of exposure to estrogenic chemicals, such as vitellogenin, vitelline envelope proteins, and the estrogen receptor alpha, were induced at every dose. Other genes were dose specific, suggesting that different doses induce distinct physiological responses. These findings demonstrate that cDNA microarrays could be used to identify both toxicant class and relative dose.

Detection technique of targets for missile defense system

NASA Astrophysics Data System (ADS)

Guo, Hua-ling; Deng, Jia-hao; Cai, Ke-rong

2009-11-01

Ballistic missile defense system (BMDS) is a weapon system for intercepting enemy ballistic missiles. It includes ballistic-missile warning system, target discrimination system, anti-ballistic-missile guidance systems, and command-control communication system. Infrared imaging detection and laser imaging detection are widely used in BMDS for surveillance, target detection, target tracking, and target discrimination. Based on a comprehensive review of the application of target-detection techniques in the missile defense system, including infrared focal plane arrays (IRFPA), ground-based radar detection technology, 3-dimensional imaging laser radar with a photon counting avalanche photodiode (APD) arrays and microchip laser, this paper focuses on the infrared and laser imaging detection techniques in missile defense system, as well as the trends for their future development.

A user-friendly workflow for analysis of Illumina gene expression bead array data available at the arrayanalysis.org portal.

PubMed

Eijssen, Lars M T; Goelela, Varshna S; Kelder, Thomas; Adriaens, Michiel E; Evelo, Chris T; Radonjic, Marijana

2015-06-30

Illumina whole-genome expression bead arrays are a widely used platform for transcriptomics. Most of the tools available for the analysis of the resulting data are not easily applicable by less experienced users. ArrayAnalysis.org provides researchers with an easy-to-use and comprehensive interface to the functionality of R and Bioconductor packages for microarray data analysis. As a modular open source project, it allows developers to contribute modules that provide support for additional types of data or extend workflows. To enable data analysis of Illumina bead arrays for a broad user community, we have developed a module for ArrayAnalysis.org that provides a free and user-friendly web interface for quality control and pre-processing for these arrays. This module can be used together with existing modules for statistical and pathway analysis to provide a full workflow for Illumina gene expression data analysis. The module accepts data exported from Illumina's GenomeStudio, and provides the user with quality control plots and normalized data. The outputs are directly linked to the existing statistics module of ArrayAnalysis.org, but can also be downloaded for further downstream analysis in third-party tools. The Illumina bead arrays analysis module is available at http://www.arrayanalysis.org . A user guide, a tutorial demonstrating the analysis of an example dataset, and R scripts are available. The module can be used as a starting point for statistical evaluation and pathway analysis provided on the website or to generate processed input data for a broad range of applications in life sciences research.

Protein inhibitor of activated STAT3 inhibits adipogenic gene expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng Jianbei; Hua Kunjie; Caveney, Erica J.

2006-01-20

Protein inhibitor of activated STAT3 (PIAS3), a cytokine-induced repressor of signal transducer and activator of transcription 3 (STAT3) and a modulator of a broad array of nuclear proteins, is expressed in white adipose tissue, but its role in adipogenesis is not known. Here, we determined that PIAS3 was constitutively expressed in 3T3-L1 cells at all stages of adipogenesis. However, it translocated from the nucleus to the cytoplasm 4 days after induction of differentiation by isobutylmethylxanthine, dexamethasone, and insulin (MDI). In ob/ob mice, PIAS3 expression was increased in white adipose tissue depots compared to lean mice and was found in themore » cytoplasm of adipocytes. Overexpression of PIAS3 in differentiating preadipocytes, which localized primarily to the nucleus, inhibited mRNA level gene expression of adipogenic transcription factors C/EBP{alpha} and PPAR{gamma}, as well as their downstream target genes aP2 and adiponectin. PIAS3 also inhibited C/EBP{alpha} promoter activation mediated specifically by insulin, but not dexamethasone or isobutylmethylxanthine. Taken together, these data suggest that PIAS3 may play an inhibitory role in adipogenesis by modulating insulin-activated transcriptional activation events. Increased PIAS3 expression in adipose tissue may play a role in the metabolic disturbances of obesity.« less

Optogenetic micro-electrocorticography for modulating and localizing cerebral cortex activity

PubMed Central

Richner, Thomas J.; Thongpang, Sanitta; Brodnick, Sarah K.; Schendel, Amelia A.; Falk, Ryan W.; Krugner-Higby, Lisa A.; Pashaie, Ramin; Williams, Justin C.

2014-01-01

Objective Spatial localization of neural activity from within the brain with electrocorticography (ECoG) and electroencephalography (EEG) remains a challenge in clinical and research settings, and while microfabricated ECoG (micro-ECoG) array technology continues to improve, complimentary methods to simultaneously modulate cortical activity while recording are needed. Approach We developed a neural interface utilizing optogenetics, cranial windowing, and micro-ECoG arrays fabricated on a transparent polymer. This approach enabled us to directly modulate neural activity at known locations around micro-ECoG arrays in mice expressing Channelrhodopsin-2 (ChR2). We applied photostimuli varying in time, space and frequency to the cortical surface, and we targeted multiple depths within the cortex using an optical fiber while recording micro-ECoG signals. Main Results Negative potentials of up to 1.5 mV were evoked by photostimuli applied to the entire cortical window, while focally applied photostimuli evoked spatially localized micro-ECoG potentials. Two simultaneously applied focal stimuli could be separated, depending on the distance between them. Photostimuli applied within the cortex with an optical fiber evoked more complex micro-ECoG potentials with multiple positive and negative peaks whose relative amplitudes depended on the depth of the fiber. Significance Optogenetic ECoG has potential applications in the study of epilepsy, cortical dynamics, and neuroprostheses. PMID:24445482

Chronic arsenic trioxide exposure leads to enhanced aggressiveness via Met oncogene addiction in cancer cells

PubMed Central

Kryeziu, Kushtrim; Pirker, Christine; Englinger, Bernhard; van Schoonhoven, Sushilla; Spitzwieser, Melanie; Mohr, Thomas; Körner, Wilfried; Weinmüllner, Regina; Tav, Koray; Grillari, Johannes; Cichna-Markl, Margit; Berger, Walter; Heffeter, Petra

2016-01-01

As an environmental poison, arsenic is responsible for many cancer deaths. Paradoxically, arsenic trioxide (ATO) presents also a powerful therapy used to treat refractory acute promyelocytic leukemia (APL) and is intensively investigated for treatment of other cancer types. Noteworthy, cancer therapy is frequently hampered by drug resistance, which is also often associated with enhancement of tumor aggressiveness. In this study, we analyzed ATO-selected cancer cells (A2780ATO) for the mechanisms underlying their enhanced tumorigenicity and aggressiveness. These cells were characterized by enhanced proliferation and spheroid growth as well as increased tumorigenicity of xenografts in SCID mice. Noteworthy, subsequent studies revealed that overexpression of Met receptor was the underlying oncogenic driver of these effects, as A2780ATO cells were characterized by collateral sensitivity against Met inhibitors. This finding was also confirmed by array comparative genomic hybridization (array CGH) and whole genome gene expression arrays, which revealed that Met overexpression by chronic ATO exposure was based on the transcriptional regulation via activation of AP-1. Finally, it was shown that treatment with the Met inhibitor crizotinib was also effective against A2780ATO cell xenografts in vivo, indicating that targeting of Met presents a promising strategy for the treatment of Met-overexpressing tumors after either arsenic exposure or failure to ATO treatment. PMID:27036042

miRNA regulation of cytotoxic effects in mouse Sertoli cells exposed to nonylphenol

PubMed Central

2011-01-01

Background It is known that some environmental chemicals affect the human endocrine system. The harmful effects of endocrine disrupting chemical (EDC) nonylphenol (NP) have been studied since the 1980s. It is known that NP adversely affects physiological functions by mimicking the natural hormone 17 beta-estradiol. In the present study, we analyzed the expression of miRNAs and their target genes in mouse Sertoli TM4 cells to better understand the regulatory roles of miRNAs on Sertoli cells after NP exposure. Methods Mouse TM4 Sertoli cells were treated with NP for 3 or 24 h, and global gene and miRNA expression were analyzed using Agilent mouse whole genome and mouse miRNA v13 arrays. Results We identified genes that were > 2-fold differentially expressed in NP-treated cells and control cells (P < 0.05) and analyzed their functions through Gene Ontology analysis. We also identified miRNAs that were differentially expressed in NP-treated and control cells. Of the 186 miRNAs the expression of which differed between NP-treated and control cells, 59 and 147 miRNAs exhibited 1.3-fold increased or decreased expression at 3 and 24 h, respectively. Network analysis of deregulated miRNAs suggested that Ppara may regulate the expression of certain miRNAs, including miR-378, miR-125a-3p miR-20a, miR-203, and miR-101a, after exposure to NP. Additionally, comprehensive analysis of predicted target genes for miRNAs showed that the expression of genes with roles in cell proliferation, the cell cycle, and cell death were regulated by miRNA in NP-treated TM4 cells. Levels of expression of the miRNAs miR-135a* and miR-199a-5p were validated by qRT-PCR. Finally, miR-135a* target gene analysis suggests that the generation of reactive oxygen species (ROS) following exposure to NP exposure may be mediated by miR-135a* through regulation of the Wnt/beta-catenin signaling pathway. Conclusions Collectively, these data help to determine NP's actions on mouse TM4 Sertoli cells and increase our understanding of the molecular mechanisms underlying the adverse effects of xenoestrogens on the reproductive system. PMID:21914226

Channel capacity of an array system for Gaussian channels with applications to combining and noise cancellation

NASA Technical Reports Server (NTRS)

Cheung, K. M.; Vilnrotter, V.

1996-01-01

A closed-form expression for the capacity of an array of correlated Gaussian channels is derived. It is shown that when signal and noise are independent, the array of observables can be replaced with a single observable without diminishing the capacity of the array channel. Examples are provided to illustrate the dependence of channel capacity on noise correlation for two- and three-channel arrays.

Channel Capacity of an Array System for Gaussian Channels With Applications to Combining and Noise Cancellation

NASA Technical Reports Server (NTRS)

Cheung, K.-M.; Vilnrotter, V.

1996-01-01

A closed-form expression for the capacity of an array of correlated Gaussian channels is derived. It is shown that when signal and noise are independent, the array of observables can be replaced with a single observable without diminishing the capacity of the array channel. Examples are provided to illustrate the dependence of channel capacity on noise correlation for two- and three-channel arrays.

Gene Expression Profiling of Multiple Leiomyomata Uteri and Matched Normal Tissue from a Single Patient

PubMed Central

Dimitrova, Irina K.; Richer, Jennifer K.; Rudolph, Michael C.; Spoelstra, Nicole S.; Reno, Elaine M.; Medina, Theresa M.; Bradford, Andrew P.

2009-01-01

Objective To identify differentially expressed genes between fibroid and adjacent normal myometrium in an identical hormonal and genetic background. Design Array analysis of 3 leiomyomata and matched adjacent normal myometrium in a single patient. Setting University of Colorado Hospital. Patient(s) A single female undergoing medically indicated hysterectomy for symptomatic fibroids. Interventions(s) mRNA isolation and microarray analysis, reverse-transcriptase polymerase chain reaction, western blotting and immunohistochemistry. Main Outcome Measure(s) Changes in mRNA and protein levels in leiomyomata and matched normal myometrium. Result(s) Expression of 197 genes was increased and 619 decreased, significantly by at least 2 fold, in leiomyomata relative to normal myometrium. Expression profiles between tumors were similar and normal myometrial samples showed minimal variation. Changes in, and variation of, expression of selected genes were confirmed in additional normal and leiomyoma samples from multiple patients. Conclusion(s) Analysis of multiple tumors from a single patient confirmed changes in expression of genes described in previous, apparently disparate, studies and identified novel targets. Gene expression profiles in leiomyomata are consistent with increased activation of mitogenic pathways and inhibition of apoptosis. Down-regulation of genes implicated in invasion and metastasis, of cancers, was observed in fibroids. This expression pattern may underlie the benign nature of uterine leiomyomata and may aid in the differential diagnosis of leiomyosarcoma. PMID:18672237

The FDA's Experience with Emerging Genomics Technologies-Past, Present, and Future.

PubMed

Xu, Joshua; Thakkar, Shraddha; Gong, Binsheng; Tong, Weida

2016-07-01

The rapid advancement of emerging genomics technologies and their application for assessing safety and efficacy of FDA-regulated products require a high standard of reliability and robustness supporting regulatory decision-making in the FDA. To facilitate the regulatory application, the FDA implemented a novel data submission program, Voluntary Genomics Data Submission (VGDS), and also to engage the stakeholders. As part of the endeavor, for the past 10 years, the FDA has led an international consortium of regulatory agencies, academia, pharmaceutical companies, and genomics platform providers, which was named MicroArray Quality Control Consortium (MAQC), to address issues such as reproducibility, precision, specificity/sensitivity, and data interpretation. Three projects have been completed so far assessing these genomics technologies: gene expression microarrays, whole genome genotyping arrays, and whole transcriptome sequencing (i.e., RNA-seq). The resultant studies provide the basic parameters for fit-for-purpose application of these new data streams in regulatory environments, and the solutions have been made available to the public through peer-reviewed publications. The latest MAQC project is also called the SEquencing Quality Control (SEQC) project focused on next-generation sequencing. Using reference samples with built-in controls, SEQC studies have demonstrated that relative gene expression can be measured accurately and reliably across laboratories and RNA-seq platforms. Besides prediction performance comparable to microarrays in clinical settings and safety assessments, RNA-seq is shown to have better sensitivity for low expression and reveal novel transcriptomic features. Future effort of MAQC will be focused on quality control of whole genome sequencing and targeted sequencing.

Impact of α-targeted radiation therapy on gene expression in a pre-clinical model for disseminated peritoneal disease when combined with paclitaxel.

PubMed

Yong, Kwon Joong; Milenic, Diane E; Baidoo, Kwamena E; Brechbiel, Martin W

2014-01-01

To better understand the molecular basis of the enhanced cell killing effected by the combined modality of paclitaxel and ²¹²Pb-trastuzumab (Pac/²¹²Pb-trastuzumab), gene expression in LS-174T i.p. xenografts was investigated 24 h after treatment. Employing a real time quantitative PCR array (qRT-PCR array), 84 DNA damage response genes were quantified. Differentially expressed genes following therapy with Pac/²¹²Pb-trastuzumab included those involved in apoptosis (BRCA1, CIDEA, GADD45α, GADD45γ, GML, IP6K3, PCBP4, PPP1R15A, RAD21, and p73), cell cycle (BRCA1, CHK1, CHK2, GADD45α, GML, GTSE1, NBN, PCBP4, PPP1R15A, RAD9A, and SESN1), and damaged DNA repair (ATRX, BTG2, EXO1, FEN1, IGHMBP2, OGG1, MSH2, MUTYH, NBN, PRKDC, RAD21, and p73). This report demonstrates that the increased stressful growth arrest conditions induced by the Pac/²¹²Pb-trastuzumab treatment suppresses cell proliferation through the regulation of genes which are involved in apoptosis and damaged DNA repair including single and double strand DNA breaks. Furthermore, the study demonstrates that ²¹²Pb-trastuzumab potentiation of cell killing efficacy results from the perturbation of genes related to the mitotic spindle checkpoint and BASC (BRCA1-associated genome surveillance complex), suggesting cross-talk between DNA damage repair and the spindle damage response.

Genome-Wide Survey of Cold Stress Regulated Alternative Splicing in Arabidopsis thaliana with Tiling Microarray

PubMed Central

Leviatan, Noam; Alkan, Noam; Leshkowitz, Dena; Fluhr, Robert

2013-01-01

Alternative splicing plays a major role in expanding the potential informational content of eukaryotic genomes. It is an important post-transcriptional regulatory mechanism that can increase protein diversity and affect mRNA stability. Alternative splicing is often regulated in a tissue-specific and stress-responsive manner. Cold stress, which adversely affects plant growth and development, regulates the transcription and splicing of plant splicing factors. This can affect the pre-mRNA processing of many genes. To identify cold regulated alternative splicing we applied Affymetrix Arabidopsis tiling arrays to survey the transcriptome under cold treatment conditions. A novel algorithm was used for detection of statistically relevant changes in intron expression within a transcript between control and cold growth conditions. A reverse transcription polymerase chain reaction (RT-PCR) analysis of a number of randomly selected genes confirmed the changes in splicing patterns under cold stress predicted by tiling array. Our analysis revealed new types of cold responsive genes. While their expression level remains relatively unchanged under cold stress their splicing pattern shows detectable changes in the relative abundance of isoforms. The majority of cold regulated alternative splicing introduced a premature termination codon (PTC) into the transcripts creating potential targets for degradation by the nonsense mediated mRNA decay (NMD) process. A number of these genes were analyzed in NMD-defective mutants by RT-PCR and shown to evade NMD. This may result in new and truncated proteins with altered functions or dominant negative effects. The results indicate that cold affects both quantitative and qualitative aspects of gene expression. PMID:23776682

The FDA’s Experience with Emerging Genomics Technologies—Past, Present, and Future

PubMed Central

Xu, Joshua; Thakkar, Shraddha; Gong, Binsheng; Tong, Weida

2016-01-01

The rapid advancement of emerging genomics technologies and their application for assessing safety and efficacy of FDA-regulated products require a high standard of reliability and robustness supporting regulatory decision-making in the FDA. To facilitate the regulatory application, the FDA implemented a novel data submission program, Voluntary Genomics Data Submission (VGDS), and also to engage the stakeholders. As part of the endeavor, for the past 10 years, the FDA has led an international consortium of regulatory agencies, academia, pharmaceutical companies, and genomics platform providers, which was named MicroArray Quality Control Consortium (MAQC), to address issues such as reproducibility, precision, specificity/sensitivity, and data interpretation. Three projects have been completed so far assessing these genomics technologies: gene expression microarrays, whole genome genotyping arrays, and whole transcriptome sequencing (i.e., RNA-seq). The resultant studies provide the basic parameters for fit-for-purpose application of these new data streams in regulatory environments, and the solutions have been made available to the public through peer-reviewed publications. The latest MAQC project is also called the SEquencing Quality Control (SEQC) project focused on next-generation sequencing. Using reference samples with built-in controls, SEQC studies have demonstrated that relative gene expression can be measured accurately and reliably across laboratories and RNA-seq platforms. Besides prediction performance comparable to microarrays in clinical settings and safety assessments, RNA-seq is shown to have better sensitivity for low expression and reveal novel transcriptomic features. Future effort of MAQC will be focused on quality control of whole genome sequencing and targeted sequencing. PMID:27116022

University of Texas Southwestern Medical Center: Functional Signature Ontology Tool: Triplicate Measurements of Reporter Gene Expression in Response to Individual Genetic and Chemical Perturbations in HCT116 Cells | Office of Cancer Genomics

Cancer.gov

The goal of this project is to use an eight-gene expression profile to define functional signatures for small molecules and natural products with heretofore undefined mechanism of action. Two genes in the eight gene set are used as internal controls and do not vary across gene expression array data collected from the public domain. The remaining six genes are found to vary independently across a large collection of publically available gene expression array datasets.  Read the abstract

Photosensitive biosensor array system using optical addressing without an addressing circuit on array biochips

NASA Astrophysics Data System (ADS)

Ahn, Chang-Geun; Ah, Chil Seong; Kim, Tae-Youb; Park, Chan Woo; Yang, Jong-Heon; Kim, Ansoon; Sung, Gun Yong

2010-09-01

This paper introduces a photosensitive biosensor array system with a simple photodiode array that detects photocurrent changes caused by reactions between probe and target molecules. Using optical addressing, the addressing circuit on the array chip is removed for low-cost application, and real cell addressing is achieved using an externally located computer-controllable light-emitting diode array module. The fabricated biosensor array chip shows a good dynamic range of 1-100 ng/mL under prostate-specific antigen detection, with an on-chip resolution of roughly 1 ng/mL.

