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Sample records for progression tissue microarray

  1. The Stanford Tissue Microarray Database.

    PubMed

    Marinelli, Robert J; Montgomery, Kelli; Liu, Chih Long; Shah, Nigam H; Prapong, Wijan; Nitzberg, Michael; Zachariah, Zachariah K; Sherlock, Gavin J; Natkunam, Yasodha; West, Robert B; van de Rijn, Matt; Brown, Patrick O; Ball, Catherine A

    2008-01-01

    The Stanford Tissue Microarray Database (TMAD; http://tma.stanford.edu) is a public resource for disseminating annotated tissue images and associated expression data. Stanford University pathologists, researchers and their collaborators worldwide use TMAD for designing, viewing, scoring and analyzing their tissue microarrays. The use of tissue microarrays allows hundreds of human tissue cores to be simultaneously probed by antibodies to detect protein abundance (Immunohistochemistry; IHC), or by labeled nucleic acids (in situ hybridization; ISH) to detect transcript abundance. TMAD archives multi-wavelength fluorescence and bright-field images of tissue microarrays for scoring and analysis. As of July 2007, TMAD contained 205 161 images archiving 349 distinct probes on 1488 tissue microarray slides. Of these, 31 306 images for 68 probes on 125 slides have been released to the public. To date, 12 publications have been based on these raw public data. TMAD incorporates the NCI Thesaurus ontology for searching tissues in the cancer domain. Image processing researchers can extract images and scores for training and testing classification algorithms. The production server uses the Apache HTTP Server, Oracle Database and Perl application code. Source code is available to interested researchers under a no-cost license. PMID:17989087

  2. The Stanford Tissue Microarray Database

    PubMed Central

    Marinelli, Robert J.; Montgomery, Kelli; Liu, Chih Long; Shah, Nigam H.; Prapong, Wijan; Nitzberg, Michael; Zachariah, Zachariah K.; Sherlock, Gavin J.; Natkunam, Yasodha; West, Robert B.; van de Rijn, Matt; Brown, Patrick O.; Ball, Catherine A.

    2008-01-01

    The Stanford Tissue Microarray Database (TMAD; http://tma.stanford.edu) is a public resource for disseminating annotated tissue images and associated expression data. Stanford University pathologists, researchers and their collaborators worldwide use TMAD for designing, viewing, scoring and analyzing their tissue microarrays. The use of tissue microarrays allows hundreds of human tissue cores to be simultaneously probed by antibodies to detect protein abundance (Immunohistochemistry; IHC), or by labeled nucleic acids (in situ hybridization; ISH) to detect transcript abundance. TMAD archives multi-wavelength fluorescence and bright-field images of tissue microarrays for scoring and analysis. As of July 2007, TMAD contained 205 161 images archiving 349 distinct probes on 1488 tissue microarray slides. Of these, 31 306 images for 68 probes on 125 slides have been released to the public. To date, 12 publications have been based on these raw public data. TMAD incorporates the NCI Thesaurus ontology for searching tissues in the cancer domain. Image processing researchers can extract images and scores for training and testing classification algorithms. The production server uses the Apache HTTP Server, Oracle Database and Perl application code. Source code is available to interested researchers under a no-cost license. PMID:17989087

  3. Tissue Microarrays in Clinical Oncology

    PubMed Central

    Voduc, David; Kenney, Challayne; Nielsen, Torsten O.

    2008-01-01

    The tissue microarray is a recently-implemented, high-throughput technology for the analysis of molecular markers in oncology. This research tool permits the rapid assessment of a biomarker in thousands of tumor samples, using commonly available laboratory assays such as immunohistochemistry and in-situ hybridization. Although introduced less than a decade ago, the TMA has proven to be invaluable in the study of tumor biology, the development of diagnostic tests, and the investigation of oncological biomarkers. This review describes the impact of TMA-based research in clinical oncology and its potential future applications. Technical aspects of TMA construction, and the advantages and disadvantages inherent to this technology are also discussed. PMID:18314063

  4. Discovering Biological Progression Underlying Microarray Samples

    PubMed Central

    Qiu, Peng; Gentles, Andrew J.; Plevritis, Sylvia K.

    2011-01-01

    In biological systems that undergo processes such as differentiation, a clear concept of progression exists. We present a novel computational approach, called Sample Progression Discovery (SPD), to discover patterns of biological progression underlying microarray gene expression data. SPD assumes that individual samples of a microarray dataset are related by an unknown biological process (i.e., differentiation, development, cell cycle, disease progression), and that each sample represents one unknown point along the progression of that process. SPD aims to organize the samples in a manner that reveals the underlying progression and to simultaneously identify subsets of genes that are responsible for that progression. We demonstrate the performance of SPD on a variety of microarray datasets that were generated by sampling a biological process at different points along its progression, without providing SPD any information of the underlying process. When applied to a cell cycle time series microarray dataset, SPD was not provided any prior knowledge of samples' time order or of which genes are cell-cycle regulated, yet SPD recovered the correct time order and identified many genes that have been associated with the cell cycle. When applied to B-cell differentiation data, SPD recovered the correct order of stages of normal B-cell differentiation and the linkage between preB-ALL tumor cells with their cell origin preB. When applied to mouse embryonic stem cell differentiation data, SPD uncovered a landscape of ESC differentiation into various lineages and genes that represent both generic and lineage specific processes. When applied to a prostate cancer microarray dataset, SPD identified gene modules that reflect a progression consistent with disease stages. SPD may be best viewed as a novel tool for synthesizing biological hypotheses because it provides a likely biological progression underlying a microarray dataset and, perhaps more importantly, the candidate genes that regulate that progression. PMID:21533210

  5. Automated evaluation and normalization of immunohistochemistry on tissue microarrays with a DNA microarray scanner.

    PubMed

    Haedicke, Wolfgang; Popper, Helmut H; Buck, Charles R; Zatloukal, Kurt

    2003-07-01

    Hundreds of tissue samples may be assembled in a tissue microarray format for simultaneous immunostaining assessment of protein expression profiling. A DNA microarray two-color laser scanner was used for automated analysis of tissue microarray indirect immunofluorescence. On sections from both a human lung adenocarcinoma and a squamous cell carcinoma tissue microarray, fluorescence intensity for two epidermal growth factor receptors (EGFR and c-erbB2) correlates with diagnostic pathologic assessment, indicating that immunohistochemistry quantitation can be achieved. Importantly, double-label indirect immunofluorescence detection with the cDNA scanner demonstrates that one reference antigen can normalize tumor marker immunosignal for the cellular content of tissue microarray tissue cores. Therefore, DNA microarray scanners and associated image analysis software provide general and efficient analysis of tissue microarray immunostaining, including estimation of specific protein expression levels. PMID:12866417

  6. A novel tissue array technique for high-throughput tissue microarray analysis -- microarray groups.

    PubMed

    Jiang, Hui-Yong; Zhang, Xue-Feng; Liu, Li; Li, Hui-Ling; Zhao, Tong

    2007-01-01

    Tissue microarrays are ordered arrays of hundreds to thousands of tissue cores in a single paraffin block. We invented a novel method to make a high-throughput microarray group. Conventional smaller tissue microarrays were made first and then sectioned. Separate paraffin films were arrayed orderly onto a regular-sized glass slide to form a larger microarray group. Sections were not floated in a water bath but, rather, were cut singly using conventional microtome, arrayed orderly onto the glass slide with forceps instead of using a tape-based tissue transfer system, and then unfolded with warm water (46 degrees C) using a micropipette. This not only lowers the difficulty in sectioning but the overall tissue disks can be included in the same section. A microarray group of 2,534 small disks (theoretically, 2,560 disks can be made; 26 fell off during the procedure), the most up to now, was successfully made and may be used in immunohistochemistry, mRNA in situ hybridization, and flourescent in situ hybridization. PMID:17514512

  7. Building "tissue" microarrays from suspension cells.

    PubMed

    Zhao, Shuchun; Natkunam, Yasodha

    2010-01-01

    Tissue microarray (TMA) is a highly efficient method that allows for large-scale measurement of -expression of RNA or protein in multiple tissue sections simultaneously. Most TMAs are made from paraffin--embedded tissues. In this chapter, we detail a method that enables construction of TMAs from small volumes of cells in suspension. A TMA is built using pellets of 1 x 10(6) to 5 x 10(7) spun cells after fixation, processing, and embedding. The entire procedure is carried out in a microcentrifuge tube and yields excellent preservation of cytomorphology and immunoreactivity from both fresh and frozen suspension cells. It is particularly useful for the study of hematopoietic neoplasms presenting in the blood and bone marrow, fine needle aspirates, and body fluids as well as cultured cells. In addition, this versatile method may facilitate the exploration of gene expression profiling and protein expression in clinical trials where regular tissue biopsies are not available. PMID:20690056

  8. Progress in the application of DNA microarrays.

    PubMed Central

    Lobenhofer, E K; Bushel, P R; Afshari, C A; Hamadeh, H K

    2001-01-01

    Microarray technology has been applied to a variety of different fields to address fundamental research questions. The use of microarrays, or DNA chips, to study the gene expression profiles of biologic samples began in 1995. Since that time, the fundamental concepts behind the chip, the technology required for making and using these chips, and the multitude of statistical tools for analyzing the data have been extensively reviewed. For this reason, the focus of this review will be not on the technology itself but on the application of microarrays as a research tool and the future challenges of the field. PMID:11673116

  9. [A novel automatic manufacture device for tissue micro-array].

    PubMed

    Wang, Chaohui; Chen, Chao; Zhang, Qunming; Jiang, Zhuangde; Wang, Teng; Meng, Tao

    2007-10-01

    A novel automatic manufacture device for tissue micro-array is introduced in this paper. Based on the analyses of task and process, the new device prototype is researched and developed. The device consists of a paraffin positioning module and a three-manipulator module. The control system is composed of accurate navigation sub-system, digital image recognition sub-system and punching-filling operating sub-system. The results of experiment demonstrate that the device can accomplish the operations such as image automatic recognition, accurate position, auto-punching and filling. It fulfills the requirements to automatic manufacture of tissue micro-array. PMID:18027675

  10. Automated prostate cancer diagnosis and Gleason grading of tissue microarrays

    NASA Astrophysics Data System (ADS)

    Tabesh, Ali; Kumar, Vinay P.; Pang, Ho-Yuen; Verbel, David; Kotsianti, Angeliki; Teverovskiy, Mikhail; Saidi, Olivier

    2005-04-01

    We present the results on the development of an automated system for prostate cancer diagnosis and Gleason grading. Images of representative areas of the original Hematoxylin-and-Eosin (H&E)-stained tissue retrieved from each patient, either from a tissue microarray (TMA) core or whole section, were captured and analyzed. The image sets consisted of 367 and 268 color images for the diagnosis and Gleason grading problems, respectively. In diagnosis, the goal is to classify a tissue image into tumor versus non-tumor classes. In Gleason grading, which characterizes tumor aggressiveness, the objective is to classify a tissue image as being from either a low- or high-grade tumor. Several feature sets were computed from the image. The feature sets considered were: (i) color channel histograms, (ii) fractal dimension features, (iii) fractal code features, (iv) wavelet features, and (v) color, shape and texture features computed using Aureon Biosciences' MAGIC system. The linear and quadratic Gaussian classifiers together with a greedy search feature selection algorithm were used. For cancer diagnosis, a classification accuracy of 94.5% was obtained on an independent test set. For Gleason grading, the achieved accuracy of classification into low- and high-grade classes of an independent test set was 77.6%.

  11. Microarrays

    ERIC Educational Resources Information Center

    Plomin, Robert; Schalkwyk, Leonard C.

    2007-01-01

    Microarrays are revolutionizing genetics by making it possible to genotype hundreds of thousands of DNA markers and to assess the expression (RNA transcripts) of all of the genes in the genome. Microarrays are slides the size of a postage stamp that contain millions of DNA sequences to which single-stranded DNA or RNA can hybridize. This…

  12. Microarrays

    ERIC Educational Resources Information Center

    Plomin, Robert; Schalkwyk, Leonard C.

    2007-01-01

    Microarrays are revolutionizing genetics by making it possible to genotype hundreds of thousands of DNA markers and to assess the expression (RNA transcripts) of all of the genes in the genome. Microarrays are slides the size of a postage stamp that contain millions of DNA sequences to which single-stranded DNA or RNA can hybridize. This

  13. Fucosyltransferase 8 expression in breast cancer patients: A high throughput tissue microarray analysis.

    PubMed

    Yue, Liling; Han, Cuicui; Li, Zubin; Li, Xin; Liu, Deshui; Liu, Shulin; Yu, Haitao

    2016-05-01

    The aim of this study was to compare the expression of fucosyltransferase 8 (FUT8) in breast cancer tissue and to investigate the relationship between this marker with tumor progression and its applicability to differential diagnosis. An immunohistochemical study was performed for FUT8 using the tissue microarray technique. In addition, the mRNA and protein levels of FUT8 in the tissue were also tested by real-time PCR and Western blot. There was a significant difference in cytoplasmic expression of FUT8 between breast cancer tissue and matched normal tissue (p<0.001). The percent of FUT8 staining in breast cancer tissues ranging from negative, weak positive, positive and strong positive were 2.7%, 40.2%, 54% and 3.2%, respectively. High FUT8 protein expression correlated with lymphatic metastasis (p=0.008) and with stage status (p=0.039). We detected that reduced FUT8 expression correlated with disease-free survival (p=0.02) and overall survival (p=0.04) of breast cancer patients. Expression of FUT8 can stratify breast cancer tissue and may be considered a prognostic marker for breast cancer patients. PMID:26596733

  14. Incidence of Epstein–Barr virus in Syrian women with breast cancer: A tissue microarray study

    PubMed Central

    Aboulkassim, Tahar; Yasmeen, Amber; Akil, Nizar; Batist, Gerald; Moustafa, Ala-Eddin Al

    2015-01-01

    Epstein–Barr virus (EBV) has been recently shown to be present in human breast cancer worldwide, which could play an important role in the initiation and progression of this cancer. In this regard, we aimed to explore the prevalence of EBV in 108 breast cancer tissues from Syrian women using polymerase chain reaction (PCR) and tissue microarray (TMA) analysis. We found that EBV is present in 56 (51.85%) of breast cancers samples. Additionally, we report that the expression of LMP1 gene of EBV is associated with a cancer invasive phenotype in the majority of the cancer samples. These data imply that EBV is present in breast cancer worldwide including Syria and its presence is associated with more aggressive cancer phenotype. Thus, future investigations are needed to elucidate the exact role of EBV in breast carcinogenesis and metastasis. PMID:25933186

  15. Immunoprofile from tissue microarrays to stratify familial breast cancer patients

    PubMed Central

    Schirosi, Laura; De Summa, Simona; Tommasi, Stefania; Paradiso, Angelo; Sambiasi, Domenico; Popescu, Ondina; Simone, Giovanni; Mangia, Anita

    2015-01-01

    Familial breast cancer (BC) is a heterogeneous disease with variable prognosis. The identification of an immunoprofile is important to predict tumor behavior for the routine clinical management of familial BC patients. Using immunohistochemistry on tissue microarrays, we studied 95 familial BCs in order to analyze the expression of some biomarkers involved in different pathways. We used unsupervised hierarchical clustering analyses (HCA), performed using the immunohistochemical score data, to define an immunoprofile able to characterize these tumors. The analyses on 95 and then on a subset of 45 tumors with all biomarkers contemporarily evaluable, revealed the same biomarker and patient clusters. Focusing on the 45 tumors we identified a group of patients characterized by the low expression of estrogen receptor (P = 0.009), progesterone receptor (P < 0.001), BRCA1 (P = 0.005), nuclear Na+/H+ exchanger regulatory factor 1 (NHERF1) (P = 0.026) and hypoxia inducible factor-1 alpha (P < 0.001), and also by the higher expression of MIB1 (P = 0.043), cytoplasmic NHERF1 (P = 0.004), cytoplasmic BRCT-repeat inhibitor of hTERT expression (P = 0.001), vascular endothelial growth factor (VEGF) (P = 0.024) and VEGF receptor-1 (P = 0.029). This immunoprofile identified a more aggressive tumor phenotype associated also with a larger tumor size (P = 0.012) and G3 grade (P = 0.006), confirmed by univariate and multivariate analyses. In conclusion, the clinical application of HCA of immunohistochemical data could allow the assessment of prognostic biomarkers to be used simultaneously. The 10 protein expression panel might be used to identify the more aggressive tumor phenotype in familial BC and to direct patients towards a different clinical therapy. PMID:26312763

  16. Immunohistochemical analysis of breast tissue microarray images using contextual classifiers

    PubMed Central

    McKenna, Stephen J.; Amaral, Telmo; Akbar, Shazia; Jordan, Lee; Thompson, Alastair

    2013-01-01

    Background: Tissue microarrays (TMAs) are an important tool in translational research for examining multiple cancers for molecular and protein markers. Automatic immunohistochemical (IHC) scoring of breast TMA images remains a challenging problem. Methods: A two-stage approach that involves localization of regions of invasive and in-situ carcinoma followed by ordinal IHC scoring of nuclei in these regions is proposed. The localization stage classifies locations on a grid as tumor or non-tumor based on local image features. These classifications are then refined using an auto-context algorithm called spin-context. Spin-context uses a series of classifiers to integrate image feature information with spatial context information in the form of estimated class probabilities. This is achieved in a rotationally-invariant manner. The second stage estimates ordinal IHC scores in terms of the strength of staining and the proportion of nuclei stained. These estimates take the form of posterior probabilities, enabling images with uncertain scores to be referred for pathologist review. Results: The method was validated against manual pathologist scoring on two nuclear markers, progesterone receptor (PR) and estrogen receptor (ER). Errors for PR data were consistently lower than those achieved with ER data. Scoring was in terms of estimated proportion of cells that were positively stained (scored on an ordinal scale of 0-6) and perceived strength of staining (scored on an ordinal scale of 0-3). Average absolute differences between predicted scores and pathologist-assigned scores were 0.74 for proportion of cells and 0.35 for strength of staining (PR). Conclusions: The use of context information via spin-context improved the precision and recall of tumor localization. The combination of the spin-context localization method with the automated scoring method resulted in reduced IHC scoring errors. PMID:23766935

  17. An alternative technology to prepare tissue microarray using frozen tissue samples.

    PubMed

    Hu, Zhongting; Chang, Elbert; Hodeib, Melissa

    2010-01-01

    Although most tissue microarray (TMA) slides are currently made from paraffin-embedded tissues, -frozen clinical tissues are also gradually being used to prepare TMAs. This is because frozen tissues contain better quality RNAs and proteins for profiling gene expressions. Here, we introduce another TMA method that is applicable to a broader range of frozen tissue samples.In this method, an agarose-gel-based array recipient block is first made using several simple instruments. Frozen donor tissues are then manually cored and arrayed into the recipient block array at -10 degrees C. After arraying, the array block can be immediately sectioned on a cryostat microtome to make TMA slides for in situ hybridization and immunocytochemistry studies. TMAs made by this method have well-defined array configurations, good tissue/cell morphology, and well-preserved proteins and mRNAs. This low-cost and time-saving method provides an alternative tool for preparing high quality TMAs for gene expression analyses. PMID:20690055

  18. TMA-Combiner, a simple software tool to permit analysis of replicate cores on tissue microarrays.

    PubMed

    Liu, Chih Long; Montgomery, Kelli D; Natkunam, Yasodha; West, Robert B; Nielsen, Torsten O; Cheang, Maggie C U; Turbin, Dmitry A; Marinelli, Robert J; van de Rijn, Matt; Higgins, John P T

    2005-12-01

    We have previously published a suite of software tools that facilitates the reformulation of tissue microarray (TMA) data so that it may be analyzed using techniques originally devised for analysis of cDNA microarray data. However, current microarray data often feature multiple scores for a given tissue sample and antibody combination. Furthermore, an efficient and systematic method for combining scores that takes into account the differing staining properties of tissue epitopes has not been described. We thus present the TMA-Combiner, a new Microsoft Excel-based macro that permits analysis of data for which tissues may have two or more scores per antibody, and permits combination of data from multiple different tissue microarrays. It accomplishes this by rendering one score per tissue per antibody from two or more scores, using one of multiple user-selectable combination rules developed to account for the differing staining properties of tissue epitopes. This greatly facilitates analysis of tissue microarrays, particularly for users with large repositories of data, and may facilitate discovery of biological trends and help refine diagnostic accuracy of tissue markers in clinical samples. PMID:16258508

  19. Application of new tissue microarrayerZM-1 without recipient paraffin block*

    PubMed Central

    Meng, Pan-qing; Hou, Gang; Zhou, Gui-ying; Peng, Jia-ping; Dong, Qi; Zheng, Shu

    2005-01-01

    The ZM-1 tissue microarrayer designed by our groups is manufactured in stainless steel and brass and contains many features that make TMA (tissue microarray) paraffin blocks construction faster and more convenient. By means of ZM-1 tissue microarrayer, biopsy needles are used to punch the donor tissue specimens respectively. All the needles with the punched specimen cylinders are arrayed into the array-board, with an array of small holes dug to fit the needles. All the specimen cylinders arraying and the TMA paraffin block shaping are finished in only one step so that the specimen cylinders and the paraffin of the TMA block can very easily be incorporated and the recipient paraffin blocks need not be made in advance, and the paraffin used is the same as that for conventional pathology purpose. ZM-1 tissue microarrayer is easy to be manufactured, does not need any precision location system, and so is much cheaper than the currently used instrument. Our methods relatively cheap and simple ZM-1 tissue microarrayer technique of constructing TMA paraffin block may facilitate popularization of the TMA technology. PMID:16130184

  20. Host Tissue and Glycan Binding Specificities of Avian Viral Attachment Proteins Using Novel Avian Tissue Microarrays

    PubMed Central

    Ambepitiya Wickramasinghe, Iresha N.; de Vries, Robert P.; Eggert, Amber M.; Wandee, Nantaporn; de Haan, Cornelis A. M.; Gröne, Andrea; Verheije, Monique H.

    2015-01-01

    The initial interaction between viral attachment proteins and the host cell is a critical determinant for the susceptibility of a host for a particular virus. To increase our understanding of avian pathogens and the susceptibility of poultry species, we developed novel avian tissue microarrays (TMAs). Tissue binding profiles of avian viral attachment proteins were studied by performing histochemistry on multi-species TMA, comprising of selected tissues from ten avian species, and single-species TMAs, grouping organ systems of each species together. The attachment pattern of the hemagglutinin protein was in line with the reported tropism of influenza virus H5N1, confirming the validity of TMAs in profiling the initial virus-host interaction. The previously believed chicken-specific coronavirus (CoV) M41 spike (S1) protein displayed a broad attachment pattern to respiratory tissues of various avian species, albeit with lower affinity than hemagglutinin, suggesting that other avian species might be susceptible for chicken CoV. When comparing tissue-specific binding patterns of various avian coronaviral S1 proteins on the single-species TMAs, chicken and partridge CoV S1 had predominant affinity for the trachea, while pigeon CoV S1 showed marked preference for lung of their respective hosts. Binding of all coronaviral S1 proteins was dependent on sialic acids; however, while chicken CoV S1 preferred sialic acids type I lactosamine (Gal(1-3)GlcNAc) over type II (Gal(1-4)GlcNAc), the fine glycan specificities of pigeon and partridge CoVs were different, as chicken CoV S1-specific sialylglycopolymers could not block their binding to tissues. Taken together, TMAs provide a novel platform in the field of infectious diseases to allow identification of binding specificities of viral attachment proteins and are helpful to gain insight into the susceptibility of host and organ for avian pathogens. PMID:26035584

  1. Microarray Evidences the Role of Pathologic Adipose Tissue in Insulin Resistance and Their Clinical Implications

    PubMed Central

    Mathur, Sandeep Kumar; Jain, Priyanka; Mathur, Prashant

    2011-01-01

    Clustering of insulin resistance and dysmetabolism with obesity is attributed to pathologic adipose tissue. The morphologic hallmarks of this pathology are adipocye hypertrophy and heightened inflammation. However, it's underlying molecular mechanisms remains unknown. Study of gene function in metabolically active tissues like adipose tissue, skeletal muscle and liver is a promising strategy. Microarray is a powerful technique of assessment of gene function by measuring transcription of large number of genes in an array. This technique has several potential applications in understanding pathologic adipose tissue. They are: (1) transcriptomic differences between various depots of adipose tissue, adipose tissue from obese versus lean individuals, high insulin resistant versus low insulin resistance, brown versus white adipose tissue, (2) transcriptomic profiles of various stages of adipogenesis, (3) effect of diet, cytokines, adipokines, hormones, environmental toxins and drugs on transcriptomic profiles, (4) influence of adipokines on transcriptomic profiles in skeletal muscle, hepatocyte, adipose tissue etc., and (5) genetics of gene expression. The microarray evidences of molecular basis of obesity and insulin resistance are presented here. Despite the limitations, microarray has potential clinical applications in finding new molecular targets for treatment of insulin resistance and classification of adipose tissue based on future risk of insulin resistance syndrome. PMID:21603273

  2. Identification of Differentially Expressed IGFBP5-Related Genes in Breast Cancer Tumor Tissues Using cDNA Microarray Experiments

    PubMed Central

    Akkiprik, Mustafa; Peker, İrem; Özmen, Tolga; Güllü Amuran, Gökçe; Güllüoğlu, Bahadır M.; Kaya, Handan; Özer, Ayşe

    2015-01-01

    IGFBP5 is an important regulatory protein in breast cancer progression. We tried to identify differentially expressed genes (DEGs) between breast tumor tissues with IGFBP5 overexpression and their adjacent normal tissues. In this study, thirty-eight breast cancer and adjacent normal breast tissue samples were used to determine IGFBP5 expression by qPCR. cDNA microarrays were applied to the highest IGFBP5 overexpressed tumor samples compared to their adjacent normal breast tissue. Microarray analysis revealed that a total of 186 genes were differentially expressed in breast cancer compared with normal breast tissues. Of the 186 genes, 169 genes were downregulated and 17 genes were upregulated in the tumor samples. KEGG pathway analyses showed that protein digestion and absorption, focal adhesion, salivary secretion, drug metabolism-cytochrome P450, and phenylalanine metabolism pathways are involved. Among these DEGs, the prominent top two genes (MMP11 and COL1A1) which potentially correlated with IGFBP5 were selected for validation using real time RT-qPCR. Only COL1A1 expression showed a consistent upregulation with IGFBP5 expression and COL1A1 and MMP11 were significantly positively correlated. We concluded that the discovery of coordinately expressed genes related with IGFBP5 might contribute to understanding of the molecular mechanism of the function of IGFBP5 in breast cancer. Further functional studies on DEGs and association with IGFBP5 may identify novel biomarkers for clinical applications in breast cancer. PMID:26569312

  3. Microarray-based gene expression profiles in multiple tissues of the domesticated silkworm, Bombyx mori.

    PubMed

    Xia, Qingyou; Cheng, Daojun; Duan, Jun; Wang, Genhong; Cheng, Tingcai; Zha, Xingfu; Liu, Chun; Zhao, Ping; Dai, Fangyin; Zhang, Ze; He, Ningjia; Zhang, Liang; Xiang, Zhonghuai

    2007-01-01

    We designed and constructed a genome-wide microarray with 22,987 70-mer oligonucleotides covering the presently known and predicted genes in the silkworm genome, and surveyed the gene expression in multiple silkworm tissues on day 3 of the fifth instar. Clusters of tissue-prevalent and tissue-specific genes and genes that are differentially expressed in different tissues were identified, and they reflect well major tissue-specific functions on the molecular level. The data presented in this study provide a new resource for annotating the silkworm genome. PMID:17683582

  4. Analysis of methylation microarray for tissue specific detection.

    PubMed

    Muangsub, Tachapol; Samsuwan, Jarunya; Tongyoo, Pumipat; Kitkumthorn, Nakarin; Mutirangura, Apiwat

    2014-12-10

    The role of human DNA methylation has been extensively studied in genomic imprinting, X-inactivation, and disease. However, studies of tissue-specific methylation remain limited. In this study, we use bioinformatics methods to analyze methylation data and reveal loci that are exclusively methylated or unmethylated in individual tissues. We collect 39 previously published DNA methylation profiles using an Illumina® HumanMethylation 27 BeadChip Kit containing 22 common tissues and involving 27,578 CpG loci across the human genome. We found 86 positions of tissue specific methylation CpG (TSM) that encompass 34 hypermethylated TSMs (31 genes) and 52 hypomethylated TSMs (47 genes). Tissues were found to contain 1 to 25 TSM loci, with the majority in the liver (25), testis (18), and brain (16). Fewer TSM loci were found in the muscle (8), ovary (7), adrenal gland (3), pancreas (2-4), kidney, spleen, and stomach (1 each). TSMs are predominantly located 0-300 base pairs in the 3' direction after the transcription start site. Similar to known promoters of methylation, hypermethylated TSM genes suppress transcription, while hypomethylated TSMs allow gene transcription. The majority of hypermethylated TSM genes encode membrane proteins and receptors, while hypomethylated TSM genes primarily encode signal peptides and tissue-specific proteins. In summary, the database of TSM loci produced herein is useful for the selection of tissue-specific DNA markers as diagnostic tools, as well as for the further study of the mechanisms and roles of TSM. PMID:25281015

  5. Microarrays of bladder cancer tissue are highly representative of proliferation index and histological grade.

    PubMed

    Nocito, A; Bubendorf, L; Tinner, E M; Süess, K; Wagner, U; Forster, T; Kononen, J; Fijan, A; Bruderer, J; Schmid, U; Ackermann, D; Maurer, R; Alund, G; Knönagel, H; Rist, M; Anabitarte, M; Hering, F; Hardmeier, T; Schoenenberger, A J; Flury, R; Jäger, P; Fehr, J L; Schraml, P; Moch, H; Mihatsch, M J; Gasser, T; Sauter, G

    2001-07-01

    The number of genes suggested to play a role in cancer biology is rapidly increasing. To be able to test a large number of molecular parameters in sufficiently large series of primary tumours, a tissue microarray (TMA) approach has been developed where samples from up to 1000 tumours can be simultaneously analysed on one glass slide. Because of the small size of the individual arrayed tissue samples (diameter 0.6 mm), the question arises of whether these specimens are representative of their donor tumours. To investigate how representative are the results obtained on TMAs, a set of 2317 bladder tumours that had been previously analysed for histological grade and Ki67 labelling index (LI) was used to construct four replica TMAs from different areas of each tumour. Clinical follow-up information was available from 1092 patients. The histological grade and the Ki67 LI were determined for every arrayed tumour sample (4x2317 analyses each). Despite discrepancies in individual cases, the grade and Ki67 information obtained on minute arrayed samples were highly similar to the data obtained on large sections (p<0.0001). Most importantly, every individual association between grade or Ki67 LI and tumour stage or prognosis (recurrence, progression, tumour-specific survival) that was observed in large section analysis could be fully reproduced on all four replica TMAs. These results show that intra-tumour heterogeneity does not significantly affect the ability to detect clinico-pathological correlations on TMAs, probably because of the large number of tumours that can be included in TMA studies. TMAs are a powerful tool for rapid identification of the biological or clinical significance of molecular alterations in bladder cancer and other tumour types. PMID:11439368

  6. Phosphoprotein Stability in Clinical Tissue and Its Relevance for Reverse Phase Protein Microarray Technology

    PubMed Central

    Espina, Virginia; Mueller, Claudius; Liotta, Lance A.

    2013-01-01

    Phosphorylated proteins reflect the activity of specific cell signaling nodes in biological kinase protein networks. Cell signaling pathways can be either activated or deactivated depending on the phosphorylation state of the constituent proteins. The state of these kinase pathways reflects the in vivo activity of the cells and tissue at any given point in time. As such, cell signaling pathway information can be extrapolated to infer which phosphorylated proteins/pathways are driving an individual tumor’s growth. Reverse Phase Protein Microarrays (RPMA) are a sensitive and precise platform that can be applied to the quantitative measurement of hundreds of phosphorylated signal proteins from a small sample of tissue. Pre-analytical variability originating from tissue procurement and preservation may cause significant variability and bias in downstream molecular analysis. Depending on the ex vivo delay time in tissue processing, and the manner of tissue handling, protein biomarkers such as signal pathway phosphoproteins will be elevated or suppressed in a manner that does not represent the biomarker levels at the time of excision. Consequently, assessment of the state of these kinase networks requires stabilization, or preservation, of the phosphoproteins immediately post tissue procurement. We have employed reverse phase protein microarray analysis of phosphoproteins to study the factors influencing stability of phosphoproteins in tissue following procurement. Based on this analysis we have established tissue procurement guidelines for clinical research with an emphasis on quantifying phosphoproteins by RPMA. PMID:21901591

  7. Optimization of gene expression microarray protocol for formalin-fixed paraffin-embedded tissues

    PubMed Central

    Belder, Nevin; Coşkun, Öznur; Erdoğan, Beyza Doğanay; Savaş, Berna; Ensari, Arzu; Özdağ, Hilal

    2016-01-01

    Formalin-fixed paraffin-embedded (FFPE) tissue is a widely available clinical specimen for retrospective studies. The possibility of long-term clinical follow-up of FFPE samples makes them a valuable source to evaluate links between molecular and clinical information. Working with FFPE samples in the molecular research area, especially using high-throughput molecular techniques such as microarray gene expression profiling, has come into prominence. Because of the harmful effects of formalin fixation process such as degradation of nucleic acids, cross-linking with proteins, and chemical modifications on DNA and RNA, there are some limitations in gene expression profiling studies using FFPE samples. To date many studies have been conducted to evaluate gene expression profiling using microarrays (Thomas et al., Thomas et al. (2013) [1]; Scicchitano et al., Scicchitano et al. (2006) [2]; Frank et al., Frank et al. (2007) [3]; Fedorowicz et al., Fedorowicz et al. (2009) [4]). However, there is still no generally accepted, efficient and standardized procedure for microarray analysis of FFPE samples. This paper describes the microarray data presented in our recently accepted to be published article showing a standard protocol from deparaffinization of FFPE tissue sections and RNA extraction to microarray gene expression analysis. Here we represent our data in detail, deposited in the gene expression omnibus (GEO) database with the accession number GSE73883. Four combinations of two different cRNA/cDNA preparation and labeling protocols with two different array platforms (Affymetrix Human Genome U133 Plus 2.0 and U133_X3P) were evaluated to determine which combination gives the best percentage of present call. The study presents a dataset for comparative analysis which has a potential in terms of providing a robust protocol for gene expression profiling with FFPE tissue samples. PMID:26981433

  8. Optimization of gene expression microarray protocol for formalin-fixed paraffin-embedded tissues.

    PubMed

    Belder, Nevin; Coşkun, Öznur; Erdoğan, Beyza Doğanay; Savaş, Berna; Ensari, Arzu; Özdağ, Hilal

    2016-03-01

    Formalin-fixed paraffin-embedded (FFPE) tissue is a widely available clinical specimen for retrospective studies. The possibility of long-term clinical follow-up of FFPE samples makes them a valuable source to evaluate links between molecular and clinical information. Working with FFPE samples in the molecular research area, especially using high-throughput molecular techniques such as microarray gene expression profiling, has come into prominence. Because of the harmful effects of formalin fixation process such as degradation of nucleic acids, cross-linking with proteins, and chemical modifications on DNA and RNA, there are some limitations in gene expression profiling studies using FFPE samples. To date many studies have been conducted to evaluate gene expression profiling using microarrays (Thomas et al., Thomas et al. (2013) [1]; Scicchitano et al., Scicchitano et al. (2006) [2]; Frank et al., Frank et al. (2007) [3]; Fedorowicz et al., Fedorowicz et al. (2009) [4]). However, there is still no generally accepted, efficient and standardized procedure for microarray analysis of FFPE samples. This paper describes the microarray data presented in our recently accepted to be published article showing a standard protocol from deparaffinization of FFPE tissue sections and RNA extraction to microarray gene expression analysis. Here we represent our data in detail, deposited in the gene expression omnibus (GEO) database with the accession number GSE73883. Four combinations of two different cRNA/cDNA preparation and labeling protocols with two different array platforms (Affymetrix Human Genome U133 Plus 2.0 and U133_X3P) were evaluated to determine which combination gives the best percentage of present call. The study presents a dataset for comparative analysis which has a potential in terms of providing a robust protocol for gene expression profiling with FFPE tissue samples. PMID:26981433

  9. Screening of differentially expressed genes in pathological scar tissues using expression microarray.

    PubMed

    Huang, L P; Mao, Z; Zhang, L; Liu, X X; Huang, C; Jia, Z S

    2015-01-01

    Pathological scar tissues and normal skin tissues were differentiated by screening for differentially expressed genes in pathologic scar tissues via gene expression microarray. The differentially expressed gene data was analyzed by gene ontology and pathway analyses. There were 5001 up- or down-regulated genes in 2-fold differentially expressed genes, 956 up- or down-regulated genes in 5-fold differentially expressed genes, and 114 up- or down-regulated genes in 20-fold differentially expressed genes. Therefore, significant differences were observed in the gene expression in pathological scar tissues and normal foreskin tissues. The development of pathological scar tissues has been correlated to changes in multiple genes and pathways, which are believed to form a dynamic network connection. PMID:26400303

  10. Microarray analysis of the AHR system: Tissue-specific flexibility in signal and target genes

    SciTech Connect

    Frericks, Markus; Meissner, Marc; Esser, Charlotte . E-mail: chesser@uni-duesseldorf.de

    2007-05-01

    Data mining published microarray experiments require that expression profiles are directly comparable. We performed linear global normalization on the data of 1967 Affymetrix U74av2 microarrays, i.e. the transcriptomes of > 100 murine tissues or cell types. The mathematical transformation effectively nullifies inter-experimental or inter-laboratory differences between microarrays. The correctness of expression values was validated by quantitative RT-PCR. Using the database we analyze components of the aryl hydrocarbon receptor (AHR) signaling pathway in various tissues. We identified lineage and differentiation specific variant expression of AHR, ARNT, and HIF1{alpha} in the T-cell lineage and high expression of CYP1A1 in immature B cells and dendritic cells. Performing co-expression analysis we found unorthodox expression of the AHR in the absence of ARNT, particularly in stem cell populations, and can reject the hypothesis that ARNT2 takes over and is highly expressed when ARNT expression is low or absent. Furthermore the AHR shows no co-expression with any other transcript present on the chip. Analysis of differential gene expression under 308 conditions revealed 53 conditions under which the AHR is regulated, numerous conditions under which an intrinsic AHR action is modified as well as conditions activating the AHR even in the absence of known AHR ligands. Thus meta-analysis of published expression profiles is a powerful tool to gain novel insights into known and unknown systems.