U.S. Marine Corps Level-Dependent Hearing Protector Assessment: Objective Measures of Hearing Protection Devices

DTIC Science & Technology

2014-01-01

12 4.2.1 Loudspeaker Array ...Instrumentation 4.2.1 Loudspeaker Array Target stimuli were presented from a spherical loudspeaker array consisting of 57 Meyer Sound MM-4XP miniature... loudspeaker array on a raised platform that placed their ears at the same elevation as the 0° loudspeaker ring. The chair was free to rotate 360° and

Prenatal diagnosis of chromosomal abnormalities using array-based comparative genomic hybridization

USDA-ARS?s Scientific Manuscript database

This study was designed to evaluate the feasibility of using a targeted array-CGH strategy for prenatal diagnosis of genomic imbalances in a clinical setting of current pregnancies. Women undergoing prenatal diagnosis were counseled and offered array-CGH (BCM V4.0) in addition to routine chromosome ...

Comparison of global brain gene expression profiles between inbred long-sleep and inbred short-sleep mice by high-density gene array hybridization.

PubMed

Xu, Y; Ehringer, M; Yang, F; Sikela, J M

2001-06-01

Inbred long-sleep (ILS) and short-sleep (ISS) mice show significant central nervous system-mediated differences in sleep time for sedative dose of ethanol and are frequently used as a rodent model for ethanol sensitivity. In this study, we have used complementary DNA (cDNA) array hybridization methodology to identify genes that are differentially expressed between the brains of ILS and ISS mice. To carry out this analysis, we used both the gene discovery array (GDA) and the Mouse GEM 1 Microarray. GDA consists of 18,378 nonredundant mouse cDNA clones on a single nylon filter. Complex probes were prepared from total brain mRNA of ILS or ISS mice by using reverse transcription and 33P labeling. The labeled probes were hybridized in parallel to the gene array filters. Data from GDA experiments were analyzed with SQL-Plus and Oracle 8. The GEM microarray includes 8,730 sequence-verified clones on a glass chip. Two fluorescently labeled probes were used to hybridize a microarray simultaneously. Data from GEM experiments were analyzed by using the GEMTools software package (Incyte). Differentially expressed genes identified from each method were confirmed by relative quantitative reverse transcription-polymerase chain reaction (RT-PCR). A total of 41 genes or expressed sequence tags (ESTs) display significant expression level differences between brains of ILS and ISS mice after GDA, GEM1 hybridization, and quantitative RT-PCR confirmation. Among them, 18 clones were expressed higher in ILS mice, and 23 clones were expressed higher in ISS mice. The individual gene or EST's function and mapping information have been analyzed. This study identified 41 genes that are differentially expressed between brains of ILS and ISS mice. Some of them may have biological relevance in mediation of phenotypic variation between ILS and ISS mice for ethanol sensitivity. This study also demonstrates that parallel gene expression comparison with high-density cDNA arrays is a rapid and efficient way to discover potential genes and pathways involved in alcoholism and alcohol-related physiologic processes.

System concepts for transmit arrays of parabolic antennas for deep space uplinks

NASA Technical Reports Server (NTRS)

Hurd, William J.

2005-01-01

Phased arrays of parabolic antennas are a potentially lower-cost way to provide uplink transmission to distant spacecraft, compared to the 34-m and 70-m antennas now used by the NASA Deep Space Network. A large transmit array could provide very high EIRP when needed for spacecraft emergencies, such as the equivalent of 1 MW radiated from a 70-m antenna. Cost-effectiveness is realized by dividing the array into smaller arrays to provide routine support to many spacecraft simultaneously. The antennas might be as small as 12-m in diameter, with as many as 100 antennas covering an area of 0.5 km to 1 km in extent. Such arrays present significant technical challenges in phase alignment, which must be maintained at close to 1 mm. The concept requires a very stable system with accurately known antenna phase center locations. The system is first calibrated by transmitting from all antennas, and observing the signals at a target located in the far fields of the individual antennas. The antennas are then pointed to the operational targets, with the signal phases and time delays set to reinforce in the target directions. This requires accurate knowledge of the target directions and calculation of the required phases. The system must be phase-stable for all directions and over the time between calibrations, which is desired to be at least one day. In this paper, a system concept is presented, the major error sources are identified, a rough error budget is established, and key elements of the system are discussed. A calibration method is recommended which uses satellites as radar targets. The performance goal is to achieve a combining loss of less than 0.2 dB in good weather, and of less than 1 dB in all but extremely bad weather.

Pseudomonas aeruginosa AES-1 exhibits increased virulence gene expression during chronic infection of cystic fibrosis lung.

PubMed

Naughton, Sharna; Parker, Dane; Seemann, Torsten; Thomas, Torsten; Turnbull, Lynne; Rose, Barbara; Bye, Peter; Cordwell, Stuart; Whitchurch, Cynthia; Manos, Jim

2011-01-01

Pseudomonas aeruginosa, the leading cause of morbidity and mortality in people with cystic fibrosis (CF), adapts for survival in the CF lung through both mutation and gene expression changes. Frequent clonal strains such as the Australian Epidemic Strain-1 (AES-1), have increased ability to establish infection in the CF lung and to superimpose and replace infrequent clonal strains. Little is known about the factors underpinning these properties. Analysis has been hampered by lack of expression array templates containing CF-strain specific genes. We sequenced the genome of an acute infection AES-1 isolate from a CF infant (AES-1R) and constructed a non-redundant micro-array (PANarray) comprising AES-1R and seven other sequenced P. aeruginosa genomes. The unclosed AES-1R genome comprised 6.254Mbp and contained 6957 putative genes, including 338 not found in the other seven genomes. The PANarray contained 12,543 gene probe spots; comprising 12,147 P. aeruginosa gene probes, 326 quality-control probes and 70 probes for non-P. aeruginosa genes, including phage and plant genes. We grew AES-1R and its isogenic pair AES-1M, taken from the same patient 10.5 years later and not eradicated in the intervening period, in our validated artificial sputum medium (ASMDM) and used the PANarray to compare gene expression of both in duplicate. 675 genes were differentially expressed between the isogenic pairs, including upregulation of alginate, biofilm, persistence genes and virulence-related genes such as dihydroorotase, uridylate kinase and cardiolipin synthase, in AES-1M. Non-PAO1 genes upregulated in AES-1M included pathogenesis-related (PAGI-5) genes present in strains PACS2 and PA7, and numerous phage genes. Elucidation of these genes' roles could lead to targeted treatment strategies for chronically infected CF patients.

Pseudomonas aeruginosa AES-1 Exhibits Increased Virulence Gene Expression during Chronic Infection of Cystic Fibrosis Lung

PubMed Central

Naughton, Sharna; Parker, Dane; Seemann, Torsten; Thomas, Torsten; Turnbull, Lynne; Rose, Barbara; Bye, Peter; Cordwell, Stuart; Whitchurch, Cynthia; Manos, Jim

2011-01-01

Pseudomonas aeruginosa, the leading cause of morbidity and mortality in people with cystic fibrosis (CF), adapts for survival in the CF lung through both mutation and gene expression changes. Frequent clonal strains such as the Australian Epidemic Strain-1 (AES-1), have increased ability to establish infection in the CF lung and to superimpose and replace infrequent clonal strains. Little is known about the factors underpinning these properties. Analysis has been hampered by lack of expression array templates containing CF-strain specific genes. We sequenced the genome of an acute infection AES-1 isolate from a CF infant (AES-1R) and constructed a non-redundant micro-array (PANarray) comprising AES-1R and seven other sequenced P. aeruginosa genomes. The unclosed AES-1R genome comprised 6.254Mbp and contained 6957 putative genes, including 338 not found in the other seven genomes. The PANarray contained 12,543 gene probe spots; comprising 12,147 P. aeruginosa gene probes, 326 quality-control probes and 70 probes for non-P. aeruginosa genes, including phage and plant genes. We grew AES-1R and its isogenic pair AES-1M, taken from the same patient 10.5 years later and not eradicated in the intervening period, in our validated artificial sputum medium (ASMDM) and used the PANarray to compare gene expression of both in duplicate. 675 genes were differentially expressed between the isogenic pairs, including upregulation of alginate, biofilm, persistence genes and virulence-related genes such as dihydroorotase, uridylate kinase and cardiolipin synthase, in AES-1M. Non-PAO1 genes upregulated in AES-1M included pathogenesis-related (PAGI-5) genes present in strains PACS2 and PA7, and numerous phage genes. Elucidation of these genes' roles could lead to targeted treatment strategies for chronically infected CF patients. PMID:21935417

Pre-amplification in the context of high-throughput qPCR gene expression experiment.

PubMed

Korenková, Vlasta; Scott, Justin; Novosadová, Vendula; Jindřichová, Marie; Langerová, Lucie; Švec, David; Šídová, Monika; Sjöback, Robert

2015-03-11

With the introduction of the first high-throughput qPCR instrument on the market it became possible to perform thousands of reactions in a single run compared to the previous hundreds. In the high-throughput reaction, only limited volumes of highly concentrated cDNA or DNA samples can be added. This necessity can be solved by pre-amplification, which became a part of the high-throughput experimental workflow. Here, we focused our attention on the limits of the specific target pre-amplification reaction and propose the optimal, general setup for gene expression experiment using BioMark instrument (Fluidigm). For evaluating different pre-amplification factors following conditions were combined: four human blood samples from healthy donors and five transcripts having high to low expression levels; each cDNA sample was pre-amplified at four cycles (15, 18, 21, and 24) and five concentrations (equivalent to 0.078 ng, 0.32 ng, 1.25 ng, 5 ng, and 20 ng of total RNA). Factors identified as critical for a success of cDNA pre-amplification were cycle of pre-amplification, total RNA concentration, and type of gene. The selected pre-amplification reactions were further tested for optimal Cq distribution in a BioMark Array. The following concentrations combined with pre-amplification cycles were optimal for good quality samples: 20 ng of total RNA with 15 cycles of pre-amplification, 20x and 40x diluted; and 5 ng and 20 ng of total RNA with 18 cycles of pre-amplification, both 20x and 40x diluted. We set up upper limits for the bulk gene expression experiment using gene expression Dynamic Array and provided an easy-to-obtain tool for measuring of pre-amplification success. We also showed that variability of the pre-amplification, introduced into the experimental workflow of reverse transcription-qPCR, is lower than variability caused by the reverse transcription step.

Expression of the membrane mucins MUC4 and MUC15, potential markers of malignancy and prognosis, in papillary thyroid carcinoma.

PubMed

Nam, Kee-Hyun; Noh, Tae-Woong; Chung, So-Hyang; Lee, So Hee; Lee, Mi Kyung; Hong, Soon Won; Chung, Woong Youn; Lee, Eun Jig; Park, Cheong Soo

2011-07-01

Papillary thyroid carcinoma (PTC) is the most frequent carcinoma of the thyroid gland and has a relatively good prognosis. However, it is important to identify PTC characteristics that indicate high risk for recurrence and metastasis. To date, overexpression of the membrane mucin, MUC1, has been investigated as a key molecular event in the pathogenesis of aggressive PTC. However, other membrane-associated mucins, matrix metalloproteinase-13 (MMP-13) and tissue inhibitor of metalloproteinase-13 (TIMP-3), have not been studied yet. The aim of this study was to evaluate the expression levels of MUC4, MUC15, MMP-13, and TIMP-3 and their prognostic significance in PTC. We analyzed MUC4, MUC15, MMP-13, and TIMP-3 expression in 10 PTC and 10 normal thyroid tissue samples using real-time reverse transcription-polymerase chain reaction. Tissue array blocks were obtained from 98 PTC cases. Tumor regions and nontumor regions were analyzed in tissue array blocks and immunohistochemistry studies were conducted using sectioned slides. Semiquantitative scores were correlated with clinicopathological factors of 98 PTC patients. MUC4- and MUC15-specific mRNA was increased by 78-fold and 4.75-fold, respectively, in PTC samples compared with normal thyroid tissues. MMP-13 and TIMP-3 gene expression levels were decreased by approximately 0.39-fold and 0.53-fold, respectively. By immunohistochemistry, MUC4 and MUC15 expression levels were increased in PTC samples compared with normal thyroid tissues (p < 0.001). MMP-13 and TIMP-3 expression levels were decreased in PTC samples compared with normal thyroid tissues (p < 0.001). High MUC4 scores were significantly correlated with small tumor size and papillary thyroid microcarcinoma subtype. High MUC15 scores were significantly correlated with age (≥45 years), distant metastasis, and multifocality. MUC4 and MUC15 were overexpressed in PTC, and high MUC15 expression was associated with high malignant potential. MUC15 may serve as a prognostic marker and potential novel therapeutic target in PTC.

Whole-exome sequencing for RH genotyping and alloimmunization risk in children with sickle cell anemia

PubMed Central

Flanagan, Jonathan M.; Vege, Sunitha; Luban, Naomi L. C.; Brown, R. Clark; Ware, Russell E.; Westhoff, Connie M.

2017-01-01

RH genes are highly polymorphic and encode the most complex of the 35 human blood group systems. This genetic diversity contributes to Rh alloimmunization in patients with sickle cell anemia (SCA) and is not avoided by serologic Rh-matched red cell transfusions. Standard serologic testing does not distinguish variant Rh antigens. Single nucleotide polymorphism (SNP)–based DNA arrays detect many RHD and RHCE variants, but the number of alleles tested is limited. We explored a next-generation sequencing (NGS) approach using whole-exome sequencing (WES) in 27 Rh alloimmunized and 27 matched non-alloimmunized patients with SCA who received chronic red cell transfusions and were enrolled in a multicenter study. We demonstrate that WES provides a comprehensive RH genotype, identifies SNPs not interrogated by DNA array, and accurately determines RHD zygosity. Among this multicenter cohort, we demonstrate an association between an altered RH genotype and Rh alloimmunization: 52% of Rh immunized vs 19% of non-immunized patients expressed variant Rh without co-expression of the conventional protein. Our findings suggest that RH allele variation in patients with SCA is clinically relevant, and NGS technology can offer a comprehensive alternative to targeted SNP-based testing. This is particularly relevant as NGS data becomes more widely available and could provide the means for reducing Rh alloimmunization in children with SCA. PMID:29296782

Progesterone Receptors: Form and Function in Brain

PubMed Central

Brinton, Roberta Diaz; Thompson, Richard F.; Foy, Michael R.; Baudry, Michel; Wang, JunMing; Finch, Caleb E; Morgan, Todd E.; Stanczyk, Frank Z.; Pike, Christian J.; Nilsen, Jon

2008-01-01

Emerging data indicate that progesterone has multiple non-reproductive functions in the central nervous system to regulate cognition, mood, inflammation, mitochondrial function, neurogenesis and regeneration, myelination and recovery from traumatic brain injury. Progesterone-regulated neural responses are mediated by an array of progesterone receptors (PR) that include the classic nuclear PRA and PRB receptors and splice variants of each, the seven transmembrane domain 7TMPRβ and the membrane-associated 25-Dx PR (PGRMC1). These PRs induce classic regulation of gene expression while also transducing signaling cascades that originate at the cell membrane and ultimately activate transcription factors. Remarkably, PRs are broadly expressed throughout the brain and can be detected in every neural cell type. The distribution of PRs beyond hypothalamic borders, suggests a much broader role of progesterone in regulating neural function. Despite the large body of evidence regarding progesterone regulation of reproductive behaviors and estrogen-inducible responses as well as effects of progesterone metabolite neurosteroids, much remains to be discovered regarding the functional outcomes resulting from activation of the complex array of PRs in brain by gonadally and / or glial derived progesterone. Moreover, the impact of clinically used progestogens and developing selective PR modulators for targeted outcomes in brain is a critical avenue of investigation as the non-reproductive functions of PRs have far-reaching implications for hormone therapy to maintain neurological health and function throughout menopausal aging. PMID:18374402

Implementation of a Multiplex and Quantitative Proteomics Platform for Assessing Protein Lysates Using DNA-Barcoded Antibodies.

PubMed

Lee, Jinho; Geiss, Gary K; Demirkan, Gokhan; Vellano, Christopher P; Filanoski, Brian; Lu, Yiling; Ju, Zhenlin; Yu, Shuangxing; Guo, Huifang; Bogatzki, Lisa Y; Carter, Warren; Meredith, Rhonda K; Krishnamurthy, Savitri; Ding, Zhiyong; Beechem, Joseph M; Mills, Gordon B

2018-06-01

Molecular analysis of tumors forms the basis for personalized cancer medicine and increasingly guides patient selection for targeted therapy. Future opportunities for personalized medicine are highlighted by the measurement of protein expression levels via immunohistochemistry, protein arrays, and other approaches; however, sample type, sample quantity, batch effects, and "time to result" are limiting factors for clinical application. Here, we present a development pipeline for a novel multiplexed DNA-labeled antibody platform which digitally quantifies protein expression from lysate samples. We implemented a rigorous validation process for each antibody and show that the platform is amenable to multiple protocols covering nitrocellulose and plate-based methods. Results are highly reproducible across technical and biological replicates, and there are no observed "batch effects" which are common for most multiplex molecular assays. Tests from basal and perturbed cancer cell lines indicate that this platform is comparable to orthogonal proteomic assays such as Reverse-Phase Protein Array, and applicable to measuring the pharmacodynamic effects of clinically-relevant cancer therapeutics. Furthermore, we demonstrate the potential clinical utility of the platform with protein profiling from breast cancer patient samples to identify molecular subtypes. Together, these findings highlight the potential of this platform for enhancing our understanding of cancer biology in a clinical translation setting. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Use of geometric properties of landmark arrays for reorientation relative to remote cities and local objects.

PubMed

Mou, Weimin; Nankoo, Jean-François; Zhou, Ruojing; Spetch, Marcia L

2014-03-01

Five experiments investigated how human adults use landmark arrays in the immediate environment to reorient relative to the local environment and relative to remote cities. Participants learned targets' directions with the presence of a proximal 4 poles forming a rectangular shape and an array of more distal poles forming a rectangular shape. Then participants were disoriented and pointed to targets with the presence of the proximal poles or the distal poles. Participants' orientation was estimated by the mean of their pointing error across targets. The targets could be 7 objects in the immediate local environment in which the poles were located or 7 cities around Edmonton (Alberta, Canada) where the experiments occurred. The directions of the 7 cities could be learned from reading a map first and then from pointing to the cities when the poles were presented. The directions of the 7 cities could also be learned from viewing labels of cities moving back and forth in the specific direction in the immediate local environment in which the poles were located. The shape of the array of the distal poles varied in salience by changing the number of poles on each edge of the rectangle (2 vs. 34). The results showed that participants regained their orientation relative to local objects using the distal poles with 2 poles on each edge; participants could not reorient relative to cities using the distal pole array with 2 poles on each edge but could reorient relative to cities using the distal pole array with 34 poles on each edge. These results indicate that use of cues in reorientation depends not only on the cue salience but also on which environment people need to reorient to.

Digital Detection of Multiple Minority Mutants and Expression Levels of Multiple Colorectal Cancer-Related Genes Using Digital-PCR Coupled with Bead-Array

PubMed Central

Huang, Huan; Li, Shuo; Sun, Lizhou; Zhou, Guohua

2015-01-01

To simultaneously analyze mutations and expression levels of multiple genes on one detection platform, we proposed a method termed “multiplex ligation-dependent probe amplification–digital amplification coupled with hydrogel bead-array” (MLPA–DABA) and applied it to diagnose colorectal cancer (CRC). CRC cells and tissues were sampled to extract nucleic acid, perform MLPA with sequence-tagged probes, perform digital emulsion polymerase chain reaction (PCR), and produce a hydrogel bead-array to immobilize beads and form a single bead layer on the array. After hybridization with fluorescent probes, the number of colored beads, which reflects the abundance of expressed genes and the mutation rate, was counted for diagnosis. Only red or green beads occurred on the chips in the mixed samples, indicating the success of single-molecule PCR. When a one-source sample was analyzed using mixed MLPA probes, beads of only one color occurred, suggesting the high specificity of the method in analyzing CRC mutation and gene expression. In gene expression analysis of a CRC tissue from one CRC patient, the mutant percentage was 3.1%, and the expression levels of CRC-related genes were much higher than those of normal tissue. The highly sensitive MLPA–DABA succeeds in the relative quantification of mutations and gene expressions of exfoliated cells in stool samples of CRC patients on the same chip platform. MLPA–DABA coupled with hydrogel bead-array is a promising method in the non-invasive diagnosis of CRC. PMID:25880764

Microarray expression profiling identifies genes with altered expression in HDL-deficient mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Callow, Matthew J.; Dudoit, Sandrine; Gong, Elaine L.

2000-05-05

Based on the assumption that severe alterations in the expression of genes known to be involved in HDL metabolism may affect the expression of other genes we screened an array of over 5000 mouse expressed sequence tags (ESTs) for altered gene expression in the livers of two lines of mice with dramatic decreases in HDL plasma concentrations. Labeled cDNA from livers of apolipoprotein AI (apo AI) knockout mice, Scavenger Receptor BI (SR-BI) transgenic mice and control mice were co-hybridized to microarrays. Two-sample t-statistics were used to identify genes with altered expression levels in the knockout or transgenic mice compared withmore » the control mice. In the SR-BI group we found 9 array elements representing at least 5 genes to be significantly altered on the basis of an adjusted p value of less than 0.05. In the apo AI knockout group 8 array elements representing 4 genes were altered compared with the control group (p < 0.05). Several of the genes identified in the SR-BI transgenic suggest altered sterol metabolism and oxidative processes. These studies illustrate the use of multiple-testing methods for the identification of genes with altered expression in replicated microarray experiments of apo AI knockout and SR-BI transgenic mice.« less

Electric crosstalk impairs spatial resolution of multi-electrode arrays in retinal implants

NASA Astrophysics Data System (ADS)

Wilke, R. G. H.; Khalili Moghadam, G.; Lovell, N. H.; Suaning, G. J.; Dokos, S.

2011-08-01

Active multi-electrode arrays are used in vision prostheses, including optic nerve cuffs and cortical and retinal implants for stimulation of neural tissue. For retinal implants, arrays with up to 1500 electrodes are used in clinical trials. The ability to convey information with high spatial resolution is critical for these applications. To assess the extent to which spatial resolution is impaired by electric crosstalk, finite-element simulation of electric field distribution in a simplified passive tissue model of the retina is performed. The effects of electrode size, electrode spacing, distance to target cells, and electrode return configuration (monopolar, tripolar, hexagonal) on spatial resolution is investigated in the form of a mathematical model of electric field distribution. Results show that spatial resolution is impaired with increased distance from the electrode array to the target cells. This effect can be partly compensated by non-monopolar electrode configurations and larger electrode diameters, albeit at the expense of lower pixel densities due to larger covering areas by each stimulation electrode. In applications where multi-electrode arrays can be brought into close proximity to target cells, as presumably with epiretinal implants, smaller electrodes in monopolar configuration can provide the highest spatial resolution. However, if the implantation site is further from the target cells, as is the case in suprachoroidal approaches, hexagonally guarded electrode return configurations can convey higher spatial resolution. This paper was originally submitted for the special issue containing contributions from the Sixth Biennial Research Congress of The Eye and the Chip.

IL-12 Can Target Human Lung Adenocarcinoma Cells and Normal Bronchial Epithelial Cells Surrounding Tumor Lesions

PubMed Central

Airoldi, Irma; Di Carlo, Emma; Cocco, Claudia; Caci, Emanuela; Cilli, Michele; Sorrentino, Carlo; Sozzi, Gabriella; Ferrini, Silvano; Rosini, Sandra; Bertolini, Giulia; Truini, Mauro; Grossi, Francesco; Galietta, Luis Juan Vicente; Ribatti, Domenico; Pistoia, Vito

2009-01-01

Background Non small cell lung cancer (NSCLC) is a leading cause of cancer death. We have shown previously that IL-12rb2 KO mice develop spontaneously lung adenocarcinomas or bronchioalveolar carcinomas. Aim of the study was to investigate i) IL-12Rβ2 expression in human primary lung adenocarcinomas and in their counterparts, i.e. normal bronchial epithelial cells (NBEC), ii) the direct anti-tumor activity of IL-12 on lung adenocarcinoma cells in vitro and vivo, and the mechanisms involved, and iii) IL-12 activity on NBEC. Methodology/Principal Findings Stage I lung adenocarcinomas showed significantly (P = 0.012) higher frequency of IL-12Rβ2 expressing samples than stage II/III tumors. IL-12 treatment of IL-12R+ neoplastic cells isolated from primary adenocarcinoma (n = 6) inhibited angiogenesis in vitro through down-regulation of different pro-angiogenic genes (e.g. IL-6, VEGF-C, VEGF-D, and laminin-5), as assessed by chorioallantoic membrane (CAM) assay and PCR array. In order to perform in vivo studies, the Calu6 NSCLC cell line was transfected with the IL-12RB2 containing plasmid (Calu6/β2). Similar to that observed in primary tumors, IL-12 treatment of Calu6/β2+ cells inhibited angiogenesis in vitro. Tumors formed by Calu6/β2 cells in SCID/NOD mice, inoculated subcutaneously or orthotopically, were significantly smaller following IL-12 vs PBS treatment due to inhibition of angiogenesis, and of IL-6 and VEGF-C production. Explanted tumors were studied by histology, immuno-histochemistry and PCR array. NBEC cells were isolated and cultured from lung specimens of non neoplastic origin. NBEC expressed IL-12R and released constitutively tumor promoting cytokines (e.g. IL-6 and CCL2). Treatment of NBEC with IL-12 down-regulated production of these cytokines. Conclusions This study demonstrates that IL-12 inhibits directly the growth of human lung adenocarcinoma and targets the adjacent NBEC. These novel anti-tumor activities of IL-12 add to the well known immune-modulatory properties of the cytokine and may provide a rational basis for the development of a clinical trial. PMID:19582164

Profiling and bioinformatics analyses reveal differential circular RNA expression in radioresistant esophageal cancer cells.

PubMed

Su, Huafang; Lin, Fuqiang; Deng, Xia; Shen, Lanxiao; Fang, Ya; Fei, Zhenghua; Zhao, Lihao; Zhang, Xuebang; Pan, Huanle; Xie, Deyao; Jin, Xiance; Xie, Congying

2016-07-28

Acquired radioresistance during radiotherapy is considered as the most important reason for local tumor recurrence or treatment failure. Circular RNAs (circRNAs) have recently been identified as microRNA sponges and involve in various biological processes. The purpose of this study is to investigate the role of circRNAs in the radioresistance of esophageal cancer. Total RNA was isolated from human parental cell line KYSE-150 and self-established radioresistant esophageal cancer cell line KYSE-150R, and hybridized to Arraystar Human circRNA Array. Quantitative real-time PCR was used to confirm the circRNA expression profiles obtained from the microarray data. Bioinformatic tools including gene ontology (GO) analysis, KEGG pathway analysis and network analysis were done for further assessment. Among the detected candidate 3752 circRNA genes, significant upregulation of 57 circRNAs and downregulation of 17 circRNAs in human radioresistant esophageal cancer cell line KYSE-150R were observed compared with the parental cell line KYSE-150 (fold change ≥2.0 and P < 0.05). There were 9 out of these candidate circRNAs were validated by real-time PCR. GO analysis revealed that numerous target genes, including most microRNAs were involved in the biological processes. There were more than 400 target genes enrichment on Wnt signaling pathway. CircRNA_001059 and circRNA_000167 were the two largest nodes in circRNA/microRNA co-expression network. Our study revealed a comprehensive expression and functional profile of differentially expressed circRNAs in radioresistant esophageal cancer cells, indicating possible involvement of these dysregulated circRNAs in the development of radiation resistance.

Dibenzoylmethane Protects Against CCl4-Induced Acute Liver Injury by Activating Nrf2 via JNK, AMPK, and Calcium Signaling.

PubMed

Cao, Mingnan; Wang, Huixia; Guo, Limei; Yang, Simin; Liu, Chun; Khor, Tin Oo; Yu, Siwang; Kong, Ah-Ng

2017-11-01

Oxidative stress is an important pathogenic factor in various hepatic diseases. Nuclear factor-erythroid 2-related factor-2 (Nrf2), which coordinates the expression of an array of antioxidant and detoxifying genes, has been proposed as a potential target for prevention and treatment of liver disease. Dibenzoylmethane (DBM) is a minor ingredient in licorice that activates Nrf2 and prevents various cancers and oxidative damage. In the present study, the mechanisms by which DBM activates Nrf2 signaling were delineated, and its protective effect against carbon tetrachloride (CCl 4 )-induced liver injury was examined. DBM potently induced the expression of HO-1 in cells and in the livers of mice, but this induction was diminished in Nrf2-deficient mice and cells. Overexpression of Nrf2 enhanced DBM-induced HO-1 expression, while overexpression of a dominant-negative fragment of Nrf2 inhibited this induction. DBM treatment resulted in dissociation from Keap1 and nuclear translocation of Nrf2. Moreover, DBM activated Akt/protein kinase B, mitogen-activated protein kinases, and AMP-activated protein kinase and increased intracellular calcium levels. Inhibition of JNK, AMPK, or intracellular calcium signaling significantly suppressed the induction of HO-1 expression by DBM. Finally, DBM treatment significantly inhibited CCl 4 -induced acute liver injury in wild-type but not in Nrf2-deficient mice. Taken together, our results revealed the mechanisms by which DBM activates Nrf2 and induces HO-1 expression, and provide molecular basis for the design and development of DBM and its derivatives for prevention or treatment of liver diseases by targeting Nrf2.

A novel interaction of PAK4 with PPARγ to regulate Nox1 and radiation-induced epithelial-to-mesenchymal transition in glioma.

PubMed

Kesanakurti, D; Maddirela, D; Banasavadi-Siddegowda, Y K; Lai, T-H; Qamri, Z; Jacob, N K; Sampath, D; Mohanam, S; Kaur, B; Puduvalli, V K

2017-09-14

Tumor recurrence in glioblastoma (GBM) is, in part, attributed to increased epithelial-to-mesenchymal transition (EMT) and enhanced tumor cell dissemination in adjacent brain parenchyma after ionizing radiation (IR). EMT is associated with aggressive behavior, increased stem-like characteristics and treatment resistance in malignancies; however, the underlying signaling mechanisms that regulate EMT are poorly understood. We identified grade-dependent p21-activated kinases 4 (PAK4) upregulation in gliomas and further determined its role in mesenchymal transition and radioresistance. IR treatment significantly elevated expression and nuclear localization of PAK4 in correlation with induction of reactive oxygen species (ROS) and mesenchymal transition in GBM cells. Stable PAK4 overexpression promoted mesenchymal transition by elevating EMT marker expression in these cells. Of note, transcription factor-DNA-binding arrays and chromatin immunoprecipitation experiments identified the formation of a novel nuclear PAK4/PPARγ complex which was recruited to the promoter of Nox1, a peroxisome proliferator-activated receptor gamma (PPARγ) target gene. In addition, IR further elevated PAK4/PPARγ complex co-recruitment to Nox1 promoter, and increased Nox1 expression and ROS levels associated with mesenchymal transition in these cells. Conversely, specific PAK4 downregulation decreased PPARγ-mediated Nox1 expression and suppressed EMT in IR-treated cells. In vivo orthotopic tumor experiments showed inhibition of growth and suppression of IR-induced PPARγ and Nox1 expression by PAK4 downregulation in tumors. Our results provide the first evidence of a novel role for PAK4 in IR-induced EMT and suggest potential therapeutic efficacy of targeting PAK4 to overcome radioresistance in gliomas.

miRNA expression in control and FSHD fetal human muscle biopsies.

PubMed

Portilho, Débora Morueco; Alves, Marcelo Ribeiro; Kratassiouk, Gueorgui; Roche, Stéphane; Magdinier, Frédérique; de Santana, Eliane Corrêa; Polesskaya, Anna; Harel-Bellan, Annick; Mouly, Vincent; Savino, Wilson; Butler-Browne, Gillian; Dumonceaux, Julie

2015-01-01

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disorder and is one of the most common forms of muscular dystrophy. We have recently shown that some hallmarks of FSHD are already expressed in fetal FSHD biopsies, thus opening a new field of investigation for mechanisms leading to FSHD. As microRNAs (miRNAs) play an important role in myogenesis and muscle disorders, in this study we compared miRNAs expression levels during normal and FSHD muscle development. Muscle biopsies were obtained from quadriceps of both healthy control and FSHD1 fetuses with ages ranging from 14 to 33 weeks of development. miRNA expression profiles were analyzed using TaqMan Human MicroRNA Arrays. During human skeletal muscle development, in control muscle biopsies we observed changes for 4 miRNAs potentially involved in secondary muscle fiber formation and 5 miRNAs potentially involved in fiber maturation. When we compared the miRNA profiles obtained from control and FSHD biopsies, we did not observe any differences in the muscle specific miRNAs. However, we identified 8 miRNAs exclusively expressed in FSHD1 samples (miR-330, miR-331-5p, miR-34a, miR-380-3p, miR-516b, miR-582-5p, miR-517* and miR-625) which could represent new biomarkers for this disease. Their putative targets are mainly involved in muscle development and morphogenesis. Interestingly, these FSHD1 specific miRNAs do not target the genes previously described to be involved in FSHD. This work provides new candidate mechanisms potentially involved in the onset of FSHD pathology. Whether these FSHD specific miRNAs cause deregulations during fetal development, or protect against the appearance of the FSHD phenotype until the second decade of life still needs to be investigated.

AMP kinase-related kinase NUAK2 affects tumor growth, migration, and clinical outcome of human melanoma.