  11. Biofunctionalization of surfaces by energetic ion implantation: Review of progress on applications in implantable biomedical devices and antibody microarrays

    NASA Astrophysics Data System (ADS)

    Bilek, Marcela M. M.

    2014-08-01

    Despite major research efforts in the field of biomaterials, rejection, severe immune responses, scar tissue and poor integration continue to seriously limit the performance of today's implantable biomedical devices. Implantable biomaterials that interact with their host via an interfacial layer of active biomolecules to direct a desired cellular response to the implant would represent a major and much sought after improvement. Another, perhaps equally revolutionary, development that is on the biomedical horizon is the introduction of cost-effective microarrays for fast, highly multiplexed screening for biomarkers on cell membranes and in a variety of analyte solutions. Both of these advances will rely on effective methods of functionalizing surfaces with bioactive molecules. After a brief introduction to other methods currently available, this review will describe recently developed approaches that use energetic ions extracted from plasma to facilitate simple, one-step covalent surface immobilization of bioactive molecules. A kinetic theory model of the immobilization process by reactions with long-lived, mobile, surface-embedded radicals will be presented. The roles of surface chemistry and microstructure of the ion treated layer will be discussed. Early progress on applications of this technology to create diagnostic microarrays and to engineer bioactive surfaces for implantable biomedical devices will be reviewed.

  12. Tissue microarrays as a tool in the discovery and validation of predictive biomarkers.

    PubMed

    Hewitt, Stephen M

    2012-01-01

    The tissue microarray (TMA) is the embodiment of high-throughput pathology. The platform combines tens to hundreds of tissue samples on a single microscope slide for interrogation with routine molecular pathology tools. TMAs have enabled the rapid and cost-effective screening of biomarkers for diagnostic, prognostic, and predictive utility. Most commonly applied to the field of oncology, the TMA has accelerated the development of new biomarkers, and is emerging as an essential tool in the discovery and validation of tissue biomarkers for use in personalized medicine. This chapter provides an overview of TMA technology and highlights the advantages of using TMAs as tools toward rapid introduction of new biomarkers for clinical use. PMID:22081347

  13. MALDI imaging mass spectrometry profiling of N-glycans in formalin-fixed paraffin embedded clinical tissue blocks and tissue microarrays.

    PubMed

    Powers, Thomas W; Neely, Benjamin A; Shao, Yuan; Tang, Huiyuan; Troyer, Dean A; Mehta, Anand S; Haab, Brian B; Drake, Richard R

    2014-01-01

    A recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method to spatially profile the location and distribution of multiple N-linked glycan species in frozen tissues has been extended and improved for the direct analysis of glycans in clinically derived formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed tissues from normal mouse kidney, human pancreatic and prostate cancers, and a human hepatocellular carcinoma tissue microarray were processed by antigen retrieval followed by on-tissue digestion with peptide N-glycosidase F. The released N-glycans were detected by MALDI-IMS analysis, and the structural composition of a subset of glycans could be verified directly by on-tissue collision-induced fragmentation. Other structural assignments were confirmed by off-tissue permethylation analysis combined with multiple database comparisons. Imaging of mouse kidney tissue sections demonstrates specific tissue distributions of major cellular N-linked glycoforms in the cortex and medulla. Differential tissue distribution of N-linked glycoforms was also observed in the other tissue types. The efficacy of using MALDI-IMS glycan profiling to distinguish tumor from non-tumor tissues in a tumor microarray format is also demonstrated. This MALDI-IMS workflow has the potential to be applied to any FFPE tissue block or tissue microarray to enable higher throughput analysis of the global changes in N-glycosylation associated with cancers. PMID:25184632

  14. MALDI Imaging Mass Spectrometry Profiling of N-Glycans in Formalin-Fixed Paraffin Embedded Clinical Tissue Blocks and Tissue Microarrays

    PubMed Central

    Powers, Thomas W.; Neely, Benjamin A.; Shao, Yuan; Tang, Huiyuan; Troyer, Dean A.; Mehta, Anand S.; Haab, Brian B.; Drake, Richard R.

    2014-01-01

    A recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method to spatially profile the location and distribution of multiple N-linked glycan species in frozen tissues has been extended and improved for the direct analysis of glycans in clinically derived formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed tissues from normal mouse kidney, human pancreatic and prostate cancers, and a human hepatocellular carcinoma tissue microarray were processed by antigen retrieval followed by on-tissue digestion with peptide N-glycosidase F. The released N-glycans were detected by MALDI-IMS analysis, and the structural composition of a subset of glycans could be verified directly by on-tissue collision-induced fragmentation. Other structural assignments were confirmed by off-tissue permethylation analysis combined with multiple database comparisons. Imaging of mouse kidney tissue sections demonstrates specific tissue distributions of major cellular N-linked glycoforms in the cortex and medulla. Differential tissue distribution of N-linked glycoforms was also observed in the other tissue types. The efficacy of using MALDI-IMS glycan profiling to distinguish tumor from non-tumor tissues in a tumor microarray format is also demonstrated. This MALDI-IMS workflow has the potential to be applied to any FFPE tissue block or tissue microarray to enable higher throughput analysis of the global changes in N-glycosylation associated with cancers. PMID:25184632

  15. Microarray Based Gene Expression Analysis of Murine Brown and Subcutaneous Adipose Tissue: Significance with Human

    PubMed Central

    Boparai, Ravneet K.; Kondepudi, Kanthi Kiran; Mantri, Shrikant; Bishnoi, Mahendra

    2015-01-01

    Background Two types of adipose tissues, white (WAT) and brown (BAT) are found in mammals. Increasingly novel strategies are being proposed for the treatment of obesity and its associated complications by altering amount and/or activity of BAT using mouse models. Methodology/Principle Findings The present study was designed to: (a) investigate the differential expression of genes in LACA mice subcutaneous WAT (sWAT) and BAT using mouse DNA microarray, (b) to compare mouse differential gene expression with previously published human data; to understand any inter- species differences between the two and (c) to make a comparative assessment with C57BL/6 mouse strain. In mouse microarray studies, over 7003, 1176 and 401 probe sets showed more than two-fold, five-fold and ten-fold change respectively in differential expression between murine BAT and WAT. Microarray data was validated using quantitative RT-PCR of key genes showing high expression in BAT (Fabp3, Ucp1, Slc27a1) and sWAT (Ms4a1, H2-Ob, Bank1) or showing relatively low expression in BAT (Pgk1, Cox6b1) and sWAT (Slc20a1, Cd74). Multi-omic pathway analysis was employed to understand possible links between the organisms. When murine two fold data was compared with published human BAT and sWAT data, 90 genes showed parallel differential expression in both mouse and human. Out of these 90 genes, 46 showed same pattern of differential expression whereas the pattern was opposite for the remaining 44 genes. Based on our microarray results and its comparison with human data, we were able to identify genes (targets) (a) which can be studied in mouse model systems to extrapolate results to human (b) where caution should be exercised before extrapolation of murine data to human. Conclusion Our study provides evidence for inter species (mouse vs human) differences in differential gene expression between sWAT and BAT. Critical understanding of this data may help in development of novel ways to engineer one form of adipose tissue to another using murine model with focus on human. PMID:26010905

  16. Production of tissue microarrays, immunohistochemistry staining and digitalization within the human protein atlas.

    PubMed

    Kampf, Caroline; Olsson, Ingmarie; Ryberg, Urban; Sjöstedt, Evelina; Pontén, Fredrik

    2012-01-01

    The tissue microarray (TMA) technology provides the means for high-throughput analysis of multiple tissues and cells. The technique is used within the Human Protein Atlas project for global analysis of protein expression patterns in normal human tissues, cancer and cell lines. Here we present the assembly of 1 mm cores, retrieved from microscopically selected representative tissues, into a single recipient TMA block. The number and size of cores in a TMA block can be varied from approximately forty 2 mm cores to hundreds of 0.6 mm cores. The advantage of using TMA technology is that large amount of data can rapidly be obtained using a single immunostaining protocol to avoid experimental variability. Importantly, only limited amount of scarce tissue is needed, which allows for the analysis of large patient cohorts (1 2). Approximately 250 consecutive sections (4 μm thick) can be cut from a TMA block and used for immunohistochemical staining to determine specific protein expression patterns for 250 different antibodies. In the Human Protein Atlas project, antibodies are generated towards all human proteins and used to acquire corresponding protein profiles in both normal human tissues from 144 individuals and cancer tissues from 216 different patients, representing the 20 most common forms of human cancer. Immunohistochemically stained TMA sections on glass slides are scanned to create high-resolution images from which pathologists can interpret and annotate the outcome of immunohistochemistry. Images together with corresponding pathology-based annotation data are made publically available for the research community through the Human Protein Atlas portal (www.proteinatlas.org) (Figure 1) (3 4). The Human Protein Atlas provides a map showing the distribution and relative abundance of proteins in the human body. The current version contains over 11 million images with protein expression data for 12.238 unique proteins, corresponding to more than 61% of all proteins encoded by the human genome. PMID:22688270

  17. Candidate Genes for Testicular Cancer Evaluated by In Situ Protein Expression Analyses on Tissue Microarrays1

    PubMed Central

    Skotheim, Rolf I; Abeler, Vera M; Nesland, Jahn M; Fosså, Sophie D; Holm, Ruth; Wagner, Urs; Flørenes, Vivi Ann; Aass, Nina; Kallioniemi, Olli P; Lothe, Ragnhild A

    2003-01-01

    Abstract By the use of high-throughput molecular technologies, the number of genes and proteins potentially relevant to testicular germ cell tumor (TGCT) and other diseases will increase rapidly. In a recent transcriptional profiling, we demonstrated the overexpression of GRB7 and JUP in TGCTs, and confirmed the reported overexpression of CCND2. We also have recent evidences for frequent genetic alterations of FHIT and epigenetic alterations of MGMT. To evaluate whether the expression of these genes is related to any clinicopathological variables, we constructed a tissue microarray with 510 testicular tissue cores from 279 patients diagnosed with TGCT, covering various histological subgroups and clinical stages. By immunohistochemistry, we found that JUP, GRB7, and CCND2 proteins were rarely present in normal testis, but frequently expressed at high levels in TGCT. Additionally, all premalignant intratubular germ cell neoplasias were JUP-immunopositive. MGMT and FHIT were expressed by normal testicular tissues, but at significantly lower frequencies in TGCT. Except for CCND2, the expressions of all markers were significantly associated with various TGCT subtypes. In summary, we have developed a high-throughput tool for the evaluation of TGCT markers, and utilized this to validate five candidate genes whose protein expressions were indeed deregulated in TGCT. PMID:14670177

  18. A metadata-aware application for remote scoring and exchange of tissue microarray images

    PubMed Central

    2013-01-01

    Background The use of tissue microarrays (TMA) and advances in digital scanning microscopy has enabled the collection of thousands of tissue images. There is a need for software tools to annotate, query and share this data amongst researchers in different physical locations. Results We have developed an open source web-based application for remote scoring of TMA images, which exploits the value of Microsoft Silverlight Deep Zoom to provide a intuitive interface for zooming and panning around digital images. We use and extend existing XML-based standards to ensure that the data collected can be archived and that our system is interoperable with other standards-compliant systems. Conclusion The application has been used for multi-centre scoring of TMA slides composed of tissues from several Phase III breast cancer trials and ten different studies participating in the International Breast Cancer Association Consortium (BCAC). The system has enabled researchers to simultaneously score large collections of TMA and export the standardised data to integrate with pathological and clinical outcome data, thereby facilitating biomarker discovery. PMID:23635078

  19. The tissue microarray data exchange specification: A document type definition to validate and enhance XML data

    PubMed Central

    Nohle, David G; Ayers, Leona W

    2005-01-01

    Background The Association for Pathology Informatics (API) Extensible Mark-up Language (XML) TMA Data Exchange Specification (TMA DES) proposed in April 2003 provides a community-based, open source tool for sharing tissue microarray (TMA) data in a common format. Each tissue core within an array has separate data including digital images; therefore an organized, common approach to produce, navigate and publish such data facilitates viewing, sharing and merging TMA data from different laboratories. The AIDS and Cancer Specimen Resource (ACSR) is a HIV/AIDS tissue bank consortium sponsored by the National Cancer Institute (NCI) Division of Cancer Treatment and Diagnosis (DCTD). The ACSR offers HIV-related malignancies and uninfected control tissues in microarrays (TMA) accompanied by de-identified clinical data to approved researchers. Exporting our TMA data into the proposed API specified format offers an opportunity to evaluate the API specification in an applied setting and to explore its usefulness. Results A document type definition (DTD) that governs the allowed common data elements (CDE) in TMA DES export XML files was written, tested and evolved and is in routine use by the ACSR. This DTD defines TMA DES CDEs which are implemented in an external file that can be supplemented by internal DTD extensions for locally defined TMA data elements (LDE). Conclusion ACSR implementation of the TMA DES demonstrated the utility of the specification and allowed application of a DTD to validate the language of the API specified XML elements and to identify possible enhancements within our TMA data management application. Improvements to the specification have additionally been suggested by our experience in importing other institution's exported TMA data. Enhancements to TMA DES to remove ambiguous situations and clarify the data should be considered. Better specified identifiers and hierarchical relationships will make automatic use of the data possible. Our tool can be used to reorder data and add identifiers; upgrading data for changes in the specification can be automatically accomplished. Using a DTD (optionally reflecting our proposed enhancements) can provide stronger validation of exported TMA data. PMID:15871741

  20. Tissue microarrays characterise the clinical significance of a VEGF-A protein expression signature in gastrointestinal stromal tumours

    PubMed Central

    Salto-Tellez, M; Nga, M E; Han, H C; Wong, A S-C; Lee, C K; Anuar, D; Ng, S S; Ho, M; Wee, A; Chan, Y H; Soong, R

    2007-01-01

    A tissue microarray analysis of 22 proteins in gastrointestinal stromal tumours (GIST), followed by an unsupervised, hierarchical monothetic cluster statistical analysis of the results, allowed us to detect a vascular endothelial growth factor (VEGF) protein overexpression signature discriminator of prognosis in GIST, and discover novel VEGF-A DNA variants that may have functional significance. PMID:17299397

  1. Microarray Expression Data Identify DCC as a Candidate Gene for Early Meningioma Progression

    PubMed Central

    Schulten, Hans-Juergen; Hussein, Deema; Al-Adwani, Fatima; Karim, Sajjad; Al-Maghrabi, Jaudah; Al-Sharif, Mona; Jamal, Awatif; Al-Ghamdi, Fahad; Baeesa, Saleh S.; Bangash, Mohammed; Chaudhary, Adeel; Al-Qahtani, Mohammed

    2016-01-01

    Meningiomas are the most common primary brain tumors bearing in a minority of cases an aggressive phenotype. Although meningiomas are stratified according to their histology and clinical behavior, the underlying molecular genetics predicting aggressiveness are not thoroughly understood. We performed whole transcript expression profiling in 10 grade I and four grade II meningiomas, three of which invaded the brain. Microarray expression analysis identified deleted in colorectal cancer (DCC) as a differentially expressed gene (DEG) enabling us to cluster meningiomas into DCC low expression (3 grade I and 3 grade II tumors), DCC medium expression (2 grade I and 1 grade II tumors), and DCC high expression (5 grade I tumors) groups. Comparison between the DCC low expression and DCC high expression groups resulted in 416 DEGs (p-value < 0.05; fold change > 2). The most significantly downregulated genes in the DCC low expression group comprised DCC, phosphodiesterase 1C (PDE1C), calmodulin-dependent 70kDa olfactomedin 2 (OLFM2), glutathione S-transferase mu 5 (GSTM5), phosphotyrosine interaction domain containing 1 (PID1), sema domain, transmembrane domain (TM) and cytoplasmic domain, (semaphorin) 6D (SEMA6D), and indolethylamine N-methyltransferase (INMT). The most significantly upregulated genes comprised chromosome 5 open reading frame 63 (C5orf63), homeodomain interacting protein kinase 2 (HIPK2), and basic helix-loop-helix family, member e40 (BHLHE40). Biofunctional analysis identified as predicted top upstream regulators beta-estradiol, TGFB1, Tgf beta complex, LY294002, and dexamethasone and as predicted top regulator effectors NFkB, PIK3R1, and CREBBP. The microarray expression data served also for a comparison between meningiomas from female and male patients and for a comparison between brain invasive and non-invasive meningiomas resulting in a number of significant DEGs and related biofunctions. In conclusion, based on its expression levels, DCC may constitute a valid biomarker to identify those benign meningiomas at risk for progression. PMID:27096627

  2. Validation of tissue microarray for molecular profiling of canine and feline mammary tumours.

    PubMed

    Muscatello, L V; Sarli, G; Beha, G; Asproni, P; Millanta, F; Poli, A; De Tolla, L J; Benazzi, C; Brunetti, B

    2015-01-01

    Tissue microarray (TMA) is a high-throughput method adopted for simultaneous molecular profiling of tissue samples from large patient cohorts. The aim of this study was to validate the TMA method for the molecular classification of canine and feline mammary tumours. Twelve samples, five feline and five canine mammary tumours and two canine haemangiosarcomas, were collected. TMA construction was based on Kononen's method of extracting a cylindrical core of paraffin wax-embedded 'donor' tissue and inserting it into a 'recipient' wax block. Seven consecutive sections from each tissue array block were subjected to immunohistochemistry (IHC) using primary antibodies specific for oestrogen receptor (OR), progesterone receptor (PR), c-erbB-2, cytokeratin (CK) 5/6, CK14, CK19 and p63. The same panel of antibodies was applied to the full sections from all cases. Comparison between full sections and TMA scores revealed different results depending on the antibodies. Labelling for OR, PR, CK19 and p63 showed total concordance, c-erbB2 (score +2, +3) was concordant in nine out of ten cases, CK5/6 and CK14 in eight out of ten cases. The TMA platform preserves the molecular profile of canine and feline mammary tumour markers, representing a useful tool for rapid and cost-effective analysis for the first phenotypic screening using OR, PR and c-erbB2 antibodies. Basal cytokeratin, used for triple negative identification, shows a multifocal 'niche' expression pattern, for which IHC of the full section or multiple core array is recommended. PMID:25670670

  3. Identification of tumor epithelium and stroma in tissue microarrays using texture analysis

    PubMed Central

    2012-01-01

    Background The aim of the study was to assess whether texture analysis is feasible for automated identification of epithelium and stroma in digitized tumor tissue microarrays (TMAs). Texture analysis based on local binary patterns (LBP) has previously been used successfully in applications such as face recognition and industrial machine vision. TMAs with tissue samples from 643 patients with colorectal cancer were digitized using a whole slide scanner and areas representing epithelium and stroma were annotated in the images. Well-defined images of epithelium (n = 41) and stroma (n = 39) were used for training a support vector machine (SVM) classifier with LBP texture features and a contrast measure C (LBP/C) as input. We optimized the classifier on a validation set (n = 576) and then assessed its performance on an independent test set of images (n = 720). Finally, the performance of the LBP/C classifier was evaluated against classifiers based on Haralick texture features and Gabor filtered images. Results The proposed approach using LPB/C texture features was able to correctly differentiate epithelium from stroma according to texture: the agreement between the classifier and the human observer was 97 per cent (kappa value = 0.934, P < 0.0001) and the accuracy (area under the ROC curve) of the LBP/C classifier was 0.995 (CI95% 0.991-0.998). The accuracy of the corresponding classifiers based on Haralick features and Gabor-filter images were 0.976 and 0.981 respectively. Conclusions The method illustrates the capability of automated segmentation of epithelial and stromal tissue in TMAs based on texture features and an SVM classifier. Applications include tissue specific assessment of gene and protein expression, as well as computerized analysis of the tumor microenvironment. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4123422336534537 PMID:22385523

  4. Biomarkers for Refractory Lupus Nephritis: A Microarray Study of Kidney Tissue

    PubMed Central

    Benjachat, Thitima; Tongyoo, Pumipat; Tantivitayakul, Pornpen; Somparn, Poorichaya; Hirankarn, Nattiya; Prom-On, Santitham; Pisitkun, Prapaporn; Leelahavanichkul, Asada; Avihingsanon, Yingyos; Townamchai, Natavudh

    2015-01-01

    The prognosis of severe lupus nephritis (LN) is very different among individual patients. None of the current biomarkers can be used to predict the development of refractory LN. Because kidney histology is the gold standard for diagnosing LN, the authors hypothesize that molecular signatures detected in kidney biopsy tissue may have predictive value in determining the therapeutic response. Sixty-seven patients with biopsy-proven severely active LN by International Society of Nephrology/Renal Pathology Society (ISN/RPS) classification III/IV were recruited. Twenty-three kidney tissue samples were used for RNA microarray analysis, while the remaining 44 samples were used for validation by real-time polymerase chain reaction (PCR) gene expression analysis. From hundreds of differential gene expressions in refractory LN, 12 candidates were selected for validation based on gene expression levels as well as relevant functions. The candidate biomarkers were members of the innate immune response molecules, adhesion molecules, calcium-binding receptors, and paracellular tight junction proteins. S100A8, ANXA13, CLDN19 and FAM46B were identified as the best kidney biomarkers for refractory LN, and COL8A1 was identified as the best marker for early loss of kidney function. These new molecular markers can be used to predict refractory LN and may eventually lead to novel molecular targets for therapy. PMID:26110394

  5. Characterizing the activation of the Wnt signaling pathway in hilar cholangiocarcinoma using a tissue microarray approach.

    PubMed

    Chen, W; Liang, J; Huang, L; Cai, J; Lei, Y; Lai, J; Liang, L; Zhang, K

    2016-01-01

    Hilar cholangiocarcinoma (HCCA) is an invasive hepatic malignancy that is difficult to biopsy; therefore, novel markers of HCCA prognosis are needed. Here, the level of canonical Wnt activation in patients with HCCA, intrahepatic cholangiocarcinoma (IHCC), and congenital choledochal cysts (CCC) was compared to understand the role of Wnt signaling in HCCA. Pathology specimens from HCCA (n=129), IHCC (n=31), and CCC (n=45) patients were used to construct tissue microarrays. Wnt2, Wnt3, β-catenin, TCF4, c-Myc, and cyclin D1 were detected by immunohistochemistry. Parallel correlation analysis was used to analyze differences in protein levels between the HCCA, IHCC, and CCC groups. Univariate and multivariate analyses were used to determine independent predictors of successful resection and prognosis in the HCCA group. The protein levels of Wnt2, β-catenin, TCF4, c-Myc, and cyclin D1 were significantly higher in HCCA compared to IHHC or CCC. Wnt signaling activation (Wnt2+, Wnt3+, nuclear β-catenin+, nuclear TCF4+) was significantly greater in HCCA tissues than CCC tissues. Univariable analyses indicated that expression of cyclin D1 as well as Wnt signaling activation, and partial Wnt activation (Wnt2+ or Wnt3+ and nuclear β-catenin+ or nuclear TCF4+) predicted successful resection, but only cyclin D1 expression remained significant in multivariable analyses. Only partial Wnt activation was an independent predictor of survival time. Proteins in the canonical Wnt signaling pathway were present at higher levels in HCCA and correlated with tumor resecility and patient prognosis. These results suggest that Wnt pathway analysis may be a useful marker for clinical outcome in HCCA. PMID:26972709

  6. Characterizing the Activation of the Wnt Signaling Pathway in Hilar Cholangiocarcinoma Using a Tissue Microarray Approach

    PubMed Central

    Chen, W.; Huang, L.; Liang, J.; Cai, J.; Lei, Y.; Lai, J.; Liang, L.; Zhang, K.

    2016-01-01

    Hilar cholangiocarcinoma (HCCA) is an invasive hepatic malignancy that is difficult to biopsy; therefore, novel markers of HCCA prognosis are needed. Here, the level of canonical Wnt activation in patients with HCCA, intrahepatic cholangiocarcinoma (IHCC), and congenital choledochal cysts (CCC) was compared to understand the role of Wnt signaling in HCCA. Pathology specimens from HCCA (n=129), IHCC (n=31), and CCC (n=45) patients were used to construct tissue microarrays. Wnt2, Wnt3, β-catenin, TCF4, c-Myc, and cyclin D1 were detected by immunohistochemistry. Parallel correlation analysis was used to analyze differences in protein levels between the HCCA, IHCC, and CCC groups. Univariate and multivariate analyses were used to determine independent predictors of successful resection and prognosis in the HCCA group. The protein levels of Wnt2, β-catenin, TCF4, c-Myc, and cyclin D1 were significantly higher in HCCA compared to IHHC or CCC. Wnt signaling activation (Wnt2+, Wnt3+, nuclear β-catenin+, nuclear TCF4+) was significantly greater in HCCA tissues than CCC tissues. Univariable analyses indicated that expression of cyclin D1 as well as Wnt signaling activation, and partial Wnt activation (Wnt2+ or Wnt3+ and nuclear β-catenin+ or nuclear TCF4+) predicted successful resection, but only cyclin D1 expression remained significant in multivariable analyses. Only partial Wnt activation was an independent predictor of survival time. Proteins in the canonical Wnt signaling pathway were present at higher levels in HCCA and correlated with tumor resecility and patient prognosis. These results suggest that Wnt pathway analysis may be a useful marker for clinical outcome in HCCA. PMID:26972709

  7. Development of a microarray for two rice subspecies: characterization and validation of gene expression in rice tissues

    PubMed Central

    2014-01-01

    Background Rice is one of the major crop species in the world helping to sustain approximately half of the global population’s diet especially in Asia. However, due to the impact of extreme climate change and global warming, rice crop production and yields may be adversely affected resulting in a world food crisis. Researchers have been keen to understand the effects of drought, temperature and other environmental stress factors on rice plant growth and development. Gene expression microarray technology represents a key strategy for the identification of genes and their associated expression patterns in response to stress. Here, we report on the development of the rice OneArray® microarray platform which is suitable for two major rice subspecies, japonica and indica. Results The rice OneArray® 60-mer, oligonucleotide microarray consists of a total of 21,179 probes covering 20,806 genes of japonica and 13,683 genes of indica. Through a validation study, total RNA isolated from rice shoots and roots were used for comparison of gene expression profiles via microarray examination. The results were submitted to NCBI’s Gene Expression Omnibus (GEO). Data can be found under the GEO accession number GSE50844 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE50844). A list of significantly differentially expressed genes was generated; 438 shoot-specific genes were identified among 3,138 up-regulated genes, and 463 root-specific genes were found among 3,845 down-regulated genes. GO enrichment analysis demonstrates these results are in agreement with the known physiological processes of the different organs/tissues. Furthermore, qRT-PCR validation was performed on 66 genes, and found to significantly correlate with the microarray results (R = 0.95, p < 0.001***). Conclusion The rice OneArray® 22 K microarray, the first rice microarray, covering both japonica and indica subspecies was designed and validated in a comprehensive study of gene expression in rice tissues. The rice OneArray® microarray platform revealed high specificity and sensitivity. Additional information for the rice OneArray® microarray can be found at http://www.phalanx.com.tw/index.php. PMID:24398116

  8. HER2 in gastric cancer: Comparative analysis of three different antibodies using whole-tissue sections and tissue microarrays

    PubMed Central

    Abraho-Machado, Lucas Faria; Jcome, Alexandre Andrade dos Anjos; Wohnrath, Durval Renato; dos Santos, Jos Sebastio; Carneseca, Estela Cristina; Fregnani, Jos Humberto Tavares Guerreiro; Scapulatempo-Neto, Cristovam

    2013-01-01

    AIM: To compare the performance of three commercially available anti-human epidermalgrowth factor receptor 2 (HER2) antibodies in whole-tissue sections and tissue microarrays (TMAs) of a series of gastric tumors. METHODS: We present a comparative analysis of three anti-HER2 antibodies (HercepTest, 4B5 and SP3) using TMA and whole-tissue sections prepared from the same paraffin blocks of 199 gastric adenocarcinomas operated upon between January 2004 and December 2008 at a Brazilian cancer hospital. The data on the patients age, sex, the anatomical location of the tumor and the Laurens histological classification were collected from clinical and pathological records. The immunohistochemical (IHC) results were examined by two pathologists and the cases were classified as positive (3+), equivocal (2+) and negative (0 or 1+), according to the criteria of the IHC scoring system of gastric cancer. TMAs and whole-tissue sections were evaluated separately and independently. All cases yielding discordant IHC results and/or scored as 2+ were subjected to dual-color in situ hybridization in order to determine the final HER2 status. Besides determining the sensitivity and predictive value for HER2-positive status, we measured the accuracy of each antibody by calculating the area under the receiver operating characteristic (ROC) curve. The agreement between the results obtained using the TMAs and those obtained using the whole-tissue sections was assessed by means of Kappa coefficient. RESULTS: Intratumoral heterogeneity of HER2 expression was observed with all antibodies. HER2-positive expression (3+) in the whole-tissue sections was observed in 23 cases (11.6%) using the 4B5 antibody, in 18 cases (9.1%) using the SP3 antibody and in 10 cases (5.1%) using the HercepTest antibody. In the TMAs, 11 positive cases (5.6%) were identified using SP3 antibody, 9 (4.6%) using the 4B5 antibody and 6 (3%) using the HercepTest antibody. The sensitivity using whole-tissue sections and TMA, respectively, was 95.2% and 42.9% with 4B5, 90.5% and 66.7% with SP3 and 47.6% and 42.9% with HercepTest. The accuracy, calculated from the area under the ROC curve, using whole-tissue sections and TMA, respectively, was 0.91 and 0.79 by 4B5, 0.86 and 0.80 by SP3 and 0.73 and 0.71 by HercepTest. The concordance of the results obtained using whole-tissue sections and TMA was 97.4% (Kappa 0.75) using HercepTest, 85.6% (Kappa 0.56) using SP3 and 84.1% (Kappa 0.38) using 4B5. CONCLUSION: The use of the 4B5 antibody on whole-tissue sections was the most accurate IHC method for evaluating HER2 expression in gastric adenocarcinoma. PMID:24151362

  9. Quantitative analysis of p53 expression in human normal and cancer tissue microarray with global normalization method

    PubMed Central

    Idikio, Halliday A

    2011-01-01

    Tissue microarray based immunohistochemical staining and proteomics are important tools to create and validate clinically relevant cancer biomarkers. Immunohistochemical stains using formalin-fixed tissue microarray sections for protein expression are scored manually and semi-quantitatively. Digital image analysis methods remove some of the drawbacks of manual scoring but may need other methods such as normalization to provide across the board utility. In the present study, quantitative proteomics-based global normalization method was used to evaluate its utility in the analysis of p53 protein expression in mixed human normal and cancer tissue microarray. Global normalization used the mean or median of β-actin to calculate ratios of individual core stain intensities, then log transformed the ratios, calculate a mean or median and subtracted the value from the log of ratios. In the absence of global normalization of p53 protein expression, 44% (42 of 95) of tissue cores were positive using the median of intensity values and 40% (38 of 95) using the mean of intensities as cut-off points. With global normalization, p53 positive cores changed to 20% (19 of 95) when using median of intensities and 15.8%(15 of 95) when the mean of intensities were used. In conclusion, the global normalization method helped to define positive p53 staining in the tissue microarray set used. The method used helped to define clear cut-off points and confirmed all negatively stained tissue cores. Such normalization methods should help to better define clinically useful biomarkers. PMID:21738821

  10. Recent progress in tissue optical clearing.

    PubMed

    Zhu, Dan; Larin, Kirill V; Luo, Qingming; Tuchin, Valery V

    2013-09-01

    Tissue optical clearing technique provides a prospective solution for the application of advanced optical methods in life sciences. This paper gives a review of recent developments in tissue optical clearing techniques. The physical, molecular and physiological mechanisms of tissue optical clearing are overviewed and discussed. Various methods for enhancing penetration of optical-clearing agents into tissue, such as physical methods, chemical-penetration enhancers and combination of physical and chemical methods are introduced. Combining the tissue optical clearing technique with advanced microscopy image or labeling technique, applications for 3D microstructure of whole tissues such as brain and central nervous system with unprecedented resolution are demonstrated. Moreover, the difference in diffusion and/or clearing ability of selected agents in healthy versus pathological tissues can provide a highly sensitive indicator of the tissue health/pathology condition. Finally, recent advances in optical clearing of soft or hard tissue for in vivo imaging and phototherapy are introduced. [Formula: see text]. PMID:24348874

  11. Recent progress in tissue optical clearing

    PubMed Central

    Zhu, Dan; Larin, Kirill V; Luo, Qingming; Tuchin, Valery V

    2013-01-01

    Tissue optical clearing technique provides a prospective solution for the application of advanced optical methods in life sciences. This paper gives a review of recent developments in tissue optical clearing techniques. The physical, molecular and physiological mechanisms of tissue optical clearing are overviewed and discussed. Various methods for enhancing penetration of optical-clearing agents into tissue, such as physical methods, chemical-penetration enhancers and combination of physical and chemical methods are introduced. Combining the tissue optical clearing technique with advanced microscopy image or labeling technique, applications for 3D microstructure of whole tissues such as brain and central nervous system with unprecedented resolution are demonstrated. Moreover, the difference in diffusion and/or clearing ability of selected agents in healthy versus pathological tissues can provide a highly sensitive indicator of the tissue health/pathology condition. Finally, recent advances in optical clearing of soft or hard tissue for in vivo imaging and phototherapy are introduced. PMID:24348874

  12. Selective invocation of shape priors for deformable segmentation and morphologic classification of prostate cancer tissue microarrays.

    PubMed

    Ali, Sahirzeeshan; Veltri, Robert; Epstein, Jonathan I; Christudass, Christhunesa; Madabhushi, Anant

    2015-04-01

    Shape based active contours have emerged as a natural solution to overlap resolution. However, most of these shape-based methods are computationally expensive. There are instances in an image where no overlapping objects are present and applying these schemes results in significant computational overhead without any accompanying, additional benefit. In this paper we present a novel adaptive active contour scheme (AdACM) that combines boundary and region based energy terms with a shape prior in a multi level set formulation. To reduce the computational overhead, the shape prior term in the variational formulation is only invoked for those instances in the image where overlaps between objects are identified; these overlaps being identified via a contour concavity detection scheme. By not having to invoke all three terms (shape, boundary, region) for segmenting every object in the scene, the computational expense of the integrated active contour model is dramatically reduced, a particularly relevant consideration when multiple objects have to be segmented on very large histopathological images. The AdACM was employed for the task of segmenting nuclei on 80 prostate cancer tissue microarray images from 40 patient studies. Nuclear shape based, architectural and textural features extracted from these segmentations were extracted and found to able to discriminate different Gleason grade patterns with a classification accuracy of 86% via a quadratic discriminant analysis (QDA) classifier. On average the AdACM model provided 60% savings in computational times compared to a non-optimized hybrid active contour model involving a shape prior. PMID:25466771

  13. Selective Invocation of Shape Priors for Deformable Segmentation and Morphologic Classification of Prostate Cancer Tissue Microarrays

    PubMed Central

    Ali, Sahirzeeshan; Veltri, Robert; Epstein, Jonathan A.; Christudass, Christhunesa; Madabhushi, Anant

    2015-01-01

    Shape based active contours have emerged as a natural solution to overlap resolution. However, most of these shape-based methods are computationally expensive. There are instances in an image where no overlapping objects are present and applying these schemes results in significant computational overhead without any accompanying, additional benefit. In this paper we present a novel adaptive active contour scheme (AdACM) that combines boundary and region based energy terms with a shape prior in a multi level set formulation. To reduce the computational overhead, the shape prior term in the variational formulation is only invoked for those instances in the image where overlaps between objects are identified; these overlaps being identified via a contour concavity detection scheme. By not having to invoke all 3 terms (shape, boundary, region) for segmenting every object in the scene, the computational expense of the integrated active contour model is dramatically reduced, a particularly relevant consideration when multiple objects have to be segmented on very large histopathological images. The AdACM was employed for the task of segmenting nuclei on 80 prostate cancer tissue microarray images from 40 patient studies. Nuclear shape based, architectural and textural features extracted from these segmentations were extracted and found to able to discriminate different Gleason grade patterns with a classification accuracy of 86% via a quadratic discriminant analysis (QDA) classifier. On average the AdACM model provided 60% savings in computational times compared to a non-optimized hybrid active contour model involving a shape prior. PMID:25466771

  14. Hierarchical normalized cuts: unsupervised segmentation of vascular biomarkers from ovarian cancer tissue microarrays.

    PubMed

    Janowczyk, Andrew; Chandran, Sharat; Singh, Rajendra; Sasaroli, Dimitra; Coukos, George; Feldman, Michael D; Madabhushi, Anant

    2009-01-01

    Research has shown that tumor vascular markers (TVMs) may serve as potential OCa biomarkers for prognosis prediction. One such TVM is ESM-1, which can be visualized by staining ovarian Tissue Microarrays (TMA) with an antibody to ESM-1. The ability to quickly and quantitatively estimate vascular stained regions may yield an image based metric linked to disease survival and outcome. Automated segmentation of the vascular stained regions on the TMAs, however, is hindered by the presence of spuriously stained false positive regions. In this paper, we present a general, robust and efficient unsupervised segmentation algorithm, termed Hierarchical Normalized Cuts (HNCut), and show its application in precisely quantifying the presence and extent of a TVM on OCa TMAs. The strength of HNCut is in the use of a hierarchically represented data structure that bridges the mean shift (MS) and the normalized cuts (NCut) algorithms. This allows HNCut to efficiently traverse a pyramid of the input image at various color resolutions, efficiently and accurately segmenting the object class of interest (in this case ESM-1 vascular stained regions) by simply annotating half a dozen pixels belonging to the target class. Quantitative and qualitative analysis of our results, using 100 pathologist annotated samples across multiple studies, prove the superiority of our method (sensitivity 81%, Positive predictive value (PPV), 80%) versus a popular supervised learning technique, Probabilistic Boosting Trees (sensitivity, PPV of 76% and 66%). PMID:20425992

  15. Comparative microarray analyses of adult female midgut tissues from feeding Rhipicephalus species.

    PubMed

    van Zyl, Willem A; Stutzer, Christian; Olivier, Nicholas A; Maritz-Olivier, Christine

    2015-02-01

    The cattle tick, Rhipicephalus microplus, has a debilitating effect on the livestock industry worldwide, owing to its being a vector of the causative agents of bovine babesiosis and anaplasmosis. In South Africa, co-infestation with R. microplus and R. decoloratus, a common vector species on local livestock, occurs widely in the northern and eastern parts of the country. An alternative to chemical control methods is sought in the form of a tick vaccine to control these tick species. However, sequence information and transcriptional data for R. decoloratus is currently lacking. Therefore, this study aimed at identifying genes that are shared between midgut tissues of feeding adult female R. microplus and R. decoloratus ticks. In this regard, a custom oligonucleotide microarray comprising of 13,477 R. microplus sequences was used for transcriptional profiling and 2476 genes were found to be shared between these Rhipicephalus species. In addition, 136 transcripts were found to be more abundantly expressed in R. decoloratus and 1084 in R. microplus. Chi-square analysis revealed that genes involved in lipid transport and metabolism are significantly overrepresented in R. microplus and R. decoloratus. This study is the first transcriptional profiling of R. decoloratus and is an additional resource that can be evaluated further in future studies for possible tick control. PMID:25448423

  16. Laser scanning cytometry and tissue microarray analysis of salinity effects on killifish chloride cells.

    PubMed

    Lima, Raquel N; Kültz, Dietmar

    2004-04-01

    The effects of salinity on chloride cells (CC) and Na(+)/K(+)-ATPase content in gill epithelium of euryhaline killifish Fundulus heteroclitus were analyzed using laser scanning cytometry (LSC) and tissue microarrays (TMAs). Salinity acclimations consisted of acute transfer from freshwater (FW) to 1x seawater (SW) and gradual transfer from FW to 2.4x SW. Suspensions of dissociated gill epithelial cells were stained with DASPMI and evaluated using LSC. CC number and volume are proportional to external salinity, being lower in FW (0.5+/-0.2 x 10(5) and 405+/-32 micro m(3), respectively) and higher after 5 weeks in 2.4x SW (3.7+/-0.9 x 10(5) and 2697+/-146 micro m(3), respectively). TMAs were constructed from fixed gill tissues and developed using antibody for Na(+)/K(+)-ATPase to visualize CCs in situ and compare their characteristics with isolated CCs. Na(+)/K(+)-ATPase content per CC increases transiently (from 2.2+/-0.5 x 10(6) to 4.8+/-1.1 x 10(6) relative fluorescence units, RFU) after 1 week of acute acclimation to 1x SW but returns to baseline values (2.4+/-0.5 x 10(6) RFU) within 5 weeks. In contrast, gradual acclimation to 2.4x SW permanently increases Na(+)/K(+)-ATPase content per CC (from 2.0+/-0.8 x 10(6) to 6.7+/-2.7 x 10(6) RFU after 5 weeks). CC size in situ did not correlate well to salinity because of basolateral membrane infoldings. Taken together, these data suggest that euryhaline fishes are capable of sensing environmental salinity to utilize transient short-term and permanent long-term adaptations for coping with salinity changes. These results also demonstrate the power of LSC and TMA for comparative biology. PMID:15073205

  17. Tissue MicroArray: a distributed Grid approach for image analysis.

    PubMed

    Viti, Federica; Merelli, Ivan; Galizia, Antonella; D'Agostino, Daniele; Clematis, Andrea; Milanesi, Luciano

    2007-01-01

    The Tissue MicroArray (TMA) technique is assuming even more importance. Digital images acquisition becomes fundamental to provide an automatic system for subsequent analysis. The accuracy of the results depends on the image resolution, which has to be very high in order to provide as many details as possible. Lossless formats are more suitable to bring information, but data file size become a critical factor researchers have to deal with. This affects not only storage methods but also computing times and performances. Pathologists and researchers who work with biological tissues, in particular with the TMA technique, need to consider a large number of case studies to formulate and validate their hypotheses. It is clear the importance of image sharing between different institutes worldwide to increase the amount of interesting data to work with. In this context, preserving the security of sensitive data is a fundamental issue. In most of the cases copying patient data in places different from the original database is forbidden by the owner institutes. Storage, computing and security are key problems of TMA methodology. In our system we tackle all these aspects using the EGEE (Enabling Grids for E-sciencE) Grid infrastructure. The Grid platform provides good storage, performance in image processing and safety of sensitive patient information: this architecture offers hundreds of Storage and Computing Elements and enables users to handle images without copying them to physical disks other than where they have been archived by the owner, giving back to end-users only the processed anonymous images. The efficiency of the TMA analysis process is obtained implementing algorithms based on functions provided by the Parallel IMAge processing Genoa Library (PIMA(GE)2 Lib). The acquisition of remotely distributed TMA images is made using specialized I/O functions based on the Grid File Access Library (GFAL) API. In our opinion this approach may represent important contribution to tele-pathology development. PMID:17476071

  18. HER2 status in gastroesophageal cancer: a tissue microarray study of 1040 cases.

    PubMed

    Cappellesso, Rocco; Fassan, Matteo; Hanspeter, Esther; Bornschein, Jan; d'Amore, Emanuele S G; Cuorvo, Lucia V; Mazzoleni, Guido; Barbareschi, Mattia; Pizzi, Marco; Guzzardo, Vincenza; Malfertheiner, Peter; Micev, Marjan; Guido, Maria; Giacomelli, Luciano; Tsukanov, Vladislav V; Zagonel, Vittorina; Nitti, Donato; Rugge, Massimo

    2015-05-01

    Among patients with gastric cancer (GC) and gastroesophageal cancer (G-EC), HER2 amplification identifies those who may benefit from trastuzumab. HER2 status assessment, however, is influenced by preanalytic, analytic, and postanalytic variables. In a series of 5426 microarray cancer tissue cores obtained from 1040 GC/G-ECs (824 GC, 216 G-EC) and 720 synchronous nodal metastases, we evaluated both the performances of 2 different immunohistochemistry (IHC) protocols and the HER2 status intratumor variability. The prevalence of HER2 amplification and protein overexpression were assessed by chromogenic in situ hybridization and by 2 IHC protocols (CB11 and 4B5). HER2 was amplified in 114 (11%) of 1040 cases; in 6 (5.3%) of 114 cases, gene amplification only involved nodal metastasis. HER2 amplification prevailed in intestinal-type (P = .001) and low-grade (P < .001) tumors, showing no correlation with patients' age/sex, tumor location, stage, and Ming histotype. Overall, 12.5% and 13.7% of cases IHC scored 2+/3+ using the CB11-IHC and the 4B5-IHC protocol, respectively. HER2 amplification was not associated with protein overexpression (score 0/1+) in 11.4% and 6.2% of cases using the CB11-IHC and the 4B5-IHC protocol, respectively. The 4B5-IHC protocol proved more sensitive than CB11-IHC (93.9% versus 88.6%) and just as specific (96.1% versus 96.9%). Tested by chromogenic in situ hybridization, intratumor HER2 status was "substantially" consistent in different tissue cores obtained from the same case (? = 0.78). Similar results were obtained for HER2 protein expression (CB11-IHC, ? = 0.78, and 4B5-IHC, ? = 0.83). Immunohistochemistry testing, however, fails in identifying about 10% of HER2-amplified cancers, potentially excluding these patients from anti-HER2 therapy. PMID:25800719

  19. The tissue micro-array data exchange specification: a web based experience browsing imported data

    PubMed Central

    Nohle, David G; Hackman, Barbara A; Ayers, Leona W

    2005-01-01

    Background The AIDS and Cancer Specimen Resource (ACSR) is an HIV/AIDS tissue bank consortium sponsored by the National Cancer Institute (NCI) Division of Cancer Treatment and Diagnosis (DCTD). The ACSR offers to approved researchers HIV infected biologic samples and uninfected control tissues including tissue cores in micro-arrays (TMA) accompanied by de-identified clinical data. Researchers interested in the type and quality of TMA tissue cores and the associated clinical data need an efficient method for viewing available TMA materials. Because each of the tissue samples within a TMA has separate data including a core tissue digital image and clinical data, an organized, standard approach to producing, navigating and publishing such data is necessary. The Association for Pathology Informatics (API) extensible mark-up language (XML) TMA data exchange specification (TMA DES) proposed in April 2003 provides a common format for TMA data. Exporting TMA data into the proposed format offers an opportunity to implement the API TMA DES. Using our public BrowseTMA tool, we created a web site that organizes and cross references TMA lists, digital "virtual slide" images, TMA DES export data, linked legends and clinical details for researchers. Microsoft Excel and Microsoft Word are used to convert tabular clinical data and produce an XML file in the TMA DES format. The BrowseTMA tool contains Extensible Stylesheet Language Transformation (XSLT) scripts that convert XML data into Hyper-Text Mark-up Language (HTML) web pages with hyperlinks automatically added to allow rapid navigation. Results Block lists, virtual slide images, legends, clinical details and exports have been placed on the ACSR web site for 14 blocks with 1623 cores of 2.0, 1.0 and 0.6 mm sizes. Our virtual microscope can be used to view and annotate these TMA images. Researchers can readily navigate from TMA block lists to TMA legends and to clinical details for a selected tissue core. Exports for 11 blocks with 3812 cores from three other institutions were processed with the BrowseTMA tool. Fifty common data elements (CDE) from the TMA DES were used and 42 more created for site-specific data. Researchers can download TMA clinical data in the TMA DES format. Conclusion Virtual TMAs with clinical data can be viewed on the Internet by interested researchers using the BrowseTMA tool. We have organized our approach to producing, sorting, navigating and publishing TMA information to facilitate such review. We have converted Excel TMA data into TMA DES XML, and imported it and TMA DES XML from another institution into BrowseTMA to produce web pages that allow us to browse through the merged data. We proposed enhancements to the TMA DES as a result of this experience. We implemented improvements to the API TMA DES as a result of using exported data from several institutions. A document type definition was written for the API TMA DES (that optionally includes proposed enhancements). Independent validators can be used to check exports against the DTD (with or without the proposed enhancements). Linking tissue core images to readily navigable clinical data greatly improves the value of the TMA. PMID:16086837

  20. A Comprehensive Inter-Tissue Crosstalk Analysis Underlying Progression and Control of Obesity and Diabetes

    PubMed Central

    Samdani, Pawan; Singhal, Meet; Sinha, Neeraj; Tripathi, Parul; Sharma, Sachin; Tikoo, Kamiya; Rao, Kanury V. S.; Kumar, Dhiraj

    2015-01-01

    Obesity is a metabolic state associated with excess of positive energy balance. While adipose tissues are considered the major contributor for complications associated with obesity, they influence a variety of tissues and inflict significant metabolic and inflammatory alterations. Unfortunately, the communication network between different cell-types responsible for such systemic alterations has been largely unexplored. Here we study the inter-tissue crosstalk during progression and cure of obesity using multi-tissue gene expression data generated through microarray analysis. We used gene expression data sets from 10 different tissues from mice fed on high-fat-high-sugar diet (HFHSD) at various stages of disease development and applied a novel analysis algorithm to deduce the tissue crosstalk. We unravel a comprehensive network of inter-tissue crosstalk that emerges during progression of obesity leading to inflammation and insulin resistance. Many of the crosstalk involved interactions between well-known modulators of obesity and associated pathology like inflammation. We then used similar datasets from mice that in addition to HFHSD were also administered with a herbal concoction known to circumvent the effects of HFHSD in the diet induced model of obesity in mice. We propose, the analysis presented here could be applied to understand systemic details of several chronic diseases. PMID:26202695

  1. Illumina next generation sequencing data and expression microarrays data from retinoblastoma and medulloblastoma tissues.

    PubMed

    García-Chequer, A J; Méndez-Tenorio, A; Olguín-López, G; Sánchez-Vallejo, C; Isa, P; Arias, C F; Torres, J; Hernández-Angeles, A; Ramírez-Ortiz, M A; Lara, C; Cabrera-Muñoz, Ma de L; Sadowinski-Pine, S; Bravo-Ortiz, J C; Ramón-García, G; Diegopérez-Ramírez, J; Ramírez-Reyes, G; Casarrubias-Islas, R; Ramírez, J; Orjuela, M; Ponce-Castañeda, M V

    2016-03-01

    Retinoblastoma (Rb) is a pediatric intraocular malignancy and probably the most robust clinical model on which genetic predisposition to develop cancer has been demonstrated. Since deletions in chromosome 13 have been described in this tumor, we performed next generation sequencing to test whether recurrent losses could be detected in low coverage data. We used Illumina platform for 13 tumor tissue samples: two pools of 4 retinoblastoma cases each and one pool of 5 medulloblastoma cases (raw data can be found at http://www.ebi.ac.uk/ena/data/view/PRJEB6630). We first created an in silico reference profile generated from a human sequenced genome (GRCh37p5). From this data we calculated an integrity score to get an overview of gains and losses in all chromosomes; we next analyzed each chromosome in windows of 40 kb length, calculating for each window the log2 ratio between reads from tumor pool and in silico reference. Finally we generated panoramic maps with all the windows whether lost or gained along each chromosome associated to its cytogenetic bands to facilitate interpretation. Expression microarrays was done for the same samples and a list of over and under expressed genes is presented here. For this detection a significance analysis was done and a log2 fold change was chosen as significant (raw data can be found at http://www.ncbi.nlm.nih.gov/geo/accession number GSE11488). The complete research article can be found at Cancer Genetics journal (Garcia-Chequer et al., in press) [1]. In summary here we provide an overview with visual graphics of gains and losses chromosome by chromosome in retinoblastoma and medulloblastoma, also the integrity score analysis and a list of genes with relevant expression associated. This material can be useful to researchers that may want to explore gains and losses in other malignant tumors with this approach or compare their data with retinoblastoma. PMID:26937470

  2. Illumina next generation sequencing data and expression microarrays data from retinoblastoma and medulloblastoma tissues

    PubMed Central

    García-Chequer, A.J.; Méndez-Tenorio, A.; Olguín-López, G.; Sánchez-Vallejo, C.; Isa, P.; Arias, C.F.; Torres, J.; Hernández-Angeles, A.; Ramírez-Ortiz, M.A.; Lara, C.; Cabrera-Muñoz, Ma.de.L.; Sadowinski-Pine, S.; Bravo-Ortiz, J.C.; Ramón-García, G.; Diegopérez-Ramírez, J.; Ramírez-Reyes, G.; Casarrubias-Islas, R.; Ramírez, J.; Orjuela, M.; Ponce-Castañeda, M.V.

    2016-01-01

    Retinoblastoma (Rb) is a pediatric intraocular malignancy and probably the most robust clinical model on which genetic predisposition to develop cancer has been demonstrated. Since deletions in chromosome 13 have been described in this tumor, we performed next generation sequencing to test whether recurrent losses could be detected in low coverage data. We used Illumina platform for 13 tumor tissue samples: two pools of 4 retinoblastoma cases each and one pool of 5 medulloblastoma cases (raw data can be found at http://www.ebi.ac.uk/ena/data/view/PRJEB6630). We first created an in silico reference profile generated from a human sequenced genome (GRCh37p5). From this data we calculated an integrity score to get an overview of gains and losses in all chromosomes; we next analyzed each chromosome in windows of 40 kb length, calculating for each window the log2 ratio between reads from tumor pool and in silico reference. Finally we generated panoramic maps with all the windows whether lost or gained along each chromosome associated to its cytogenetic bands to facilitate interpretation. Expression microarrays was done for the same samples and a list of over and under expressed genes is presented here. For this detection a significance analysis was done and a log2 fold change was chosen as significant (raw data can be found at http://www.ncbi.nlm.nih.gov/geo/accession number GSE11488). The complete research article can be found at Cancer Genetics journal (Garcia-Chequer et al., in press) [1]. In summary here we provide an overview with visual graphics of gains and losses chromosome by chromosome in retinoblastoma and medulloblastoma, also the integrity score analysis and a list of genes with relevant expression associated. This material can be useful to researchers that may want to explore gains and losses in other malignant tumors with this approach or compare their data with retinoblastoma. PMID:26937470

  3. A Comparison between Manual and Automated Evaluations of Tissue Microarray Patterns of Protein Expression

    PubMed Central

    Alvarenga, Arthur W.; Coutinho-Camillo, Claudia M.; Rodrigues, Bruna R.; Rocha, Rafael M.; Torres, Luiz Fernando B.; Martins, Vilma R.; da Cunha, Isabela W.

    2013-01-01

    Tissue microarray technology enables us to evaluate the pattern of protein expression in large numbers of samples. However, manual data acquisition and analysis still represent a challenge because they are subjective and time-consuming. Automated analysis may thus increase the speed and reproducibility of evaluation. However, the reliability of automated analysis systems should be independently evaluated. Herein, the expression of phosphorylated AKT and mTOR was determined by ScanScope XT (Aperio; Vista, CA) and ACIS III (Dako; Glostrup, Denmark) and compared with the manual analysis by two observers. The percentage of labeled pixels or nuclei analysis had a good correlation between human observers and automated systems (κ = 0.855 and 0.879 for ScanScope vs. observers and κ = 0.765 and 0.793 for ACIS III vs. observers). The intensity of labeling determined by ScanScope was also correlated with that found by the human observers (correlation index of 0.946 and 0.851 for pAKT and 0.851 and 0.875 for pmTOR). However, the correlation between ACIS III and human observation varied for labeling intensity and was considered poor in some cases (correlation index of 0.718 and 0.680 for pAKT and 0.223 and 0.225 for pmTOR). Thus, the percentage of positive pixels or nuclei determination was satisfactorily performed by both systems; however, labeling intensity was better identified by ScanScope XT. PMID:23340270

  4. Simultaneous EGFR and VEGF Alterations in Non-Small Cell Lung Carcinoma Based on Tissue Microarrays

    PubMed Central

    Tsiambas, Evangelos; Stamatelopoulos, Athanasios; Karameris, Andreas; Panagiotou, Ioannis; Rigopoulos, Dimitrios; Chatzimichalis, Antonios; Bouros, Demosthenes; Patsouris, Efstratios

    2007-01-01

    Background: Epidermal growth factor receptor (EGFR) overexpression is observed in significant proportions of non-small cell lung carcinomas (NSCLC). Furthermore, overactivation of vascular endothelial growth factor (VEGF) leads to increased angiogenesis implicated as an important factor in vascularization of those tumors. Patients and Methods: Using tissue microarray technology, forty-paraffin (n = 40) embedded, histologically confirmed primary NSCLCs were cored and re-embedded into a recipient block. Immunohistochemistry was performed for the determination of EGFR and VEGF protein levels which were evaluated by the performance of computerized image analysis. EGFR gene amplification was studied by chromogenic in situ hybridization based on the use of EGFR gene and chromosome 7 centromeric probes. Results: EGFR overexpression was observed in 23/40 (57.5%) cases and was correlated to the stage of the tumors (p = 0.001), whereas VEGF was overexpressed in 35/40 (87.5%) cases and was correlated to the stage of the tumors (p = 0.005) and to the smoking history of the patients (p = 0.016). Statistical significance was assessed comparing the protein levels of EGFR and VEGF (p = 0.043, k = 0.846). EGFR gene amplification was identified in 2/40 (5%) cases demonstrating no association to its overall protein levels (p = 0.241), whereas chromosome 7 aneuploidy was detected in 7/40 (17.5%) cases correlating to smoking history of the patients (p = 0.013). Conclusions: A significant subset of NSCLC is characterized by EGFR and VEGF simultaneous overexpression and maybe this is the eligible target group for the application of combined anti-EGFR/VEGF targeted therapies at the basis of genetic deregulation (especially gene amplification for EGFR). PMID:19455247

  5. Tissue microarray analysis of connexin expression and its prognostic significance in human breast cancer.

    PubMed

    Conklin, Chris; Huntsman, David; Yorida, Erika; Makretsov, Nikita; Turbin, Dmitry; Bechberger, John F; Sin, Wun Chey; Naus, Christian C

    2007-10-01

    Breast cancer accounts for approximately 15% of all cancer deaths. Currently, axillary nodal status is the most reliable prognostic indicator for breast cancer. Tumor size and histological grade are used to stage breast cancer. Estrogen receptor/progesterone receptor (ER/PR) and HER-2/neu status are useful in predicting patient survival and relapse. Ki67, an indicator of proliferative activity, also correlates well with prognosis. Connexin proteins form gap junction channels, permitting intercellular exchange of ions and small molecules. Reduced connexin protein levels and impaired gap junctional intercellular communication are associated with tumor phenotypes. This study investigated the prognostic value of connexin proteins as breast cancer markers. Tissue microarrays, containing 438 cases of invasive breast carcinoma, were stained with Cx26, Cx32, and Cx43 antibodies. The degree of connexin immunoreactivity was determined and then correlated with patient outcome, tumor grade, tumor size, lymph node status, and immunohistochemical markers, such as p53, ER/PR status, Ki67 and c-erbB-2 expression. Cx26, Cx32, or Cx43 did not correlate well with tumor grade, tumor size, p53 or c-erbB-2 status. There was an inverse correlation between Cx32 and lymph node status (P <0.05) and a positive correlation between Cx43 and PR status (P <0.01). Cx32 and Cx43 correlated positively with ER status (P <0.01). Cx43 correlated negatively with Ki67 expression (P <0.01). Cx26, Cx32, and Cx43 did not correlate with patient outcome. Based on our observations in this study, connexin proteins do not appear to be reliable indicators of breast cancer prognosis. PMID:17583422

  6. Tissue microarray immunohistochemical profiles of p53 and pRB in hepatocellular carcinoma and hepatoblastoma.

    PubMed

    Azlin, Abdul Hadi; Looi, Lai Meng; Cheah, Phaik Leng

    2014-01-01

    The tumour suppressor genes, p53 and pRb, are known to play important roles in neoplastic transformation. While molecular routes to the uncontrolled growth of hepatocytes, leading to primary liver cancer have generated considerable interest, the roles of p53 and pRb mutations in hepatocellular carcinoma (HCC) and hepatoblastoma (HB) remain to be clarified. We examined the immunohistochemical expression of p53 and pRb gene products in 26 HCC and 9 HB, sampled into tissue microarray blocks. 10 (38%) of 26 HCC showed > 10% tumour nuclear staining for p53 protein, 3 of these also being HbsAg positive. Conversely, none of 9 HB expressed nuclear p53 immunopositivity. Some 24 (92%) HCC and 8 (89%) HB showed loss of pRb nuclear expression. Two of the 26 HCC and one of the 9 HB showed >10% tumour nuclear staining for pRb protein. Our results suggest that p53 does not have an important role in the development of HB but may contribute in HCC. There is also loss of pRb expression in the majority of HCC and HB, supporting loss of pRb gene function in the hepatocarcinogenesis pathway. However, a comparison of the staining profiles of p53 and pRb proteins in HCC and HB did not reveal a consistent pattern to differentiate between the two types of tumours immunohistochemically. Hence the use of p53 and pRB protein expression has no contribution in the situation where there is a diagnostic difficulty in deciding between HCC and HB. PMID:24935581

  7. Tissue microarray in a subset of South African patients with DLBCL.

    PubMed

    Sissolak, Gerhard; Wood, Lucille; Smith, Lynette; Chan, John Wing C; Armitage, James; Jacobs, Peter

    2013-10-01

    Tissue samples from 93 de novo diffuse large B-cell lymphoma patients seen between 1995 and 2009 randomly receiving either standard combination chemotherapy (CHOP, n=48) or the identical program with rituximab (n=45) were subtyped using an investigational immunohistochemical (IHC) based tissue microarray (TMA) and contrasted to the approximately corresponding categories as defined either by Hans and associates using a three marker panel into germinal or non-germinal centre subtypes or by Choi and colleagues with two additional antibodies into germinal centre (GCB) or activated B-cells (ABC). Each of these primary subdivisions was further evaluated for expression of BCL2 and LMO2 both of which are recognised to predicate response. The addition of rituximab to the uniform drug regimen did not show any significant improvement in 5 years overall (63% versus 59%, p 0.68) or event-free survival (42% versus 39%, p 0.94), for CHOP versus R-CHOP comparisons. Similarly no differences were evident in subtype analysis. Interestingly however, when segregated on the Choi criteria, cytotoxic drugs alone showed a non-significant trend in improved survival (74% versus 55%, p 0.32) as well as event-free survival (44% versus 40%, p 0.42) for the germinal centre as opposed to the activated B-cell subtype. Nevertheless not even a small difference could be demonstrated in the presence of the anti CD 20 monoclonal antibody. According to Choi, both regimens (chemotherapy or immunotherapy antibody) revealed similar results to the Hans algorithm on 5 years OS as well as 3 year EFS when comparing GCB versus ABC or non-GCB subgroups. BCL2 and LMO2 marker expression of the respective immunohistochemical (IHC) subtype, despite small sample size, revealed the following. Analysis by Choi criteria on survival for BCL2, no matter for which subsets (GCB or ABC) or treatment modality (chemotherapy with or without the addition of rituximab) showed no difference in 5 years OS or EFS. In contrast, a significant difference for better EFS (p=0.0015) in the BCL2 positive group of the ABC subgroups subtypes treated with rituximab containing chemotherapy. For LMO2 similar results on survival outcome were seen thus showing no difference in 5 years OS or EFS - regardless of subtype or treatment modality. Also here, this was contrasted by better EFS (p=0.039) in the LMO2 positive group of ABC subtypes when treated with the rituximab containing regimen. The use of the IHC based TMA methodology has shown to be a simple, cost effective and a robust alternative to gene expression profiling (GEP) which is currently regarded as the gold standard for the classification in lymphomas. It provides a useful prognostic tool in stratifying DLBCL or other entities in future, even when frozen tissue samples are not available for GEP analysis. With the current budgetary limitations in South African public hospitals chemotherapy protocols for lymphoproliferative disorders exclude agents such as rituximab. Local therapeutic drug committees consider the approximately 15% overall survival benefit seen at 5 years for DLBCL when rituximab is added to combination chemotherapy as too marginal for justifying the arising additional expenses. Accordingly, demonstration that a specific molecular subtype accounts for superior outcome, when using these regimens, is needed. Such an option would provide convincing evidence for the use of immunochemotherapy in a resource constrained setting. PMID:23942329

  8. Tissue microarrays analysis in chondrosarcomas: light microscopy, immunohistochemistry and xenograft study

    PubMed Central

    Machado, Isidro; Giner, Francisco; Mayordomo, Empar; Carda, Carmen; Navarro, Samuel; Llombart-Bosch, Antonio

    2008-01-01

    Background Chondrosarcoma (Chs) is the third most frequent primary malignant tumour of bone and can be primary or secondary, the latter results mainly from the malignant transformation of a benign pre-existing tumour. Methods All the cases diagnosed as Chs (primary tumours, recurrences and/or metastasis and xenotransplanted Chs) from the files of our Department were collected. Only cases with paraffin blocks available were selected (Total 32 cases). Six Tissue Microarrays (TMAs) were performed and all the cases and biopsies were distributed into the following groups: a) only paraffin block available from primary and/or metastatic tumours (3 TMAs), b) paraffin block available from primary and/or metastatic tumours as well as from the corresponding Nude mice xenotransplant (2 TMAs), c) only paraffin block available from xenotransplanted Chs (1 TMA). A reclassification of all the cases was performed; in addition, conventional hematoxylin-eosin as well as immunohistochemistry staining (S100, SOX-9, Ki-67, BCL-2, p53, p16, CK, CD99, Survivin and Caveolin) was analyzed in all the TMA. Results The distribution of the cases according to the histopathological pattern and the location of tumours were as follows: fourteen Grade I Chs (all primaries), two primary Grade II Chs, ten Grade III Chs (all primaries), five dedifferentiated Chs (four primaries and one primary with metastasis), and two Chs from cell cultures (Ch grade III). One recurrent extraskeletal myxoid Chs was included as a control in the TMA. Although there was heterogeneity in immunohistochemistry results of the different material analyzed, S100, SOX-9, Caveolin and Survivin were more expressed. The number of passages in xenotransplants fluctuated between 1 and 13. Curiously, in Grade I Chs, these implanted tumours hardly grew, and the number of passages did not exceed one. Conclusion The study of Chs by means of TMA techniques is very important because it will improve the assessment of different antibodies applied in the immunohistochemical assays. Xenotransplanted tumours in TMA improve knowledge concerning the variability in the morphological pattern shown by these tumours during the evolution in nudes. PMID:18673514

  9. A novel microarray approach reveals new tissue-specific signatures of known and predicted mammalian microRNAs

    PubMed Central

    Beuvink, Iwan; Kolb, Fabrice A.; Budach, Wolfgang; Garnier, Arlette; Lange, Joerg; Natt, Francois; Dengler, Uwe; Hall, Jonathan; Weiler, Jan

    2007-01-01

    Microarrays to examine the global expression levels of microRNAs (miRNAs) in a systematic in-parallel manner have become important tools to help unravel the functions of miRNAs and to understand their roles in RNA-based regulation and their implications in human diseases. We have established a novel miRNA-specific microarray platform that enables the simultaneous expression analysis of both known and predicted miRNAs obtained from human or mouse origin. Chemically modified 2′-O-(2-methoxyethyl)-(MOE) oligoribonucleotide probes were arrayed onto Evanescent Resonance (ER) microchips by robotic spotting. Supplementing the complementary probes against miRNAs with carefully designed mismatch controls allowed for accurate sequence-specific determination of miRNA expression profiles obtained from a panel of mouse tissues. This revealed new expression signatures of known miRNAs as well as of novel miRNAs previously predicted using bioinformatic methods. Systematic confirmation of the array data with northern blotting and, in particular, real-time PCR suggests that the described microarray platform is a powerful tool to analyze miRNA expression patterns with rapid throughput and high fidelity. PMID:17355992

  10. Tissue microarrays from bone marrow aspirates for high-throughput assessment of immunohistologic markers in pediatric acute leukemia.

    PubMed

    Hazard, Florette K; Zhao, Shuchun; Schiffman, Joshua D; Lacayo, Norman J; Dahl, Gary V; Natkunam, Yasodha

    2008-01-01

    Gene expression profiling studies have been employed to investigate prognostic subgroups in pediatric acute leukemia. Tissue microarrays (TMAs) are useful for high-throughput analysis of protein expression of target genes in acute leukemia samples and for validation of gene microarray analysis. Using cryopreserved samples of pediatric acute leukemia bone marrow aspirates, we constructed TMA from as few as 1 million cells. Bone marrow core biopsies from the same patients were included on the same TMA for comparison. A panel of 15 immunohistochemical markers typically used for diagnosis as well as those targeting recently characterized, prognostically relevant molecules of interest in pediatric acute leukemia was used to evaluate protein expression. Staining results confirm that suspension cells from bone marrow aspirates can be effectively used to derive protein expression data from multiple cases simultaneously with comparable efficacy to that of biopsy tissue. This method allows for new markers of diagnostic, prognostic, or therapeutic importance to be screened on large numbers of study patients. Furthermore, this technique may facilitate the inclusion of small samples, aspirates, and body fluids in large-scale studies of protein expression in clinical trials and protocols in which tissue biopsies are often unavailable. PMID:17990919

  11. JMJD2B as a potential diagnostic immunohistochemical marker for hepatocellular carcinoma: a tissue microarray-based study.