PubMed

Namiki, Takeshi; Tanemura, Atsushi; Valencia, Julio C; Coelho, Sergio G; Passeron, Thierry; Kawaguchi, Masakazu; Vieira, Wilfred D; Ishikawa, Masashi; Nishijima, Wataru; Izumo, Toshiyuki; Kaneko, Yasuhiko; Katayama, Ichiro; Yamaguchi, Yuji; Yin, Lanlan; Polley, Eric C; Liu, Hongfang; Kawakami, Yutaka; Eishi, Yoshinobu; Takahashi, Eishi; Yokozeki, Hiroo; Hearing, Vincent J

2011-04-19

The identification of genes that participate in melanomagenesis should suggest strategies for developing therapeutic modalities. We used a public array comparative genomic hybridization (CGH) database and real-time quantitative PCR (qPCR) analyses to identify the AMP kinase (AMPK)-related kinase NUAK2 as a candidate gene for melanomagenesis, and we analyzed its functions in melanoma cells. Our analyses had identified a locus at 1q32 where genomic gain is strongly associated with tumor thickness, and we used real-time qPCR analyses and regression analyses to identify NUAK2 as a candidate gene at that locus. Associations of relapse-free survival and overall survival of 92 primary melanoma patients with NUAK2 expression measured using immunohistochemistry were investigated using Kaplan-Meier curves, log rank tests, and Cox regression models. Knockdown of NUAK2 induces senescence and reduces S-phase, decreases migration, and down-regulates expression of mammalian target of rapamycin (mTOR). In vivo analysis demonstrated that knockdown of NUAK2 suppresses melanoma tumor growth in mice. Survival analysis showed that the risk of relapse is greater in acral melanoma patients with high levels of NUAK2 expression than in acral melanoma patients with low levels of NUAK2 expression (hazard ratio = 3.88; 95% confidence interval = 1.44-10.50; P = 0.0075). These data demonstrate that NUAK2 expression is significantly associated with the oncogenic features of melanoma cells and with the survival of acral melanoma patients. NUAK2 may provide a drug target to suppress melanoma progression. This study further supports the importance of NUAK2 in cancer development and tumor progression, while AMPK has antioncogenic properties.

Titanium and Zirconium Levels Are Associated with Changes in MicroRNAs Expression: Results from a Human Cross-Sectional Study on Obese Population

PubMed Central

Dioni, Laura; Angelici, Laura; Vigna, Luisella; Farronato, Giampietro; Pesatori, Angela Cecilia; Bollati, Valentina

2016-01-01

Objectives In this study on 90 individuals we aimed at evaluating the microRNAs (miRNAs) expression profile associated with personal levels of Titanium (Ti) and Zirconium (Zr) traced in hair samples. Ti and Zr materials are broadly used for dental implants but the biological reactions triggered by a long term presence of these materials in the oral cavity still need to be assessed. MiRNAs are mechanisms that need to be investigated as they play a fundamental role in the control of gene expression following external stimuli and contribute to a wide range of pathophysiological processes. Methods Using the TaqMan® Low-Density Array, we assessed the expression levels of 377 human miRNAs in peripheral blood of 90 subjects. Hair samples were analyzed for Ti and Zr content using Inductively Coupled Plasma-Mass Spectrometry. We performed multivariable regression analysis to investigate the effects of Ti and Zr exposure on miRNA expression levels. We used the Ingenuity Pathway Analysis (IPA) software to explore the functional role of the investigated miRNAs and the related target genes. Results Seven miRNAs (miR-99b, miR-142-5p, miR-152, miR-193a-5p, miR-323-3p, miR-335, miR-494) resulted specifically associated with Zr levels. The functional target analysis showed that miRNAs are involved in mechanisms such as inflammation, skeletal and connective tissue disorders. Conclusions Our data suggest that Zr is more bioactive than Ti and show that miRNAs are relevant molecular mechanisms sensitive to Zr exposure. PMID:27611787

Nanoscale Surface Plasmonics Sensor With Nanofluidic Control

NASA Technical Reports Server (NTRS)

Wei, Jianjun; Singhal, Sameer; Waldeck, David H.; Kofke, Matthew

2013-01-01

Conventional quantitative protein assays of bodily fluids typically involve multiple steps to obtain desired measurements. Such methods are not well suited for fast and accurate assay measurements in austere environments such as spaceflight and in the aftermath of disasters. Consequently, there is a need for a protein assay technology capable of routinely monitoring proteins in austere environments. For example, there is an immediate need for a urine protein assay to assess astronaut renal health during spaceflight. The disclosed nanoscale surface plasmonics sensor provides a core detection method that can be integrated to a lab-on-chip device that satisfies the unmet need for such a protein assay technology. Assays based upon combinations of nanoholes, nanorings, and nanoslits with transmission surface plasmon resonance (SPR) are used for assays requiring extreme sensitivity, and are capable of detecting specific analytes at concentrations as low as picomole to femtomole level in well-controlled environments. The device operates in a transmission mode configuration in which light is directed at one planar surface of the array, which functions as an optical aperture. The incident light induces surface plasmon light transmission from the opposite surface of the array. The presence of a target analyte is detected by changes in the spectrum of light transmitted by the array when a target analyte induces a change in the refractive index of the fluid within the nanochannels. This occurs, for example, when a target analyte binds to a receptor fixed to the walls of the nanochannels in the array. Independent fluid handling capability for individual nanoarrays on a nanofluidic chip containing a plurality of nanochannel arrays allows each array to be used to sense a different target analyte and/or for paired arrays to analyze control and test samples simultaneously in parallel. The present invention incorporates transmission mode nanoplasmonics and nanofluidics into a single, microfluidically controlled device. The device comprises one or more arrays of aligned nanochannels that are in fluid communication with inflowing and outflowing fluid handling manifolds that control the flow of fluid through the arrays. The array acts as an aperture in a plasmonic sensor. Fluid, in the form of a liquid or a gas and comprising a sample for analysis, is moved from an inlet manifold through the nanochannel array, and out through an exit manifold. The fluid may also contain a reagent used to modify the interior surfaces of the nanochannels, and/or a reagent required for the detection of an analyte.

Functionalization of Carbon Nanotubes

NASA Technical Reports Server (NTRS)

Khare, Bishun N. (Inventor); Meyyappan, Meyya (Inventor)

2009-01-01

Method and system for functionalizing a collection of carbon nanotubes (CNTs). A selected precursor gas (e.g., H2 or F2 or CnHm) is irradiated to provide a cold plasma of selected target species particles, such as atomic H or F, in a first chamber. The target species particles are d irected toward an array of CNTs located in a second chamber while suppressing transport of ultraviolet radiation to the second chamber. A CNT array is functionalized with the target species particles, at or below room temperature, to a point of saturation, in an exposure time interval no longer than about 30 sec. *Discrimination against non-target species is provided by (i) use of a target species having a lifetime that is much greater than a lifetime of a non-target species and/or (2) use of an applied magnetic field to discriminate between charged particle trajectories for target species and for non-target species.

Evaluation of Genomic Instability in the Abnormal Prostate

DTIC Science & Technology

2006-12-01

array CGH maps copy number aberrations relative to the genome sequence by using arrays of BAC or cDNA clones as the hybridization target instead of...data produced from these analyses complicate the interpretation of results . For these reasons, and as outlined by Davies et al., 22 it is desirable...There have been numerous studies of these abnormalities and several techniques, including 9 chromosome painting, array CGH and SNP arrays , have

Enhanced Antitumorigenic Effects in Glioblastoma on Double Targeting of Pleiotrophin and Its Receptor ALK1

PubMed Central

Grzelinski, Marius; Steinberg, Florian; Martens, Tobias; Czubayko, Frank; Lamszus, Katrin; Aigner, Achim

2009-01-01

In adults, glioblastomas are the most lethal and most frequent malignant brain tumors, and the poor prognosis despite aggressive treatment indicates the need to establish novel targets for molecular intervention. The secreted growth factor pleiotrophin (PTN, HB-GAM, HBNF, OSF-1) shows mitogenic, chemotactic, and transforming activity. Whereas PTN expression is tightly regulated during embryogenesis and is very limited in normal adult tissues, a marked PTN up-regulation is seen in tumors including glioblastomas. Likewise, the PTN receptor anaplastic lymphoma kinase (ALK) has been shown previously to be upregulated and functionally relevant in glioblastoma. In this study, we explore the antitumorigenic effects of the simultaneous ribozyme-mediated knockdown of both receptor and ligand. Various glioblastoma cell lines are analyzed for PTN and ALK expression. Beyond the individual efficacies of several specific ribozymes against PTN or ALK, respectively, antiproliferative and proapoptotic effects of a single gene targeting approach are strongly enhanced on double knockdown of both genes in vitro. More importantly, this results in the abolishment of tumor growth in an in vivo subcutaneous tumor xenograft model. Finally, the analysis of various downstream signaling pathways by antibody arrays reveals a distinct pattern of changes in the activation of signal transduction molecules on PTN/ALK double knockdown. Beyond the already known ones, it identifies additional pathways relevant for PTN/ALK signaling. We conclude that double targeting of PTN and ALK leads to enhanced antitumorigenic effects over single knockdown approaches, which offers novel therapeutic options owing to increased efficacy also after prolonged knockdown. PMID:19177199

Method for the electro-addressable functionalization of electrode arrays

DOEpatents

Harper, Jason C.; Polsky, Ronen; Dirk, Shawn M.; Wheeler, David R.; Arango, Dulce C.; Brozik, Susan M.

2015-12-15

A method for preparing an electrochemical biosensor uses bias-assisted assembly of unreactive -onium molecules on an electrode array followed by post-assembly electro-addressable conversion of the unreactive group to a chemical or biological recognition group. Electro-addressable functionalization of electrode arrays enables the multi-target electrochemical sensing of biological and chemical analytes.

Transcription-dependent radial distribution of TCF7L2 regulated genes in chromosome territories.

PubMed

Torabi, Keyvan; Wangsa, Darawalee; Ponsa, Immaculada; Brown, Markus; Bosch, Anna; Vila-Casadesús, Maria; Karpova, Tatiana S; Calvo, Maria; Castells, Antoni; Miró, Rosa; Ried, Thomas; Camps, Jordi

2017-10-01

Human chromosomes occupy distinct territories in the interphase nucleus. Such chromosome territories (CTs) are positioned according to gene density. Gene-rich CTs are generally located in the center of the nucleus, while gene-poor CTs are positioned more towards the nuclear periphery. However, the association between gene expression levels and the radial positioning of genes within the CT is still under debate. In the present study, we performed three-dimensional fluorescence in situ hybridization experiments in the colorectal cancer cell lines DLD-1 and LoVo using whole chromosome painting probes for chromosomes 8 and 11 and BAC clones targeting four genes with different expression levels assessed by gene expression arrays and RT-PCR. Our results confirmed that the two over-expressed genes, MYC on chromosome 8 and CCND1 on chromosome 11, are located significantly further away from the center of the CT compared to under-expressed genes on the same chromosomes, i.e., DLC1 and SCN3B. When CCND1 expression was reduced after silencing the major transcription factor of the WNT/β-catenin signaling pathway, TCF7L2, the gene was repositioned and mostly detected in the interior of the CT. Thus, we suggest a non-random distribution in which over-expressed genes are located more towards the periphery of the respective CTs.

In situ analysis of integrin and growth factor receptor signaling pathways in human glioblastomas suggests overlapping relationships with focal adhesion kinase activation.

PubMed

Riemenschneider, Markus J; Mueller, Wolf; Betensky, Rebecca A; Mohapatra, Gayatry; Louis, David N

2005-11-01

Deregulated integrin signaling is common in cancers, including glioblastoma. Integrin binding and growth factor receptor signaling activate focal adhesion kinase (FAK) and subsequently up-regulate extracellular regulated kinases (ERK-1/2), leading to cell-cycle progression and cell migration. Most studies of this pathway have used in vitro systems or tumor lysate-based approaches. We examined these pathways primarily in situ using a panel of 30 glioblastomas and gene expression arrays, immunohistochemistry, and fluorescence in situ hybridization, emphasizing the histological distribution of molecular changes. Within individual tumors, increased expression of FAK, p-FAK, paxillin, ERK-1/2, and p-ERK-1/2 occurred in regions of elevated EGFR and/or PDGFRA expression. Moreover, FAK activation levels correlated with EGFR and PDGFRA expression, and p-FAK and EGFR expression co-localized at the single-cell level. In addition, integrin expression was enriched in EGFR/PDGFRA-overexpressing areas but was more regionally confined than FAK, p-FAK, and paxillin. Integrins beta8 and alpha5beta1 were most commonly expressed, often in a perinecrotic or perivascular pattern. Taken together, our data suggest that growth factor receptor overexpression facilitates alterations in the integrin signaling pathway. Thus, FAK may act in glioblastoma as a downstream target of growth factor signaling, with integrins enhancing the impact of such signaling in the tumor microenvironment.

Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

PubMed Central

Dorfman, Mauricio D.; Kerr, Bredford; Garcia-Rudaz, Cecilia; Paredes, Alfonso H.; Dissen, Gregory A.

2011-01-01

Tropomyosin-related kinase (TRK) receptor B (TRKB) mediates the supportive actions of neurotrophin 4/5 and brain-derived neurotrophic factor on early ovarian follicle development. Absence of TRKB receptors reduces granulosa cell (GC) proliferation and delays follicle growth. In the present study, we offer mechanistic insights into this phenomenon. DNA array and quantitative PCR analysis of ovaries from TrkB-null mice revealed that by the end of the first week of postnatal life, Jagged1, Hes1, and Hey2 mRNA abundance is reduced in the absence of TRKB receptors. Although Jagged1 encodes a NOTCH receptor ligand, Hes1 and Hey2 are downstream targets of the JAGGED1-NOTCH2 signaling system. Jagged1 is predominantly expressed in oocytes, and the abundance of JAGGED1 is decreased in TrkB−/− oocytes. Lack of TRKB receptors also resulted in reduced expression of c-Myc, a NOTCH target gene that promotes entry into the cell cycle, but did not alter the expression of genes encoding core regulators of cell-cycle progression. Selective restoration of JAGGED1 synthesis in oocytes of TrkB−/− ovaries via lentiviral-mediated transfer of the Jagged1 gene under the control of the growth differentiation factor 9 (Gdf9) promoter rescued c-Myc expression, GC proliferation, and follicle growth. These results suggest that neurotrophins acting via TRKB receptors facilitate early follicle growth by supporting a JAGGED1-NOTCH2 oocyte-to-GC communication pathway, which promotes GC proliferation via a c-MYC-dependent mechanism. PMID:22028443

Overexpression of Rab11-FIP2 in colorectal cancer cells promotes tumor migration and angiogenesis through increasing secretion of PAI-1.

PubMed

Dong, Wenjie; Wu, Xinai

2018-01-01

Rab11 family-interacting protein 2 (Rab11-FIP2) can interact with MYO5B and plays an important role in regulating plasma membrane recycling. However, little is known about the clinical significance of DUSP2 in colorectal cancer (CRC). In this study, we investigated Rab11-FIP2 expression by immunohistochemistry in 125 patients with colorectal cancer. Conditioned media containing all secreted factors was harvested. Chemokine secretion and expression were analyzed by Chemi-array. We found that the expression level of Rab11-FIP2 was significantly increased in colorectal cancer tissues and high expression of Rab11-FIP2 was closely correlated with nodal metastasis in colorectal cancer patients. Rab11-FIP2 overexpression promoted colorectal cancer metastasis in vitro and in vivo. Finally, we demonstrated that Rab11-FIP2 overexpression may contribute to increased secretion of PAI-1 in human colorectal cancer cells. Our findings reveal a novel mechanism underlying the role of Rab11-FIP2 in colorectal cancer dissemination, suggesting that targeting Rab11-FIP2 might be a promising therapeutic strategy for CRC.

A second trigeminal CGRP receptor: function and expression of the AMY1 receptor

PubMed Central

Walker, Christopher S; Eftekhari, Sajedeh; Bower, Rebekah L; Wilderman, Andrea; Insel, Paul A; Edvinsson, Lars; Waldvogel, Henry J; Jamaluddin, Muhammad A; Russo, Andrew F; Hay, Debbie L

2015-01-01

Objective The trigeminovascular system plays a central role in migraine, a condition in need of new treatments. The neuropeptide, calcitonin gene-related peptide (CGRP), is proposed as causative in migraine and is the subject of intensive drug discovery efforts. This study explores the expression and functionality of two CGRP receptor candidates in the sensory trigeminal system. Methods Receptor expression was determined using Taqman G protein-coupled receptor arrays and immunohistochemistry in trigeminal ganglia (TG) and the spinal trigeminal complex of the brainstem in rat and human. Receptor pharmacology was quantified using sensitive signaling assays in primary rat TG neurons. Results mRNA and histological expression analysis in rat and human samples revealed the presence of two CGRP-responsive receptors (AMY1: calcitonin receptor/receptor activity-modifying protein 1 [RAMP1]) and the CGRP receptor (calcitonin receptor-like receptor/RAMP1). In support of this finding, quantification of agonist and antagonist potencies revealed a dual population of functional CGRP-responsive receptors in primary rat TG neurons. Interpretation The unexpected presence of a functional non-canonical CGRP receptor (AMY1) at neural sites important for craniofacial pain has important implications for targeting the CGRP axis in migraine. PMID:26125036

TIP60 represses telomerase expression by inhibiting Sp1 binding to the TERT promoter

PubMed Central

Pandey, Amit Kumar; Xiuzhen, Magdalene Claire; Lee, Kwok Kin; Hora, Shainan; Zhang, Yanzhou; Kwok, Hui Si; Deng, Lih Wen; Tenen, Daniel G.; Kappei, Dennis

2017-01-01

HIV1-TAT interactive protein (TIP60) is a haploinsufficient tumor suppressor. However, the potential mechanisms endowing its tumor suppressor ability remain incompletely understood. It plays a vital role in virus-induced cancers where TIP60 down-regulates the expression of human papillomavirus (HPV) oncoprotein E6 which in turn destabilizes TIP60. This intrigued us to identify the role of TIP60, in the context of a viral infection, where it is targeted by oncoproteins. Through an array of molecular biology techniques such as Chromatin immunoprecipitation, expression analysis and mass spectrometry, we establish the hitherto unknown role of TIP60 in repressing the expression of the catalytic subunit of the human telomerase complex, TERT, a key driver for immortalization. TIP60 acetylates Sp1 at K639, thus inhibiting Sp1 binding to the TERT promoter. We identified that TIP60-mediated growth suppression of HPV-induced cervical cancer is mediated in part due to TERT repression through Sp1 acetylation. In summary, our study has identified a novel substrate for TIP60 catalytic activity and a unique repressive mechanism acting at the TERT promoter in virus-induced malignancies. PMID:29045464

Polymer-carbon black composite sensors in an electronic nose for air-quality monitoring

NASA Technical Reports Server (NTRS)

Ryan, M. A.; Shevade, A. V.; Zhou, H.; Homer, M. L.