    PubMed

    Lu, Jeng-Wei; Ho, Yi-Jung; Lin, Liang-In; Huang, Yen-Chi; Yeh, Kun-Tu; Lin, Yu-Hsiang; Lin, Yueh-Min; Tzeng, Tsai-Yu

    2015-01-01

    The purpose of this study was to examine JMJD2B expression in human hepatocellular carcinoma (HCC) and elucidate relationships between various expression patterns and clinicopathological parameters of HCC patients. Immunohistochemical techniques were performed to detect JMJD2B expression in a tissue microarray from patients with breast, cerebrum, colon, esophagus, kidney, liver, lung, prostate, stomach, and uterus cancers. We performed immunohistochemical staining of a multiple tissue array to examine the expression profile of JMJD2B. Our results demonstrate that JMJD2B protein levels were upregulated in malignant human tumors, including breast, colon, liver, and lung. Immunohistochemistry staining examination of liver tumor tissue microarray revealed that the expression of JMJD2B is significant according to the histological grade and TNM stage of liver tumor. Moreover, JMJD2B was also correlated with Ki-67 expression in HCC samples. These results reveal that JMJD2B is dramatically upregulated in HCC, making it a potential diagnostic marker for the further development of HCC treatment therapies. PMID:25533242

  12. Construction of High-Density Tissue Microarrays at Low Cost by Using Self-Made Manual Microarray Kits and Recipient Paraffin Blocks

    PubMed Central

    Choi, Chang Hwan; Kim, Kyu Ho; Song, Ju Young; Kim, Lucia; Park, In Suh; Han, Jee Young; Kim, Joon Mee; Chu, Young Chae

    2012-01-01

    Background Advances of tissue microarray (TMA) technology have enabled simultaneous in situ analysis of biomarker expression in a large number of archived pathology specimens. However, the relatively high cost of TMA construction may hamper many researchers from using this essential tool of modern pathology research. We discuss methods for making TMA kits and recipient blocks for manual construction of high-density TMAs at low cost. Methods Ordinary cannula piercing needles, hypodermic needles, bone marrow biopsy needles, metallic ink cartridges of ballpoint pens, and disposable skin biopsy punches were used to construct self-made manual TMA kits. The recipient blocks were manufactured by boring holes in the conventional bare paraffin blocks. A mini electric hand drill and a microcompound table assembled on a drill stand were used to maximize the capacity of the recipient blocks. Results By using TMA kits made from cannula piercing needles (16- and 18-gauge), it was possible to construct TMAs with 1 mm×140 cores, 0.6 mm×320 cores, 2 mm×70 cores, 3 mm×35 cores, and 5 mm×12 cores. The capacity of the recipient blocks could be dramatically increased by drilling holes. Conclusions Construction of TMAs using self-made TMA kits is an inexpensive alternative to construction of TMAs using commercial devices. PMID:23323107

  13. Quantitative assessment of Tn antigen in breast tissue micro-arrays using CdSe aqueous quantum dots.

    PubMed

    Au, Giang H T; Mejias, Linette; Swami, Vanlila K; Brooks, Ari D; Shih, Wan Y; Shih, Wei-Heng

    2014-03-01

    In this study, we examined the use of CdSe aqueous quantum dots (AQDs) each conjugated to three streptavidin as a fluorescent label to image Tn antigen expression in various breast tissues via a sandwich staining procedure where the primary monoclonal anti-Tn antibody was bound to the Tn antigen on the tissue, a biotin-labeled secondary antibody was bound to the primary anti-Tn antibody, and finally the streptavidin-conjugated AQDs were bound to the biotin on the secondary antibody. We evaluated the AQD staining of Tn antigen on tissue microarrays consisting of 395 cores from 115 cases including three tumor cores and one normal-tissue core from each breast cancer case and three tumor cores from each benign case. The results indicated AQD-Tn staining was positive in more than 90% of the cells in the cancer cores but not the cells in the normal-tissue cores and the benign tumor cores. As a result, AQD-Tn staining exhibited 95% sensitivity and 90% specificity in differentiating breast cancer against normal breast tissues and benign breast conditions. These results were better than the 90% sensitivity and 80% specificity exhibited by the corresponding horse radish peroxidase (HRP) staining using the same antibodies on the same tissues and those of previous studies that used different fluorescent labels to image Tn antigen. In addition to sensitivity and specificity, the current AQD-Tn staining with a definitive threshold was quantitative. PMID:24411673

  14. Construction Strategy and Progress of Whole Intervertebral Disc Tissue Engineering.

    PubMed

    Yang, Qiang; Xu, Hai-Wei; Hurday, Sookesh; Xu, Bao-Shan

    2016-02-01

    Degenerative disc disease (DDD) is the major cause of low back pain, which usually leads to work absenteeism, medical visits and hospitalization. Because the current conservative procedures and surgical approaches to treatment of DDD only aim to relieve the symptoms of disease but not to regenerate the diseased disc, their long-term efficiency is limited. With the rapid developments in medical science, tissue engineering techniques have progressed markedly in recent years, providing a novel regenerative strategy for managing intervertebral disc disease. However, there are as yet no ideal methods for constructing tissue-engineered intervertebral discs. This paper reviews published reports pertaining to intervertebral disc tissue engineering and summarizes data concerning the seed cells and scaffold materials for tissue-engineered intervertebral discs, construction of tissue-engineered whole intervertebral discs, relevant animal experiments and effects of mechanics on the construction of tissue-engineered intervertebral disc and outlines the existing problems and future directions. Although the perfect regenerative strategy for treating DDD has not yet been developed, great progress has been achieved in the construction of tissue-engineered intervertebral discs. It is believed that ongoing research on intervertebral disc tissue engineering will result in revolutionary progress in the treatment of DDD. PMID:27028376

  15. Extraction and labeling methods for microarrays using small amounts of plant tissue.

    PubMed

    Stimpson, Alexander J; Pereira, Rhea S; Kiss, John Z; Correll, Melanie J

    2009-03-01

    Procedures were developed to maximize the yield of high-quality RNA from small amounts of plant biomass for microarrays. Two disruption techniques (bead milling and pestle and mortar) were compared for the yield and the quality of RNA extracted from 1-week-old Arabidopsis thaliana seedlings (approximately 0.5-30 mg total biomass). The pestle and mortar method of extraction showed enhanced RNA quality at the smaller biomass samples compared with the bead milling technique, although the quality in the bead milling could be improved with additional cooling steps. The RNA extracted from the pestle and mortar technique was further tested to determine if the small quantity of RNA (500 ng-7 microg) was appropriate for microarray analyses. A new method of low-quantity RNA labeling for microarrays (NuGEN Technologies, Inc.) was used on five 7-day-old seedlings (approximately 2.5 mg fresh weight total) of Arabidopsis that were grown in the dark and exposed to 1 h of red light or continued dark. Microarray analyses were performed on a small plant sample (five seedlings; approximately 2.5 mg) using these methods and compared with extractions performed with larger biomass samples (approximately 500 roots). Many well-known light-regulated genes between the small plant samples and the larger biomass samples overlapped in expression changes, and the relative expression levels of selected genes were confirmed with quantitative real-time polymerase chain reaction, suggesting that these methods can be used for plant experiments where the biomass is extremely limited (i.e. spaceflight studies). PMID:19140889

  16. Overexpression of α2,3sialyl T-antigen in breast cancer determined by miniaturized glycosyltransferase assays and confirmed using tissue microarray immunohistochemical analysis

    PubMed Central

    Patil, Shilpa A.; Bshara, Wiam; Morrison, Carl; Chandrasekaran, E. V.; Matta, Khushi L.; Neelamegham, Sriram

    2014-01-01

    Glycan structure alterations during cancer regulate disease progression and represent clinical biomarkers. The study determined the degree to which changes in glycosyl transferase activities during cancer can be related to aberrant cell-surface tumor associated carbohydrate structures (TACA). To this end, changes in sialyltransferase (sialylT), fucosyltransferase (fucT) and galactosyltransferase (galT) activity were measured in normal and tumor tissue using a miniaturized enzyme activity assay and synthetic glycoconjugates bearing terminal LacNAc Type-I (Galβ1,3GlcNAc), LacNAc Type-II (Galβ1,4GlcNAc), and mucin core-1/Type-III (Galβ1,3GalNAc) structures. These data were related to TACA using tissue microarrays containing 115 breast and 26 colon cancer specimen. The results show that primary human breast and colon tumors, but not adjacent normal tissue, express elevated β1,3 galT and α2,3sialylT activity that can form α2,3sialylated Type-III glycans (Siaα2,3Galβ1,3GalNAc). Prostate tumors did not exhibit such elevated enzymatic activities. α1,3/4fucT activity was higher in breast, but not colon tissue. The enzymology based prediction of enhanced α2,3sialylated Type-III structures in breast tumors was verified using histochemical analysis of tissue sections and tissue microarrays. Here, the binding of two markers that recognize Galβ1,3GalNAc (peanut lectin and mAb A78-G/A7) was elevated in breast tumor, but not normal control, only upon sialidase treatment. These antigens were also upregulated in colon tumors though to a lesser extent. α2,3sialylated Type-III expression correlated inversely with patient HER2 expression and breast metastatic potential. Overall, enzymology measurements of glycoT activity predict glycan structure changes during cancer. High expression of the α2,3sialylated T-antigen O-glycans occur in breast tumors. A transformation from linear core-1 glycan to other epitopes may accompany metastasis. PMID:25142811

  17. No-cost manual method for preparation of tissue microarrays having high quality comparable to semiautomated methods.

    PubMed

    Foda, Abd Al-Rahman Mohammad

    2013-05-01

    Manual tissue microarray (TMA) construction had been introduced to avoid the high cost of automated and semiautomated techniques. The cheapest and simplest technique for constructing manual TMA was that of using mechanical pencil tips. This study was carried out to modify this method, aiming to raise its quality to reach that of expensive ones. Some modifications were introduced to Shebl's technique. Two conventional mechanical pencil tips of different diameters were used to construct the recipient blocks. A source of mild heat was used, and blocks were incubated at 38°C overnight. With our modifications, 3 high-density TMA blocks were constructed. We successfully performed immunostaining without substantial tissue loss. Our modifications increased the number of cores per block and improved the stability of the cores within the paraffin block. This new, modified technique is a good alternative for expensive machines in many laboratories. PMID:23235346

  18. Tissue stiffness dictates development, homeostasis, and disease progression.

    PubMed

    Handorf, Andrew M; Zhou, Yaxian; Halanski, Matthew A; Li, Wan-Ju

    2015-01-01

    Tissue development is orchestrated by the coordinated activities of both chemical and physical regulators. While much attention has been given to the role that chemical regulators play in driving development, researchers have recently begun to elucidate the important role that the mechanical properties of the extracellular environment play. For instance, the stiffness of the extracellular environment has a role in orienting cell division, maintaining tissue boundaries, directing cell migration, and driving differentiation. In addition, extracellular matrix stiffness is important for maintaining normal tissue homeostasis, and when matrix mechanics become imbalanced, disease progression may ensue. In this article, we will review the important role that matrix stiffness plays in dictating cell behavior during development, tissue homeostasis, and disease progression. PMID:25915734

  19. Tissue Stiffness Dictates Development, Homeostasis, and Disease Progression

    PubMed Central

    Handorf, Andrew M; Zhou, Yaxian; Halanski, Matthew A; Li, Wan-Ju

    2015-01-01

    Abstract Tissue development is orchestrated by the coordinated activities of both chemical and physical regulators. While much attention has been given to the role that chemical regulators play in driving development, researchers have recently begun to elucidate the important role that the mechanical properties of the extracellular environment play. For instance, the stiffness of the extracellular environment has a role in orienting cell division, maintaining tissue boundaries, directing cell migration, and driving differentiation. In addition, extracellular matrix stiffness is important for maintaining normal tissue homeostasis, and when matrix mechanics become imbalanced, disease progression may ensue. In this article, we will review the important role that matrix stiffness plays in dictating cell behavior during development, tissue homeostasis, and disease progression. PMID:25915734

  20. A Texture Based Pattern Recognition Approach to Distinguish Melanoma from Non-Melanoma Cells in Histopathological Tissue Microarray Sections

    PubMed Central

    Rexhepaj, Elton; Agnarsdóttir, Margrét; Bergman, Julia; Edqvist, Per-Henrik; Bergqvist, Michael; Uhlén, Mathias; Gallagher, William M.; Lundberg, Emma; Ponten, Fredrik

    2013-01-01

    Aims Immunohistochemistry is a routine practice in clinical cancer diagnostics and also an established technology for tissue-based research regarding biomarker discovery efforts. Tedious manual assessment of immunohistochemically stained tissue needs to be fully automated to take full advantage of the potential for high throughput analyses enabled by tissue microarrays and digital pathology. Such automated tools also need to be reproducible for different experimental conditions and biomarker targets. In this study we present a novel supervised melanoma specific pattern recognition approach that is fully automated and quantitative. Methods and Results Melanoma samples were immunostained for the melanocyte specific target, Melan-A. Images representing immunostained melanoma tissue were then digitally processed to segment regions of interest, highlighting Melan-A positive and negative areas. Color deconvolution was applied to each region of interest to separate the channel containing the immunohistochemistry signal from the hematoxylin counterstaining channel. A support vector machine melanoma classification model was learned from a discovery melanoma patient cohort (n = 264) and subsequently validated on an independent cohort of melanoma patient tissue sample images (n = 157). Conclusion Here we propose a novel method that takes advantage of utilizing an immuhistochemical marker highlighting melanocytes to fully automate the learning of a general melanoma cell classification model. The presented method can be applied on any protein of interest and thus provides a tool for quantification of immunohistochemistry-based protein expression in melanoma. PMID:23690928

  1. Effects of Long-Term Storage on the Detection of Proteins, DNA, and mRNA in Tissue Microarray Slides

    PubMed Central

    Karlsson, Mats G.

    2011-01-01

    Storage of tissue slides has been claimed to induce dramatically reduced antigen detection particularly for immunohistochemistry (IHC). With tissue microarrays, the necessity to serially cut blocks in order to obtain as much material as possible is obvious. The presumed adverse effect of storage might hamper such an approach. The authors designed an experimental setting consisting of four different storage conditions with storage time of tissue slides of up to 1 year. Detection of proteins, DNA, and mRNA was performed using IHC and in situ hybridization techniques. Slight but significant changes in IHC occurred over time. The most important factor is the primary antibody used: four showed no significant changes, whereas limited decreases in 8 antibodies could be detected by image analysis. Whether the antigen was nuclear or cytoplasmic/membranous did not matter. No major differences between different storage conditions could be shown, but storage at 4C was overall the best procedure. Furthermore, gene copy number aberrations, chromosomal translocations, and the presence of mRNA could be detected on slides stored up to 1 year. In conclusion, in tissues optimally formalin fixed and using modern histological techniques, only minute changes in tissue antigenicity are induced by long-term storage. PMID:22147608

  2. Microarray analysis of differentially expressed genes regulating lipid metabolism during melanoma progression.

    PubMed

    Sumantran, Venil N; Mishra, Pratik; Sudhakar, N

    2015-04-01

    A new hallmark of cancer involves acquisition of a lipogenic phenotype which promotes tumorigenesis. Little is known about lipid metabolism in melanomas. Therefore, we used BRB (Biometrics Research Branch) class comparison tool with multivariate analysis to identify differentially expressed genes in human cutaneous melanomas, compared with benign nevi and normal skin derived from the microarray dataset (GDS1375). The methods were validated by identifying known melanoma biomarkers (CITED1, FGFR2, PTPRF, LICAM, SPP1 and PHACTR1) in our results. Eighteen genes regulating metabolism of fatty acids, lipid second messengers and gangliosides were 2-9 fold upregulated in melanomas of GDS-1375. Out of the 18 genes, 13 were confirmed by KEGG pathway analysis and 10 were also significantly upregulated in human melanoma cell lines of NCI-60 Cell Miner database. Results showed that melanomas upregulated PPARGC1A transcription factor and its target genes regulating synthesis of fatty acids (SCD) and complex lipids (FABP3 and ACSL3). Melanoma also upregulated genes which prevented lipotoxicity (CPT2 and ACOT7) and regulated lipid second messengers, such as phosphatidic acid (AGPAT-4, PLD3) and inositol triphosphate (ITPKB, ITPR3). Genes for synthesis of pro-tumorigenic GM3 and GD3 gangliosides (UGCG, HEXA, ST3GAL5 and ST8SIA1) were also upregulated in melanoma. Overall, the microarray analysis of GDS-1375 dataset indicated that melanomas can become lipogenic by upregulating genes, leading to increase in fatty acid metabolism, metabolism of specific lipid second messengers, and ganglioside synthesis. PMID:26118123

  3. Heterogeneity of ERBB2 in gastric carcinomas: a study of tissue microarray and matched primary and metastatic carcinomas.

    PubMed

    Cho, Eun Yoon; Park, Kyeongmee; Do, Ingu; Cho, Junhun; Kim, Jiyun; Lee, Jeeyun; Kim, Seonwoo; Kim, Kyoung-Mee; Sohn, Tae Sung; Kang, Won Ki; Kim, Sung

    2013-05-01

    Trastuzumab in association with systemic cytotoxic chemotherapy is a therapeutic option for patients with advanced or metastatic ERBB2+ gastric carcinoma. The status of the ERBB2 overexpression or gene amplification is an important predictive marker in gastric cancer. However, it is controversial whether the primary tumor is representative of distant metastases in terms of ERBB2 status. Quadruplicated tissue microarrays from formalin-fixed paraffin-embedded tissues from 498 advanced primary gastric carcinomas and 97 matched metastatic lymph nodes were investigated by immunohistochemistry with HercepTest and silver in situ hybridization. For further comparison, another set of 41 paired primary and distant metastatic gastric carcinomas were also tested. Intratumoral heterogeneity was defined as different results between tissue microarray cores. ERBB2-positivity was observed in 52 gastric carcinomas (10%) and was not associated with recurrence of disease or survival of patients. In ERBB2-positive primary gastric carcinomas, heterogeneous ERBB2 overexpression was observed in 21/63 (33%) gastric carcinomas and heterogeneous ERBB2 gene amplification in 14/62 (23%) cases. Repeated immunohistochemistry and silver in situ hybridization in representative paraffin tumor blocks confirmed focal ERBB2 overexpression and ERBB2 gene amplification and did not change the final results. Discrepancies in ERBB2 results between primary and paired metastatic lymph nodes were observed in 11% of cases by immunohistochemistry and 7% by silver in situ hybridization. Out of the 41 paired primary and distant metastases, 5 (12%) cases were ERBB2-positive, and discrepancy was observed in one case. Intratumoral heterogeneity and discrepant ERBB2 results in primary and metastatic tumor are not uncommon in gastric carcinoma. Results of silver in situ hybridization showed less frequent heterogeneity compared with immunohistochemistry. Wherever possible, ERBB2 immunohistochemistry testing should be performed in both primary and distant metastatic sites. PMID:23238628

  4. [Recent progress of researches on scaffolds for tissue engineered meniscus].

    PubMed

    Fu, Weili; Wang, Jianyun; Wan, Changxiu; Li, Jian

    2010-04-01

    Tissue engineered meniscus provides a novel approach for the treatment of severe meniscus injury. The researches on scaffold for tissue engineered meniscus is an essential element. The researches on scaffolds for tissue engineered meniscus, including natural biomaterials and synthetic polymer, have made great advances in recent years. At present, collagen meniscus implants have been used in clinical practice. As hydrogel is characterized by having high water content and good biocompatibility, being similar to extracellular matrix in structure, and being able to provide access to microsurgery, it has become the hotspot field in recent years. Meanwhile, the combinatorial scaffold material advantages of biological composite tissue engineered scaffolds also illuminate the key point of research on meniscal scaffolds. This paper reviews the recent progress in betterment of the scaffolds so as to provide a theoretical and practical basis for further researches of tissue engineered meniscus. PMID:20481339

  5. Evaluation of cytokeratin-19 in breast cancer tissue samples: a comparison of automatic and manual evaluations of scanned tissue microarray cylinders

    PubMed Central

    2015-01-01

    Background Digital image (DI) analysis avoids visual subjectivity in interpreting immunohistochemical stains and provides more reproducible results. An automated procedure consisting of two variant methods for quantifying the cytokeratin-19 (CK19) marker in breast cancer tissues is presented. Methods The first method (A) excludes the holes inside selected CK19 stained areas, and the second (B) includes them. 93 DIs scanned from complete cylinders of tissue microarrays were evaluated visually by two pathologists and by the automated procedures. Results and conclusions There was good concordance between the two automated methods, both of which tended to identify a smaller CK19-positive area than did the pathologists. The results obtained with method B were more similar to those of the pathologists; probably because it takes into account the entire positive tumoural area, including the holes. However, the pathologists overestimated the positive area of CK19. Further studies are needed to confirm the utility of this automated procedure in prognostic studies. PMID:26329009

  6. Quality Control of RNA Preservation and Extraction from Paraffin-Embedded Tissue: Implications for RT-PCR and Microarray Analysis

    PubMed Central

    Pichler, Martin; Zatloukal, Kurt

    2013-01-01

    Analysis of RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathological diagnostics. We have performed a comprehensive and systematic investigation of the impact of factors in the pre-analytical workflow, such as different fixatives, fixation time, RNA extraction method and storage of tissues in paraffin blocks, on several downstream reactions including complementary DNA (cDNA) synthesis, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microarray hybridization. We compared the effects of routine formalin fixation with the non-crosslinking, alcohol-based Tissue Tek Xpress Molecular Fixative (TTXMF, Sakura Finetek), and cryopreservation as gold standard for molecular analyses. Formalin fixation introduced major changes into microarray gene expression data and led to marked gene-to-gene variations in delta-ct values of qRT-PCR. We found that qRT-PCR efficiency and gene-to-gene variations were mainly attributed to differences in the efficiency of cDNA synthesis as the most sensitive step. These differences could not be reliably detected by quality assessment of total RNA isolated from formalin-fixed tissues by electrophoresis or spectrophotometry. Although RNA from TTXMF fixed samples was as fragmented as RNA from formalin fixed samples, much higher cDNA yield and lower ct-values were obtained in qRT-PCR underlining the negative impact of crosslinking by formalin. In order to better estimate the impact of pre-analytical procedures such as fixation on the reliability of downstream analysis, we applied a qRT-PCR-based assay using amplicons of different length and an assay measuring the efficiency of cDNA generation. Together these two assays allowed better quality assessment of RNA extracted from fixed and paraffin-embedded tissues and should be used to supplement quality scores derived from automated electrophoresis. A better standardization of the pre-analytical workflow, application of additional quality controls and detailed sample information would markedly improve the comparability and reliability of molecular studies based on formalin-fixed and paraffin-embedded tissue samples. PMID:23936242

  7. Identification of Genes Associated With Progression and Metastasis of Advanced Cervical Cancers After Radiotherapy by cDNA Microarray Analysis

    SciTech Connect

    Harima, Yoko; Ikeda, Koshi; Utsunomiya, Keita; Shiga, Toshiko; Komemushi, Atsushi; Kojima, Hiroyuki; Nomura, Motoo; Kamata, Minoru; Sawada, Satoshi

    2009-11-15

    Purpose: To identify a set of genes related to the progression and metastasis of advanced cervical cancer after radiotherapy and to establish a predictive method. Methods and Materials: A total of 28 patients with cervical cancer (15 stage IIIB, 13 stage IVA patients) who underwent definitive radiotherapy between May 1995 and April 2001 were included in this study. All patients were positive for human papillomavirus infection and harbored the wild-type p53 gene. The expression profiles of 14 tumors with local failure and multiple distant metastasis and 14 tumors without metastasis (cancer free) obtained by punch biopsy were compared before treatment, using a cDNA microarray consisting of 23,040 human genes. Results: Sixty-three genes were selected on the basis of a clustering analysis, and the validity of these genes was confirmed using a cross-validation test. The most accurate prediction was achieved for 63 genes (sensitivity, 78.8%; specificity, 38.1%). Some of these genes were already known to be associated with metastasis via chromosomal instability (TTK, BUB1B), extracellular matrix components (matrix metalloproteinase 1 [MMP-1]), and carcinogenesis (protein phosphatase 1 regulatory subunit 7 [PPP1R7]). A 'predictive score' system was developed that could predict the probability for development of metastases using leave-one-out cross-validation methods. Conclusions: The present results may provide valuable information for identified predictive markers and novel therapeutic target molecules for progression and metastasis of advanced cervical cancer.

  8. Comparative study of gene expression by cDNA microarray in human colorectal cancer tissues and normal mucosa.

    PubMed

    Bianchini, Michele; Levy, Estrella; Zucchini, Cinzia; Pinski, Victor; Macagno, Carlos; De Sanctis, Paola; Valvassori, Luisa; Carinci, Paolo; Mordoh, Jos

    2006-07-01

    The causative molecular pathways underlying the pathogenesis of colorectal cancer (CRC) need to be better characterized. The purpose of our study was to better understand the genetic mechanism of oncogenesis for human colorectal cancer and to identify new potential tumor markers of use in clinical practice. We used cDNA microarrays to compare gene expression profiles of colorectal biopsies from 25 CRC patients and 13 normal mucosa from adjacent non-cancerous tissues. Findings were validated by real-time PCR; in addition, western blotting and immunochemistry analysis were carried out as further confirmation of differential expression at a protein level. Comparing cancerous tissues with normal colonic mucosa we identified 584 known genes differentially expressed to a significant degree (p<0.001). Many of the transcripts that were more abundant in tumors than in non-neoplastic tissues appear to reflect important events for colon carcinogenesis. For example, a significant number of these genes serve as apoptotic inhibitors (e.g. BFAR, BIRC1, BIRC6). Furthermore, we observed the simultaneous up-regulation of HLA-E and the down-regulation of beta2-microglobulin; these genes strongly support a potential tumor escape strategy from immune surveillance in colon cancer tissues. Our study provides new gene candidates in the pathogenesis of human CRC disease. From our results we hypothesize that CRC cells escape immune surveillance through a specific gene expression alteration; moreover, over-expression of several survival genes seems to confer a more anti-apoptotic phenotype. These genes are involved in pathways not previously implicated in CRC pathogenesis and they may provide new targets for therapy. PMID:16773188

  9. Tenascin-C: A Novel Candidate Marker for Cancer Stem Cells in Glioblastoma Identified by Tissue Microarrays

    PubMed Central

    2015-01-01

    Glioblastoma multiforme (GBM) is a highly aggressive brain tumor, with dismal survival outcomes. Recently, cancer stem cells (CSCs) have been demonstrated to play a role in therapeutic resistance and are considered to be the most likely cause of cancer relapse. The identification of CSCs is an important step toward finding new and effective ways to treat GBM. Tenascin-C (TNC) protein has been identified as a potential marker for CSCs in gliomas based on previous work. Here, we have investigated the expression of TNC in tissue microarrays including 17 GBMs, 18 WHO grade III astrocytomas, 15 WHO grade II astrocytomas, 4 WHO grade I astrocytomas, and 7 normal brain tissue samples by immunohistochemical staining. TNC expression was found to be highly associated with the grade of astrocytoma. It has a high expression level in most of the grade III astrocytomas and GBMs analyzed and a very low expression in most grade II astrocytomas, whereas it is undetectable in grade I astrocytomas and normal brain tissues. Double-immunofluorescence staining for TNC and CD133 in GBM tissues revealed that there was a high overlap between theses two positive populations. The results were further confirmed by flow cytometry analysis of TNC and CD133 in GBM-derived stem-like neurospheres in vitro. A limiting dilution assay demonstrated that the sphere formation ability of CD133+/TNC+ and CD133–/TNC+ cell populations is much higher than that of the CD133+/TNC– and CD133–/TNC– populations. These results suggest that TNC is not only a potential prognostic marker for GBM but also a potential marker for glioma CSCs, where the TNC+ population is identified as a CSC population overlapping with part of the CD133– cell population. PMID:25469866

  10. Software Tools for High-Throughput Analysis and Archiving of Immunohistochemistry Staining Data Obtained with Tissue Microarrays

    PubMed Central

    Liu, Chih Long; Prapong, Wijan; Natkunam, Yasodha; Alizadeh, Ash; Montgomery, Kelli; Gilks, C. Blake; van de Rijn, Matt

    2002-01-01

    The creation of tissue microarrays (TMAs) allows for the rapid immunohistochemical analysis of thousands of tissue samples, with numerous different antibodies per sample. This technical development has created a need for tools to aid in the analysis and archival storage of the large amounts of data generated. We have developed a comprehensive system for high-throughput analysis and storage of TMA immunostaining data, using a combination of commercially available systems and novel software applications developed in our laboratory specifically for this purpose. Staining results are recorded directly into an Excel worksheet and are reformatted by a novel program (TMA-Deconvoluter) into a format suitable for hierarchical clustering analysis or other statistical analysis. Hierarchical clustering analysis is a powerful means of assessing relatedness within groups of tumors, based on their immunostaining with a panel of antibodies. Other analyses, such as generation of survival curves, construction of Cox regression models, or assessment of intra- or interobserver variation, can also be done readily on the reformatted data. Finally, the immunoprofile of a specific case can be rapidly retrieved from the archives and reviewed through the use of Stainfinder, a novel web-based program that creates a direct link between the clustered data and a digital image database. An on-line demonstration of this system is available at http://genome-www.stanford.edu/TMA/explore.shtml. PMID:12414504

  11. Quantitative multiplex quantum dot in-situ hybridisation based gene expression profiling in tissue microarrays identifies prognostic genes in acute myeloid leukaemia

    SciTech Connect

    Tholouli, Eleni; MacDermott, Sarah; Hoyland, Judith; Yin, John Liu; Byers, Richard

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer Development of a quantitative high throughput in situ expression profiling method. Black-Right-Pointing-Pointer Application to a tissue microarray of 242 AML bone marrow samples. Black-Right-Pointing-Pointer Identification of HOXA4, HOXA9, Meis1 and DNMT3A as prognostic markers in AML. -- Abstract: Measurement and validation of microarray gene signatures in routine clinical samples is problematic and a rate limiting step in translational research. In order to facilitate measurement of microarray identified gene signatures in routine clinical tissue a novel method combining quantum dot based oligonucleotide in situ hybridisation (QD-ISH) and post-hybridisation spectral image analysis was used for multiplex in-situ transcript detection in archival bone marrow trephine samples from patients with acute myeloid leukaemia (AML). Tissue-microarrays were prepared into which white cell pellets were spiked as a standard. Tissue microarrays were made using routinely processed bone marrow trephines from 242 patients with AML. QD-ISH was performed for six candidate prognostic genes using triplex QD-ISH for DNMT1, DNMT3A, DNMT3B, and for HOXA4, HOXA9, Meis1. Scrambled oligonucleotides were used to correct for background staining followed by normalisation of expression against the expression values for the white cell pellet standard. Survival analysis demonstrated that low expression of HOXA4 was associated with poorer overall survival (p = 0.009), whilst high expression of HOXA9 (p < 0.0001), Meis1 (p = 0.005) and DNMT3A (p = 0.04) were associated with early treatment failure. These results demonstrate application of a standardised, quantitative multiplex QD-ISH method for identification of prognostic markers in formalin-fixed paraffin-embedded clinical samples, facilitating measurement of gene expression signatures in routine clinical samples.

  12. Assessment of Automated Image Analysis of Breast Cancer Tissue Microarrays for Epidemiologic Studies

    PubMed Central

    Bolton, Kelly L.; Garcia-Closas, Montserrat; Pfeiffer, Ruth M.; Duggan, Máire A.; Howat, William J.; Hewitt, Stephen M.; Yang, Xiaohong R.; Cornelison, Robert; Anzick, Sarah L.; Meltzer, Paul; Davis, Sean; Lenz, Petra; Figueroa, Jonine D.; Pharoah, Paul D.P.; Sherman, Mark E.

    2010-01-01

    A major challenge in studies of etiologic heterogeneity in breast cancer has been the limited throughput, accuracy and reproducibility of measuring tissue markers. Computerized image analysis systems may help address these concerns but published reports of their use are limited. We assessed agreement between automated and pathologist scores of a diverse set of immunohistochemical (IHC) assays performed on breast cancer TMAs. TMAs of 440 breast cancers previously stained for ER-α, PR, HER-2, ER-β and aromatase were independently scored by two pathologists and three automated systems (TMALabII, TMAx, Ariol). Agreement between automated and pathologist scores of negative/positive was measured using the area under the receiver operator characteristics curve (AUC) and weighted kappa statistics (κ) for categorical scores. We also investigated the correlation between IHC scores and mRNA expression levels. Agreement between pathologist and automated negative/positive and categorical scores was excellent for ER-α and PR (AUC range =0.98-0.99; κ range =0.86-0.91). Lower levels of agreement were seen for ER-β categorical scores (AUC=0.99-1.0; κ=0.80-0.86) and both negative/positive and categorical scores for aromatase (AUC=0.85-0.96; κ=0.41-0.67) and HER2 (AUC=0.94-0.97; κ=0.53-0.72). For ER-α and PR, there was strong correlation between mRNA levels and automated (ρ=0.67-0.74) and pathologist IHC scores (ρ=0.67-0.77). HER2 mRNA levels were more strongly correlated with pathologist (ρ=0.63) than automated IHC scores (ρ=0.41-0.49). Automated analysis of IHC markers is a promising approach for scoring large numbers of breast cancer tissues in epidemiologic investigations. This would facilitate studies of etiologic heterogeneity which ultimately may allow improved risk prediction and better prevention approaches. PMID:20332278

  13. Cathepsin D Expression in Colorectal Cancer: From Proteomic Discovery through Validation Using Western Blotting, Immunohistochemistry, and Tissue Microarrays

    PubMed Central

    Kirana, Chandra; Shi, Hongjun; Laing, Emma; Hood, Kylie; Miller, Rose; Bethwaite, Peter; Keating, John; Jordan, T. William; Hayes, Mark; Stubbs, Richard

    2012-01-01

    Despite recent advances in surgical techniques and therapeutic treatments, survival from colorectal cancer (CRC) remains disappointing with some 4050% of newly diagnosed patients ultimately dying of metastatic disease. Current staging by light microscopy alone is not sufficiently predictive of prognosis and would benefit from additional support from biomarkers in order to stratify patients appropriately for adjuvant therapy. We have identified that cathepsin D expression was significantly greater in cells from invasive front (IF) area and liver metastasis (LM) than those from main tumour body (MTB). Cathepsin D expression was subsequently examined by immunohistochemistry in tissue microarrays from 119 patients with CRC. Strong expression in tumour cells at the IF did not correlate significantly with any clinico-pathological parameters examined or patient survival. However, cathepsin D expression in cells from the MTB was highly elevated in late stage CRC and showed significant correlation with subsequent distant metastasis and shorter cancer-specific survival. We also found that macrophages surrounding tumour cells stained strongly for cathepsin D but there was no significant correlation found between cathepsin D in macrophages at IF and MTB of CRC patient with the clinic-pathological parameters examined. PMID:22919486

  14. Tissue microarray design and construction for scientific, industrial and diagnostic use

    PubMed Central

    Pilla, Daniela; Bosisio, Francesca M.; Marotta, Roberto; Faggi, Stefano; Forlani, Paolo; Falavigna, Maurizio; Biunno, Ida; Martella, Emanuele; De Blasio, Pasquale; Borghesi, Simone; Cattoretti, Giorgio

    2012-01-01

    Context: In 2013 the high throughput technology known as Tissue Micro Array (TMA) will be fifteen years old. Its elements (design, construction and analysis) are intuitive and the core histopathology technique is unsophisticated, which may be a reason why has eluded a rigorous scientific scrutiny. The source of errors, particularly in specimen identification and how to control for it is unreported. Formal validation of the accuracy of segmenting (also known as de-arraying) hundreds of samples, pairing with the sample data is lacking. Aims: We wanted to address these issues in order to bring the technique to recognized standards of quality in TMA use for research, diagnostics and industrial purposes. Results: We systematically addressed the sources of error and used barcode-driven data input throughout the whole process including matching the design with a TMA virtual image and segmenting that image back to individual cases, together with the associated data. In addition we demonstrate on mathematical grounds that a TMA design, when superimposed onto the corresponding whole slide image, validates on each and every sample the correspondence between the image and patient's data. Conclusions: High throughput use of the TMA technology is a safe and efficient method for research, diagnosis and industrial use if all sources of errors are identified and addressed. PMID:23372983

  15. Mechanisms of benzene-induced hematotoxicity and leukemogenicity: cDNA microarray analyses using mouse bone marrow tissue.

    PubMed Central

    Yoon, Byung-Il; Li, Guang-Xun; Kitada, Kunio; Kawasaki, Yasushi; Igarashi, Katsuhide; Kodama, Yukio; Inoue, Tomoaki; Kobayashi, Kazuko; Kanno, Jun; Kim, Dae-Yong; Inoue, Tohru; Hirabayashi, Yoko

    2003-01-01

    Although the mechanisms underlying benzene-induced toxicity and leukemogenicity are not yet fully understood, they are likely to be complicated by various pathways, including those of metabolism, growth factor regulation, oxidative stress, DNA damage, cell cycle regulation, and programmed cell death. With this as a background, we performed cDNA microarray analyses on mouse bone marrow tissue during and after a 2-week benzene exposure by inhalation. Our goal was to clarify the mechanisms underlying the hematotoxicity and leukemogenicity induced by benzene at the level of altered multigene expression. Because a few researchers have postulated that the cell cycle regulation mediated by p53 is a critical event for benzene-induced hematotoxicity, the present study was carried out using p53-knockout (KO) mice and C57BL/6 mice. On the basis of the results of large-scale gene expression studies, we conclude the following: (a) Benzene induces DNA damage in cells at any phase of the cell cycle through myeloperoxidase and in the redox cycle, resulting in p53 expression through Raf-1 and cyclin D-interacting myb-like protein 1. (b) For G1/S cell cycle arrest, the p53-mediated pathway through p21 is involved, as well as the pRb gene-mediated pathway. (c) Alteration of cyclin G1 and Wee-1 kinase genes may be related to the G2/M arrest induced by benzene exposure. (d) DNA repair genes such as Rad50 and Rad51 are markedly downregulated in p53-KO mice. (e) p53-mediated caspase 11 activation, aside from p53-mediated Bax gene induction, may be an important pathway for cellular apoptosis after benzene exposure. Our results strongly suggest that the dysfunction of the p53 gene, possibly caused by strong and repeated genetic and epigenetic effects of benzene on candidate leukemia cells, may induce fatal problems such as those of cell cycle checkpoint, apoptosis, and the DNA repair system, finally resulting in hemopoietic malignancies. Our cDNA microarray data provide valuable information for future investigations of the mechanisms underlying the toxicity and leukemogenicity of benzene. PMID:12928149

  16. Comparison of Nanostring nCounter® Data on FFPE Colon Cancer Samples and Affymetrix Microarray Data on Matched Frozen Tissues

    PubMed Central

    Chen, Xi; Deane, Natasha G.; Lewis, Keeli B.; Li, Jiang; Zhu, Jing; Washington, M. Kay; Beauchamp, R. Daniel

    2016-01-01

    The prognosis of colorectal cancer (CRC) stage II and III patients remains a challenge due to the difficulties of finding robust biomarkers suitable for testing clinical samples. The majority of published gene signatures of CRC have been generated on fresh frozen colorectal tissues. Because collection of frozen tissue is not practical for routine surgical pathology practice, a clinical test that improves prognostic capabilities beyond standard pathological staging of colon cancer will need to be designed for formalin-fixed paraffin-embedded (FFPE) tissues. The NanoString nCounter® platform is a gene expression analysis tool developed for use with FFPE-derived samples. We designed a custom nCounter® codeset based on elements from multiple published fresh frozen tissue microarray-based prognostic gene signatures for colon cancer, and we used this platform to systematically compare gene expression data from FFPE with matched microarray array data from frozen tissues. Our results show moderate correlation of gene expression between two platforms and discovery of a small subset of genes as candidate biomarkers for colon cancer prognosis that are detectable and quantifiable in FFPE tissue sections. PMID:27176004

  17. Expression of ITGB1 predicts prognosis in colorectal cancer: a large prospective study based on tissue microarray

    PubMed Central

    Liu, Qi-Zhi; Gao, Xian-Hua; Chang, Wen-Jun; Gong, Hai-Feng; Fu, Chuan-Gang; Zhang, Wei; Cao, Guang-Wen

    2015-01-01

    Background: ITGB1 is a heterodimeric cell-surface receptor involved in cell functions such as proliferation, migration, invasion and survival. The aim of this study was to assess ITGB1 expression in colorectal cancer and correlate it with clinicopathological features, as well as to evaluate its potential prognostic significance. Materials and methods: In this study, we examined the expression of ITGB1 using tissue microarrays containing analyzed specimens by immunohistochemistry. ITGB1 expression was further correlated with clinicopathological and prognostic data. The prognostic significance was assessed using Kaplan-Meier survival estimates and log-rank tests. A multivariate study with the Coxs proportional hazard model was used to evaluate the prognostic aspects. Results: ITGB1 expression was present in 88.5% of the analyzed specimens. Significant differences in ITGB1 expression were found between normal mucosa and carcinomas (P<0.001). High ITGB1 expression was associated with poor prognosis, and it independently correlated with shortened overall survival and disease-free survival in colorectal cancer patients (P<0.001). More so, ITGB1 expression, bowel wall invasion, lymph node metastasis and distant metastasis were independent prognostic factors for overall survival. Additionally, significant differences in ITGB1 expression were observed in adenomas and tumors from patients with familial adenomatous polyposis compared to normal colon mucosa (P<0.05) Conclusion: The results of this study indicate that ITGB1 overexpression in colorectal tumors is associated with poor prognosis, as well as aggressive clinicopathological features. Therefore, ITGB1 expression could be used as potential prognostic predictor in colorectal cancer patients. PMID:26722470

  18. Using Ambystoma mexicanum (Mexican axolotl) embryos, chemical genetics, and microarray analysis to identify signaling pathways associated with tissue regeneration.

    PubMed

    Ponomareva, Larissa V; Athippozhy, Antony; Thorson, Jon S; Voss, S Randal

    2015-12-01

    Amphibian vertebrates are important models in regenerative biology because they present exceptional regenerative capabilities throughout life. However, it takes considerable effort to rear amphibians to juvenile and adult stages for regeneration studies, and the relatively large sizes that frogs and salamanders achieve during development make them difficult to use in chemical screens. Here, we introduce a new tail regeneration model using late stage Mexican axolotl embryos. We show that axolotl embryos completely regenerate amputated tails in 7days before they exhaust their yolk supply and begin to feed. Further, we show that axolotl embryos can be efficiently reared in microtiter plates to achieve moderate throughput screening of soluble chemicals to investigate toxicity and identify molecules that alter regenerative outcome. As proof of principle, we identified integration 1 / wingless (Wnt), transforming growth factor beta (Tgf-β), and fibroblast growth factor (Fgf) pathway antagonists that completely block tail regeneration and additional chemicals that significantly affected tail outgrowth. Furthermore, we used microarray analysis to show that inhibition of Wnt signaling broadly affects transcription of genes associated with Wnt, Fgf, Tgf-β, epidermal growth factor (Egf), Notch, nerve growth factor (Ngf), homeotic gene (Hox), rat sarcoma/mitogen-activated protein kinase (Ras/Mapk), myelocytomatosis viral oncogene (Myc), tumor protein 53 (p53), and retinoic acid (RA) pathways. Punctuated changes in the expression of genes known to regulate vertebrate development were observed; this suggests the tail regeneration transcriptional program is hierarchically structured and temporally ordered. Our study establishes the axolotl as a chemical screening model to investigate signaling pathways associated with tissue regeneration. PMID:26092703

  19. HER2 in gastric cancer: an immunohistochemical study on tissue microarrays and the corresponding whole-tissue sections with a supplemental fish study.