2004-01-01

An electronic nose that uses an array of 32 polymer-carbon black composite sensors has been developed, trained, and tested. By selecting a variety of chemical functionalities in the polymers used to make sensors, it is possible to construct an array capable of identifying and quantifying a broad range of target compounds, such as alcohols and aromatics, and distinguishing isomers and enantiomers (mirror-image isomers). A model of the interaction between target molecules and the polymer-carbon black composite sensors is under development to aid in selecting the array members and to enable identification of compounds with responses not stored in the analysis library.

ELECTRICAL TECHNIQUES FOR ENGINEERING APPLICATIONS.

USGS Publications Warehouse

Bisdorf, Robert J.

1985-01-01

Surface electrical geophysical methods have been used in such engineering applications as locating and delineating shallow gravel deposits, depth to bedrock, faults, clay zones, and other geological phenomena. Other engineering applications include determining water quality, tracing ground water contaminant plumes and locating dam seepages. Various methods and electrode arrays are employed to solve particular geological problems. The sensitivity of a particular method or electrode array depends upon the physics on which the method is based, the array geometry, the electrical contrast between the target and host materials, and the depth to the target. Each of the available electrical methods has its own particular advantages and applications which the paper discusses.

Preclinical evaluation of dasatinib alone and in combination with cabozantinib for the treatment of diffuse intrinsic pontine glioma

PubMed Central

Philippe, Cathy; Paulsson, Janna; Andreiuolo, Felipe; Guerrini-Rousseau, Léa; Cornilleau, Gaétan; Le Dret, Ludivine; Richon, Catherine; Lacroix, Ludovic; Puget, Stéphanie; Geoerger, Birgit; Vassal, Gilles; Östman, Arne; Grill, Jacques

2015-01-01

Abstract Background Platelet-derived growth factor receptor A is altered by amplification and/or mutation in diffuse intrinsic pontine glioma (DIPG). We explored in vitro on new DIPG models the efficacy of dasatinib, a multi-tyrosine kinase inhibitor targeting this receptor. Methods Gene expression profiles were generated from 41 DIPGs biopsied at diagnosis and compared with the signature associated with sensitivity/resistance to dasatinib. A panel of 12 new DIPG cell lines were established from biopsy at diagnosis, serially passaged, and characterized by gene expression analyses. Effects of dasatinib (1–10 μM) on proliferation, invasion, and cytotoxicity were determined on 4 of these cell lines using live-cell imaging and flow cytometry assays. Downstream signaling and receptor tyrosine kinases (RTKs) were assessed by western blot and phospho-RTK array. The effect of the combination with the c-Met inhibitor cabozantinib was studied on cellular growth and invasion analyzed by the Chou–Talaly method. Results DIPG primary tumors and cell lines exhibited the gene expression signature of sensitivity to dasatinib. Dasatinib reduced proliferation (half-maximal inhibitory concentration = 10–100 nM) and invasion (30%–60% reduction) at 100 nM in 4/4 cultures and induced apoptosis in 1 of 4 DIPG cell lines. Activity of downstream effectors of dasatinib targets including activin receptor 1 was strongly reduced. Since multiple RTKs were activated simultaneously in DIPG cell lines, including c-Met, which can be also amplified in DIPG, the benefit of the combination of dasatinib with cabozantinib was explored for its synergistic effects on proliferation and migration/invasion in these cell lines. Conclusion Dasatinib exhibits antitumor effects in vitro that could be increased by the combination with another RTK inhibitor targeting c-Met. PMID:25534822

Small interfering RNA-mediated silencing of G-protein-coupled receptor 137 inhibits growth of osteosarcoma cells.

PubMed

Li, Hao; Fu, Xiaodong; Gao, Yingjian; Li, Xiaomiao; Shen, Yi; Wang, Weili

2018-06-01

Osteosarcoma is the most widespread primary carcinoma in bones. Osteosarcoma cells are highly metastatic and frequently develop resistance to chemotherapy making this disease harder to treat. This identifies an urgent need of novel therapeutic strategies for osteosarcoma. G-Protein-coupled receptor 137 (GPR137) is involved in several human cancers and may be a novel therapeutic target. The expression of GPR137 was assessed in one osteoblast and three human osteosarcoma cell lines via the quantitative real-time polymerase chain reaction and western blot assays. Stable GPR137 knockdown cell lines were established using an RNA interference lentivirus system. Viability, colony formation, and flow cytometry assays were performed to measure the effects of GPR137 depletion on cell growth. The underlying molecular mechanism was determined using signaling array analysis and western blot assays. GPR137 expression was higher in the three human osteosarcoma cell lines, Saos-2, U2OS, and SW1353, than in osteoblast hFOB 1.19 cells. Lentivirus-mediated small interfering RNA targeting GPR137 successfully knocked down GPR137 mRNA and protein expression in both Saos-2 and U2OS cells. In the absence of GPR137, cell viability and colony formation ability were seriously impaired. The extent of apoptosis was also increased in both cell lines. Moreover, AMP-activated protein kinase α, proline-rich AKT substrate of 40 kDa, AKT, and extracellular signal-regulated kinase phosphorylation levels were down-regulated in GPR137 knockdown cells. The results of this study highlight the crucial role of GPR137 in promoting osteosarcoma cell growth in vitro . GPR137 could serve as a potential therapeutic target against osteosarcoma.

Systemic approaches identify a garlic-derived chemical, Z-ajoene, as a glioblastoma multiforme cancer stem cell-specific targeting agent.

PubMed

Jung, Yuchae; Park, Heejoo; Zhao, Hui-Yuan; Jeon, Raok; Ryu, Jae-Ha; Kim, Woo-Young

2014-07-01

Glioblastoma multiforme (GBM) is one of the most common brain malignancies and has a very poor prognosis. Recent evidence suggests that the presence of cancer stem cells (CSC) in GBM and the rare CSC subpopulation that is resistant to chemotherapy may be responsible for the treatment failure and unfavorable prognosis of GBM. A garlic-derived compound, Z-ajoene, has shown a range of biological activities, including anti-proliferative effects on several cancers. Here, we demonstrated for the first time that Z-ajoene specifically inhibits the growth of the GBM CSC population. CSC sphere-forming inhibition was achieved at a concentration that did not exhibit a cytotoxic effect in regular cell culture conditions. The specificity of this inhibitory effect on the CSC population was confirmed by detecting CSC cell surface marker CD133 expression and biochemical marker ALDH activity. In addition, stem cell-related mRNA profiling and real-time PCR revealed the differential expression of CSC-specific genes, including Notch, Wnt, and Hedgehog, upon treatment with Z-ajoene. A proteomic approach, i.e., reverse-phase protein array (RPPA) and Western blot analysis, showed decreased SMAD4, p-AKT, 14.3.3 and FOXO3A expression. The protein interaction map (http://string-db.org/) of the identified molecules suggested that the AKT, ERK/p38 and TGFβ signaling pathways are key mediators of Z-ajoene's action, which affects the transcriptional network that includes FOXO3A. These biological and bioinformatic analyses collectively demonstrate that Z-ajoene is a potential candidate for the treatment of GBM by specifically targeting GBM CSCs. We also show how this systemic approach strengthens the identification of new therapeutic agents that target CSCs.

Efficient Feature Extraction and Likelihood Fusion for Vehicle Tracking in Low Frame Rate Airborne Video

DTIC Science & Technology

2010-07-01

imagery, persistent sensor array I. Introduction New device fabrication technologies and heterogeneous embedded processors have led to the emergence of a...geometric occlusions between target and sensor , motion blur, urban scene complexity, and high data volumes. In practical terms the targets are small...distributed airborne narrow-field-of-view video sensor networks. Airborne camera arrays combined with com- putational photography techniques enable the

Ground-Based Radar (GBR): Environmental Assessment.

DTIC Science & Technology

1989-03-01

control of the antenna’s radiation pattern, the narrow, pencil-shaped main beam can be directed essentially instantaneously at incoming targets in any...Public Law 99-239: Compact of Free Association Act of 1M, January 14. 108. U.S. Department of Defense, Office of Economic Adjustment, 1984. Ecnomic ...may be directed essentially instantaneously at incoming targets. Phased-Array Technology The GBR will use a phased-array antenna for radiating the

Beamforming using subspace estimation from a diagonally averaged sample covariance.

PubMed

Quijano, Jorge E; Zurk, Lisa M

2017-08-01

The potential benefit of a large-aperture sonar array for high resolution target localization is often challenged by the lack of sufficient data required for adaptive beamforming. This paper introduces a Toeplitz-constrained estimator of the clairvoyant signal covariance matrix corresponding to multiple far-field targets embedded in background isotropic noise. The estimator is obtained by averaging along subdiagonals of the sample covariance matrix, followed by covariance extrapolation using the method of maximum entropy. The sample covariance is computed from limited data snapshots, a situation commonly encountered with large-aperture arrays in environments characterized by short periods of local stationarity. Eigenvectors computed from the Toeplitz-constrained covariance are used to construct signal-subspace projector matrices, which are shown to reduce background noise and improve detection of closely spaced targets when applied to subspace beamforming. Monte Carlo simulations corresponding to increasing array aperture suggest convergence of the proposed projector to the clairvoyant signal projector, thereby outperforming the classic projector obtained from the sample eigenvectors. Beamforming performance of the proposed method is analyzed using simulated data, as well as experimental data from the Shallow Water Array Performance experiment.

The therapeutic application of CRISPR/Cas9 technologies for HIV

PubMed Central

Saayman, Sheena; Ali, Stuart A.; Morris, Kevin V.; Weinberg, Marc S.

2015-01-01

Introduction The use of antiretroviral therapy (ART) has led to a significant decrease in morbidity and mortality in HIV-infected individuals. Nevertheless gene-based therapies represent a promising therapeutic paradigm for HIV-1, as they have the potential for sustained viral inhibition and reduced treatment interventions. One new method amendable to a gene-based therapy is the clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 gene editing system. Areas covered CRISPR/Cas9 can be engineered to successfully modulate an array of disease-causing genetic elements. We discuss the diverse roles that CRISPR/Cas9 may play in targeting HIV and eradicating infection. The Cas9 nuclease coupled with one or more small guide RNAs (sgRNAs) can target the provirus to mediate excision of the integrated viral genome. Moreover, a modified nuclease deficient Cas9 fused to transcription activating domains may induce targeted activation of proviral gene expression allowing for the purging of the latent reservoirs. These technologies can also be exploited to target host dependency factors such as the co-receptor CCR5, thus preventing cellular entry of the virus. Expert opinion The diversity of the CRISPR/Cas9 technologies hold great promise for targeting different stages of the viral life cycle, and have the capacity for mediating an effective and sustained genetic therapy against HIV. PMID:25865334

Serine, Glycine and One-carbon Metabolism in Colorectal Cancer Cell in Heterogeneous Microenvironment

NASA Astrophysics Data System (ADS)

Lin, Ke-Chih; Austin, Robert; Ducker, Greg; Sturm, James; Sturm, James

The up-regulation of serine metabolism associated with one-carbon metabolism has been identified to support cellular biosynthesis and redox maintenance of tumors. The consistently over-expressed one-carbon genes have been targeted for potential drug development. To investigate the biological function of specific enzymes, we had genetic engineered HCT116 cell lines, methylenetetrahydrofolate dehydrogenase (MTHFD) and phosphoglycerate dehydrogenase (PHGDH) deleted cell lines, growing in the artificial microhabitats array with serine and glycine gradient across. The impact of depletion of serine and the blocking of biosynthesis pathway will be shown in terms of cell morphology, proliferation rate, and cell motility. The evolution dynamic and migration rate can also be tracked throughout the experiments.

Probing the function of neuronal populations: combining micromirror-based optogenetic photostimulation with voltage-sensitive dye imaging.

PubMed

Tsuda, Sachiko; Kee, Michelle Z L; Cunha, Catarina; Kim, Jinsook; Yan, Ping; Loew, Leslie M; Augustine, George J

2013-01-01

Recent advances in our understanding of brain function have come from using light to either control or image neuronal activity. Here we describe an approach that combines both techniques: a micromirror array is used to photostimulate populations of presynaptic neurons expressing channelrhodopsin-2, while a red-shifted voltage-sensitive dye allows optical detection of resulting postsynaptic activity. Such technology allowed us to control the activity of cerebellar interneurons while simultaneously recording inhibitory responses in multiple Purkinje neurons, their postsynaptic targets. This approach should substantially accelerate our understanding of information processing by populations of neurons within brain circuits. Copyright © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Mapping the neglected space: gradients of detection revealed by virtual reality.

PubMed

Dvorkin, Assaf Y; Bogey, Ross A; Harvey, Richard L; Patton, James L

2012-02-01

Spatial neglect affects perception along different dimensions. However, there is limited availability of 3-dimensional (3D) methods that fully map out a patient's volume of deficit, although this could guide clinical management. To test whether patients with neglect exhibit simple contralesional versus complex perceptual deficits and whether deficits are best described using Cartesian (rectangular) or polar coordinates. Seventeen right-hemisphere persons with stroke (8 with a history of neglect) and 9 healthy controls were exposed to a 3D virtual environment. Targets placed in a dense array appeared one at a time in various locations. When tested using rectangular array of targets, subjects in the neglect group exhibited complex asymmetries across several dimensions in both reaction time and target detection rates. Paper-and-pencil tests only detected neglect in 4 of 8 of these patients. When tested using polar array of targets, 2 patients who initially appeared to perform poorly in both left and near space only showed a simple left-side asymmetry that depended almost entirely on the angle from the sagittal plane. A third patient exhibited left neglect irrespective of the arrangements of targets used. An idealized model with pure dependence on the polar angle demonstrated how such deficits could be misconstrued as near neglect if one uses a rectangular array. Such deficits may be poorly detected by paper-and-pencil tests and even by computerized tests that use regular screens. Assessments that incorporate 3D arrangements of targets enable precise mapping of deficient areas and detect subtle forms of neglect whose identification may be relevant to treatment strategies.

University of Texas Southwestern Medical Center (UTSW): Functional Signature Ontology Tool: Triplicate Measurements of Reporter Gene Expression in Response to Individual Genetic and Chemical Perturbations in HCT116 Cells | Office of Cancer Genomics

Cancer.gov

The goal of this project is to use an eight-gene expression profile to define functional signatures for small molecules and natural products with heretofore undefined mechanism of action. Two genes in the eight gene set are used as internal controls and do not vary across gene expression array data collected from the public domain. The remaining six genes are found to vary independently across a large collection of publically available gene expression array datasets.  Read the abstract

Dose–response relationships in gene expression profiles in rainbow trout, Oncorhyncus mykiss, exposed to ethynylestradiol

PubMed Central

Hook, Sharon E.; Skillman, Ann D.; Small, Jack A.; Schultz, Irvin R.

2008-01-01

Determining how gene expression profiles change with toxicant dose will improve the utility of arrays in identifying biomarkers and modes of toxic action. Isogenic rainbow trout, Oncorhyncus mykiss, were exposed to 10, 50 or 100 ng/L ethynylestradiol (a xeno-estrogen) for 7 days. Following exposure hepatic RNA was extracted. Fluorescently labeled cDNA were generated and hybridized against a commercially available Atlantic Salmon/Trout array (GRASP project, University of Victoria) spotted with 16,000 cDNAs. Transcript expression in treated vs control fish was analyzed via Genespring (Silicon Genetics) to identify genes with altered expression, as well as to determine gene clustering patterns that can be used as “expression signatures”. Array results were confirmed via qRT PCR. Our analysis indicates that gene expression profiles varied somewhat with dose. Established biomarkers of exposure to estrogenic chemicals, such as vitellogenin, vitelline envelope proteins, and the estrogen receptor alpha, were induced at every dose. Other genes were dose specific, suggesting that diffierent doses induce distinct physiological responses. These findings demonstrate that cDNA microarrays could be used to identify both toxicant class and relative dose. PMID:16725192

FPGA-accelerated algorithm for the regular expression matching system

NASA Astrophysics Data System (ADS)

Russek, P.; Wiatr, K.

2015-01-01

This article describes an algorithm to support a regular expressions matching system. The goal was to achieve an attractive performance system with low energy consumption. The basic idea of the algorithm comes from a concept of the Bloom filter. It starts from the extraction of static sub-strings for strings of regular expressions. The algorithm is devised to gain from its decomposition into parts which are intended to be executed by custom hardware and the central processing unit (CPU). The pipelined custom processor architecture is proposed and a software algorithm explained accordingly. The software part of the algorithm was coded in C and runs on a processor from the ARM family. The hardware architecture was described in VHDL and implemented in field programmable gate array (FPGA). The performance results and required resources of the above experiments are given. An example of target application for the presented solution is computer and network security systems. The idea was tested on nearly 100,000 body-based viruses from the ClamAV virus database. The solution is intended for the emerging technology of clusters of low-energy computing nodes.

microRNA and Autism.

PubMed

Anitha, Ayyappan; Thanseem, Ismail

2015-01-01

Autism is a complex neurodevelopmental disorder characterized by deficiencies in social interaction and communication, and by repetitive and stereotyped behaviors. According to a recent report, the prevalence of this pervasive developmental disorder has risen to 1 in 88. This will have enormous public health implications in the future, and has necessitated the need to discover predictive biomarkers that could index for autism before the onset of symptoms. microRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression at the posttranscriptional level. They have recently emerged as prominent epigenetic regulators of various cellular processes including neurodevelopment. They are abundantly present in the brain, and their dysfunction has been implicated in an array of neuropathological conditions including autism. miRNAs, previously known to be expressed only in cells and tissues, have also been detected in extracellular body fluids such as serum, plasma, saliva, and urine. Altered expression of cellular and circulating miRNAs have been observed in autistic individuals compared to healthy controls. miRNAs are now being considered as potential targets for the development of novel therapeutic strategies for autism.