    PubMed

    Gasljevic, Gorana; Lamovec, Janez; Contreras, Juan Antonio; Zadnik, Vesna; Blas, Mateja; Gasparov, Slavko

    2013-10-01

    Since focal HER2 expression is an issue in GC, TMA construction from the paraffin-embedded surgically-obtained tissue may not reflect its real status. The aim of this study was to assess the HER2 status in tissue microarrays (TMAs) and the corresponding whole sections using HercepTest immunohistochemistry (IHC), and to correlate it and to assess the concordance of HER2 IHC and fluorescence in situ hybridization (FISH) in TMAs. Concordance of the HER2 expression status for 302 cases of gastric cancer using 9 paired TMAs was evaluated using a 2-mm core size and 305 corresponding whole sections. Concordance of the IHC and FISH HER2 status was compared. In addition,, the HER2 status was compared to clinicopathological characteristics and patients' survival. Using the whole-section approach, HER2 over-expression was found in 25.2 % (HER2 3+ 6.6 %, HER2 2+ 18.7 %) of tumours. The overall concordance of IHC between the cores and the whole section was 84.9 %; 15.1 % of the tumours showed HER2 amplification. The overall concordance of IHC and FISH on cores was 75.7 %. The level of amplification correlated with the IHC score. Relationship between the intestinal and papillary types and tumour grade was observed for tumours with over-expression and amplification, whereas tumour location was related only to over-expression. There was a statistically significant difference in the overall survival of the patients, which was related to HER2 amplification. In conclusion, good concordance of the IHC HER2 results between tissue cores in TMA and whole sections, and excellent concordance of the IHC and FISH results on tissue cores was found. At least a part of the observed IHC HER2 heterogeneity could very likely be explained by fixation artifacts. With adequate fixation, a higher concordance of IHC HER2 between the cores and the whole sections can be expected. The TMA approach could enable an easier analysis of more than one representative tumour block. PMID:23800891

  20. Genome-wide effects of acute progressive feed restriction in liver and white adipose tissue

    SciTech Connect

    Pohjanvirta, Raimo Boutros, Paul C.; Moffat, Ivy D.; Linden, Jere; Wendelin, Dominique; Okey, Allan B.

    2008-07-01

    Acute progressive feed restriction (APFR) represents a specific form of caloric restriction in which feed availability is increasingly curtailed over a period of a few days to a few weeks. It is often used for control animals in toxicological and pharmacological studies on compounds causing body weight loss to equalize weight changes between experimental and control groups and thereby, intuitively, to also set their metabolic states to the same phase. However, scientific justification for this procedure is lacking. In the present study, we analyzed by microarrays the impact on hepatic gene expression in rats of two APFR regimens that caused identical diminution of body weight (19%) but differed slightly in duration (4 vs. 10 days). In addition, white adipose tissue (WAT) was also subjected to the transcriptomic analysis on day-4. The data revealed that the two regimens led to distinct patterns of differentially expressed genes in liver, albeit some major pathways of energy metabolism were similarly affected (particularly fatty acid and amino acid catabolism). The reason for the divergence appeared to be entrainment by the longer APFR protocol of peripheral oscillator genes, which resulted in derailment of circadian rhythms and consequent interaction of altered diurnal fluctuations with metabolic adjustments in gene expression activities. WAT proved to be highly unresponsive to the 4-day APFR as only 17 mRNA levels were influenced by the treatment. This study demonstrates that body weight is a poor proxy of metabolic state and that the customary protocols of feed restriction can lead to rhythm entrainment.

  1. Development of a Pacific oyster (Crassostrea gigas) 31,918-feature microarray: identification of reference genes and tissue-enriched expression patterns

    PubMed Central

    2011-01-01

    Background Research using the Pacific oyster Crassostrea gigas as a model organism has experienced rapid growth in recent years due to the development of high-throughput molecular technologies. As many as 56,268 EST sequences have been sequenced to date, representing a genome-wide resource that can be used for transcriptomic investigations. Results In this paper, we developed a Pacific oyster microarray containing oligonucleotides representing 31,918 transcribed sequences selected from the publicly accessible GigasDatabase. This newly designed microarray was used to study the transcriptome of male and female gonads, mantle, gills, posterior adductor muscle, visceral ganglia, hemocytes, labial palps and digestive gland. Statistical analyses identified genes differentially expressed among tissues and clusters of tissue-enriched genes. These genes reflect major tissue-specific functions at the molecular level, such as tissue formation in the mantle, filtering in the gills and labial palps, and reproduction in the gonads. Hierarchical clustering predicted the involvement of unannotated genes in specific functional pathways such as the insulin/NPY pathway, an important pathway under study in our model species. Microarray data also accurately identified reference genes whose mRNA level appeared stable across all the analyzed tissues. Adp-ribosylation factor 1 (arf1) appeared to be the most robust reference for normalizing gene expression data across different tissues and is therefore proposed as a relevant reference gene for further gene expression analysis in the Pacific oyster. Conclusions This study provides a new transcriptomic tool for studies of oyster biology, which will help in the annotation of its genome and which identifies candidate reference genes for gene expression analysis. PMID:21951653

  2. Clinicopathologic Significance of HNF-1?, AIRD1A, and PIK3CA Expression in Ovarian Clear Cell Carcinoma: A Tissue Microarray Study of 130 Cases.

    PubMed

    Ye, Shuang; Yang, Jiaxin; You, Yan; Cao, Dongyan; Huang, Huifang; Wu, Ming; Chen, Jie; Lang, Jinghe; Shen, Keng

    2016-03-01

    Ovarian clear cell carcinoma (CCC) is a distinct histologic subtype with relatively poor survival. No prognostic or predictive molecular marker is currently available. Recent studies have shown that AT-rich interactive domain 1A (ARID1A) and phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutations are common genetic changes in ovarian CCC. Hepatocyte nuclear factor-1? (HNF-1?) expression has been proven to be highly sensitive and specific for clear cell histology. However, the correlations between these biomarkers and clinicopathologic variables and survival outcomes are controversial.The immunohistochemical analysis for HNF-1?, ARID1A, and PIK3CA was performed on a tissue microarray (TMA) consisting of 130 cases of ovarian CCC (237 tissue blocks) linked with clinical information. The immunostaining results were interpreted in a manner consistent with previous publications. The associations between biomarker expression and clinical and prognostic features were examined. All statistical analyses were conducted using 2-sided tests, and a value of P?progression-free survival (PFS) (P?=?0.03 and P?=?0.01, respectively). On the contrary, patients with high-level HNF-1? were associated with good prognosis (P?=?0.02 for OS and P?=?0.01 for PFS). PIK3CA expression had no impact on survival. For univariate and multivariate analyses, only HNF-1? expression seemed to be a prognostic factor for favorable OS (P?=?0.04).The loss of ARID1A was correlated with late-stage and endometriosis-associated tumors. The measurement of ARID1A expression might be a method to predict the risk of recurrence. Among the 3 biomarkers, only high-level HNF-1? expression proved to be a positive predictor for OS. PMID:26945423

  3. Determining sensitivity and specificity of HER2 testing in breast cancer using a tissue micro-array approach

    PubMed Central

    2012-01-01

    Introduction Overexpression of the human epidermal growth factor receptor 2 (HER2) as a result of HER2 gene amplification is associated with a relatively poor prognosis in breast cancer and is predictive of HER2-targeting therapy response. False-positive rates of up to 20% for HER2 testing have been described. HER2-testing laboratories are therefore encouraged to participate in external quality control schemes in order to improve HER2-testing standardization. Methods This study investigated the feasibility of retesting large numbers of invasive breast cancers for HER2 status on tissue micro-array (TMA) as part of a quality control scheme. For this assessment different HER2 testing methods were used including HER2 detecting antibodies SP3, 4B5, Herceptest and mono color silver in situ hybridization (SISH) and dual color SISH. Final HER2 status for each tumor on the TMA was compared to the local testing result for the same tumor. Discordances between these two results were investigated further by staining whole tumor sections. Results For this study, 1,210 invasive breast carcinomas of patients treated in six hospitals between 2006 and 2008 were evaluated. Results from the three immunohistochemistry (IHC) and two in situ hybridization (ISH) assays performed on the TMAs were compared. The final HER2 status on TMA was determined with SP3, 4B5 and mono color SISH. Concordance between local HER2 test results and TMA retesting was 98.0%. Discordant results between local and TMA retesting were found in 20 tumors (2.0%). False positive HER2 IHC results were identified in 13 (1.3%) tumors; false negative IHC results in seven (0.7%) tumors. Conclusions Retesting large volumes of HER2 classified breast carcinomas was found to be feasible and can be reliably performed by staining TMAs with SP3, 4B5 and mono color SISH in combination with full-sized slides for discordant cases. The frequency of false-positive results was lower than previously reported in the literature. This method is now offered to other HER2-testing laboratories. PMID:22694844

  4. Tissue Array Research Program (TARP)

    Cancer.gov

    New technologies, including: Tissue microarray construction using an automated tissue microarray tool. Digital imaging of tissue microarrays for automated or manual interpretation. Standard histology techniques, including: Tissue fixation and processing.

  5. DNA Microarrays

    NASA Astrophysics Data System (ADS)

    Nguyen, C.; Gidrol, X.

    Genomics has revolutionised biological and biomedical research. This revolution was predictable on the basis of its two driving forces: the ever increasing availability of genome sequences and the development of new technology able to exploit them. Up until now, technical limitations meant that molecular biology could only analyse one or two parameters per experiment, providing relatively little information compared with the great complexity of the systems under investigation. This gene by gene approach is inadequate to understand biological systems containing several thousand genes. It is essential to have an overall view of the DNA, RNA, and relevant proteins. A simple inventory of the genome is not sufficient to understand the functions of the genes, or indeed the way that cells and organisms work. For this purpose, functional studies based on whole genomes are needed. Among these new large-scale methods of molecular analysis, DNA microarrays provide a way of studying the genome and the transcriptome. The idea of integrating a large amount of data derived from a support with very small area has led biologists to call these chips, borrowing the term from the microelectronics industry. At the beginning of the 1990s, the development of DNA chips on nylon membranes [1, 2], then on glass [3] and silicon [4] supports, made it possible for the first time to carry out simultaneous measurements of the equilibrium concentration of all the messenger RNA (mRNA) or transcribed RNA in a cell. These microarrays offer a wide range of applications, in both fundamental and clinical research, providing a method for genome-wide characterisation of changes occurring within a cell or tissue, as for example in polymorphism studies, detection of mutations, and quantitative assays of gene copies. With regard to the transcriptome, it provides a way of characterising differentially expressed genes, profiling given biological states, and identifying regulatory channels.

  6. Pulp and dentin tissue engineering and regeneration: current progress.

    PubMed

    Huang, George T J

    2009-09-01

    Dental pulp tissue is vulnerable to infection. Entire pulp amputation followed by pulp-space disinfection and filling with an artificial rubber-like material is employed to treat the infection - commonly known as root-canal therapy. Regeneration of pulp tissue has been difficult as the tissue is encased in dentin without collateral blood supply except from the root apical end. However, with the advent of the concept of modern tissue engineering and the discovery of dental stem cells, regeneration of pulp and dentin has been tested. This article will review the early attempts to regenerate pulp tissue and the current endeavor of pulp and dentin tissue engineering, and regeneration. The prospective outcome of the current advancement in this line of research will be discussed. PMID:19761395

  7. Pulp and dentin tissue engineering and regeneration: current progress

    PubMed Central

    Huang, George TJ

    2009-01-01

    Dental pulp tissue is vulnerable to infection. Entire pulp amputation followed by pulp-space disinfection and filling with an artificial rubber-like material is employed to treat the infection – commonly known as root-canal therapy. Regeneration of pulp tissue has been difficult as the tissue is encased in dentin without collateral blood supply except from the root apical end. However, with the advent of the concept of modern tissue engineering and the discovery of dental stem cells, regeneration of pulp and dentin has been tested. This article will review the early attempts to regenerate pulp tissue and the current endeavor of pulp and dentin tissue engineering, and regeneration. The prospective outcome of the current advancement in this line of research will be discussed. PMID:19761395

  8. Hydrogel scaffolds for tissue engineering: Progress and challenges

    PubMed Central

    El-Sherbiny, Ibrahim M.; Yacoub, Magdi H.

    2013-01-01

    Designing of biologically active scaffolds with optimal characteristics is one of the key factors for successful tissue engineering. Recently, hydrogels have received a considerable interest as leading candidates for engineered tissue scaffolds due to their unique compositional and structural similarities to the natural extracellular matrix, in addition to their desirable framework for cellular proliferation and survival. More recently, the ability to control the shape, porosity, surface morphology, and size of hydrogel scaffolds has created new opportunities to overcome various challenges in tissue engineering such as vascularization, tissue architecture and simultaneous seeding of multiple cells. This review provides an overview of the different types of hydrogels, the approaches that can be used to fabricate hydrogel matrices with specific features and the recent applications of hydrogels in tissue engineering. Special attention was given to the various design considerations for an efficient hydrogel scaffold in tissue engineering. Also, the challenges associated with the use of hydrogel scaffolds were described. PMID:24689032

  9. [Progress on strategies to promote vascularization in bone tissue engineering].

    PubMed

    Chen, Kai; Zhang, Chao; Wang, Lu; Mao, Yu-Yan; Lu, Jian-Xi; Chen, Lei

    2015-04-01

    With the continuous development of bone tissue engineering, a variety of emerging bone graft materials provided various methods for repairing bone defects. Early and rapid accomplishment of revascularization of materials interior after implantation of bone transplantation materials is a difficulty faced to bone tissue engineering. Blood vessels ingrowth provides the requisite netritional support for the regeneration reconstruction of bone tissue, for this reason, vascularization plays a significant role in bone tissue engineering. However,there is not a golden standard strategy of vascularization at present. Scaffold materials, cells and growth factors still are three indispensable elements in tissue engineering, and are cardinal points of the promoting vascularization strategies. Multiple growth factors or multiple cells combined with scaffolds, which are hot spots, have obtained excellent vascularization. This review focused on the comprehensive strategies for promoting the successful vascularization of tissue engineered scaffolds. PMID:26072627

  10. Microarray analysis of spaceflown murine thymus tissue reveals changes in gene expression regulating stress and glucocorticoid receptors.

    PubMed

    Lebsack, Ty W; Fa, Vuna; Woods, Chris C; Gruener, Raphael; Manziello, Ann M; Pecaut, Michael J; Gridley, Daila S; Stodieck, Louis S; Ferguson, Virginia L; Deluca, Dominick

    2010-05-15

    The detrimental effects of spaceflight and simulated microgravity on the immune system have been extensively documented. We report here microarray gene expression analysis, in concert with quantitative RT-PCR, in young adult C57BL/6NTac mice at 8 weeks of age after exposure to spaceflight aboard the space shuttle (STS-118) for a period of 13 days. Upon conclusion of the mission, thymus lobes were extracted from space flown mice (FLT) as well as age- and sex-matched ground control mice similarly housed in animal enclosure modules (AEM). mRNA was extracted and an automated array analysis for gene expression was performed. Examination of the microarray data revealed 970 individual probes that had a 1.5-fold or greater change. When these data were averaged (n = 4), we identified 12 genes that were significantly up- or down-regulated by at least 1.5-fold after spaceflight (P < or = 0.05). The genes that significantly differed from the AEM controls and that were also confirmed via QRT-PCR were as follows: Rbm3 (up-regulated) and Hsph110, Hsp90aa1, Cxcl10, Stip1, Fkbp4 (down-regulated). QRT-PCR confirmed the microarray results and demonstrated additional gene expression alteration in other T cell related genes, including: Ctla-4, IFN-alpha2a (up-regulated) and CD44 (down-regulated). Together, these data demonstrate that spaceflight induces significant changes in the thymic mRNA expression of genes that regulate stress, glucocorticoid receptor metabolism, and T cell signaling activity. These data explain, in part, the reported systemic compromise of the immune system after exposure to the microgravity of space. PMID:20213684

  11. Manual evaluation of tissue microarrays in a high-throughput research project: The contribution of Indian surgical pathology to the Human Protein Atlas (HPA) project.

    PubMed

    Navani, Sanjay

    2016-04-01

    The Human Protein Atlas (HPA) program (www.proteinatlas.org) is an international program that has been set up to allow for a systematic exploration of the human proteome using antibody-based proteomics. This is accomplished by combining high-throughput generation of affinity-purified (mono-specific) antibodies with protein profiling in a multitude of tissues/cell types assembled in tissue microarrays. Twenty-six surgical pathologists over a seven-and-half year period have annotated and curated approximately sixteen million tissue images derived from immunostaining of normal and cancer tissues by approximately 23 000 antibodies. Web-based annotation software that allows for a basic and rapid evaluation of immunoreactivity in tissues has been utilized. Intensity, fraction of immunoreactive cells and subcellular localization were recorded for each given cell population. A text comment summarizing the characteristics for each antibody was added. The methods used and the challenges encountered for this exercise, the largest effort ever by a single group of surgical pathologists, are discussed. Manual annotation of digital images is an important tool that may be successfully utilized in high-throughput research projects. This is the first time an Indian private pathology laboratory has been associated with cutting-edge research internationally providing a classic example of developed and emerging nation collaboration. PMID:26748468

  12. Fiber-Based Tissue Engineering: Progress, Challenges, and Opportunities

    PubMed Central

    Tamayol, Ali; Akbari, Mohsen; Annabi, Nasim; Paul, Arghya; Khademhosseini, Ali; Juncker, David

    2013-01-01

    Tissue engineering aims to improve the function of diseased or damaged organs by creating biological substitutes. To fabricate a functional tissue, the engineered construct should mimic the physiological environment including its structural, topographical, and mechanical properties. Moreover, the construct should facilitate nutrients and oxygen diffusion as well as removal of metabolic waste during tissue regeneration. In the last decade, fiber-based techniques such as weaving, knitting, braiding, as well as electrospinning, and direct writing have emerged as promising platforms for making 3D tissue constructs that can address the above mentioned challenges. Here, we critically review the techniques used to form cell-free and cell-laden fibers and to assemble them into scaffolds. We compare their mechanical properties, morphological features and biological activity. We discuss current challenges and future opportunities of fiber-based tissue engineering (FBTE) for use in research and clinical practice. PMID:23195284

  13. Progress and opportunities for tissue-engineered skin

    NASA Astrophysics Data System (ADS)

    MacNeil, Sheila

    2007-02-01

    Tissue-engineered skin is now a reality. For patients with extensive full-thickness burns, laboratory expansion of skin cells to achieve barrier function can make the difference between life and death, and it was this acute need that drove the initiation of tissue engineering in the 1980s. A much larger group of patients have ulcers resistant to conventional healing, and treatments using cultured skin cells have been devised to restart the wound-healing process. In the laboratory, the use of tissue-engineered skin provides insight into the behaviour of skin cells in healthy skin and in diseases such as vitiligo, melanoma, psoriasis and blistering disorders.

  14. Normal morphogenesis of epithelial tissues and progression of epithelial tumors

    PubMed Central

    Wang, Chun-Chao; Jamal, Leen; Janes, Kevin A.

    2011-01-01

    Epithelial cells organize into various tissue architectures that largely maintain their structure throughout the life of an organism. For decades, the morphogenesis of epithelial tissues has fascinated scientists at the interface of cell, developmental, and molecular biology. Systems biology offers ways to combine knowledge from these disciplines by building integrative models that are quantitative and predictive. Can such models be useful for gaining a deeper understanding of epithelial morphogenesis? Here, we take inventory of some recurring themes in epithelial morphogenesis that systems approaches could strive to capture. Predictive understanding of morphogenesis at the systems level would prove especially valuable for diseases such as cancer, where epithelial tissue architecture is profoundly disrupted. PMID:21898857

  15. Robotic multimodality stereotactic brain tissue identification: work in progress.

    PubMed

    Andrews, R; Mah, R; Galvagni, A; Guerrero, M; Papasin, R; Wallace, M; Winters, J

    1997-01-01

    Real-time identification of tissue would improve procedures such as stereotactic brain biopsy (SBX), functional and implantation neurosurgery, and brain tumor excision. To standard SBX equipment has been added: (1) computer-controlled stepper motors to drive the biopsy needle/probe precisely; (2) multiple microprobes to track tissue density, detect blood vessels and changes in blood flow, and distinguish the various tissues being penetrated; (3) neural net learning programs to allow real-time comparisons of current data with a normative data bank; (4) three-dimensional graphic displays to follow the probe as it traverses brain tissue. The probe can differentiate substances such as pig brain, differing consistencies of the 'brain-like' foodstuff tofu, and gels made to simulate brain, as well as detect blood vessels imbedded in these substances. Multimodality probes should improve the safety, efficacy, and diagnostic accuracy of SBX and other neurosurgical procedures. PMID:9711699

  16. Robotic multimodality stereotactic brain tissue identification: work in progress

    NASA Technical Reports Server (NTRS)

    Andrews, R.; Mah, R.; Galvagni, A.; Guerrero, M.; Papasin, R.; Wallace, M.; Winters, J.

    1997-01-01

    Real-time identification of tissue would improve procedures such as stereotactic brain biopsy (SBX), functional and implantation neurosurgery, and brain tumor excision. To standard SBX equipment has been added: (1) computer-controlled stepper motors to drive the biopsy needle/probe precisely; (2) multiple microprobes to track tissue density, detect blood vessels and changes in blood flow, and distinguish the various tissues being penetrated; (3) neural net learning programs to allow real-time comparisons of current data with a normative data bank; (4) three-dimensional graphic displays to follow the probe as it traverses brain tissue. The probe can differentiate substances such as pig brain, differing consistencies of the 'brain-like' foodstuff tofu, and gels made to simulate brain, as well as detect blood vessels imbedded in these substances. Multimodality probes should improve the safety, efficacy, and diagnostic accuracy of SBX and other neurosurgical procedures.

  17. Tendon tissue engineering: progress, challenges, and translation to the clinic.

    PubMed

    Shearn, J T; Kinneberg, K R; Dyment, N A; Galloway, M T; Kenter, K; Wylie, C; Butler, D L

    2011-06-01

    The tissue engineering field has made great strides in understanding how different aspects of tissue engineered constructs (TECs) and the culture process affect final tendon repair. However, there remain significant challenges in developing strategies that will lead to a clinically effective and commercially successful product. In an effort to increase repair quality, a better understanding of normal development, and how it differs from adult tendon healing, may provide strategies to improve tissue engineering. As tendon tissue engineering continues to improve, the field needs to employ more clinically relevant models of tendon injury such as degenerative tendons. We need to translate successes to larger animal models to begin exploring the clinical implications of our treatments. By advancing the models used to validate our TECs, we can help convince our toughest customer, the surgeon, that our products will be clinically efficacious. As we address these challenges in musculoskeletal tissue engineering, the field still needs to address the commercialization of products developed in the laboratory. TEC commercialization faces numerous challenges because each injury and patient is unique. This review aims to provide tissue engineers with a summary of important issues related to engineering tendon repairs and potential strategies for producing clinically successful products. PMID:21625053

  18. Tendon Tissue Engineering: Progress, Challenges, and Translation to the Clinic

    PubMed Central

    Shearn, Jason T.; Kinneberg, Kirsten R.C.; Dyment, Nathaniel A.; Galloway, Marc T.; Kenter, Keith; Wylie, Christopher; Butler, David L.

    2013-01-01

    The tissue engineering field has made great strides in understanding how different aspects of tissue engineered constructs (TECs) and the culture process affect final tendon repair. However, there remain significant challenges in developing strategies that will lead to a clinically effective and commercially successful product. In an effort to increase repair quality, a better understanding of normal development, and how it differs from adult tendon healing, may provide strategies to improve tissue engineering. As tendon tissue engineering continues to improve, the field needs to employ more clinically relevant models of tendon injury such as degenerative tendons. We need to translate successes to larger animal models to begin exploring the clinical implications of our treatments. By advancing the models used to validate our TECs, we can help convince our toughest customer, the surgeon, that our products will be clinically efficacious. As we address these challenges in musculoskeletal tissue engineering, the field still needs to address the commercialization of products developed in the laboratory. TEC commercialization faces numerous challenges because each injury and patient is unique. This review aims to provide tissue engineers with a summary of important issues related to engineering tendon repairs and potential strategies for producing clinically successful products. PMID:21625053

  19. ImageMiner: a software system for comparative analysis of tissue microarrays using content-based image retrieval, high-performance computing, and grid technology

    PubMed Central

    Foran, David J; Yang, Lin; Hu, Jun; Goodell, Lauri A; Reiss, Michael; Wang, Fusheng; Kurc, Tahsin; Pan, Tony; Sharma, Ashish; Saltz, Joel H

    2011-01-01

    Objective and design The design and implementation of ImageMiner, a software platform for performing comparative analysis of expression patterns in imaged microscopy specimens such as tissue microarrays (TMAs), is described. ImageMiner is a federated system of services that provides a reliable set of analytical and data management capabilities for investigative research applications in pathology. It provides a library of image processing methods, including automated registration, segmentation, feature extraction, and classification, all of which have been tailored, in these studies, to support TMA analysis. The system is designed to leverage high-performance computing machines so that investigators can rapidly analyze large ensembles of imaged TMA specimens. To support deployment in collaborative, multi-institutional projects, ImageMiner features grid-enabled, service-based components so that multiple instances of ImageMiner can be accessed remotely and federated. Results The experimental evaluation shows that: (1) ImageMiner is able to support reliable detection and feature extraction of tumor regions within imaged tissues; (2) images and analysis results managed in ImageMiner can be searched for and retrieved on the basis of image-based features, classification information, and any correlated clinical data, including any metadata that have been generated to describe the specified tissue and TMA; and (3) the system is able to reduce computation time of analyses by exploiting computing clusters, which facilitates analysis of larger sets of tissue samples. PMID:21606133

  20. Microarrays for Undergraduate Classes

    ERIC Educational Resources Information Center

    Hancock, Dale; Nguyen, Lisa L.; Denyer, Gareth S.; Johnston, Jill M.

    2006-01-01

    A microarray experiment is presented that, in six laboratory sessions, takes undergraduate students from the tissue sample right through to data analysis. The model chosen, the murine erythroleukemia cell line, can be easily cultured in sufficient quantities for class use. Large changes in gene expression can be induced in these cells by…

  1. Microarrays for Undergraduate Classes

    ERIC Educational Resources Information Center

    Hancock, Dale; Nguyen, Lisa L.; Denyer, Gareth S.; Johnston, Jill M.

    2006-01-01

    A microarray experiment is presented that, in six laboratory sessions, takes undergraduate students from the tissue sample right through to data analysis. The model chosen, the murine erythroleukemia cell line, can be easily cultured in sufficient quantities for class use. Large changes in gene expression can be induced in these cells by

  2. [Progress on cervical muscle strength and soft tissue stiffness testing].

    PubMed

    Ma, Ming; Zhang, Shi-min

    2015-08-01

    Biomechanical evaluation of neck muscles has important significance in the diagnosis and treatment for cervical spondylosis, the neck muscle strength and soft tissue stiffness test is two aspects of biomechanical testing. Isometric muscle testing operation is relatively simple, the cost is lower, which can evaluate the muscle force below grade 3. However, isokinetic muscle strength testing can assess the muscle strength of joint motion in any position. It is hard to distinguish stiffness difference in different soft tissues when the load-displacement curve is used to evaluate the local soft tissue stiffness. Elasticity imaging technique can not only show the elastic differences of different tissues by images, but also quantify the elastic modulus of subcutaneous tissues and muscles respectively. Nevertheless, it is difficult to observe the flexibility of the cervical spine by means of the analysis of the whole neck stiffness. In a word, a variety of test method will conduce not only the biomechanical evaluation of neck muscles, but also making an effective biomechanics mathematical model of neck muscles. Besides, isokinetic muscle testing and the elasticity imaging technology still need further validation and optimization before they are better applied to neck muscles biomechanical testing. PMID:26502535

  3. A PROGRESSIVE RUPTURE MODEL OF SOFT TISSUE STRESS RELAXATION

    PubMed Central

    Bates, Jason H.T.; Ma, Baoshun

    2013-01-01

    A striking feature of stress relaxation in biological soft tissue is that it frequently follows a power law in time with an exponent that is independent of strain even when the elastic properties of the tissue are highly nonlinear. This kind of behavior is an example of quasi-linear viscoelasticity, and is usually modeled in a purely empirical fashion. The goal of the present study was to account for quasi-linear viscoelasticity in mechanistic terms based on our previously developed hypothesis that it arises as a result of isolated micro-yield events occurring in sequence throughout the tissue, each event passing the stress it was sustaining on to other regions of the tissue until they themselves yield. We modeled stress relaxation computationally in a collection of stress-bearing elements. Each element experiences a stochastic sequence of either increases in elastic equilibrium length or decreases in stiffness according to the stress imposed upon it. This successfully predicts quasi-linear viscoelastic behavior, and in addition predicts power-law stress relaxation that proceeds at the same slow rate as observed in real biological soft tissue. PMID:23508634

  4. Tissue engineering and regenerative medicine: history, progress, and challenges.

    PubMed

    Berthiaume, Franois; Maguire, Timothy J; Yarmush, Martin L

    2011-01-01

    The past three decades have seen the emergence of an endeavor called tissue engineering and regenerative medicine in which scientists, engineers, and physicians apply tools from a variety of fields to construct biological substitutes that can mimic tissues for diagnostic and research purposes and can replace (or help regenerate) diseased and injured tissues. A significant portion of this effort has been translated to actual therapies, especially in the areas of skin replacement and, to a lesser extent, cartilage repair. A good amount of thoughtful work has also yielded prototypes of other tissue substitutes such as nerve conduits, blood vessels, liver, and even heart. Forward movement to clinical product, however, has been slow. Another offshoot of these efforts has been the incorporation of some new exciting technologies (e.g., microfabrication, 3D printing) that may enable future breakthroughs. In this review we highlight the modest beginnings of the field and then describe three application examples that are in various stages of development, ranging from relatively mature (skin) to ongoing proof-of-concept (cartilage) to early stage (liver). We then discuss some of the major issues that limit the development of complex tissues, some of which are fundamentals-based, whereas others stem from the needs of the end users. PMID:22432625

  5. Adipose Tissue in Metabolic Syndrome: Onset and Progression of Atherosclerosis.

    PubMed

    Luna-Luna, María; Medina-Urrutia, Aida; Vargas-Alarcón, Gilberto; Coss-Rovirosa, Fernanda; Vargas-Barrón, Jesús; Pérez-Méndez, Óscar

    2015-07-01

    Metabolic syndrome (MetS) should be considered a clinical entity when its different symptoms share a common etiology: obesity/insulin resistance as a result of a multi-organ dysfunction. The main interest in treating MetS as a clinical entity is that the addition of its components drastically increases the risk of atherosclerosis. In MetS, the adipose tissue plays a central role along with an unbalanced gut microbiome, which has become relevant in recent years. Once visceral adipose tissue (VAT) increases, dyslipidemia and endothelial dysfunction follow as additive risk factors. However, when the nonalcoholic fatty liver is present, risk of a cardiovascular event is highly augmented. Epicardial adipose tissue (EAT) seems to increase simultaneously with the VAT. In this context, the former may play a more important role in the development of the atherosclerotic plaque than the latter. Hence, EAT may act as a paracrine tissue vis-à-vis the coronary arteries favoring the local inflammation and the atheroma calcification. PMID:26009250

  6. A microarray-based method for the parallel analysis of genotypes and expression profiles of wood-forming tissues in Eucalyptus grandis

    PubMed Central

    Barros, Eugenia; van Staden, Carol-Ann; Lezar, Sabine

    2009-01-01

    Background Fast-growing Eucalyptus grandis trees are one of the most efficient producers of wood in South Africa. The most serious problem affecting the quality and yield of solid wood products is the occurrence of end splitting in logs. Selection of E. grandis planting stock that exhibit preferred wood qualities is thus a priority of the South African forestry industry. We used microarray-based DNA-amplified fragment length polymorphism (AFLP) analysis in combination with expression profiling to develop fingerprints and profile gene expression of wood-forming tissue of seven different E. grandis trees. Results A 1578-probe cDNA microarray was constructed by arraying 768 cDNA-AFLP clones and 810 cDNA library clones from seven individual E. grandis trees onto silanised slides. The results revealed that 32% of the spotted fragments showed distinct expression patterns (with a fold change of at least 1.4 or -1.4 and a p value of 0.01) could be grouped into clusters representing co-expressed genes. Evaluation of the binary distribution of cDNA-AFLP fragments on the array showed that the individual genotypes could be discriminated. Conclusion A simple, yet general method was developed for genotyping and expression profiling of wood-forming tissue of E. grandis trees differing in their splitting characteristics and in their lignin contents. Evaluation of gene expression profiles and the binary distribution of cDNA-AFLP fragments on the chip suggest that the prototype chip developed could be useful for transcript profiling and for the identification of Eucalyptus trees with preferred wood quality traits in commercial breeding programmes. PMID:19473481

  7. Numerical and Structural Genomic Aberrations Are Reliably Detectable in Tissue Microarrays of Formalin-Fixed Paraffin-Embedded Tumor Samples by Fluorescence In-Situ Hybridization

    PubMed Central

    Horn, Heike; Bausinger, Julia; Staiger, Annette M.; Sohn, Maximilian; Schmelter, Christopher; Gruber, Kim; Kalla, Claudia; Ott, M. Michaela; Rosenwald, Andreas; Ott, German

    2014-01-01

    Few data are available regarding the reliability of fluorescence in-situ hybridization (FISH), especially for chromosomal deletions, in high-throughput settings using tissue microarrays (TMAs). We performed a comprehensive FISH study for the detection of chromosomal translocations and deletions in formalin-fixed and paraffin-embedded (FFPE) tumor specimens arranged in TMA format. We analyzed 46 B-cell lymphoma (B-NHL) specimens with known karyotypes for translocations of IGH-, BCL2-, BCL6- and MYC-genes. Locus-specific DNA probes were used for the detection of deletions in chromosome bands 6q21 and 9p21 in 62 follicular lymphomas (FL) and six malignant mesothelioma (MM) samples, respectively. To test for aberrant signals generated by truncation of nuclei following sectioning of FFPE tissue samples, cell line dilutions with 9p21-deletions were embedded into paraffin blocks. The overall TMA hybridization efficiency was 94%. FISH results regarding translocations matched karyotyping data in 93%. As for chromosomal deletions, sectioning artefacts occurred in 17% to 25% of cells, suggesting that the proportion of cells showing deletions should exceed 25% to be reliably detectable. In conclusion, FISH represents a robust tool for the detection of structural as well as numerical aberrations in FFPE tissue samples in a TMA-based high-throughput setting, when rigorous cut-off values and appropriate controls are maintained, and, of note, was superior to quantitative PCR approaches. PMID:24733537

  8. Pax‐5 immunoexpression in various types of benign and malignant tumours: a high‐throughput tissue microarray analysis

    PubMed Central

    Mhawech‐Fauceglia, Paulette; Saxena, Rhakee; Zhang, Shaozeng; Terracciano, Luigi; Sauter, Guido; Chadhuri, Arundhuti; Herrmann, Francois R; Penetrante, Remedios

    2007-01-01

    Background Pax‐5 belongs to the Pax gene family transcription factors that play an important role in organogenesis and in B cell ontogeny. It is expressed in B cell non‐Hodgkin's lymphoma (B‐NHL), Hodgkin's lymphoma (HL) and neuroendocrine carcinomas. However, its expression in other tumour types is not fully explored. Aims and methods To determine Pax‐5 expression in other tumour types, immunohistochemistry was performed on 3758 benign and malignant tumours using multiple tumour microarrays, as well as on whole sections. Results Pax‐5 was expressed in 108/118 (91.5%) B‐NHLs, in 60/70 (85.7%) HLs and in 0/7 T cell lymphomas. In addition, Pax‐5 was seen in 24/34 (70.6%) Merkel cell carcinomas, 42/53 (79.2%) small cell carcinomas, 1/164 (0.6%) breast carcinomas, 2/204 (1%) endometrial adenocarcinomas and 1/452 (0.2%) urothelial carcinoma of the bladder. Conclusion Despite its expression in a small subset of malignancies of epithelial origin, Pax‐5 is still a good and reliable immunomarker in diagnosing B‐NHL, HL and neuroendocrine carcinomas. PMID:16837628

  9. Tissue Engineering of Blood Vessels: Functional Requirements, Progress, and Future Challenges

    PubMed Central

    Kumar, Vivek A.; Brewster, Luke P.; Caves, Jeffrey M.; Chaikof, Elliot L.

    2012-01-01

    Vascular disease results in the decreased utility and decreased availability of autologus vascular tissue for small diameter (< 6 mm) vessel replacements. While synthetic polymer alternatives to date have failed to meet the performance of autogenous conduits, tissue-engineered replacement vessels represent an ideal solution to this clinical problem. Ongoing progress requires combined approaches from biomaterials science, cell biology, and translational medicine to develop feasible solutions with the requisite mechanical support, a non-fouling surface for blood flow, and tissue regeneration. Over the past two decades interest in blood vessel tissue engineering has soared on a global scale, resulting in the first clinical implants of multiple technologies, steady progress with several other systems, and critical lessons-learned. This review will highlight the current inadequacies of autologus and synthetic grafts, the engineering requirements for implantation of tissue-engineered grafts, and the current status of tissue-engineered blood vessel research. PMID:23181145

  10. DNA Microarray-Based Diagnostics.

    PubMed

    Marzancola, Mahsa Gharibi; Sedighi, Abootaleb; Li, Paul C H

    2016-01-01

    The DNA microarray technology is currently a useful biomedical tool which has been developed for a variety of diagnostic applications. However, the development pathway has not been smooth and the technology has faced some challenges. The reliability of the microarray data and also the clinical utility of the results in the early days were criticized. These criticisms added to the severe competition from other techniques, such as next-generation sequencing (NGS), impacting the growth of microarray-based tests in the molecular diagnostic market.Thanks to the advances in the underlying technologies as well as the tremendous effort offered by the research community and commercial vendors, these challenges have mostly been addressed. Nowadays, the microarray platform has achieved sufficient standardization and method validation as well as efficient probe printing, liquid handling and signal visualization. Integration of various steps of the microarray assay into a harmonized and miniaturized handheld lab-on-a-chip (LOC) device has been a goal for the microarray community. In this respect, notable progress has been achieved in coupling the DNA microarray with the liquid manipulation microsystem as well as the supporting subsystem that will generate the stand-alone LOC device.In this chapter, we discuss the major challenges that microarray technology has faced in its almost two decades of development and also describe the solutions to overcome the challenges. In addition, we review the advancements of the technology, especially the progress toward developing the LOC devices for DNA diagnostic applications. PMID:26614075

  11. A Decade of Progress in Adipose Tissue Macrophage Biology

    PubMed Central

    Hill, Andrea A.; Bolus, W. Reid; Hasty, Alyssa H.

    2014-01-01

    Summary One decade has passed since seminal publications described macrophage infiltration into adipose tissue (AT) as a key contributor to inflammation and obesity-related insulin resistance. Currently, a PubMed search for ‘adipose tissue inflammation’ reveals over 3500 entries since these original reports. We now know that resident macrophages in lean AT are alternatively activated, M2-like, and play a role in AT homeostasis. In contrast, the macrophages in obese AT are dramatically increased in number and are predominantly classically activated, M1-like, and promote inflammation and insulin resistance. Mediators of AT macrophage (ATM) phenotype include adipokines and fatty acids secreted from adipocytes as well as cytokines secreted from other immune cells in AT. There are several mechanisms that could explain the large increase in ATMs in obesity. These include recruitment-dependent mechanisms such as adipocyte death, chemokine release, and lipolysis of fatty acids. Newer evidence also points to recruitment-independent mechanisms such as impaired apoptosis, increased proliferation, and decreased egress. Although less is known about the homeostatic function of M2-like resident ATMs, recent evidence suggests roles in AT expansion, thermoregulation, antigen presentation, and iron homeostasis. The field of immunometabolism has come a long way in the past decade, and many exciting new discoveries are bound to be made in the coming years that will expand our understanding of how AT stands at the junction of immune and metabolic co-regulation. PMID:25319332

  12. Analyzing Illumina Gene Expression Microarray Data from Different Tissues: Methodological Aspects of Data Analysis in the MetaXpress Consortium

    PubMed Central

    Blankenberg, Stefan; Carstensen, Maren; Dörr, Marcus; Endlich, Karlhans; Felix, Stephan B.; Gieger, Christian; Grallert, Harald; Herder, Christian; Hoffmann, Wolfgang; Homuth, Georg; Illig, Thomas; Kruppa, Jochen; Meitinger, Thomas; Müller, Christian; Nauck, Matthias; Peters, Annette; Rettig, Rainer; Roden, Michael; Strauch, Konstantin; Völker, Uwe; Völzke, Henry; Wahl, Simone; Wallaschofski, Henri; Wild, Philipp S.; Zeller, Tanja; Teumer, Alexander; Prokisch, Holger; Ziegler, Andreas

    2012-01-01

    Microarray profiling of gene expression is widely applied in molecular biology and functional genomics. Experimental and technical variations make meta-analysis of different studies challenging. In a total of 3358 samples, all from German population-based cohorts, we investigated the effect of data preprocessing and the variability due to sample processing in whole blood cell and blood monocyte gene expression data, measured on the Illumina HumanHT-12 v3 BeadChip array. Gene expression signal intensities were similar after applying the log2 or the variance-stabilizing transformation. In all cohorts, the first principal component (PC) explained more than 95% of the total variation. Technical factors substantially influenced signal intensity values, especially the Illumina chip assignment (33–48% of the variance), the RNA amplification batch (12–24%), the RNA isolation batch (16%), and the sample storage time, in particular the time between blood donation and RNA isolation for the whole blood cell samples (2–3%), and the time between RNA isolation and amplification for the monocyte samples (2%). White blood cell composition parameters were the strongest biological factors influencing the expression signal intensities in the whole blood cell samples (3%), followed by sex (1–2%) in both sample types. Known single nucleotide polymorphisms (SNPs) were located in 38% of the analyzed probe sequences and 4% of them included common SNPs (minor allele frequency >5%). Out of the tested SNPs, 1.4% significantly modified the probe-specific expression signals (Bonferroni corrected p-value<0.05), but in almost half of these events the signal intensities were even increased despite the occurrence of the mismatch. Thus, the vast majority of SNPs within probes had no significant effect on hybridization efficiency. In summary, adjustment for a few selected technical factors greatly improved reliability of gene expression analyses. Such adjustments are particularly required for meta-analyses. PMID:23236413

  13. Regulation of Gene Expression in Brain Tissues of Rats Repeatedly Treated by the Highly Abused Opioid Agonist, Oxycodone: Microarray Profiling and Gene Mapping Analysis

    PubMed Central

    Hassan, Hazem E.; Myers, Alan L.; Lee, Insong J.; Chen, Hegang; Coop, Andrew

    2010-01-01

    Although oxycodone is the most often used opioid agonist, it remains one of the most understudied drugs. We used microarray analysis to better understand the global changes in gene expression in brain tissues of rats repeatedly treated with oxycodone. Many genes were significantly regulated by oxycodone (e.g., Fkbp5, Per2, Rt1.Dα, Slc16a1, and Abcg2). Validation of the microarray data by quantitative real-time-polymerase chain reaction (Q-PCR) indicated that there was a strong significant correlation (r = 0.979, p < 0.0000001) between the Q-PCR and the microarray data. Using MetaCore (a computational platform), many biological processes were identified [e.g., organic anion transport (p = 7.251 × 10−4) and regulation of immune response (p = 5.090 × 10−4)]. Among the regulated genes, Abcg2 mRNA was up-regulated by 2.1-fold, which was further confirmed by immunoblotting (1.8-fold up-regulation). Testing the Abcg2 affinity status of oxycodone using an Abcg2 ATPase assay suggests that oxycodone behaves as an Abcg2 substrate only at higher concentrations (≥500 μM). Furthermore, brain uptake studies demonstrated that oxycodone-induced Abcg2 up-regulation resulted in a significant (p < 0.05) decrease (∼2-fold) in brain/plasma ratios of mitoxantrone. These results highlight markers/mediators of neuronal responses and identify regulatory pathways involved in the pharmacological action of oxycodone. These results also identify genes that potentially modulate tolerance, dependence, immune response, and drug-drug interactions. Finally, our findings suggest that oxycodone-induced up-regulation of Abcg2 enhanced the efflux of the Abcg2 substrate, mitoxantrone, limiting its brain accumulation and resulting in an undesirable drug-drug interaction. Extrapolating these results to other Abcg2 substrates (e.g., daunorubicin and doxorubicin) indicates that the brain uptake of these agents may be affected if they are administered concomitantly with oxycodone. PMID:19786507

  14. Evaluation of Ki67 Expression across Distinct Categories of Breast Cancer Specimens: A Population-Based Study of Matched Surgical Specimens, Core Needle Biopsies and Tissue Microarrays

    PubMed Central

    Knutsvik, Gril; Stefansson, Ingunn M.; Aziz, Sura; Arnes, Jarle; Eide, Johan; Collett, Karin; Akslen, Lars A.

    2014-01-01

    Introduction Tumor cell proliferation in breast cancer is strongly prognostic and may also predict response to chemotherapy. However, there is no consensus on counting areas or cut-off values for patient stratification. Our aim was to assess the matched level of proliferation by Ki67 when using different tissue categories (whole sections, WS; core needle biopsies, CNB; tissue microarrays, TMA), and the corresponding prognostic value. Methods We examined a retrospective, population-based series of breast cancer (n?=?534) from the Norwegian Breast Cancer Screening Program. The percentage of Ki67 positive nuclei was evaluated by visual counting on WS (n?=?534), CNB (n?=?154) and TMA (n?=?459). Results The median percentage of Ki67 expression was 18% on WS (hot-spot areas), 13% on CNB, and 7% on TMA, and this difference was statistically significant in paired cases. Increased Ki67 expression by all evaluation methods was associated with aggressive tumor features (large tumor diameter, high histologic grade, ER negativity) and reduced patient survival. Conclusion There is a significant difference in tumor cell proliferation by Ki67 across different sample categories. Ki67 is prognostic over a wide range of cut-off points and for different sample types, although Ki67 results derived from TMA sections are lower compared with those obtained using specimens from a clinical setting. Our findings indicate that specimen specific cut-off values should be applied for practical use. PMID:25375149

  15. Measuring brain lesion progression with a supervised tissue classification system.

    PubMed

    Zacharaki, Evangelia I; Kanterakis, Stathis; Bryan, R Nick; Davatzikos, Christos

    2008-01-01

    Brain lesions, especially White Matter Lesions (WMLs), are associated with cardiac and vascular disease, but also with normal aging. Quantitative analysis of WML in large clinical trials is becoming more and more important. In this paper, we present a computer-assisted WML segmentation method, based on local features extracted from conventional multi-parametric Magnetic Resonance Imaging (MRI) sequences. A framework for preprocessing the temporal data by jointly equalizing histograms reduces the spatial and temporal variance of data, thereby improving the longitudinal stability of such measurements and hence the estimate of lesion progression. A Support Vector Machine (SVM) classifier trained on expert-defined WML's is applied for lesion segmentation on each scan using the AdaBoost algorithm. Validation on a population of 23 patients from 3 different imaging sites with follow-up studies and WMLs of varying sizes, shapes and locations tests the robustness and accuracy of the proposed segmentation method, compared to the manual segmentation results from an experienced neuroradiologist. The results show that our CAD-system achieves consistent lesion segmentation in the 4D data facilitating the disease monitoring. PMID:18979798

  16. Tissue hemoglobin monitoring of progressive central hypovolemia in humans using broadband diffuse optical spectroscopy

    PubMed Central

    Lee, Jangwoen; Kim, Jae G.; Mahon, Sari; Tromberg, Bruce J.; Ryan, Kathy L.; Convertino, Victor A.; Rickards, Caroline A.; Osann, Kathryn; Brenner, Matthew

    2014-01-01

    We demonstrate noninvasive near-infrared diffuse optical spectroscopy (DOS) measurements of tissue hemoglobin contents that can track progressive reductions in central blood volume in human volunteers. Measurements of mean arterial blood pressure (MAP), heart rate (HR), stroke volume (SV), and cardiac output (Q) are obtained in ten healthy human subjects during baseline supine rest and exposure to progressive reductions of central blood volume produced by application of lower body negative pressure (LBNP). Simultaneous quantitative noninvasive measurements of tissue oxyhemoglobin (OHb), deoxyhemoglobin (RHb), total hemoglobin concentration (THb), and tissue hemoglobin oxygen saturation (StO2) are performed throughout LBNP application using broadband DOS. As progressively increasing amounts of LBNP are applied, HR increases, and MAP, SV, and Q decrease (p<0.001). OHb, StO2, and THb decrease (p <0.001) in correlation with progressive increases in LBNP, while tissue RHb remained relatively constant (p=0.378). The average fractional changes from baseline values in DOS OHb (fOHb) correlate closely with independently measured changes in SV (r2=0.95) and Q (r2=0.98) during LBNP. Quantitative noninvasive broadband DOS measurements of tissue hemoglobin parameters of peripheral perfusion are capable of detecting progressive reductions in central blood volume, and appear to be sensitive markers of early hypoperfusion associated with hemorrhage as simulated by LBNP. PMID:19123673

  17. Potential Upstream Regulators of Cannabinoid Receptor 1 Signaling in Prostate Cancer: A Bayesian Network Analysis of Data From a Tissue Microarray

    PubMed Central

    Häggström, Jenny; Cipriano, Mariateresa; Forshell, Linus Plym; Persson, Emma; Hammarsten, Peter; Stella, Nephi; Fowler, Christopher J

    2014-01-01

    BACKGROUND The endocannabinoid system regulates cancer cell proliferation, and in prostate cancer a high cannabinoid CB1 receptor expression is associated with a poor prognosis. Down-stream mediators of CB1 receptor signaling in prostate cancer are known, but information on potential upstream regulators is lacking. RESULTS Data from a well-characterized tumor tissue microarray were used for a Bayesian network analysis using the max-min hill-climbing method. In non-malignant tissue samples, a directionality of pEGFR (the phosphorylated form of the epidermal growth factor receptor) → CB1 receptors were found regardless as to whether the endocannabinoid metabolizing enzyme fatty acid amide hydrolase (FAAH) was included as a parameter. A similar result was found in the tumor tissue, but only when FAAH was included in the analysis. A second regulatory pathway, from the growth factor receptor ErbB2 → FAAH was also identified in the tumor samples. Transfection of AT1 prostate cancer cells with CB1 receptors induced a sensitivity to the growth-inhibiting effects of the CB receptor agonist CP55,940. The sensitivity was not dependent upon the level of receptor expression. Thus a high CB1 receptor expression alone does not drive the cells towards a survival phenotype in the presence of a CB receptor agonist. CONCLUSIONS The data identify two potential regulators of the endocannabinoid system in prostate cancer and allow the construction of a model of a dysregulated endocannabinoid signaling network in this tumor. Further studies should be designed to test the veracity of the predictions of the network analysis in prostate cancer and other solid tumors. Prostate 74:1107–1117, 2014. © 2014 The Authors. The Prostate published by Wiley Periodicals, Inc. PMID:24913716

  18. Matrix Metalloproteases and Tissue Inhibitors of Metalloproteinases in Medial Plica and Pannus-like Tissue Contribute to Knee Osteoarthritis Progression

    PubMed Central

    Yang, Chih-Chang; Lin, Cheng-Yu; Wang, Hwai-Shi; Lyu, Shaw-Ruey

    2013-01-01

    Osteoarthritis (OA) is characterized by degradation of the cartilage matrix, leading to pathologic changes in the joints. However, the pathogenic effects of synovial tissue inflammation on OA knees are not clear. To investigate whether the inflammation caused by the medial plica is involved in the pathogenesis of osteoarthritis, we examined the expression of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), interleukin (IL)-1β, and tumor necrosis factor (TNF)-α in the medial plica and pannus-like tissue in the knees of patients with medial compartment OA who underwent either arthroscopic medial release (stage II; 15 knee joints from 15 patients) or total knee replacement (stage IV; 18 knee joints from 18 patients). MMP-2, MMP-3, MMP-9, IL-1β, and TNF-α mRNA and protein levels measured, respectively, by quantitative real-time PCR and Quantibody human MMP arrays, were highly expressed in extracts of medial plica and pannus-like tissue from stage IV knee joints. Immunohistochemical staining also demonstrated high expression of MMP-2, MMP-3, and MMP-9 in plica and pannus-like tissue of stage IV OA knees and not in normal cartilage. Some TIMP/MMP ratios decreased significantly in both medial plica and pannus-like tissue as disease progressed from stage II to stage IV. Furthermore, the migration of cells from the pannus-like tissue was enhanced by IL-1β, while plica cell migration was enhanced by TNF-α. The results suggest that medial plica and pannus-like tissue may be involved in the process of cartilage degradation in medial compartment OA of the knee. PMID:24223987

  19. Immunohistochemical characterization of nasal-type extranodal NK/T-cell lymphoma using a tissue microarray: an analysis of 84 cases.

    PubMed

    Schwartz, Erich J; Molina-Kirsch, Hernan; Zhao, Shuchun; Marinelli, Robert J; Warnke, Roger A; Natkunam, Yasodha

    2008-09-01

    Nasal-type extranodal natural killer (NK)/T-cell lymphoma is an uncommon malignancy. By using a tissue microarray, we characterized 84 cases of extranodal NK/T-cell lymphoma with regard to expression of 18 immunohistochemical markers and the presence of Epstein-Barr virus (EBV) RNA. In our series, CD2 was positive in 69 (93%) of 74 cases, CD3 in 68 (84%) of 81, CD5 in 22 (27%) of 81, CD20 in 0 (0%) of 82, CD29 in 75 (91%) of 82, CD30 in 29 (35%) of 84, CD43 in 81 (96%) of 84, CD54 in 58 (72%) of 81, CD56 in 46 (58%) of 79, CD62L in 23 (28%) of 83, CD183 in 66 (80%) of 83, BCL2 in 33 (39%) of 84, cutaneous lymphocyte antigen in 21 (25%) of 84, granzyme B in 70 (83%) of 84, Ki-67 in 59 (71%) of 83, linker for activation of T cells in 60 (71%) of 84, perforin in 66 (86%) of 77, TIA1 in 76 (90%) of 84, and EBV in 73 (87%) of 84. Hierarchical cluster analysis separated primary cutaneous cases from cases manifesting in other sites based on lower expression of the cell adhesion molecule CD54. PMID:18701406

  20. A high-density tissue microarray from patients with clinically localized prostate cancer reveals ERG and TATI exclusivity in tumor cells

    PubMed Central

    Lippolis, G; Edsjö, A; Stenman, U-H; Bjartell, A

    2013-01-01

    Background: Prostate cancer (PCa) is characterized by high tumor heterogeneity. In 2005, the fusion between the androgen-regulated gene TMPRSS2 and members of the ETS family was discovered in prostate cancer. In particular, fusion of TMPRSS2 with ERG was found in approximately 50% of prostate cancers and considered as an early event in the onset of the disease. The prognostic value of this fusion is still contradictory. Bioinformatics showed that overexpression of SPINK1 gene in a subset of fusion-gene-negative prostate cancers was associated with a poor prognosis. In theory, overexpression of the tumor-associated trypsin inhibitor (TATI) protein encoded by SPINK1 in fusion-gene-negative tumor cells opens the way to selected treatments for genotypically different cases. However, their expression has never been assessed at the cellular level in the same tissue samples. Methods: As ERG expression has been shown to be a surrogate of fusion gene occurrence in prostate cancer, we have used double immunohistochemical staining to assess expression of ERG and TATI on a large tissue microarray comprising 4177 cases of localized prostate cancer. Results: We did not detect any co-expression of ERG and TATI in the same cancer cells, which confirms previous suggestions from in silico studies. ERG was associated with Gleason score (GS), surgical margins and pathological stage, but had no prognostic value in this cohort. TATI was weakly associated with pathological stage but had no significant association with outcome. Conclusions: We here provide a morphological basis for ERG and TATI exclusivity in prostate cancer cells. Future therapies should be based on a combination of different targets in order to eradicate tumor cells with gene fusions and cells expressing other tumor-associated antigens. Further studies are needed to understand why ERG and TATI are not co-expressed in the same prostatic tumor cells. PMID:23459095

  1. Microarray studies of psychostimulant-induced changes in gene expression.

    PubMed

    Yuferov, Vadim; Nielsen, David; Butelman, Eduardo; Kreek, Mary Jeanne

    2005-03-01

    Alterations in the expression of multiple genes in many brain regions are likely to contribute to psychostimulant-induced behaviours. Microarray technology provides a powerful tool for the simultaneous interrogation of gene expression levels of a large number of genes. Several recent experimental studies, reviewed here, demonstrate the power, limitations and progress of microarray technology in the field of psychostimulant addiction. These studies vary in the paradigms of cocaine or amphetamine administration, drug doses, route and also mode of administration, duration of treatment, animal species, brain regions studied and time of tissue collection after final drug administration. The studies also utilize different microarray platforms and statistical techniques for analysis of differentially expressed genes. These variables influence substantially the results of these studies. It is clear that current microarray techniques cannot detect small changes reliably in gene expression of genes with low expression levels, including functionally significant changes in components of major neurotransmission systems such as glutamate, dopamine, opioid and GABA receptors, especially those that may occur after chronic drug administration or drug withdrawal. However, the microarray studies reviewed here showed cocaine- or amphetamine-induced alterations in the expression of numerous genes involved in the modulation of neuronal growth, cytoskeletal structures, synaptogenesis, signal transduction, apoptosis and cell metabolism. Application of laser capture microdissection and single-cell cDNA amplification may greatly enhance microarray studies of gene expression profiling. The combination of rapidly evolving microarray technology with established methods of neuroscience, molecular biology and genetics, as well as appropriate behavioural models of drug reinforcement, may provide a productive approach for delineating the neurobiological underpinnings of drug responses that lead to addiction. PMID:15849024

  2. In vitro-differentiated neural cell cultures progress towards donor-identical brain tissue

    PubMed Central

    Hjelm, Brooke E.; Salhia, Bodour; Kurdoglu, Ahmet; Szelinger, Szabolcs; Reiman, Rebecca A.; Sue, Lucia I.; Beach, Thomas G.; Huentelman, Matthew J.; Craig, David W.

    2013-01-01

    Multiple research groups have observed neuropathological phenotypes and molecular symptoms in vitro using induced pluripotent stem cell (iPSC)-derived neural cell cultures (i.e. patient-specific neurons and glia). However, the global differences/similarities that may exist between in vitro neural cells and their tissue-derived counterparts remain largely unknown. In this study, we compared temporal series of iPSC-derived in vitro neural cell cultures to endogenous brain tissue from the same autopsy donor. Specifically, we utilized RNA sequencing (RNA-Seq) to evaluate the transcriptional progression of in vitro-differentiated neural cells (over a timecourse of 0, 35, 70, 105 and 140 days), and compared this with donor-identical temporal lobe tissue. We observed in vitro progression towards the reference brain tissue, and the following three results support this conclusion: (i) there was a significant increasing monotonic correlation between the days of our timecourse and the number of actively transcribed protein-coding genes and long intergenic non-coding RNAs (lincRNAs) (P < 0.05), consistent with the transcriptional complexity of the brain; (ii) there was an increase in CpG methylation after neural differentiation that resembled the epigenomic signature of the endogenous tissue; and (iii) there was a significant decreasing monotonic correlation between the days of our timecourse and the percent of in vitro to brain-tissue differences (P < 0.05) for tissue-specific protein-coding genes and all putative lincRNAs. Taken together, these results are consistent with in vitro neural development and physiological progression occurring predominantly by transcriptional activation of downregulated genes rather than deactivation of upregulated genes. PMID:23666530

  3. COMPARISON OF TRANSCRIPTIONAL RESONSES FROM AVIAN GUT TISSUES AFTER EIMERIA ACERVULINA AND E. MAXIMA INFECTIONS USING cDNA MICROARRAY TECHNOLOGY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Understanding the host response during pathogen infection will extend our knowledge of pathogenesis and enhance the development of novel preventive methodologies against important infectious diseases. Microarray technology is a powerful tool to analyze host transcriptional responses. Coccidiosis re...

  4. A comprehensive immunophenotypic marker analysis of hairy cell leukemia in paraffin-embedded bone marrow trephine biopsies--a tissue microarray study.

    PubMed

    Tth-Liptk, Judit; Piukovics, Klra; Borbnyi, Zita; Demeter, Judit; Bagdi, Enik?; Krencs, Lszl

    2015-01-01

    Hairy cell leukemia (HCL) is an uncommon B cell lymphoproliferation characterized by a unique immunophenotype. Due to low number of circulating neoplastic cells and 'dry tap' aspiration, the diagnosis is often based on BM trephine biopsy. We have performed a consecutive immunohistochemical analysis to evaluate diagnostic usefulness of various HCL markers (CD11c, CD25, CD68, CD103, CD123, CD200, annexin A1, cyclin D1, DBA.44, HBME-1, phospho-ERK1/2, TRAP, and T-bet) currently available against fixation resistant epitopes. We analyzed tissue microarrays consisting of samples gained from 73 small B-cell lymphoma cases, including hairy cell leukemia (HCL) (n = 32), HCL variant (HCL-v) (n = 4), B-cell chronic lymphocytic leukemia (B-CLL) (n = 11), lymphoplasmacytic lymphoma (LPL) (n = 3), mantle cell lymphoma (MCL) (n = 10), splenic diffuse red pulp small B cell lymphoma (SDRPL) (n = 2), splenic B cell marginal zone lymphoma (SMZL) (n = 8), and splenic B cell lymphoma/leukemia, unclassifiable (SBCL) (n = 3) cases. The HCL cases were 100% positive for all but 2 (DBA.44 and CD123) of these markers. Annexin A1 showed 100% specificity and accuracy, which was followed by CD123, pERK, CD103, HBME-1, CD11c, CD25, CD68, cyclin D1, CD200, T-bet, DBA.44, and TRAP, in decreasing order. In conclusion, our results reassured the high specificity of annexin A1 and pERK, as well as the diagnostic value of standard HCL markers of CD11c, CD25, CD103, and CD123 also in paraffin-embedded BM samples. Additional markers, including HBME-1, cyclin D1, CD200, and T-bet also represent valuable tools in the differential diagnosis of HCL and its mimics. PMID:24903677

  5. Triple-negative breast carcinoma in women from Vietnam and the United States: characterization of differential marker expression by tissue microarray.

    PubMed

    Williams, Daron J; Cohen, Cynthia; To, Ta Van; Page, Andrew J; Lawson, Diane; Sussman, Zachary M; Nassar, Aziza

    2009-08-01

    Triple-negative breast carcinoma accounts for approximately 15% of all breast cancers. It is characterized by an aggressive clinical history, high rate of local relapse, and association with the basal epithelial-like subtype. Variations in breast cancer subtype and clinical outcome often exist across racial and ethnic lines. Therefore, the aim of this study was to compare the immunohistochemical and clinicopathologic characteristics of triple-negative breast carcinoma in women living in Vietnam with those from the United States. Invasive triple-negative breast carcinoma of patients from the 2 populations was characterized by tissue microarray for the expression of basal cytokeratins (CK5/6, CK7, CK14), luminal cytokeratins (CK8, CK18, CK19), and markers associated with the basal phenotype (cKit, epithelial growth factor receptor, P-cadherin, p53, and p63). Significant differences in expression between the 2 populations were not observed for the basal cytokeratins. However, epithelial growth factor receptor and P-cadherin, markers associated with the basal phenotype, were underexpressed in Vietnamese patients. Of the luminal cytokeratins, CK8 was overexpressed and CK18 was underexpressed in the Vietnamese women. Significant differences were also observed regarding the clinicopathologic characteristics. Triple-negative breast carcinoma in Vietnamese women was smaller and less likely to be grade III. In addition, it was more frequently of ductal histologic type and less often medullary or metaplastic. These differences in histology and marker expression suggest that triple-negative breast carcinoma has unique biological characteristics in women from Vietnam and the United States, and may follow a unique clinical course in each of the 2 populations. PMID:19368951

  6. A Seven-Marker Signature and Clinical Outcome in Malignant Melanoma: A Large-Scale Tissue-Microarray Study with Two Independent Patient Cohorts

    PubMed Central

    Bosserhoff, Anja K.; Hofstädter, Ferdinand; Pauer, Armin; Roth, Volker; Buhmann, Joachim M.; Moll, Ingrid; Anagnostou, Nikos; Brandner, Johanna M.; Ikenberg, Kristian; Moch, Holger; Landthaler, Michael; Vogt, Thomas; Wild, Peter J.

    2012-01-01

    Background Current staging methods such as tumor thickness, ulceration and invasion of the sentinel node are known to be prognostic parameters in patients with malignant melanoma (MM). However, predictive molecular marker profiles for risk stratification and therapy optimization are not yet available for routine clinical assessment. Methods and Findings Using tissue microarrays, we retrospectively analyzed samples from 364 patients with primary MM. We investigated a panel of 70 immunohistochemical (IHC) antibodies for cell cycle, apoptosis, DNA mismatch repair, differentiation, proliferation, cell adhesion, signaling and metabolism. A marker selection procedure based on univariate Cox regression and multiple testing correction was employed to correlate the IHC expression data with the clinical follow-up (overall and recurrence-free survival). The model was thoroughly evaluated with two different cross validation experiments, a permutation test and a multivariate Cox regression analysis. In addition, the predictive power of the identified marker signature was validated on a second independent external test cohort (n = 225). A signature of seven biomarkers (Bax, Bcl-X, PTEN, COX-2, loss of β-Catenin, loss of MTAP, and presence of CD20 positive B-lymphocytes) was found to be an independent negative predictor for overall and recurrence-free survival in patients with MM. The seven-marker signature could also predict a high risk of disease recurrence in patients with localized primary MM stage pT1-2 (tumor thickness ≤2.00 mm). In particular, three of these markers (MTAP, COX-2, Bcl-X) were shown to offer direct therapeutic implications. Conclusions The seven-marker signature might serve as a prognostic tool enabling physicians to selectively triage, at the time of diagnosis, the subset of high recurrence risk stage I–II patients for adjuvant therapy. Selective treatment of those patients that are more likely to develop distant metastatic disease could potentially lower the burden of untreatable metastatic melanoma and revolutionize the therapeutic management of MM. PMID:22685558

  7. Early progressive changes in tissue viability in the seated spinal cord injured subject.

    PubMed

    Bogie, K M; Nuseibeh, I; Bader, D L

    1995-03-01

    The patient with spinal cord injury is at high risk of tissue breakdown at all times due to a number of adverse factors, such as reduced mobility and anaesthesia. It is therefore essential that each patient is prescribed appropriate support media during initial rehabilitation. In this study, the effectiveness of prescribed wheelchair cushions has been assessed in terms of tissue response at the ischial tuberosities. A total of 42 subjects who had sustained traumatic spinal cord injury within 1 year were monitored on at least two occasions during initial rehabilitation. Changes in transcutaneous gas response (TcPO2 and TcPCO2) were monitored concurrently with regional interface pressures. A series of six transcutaneous gas variables were established, as markers of tissue viability. Non-parametric statistical analyses revealed some significant correlations between these variables. The results of this study also indicate that (1) spinal cord injury subjects with lesions below T6 show a progressive decrease in ability to maintain blood flow in sitting on prescribed support cushions and (2) SCI subjects with lesions above T6 show a progressive improvement in tissue viability status at the seating support interface. Therefore results imply that paraplegics are at a potentially higher risk of tissue breakdown than tetraplegics and thus require effective support cushions with strict adherence to a pressure relief regime. PMID:7784116

  8. Clinical Utility of Microarrays: Current Status, Existing Challenges and Future Outlook

    PubMed Central

    Li, Xinmin; Quigg, Richard J; Zhou, Jian; Gu, Weikuan; Nagesh Rao, P; Reed, Elaine F

    2008-01-01

    Microarray-based clinical tests have become powerful tools in the diagnosis and treatment of diseases. In contrast to traditional DNA-based tests that largely focus on single genes associated with rare conditions, microarray-based tests are ideal for the study of diseases with underlying complex genetic causes. Several microarray based tests have been translated into clinical practice such as MammaPrint and AmpliChip CYP450. Additional cancer-related microarray-based tests are either in the process of FDA review or under active development, including Tissue of Tumor Origin and AmpliChip p53. All diagnostic microarray testing is ordered by physicians and tested by a Clinical Laboratories Improvement Amendment-certified (CLIA) reference laboratory. Recently, companies offering consumer based microarray testing have emerged. Individuals can order tests online and service providers deliver the results directly to the clients via a password-protected secure website. Navigenics, 23andMe and deCODE Genetics represent pioneering companies in this field. Although the progress of these microarray-based tests is extremely encouraging with the potential to revolutionize the recognition and treatment of common diseases, these tests are still in their infancy and face technical, clinical and marketing challenges. In this article, we review microarray-based tests which are currently approved or under review by the FDA, as well as the consumer-based testing. We also provide a summary of the challenges and strategic solutions in the development and clinical use of the microarray-based tests. Finally, we present a brief outlook for the future of microarray-based clinical applications. PMID:19506735

  9. Relation of Doppler Tissue Imaging Parameters With Heart Failure Progression in Hypertrophic Cardiomyopathy.

    PubMed

    Kalra, Ankur; Harris, Kevin M; Maron, Bradley A; Maron, Martin S; Garberich, Ross F; Haas, Tammy S; Lesser, John R; Maron, Barry J

    2016-06-01

    Refractory progressive heart failure (HF) is becoming the predominant cause of mortality in nonobstructive hypertrophic cardiomyopathy (HC). To anticipate development of this important and often unpredictable clinical course, we investigated whether left ventricular diastolic dysfunction, assessed by echocardiographic Doppler parameters, could identify a subset of patients with HC without obstruction at rest who would experience progression of HF. Diastolic function parameters, assessed by Doppler tissue imaging (DTI), mitral inflow, and pulmonary venous flow were measured in 274 consecutive adult patients with HC evaluated from 2003 to 2007. DTI and other diastolic and clinical/demographic parameters were measured against the composite end point of HF/death, heart transplantation, or progression to advanced New York Heart Association functional class III/IV symptoms and sudden death (SD)/implantable defibrillator (ICD) interventions. HF end points were reached in 19 of 274 patients (7%) over a follow-up period of 4.0 ± 2.3 years. Variables significantly associated with HF outcome by univariate analysis included male gender, initial New York Heart Association class II, lower ejection fraction, and reduced septal and lateral e' mitral annular tissue velocities. Multivariable analysis showed only a reduced lateral e' mitral annular tissue velocity to be independently associated with the composite HF end points (HR 0.77; 95% CI 0.65 to 0.91; p = 0.003). In addition, estimated pulmonary arterial systolic pressure and extensive late gadolinium enhancement by magnetic resonance were also associated with HF outcome (p = 0.04 and p <0.001, respectively). No Doppler (or clinical) variable was associated with SD/appropriate ICD interventions. In conclusion, in HC without outflow obstruction at rest, diastolic dysfunction, evidenced by DTI-reduced lateral e' mitral annular tissue velocity, was associated with adverse long-term HF outcome but was unrelated to SD. This echocardiographic marker provides a potential noninvasive strategy for anticipating progressive HF in this HC patient group. PMID:27089980

  10. Tissue transglutaminase-2 promotes gastric cancer progression via the ERK1/2 pathway

    PubMed Central

    Zhou, Quan; Wu, Xiongyan; Chen, Xuehua; Li, Jianfang; Zhu, Zhenggang; Liu, Bingya; Su, Liping

    2016-01-01

    Gastric cancer (GC) is one of the most common tumors worldwide and involves extensive local tumor invasion, metastasis, and poor prognosis. Understanding mechanisms regulating progression of GC is necessary for developing effective therapeutic strategies. Tissue transglutaminase-2 (TG2), a multifunctional member of the transglutaminase family, has been shown to be critical for tumor initiation and progression. However, how TG2 promotes the progression of GC is unknown. We report that TG2 was highly expressed in GC tissues and positively associated with depth of tumor invasion and late TNM stage. With gain- and loss-of-function approaches, we observed that TG2 promoted GC cell proliferation, migration, invasion, as well as tumorigenesis and peritoneal metastasis in vivo. These events were associated with the ERK1/2 pathway activation and an ERK1/2 inhibitor (U0126) inhibited cell proliferation, migration, and invasion induced by overexpression of TG2. In summary, TG2 contributes to tumorigenesis and progression of GC by activating the ERK1/2 signaling pathway and is a potential therapeutic target of metastatic gastric cancer. PMID:26771235

  11. [Research Progress of Collagen-based Three-dimensional Porous Scaffolds Used in Skin Tissue Engineering].

    PubMed

    Zhang, Jing; Tang, Qiwei; Zhou, Aimei; Yang, Shulin

    2015-08-01

    Collagen is a kind of natural biomedical material and collagen based three-dimensional porous scaffolds have been widely used in skin tissue engineering. However, these scaffolds do not meet the requirements for artificial skin substitutes in terms of their poor mechanical properties, short supply, and rejection in the bodies. All of these factors limit their further application in skin tissue engineering. A variety of methods have been chosen to meliorate the situation, such as cross linking and blending other substance for improving mechanical properties. The highly biomimetic scaffolds either in structure or in function can be prepared through culturing cells and loading growth factors. To avoid the drawbacks of unsafety attributing to animals, investigators have fixed their eyes on the recombinant collagen. This paper reviews the the progress of research and application of collagen-based 3-dimensional porous scaffolds in skin tissue engineering. PMID:26710471

  12. Integration of diverse microarray data types.

    PubMed

    Salari, Keyan; Pollack, Jonathan R

    2009-01-01

    Over the past decade, DNA microarrays have proven to be a powerful tool in biological research for the molecular surveillance of cells and tissues. The expansive utility of DNA microarrays owes its nascence to the development of a multitude of microarray platforms that enable the systematic and comprehensive exploration of diverse genomic properties and processes. Concomitant with the explosive generation of microarray data over the last several years has been an increasing interest in the integration of such diverse data types, thus spurring the development of novel statistical techniques and integrative bioinformatics tools. This chapter will outline general approaches to microarray data integration and provide an introduction to DR-Integrator, a broadly useful analysis tool for the integration of DNA copy number and gene-expression microarray data. PMID:19488881

  13. Aptamer Microarrays

    NASA Astrophysics Data System (ADS)

    Syrett, Heather Angel; Collett, James R.; Ellington, Andrew D.