Identification and pathway analysis of microRNAs with no previous involvement in breast cancer.

PubMed

Romero-Cordoba, Sandra; Rodriguez-Cuevas, Sergio; Rebollar-Vega, Rosa; Quintanar-Jurado, Valeria; Maffuz-Aziz, Antonio; Jimenez-Sanchez, Gerardo; Bautista-Piña, Veronica; Arellano-Llamas, Rocio; Hidalgo-Miranda, Alfredo

2012-01-01

microRNA expression signatures can differentiate normal and breast cancer tissues and can define specific clinico-pathological phenotypes in breast tumors. In order to further evaluate the microRNA expression profile in breast cancer, we analyzed the expression of 667 microRNAs in 29 tumors and 21 adjacent normal tissues using TaqMan Low-density arrays. 130 miRNAs showed significant differential expression (adjusted P value = 0.05, Fold Change = 2) in breast tumors compared to the normal adjacent tissue. Importantly, the role of 43 of these microRNAs has not been previously reported in breast cancer, including several evolutionary conserved microRNA*, showing similar expression rates to that of their corresponding leading strand. The expression of 14 microRNAs was replicated in an independent set of 55 tumors. Bioinformatic analysis of mRNA targets of the altered miRNAs, identified oncogenes like ERBB2, YY1, several MAP kinases, and known tumor-suppressors like FOXA1 and SMAD4. Pathway analysis identified that some biological process which are important in breast carcinogenesis are affected by the altered microRNA expression, including signaling through MAP kinases and TP53 pathways, as well as biological processes like cell death and communication, focal adhesion and ERBB2-ERBB3 signaling. Our data identified the altered expression of several microRNAs whose aberrant expression might have an important impact on cancer-related cellular pathways and whose role in breast cancer has not been previously described.

An EDMD mutation in C. elegans lamin blocks muscle-specific gene relocation and compromises muscle integrity.

PubMed

Mattout, Anna; Pike, Brietta L; Towbin, Benjamin D; Bank, Erin M; Gonzalez-Sandoval, Adriana; Stadler, Michael B; Meister, Peter; Gruenbaum, Yosef; Gasser, Susan M

2011-10-11

In worms, as in other organisms, many tissue-specific promoters are sequestered at the nuclear periphery when repressed and shift inward when activated. It has remained unresolved, however, whether the association of facultative heterochromatin with the nuclear periphery, or its release, has functional relevance for cell or tissue integrity. Using ablation of the unique lamin gene in C. elegans, we show that lamin is necessary for the perinuclear positioning of heterochromatin. We then express at low levels in otherwise wild-type worms a lamin carrying a point mutation, Y59C, which in humans is linked to an autosomal-dominant form of Emery-Dreifuss muscular dystrophy. Using embryos and differentiated tissues, we track the subnuclear position of integrated heterochromatic arrays and their expression. In LMN-1 Y59C-expressing worms, we see abnormal retention at the nuclear envelope of a gene array bearing a muscle-specific promoter. This correlates with impaired activation of the array-borne myo-3 promoter and altered expression of a number of muscle-specific genes. However, an equivalent array carrying the intestine-specific pha-4 promoter is expressed normally and shifts inward when activated in gut cells of LMN-1 Y59C worms. Remarkably, adult LMN-1 Y59C animals have selectively perturbed body muscle ultrastructure and reduced muscle function. Lamin helps sequester heterochromatin at the nuclear envelope, and wild-type lamin permits promoter release following tissue-specific activation. A disease-linked point mutation in lamin impairs muscle-specific reorganization of a heterochromatic array during tissue-specific promoter activation in a dominant manner. This dominance and the correlated muscle dysfunction in LMN-1 Y59C worms phenocopies Emery-Dreifuss muscular dystrophy. Copyright © 2011 Elsevier Ltd. All rights reserved.

MicroRNA array normalization: an evaluation using a randomized dataset as the benchmark.

PubMed

Qin, Li-Xuan; Zhou, Qin

2014-01-01

MicroRNA arrays possess a number of unique data features that challenge the assumption key to many normalization methods. We assessed the performance of existing normalization methods using two microRNA array datasets derived from the same set of tumor samples: one dataset was generated using a blocked randomization design when assigning arrays to samples and hence was free of confounding array effects; the second dataset was generated without blocking or randomization and exhibited array effects. The randomized dataset was assessed for differential expression between two tumor groups and treated as the benchmark. The non-randomized dataset was assessed for differential expression after normalization and compared against the benchmark. Normalization improved the true positive rate significantly in the non-randomized data but still possessed a false discovery rate as high as 50%. Adding a batch adjustment step before normalization further reduced the number of false positive markers while maintaining a similar number of true positive markers, which resulted in a false discovery rate of 32% to 48%, depending on the specific normalization method. We concluded the paper with some insights on possible causes of false discoveries to shed light on how to improve normalization for microRNA arrays.

MicroRNA Array Normalization: An Evaluation Using a Randomized Dataset as the Benchmark

PubMed Central

Qin, Li-Xuan; Zhou, Qin

2014-01-01

MicroRNA arrays possess a number of unique data features that challenge the assumption key to many normalization methods. We assessed the performance of existing normalization methods using two microRNA array datasets derived from the same set of tumor samples: one dataset was generated using a blocked randomization design when assigning arrays to samples and hence was free of confounding array effects; the second dataset was generated without blocking or randomization and exhibited array effects. The randomized dataset was assessed for differential expression between two tumor groups and treated as the benchmark. The non-randomized dataset was assessed for differential expression after normalization and compared against the benchmark. Normalization improved the true positive rate significantly in the non-randomized data but still possessed a false discovery rate as high as 50%. Adding a batch adjustment step before normalization further reduced the number of false positive markers while maintaining a similar number of true positive markers, which resulted in a false discovery rate of 32% to 48%, depending on the specific normalization method. We concluded the paper with some insights on possible causes of false discoveries to shed light on how to improve normalization for microRNA arrays. PMID:24905456

Carbon Nanotube Nanoelectrode Array for Ultrasensitive DNA Detection

NASA Technical Reports Server (NTRS)

Li, Jun; Koehne, Jessica; Chen, Hua; Cassell, Alan; Ng, Hou Tee; Fan, Wendy; Ye, Qi; Han, Jie; Meyyappan, M.

2003-01-01

A reliable nanoelectrode array based on vertically aligned multi-walled carbon nanotubes (MWNTs) embedded in SiO2 is used for ultrasensitive DNA detection. Characteristic nanoelectrode behavior is observed using low-density MWNT arrays for measuring both bulk and surface immobilized redox species such as K4Fe(CN)6. The open-end of MWNTs present similar properties as graphite edge-plane electrodes with wide potential window, flexible chemical functionalities, and good biocompatibility. Oligonucleotide probes are selectively functionalized at the open ends cf the nanotube array and specifically hybridized with oligonucleotide targets. The guanine groups are employed as the signal moieties in the electrochemical measurements. Ru(bpy)3(2+) mediator is used to further amplify the guanine oxidation signal. The hybridization of subattomoles of PCR amplified DNA targets is detected electrochemically by combining the MWNT nanoelectrode array with the Ru(bpy)32' amplification mechanism. This system provides a general platform of molecular diagnostics for applications requiring ultrahigh sensitivity, high-degree of miniaturization, and simple sample preparations.

Solar array flight dynamic experiment

NASA Technical Reports Server (NTRS)

Schock, R. W.

1986-01-01

The purpose of the Solar Array Flight Dynamic Experiment (SAFDE) is to demonstrate the feasibility of on-orbit measurement and ground processing of large space structures dynamic characteristics. Test definition or verification provides the dynamic characteristic accuracy required for control systems use. An illumination/measurement system was developed to fly on space shuttle flight STS-31D. The system was designed to dynamically evaluate a large solar array called the Solar Array Flight Experiment (SAFE) that had been scheduled for this flight. The SAFDE system consisted of a set of laser diode illuminators, retroreflective targets, an intelligent star tracker receiver and the associated equipment to power, condition, and record the results. In six tests on STS-41D, data was successfully acquired from 18 retroreflector targets and ground processed, post flight, to define the solar array's dynamic characteristic. The flight experiment proved the viability of on-orbit test definition of large space structures dynamic characteristics. Future large space structures controllability should be greatly enhanced by this capability.

Solar array flight dynamic experiment

NASA Technical Reports Server (NTRS)

Schock, Richard W.

1986-01-01

The purpose of the Solar Array Flight Dynamic Experiment (SAFDE) is to demonstrate the feasibility of on-orbit measurement and ground processing of large space structures dynamic characteristics. Test definition or verification provides the dynamic characteristic accuracy required for control systems use. An illumination/measurement system was developed to fly on Space Shuttle flight STS-31D. The system was designed to dynamically evaluate a large solar array called the Solar Array Flight Experiment (SAFE) that had been scheduled for this flight. The SAFDE system consisted of a set of laser diode illuminators, retroreflective targets, an intelligent star tracker receiver and the associated equipment to power, condition, and record the results. In six tests on STS-41D, data was successfully acquired from 18 retroreflector targets and ground processed, post flight, to define the solar array's dynamic characteristic. The flight experiment proved the viability of on-orbit test definition of large space structures dynamic characteristics. Future large space structures controllability should be greatly enhanced by this capability.

Solar array flight dynamic experiment

NASA Technical Reports Server (NTRS)

Schock, Richard W.

1987-01-01

The purpose of the Solar Array Flight Dynamic Experiment (SAFDE) is to demonstrate the feasibility of on-orbit measurement and ground processing of large space structures' dynamic characteristics. Test definition or verification provides the dynamic characteristic accuracy required for control systems use. An illumination/measurement system was developed to fly on space shuttle flight STS-41D. The system was designed to dynamically evaluate a large solar array called the Solar Array Flight Experiment (SAFE) that had been scheduled for this flight. The SAFDE system consisted of a set of laser diode illuminators, retroreflective targets, an intelligent star tracker receiver and the associated equipment to power, condition, and record the results. In six tests on STS-41D, data was successfully acquired from 18 retroreflector targets and ground processed, post flight, to define the solar array's dynamic characteristic. The flight experiment proved the viability of on-orbit test definition of large space structures dynamic characteristics. Future large space structures controllability should be greatly enhanced by this capability.

Dual RNA regulatory control of a Staphylococcus aureus virulence factor.

PubMed

Chabelskaya, Svetlana; Bordeau, Valérie; Felden, Brice

2014-04-01

In pathogens, the accurate programming of virulence gene expression is essential for infection. It is achieved by sophisticated arrays of regulatory proteins and ribonucleic acids (sRNAs), but in many cases their contributions and connections are not yet known. Based on genetic, biochemical and structural evidence, we report that the expression pattern of a Staphylococcus aureus host immune evasion protein is enabled by the collaborative actions of RNAIII and small pathogenicity island RNA D (SprD). Their combined expression profiles during bacterial growth permit early and transient synthesis of Sbi to avoid host immune responses. Together, these two sRNAs use antisense mechanisms to monitor Sbi expression at the translational level. Deletion analysis combined with structural analysis of RNAIII in complex with its novel messenger RNA (mRNA) target indicate that three distant RNAIII domains interact with distinct sites of the sbi mRNA and that two locations are deep in the sbi coding region. Through distinct domains, RNAIII lowers production of two proteins required for avoiding innate host immunity, staphylococcal protein A and Sbi. Toeprints and in vivo mutational analysis reveal a novel regulatory module within RNAIII essential for attenuation of Sbi translation. The sophisticated translational control of mRNA by two differentially expressed sRNAs ensures supervision of host immune escape by a major pathogen.

Gene expression profiles reveal that DCN, DIO1, and DIO2 are underexpressed in benign and malignant thyroid tumors.

PubMed

Arnaldi, L A T; Borra, R C; Maciel, R M B; Cerutti, J M

2005-03-01

To investigate the molecular events involved in the pathogenesis and/or progression of thyroid tumors, we compared the gene expression profiles of three thyroid carcinoma cell lines, which represent major tumor subtypes of thyroid cancer and normal thyroid tissue. Using cDNA array methodology, we investigated the expression of 1807 open reading frame expressed sequence tags (ORESTES), selected from head and neck tumor libraries generated through the Brazilian Human Cancer Project-LICR/FAPESP. We found that 505 transcripts were differentially expressed in the thyroid carcinoma cell lines. Using a more stringent criterion, transcripts underexpressed or overexpressed more than fivefold in 1 of 3 or 3 of 3 carcinoma cell lines, a list of 55 ESTs were detected. Five candidate genes were further validated by quantitative polymerase chain reaction (qPCR) in an independent set of 52 thyroid tumors and 22 matched normal thyroid tissues. DCN was found underexpressed in a high percentage of the follicular thyroid adenomas, follicular thyroid carcinomas, and follicular variant of papillary thyroid carcinomas. DIO1 and DIO2 were underexpressed in nearly all papillary thyroid carcinomas. These genes not only could help to better define a tumor signature for thyroid tumors, but may, in part, also become useful as potential targets for thyroid tumor treatment.

Preclinical Assessment of CD171-Directed CAR T-cell Adoptive Therapy for Childhood Neuroblastoma: CE7 Epitope Target Safety and Product Manufacturing Feasibility.

PubMed

Künkele, Annette; Taraseviciute, Agne; Finn, Laura S; Johnson, Adam J; Berger, Carolina; Finney, Olivia; Chang, Cindy A; Rolczynski, Lisa S; Brown, Christopher; Mgebroff, Stephanie; Berger, Michael; Park, Julie R; Jensen, Michael C

2017-01-15

The identification and vetting of cell surface tumor-restricted epitopes for chimeric antigen receptor (CAR)-redirected T-cell immunotherapy is the subject of intensive investigation. We have focused on CD171 (L1-CAM), an abundant cell surface molecule on neuroblastomas and, specifically, on the glycosylation-dependent tumor-specific epitope recognized by the CE7 monoclonal antibody. CD171 expression was assessed by IHC using CE7 mAb in tumor microarrays of primary, metastatic, and recurrent neuroblastoma, as well as human and rhesus macaque tissue arrays. The safety of targeting the CE7 epitope of CD171 with CE7-CAR T cells was evaluated in a preclinical rhesus macaque trial on the basis of CD171 homology and CE7 cross reactivity. The feasibility of generating bioactive CAR T cells from heavily pretreated pediatric patients with recurrent/refractory disease was assessed. CD171 is uniformly and abundantly expressed by neuroblastoma tumor specimens obtained at diagnoses and relapse independent of patient clinical risk group. CD171 expression in normal tissues is similar in humans and rhesus macaques. Infusion of up to 1 × 10 8 /kg CE7-CAR + CTLs in rhesus macaques revealed no signs of specific on-target off-tumor toxicity. Manufacturing of lentivirally transduced CD4 + and CD8 + CE7-CAR T-cell products under GMP was successful in 4 out of 5 consecutively enrolled neuroblastoma patients in a phase I study. All four CE7-CAR T-cell products demonstrated in vitro and in vivo antitumor activity. Our preclinical assessment of the CE7 epitope on CD171 supports its utility and safety as a CAR T-cell target for neuroblastoma immunotherapy. Clin Cancer Res; 23(2); 466-77. ©2016 AACR. ©2016 American Association for Cancer Research.

Optical demodulation system for digitally encoded suspension array in fluoroimmunoassay

NASA Astrophysics Data System (ADS)

He, Qinghua; Li, Dongmei; He, Yonghong; Guan, Tian; Zhang, Yilong; Shen, Zhiyuan; Chen, Xuejing; Liu, Siyu; Lu, Bangrong; Ji, Yanhong

2017-09-01

A laser-induced breakdown spectroscopy and fluorescence spectroscopy-coupled optical system is reported to demodulate digitally encoded suspension array in fluoroimmunoassay. It takes advantage of the plasma emissions of assembled elemental materials to digitally decode the suspension array, providing a more stable and accurate recognition to target biomolecules. By separating the decoding procedure of suspension array and adsorption quantity calculation of biomolecules into two independent channels, the cross talk between decoding and label signals in traditional methods had been successfully avoided, which promoted the accuracy of both processes and realized more sensitive quantitative detection of target biomolecules. We carried a multiplexed detection of several types of anti-IgG to verify the quantitative analysis performance of the system. A limit of detection of 1.48×10-10 M was achieved, demonstrating the detection sensitivity of the optical demodulation system.

Fast photoacoustic imaging system based on 320-element linear transducer array.

PubMed

Yin, Bangzheng; Xing, Da; Wang, Yi; Zeng, Yaguang; Tan, Yi; Chen, Qun

2004-04-07

A fast photoacoustic (PA) imaging system, based on a 320-transducer linear array, was developed and tested on a tissue phantom. To reconstruct a test tomographic image, 64 time-domain PA signals were acquired from a tissue phantom with embedded light-absorption targets. A signal acquisition was accomplished by utilizing 11 phase-controlled sub-arrays, each consisting of four transducers. The results show that the system can rapidly map the optical absorption of a tissue phantom and effectively detect the embedded light-absorbing target. By utilizing the multi-element linear transducer array and phase-controlled imaging algorithm, we thus can acquire PA tomography more efficiently, compared to other existing technology and algorithms. The methodology and equipment thus provide a rapid and reliable approach to PA imaging that may have potential applications in noninvasive imaging and clinic diagnosis.