    In vitro selection can yield specific, high-affinity aptamers. We and others have devised methods for the automated selection of aptamers and have begun to use these reagents for the construction of arrays. Arrayed aptamers have proven to be almost as sensitive as their solution-phase counterparts and when ganged together can provide both specific and general diagnostic signals for proteins and other ana-lytes. We describe here technical details regarding the production and processing of aptamer microarrays, including blocking, washing, drying, and scanning. We also discuss the challenges involved in developing standardized and reproducible methods for binding and quantitating protein targets. Although signals from fluorescent analytes or sandwiches are typically captured, it has proven possible for immobilized aptamers to be uniquely coupled to amplification methods not available to protein reagents, thus allowing for protein-binding signals to be greatly amplified. Into the future, many of the biosensor methods described in this book can potentially be adapted to array formats, thus further expanding the their utility and applications for aptamer arrays.

  14. Aptamer Microarrays

    SciTech Connect

    Angel-Syrett, Heather; Collett, Jim; Ellington, Andrew D.

    2009-01-02

    In vitro selection can yield specific, high-affinity aptamers. We and others have devised methods for the automated selection of aptamers, and have begun to use these reagents for the construction of arrays. Arrayed aptamers have proven to be almost as sensitive as their solution phase counterparts, and when ganged together can provide both specific and general diagnostic signals for proteins and other analytes. We describe here technical details regarding the production and processing of aptamer microarrays, including blocking, washing, drying, and scanning. We will also discuss the challenges involved in developing standardized and reproducible methods for binding and quantitating protein targets. While signals from fluorescent analytes or sandwiches are typically captured, it has proven possible for immobilized aptamers to be uniquely coupled to amplification methods not available to protein reagents, thus allowing for protein-binding signals to be greatly amplified. Into the future, many of the biosensor methods described in this book can potentially be adapted to array formats, thus further expanding the utility of and applications for aptamer arrays.

  15. Microarray analysis of thioacetamide-treated type 1 diabetic rats

    SciTech Connect

    Devi, Sachin S.; Mehendale, Harihara M. . E-mail: mehendale@ulm.edu

    2006-04-01

    It is well known that diabetes imparts high sensitivity to numerous hepatotoxicants. Previously, we have shown that a normally non-lethal dose of thioacetamide (TA, 300 mg/kg) causes 90% mortality in type 1 diabetic (DB) rats due to inhibited tissue repair allowing progression of liver injury. On the other hand, DB rats exposed to 30 mg TA/kg exhibit delayed tissue repair and delayed recovery from injury. The objective of this study was to investigate the mechanism of impaired tissue repair and progression of liver injury in TA-treated DB rats by using cDNA microarray. Gene expression pattern was examined at 0, 6, and 12 h after TA challenge, and selected mechanistic leads from microarray experiments were confirmed by real-time RT-PCR and further investigated at protein level over the time course of 0 to 36 h after TA treatment. Diabetic condition itself increased gene expression of proteases and decreased gene expression of protease inhibitors. Administration of 300 mg TA/kg to DB rats further elevated gene expression of proteases and suppressed gene expression of protease inhibitors, explaining progression of liver injury in DB rats after TA treatment. Inhibited expression of genes involved in cell division cycle (cyclin D1, IGFBP-1, ras, E2F) was observed after exposure of DB rats to 300 mg TA/kg, explaining inhibited tissue repair in these rats. On the other hand, DB rats receiving 30 mg TA/kg exhibit delayed expression of genes involved in cell division cycle, explaining delayed tissue repair in these rats. In conclusion, impaired cyclin D1 signaling along with increased proteases and decreased protease inhibitors may explain impaired tissue repair that leads to progression of liver injury initiated by TA in DB rats.

  16. Tissue mechanics modulate microRNA-dependent PTEN expression to regulate malignant progression.

    PubMed

    Mouw, Janna K; Yui, Yoshihiro; Damiano, Laura; Bainer, Russell O; Lakins, Johnathon N; Acerbi, Irene; Ou, Guanqing; Wijekoon, Amanda C; Levental, Kandice R; Gilbert, Penney M; Hwang, E Shelley; Chen, Yunn-Yi; Weaver, Valerie M

    2014-04-01

    Tissue mechanics regulate development and homeostasis and are consistently modified in tumor progression. Nevertheless, the fundamental molecular mechanisms through which altered mechanics regulate tissue behavior and the clinical relevance of these changes remain unclear. We demonstrate that increased matrix stiffness modulates microRNA expression to drive tumor progression through integrin activation of β-catenin and MYC. Specifically, in human and mouse tissue, increased matrix stiffness induced miR-18a to reduce levels of the tumor suppressor phosphatase and tensin homolog (PTEN), both directly and indirectly by decreasing levels of homeobox A9 (HOXA9). Clinically, extracellular matrix stiffness correlated directly and significantly with miR-18a expression in human breast tumor biopsies. miR-18a expression was highest in basal-like breast cancers in which PTEN and HOXA9 levels were lowest, and high miR-18a expression predicted poor prognosis in patients with luminal breast cancers. Our findings identify a mechanically regulated microRNA circuit that can promote malignancy and suggest potential prognostic roles for HOXA9 and miR-18a levels in stratifying patients with luminal breast cancers. PMID:24633304

  17. Tissue mechanics modulate microRNA-dependent PTEN expression to regulate malignant progression

    PubMed Central

    Mouw, Janna K; Yui, Yoshihiro; Damiano, Laura; Bainer, Russell O; Lakins, Johnathan N; Acerbi, Irene; Ou, Guanqing; Wijekoon, Amanda C; Levental, Kandice R; Gilbert, Penney M; Chen, Yunn-Yi; Weaver, Valerie M

    2014-01-01

    Tissue mechanics regulate development and homeostasis and are consistently modified in tumor progression. Nevertheless, the fundamental molecular mechanisms through which altered mechanics regulate tissue behavior and the clinical relevance of these changes remain unclear. We demonstrate that increased matrix stiffness modulates microRNA expression to drive tumor progression through integrin activation of β-catenin and MYC. Specifically, in human and mouse tissue, increased matrix stiffness induced miR-18a to reduce levels of the tumor suppressor PTEN, both directly and indirectly by decreasing levels of HOXA9. Clinically, extracellular matrix stiffness correlated significantly with miR-18a in human breast tumor biopsies. miR-18a expression was highest in basal-like breast cancers in which PTEN and HOXA9 levels were lowest and predicted for poor prognosis in patients with luminal breast cancers. Our findings identify a mechanically-regulated microRNA circuit that can promote malignancy and suggest potential prognostic roles for HOXA9 and miR-18a levels in stratifying patients with luminal breast cancers. PMID:24633304

  18. Salivary gland morphology, tissue tropism and the progression of tospovirus infection in Frankliniella occidentalis.

    PubMed

    Montero-Astúa, Mauricio; Ullman, Diane E; Whitfield, Anna E

    2016-06-01

    Tomato spotted wilt virus (TSWV) is transmitted by thrips in a propagative manner; however, progression of virus infection in the insect is not fully understood. The goal of this work was to study the morphology and infection of thrips salivary glands. The primary salivary glands (PSG) are complex, with three distinct regions that may have unique functions. Analysis of TSWV progression in thrips revealed the presence of viral proteins in the foregut, midgut, ligaments, tubular salivary glands (TSG), and efferent duct and filament structures connecting the TSG and PSG of first and second instar larvae. The primary site of virus infection shifted from the midgut and TSG in the larvae to the PSG in adults, suggesting that tissue tropism changes with insect development. TSG infection was detected in advance of PSG infection. These findings support the hypothesis that the TSG are involved in trafficking of TSWV to the PSG. PMID:26999025

  19. Progress of tissue culture and genetic transformation research in pigeon pea [Cajanus cajan (L.) Millsp.].

    PubMed

    Krishna, Gaurav; Reddy, P Sairam; Ramteke, P W; Bhattacharya, P S

    2010-10-01

    Pigeon pea [Cajanus cajan (L.) Millsp.] (Family: Fabaceae) is an important legume crop cultivated across 50 countries in Asia, Africa, and the Americas; and ranks fifth in area among pulses after soybean, common bean, peanut, and chickpea. It is consumed as a major source of protein (21%) to the human population in many developing countries. In India, it is the second important food legume contributing to 80% of the global production. Several biotic and abiotic stresses are posing a big threat to its production and productivity. Attempts to address these problems through conventional breeding methods have met with partial success. This paper reviews the chronological progress made in tissue culture through organogenesis and somatic embryogenesis, including the influence of factors such as genotypes, explant sources, and culture media including the supplementation of plant growth regulators. Comprehensive lists of morphogenetic pathways involved in in vitro regeneration through organogenesis and somatic embryogenesis using different explant tissues of diverse pigeon pea genotypes are presented. Similarly, the establishment of protocols for the production of transgenics via particle bombardment and Agrobacterium-mediated transformation using different explant tissues, Agrobacterium strains, Ti plasmids, and plant selectable markers, as well as their interactions on transformation efficiency have been discussed. Future research thrusts on the use of different promoters and stacking of genes for various biotic and abiotic stresses in pigeon pea are suggested. PMID:20652570

  20. Identifying Cancer Biomarkers From Microarray Data Using Feature Selection and Semisupervised Learning

    PubMed Central

    Maulik, Ujjwal

    2014-01-01

    Microarrays have now gone from obscurity to being almost ubiquitous in biological research. At the same time, the statistical methodology for microarray analysis has progressed from simple visual assessments of results to novel algorithms for analyzing changes in expression profiles. In a micro-RNA (miRNA) or gene-expression profiling experiment, the expression levels of thousands of genes/miRNAs are simultaneously monitored to study the effects of certain treatments, diseases, and developmental stages on their expressions. Microarray-based gene expression profiling can be used to identify genes, whose expressions are changed in response to pathogens or other organisms by comparing gene expression in infected to that in uninfected cells or tissues. Recent studies have revealed that patterns of altered microarray expression profiles in cancer can serve as molecular biomarkers for tumor diagnosis, prognosis of disease-specific outcomes, and prediction of therapeutic responses. Microarray data sets containing expression profiles of a number of miRNAs or genes are used to identify biomarkers, which have dysregulation in normal and malignant tissues. However, small sample size remains a bottleneck to design successful classification methods. On the other hand, adequate number of microarray data that do not have clinical knowledge can be employed as additional source of information. In this paper, a combination of kernelized fuzzy rough set (KFRS) and semisupervised support vector machine (S3VM) is proposed for predicting cancer biomarkers from one miRNA and three gene expression data sets. Biomarkers are discovered employing three feature selection methods, including KFRS. The effectiveness of the proposed KFRS and S3VM combination on the microarray data sets is demonstrated, and the cancer biomarkers identified from miRNA data are reported. Furthermore, biological significance tests are conducted for miRNA cancer biomarkers. PMID:27170887

  1. Identification of novel candidate circulating biomarkers for malignant soft tissue sarcomas: Correlation with metastatic progression.

    PubMed

    Conti, Amalia; Fredolini, Claudia; Tamburro, Davide; Magagnoli, Giovanna; Zhou, Weidong; Liotta, Lance A; Picci, Piero; Luchini, Alessandra; Benassi, Maria Serena

    2016-02-01

    Soft tissue sarcomas (STS) are a heterogeneous group of rare tumors for which identification and validation of biological markers may improve clinical management. The fraction of low-molecular-weight (LMW) circulating proteins and fragments of proteins is a rich source of new potential biomarkers. To identify circulating biomarkers useful for STS early diagnosis and prognosis, we analyzed 53 high-grade STS sera using hydrogel core-shell nanoparticles that selectively entrap LMW proteins by size exclusion and affinity chromatography, protect them from degradation and amplify their concentration for mass spectrometry detection. Twenty-two analytes mostly involved in inflammatory and immunological response, showed a progressive increase from benign to malignant STS with a relative difference in abundance, more than 50% when compared to healthy control. 16 of these were higher in metastatic compared to non-metastatic tumors. Cox's regression analysis revealed a statistical significant association between the abundance of lactotransferrin (LTF) and complement factor H-related 5 (CFHR5) and risk of metastasis. In particular, CFHR5 was associated with the risk of metastasis. The role of circulating proteins involved in metastatic progression will be crucial for a better understanding of STS biology and patient management. PMID:26699407

  2. Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular fibrosis and tumor progression.

    PubMed

    Laklai, Hanane; Miroshnikova, Yekaterina A; Pickup, Michael W; Collisson, Eric A; Kim, Grace E; Barrett, Alex S; Hill, Ryan C; Lakins, Johnathon N; Schlaepfer, David D; Mouw, Janna K; LeBleu, Valerie S; Roy, Nilotpal; Novitskiy, Sergey V; Johansen, Julia S; Poli, Valeria; Kalluri, Raghu; Iacobuzio-Donahue, Christine A; Wood, Laura D; Hebrok, Matthias; Hansen, Kirk; Moses, Harold L; Weaver, Valerie M

    2016-05-01

    Fibrosis compromises pancreatic ductal carcinoma (PDAC) treatment and contributes to patient mortality, yet antistromal therapies are controversial. We found that human PDACs with impaired epithelial transforming growth factor-β (TGF-β) signaling have high epithelial STAT3 activity and develop stiff, matricellular-enriched fibrosis associated with high epithelial tension and shorter patient survival. In several KRAS-driven mouse models, both the loss of TGF-β signaling and elevated β1-integrin mechanosignaling engaged a positive feedback loop whereby STAT3 signaling promotes tumor progression by increasing matricellular fibrosis and tissue tension. In contrast, epithelial STAT3 ablation attenuated tumor progression by reducing the stromal stiffening and epithelial contractility induced by loss of TGF-β signaling. In PDAC patient biopsies, higher matricellular protein and activated STAT3 were associated with SMAD4 mutation and shorter survival. The findings implicate epithelial tension and matricellular fibrosis in the aggressiveness of SMAD4 mutant pancreatic tumors and highlight STAT3 and mechanics as key drivers of this phenotype. PMID:27089513

  3. Manufacturing of microarrays.

    PubMed

    Petersen, David W; Kawasaki, Ernest S

    2007-01-01

    DNA microarray technology has become a powerful tool in the arsenal of the molecular biologist. Capitalizing on high precision robotics and the wealth of DNA sequences annotated from the genomes of a large number of organisms, the manufacture of microarrays is now possible for the average academic laboratory with the funds and motivation. Microarray production requires attention to both biological and physical resources, including DNA libraries, robotics, and qualified personnel. While the fabrication of microarrays is a very labor-intensive process, production of quality microarrays individually tailored on a project-by-project basis will help researchers shed light on future scientific questions. PMID:17265711

  4. Role of mesenchymal stem cell-derived fibrinolytic factor in tissue regeneration and cancer progression.

    PubMed

    Heissig, Beate; Dhahri, Douaa; Eiamboonsert, Salita; Salama, Yousef; Shimazu, Hiroshi; Munakata, Shinya; Hattori, Koichi

    2015-12-01

    Tissue regeneration during wound healing or cancer growth and progression depends on the establishment of a cellular microenvironment. Mesenchymal stem cells (MSC) are part of this cellular microenvironment, where they functionally modulate cell homing, angiogenesis, and immune modulation. MSC recruitment involves detachment of these cells from their niche, and finally MSC migration into their preferred niches; the wounded area, the tumor bed, and the BM, just to name a few. During this recruitment phase, focal proteolysis disrupts the extracellular matrix (ECM) architecture, breaks cell-matrix interactions with receptors, and integrins, and causes the release of bioactive fragments from ECM molecules. MSC produce a broad array of proteases, promoting remodeling of the surrounding ECM through proteolytic mechanisms. The fibrinolytic system, with its main player plasmin, plays a crucial role in cell migration, growth factor bioavailability, and the regulation of other protease systems during inflammation, tissue regeneration, and cancer. Key components of the fibrinolytic cascade, including the urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1), are expressed in MSC. This review will introduce general functional properties of the fibrinolytic system, which go beyond its known function of fibrin clot dissolution (fibrinolysis). We will focus on the role of the fibrinolytic system for MSC biology, summarizing our current understanding of the role of the fibrinolytic system for MSC recruitment and the functional consequences for tissue regeneration and cancer. Aspects of MSC origin, maintenance, and the mechanisms by which these cells contribute to altered protease activity in the microenvironment under normal and pathological conditions will also be discussed. PMID:26350342

  5. Evaluation of a Gene Expression Microarray-based Assay to Determine Tissue Type of Origin on a Diverse Set of 49 Malignancies

    PubMed Central

    Beck, Andrew H.; Rodriguez-Paris, Juan; Zehnder, James; Schrijver, Iris

    2016-01-01

    The Tissue of Origin Frozen (TOO-FRZ) assay from Pathwork Diagnostics has been cleared by the Food and Drug Administration as a diagnostic study for malignancies of unknown primary. The goal of this study was to evaluate the performance of TOO-FRZ on a diverse collection of malignancies. We collected a diverse set of 49 malignancies. We classified each case into 1 of 4 groups: common morphology from a tissue type included in the TOO-FRZ assay (n=29), uncommon morphology from a tissue type included in the TOO-FRZ assay (n=10), tumor from a tissue type not included in the TOO-FRZ assay (n=3), and malignancies of unknown primary (n=7). We found strong diagnostic performance for common morphologies from tissue types on the TOO-FRZ [overall accuracy=26 of 29 (90%, 95% CI, 73% to 97%)], with perfect performance in all tissue types except gastric (0 of 2) and pancreatic (1 of 2) tissues. There was a significant decline in performance for uncommon morphologies from tissue types included in the TOO-FRZ assay [6 of 10 (60%) cases with an indeterminate result, 1 of 10 (10%) cases with an incorrect prediction, and 3 of 10 (30%) with a correct prediction] and for tumors from tissue types not included in the assay (incorrect prediction in 2 of 3 cases). For the 7 malignancies of unknown primary in our study set, the TOO-FRZ provided a likely clinically useful result in only 2 of 7 cases. These results provide an insight into the strengths and limitations of this molecular assay for the surgical pathologist, and our findings suggest future directions for research in this area. PMID:21602661

  6. IMPROVING THE RELIABILITY OF MICROARRAYS FOR TOXICOLOGY RESEARCH: A COLLABORATIVE APPROACH

    EPA Science Inventory

    Microarray-based gene expression profiling is a critical tool to identify molecular biomarkers of specific chemical stressors. Although current microarray technologies have progressed from their infancy, biological and technical repeatability and reliability are often still limit...

  7. Glycan gene expression signatures in normal and malignant breast tissue; possible role in diagnosis and progression.

    PubMed

    Potapenko, Ivan O; Haakensen, Vilde D; Lüders, Torben; Helland, Aslaug; Bukholm, Ida; Sørlie, Therese; Kristensen, Vessela N; Lingjaerde, Ole C; Børresen-Dale, Anne-Lise

    2010-04-01

    Glycosylation is the stepwise procedure of covalent attachment of oligosaccharide chains to proteins or lipids, and alterations in this process have been associated with malignant transformation. Simultaneous analysis of the expression of all glycan-related genes clearly gives the advantage of enabling a comprehensive view of the genetic background of the glycobiological changes in cancer cells. Studies focusing on the expression of the whole glycome have now become possible, which prompted us to review the present knowledge on glycosylation in relation to breast cancer diagnosis and progression, in the light of available expression data from tumors and breast tissue of healthy individuals. We used various data resources to select a set of 419 functionally relevant genes involved in synthesis, degradation and binding of N-linked and O-linked glycans, Lewis antigens, glycosaminoglycans (chondroitin, heparin and keratan sulfate in addition to hyaluronan) and glycosphingolipids. Such glycans are involved in a number of processes relevant to carcinogenesis, including regulation of growth factors/growth factor receptors, cell-cell adhesion and motility as well as immune system modulation. Expression analysis of these glycan-related genes revealed that mRNA levels for many of them differ significantly between normal and malignant breast tissue. An associative analysis of these genes in the context of current knowledge of their function in protein glycosylation and connection(s) to cancer indicated that synthesis, degradation and adhesion mediated by glycans may be altered drastically in mammary carcinomas. Although further analysis is needed to assess how changes in mRNA levels of glycan genes influence a cell's glycome and the precise role that such altered glycan structures play in the pathogenesis of the disease, lessons drawn from this study may help in determining directions for future research in the rapidly-developing field of glycobiology. PMID:20060370

  8. Microarrays in hematology.

    PubMed

    Walker, Josef; Flower, Darren; Rigley, Kevin

    2002-01-01

    Microarrays are fast becoming routine tools for the high-throughput analysis of gene expression in a wide range of biologic systems, including hematology. Although a number of approaches can be taken when implementing microarray-based studies, all are capable of providing important insights into biologic function. Although some technical issues have not been resolved, microarrays will continue to make a significant impact on hematologically important research. PMID:11753074

  9. SNP microarray analyses reveal copy number alterations and progressive genome reorganization during tumor development in SVT/t driven mice breast cancer

    PubMed Central

    2012-01-01

    Background Tumor development is known to be a stepwise process involving dynamic changes that affect cellular integrity and cellular behavior. This complex interaction between genomic organization and gene, as well as protein expression is not yet fully understood. Tumor characterization by gene expression analyses is not sufficient, since expression levels are only available as a snapshot of the cell status. So far, research has mainly focused on gene expression profiling or alterations in oncogenes, even though DNA microarray platforms would allow for high-throughput analyses of copy number alterations (CNAs). Methods We analyzed DNA from mouse mammary gland epithelial cells using the Affymetrix Mouse Diversity Genotyping array (MOUSEDIVm520650) and calculated the CNAs. Segmental copy number alterations were computed based on the probeset CNAs using the circular binary segmentation algorithm. Motif search was performed in breakpoint regions (inter-segment regions) with the MEME suite to identify common motif sequences. Results Here we present a four stage mouse model addressing copy number alterations in tumorigenesis. No considerable changes in CNA were identified for non-transgenic mice, but a stepwise increase in CNA was found during tumor development. The segmental copy number alteration revealed informative chromosomal fragmentation patterns. In inter-segment regions (hypothetical breakpoint sides) unique motifs were found. Conclusions Our analyses suggest genome reorganization as a stepwise process that involves amplifications and deletions of chromosomal regions. We conclude from distinctive fragmentation patterns that conserved as well as individual breakpoints exist which promote tumorigenesis. PMID:22935085

  10. Peripheral Ovine Progressive Pneumonia Provirus Levels Correlate with and Predict Histological Tissue Lesion Severity in Naturally Infected Sheep

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Studies were undertaken to determine whether host immune responses in the form of serum anti-ovine progressive pneumonia virus (OPPV) antibody responses or virus replication in the form of peripheral OPP provirus levels associate with the degree of histological tissue lesions in naturally OPPV infec...

  11. cDNA microarray analysis of esophageal cancer: discoveries and prospects.

    PubMed

    Shimada, Yutaka; Sato, Fumiaki; Shimizu, Kazuharu; Tsujimoto, Gozoh; Tsukada, Kazuhiro

    2009-07-01

    Recent progress in molecular biology has revealed many genetic and epigenetic alterations that are involved in the development and progression of esophageal cancer. Microarray analysis has also revealed several genetic networks that are involved in esophageal cancer. However, clinical application of microarray techniques and use of microarray data have not yet occurred. In this review, we focus on the recent developments and problems with microarray analysis of esophageal cancer. PMID:19597924

  12. Progress on ThermoBrachytherapy Surface Applicator for Superficial Tissue Diseases

    PubMed Central

    Arunachalam, Kavitha; Craciunescu, Oana I.; Maccarini, Paolo F.; Schlorff, Jaime L.; Markowitz, Edward; Stauffer, Paul R.

    2013-01-01

    This work reports the ongoing development of a combination applicator for simultaneous heating of superficial tissue disease using a 915 MHz DCC (dual concentric conductor) array and High Dose Rate (HDR) brachytherapy delivered via an integrated conformal catheter array. The progress includes engineering design changes in the waterbolus, DCC configurations and fabrication techniques of the conformal multilayer applicator. The dosimetric impact of the thin copper DCC array is also assessed. Steady state fluid dynamics of the new waterbolus bag indicates nearly uniform flow with less than 1C variation across a large (1932cm) bolus. Thermometry data of the torso phantom acquired with computer controlled movement of fiberoptic temperature probes inside thermal mapping catheters indicate feasibility of real time feedback control for the DCC array. MR (magnetic resonance) scans of a torso phantom indicate that the waterbolus thickness across the treatment area is controlled by the pressure applied by the surrounding inflatable airbladder and applicator securing straps. The attenuation coefficient of the DCC array was measured as 3 0.001% and 2.950.03 % using an ion chamber and OneDose dosimeters respectively. The performance of the combination applicator on patient phantoms provides valuable feedback to optimize the applicator prior use in the patient clinic. PMID:24392196

  13. Microarrays, antiobesity and the liver

    PubMed Central

    Castro-Chávez, Fernando

    2013-01-01

    In this review, the microarray technology and especially oligonucleotide arrays are exemplified with a practical example taken from the perilipin−/− mice and using the dChip software, available for non-lucrative purposes. It was found that the liver of perilipin−/− mice was healthy and normal, even under high-fat diet when compared with the results published for the scd1−/− mice, which under high-fat diets had a darker liver, suggestive of hepatic steatosis. Scd1 is required for the biosynthesis of monounsaturated fatty acids and plays a key role in the hepatic synthesis of triglycerides and of very-low-density lipoproteins. Both models of obesity resistance share many similar phenotypic antiobesity features, however, the perilipin−/− mice had a significant downregulation of stearoyl CoA desaturases scd1 and scd2 in its white adipose tissue, but a normal level of both genes inside the liver, even under high-fat diet. Here, different microarray methodologies are discussed, and also some of the most recent discoveries and perspectives regarding the use of microarrays, with an emphasis on obesity gene expression, and a personal remark on my findings of increased expression for hemoglobin transcripts and other hemo related genes (hemo-like), and for leukocyte like (leuko-like) genes inside the white adipose tissue of the perilipin−/− mice. In conclusion, microarrays have much to offer in comparative studies such as those in antiobesity, and also they are methodologies adequate for new astounding molecular discoveries [free full text of this article PMID:15657555

  14. [Research progress in peri-implant soft tissue engineering augmentation method].

    PubMed

    Pei, T T; Yu, H Q; Wen, C J; Guo, T Q; Zhou, Y M; Peng, H M

    2016-05-01

    The sufficiency of hard and soft tissue at the implant site is the guarantee of long-term function, health and the appearance of implant denture. Problem of soft tissue recession at the implant site has always been bothering dentists. Traditional methods for augmentation of soft tissue such as gingival transplantation have disadvantages of instability of the increased soft-tissue and more trauma. Lately the methods that base on tissue engineering to increase the soft tissue of peri-implant sites have drawn great attention. This review focuses on the current methods of peri-implant restoration through tissue engineering, seed cells, biological scaffolds and cytokines. PMID:27220393

  15. Pineal Function: Impact of Microarray Analysis

    PubMed Central

    Klein, David C.; Bailey, Michael J.; Carter, David A.; Kim, Jong-so; Shi, Qiong; Ho, Anthony; Chik, Constance; Gaildrat, Pascaline; Morin, Fabrice; Ganguly, Surajit; Rath, Martin F.; Møller, Morten; Sugden, David; Rangel, Zoila G.; Munson, Peter J.; Weller, Joan L.; Coon, Steven L.

    2009-01-01

    Microarray analysis has provided a new understanding of pineal function by identifying genes that are highly expressed in this tissue relative to other tissues and also by identifying over 600 genes that are expressed on a 24-hour schedule. This effort has highlighted surprising similarity to the retina and has provided reason to explore new avenues of study including intracellular signaling, signal transduction, transcriptional cascades, thyroid/retinoic acid hormone signaling, metal biology, RNA splicing, and the role the pineal gland plays in the immune/inflammation response. The new foundation that microarray analysis has provided will broadly support future research on pineal function. PMID:19622385

  16. Microarray technology: basic methodology and application in clinical research for biomarker discovery in vascular diseases.

    PubMed

    Raghavachari, Nalini

    2013-01-01

    Microarray technology is a novel tool in molecular biology, capable of quantitating hundreds or thousands of gene transcripts from a given cell or tissue sample simultaneously. A microarray has thousands of DNA fragments or oligonucleotides of known sequence arrayed in a known sequence of rows and columns on a chip. Hybridization of sample RNA that has been reverse-transcribed and labeled enables the detection and quantitation of specific transcripts. The ability to quantitate systemic gene changes in normal vs. diseased states has led to significant progress in many biomedical disciplines, including lipoprotein and atherosclerosis research, and can be used for discovery of diagnostic/prognostic and predictive biomarkers and to test the effectiveness of potential therapeutic agents. The design and analysis of microarray experiments present some unique problems to clinical medicine due to inherent issues related to biological sample procurement and processing, sensitivity and specificity of the assay, reliability and reproducibility of data, and applicability of the technology in multicenter-based clinical studies. This chapter will provide details on the methodologies that address these problems for successful microarray-based transcriptome analysis of tissues, whole blood, cell subpopulations, and cultured cells. PMID:23912982

  17. New hypotheses on the function of the avian shell gland derived from microarray analysis comparing tissue from juvenile and sexually mature hens.

    PubMed

    Dunn, I C; Wilson, P W; Lu, Z; Bain, M M; Crossan, C L; Talbot, R T; Waddington, D

    2009-09-01

    Activation of the shell gland region of the avian oviduct is mediated by ovarian steroids. To understand more extensively how shell glands are maintained and function, we have compared gene expression in the shell glands from juvenile and laying hens using a chicken cDNA microarray. Average expression profiles of juvenile and sexually mature shell glands were compared resulting in the identification of 266 differentially regulated genes. Reverse transcription quantitative polymerase chain reaction confirmed expression differences. The differentially expressed genes included several with known involvement in shell gland function, including ion transport and shell matrix proteins. There were also many unpredicted differentially expressed genes, and for some we propose hypotheses for their functions. These include those encoding (a) osteoprotegerin, a decoy death receptor for receptor activator of nuclear factor NFkB ligand (RANKL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), that in the shell gland, may prevent apoptosis and/or may have an endocrine effect by preventing RANKL's action on bone osteoclasts that mobilize stored calcium; (b) prostatic acid phosphatase (ACPP) and prostate stem cell antigen (PSCA) that could play a role in sperm physiology within the shell gland; (c) urea transporter (SLC14A2) that could provide a novel anti-microbial defence; (d) bactericidal/permeability-increasing protein-like 2 (BPIL2), and other potential anti-microbials that have not previously been documented in the chicken. These new hypotheses, if borne out experimentally, will lead to a greater understanding of shell gland function including the processes involved in eggshell formation and anti-microbial activity. PMID:19303879

  18. Using Pharmacokinetic Profiles and Digital Quantification of Stained Tissue Microarrays as a Medium-Throughput, Quantitative Method for Measuring the Kinetics of Early Signaling Changes Following Integrin-Linked Kinase Inhibition in an In Vivo Model of Cancer.

    PubMed

    Kalra, Jessica; Dragowska, Weislawa H; Bally, Marcel B

    2015-09-01

    A small molecule inhibitor (QLT0267) targeting integrin-linked kinase is able to slow breast tumor growth in vivo; however, the mechanism of action remains unknown. Understanding how targeting molecules involved in intersecting signaling pathways impact disease is challenging. To facilitate this understanding, we used tumor tissue microarrays (TMA) and digital image analysis for quantification of immunohistochemistry (IHC) in order to investigate how QLT0267 affects signaling pathways in an orthotopic model of breast cancer over time. Female NCR nude mice were inoculated with luciferase-positive human breast tumor cells (LCC6(Luc)) and tumor growth was assessed by bioluminescent imaging (BLI). The plasma levels of QLT0267 were determined by LC-MS/MS methods following oral dosing of QLT0267 (200 mg/kg). A TMA was constructed using tumor tissue collected at 2, 4, 6, 24, 78 and 168 hr after treatment. IHC methods were used to assess changes in ILK-related signaling. The TMA was digitized, and Aperio ScanScope and ImageScope software were used to provide semi-quantitative assessments of staining levels. Using medium-throughput IHC quantitation, we show that ILK targeting by QLT0267 in vivo influences tumor physiology through transient changes in pathways involving AKT, GSK-3 and TWIST accompanied by the translocation of the pro-apoptotic protein BAD and an increase in Caspase-3 activity. PMID:25940338

  19. [Research progress of cell sheet technology and its applications in tissue engineering and regenerative medicine].

    PubMed

    Ma, Dongyang; Ren, Liling; Mao, Tianqiu

    2014-10-01

    Cell sheet engineering is an important technology to harvest the cultured cells in the form of confluent monolayers using a continuous culture method and a physical approach. Avoiding the use of enzymes, expended cells can be harvested together with endogenous extracellular matrix, cell-matrix contacts, and cell-cell contacts. With high efficiency of cell loading ability and without using exogenous scaffolds, cell sheet engineering has several advantages over traditional tissue engineering methods. In this article, we give an overview on cell sheet technology about its applications in the filed of tissue regeneration, including the construction of soft tissues (corneal, mucous membrane, myocardium, blood vessel, pancreas islet, liver, bladder and skin) and hard tissues (bone, cartilage and tooth root). This techonoly is promising to provide a novel strategy for the development of tissue engineering and regenerative medicine. And further works should be carried out on the operability of this technology and its feasibility to construct thick tissues. PMID:25764743

  20. Early Prediction of Cancer Progression by Depth-Resolved Nanoscale Mapping of Nuclear Architecture from Unstained Tissue Specimens.

    PubMed

    Uttam, Shikhar; Pham, Hoa V; LaFace, Justin; Leibowitz, Brian; Yu, Jian; Brand, Randall E; Hartman, Douglas J; Liu, Yang

    2015-11-15

    Early cancer detection currently relies on screening the entire at-risk population, as with colonoscopy and mammography. Therefore, frequent, invasive surveillance of patients at risk for developing cancer carries financial, physical, and emotional burdens because clinicians lack tools to accurately predict which patients will actually progress into malignancy. Here, we present a new method to predict cancer progression risk via nanoscale nuclear architecture mapping (nanoNAM) of unstained tissue sections based on the intrinsic density alteration of nuclear structure rather than the amount of stain uptake. We demonstrate that nanoNAM detects a gradual increase in the density alteration of nuclear architecture during malignant transformation in animal models of colon carcinogenesis and in human patients with ulcerative colitis, even in tissue that appears histologically normal according to pathologists. We evaluated the ability of nanoNAM to predict "future" cancer progression in patients with ulcerative colitis who did and did not develop colon cancer up to 13 years after their initial colonoscopy. NanoNAM of the initial biopsies correctly classified 12 of 15 patients who eventually developed colon cancer and 15 of 18 who did not, with an overall accuracy of 85%. Taken together, our findings demonstrate great potential for nanoNAM in predicting cancer progression risk and suggest that further validation in a multicenter study with larger cohorts may eventually advance this method to become a routine clinical test. PMID:26383164

  1. Tumor-induced inflammation in mammary adipose tissue stimulates a vicious cycle of autotaxin expression and breast cancer progression.

    PubMed

    Benesch, Matthew G K; Tang, Xiaoyun; Dewald, Jay; Dong, Wei-Feng; Mackey, John R; Hemmings, Denise G; McMullen, Todd P W; Brindley, David N

    2015-09-01

    Compared to normal tissues, many cancer cells overexpress autotaxin (ATX). This secreted enzyme produces extracellular lysophosphatidate, which signals through 6 GPCRs to drive cancer progression. Our previous work showed that ATX inhibition decreases 4T1 breast tumor growth in BALB/c mice by 60% for about 11 d. However, 4T1 cells do not produce significant ATX. Instead, the ATX is produced by adjacent mammary adipose tissue. We investigated the molecular basis of this interaction in human and mouse breast tumors. Inflammatory mediators secreted by breast cancer cells increased ATX production in adipose tissue. The increased lysophosphatidate signaling further increased inflammatory mediator production in adipose tissue and tumors. Blocking ATX activity in mice bearing 4T1 tumors with 10 mg/kg/d ONO-8430506 (a competitive ATX inhibitor, IC90 = 100 nM; Ono Pharma Co., Ltd., Osaka, Japan) broke this vicious inflammatory cycle by decreasing 20 inflammatory mediators by 1.5-8-fold in cancer-inflamed adipose tissue. There was no significant decrease in inflammatory mediator levels in fat pads that did not bear tumors. ONO-8430506 also decreased plasma TNF-α and G-CSF cytokine levels by >70% and leukocyte infiltration in breast tumors and adjacent adipose tissue by >50%. Hence, blocking tumor-driven inflammation by ATX inhibition is effective in decreasing tumor growth in breast cancers where the cancer cells express negligible ATX. PMID:26071407

  2. Tissue factor associates with survival and regulates tumour progression in osteosarcoma.

    PubMed

    Tieken, Chris; Verboom, Michiel C; Ruf, Wolfram; Gelderblom, Hans; Bovée, Judith V M G; Reitsma, Pieter H; Cleton-Jansen, Anne-Marie; Versteeg, Henri H

    2016-05-01

    Osteosarcoma is the most common primary malignant bone tumour. Patients often develop lung metastasis and have a poor prognosis despite extensive chemotherapy and surgical resections. Tissue Factor is associated with poor clinical outcome in a wide range of cancer types, and promotes angiogenesis and metastasis. The role of Tissue Factor in OS tumourigenesis is unknown. Fifty-three osteosarcoma pre-treatment biopsies and four osteosarcoma cell lines were evaluated for Tissue Factor expression, and a possible association with clinical parameters was investigated. Tissue Factor function was inhibited in an osteosarcoma cell line (143B) by shRNA knockdown or specific antibodies, and pro-tumourigenic gene expression, proliferation, matrigel invasion and transwell migration was examined. 143B cells were implanted in mice in the presence of Tissue Factor-blocking antibodies, and tumour volume, micro-vessel density and metastases in the lung were evaluated. Tissue Factor was highly expressed in 73.6 % of osteosarcoma biopsies, and expression associated significantly with disease-free survival. Tissue Factor was expressed in all four investigated cell lines. Tissue Factor was knocked down in 143B cells, which led to reduced expression of IL-8, CXCL-1, SNAIL and MMP2, but not MMP9. Tissue Factor knockdown or inhibition with antibodies reduced matrigel invasion. Tissue Factor antibodies limited 143B tumour growth in vivo, and resulted in decreased intra-tumoural micro-vessel density. Furthermore, lung metastasis from the primary tumour was significantly reduced. Thus, Tissue Factor expression in osteosarcoma reduces metastasis-free survival in patients, and increases pro-tumourigenic behaviour both in vitro and in vivo. PMID:26763081

  3. Microarrays in Glycoproteomics Research

    PubMed Central

    Yue, Tingting; Haab, Brian B.