Combined array CGH plus SNP genome analyses in a single assay for optimized clinical testing

PubMed Central

Wiszniewska, Joanna; Bi, Weimin; Shaw, Chad; Stankiewicz, Pawel; Kang, Sung-Hae L; Pursley, Amber N; Lalani, Seema; Hixson, Patricia; Gambin, Tomasz; Tsai, Chun-hui; Bock, Hans-Georg; Descartes, Maria; Probst, Frank J; Scaglia, Fernando; Beaudet, Arthur L; Lupski, James R; Eng, Christine; Wai Cheung, Sau; Bacino, Carlos; Patel, Ankita

2014-01-01

In clinical diagnostics, both array comparative genomic hybridization (array CGH) and single nucleotide polymorphism (SNP) genotyping have proven to be powerful genomic technologies utilized for the evaluation of developmental delay, multiple congenital anomalies, and neuropsychiatric disorders. Differences in the ability to resolve genomic changes between these arrays may constitute an implementation challenge for clinicians: which platform (SNP vs array CGH) might best detect the underlying genetic cause for the disease in the patient? While only SNP arrays enable the detection of copy number neutral regions of absence of heterozygosity (AOH), they have limited ability to detect single-exon copy number variants (CNVs) due to the distribution of SNPs across the genome. To provide comprehensive clinical testing for both CNVs and copy-neutral AOH, we enhanced our custom-designed high-resolution oligonucleotide array that has exon-targeted coverage of 1860 genes with 60 000 SNP probes, referred to as Chromosomal Microarray Analysis – Comprehensive (CMA-COMP). Of the 3240 cases evaluated by this array, clinically significant CNVs were detected in 445 cases including 21 cases with exonic events. In addition, 162 cases (5.0%) showed at least one AOH region >10 Mb. We demonstrate that even though this array has a lower density of SNP probes than other commercially available SNP arrays, it reliably detected AOH events >10 Mb as well as exonic CNVs beyond the detection limitations of SNP genotyping. Thus, combining SNP probes and exon-targeted array CGH into one platform provides clinically useful genetic screening in an efficient manner. PMID:23695279

A Targeted RNAi Screen of the Breast Cancer Genome Identifies KIF14 and TLN1 as Genes That Modulate Docetaxel Chemosensitivity in Triple-Negative Breast Cancer

PubMed Central

Singel, Stina Mui; Cornelius, Crystal; Batten, Kimberly; Fasciani, Gail; Wright, Woodring E.; Lum, Lawrence; Shay, Jerry W.

2015-01-01

Purpose To identify biomarkers within the breast cancer genome that may predict chemosensitivity in breast cancer. Experimental Design We conducted an RNA interference (RNAi) screen within the breast cancer genome for genes whose loss-of-function enhanced docetaxel chemosensitivity in an estrogen receptor–negative, progesterone receptor–negative, and Her2-negative (ER−, PR−, and Her2−, respectively) breast cancer cell line, MDA-MB-231. Top candidates were tested for their ability to modulate chemosensitivity in 8 breast cancer cell lines and to show in vivo chemosensitivity in a mouse xenograft model. Results From ranking chemosensitivity of 328 short hairpin RNA (shRNA) MDA-MB-231 cell lines (targeting 133 genes with known somatic mutations in breast cancer), we focused on the top two genes, kinesin family member 14 (KIF14) and talin 1 (TLN1). KIF14 and TLN1 loss-of-function significantly enhanced chemosensitivity in four triple-negative breast cancer (TNBC) cell lines (MDA-MB-231, HCC38, HCC1937, and Hs478T) but not in three hormone receptor–positive cell lines (MCF7, T47D, and HCC1428) or normal human mammary epithelial cells (HMEC). Decreased expression of KIF14, but not TLN1, also enhanced docetaxel sensitivity in a Her2-amplified breast cancer cell line, SUM190PT. Higher KIF14 and TLN1 expressions are found in TNBCs compared with the other clinical subtypes. Mammary fat pad xenografts of KIF14- and TLN1-deficient MDA-MB-231 cells revealed reduced tumor mass compared with control MDA-MB-231 cells after chemotherapy. KIF14 expression is also prognostic of relapse-free and overall survival in representative breast cancer expression arrays. Conclusion KIF14 and TLN1 are modulators of response to docetaxel and potential therapeutic targets in TNBC. PMID:23479679

Interplay between estrogen receptor and AKT in Estradiol-induced alternative splicing

PubMed Central

2013-01-01

Background Alternative splicing is critical for generating complex proteomes in response to extracellular signals. Nuclear receptors including estrogen receptor alpha (ERα) and their ligands promote alternative splicing. The endogenous targets of ERα:estradiol (E2)-mediated alternative splicing and the influence of extracellular kinases that phosphorylate ERα on E2-induced splicing are unknown. Methods MCF-7 and its anti-estrogen derivatives were used for the majority of the assays. CD44 mini gene was used to measure the effect of E2 and AKT on alternative splicing. ExonHit array analysis was performed to identify E2 and AKT-regulated endogenous alternatively spliced apoptosis-related genes. Quantitative reverse transcription polymerase chain reaction was performed to verify alternative splicing. ERα binding to alternatively spliced genes was verified by chromatin immunoprecipitation assay. Bromodeoxyuridine incorporation-ELISA and Annexin V labeling assays were done to measure cell proliferation and apoptosis, respectively. Results We identified the targets of E2-induced alternative splicing and deconstructed some of the mechanisms surrounding E2-induced splicing by combining splice array with ERα cistrome and gene expression array. E2-induced alternatively spliced genes fall into at least two subgroups: coupled to E2-regulated transcription and ERα binding to the gene without an effect on rate of transcription. Further, AKT, which phosphorylates both ERα and splicing factors, influenced ERα:E2 dependent splicing in a gene-specific manner. Genes that are alternatively spliced include FAS/CD95, FGFR2, and AXIN-1. E2 increased the expression of FGFR2 C1 isoform but reduced C3 isoform at mRNA level. E2-induced alternative splicing of FAS and FGFR2 in MCF-7 cells correlated with resistance to FAS activation-induced apoptosis and response to keratinocyte growth factor (KGF), respectively. Resistance of MCF-7 breast cancer cells to the anti-estrogen tamoxifen was associated with ERα-dependent overexpression of FGFR2, whereas resistance to fulvestrant was associated with ERα-dependent isoform switching, which correlated with altered response to KGF. Conclusion E2 may partly alter cellular proteome through alternative splicing uncoupled to its effects on transcription initiation and aberration in E2-induced alternative splicing events may influence response to anti-estrogens. PMID:23758675

Arrays of nucleic acid probes on biological chips

DOEpatents

Chee, Mark; Cronin, Maureen T.; Fodor, Stephen P. A.; Huang, Xiaohua X.; Hubbell, Earl A.; Lipshutz, Robert J.; Lobban, Peter E.; Morris, MacDonald S.; Sheldon, Edward L.

1998-11-17

DNA chips containing arrays of oligonucleotide probes can be used to determine whether a target nucleic acid has a nucleotide sequence identical to or different from a specific reference sequence. The array of probes comprises probes exactly complementary to the reference sequence, as well as probes that differ by one or more bases from the exactly complementary probes.

Optimal expression evaluation for data parallel architectures

NASA Technical Reports Server (NTRS)

Gilbert, John R.; Schreiber, Robert

1990-01-01

A data parallel machine represents an array or other composite data structure by allocating one processor (at least conceptually) per data item. A pointwise operation can be performed between two such arrays in unit time, provided their corresponding elements are allocated in the same processors. If the arrays are not aligned in this fashion, the cost of moving one or both of them is part of the cost of the operation. The choice of where to perform the operation then affects this cost. If an expression with several operands is to be evaluated, there may be many choices of where to perform the intermediate operations. An efficient algorithm is given to find the minimum-cost way to evaluate an expression, for several different data parallel architectures. This algorithm applies to any architecture in which the metric describing the cost of moving an array is robust. This encompasses most of the common data parallel communication architectures, including meshes of arbitrary dimension and hypercubes. Remarks are made on several variations of the problem, some of which are solved and some of which remain open.

Mining the nucleus accumbens proteome for novel targets of alcohol self-administration in male C57BL/6J mice.

PubMed

Faccidomo, Sara; Swaim, Katarina S; Saunders, Briana L; Santanam, Taruni S; Taylor, Seth M; Kim, Michelle; Reid, Grant T; Eastman, Vallari R; Hodge, Clyde W

2018-06-01

There is a clear need for discovery of effective medications to treat behavioral pathologies associated with alcohol addiction, such as chronic drinking. The goal of this preclinical study was to assess effects of chronic alcohol drinking on the nucleus accumbens (NAcb) proteome to identify and validate novel targets for medications development. Two-dimensional difference in-gel electrophoresis (2D-DIGE) with matrix-assisted laser desorption ionization tandem time-of-flight (MALDI-TOF/TOF) was used to assess effects of chronic voluntary home-cage (24-h access) alcohol drinking on the NAcb proteome of C57BL/6J mice. To extend these findings to a model of alcohol self-administration and reinforcement, we investigated potential regulation of the positive reinforcing effects of alcohol by the target protein glutathione S-transferase Pi 1 (GSTP1) using a pharmacological inhibition strategy in mice trained to self-administer alcohol or sucrose. Expression of 52 unique proteins in the NAcb was changed by chronic alcohol drinking relative to water control (23 upregulated, 29 downregulated). Ingenuity Pathway Analysis showed that alcohol drinking altered an array of protein networks associated with neurological and psychological disorders, molecular and cellular functions, and physiological systems and development. DAVID functional annotation analysis identified 9 proteins (SNCA, GSTP1, PRDX3, PPP3R1, EIF5A, PHB, PEBP1/RKIP, GAPDH, AND SOD1) that were significantly overrepresented in a functional cluster that included the Gene Ontology categories "response to alcohol" and "aging." Immunoblots confirmed changes in Pebp1 (RKIP) and GSTP1 in NAcb with no change in amygdala or frontal cortex, suggesting anatomical specificity. Systemic inhibition of GSTP1 with Ezatiostat (0-30 mg/kg, i.p.) dose-dependently reduced the reinforcing effects of alcohol as measured by operant self-administration, in the absence of motor effects. Sucrose self-administration was also reduced but in a manner associated with nonspecific motor inhibition. Protein expression profiling identified an array of proteins and networks in the NAcb, including GSTP1, that are novel molecular targets of chronic alcohol drinking. Pharmacological inhibition of GSTP1 significantly reduced the positive reinforcing effects of alcohol, which regulate repetitive use and abuse liability. The observation that this protein was both upregulated after chronic drinking and that its inhibition could modulate the reinforcing properties of alcohol suggests that it is a key target for alcohol-related pathologies. Proteomic strategies combined with specific preclinical models has potential to identify and validate novel targets of alcohol that may be useful in the medical management of alcohol addiction.

Functionalization of Carbon Nanotubes

NASA Technical Reports Server (NTRS)

Khare, Bishun N. (Inventor); Meyyappan, Meyya (Inventor)

2007-01-01

Method and system for functionalizing a collection of carbon nanotubes (CNTs). A selected precursor gas (e.g., H2, or F2, or CnHm) is irradiated to provide a cold plasma of selected target particles, such as atomic H or F, in a first chamber. The target particles are directed toward an array of CNTs located in a second chamber while suppressing transport of ultraviolet radiation to the second chamber. A CNT array is functionalized with the target particles, at or below room temperature, to a point of saturation, in an exposure time interval no longer than about 30 sec.

Functionalization of carbon nanotubes

NASA Technical Reports Server (NTRS)

Khare, Bishun N. (Inventor); Meyyappan, Meyya (Inventor)

2007-01-01

Method and system for functionalizing a collection of carbon nanotubes (CNTs). A selected precursor gas (e.g., H.sub.2 or F.sub.2 or C.sub.nH.sub.m) is irradiated to provide a cold plasma of selected target particles, such as atomic H or F, in a first chamber. The target particles are directed toward an array of CNTs located in a second chamber while suppressing transport of ultraviolet radiation to the second chamber. A CNT array is functionalized with the target particles, at or below room temperature, to a point of saturation, in an exposure time interval no longer than about 30 sec.

Changes in Hepatic TRβ Protein Expression, Lipogenic Gene Expression, and Long-Chain Acylcarnitine Levels During Chronic Hyperthyroidism and Triiodothyronine Withdrawal in a Mouse Model.

PubMed

Ohba, Kenji; Sinha, Rohit Anthony; Singh, Brijesh Kumar; Iannucci, Liliana Felicia; Zhou, Jin; Kovalik, Jean-Paul; Liao, Xiao-Hui; Refetoff, Samuel; Sng, Judy Chia Ghee; Leow, Melvin Khee-Shing; Yen, Paul Michael

2017-06-01

Thyroid hormone (TH) has important roles in regulating hepatic metabolism. It was previously reported that most hepatic genes activated by a single triiodothyronine (T3) injection became desensitized after multiple injections, and that approximately 10% of target genes did not return to basal expression levels after T3 withdrawal, despite normalization of serum TH and thyrotropin (TSH) levels. To determine the possible mechanism(s) for desensitization and incomplete recovery of hepatic target gene transcription and their effects on metabolism, mRNA and/or protein expression levels of key regulators of TH action were measured, as well as metabolomic changes after chronic T3 treatment and withdrawal. Adult male mice were treated with daily injections of T3 (20 μg/100 g body weight) for 14 days followed by the cessation of T3 for 10 days. Livers were harvested at 6 hours, 24 hours, and 14 days after the first T3 injection, and at 10 days after withdrawal, and then analyzed by quantitative reverse transcription polymerase chain reaction, Western blotting, and metabolomics. Although TH receptor (TRα and TRβ) mRNAs decreased slightly after chronic T3 treatment, only TRβ protein decreased before returning to basal expression level after withdrawal. The expression of other regulators of TH action was unchanged. TRβ protein expression was also decreased in adult male monocarboxylate transporter-8 (Mct8)-knockout mice, an in vivo model of chronic intrahepatic hyperthyroidism. Previously, increased hepatic long-chain acylcarnitine levels were found after acute TH treatment. However, in this study, long-chain acylcarnitine levels were unchanged after chronic T3, and paradoxically increased after T3 withdrawal. Pathway analyses of the previous microarray results showed upregulation of lipogenic genes after acute T3 treatment and withdrawal. Phosphorylation of acetyl-CoA carboxylase also decreased after T3 withdrawal. Decreased hepatic TRβ protein expression occurred after chronic T3 exposure in adult male wild-type and Mct8-knockout mice. Gene array pathway and metabolomics analyses showed abnormalities in hepatic lipogenic gene expression and acylcarnitine levels, respectively, after withdrawal, despite normalization of serum TSH and TH levels. These findings may help explain the variable clinical presentations of some patients during hyperthyroidism and recovery, since TRβ protein, target gene expression, and metabolic adaptive changes can occur in individual tissues without necessarily being reflected by circulating TH and TSH concentrations.

Changes in Hepatic TRβ Protein Expression, Lipogenic Gene Expression, and Long-Chain Acylcarnitine Levels During Chronic Hyperthyroidism and Triiodothyronine Withdrawal in a Mouse Model

PubMed Central

Ohba, Kenji; Sinha, Rohit Anthony; Singh, Brijesh Kumar; Iannucci, Liliana Felicia; Zhou, Jin; Kovalik, Jean-Paul; Liao, Xiao-Hui; Refetoff, Samuel; Sng, Judy Chia Ghee; Leow, Melvin Khee-Shing; Yen, Paul Michael

2017-01-01

Background: Thyroid hormone (TH) has important roles in regulating hepatic metabolism. It was previously reported that most hepatic genes activated by a single triiodothyronine (T3) injection became desensitized after multiple injections, and that approximately 10% of target genes did not return to basal expression levels after T3 withdrawal, despite normalization of serum TH and thyrotropin (TSH) levels. To determine the possible mechanism(s) for desensitization and incomplete recovery of hepatic target gene transcription and their effects on metabolism, mRNA and/or protein expression levels of key regulators of TH action were measured, as well as metabolomic changes after chronic T3 treatment and withdrawal. Methods: Adult male mice were treated with daily injections of T3 (20 μg/100 g body weight) for 14 days followed by the cessation of T3 for 10 days. Livers were harvested at 6 hours, 24 hours, and 14 days after the first T3 injection, and at 10 days after withdrawal, and then analyzed by quantitative reverse transcription polymerase chain reaction, Western blotting, and metabolomics. Results: Although TH receptor (TRα and TRβ) mRNAs decreased slightly after chronic T3 treatment, only TRβ protein decreased before returning to basal expression level after withdrawal. The expression of other regulators of TH action was unchanged. TRβ protein expression was also decreased in adult male monocarboxylate transporter-8 (Mct8)-knockout mice, an in vivo model of chronic intrahepatic hyperthyroidism. Previously, increased hepatic long-chain acylcarnitine levels were found after acute TH treatment. However, in this study, long-chain acylcarnitine levels were unchanged after chronic T3, and paradoxically increased after T3 withdrawal. Pathway analyses of the previous microarray results showed upregulation of lipogenic genes after acute T3 treatment and withdrawal. Phosphorylation of acetyl-CoA carboxylase also decreased after T3 withdrawal. Conclusions: Decreased hepatic TRβ protein expression occurred after chronic T3 exposure in adult male wild-type and Mct8-knockout mice. Gene array pathway and metabolomics analyses showed abnormalities in hepatic lipogenic gene expression and acylcarnitine levels, respectively, after withdrawal, despite normalization of serum TSH and TH levels. These findings may help explain the variable clinical presentations of some patients during hyperthyroidism and recovery, since TRβ protein, target gene expression, and metabolic adaptive changes can occur in individual tissues without necessarily being reflected by circulating TH and TSH concentrations. PMID:28457184

Target-in-the-loop high-power adaptive phase-locked fiber laser array using single-frequency dithering technique

NASA Astrophysics Data System (ADS)

Tao, R.; Ma, Y.; Si, L.; Dong, X.; Zhou, P.; Liu, Z.

2011-11-01

We present a theoretical and experimental study of a target-in-the-loop (TIL) high-power adaptive phase-locked fiber laser array. The system configuration of the TIL adaptive phase-locked fiber laser array is introduced, and the fundamental theory for TIL based on the single-dithering technique is deduced for the first time. Two 10-W-level high-power fiber amplifiers are set up and adaptive phase locking of the two fiber amplifiers is accomplished successfully by implementing a single-dithering algorithm on a signal processor. The experimental results demonstrate that the optical phase noise for each beam channel can be effectively compensated by the TIL adaptive optics system under high-power applications and the fringe contrast on a remotely located extended target is advanced from 12% to 74% for the two 10-W-level fiber amplifiers.

Sensor modeling and demonstration of a multi-object spectrometer for performance-driven sensing

NASA Astrophysics Data System (ADS)

Kerekes, John P.; Presnar, Michael D.; Fourspring, Kenneth D.; Ninkov, Zoran; Pogorzala, David R.; Raisanen, Alan D.; Rice, Andrew C.; Vasquez, Juan R.; Patel, Jeffrey P.; MacIntyre, Robert T.; Brown, Scott D.

2009-05-01

A novel multi-object spectrometer (MOS) is being explored for use as an adaptive performance-driven sensor that tracks moving targets. Developed originally for astronomical applications, the instrument utilizes an array of micromirrors to reflect light to a panchromatic imaging array. When an object of interest is detected the individual micromirrors imaging the object are tilted to reflect the light to a spectrometer to collect a full spectrum. This paper will present example sensor performance from empirical data collected in laboratory experiments, as well as our approach in designing optical and radiometric models of the MOS channels and the micromirror array. Simulation of moving vehicles in a highfidelity, hyperspectral scene is used to generate a dynamic video input for the adaptive sensor. Performance-driven algorithms for feature-aided target tracking and modality selection exploit multiple electromagnetic observables to track moving vehicle targets.

Novel Permissive Cell Lines for Complete Propagation of Hepatitis C Virus

PubMed Central

Shiokawa, Mai; Fukuhara, Takasuke; Ono, Chikako; Yamamoto, Satomi; Okamoto, Toru; Watanabe, Noriyuki; Wakita, Takaji

2014-01-01

ABSTRACT Hepatitis C virus (HCV) is a major etiologic agent of chronic liver diseases. Although the HCV life cycle has been clarified by studying laboratory strains of HCV derived from the genotype 2a JFH-1 strain (cell culture-adapted HCV [HCVcc]), the mechanisms of particle formation have not been elucidated. Recently, we showed that exogenous expression of a liver-specific microRNA, miR-122, in nonhepatic cell lines facilitates efficient replication but not particle production of HCVcc, suggesting that liver-specific host factors are required for infectious particle formation. In this study, we screened human cancer cell lines for expression of the liver-specific α-fetoprotein by using a cDNA array database and identified liver-derived JHH-4 cells and stomach-derived FU97 cells, which express liver-specific host factors comparable to Huh7 cells. These cell lines permit not only replication of HCV RNA but also particle formation upon infection with HCVcc, suggesting that hepatic differentiation participates in the expression of liver-specific host factors required for HCV propagation. HCV inhibitors targeting host and viral factors exhibited different antiviral efficacies between Huh7 and FU97 cells. Furthermore, FU97 cells exhibited higher susceptibility for propagation of HCVcc derived from the JFH-2 strain than Huh7 cells. These results suggest that hepatic differentiation participates in the expression of liver-specific host factors required for complete propagation of HCV. IMPORTANCE Previous studies have shown that liver-specific host factors are required for efficient replication of HCV RNA and formation of infectious particles. In this study, we screened human cancer cell lines for expression of the liver-specific α-fetoprotein by using a cDNA array database and identified novel permissive cell lines for complete propagation of HCVcc without any artificial manipulation. In particular, gastric cancer-derived FU97 cells exhibited a much higher susceptibility to HCVcc/JFH-2 infection than observed in Huh7 cells, suggesting that FU97 cells would be useful for further investigation of the HCV life cycle, as well as the development of therapeutic agents for chronic hepatitis C. PMID:24599999

Identification of Cytokines and Signaling Proteins Differentially Regulated by Sumatriptan/Naproxen

PubMed Central

Vause, Carrie V; Durham, Paul L

2011-01-01

Summary Objectives The goal of this study was to use protein array analysis to investigate temporal regulation of stimulated cytokine expression in trigeminal ganglia and spinal trigeminal nuclei in response to cotreatment of sumatriptan and naproxen sodium or individual drug. Background Activation of neurons and glia in trigeminal ganglia and spinal trigeminal nuclei leads to increased levels of cytokines that promote peripheral and central sensitization, which are key events in migraine pathology. While recent clinical studies have provided evidence that a combination of sumatriptan and naproxen sodium is more efficacious in treating migraine than either drug alone, it is not well understood why the combination therapy is superior to monotherapy. Methods Male Sprague Dawley rats were left untreated (control), injected with capsaicin, or pre-treated with sumatriptan/naproxen, sumatriptan, or naproxen for 1 hour prior to capsaicin. Trigeminal ganglia and spinal trigeminal nuclei were isolated 2 and 24 hours after capsaicin or drug treatment and levels of 90 proteins were determined using a RayBio® Label-Based Rat Antibody Array. Results Capsaicin stimulated a >3-fold increase in expression of the majority of cytokines in trigeminal ganglia at 2 hours that was sustained at 24 hours. Significantly, treatment with sumatriptan/naproxen almost completely abolished the stimulatory effects of capsaicin at 2 and 24 hours. Capsaicin stimulated >3-fold expression of more proteins in spinal trigeminal nuclei at 24 hours when compared to 2 hours. Similarly, sumatriptan/naproxen abolished capsaicin stimulation of proteins in spinal trigeminal nuclei at 2 hours and greatly suppressed protein expression 24 hours post capsaicin injection. Interestingly, treatment with sumatriptan alone suppressed expression of different cytokines in trigeminal ganglia and spinal trigeminal nuclei than repressed by naproxen sodium. Conclusion We found that the combination of sumatriptan/naproxen was effective in blocking capsaicin stimulation of pro-inflammatory proteins implicated in the development of peripheral and central sensitization in response to capsaicin activation of trigeminal neurons. Based on our findings that sumatriptan and naproxen regulate expression of different proteins in trigeminal ganglia and spinal trigeminal nuclei, we propose that these drugs function on therapeutically distinct cellular targets to suppress inflammation and pain associated with migraine. PMID:22150557

Role of miR-452-5p in the tumorigenesis of prostate cancer: A study based on the Cancer Genome Atl(TCGA), Gene Expression Omnibus (GEO), and bioinformatics analysis.

PubMed

Gao, Li; Zhang, Li-Jie; Li, Sheng-Hua; Wei, Li-Li; Luo, Bin; He, Rong-Quan; Xia, Shuang

2018-03-06

MiR-452-5p has been reported to be down-regulated in prostate cancer, affecting the development of this type of cancer. However, the molecular mechanism of miR-452-5p in prostate cancer remains unclear. Therefore, we investigated the network of target genes of miR-452-5p in prostate cancer using bioinformatics analyses. We first analyzed the expression profiles and prognostic value of miR-452-5p in prostate cancer tissues from a public database. Gene Ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG), PANTHER pathway analyses, and a disease ontology (DG) analysis were performed to find the molecular functions of the target genes from GSE datasets and miRWalk. Finally, we validated hub genes from the protein-protein interaction (PPI) networks of the target genes in the Human Protein Atlas (HPA) database and Gene Expression Profiling Interactive Analysis (GEPIA). Narrowing down the optimal target genes was conducted by seeking the common parts of up-regulated genes from GEPIA, down-regulated genes from GSE datasets, and predicted genes in miRWalk. Based on mining of GEO and ArrayExpress microarray chips and miRNA-Seq data in the TCGA database, which includes 1007 prostate cancer samples and 387 non-cancer samples, miR-452-5p is shown to be down-regulated in prostate cancer. GO, KEGG, and PANTHER pathway analyses suggested that the target genes might participate in important biological processes, such as transforming growth factor beta signaling and the positive regulation of brown fat cell differentiation and mesenchymal cell differentiation, as well as the Ras signaling pathway and pathways regulating the pluripotency of stem cells and arrhythmogenic right ventricular cardiomyopathy (ARVC). Nine genes-GABBR, PNISR, NTSR1, DOCK1, EREG, SFRP1, PTGS2, LEF1, and BMP2-were defined as hub genes in the PPI network. Three genes-FAM174B, SLC30A4, and SLIT1-were jointly shared by GEPIA, the GSE datasets, and miRWalk. Down-regulated miR-452-5p might play an essential role in the tumorigenesis of prostate cancer. Copyright © 2018. Published by Elsevier GmbH.

Validation of RNAi Silencing Efficiency Using Gene Array Data shows 18.5% Failure Rate across 429 Independent Experiments.

PubMed

Munkácsy, Gyöngyi; Sztupinszki, Zsófia; Herman, Péter; Bán, Bence; Pénzváltó, Zsófia; Szarvas, Nóra; Győrffy, Balázs

2016-09-27

No independent cross-validation of success rate for studies utilizing small interfering RNA (siRNA) for gene silencing has been completed before. To assess the influence of experimental parameters like cell line, transfection technique, validation method, and type of control, we have to validate these in a large set of studies. We utilized gene chip data published for siRNA experiments to assess success rate and to compare methods used in these experiments. We searched NCBI GEO for samples with whole transcriptome analysis before and after gene silencing and evaluated the efficiency for the target and off-target genes using the array-based expression data. Wilcoxon signed-rank test was used to assess silencing efficacy and Kruskal-Wallis tests and Spearman rank correlation were used to evaluate study parameters. All together 1,643 samples representing 429 experiments published in 207 studies were evaluated. The fold change (FC) of down-regulation of the target gene was above 0.7 in 18.5% and was above 0.5 in 38.7% of experiments. Silencing efficiency was lowest in MCF7 and highest in SW480 cells (FC = 0.59 and FC = 0.30, respectively, P = 9.3E-06). Studies utilizing Western blot for validation performed better than those with quantitative polymerase chain reaction (qPCR) or microarray (FC = 0.43, FC = 0.47, and FC = 0.55, respectively, P = 2.8E-04). There was no correlation between type of control, transfection method, publication year, and silencing efficiency. Although gene silencing is a robust feature successfully cross-validated in the majority of experiments, efficiency remained insufficient in a significant proportion of studies. Selection of cell line model and validation method had the highest influence on silencing proficiency.

Decreased miR-128 and increased miR-21 synergistically cause podocyte injury in sepsis.

PubMed

Wang, Shanshan; Wang, Jun; Zhang, Zengdi; Miao, Hongjun

2017-08-01

Glomerular podocytes are injured in sepsis. We studied, in a sepsis patient, whether microRNAs (miRNAs) play a role in the podocyte injury. Podocytes were cultured and treated with lipopolysaccharide (LPS). Filtration barrier function of podocyte was analyzed with albumin influx assay. Nephrin level was analyzed with reverse transcription polymerase chain reaction (RT-PCR) and western blot. MiRNAs were detected using miRNAs PCR Array and in situ hybridization. MiRNA target sites were evaluated with luciferase reporter assays. LPS impaired the filtration barrier function of podocytes. MiR-128 level was decreased and miR-21 level was increased in podocytes in vitro and in the sepsis patient. The decrease in miR-128 was sufficient to induce the loss of nephrin and the impairment of filtration barrier function, while the increase of miR-21 exacerbated the process. Snail and phosphatase and tensin homolog (PTEN) were identified as the targets of miR-128 and miR-21. Decreased miR-128 induced Snail expression, and the increased miR-21 stabilized Snail by regulating the PTEN/Akt/GSK3β pathway. Supplementation of miR-128 and inhibition of miR-21 suppressed Snail expression and prevented the podocyte injury induced by LPS. Our study suggests that decreased miR-128 and increased miR-21 synergistically cause podocyte injury and are the potential therapeutic targets in sepsis.

Elevated expression of Thoc1 is associated with aggressive phenotype and poor prognosis in colorectal cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Chenchen; Yue, Ben; Yuan, Chenwei

The THO complex 1 (Thoc1) is a nuclear matrix protein playing vital roles in transcription elongation and mRNA export. Recently, aberrant expression of Thoc1 has been reported in an increasing array of tumor types. However, the clinical significance of Thoc1 expression in colorectal cancer (CRC) is still unknown. The present study aimed to characterize the expression of Thoc1 in human CRC and evaluate its clinical significance. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting analyses showed that the mRNA and protein expression of Thoc1 in CRC specimens was significantly higher than that in adjacent normal colon mucosae. Immunohistochemistry (IHC)more » was conducted to characterize the expression pattern of Thoc1 in 185 archived paraffin-embedded CRC specimens. Statistical analyses revealed that high levels of Thoc1 expression were associated with the clinical stages and tumor differentiation. CRC patients with high levels of Thoc1 expression had poorer overall-survival and disease-free survival, whereas those with lower levels of Thoc1 expression survived longer. Furthermore, multivariate Cox regression analyses demonstrated that Thoc1 expression remained an independent prognostic factor for increased disease recurrence and decreased survival. Our results suggest for the first time that Thoc1 is involved in the development and progression of CRC, and elevated expression of Thoc1 is associated with aggressive phenotype and poor prognosis in CRC. These findings may prove to be clinically useful for developing a new therapeutic target of CRC treatment.« less

Analytic solution of field distribution and demagnetization function of ideal hollow cylindrical field source

NASA Astrophysics Data System (ADS)

Xu, Xiaonong; Lu, Dingwei; Xu, Xibin; Yu, Yang; Gu, Min

2017-09-01

The Halbach type hollow cylindrical permanent magnet array (HCPMA) is a volume compact and energy conserved field source, which have attracted intense interests in many practical applications. Here, using the complex variable integration method based on the Biot-Savart Law (including current distributions inside the body and on the surfaces of magnet), we derive analytical field solutions to an ideal multipole HCPMA in entire space including the interior of magnet. The analytic field expression inside the array material is used to construct an analytic demagnetization function, with which we can explain the origin of demagnetization phenomena in HCPMA by taking into account an ideal magnetic hysteresis loop with finite coercivity. These analytical field expressions and demagnetization functions provide deeper insight into the nature of such permanent magnet array systems and offer guidance in designing optimized array system.

A cMUT probe for ultrasound-guided focused ultrasound targeted therapy.

PubMed

Gross, Dominique; Coutier, Caroline; Legros, Mathieu; Bouakaz, Ayache; Certon, Dominique

2015-06-01

Ultrasound-mediated targeted therapy represents a promising strategy in the arsenal of modern therapy. Capacitive micromachined ultrasonic transducer (cMUT) technology could overcome some difficulties encountered by traditional piezoelectric transducers. In this study, we report on the design, fabrication, and characterization of an ultrasound-guided focused ultrasound (USgFUS) cMUT probe dedicated to preclinical evaluation of targeted therapy (hyperthermia, thermosensitive liposomes activation, and sonoporation) at low frequency (1 MHz) with simultaneous ultrasonic imaging and guidance (15 to 20 MHz). The probe embeds two types of cMUT arrays to perform the modalities of targeted therapy and imaging respectively. The wafer-bonding process flow employed for the manufacturing of the cMUTs is reported. One of its main features is the possibility of implementing two different gap heights on the same wafer. All the design and characterization steps of the devices are described and discussed, starting from the array design up to the first in vitro measurements: optical (microscopy) and electrical (impedance) measurements, arrays' electroacoustic responses, focused pressure field mapping (maximum peak-to-peak pressure = 2.5 MPa), and the first B-scan image of a wire-target phantom.

Qualitative assessment of gene expression in affymetrix genechip arrays

NASA Astrophysics Data System (ADS)

Nagarajan, Radhakrishnan; Upreti, Meenakshi

2007-01-01

Affymetrix Genechip microarrays are used widely to determine the simultaneous expression of genes in a given biological paradigm. Probes on the Genechip array are atomic entities which by definition are randomly distributed across the array and in turn govern the gene expression. In the present study, we make several interesting observations. We show that there is considerable correlation between the probe intensities across the array which defy the independence assumption. While the mechanism behind such correlations is unclear, we show that scaling behavior and the profiles of perfect match (PM) as well as mismatch (MM) probes are similar and immune-to-background subtraction. We believe that the observed correlations are possibly an outcome of inherent non-stationarities or patchiness in the array devoid of biological significance. This is demonstrated by inspecting their scaling behavior and profiles of the PM and MM probe intensities obtained from publicly available Genechip arrays from three eukaryotic genomes, namely: Drosophila melanogaster (fruit fly), Homo sapiens (humans) and Mus musculus (house mouse) across distinct biological paradigms and across laboratories, with and without background subtraction. The fluctuation functions were estimated using detrended fluctuation analysis (DFA) with fourth-order polynomial detrending. The results presented in this study provide new insights into correlation signatures of PM and MM probe intensities and suggests the choice of DFA as a tool for qualitative assessment of Affymetrix Genechip microarrays prior to their analysis. A more detailed investigation is necessary in order to understand the source of these correlations.

Fiber-optic microsphere-based arrays for multiplexed biological warfare agent detection.

PubMed

Song, Linan; Ahn, Soohyoun; Walt, David R

2006-02-15

We report a multiplexed high-density DNA array capable of rapid, sensitive, and reliable identification of potential biological warfare agents. An optical fiber bundle containing 6000 individual 3.1-mum-diameter fibers was chemically etched to yield microwells and used as the substrate for the array. Eighteen different 50-mer single-stranded DNA probes were covalently attached to 3.1-mum microspheres. Probe sequences were designed for Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella melitensis, Clostridium botulinum, Vaccinia virus, and one biological warfare agent (BWA) simulant, Bacillus thuringiensis kurstaki. The microspheres were distributed into the microwells to form a randomized multiplexed high-density DNA array. A detection limit of 10 fM in a 50-microL sample volume was achieved within 30 min of hybridization for B. anthracis, Y. pestis, Vaccinia virus, and B. thuringiensis kurstaki. We used both specific responses of probes upon hybridization to complementary targets as well as response patterns of the multiplexed array to identify BWAs with high accuracy. We demonstrated the application of this multiplexed high-density DNA array for parallel identification of target BWAs in spiked sewage samples after PCR amplification. The array's miniaturized feature size, fabrication flexibility, reusability, and high reproducibility may enable this array platform to be integrated into a highly sensitive, specific, and reliable portable instrument for in situ BWA detection.