    2009-01-01

    Microarrays have been extremely useful for investigating binding interactions among diverse types of molecular species, with the main advantage being the ability to examine many interactions using small amount of samples and reagents. Microarrays are increasingly being used to advance research in the field of glycobiology, which is the study of the nature and function and carbohydrates in health and disease. Several types of microarrays are being used in the study of glycans and proteins in glycobiology, including glycan arrays to study the recognition of carbohydrates, lectin arrays to determine carbohydrate expression on purified proteins or on cells, and antibody arrays to examine the variation in particular glycan structures on specific proteins. This review will cover the technology and applications of these types of microarrays, as well as their use for obtaining complementary information on various aspects of glycobiology. PMID:19389548

  4. Bone tissue engineering using silica-based mesoporous nanobiomaterials:Recent progress.

    PubMed

    Shadjou, Nasrin; Hasanzadeh, Mohammad

    2015-10-01

    Bone disorders are of significant concern due to increase in the median age of our population. It is in this context that tissue engineering has been emerging as a valid approach to the current therapies for bone regeneration/substitution. Tissue-engineered bone constructs have the potential to alleviate the demand arising from the shortage of suitable autograft and allograft materials for augmenting bone healing. Silica based mesostructured nanomaterials possessing pore sizes in the range 2-50 nm and surface reactive functionalities have elicited immense interest due to their exciting prospects in bone tissue engineering. In this review we describe application of silica-based mesoporous nanomaterials for bone tissue engineering. We summarize the preparation methods, the effect of mesopore templates and composition on the mesopore-structure characteristics, and different forms of these materials, including particles, fibers, spheres, scaffolds and composites. Also, the effect of structural and textural properties of mesoporous materials on development of new biomaterials for production of bone implants and bone cements was discussed. Also, application of different mesoporous materials on construction of manufacture 3-dimensional scaffolds for bone tissue engineering was discussed. It begins by giving the reader a brief background on tissue engineering, followed by a comprehensive description of all the relevant components of silica-based mesoporous biomaterials on bone tissue engineering, going from materials to scaffolds and from cells to tissue engineering strategies that will lead to "engineered" bone. PMID:26117771

  5. Chromosomal aberrations in bladder cancer: fresh versus formalin fixed paraffin embedded tissue and targeted FISH versus wide microarray-based CGH analysis.

    PubMed

    Panzeri, Elena; Conconi, Donatella; Antolini, Laura; Redaelli, Serena; Valsecchi, Maria Grazia; Bovo, Giorgio; Pallotti, Francesco; Viganò, Paolo; Strada, Guido; Dalprà, Leda; Bentivegna, Angela

    2011-01-01

    Bladder carcinogenesis is believed to follow two alternative pathways driven by the loss of chromosome 9 and the gain of chromosome 7, albeit other nonrandom copy number alterations (CNAs) were identified. However, confirmation studies are needed since many aspects of this model remain unclear and considerable heterogeneity among cases has emerged. One of the purposes of this study was to evaluate the performance of a targeted test (UroVysion assay) widely used for the detection of Transitional Cell Carcinoma (TCC) of the bladder, in two different types of material derived from the same tumor. We compared the results of UroVysion test performed on Freshly Isolated interphasic Nuclei (FIN) and on Formalin Fixed Paraffin Embedded (FFPE) tissues from 22 TCCs and we didn't find substantial differences. A second goal was to assess the concordance between array-CGH profiles and the targeted chromosomal profiles of UroVysion assay on an additional set of 10 TCCs, in order to evaluate whether UroVysion is an adequately sensitive method for the identification of selected aneuploidies and nonrandom CNAs in TCCs. Our results confirmed the importance of global genomic screening methods, that is array based CGH, to comprehensively determine the genomic profiles of large series of TCCs tumors. However, this technique has yet some limitations, such as not being able to detect low level mosaicism, or not detecting any change in the number of copies for a kind of compensatory effect due to the presence of high cellular heterogeneity. Thus, it is still advisable to use complementary techniques such as array-CGH and FISH, as the former is able to detect alterations at the genome level not excluding any chromosome, but the latter is able to maintain the individual data at the level of single cells, even if it focuses on few genomic regions. PMID:21909424

  6. Chromosomal Aberrations in Bladder Cancer: Fresh versus Formalin Fixed Paraffin Embedded Tissue and Targeted FISH versus Wide Microarray-Based CGH Analysis

    PubMed Central

    Antolini, Laura; Redaelli, Serena; Valsecchi, Maria Grazia; Bovo, Giorgio; Pallotti, Francesco; Viganò, Paolo; Strada, Guido; Dalprà, Leda; Bentivegna, Angela

    2011-01-01

    Bladder carcinogenesis is believed to follow two alternative pathways driven by the loss of chromosome 9 and the gain of chromosome 7, albeit other nonrandom copy number alterations (CNAs) were identified. However, confirmation studies are needed since many aspects of this model remain unclear and considerable heterogeneity among cases has emerged. One of the purposes of this study was to evaluate the performance of a targeted test (UroVysion assay) widely used for the detection of Transitional Cell Carcinoma (TCC) of the bladder, in two different types of material derived from the same tumor. We compared the results of UroVysion test performed on Freshly Isolated interphasic Nuclei (FIN) and on Formalin Fixed Paraffin Embedded (FFPE) tissues from 22 TCCs and we didn't find substantial differences. A second goal was to assess the concordance between array-CGH profiles and the targeted chromosomal profiles of UroVysion assay on an additional set of 10 TCCs, in order to evaluate whether UroVysion is an adequately sensitive method for the identification of selected aneuploidies and nonrandom CNAs in TCCs. Our results confirmed the importance of global genomic screening methods, that is array based CGH, to comprehensively determine the genomic profiles of large series of TCCs tumors. However, this technique has yet some limitations, such as not being able to detect low level mosaicism, or not detecting any change in the number of copies for a kind of compensatory effect due to the presence of high cellular heterogeneity. Thus, it is still advisable to use complementary techniques such as array-CGH and FISH, as the former is able to detect alterations at the genome level not excluding any chromosome, but the latter is able to maintain the individual data at the level of single cells, even if it focuses on few genomic regions. PMID:21909424

  7. Hidden Treasures in “Ancient” Microarrays: Gene-Expression Portrays Biology and Potential Resistance Pathways of Major Lung Cancer Subtypes and Normal Tissue

    PubMed Central

    Kerkentzes, Konstantinos; Lagani, Vincenzo; Tsamardinos, Ioannis; Vyberg, Mogens; Røe, Oluf Dimitri

    2014-01-01

    Objective: Novel statistical methods and increasingly more accurate gene annotations can transform “old” biological data into a renewed source of knowledge with potential clinical relevance. Here, we provide an in silico proof-of-concept by extracting novel information from a high-quality mRNA expression dataset, originally published in 2001, using state-of-the-art bioinformatics approaches. Methods: The dataset consists of histologically defined cases of lung adenocarcinoma (AD), squamous (SQ) cell carcinoma, small-cell lung cancer, carcinoid, metastasis (breast and colon AD), and normal lung specimens (203 samples in total). A battery of statistical tests was used for identifying differential gene expressions, diagnostic and prognostic genes, enriched gene ontologies, and signaling pathways. Results: Our results showed that gene expressions faithfully recapitulate immunohistochemical subtype markers, as chromogranin A in carcinoids, cytokeratin 5, p63 in SQ, and TTF1 in non-squamous types. Moreover, biological information with putative clinical relevance was revealed as potentially novel diagnostic genes for each subtype with specificity 93–100% (AUC = 0.93–1.00). Cancer subtypes were characterized by (a) differential expression of treatment target genes as TYMS, HER2, and HER3 and (b) overrepresentation of treatment-related pathways like cell cycle, DNA repair, and ERBB pathways. The vascular smooth muscle contraction, leukocyte trans-endothelial migration, and actin cytoskeleton pathways were overexpressed in normal tissue. Conclusion: Reanalysis of this public dataset displayed the known biological features of lung cancer subtypes and revealed novel pathways of potentially clinical importance. The findings also support our hypothesis that even old omics data of high quality can be a source of significant biological information when appropriate bioinformatics methods are used. PMID:25325012

  8. High-throughput protein expression analysis using tissue microarray technology of a large well-characterised series identifies biologically distinct classes of breast cancer confirming recent cDNA expression analyses.

    PubMed

    Abd El-Rehim, Dalia M; Ball, Graham; Pinder, Sarah E; Rakha, Emad; Paish, Claire; Robertson, John F R; Macmillan, Douglas; Blamey, Roger W; Ellis, Ian O

    2005-09-01

    Recent studies on gene molecular profiling using cDNA microarray in a relatively small series of breast cancer have identified biologically distinct groups with apparent clinical and prognostic relevance. The validation of such new taxonomies should be confirmed on larger series of cases prior to acceptance in clinical practice. The development of tissue microarray (TMA) technology provides methodology for high-throughput concomitant analyses of multiple proteins on large numbers of archival tumour samples. In our study, we have used immunohistochemistry techniques applied to TMA preparations of 1,076 cases of invasive breast cancer to study the combined protein expression profiles of a large panel of well-characterized commercially available biomarkers related to epithelial cell lineage, differentiation, hormone and growth factor receptors and gene products known to be altered in some forms of breast cancer. Using hierarchical clustering methodology, 5 groups with distinct patterns of protein expression were identified. A sixth group of only 4 cases was also identified but deemed too small for further detailed assessment. Further analysis of these clusters was performed using multiple layer perceptron (MLP)-artificial neural network (ANN) with a back propagation algorithm to identify key biomarkers driving the membership of each group. We have identified 2 large groups by their expression of luminal epithelial cell phenotypic characteristics, hormone receptors positivity, absence of basal epithelial phenotype characteristics and lack of c-erbB-2 protein overexpression. Two additional groups were characterized by high c-erbB-2 positivity and negative or weak hormone receptors expression but showed differences in MUC1 and E-cadherin expression. The final group was characterized by strong basal epithelial characteristics, p53 positivity, absent hormone receptors and weak to low luminal epithelial cytokeratin expression. In addition, we have identified significant differences between clusters identified in this series with respect to established prognostic factors including tumour grade, size and histologic tumour type as well as differences in patient outcomes. The different protein expression profiles identified in our study confirm the biologic heterogeneity of breast cancer and demonstrate the clinical relevance of classification in this manner. These observations could form the basis of revision of existing traditional classification systems for breast cancer. PMID:15818618

  9. Effects of Moderate and Subsequent Progressive Weight Loss on Metabolic Function and Adipose Tissue Biology in Humans with Obesity.

    PubMed

    Magkos, Faidon; Fraterrigo, Gemma; Yoshino, Jun; Luecking, Courtney; Kirbach, Kyleigh; Kelly, Shannon C; de Las Fuentes, Lisa; He, Songbing; Okunade, Adewole L; Patterson, Bruce W; Klein, Samuel

    2016-04-12

    Although 5%-10% weight loss is routinely recommended for people with obesity, the precise effects of 5% and further weight loss on metabolic health are unclear. We conducted a randomized controlled trial that evaluated the effects of 5.1% ± 0.9% (n = 19), 10.8% ± 1.3% (n = 9), and 16.4% ± 2.1% (n = 9) weight loss and weight maintenance (n = 14) on metabolic outcomes. 5% weight loss improved adipose tissue, liver and muscle insulin sensitivity, and β cell function, without a concomitant change in systemic or subcutaneous adipose tissue markers of inflammation. Additional weight loss further improved β cell function and insulin sensitivity in muscle and caused stepwise changes in adipose tissue mass, intrahepatic triglyceride content, and adipose tissue expression of genes involved in cholesterol flux, lipid synthesis, extracellular matrix remodeling, and oxidative stress. These results demonstrate that moderate 5% weight loss improves metabolic function in multiple organs simultaneously, and progressive weight loss causes dose-dependent alterations in key adipose tissue biological pathways. PMID:26916363

  10. Chromosomal Microarray versus Karyotyping for Prenatal Diagnosis

    PubMed Central

    Wapner, Ronald J.; Martin, Christa Lese; Levy, Brynn; Ballif, Blake C.; Eng, Christine M.; Zachary, Julia M.; Savage, Melissa; Platt, Lawrence D.; Saltzman, Daniel; Grobman, William A.; Klugman, Susan; Scholl, Thomas; Simpson, Joe Leigh; McCall, Kimberly; Aggarwal, Vimla S.; Bunke, Brian; Nahum, Odelia; Patel, Ankita; Lamb, Allen N.; Thom, Elizabeth A.; Beaudet, Arthur L.; Ledbetter, David H.; Shaffer, Lisa G.; Jackson, Laird

    2013-01-01

    Background Chromosomal microarray analysis has emerged as a primary diagnostic tool for the evaluation of developmental delay and structural malformations in children. We aimed to evaluate the accuracy, efficacy, and incremental yield of chromosomal microarray analysis as compared with karyotyping for routine prenatal diagnosis. Methods Samples from women undergoing prenatal diagnosis at 29 centers were sent to a central karyotyping laboratory. Each sample was split in two; standard karyotyping was performed on one portion and the other was sent to one of four laboratories for chromosomal microarray. Results We enrolled a total of 4406 women. Indications for prenatal diagnosis were advanced maternal age (46.6%), abnormal result on Down’s syndrome screening (18.8%), structural anomalies on ultrasonography (25.2%), and other indications (9.4%). In 4340 (98.8%) of the fetal samples, microarray analysis was successful; 87.9% of samples could be used without tissue culture. Microarray analysis of the 4282 nonmosaic samples identified all the aneuploidies and unbalanced rearrangements identified on karyotyping but did not identify balanced translocations and fetal triploidy. In samples with a normal karyotype, microarray analysis revealed clinically relevant deletions or duplications in 6.0% with a structural anomaly and in 1.7% of those whose indications were advanced maternal age or positive screening results. Conclusions In the context of prenatal diagnostic testing, chromosomal microarray analysis identified additional, clinically significant cytogenetic information as compared with karyotyping and was equally efficacious in identifying aneuploidies and unbalanced rearrangements but did not identify balanced translocations and triploidies. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and others; ClinicalTrials.gov number, NCT01279733.) PMID:23215555

  11. Applications of Functional Protein Microarrays in Basic and Clinical Research

    PubMed Central

    Zhu, Heng; Qian, Jiang

    2013-01-01

    The protein microarray technology provides a versatile platform for characterization of hundreds of thousands of proteins in a highly parallel and high-throughput manner. It is viewed as a new tool that overcomes the limitation of DNA microarrays. On the basis of its application, protein microarrays fall into two major classes: analytical and functional protein microarrays. In addition, tissue or cell lysates can also be directly spotted on a slide to form the so-called reverse-phase protein microarray. In the last decade, applications of functional protein microarrays in particular have flourished in studying protein function and construction of networks and pathways. In this chapter, we will review the recent advancements in the protein microarray technology, followed by presenting a series of examples to illustrate the power and versatility of protein microarrays in both basic and clinical research. As a powerful technology platform, it would not be surprising if protein microarrays will become one of the leading technologies in proteomic and diagnostic fields in the next decade. PMID:22989767

  12. [The application progress of human urine derived stem cells in bone tissue engineering].

    PubMed

    Gao, P; Jiang, D P; Li, Z Z

    2016-04-01

    The research of bone tissue engineering bases on three basic directions of seed cells, scaffold materials and growth information. Stem cells have been widely studied as seed cells. Human urine-derived stem cell(hUSC) is extracted from urine and described to be adhesion growth, cloning, expression of the majority of mesenchymal stem cell markers and peripheral cell markers, multi-potential and no tumor but stable karyotype with passaging many times. Some researches proposed that hUSC might be a new source of seed cells in tissue engineering because of their invasive and convenient obtention, stable culture and multiple differentiation potential. PMID:27029208

  13. [Research progress on real-time deformable models of soft tissues for surgery simulation].

    PubMed

    Xu, Shaoping; Liu, Xiaoping; Zhang, Hua; Luo, Jie

    2010-04-01

    Biological tissues generally exhibit nonlinearity, anisotropy, quasi-incompressibility and viscoelasticity about material properties. Simulating the behaviour of elastic objects in real time is one of the current objectives of virtual surgery simulation which is still a challenge for researchers to accurately depict the behaviour of human tissues. In this paper, we present a classification of the different deformable models that have been developed. We present the advantages and disadvantages of each one. Finally, we make a comparison of deformable models and perform an evaluation of the state of the art and the future of deformable models. PMID:20481334

  14. Disease progression in iridocorneal angle tissues of BMP2-induced ocular hypertensive mice with optical coherence tomography

    PubMed Central

    Li, Guorong; Farsiu, Sina; Qiu, Jianming; Dixon, Angela; Song, Chunwei; McKinnon, Stuart J.; Yuan, Fan; Gonzalez, Pedro

    2014-01-01

    Purpose The goal of the present study was to test for the first time whether glaucomatous-like disease progression in a mouse can be assessed morphologically and functionally with spectral domain optical coherence tomography (SD-OCT). Methods We monitored progressive changes in conventional outflow tissues of living mice overexpressing human bone morphogenetic protein 2 (BMP2), a model for glaucoma. Intraocular pressure (IOP) and outflow tissue morphology/Young's modulus were followed in mice for 36 days with rebound tonometry and SD-OCT, respectively. Results were compared to standard histological methods. Outflow facility was calculated from flow measurements with direct cannulation of anterior chambers subjected to three sequential pressure steps. Results Overexpression of BMP2 significantly elevated IOP in a biphasic manner over time compared to mice that overexpressed green fluorescent protein in outflow cells and naïve controls. SD-OCT revealed changes in outflow tissues overexpressing BMP2 that corresponded with the timing of the IOP phases and decreased outflow facility. In the first phase, the angle was open, but the trabecular meshwork and the cornea were thickened. OCT detected increased trabecular meshwork stiffness after provocative IOP challenges of the BMP2 eyes, which corresponded to increased collagen deposition with transmission electron microscopy. In contrast, the angle was closed in the second phase. IOP elevation over 36 days due to BMP2 overexpression resulted in significant retinal ganglion cell and axon loss. Conclusions Although not a feasible open-angle glaucoma model, the BMP2 mice were useful for demonstrating the utility of SD-OCT in following disease progression and differentiating between two forms of ocular pathology over time that resulted in ocular hypertension. PMID:25558173

  15. The changes in various hydroxyproline fractions in aortic tissue of rabbits are closely related to the progression of atherosclerosis

    PubMed Central

    2010-01-01

    Background The most important function of collagen and elastin is to induce several mechanical parameters which are known to play a dominant role in governing mechanical properties of the blood vessels. The aortic tissue of rabbit is one of the important sources of collagen and elastin. The effects of high fat diet (HFD) on the hydroxyproline (Hyp) fractions in serum and aortic tissues of rabbits and collagen content in the aortic tissues of rabbits have not been documented before. The present study was undertaken to investigate the changes in Hyp fractions in serum and aortic tissues of rabbits and collagen content in the aortic tissues of rabbits during the progression of atherosclerosis. The atherosclerotic model used in this study was the New Zealand white rabbit (male; 12 weeks old). Twenty five rabbits were individually caged, and divided into control group (NOR; n = 10) and HFD group (CHO; n = 15). The control group was fed (100 g/day) of normal (NOR) diet for a period of 15 weeks. The HFD group was fed normal diet supplemented with 1.0% cholesterol plus 1.0% olive oil (100 g/day) for the same period of time. Results We found that the TC, LDLC, and TG (mg/dl) were significantly (p < 0.001) increased in HFD rabbits compared with control rabbits with percentage normalized changes of 1198%, 1591%, and 710%, respectively. The peptide-bound Hyp in the serum was significantly (P < 0.05) increased in HFD rabbits compared with control rabbits with percentage normalized change of 517% while it significantly (P < 0.01) decreased in aortic tissues of HFD rabbits compared with control rabbits with percentage normalized change of 65%. The protein-bound Hyp in the serum was significantly (P < 0.01) increased in HFD rabbits compared with control rabbits with percentage normalized change of 100%; the protein-bound Hyp in the aortic tissues of control rabbits was 235.30 ± 55.14 (Mean ± SD) while it was not detectable (ND) in HFD rabbits. Total serum Hyp showed no significant (P < 0.05) change in HFD rabbits compared with control rabbits while it was significantly (P < 0.05) decreased in aortic tissues of HFD rabbits compared with control rabbits with percentage normalized change of 73%. The total collagen was significantly (p < 0.01) decreased in aortic tissues of HFD rabbits compared with control rabbits with percentage normalized change of 73% which was supported by histological study. Conclusions These results suggest that percentage decrease in various Hyp fractions in aortic tissue of HFD rabbits are closely related to percentage decrease of collagen content in aortic tissues of HFD rabbits. These results also suggest that it may be possible to use the changes in various Hyp fractions in aortic tissues of rabbits as an important risk factor during the progression of atherosclerosis. PMID:20214825

  16. Progress toward automatic classification of human brown adipose tissue using biomedical imaging

    NASA Astrophysics Data System (ADS)

    Gifford, Aliya; Towse, Theodore F.; Walker, Ronald C.; Avison, Malcom J.; Welch, E. B.

    2015-03-01

    Brown adipose tissue (BAT) is a small but significant tissue, which may play an important role in obesity and the pathogenesis of metabolic syndrome. Interest in studying BAT in adult humans is increasing, but in order to quantify BAT volume in a single measurement or to detect changes in BAT over the time course of a longitudinal experiment, BAT needs to first be reliably differentiated from surrounding tissue. Although the uptake of the radiotracer 18F-Fluorodeoxyglucose (18F-FDG) in adipose tissue on positron emission tomography (PET) scans following cold exposure is accepted as an indication of BAT, it is not a definitive indicator, and to date there exists no standardized method for segmenting BAT. Consequently, there is a strong need for robust automatic classification of BAT based on properties measured with biomedical imaging. In this study we begin the process of developing an automated segmentation method based on properties obtained from fat-water MRI and PET-CT scans acquired on ten healthy adult subjects.

  17. Chromatin immunoprecipitation using microarrays.

    PubMed

    Durand-Dubief, Mickaël; Ekwall, Karl

    2009-01-01

    Chromatin immunoprecipitation (ChIP) is a powerful procedure to investigate the interactions between proteins and DNA. ChIP-chip combines chromatin immunoprecipitation and DNA microarray analysis to identify protein-DNA interactions that occur in vivo. This genome-wide analysis of protein-DNA association is carried out in several steps including chemical cross-linking, cell lysis, DNA fragmentation and immunoaffinity purification that allow the identification of DNA interactions and provide a powerful tool for genome-wide investigations. Immunoprecipitated DNA fragments associated with the desired protein are amplified, labelled and hybridized to DNA microarrays to detect enriched signals compared to a labelled reference sample. PMID:19381973

  18. Microarray analysis of laser-microdissected tissues indicates the biosynthesis of suberin in the outer part of roots during formation of a barrier to radial oxygen loss in rice (Oryza sativa).

    PubMed

    Shiono, Katsuhiro; Yamauchi, Takaki; Yamazaki, So; Mohanty, Bijayalaxmi; Malik, Al Imran; Nagamura, Yoshiaki; Nishizawa, Naoko K; Tsutsumi, Nobuhiro; Colmer, Timothy D; Nakazono, Mikio

    2014-09-01

    Internal aeration is crucial for root growth in waterlogged soil. A barrier to radial oxygen loss (ROL) can enhance long-distance oxygen transport via the aerenchyma to the root tip; a higher oxygen concentration at the apex enables root growth into anoxic soil. The ROL barrier is formed within the outer part of roots (OPR). Suberin and/or lignin deposited in cell walls are thought to contribute to the barrier, but it is unclear which compound is the main constituent. This study describes gene expression profiles during ROL barrier formation in rice roots to determine the relative responses of suberin and/or lignin biosyntheses for the barrier. OPR tissues were isolated by laser microdissection and their transcripts were analysed by microarray. A total of 128 genes were significantly up- or downregulated in the OPR during the barrier formation. Genes associated with suberin biosynthesis were strongly upregulated, whereas genes associated with lignin biosynthesis were not. By an ab initio analysis of the promoters of the upregulated genes, the putative cis-elements that could be associated with transcription factors, WRKY, AP2/ERF, NAC, bZIP, MYB, CBT/DREB, and MADS, were elucidated. They were particularly associated with the expression of transcription factor genes containing WRKY, AP2, and MYB domains. A semiquantitative reverse-transcription PCR analysis of genes associated with suberin biosynthesis (WRKY, CYP, and GPAT) confirmed that they were highly expressed during ROL barrier formation. Overall, these results suggest that suberin is a major constituent of the ROL barrier in roots of rice. PMID:24913626

  19. Inflammation and adipose tissue: effects of progressive load training in rats

    PubMed Central

    2010-01-01

    Introduction Cytokines (IL-6, IL-10 and TNF-α) are increased after exhaustive exercise in the rat retroperitoneal (RPAT) and mesenteric adipose tissue (MEAT) pads. On the other hand, these cytokines show decreased expression in these depots in response to a chronic exercise protocol. However, the effect of exercise with overload combined with a short recovery period on pro- and anti-inflammatory cytokine expression is unknown. In the present study, we investigated the regulation of cytokine production in the adipose tissue of rats after an overtraining-inducing exercise protocol. Methods Male Wistar rats were divided into four groups: Control (C), Trained (Tr), Overtrained (OT) and recovered overtrained (R). Cytokines (IL-6, TNF-α and IL-10) levels and Toll Like Receptor 4 (TLR4), Nuclear Factor kBp65 (NF-kBp65), Hormone Sensitive Lipase (HSL) and, Perilipin protein expression were assessed in the adipose tissue. Furthermore, we analysed plasma lipid profile, insulin, testosterone, corticosterone and endotoxin levels, and liver triacylglycerol, cytokine content, as well as apolipoprotein B (apoB) and TLR4 expression in the liver. Results OT and R groups exhibited reduced performance accompanied by lower testosterone and increased corticosterone and endotoxin levels when compared with the control and trained groups. IL-6 and IL-10 protein levels were increased in the adipose tissue of the group allowed to recover, in comparison with all the other studied groups. TLR-4 and NF-kBp65 were increased in this same group when compared with both control and trained groups. The protein expression of HSL was increased and that of Perilipin, decreased in the adipose in R in relation to the control. In addition, we found increased liver and serum TAG, along with reduced apoB protein expression and IL-6 and IL-10 levels in the of R in relation to the control and trained groups. Conclusion In conclusion, we have shown that increases in pro-inflammatory cytokines in the adipose tissue after an overtraining protocol may be mediated via TLR-4 and NF-kBp65 signalling, leading to an inflammatory state in this tissue. PMID:20920329

  20. Evolution of normal and neoplastic tissue stem cells: progress after Robert Hooke.

    PubMed

    Weissman, Irving

    2015-10-19

    The appearance of stem cells coincides with the transition from single-celled organisms to metazoans. Stem cells are capable of self-renewal as well as differentiation. Each tissue is maintained by self-renewing tissue-specific stem cells. The accumulation of mutations that lead to preleukaemia are in the blood-forming stem cell, while the transition to leukaemia stem cells occurs in the clone at a progenitor stage. All leukaemia and cancer cells escape being removed by scavenger macrophages by expressing the 'don't eat me' signal CD47. Blocking antibodies to CD47 are therapeutics for all cancers, and are currently being tested in clinical trials in the US and UK. PMID:26416675

  1. LDRD Progress Report: Radioimmunotherapy using oxide nanoparticles: Radionuclide contaiment and mitigation of normal tissue toxicity.

    SciTech Connect

    Rondinone, Adam Justin; Dai, Sheng; Mirzadeh, Saed; Kennel, Steve J

    2005-10-01

    Radionuclides with specific emission properties can be incorporated into metal-chalcogenide and metal-oxide nanoparticles. Coupled to antibodies, these conjugates could be injected into the bloodstream to target and destroy non-solid tumors or target organs for radioimaging. In the first year of this project, two types of radioactive nanoparticles, CdTe: {sup 125m}Te and Y{sub 2}O{sub 3}: {sup 170}Tm were synthesized and coupled to antibodies specific to murine epithelial lung tissue. The nanoparticles successfully target the lung tissue in vivo. Some leaching of the radioisotope was observed. The coming year will explore other types of nanoparticles (other crystal chemistries) in order to minimize leaching.

  2. Hybridization and Selective Release of DNA Microarrays

    SciTech Connect

    Beer, N R; Baker, B; Piggott, T; Maberry, S; Hara, C M; DeOtte, J; Benett, W; Mukerjee, E; Dzenitis, J; Wheeler, E K

    2011-11-29

    DNA microarrays contain sequence specific probes arrayed in distinct spots numbering from 10,000 to over 1,000,000, depending on the platform. This tremendous degree of multiplexing gives microarrays great potential for environmental background sampling, broad-spectrum clinical monitoring, and continuous biological threat detection. In practice, their use in these applications is not common due to limited information content, long processing times, and high cost. The work focused on characterizing the phenomena of microarray hybridization and selective release that will allow these limitations to be addressed. This will revolutionize the ways that microarrays can be used for LLNL's Global Security missions. The goals of this project were two-fold: automated faster hybridizations and selective release of hybridized features. The first study area involves hybridization kinetics and mass-transfer effects. the standard hybridization protocol uses an overnight incubation to achieve the best possible signal for any sample type, as well as for convenience in manual processing. There is potential to significantly shorten this time based on better understanding and control of the rate-limiting processes and knowledge of the progress of the hybridization. In the hybridization work, a custom microarray flow cell was used to manipulate the chemical and thermal environment of the array and autonomously image the changes over time during hybridization. The second study area is selective release. Microarrays easily generate hybridization patterns and signatures, but there is still an unmet need for methodologies enabling rapid and selective analysis of these patterns and signatures. Detailed analysis of individual spots by subsequent sequencing could potentially yield significant information for rapidly mutating and emerging (or deliberately engineered) pathogens. In the selective release work, optical energy deposition with coherent light quickly provides the thermal energy to single spots to release hybridized DNA. This work leverages LLNL expertise in optics, microfluids, and bioinformatics.

  3. JC Polyomavirus Abundance and Distribution in Progressive Multifocal Leukoencephalopathy (PML) Brain Tissue Implicates Myelin Sheath in Intracerebral Dissemination of Infection

    PubMed Central

    Wharton, Keith A.; Quigley, Catherine; Themeles, Marian; Dunstan, Robert W.; Doyle, Kathryn; Cahir-McFarland, Ellen; Wei, Jing; Buko, Alex; Reid, Carl E.; Sun, Chao; Carmillo, Paul; Sur, Gargi; Carulli, John P.; Mansfield, Keith G.; Westmoreland, Susan V.; Staugaitis, Susan M.; Fox, Robert J.; Meier, Werner; Goelz, Susan E.

    2016-01-01

    Over half of adults are seropositive for JC polyomavirus (JCV), but rare individuals develop progressive multifocal leukoencephalopathy (PML), a demyelinating JCV infection of the central nervous system. Previously, PML was primarily seen in immunosuppressed patients with AIDS or certain cancers, but it has recently emerged as a drug safety issue through its association with diverse immunomodulatory therapies. To better understand the relationship between the JCV life cycle and PML pathology, we studied autopsy brain tissue from a 70-year-old psoriasis patient on the integrin alpha-L inhibitor efalizumab following a ~2 month clinical course of PML. Sequence analysis of lesional brain tissue identified PML-associated viral mutations in regulatory (non-coding control region) DNA, capsid protein VP1, and the regulatory agnoprotein, as well as 9 novel mutations in capsid protein VP2, indicating rampant viral evolution. Nine samples, including three gross PML lesions and normal-appearing adjacent tissues, were characterized by histopathology and subject to quantitative genomic, proteomic, and molecular localization analyses. We observed a striking correlation between the spatial extent of demyelination, axonal destruction, and dispersion of JCV along white matter myelin sheath. Our observations in this case, as well as in a case of PML-like disease in an immunocompromised rhesus macaque, suggest that long-range spread of polyomavirus and axonal destruction in PML might involve extracellular association between virus and the white matter myelin sheath. PMID:27191595

  4. Appearance of paired nucleated, Tau-positive glia in patients with progressive supranuclear palsy brain tissue.

    PubMed

    Yamada, T; McGeer, P L; McGeer, E G

    1992-01-20

    Many Tau-positive glia with paired nuclei and astrocyte type morphology were identified in three brains from patients with progressive supranuclear palsy (PSP). They were positive by Bielschowsky's and Bodian's silver staining as well as by immunostaining with Tau-2, Alz-50, anti-GFAP and anti-paired helical filament antibodies, but not with anti-ubiquitin antibody. They were predominantly localized in the striatum, thalamus and frontal cortex but were not seen in white matter and were not plentiful in areas of heavy neuronal degeneration. Electron microscopy clearly showed the nuclear pairing and localized the Tau protein to bundles suggestive of microtubules in the cytoplasm and proximal processes. Such glial cells were rarely seen in cases of other neurodegenerative diseases or neurologically normal controls. These data suggest that there is an unusual gliotic reaction in PSP in brain areas which show relatively little neuronal loss. PMID:1371861

  5. Microarrays under the microscope

    PubMed Central

    Wildsmith, S E; Elcock, F J

    2001-01-01

    Microarray technology is a rapidly advancing area, which is gaining popularity in many biological disciplines from drug target identification to predictive toxicology. Over the past few years, there has been a dramatic increase in the number of methods and techniques available for carrying out this form of gene expression analysis. The techniques and associated peripherals, such as slide types, deposition methods, robotics, and scanning equipment, are undergoing constant improvement, helping to drive the technology forward in terms of robustness and ease of use. These rapid developments, combined with the number of options available and the associated hyperbole, can prove daunting for the new user. This review aims to guide the researcher through the various steps of conducting microarray experiments, from initial strategy to analysing the data, with critical examination of the benefits and disadvantages along the way. PMID:11212888

  6. Microarray data analysis.

    PubMed

    Mohapatra, Saroj K; Krishnan, Arjun

    2011-01-01

    Gene expression profiling has revolutionized functional genomics research by providing a quick handle on all the transcriptional changes that occur in the cell in response to internal or external perturbations or developmental programs. Microarrays have become the most popular technology for recording gene expression profiles. This chapter describes all the necessary steps for analyzing Affymetrix microarray data using the open-source statistical tools (R and bioconductor). The reader is walked through all the basic steps of data analysis: reading raw data, assessing quality, preprocessing/normalization, discovery of differentially expressed genes, comparison of gene lists, functional enrichment analysis, and saving results to files for future reference. Some familiarity with computer is assumed. This chapter is self-contained with installation instructions for R and bioconductor packages along with links to downloadable data and code for reproducing the examples. PMID:20931370

  7. Gene microarray analysis of lncRNA and mRNA expression profiles in patients with hypopharyngeal squamous cell carcinoma

    PubMed Central

    Zhou, Jieyu; Li, Wenming; Jin, Tong; Xiang, Xuan; Li, Maocai; Wang, Juan; Li, Guojun; Pan, Xinliang; Lei, Dapeng

    2015-01-01

    Background: Studies have shown that long noncoding RNAs (lncRNAs) are involved in the development and progression of many types of cancer. However, the mechanisms by which lncRNAs influence development and progression of hypopharyngeal squamous cell carcinoma (HSCC) are unclear. Method: We investigated differences in lncRNA and mRNA expression profiles between 3 pairs of HSCC tissues and adjacent nontumor tissues by microarray analysis. Results: In HSCC tissues, 1299 lncRNAs were significantly upregulated (n=669) or downregulated (n=630) compared to levels in adjacent nontumor tissues. Moreover, 1432 mRNAs were significantly upregulated (n=684) or downregulated (n=748) in HSCC tissues. We randomly selected 2 differentially expressed lncRNAs (AB209630, AB019562) and 2 differentially expressed mRNAs (SPP1, TJP2) for confirmation of microarray results using qRT-PCR. The qRT-PCR results matched well with the microarray data. The differentially expressed lncRNAs and mRNAs were distributed on each of the chromosomes, including the X and Y chromosomes. Pathway analysis indicated that the biological functions of differentially expressed mRNAs were related to 48 cellular pathways that may be associated with HSCC development. GO analysis revealed that 593 mRNAs involved in biological processes, 50 mRNAs involved in cellular components, and 46 mRNAs involved in molecular functions were upregulated in the carcinomas; 280 mRNAs involved in biological processes, 58 mRNAs involved in cellular components, and 71 mRNAs involved in molecular functions were downregulated in the carcinomas. In addition, 8 enhancer-like lncRNAs and 21 intergenic lncRNAs with their adjacent mRNA pairs were identified as coregulated transcripts. Conclusion: These findings provide insight into the mechanisms underlying HSCC tumorigenesis and will facilitate identification of new therapeutic targets and diagnostic biomarkers for this disease. PMID:26131061

  8. Tissue Array Research Program (TARP)

    Cancer.gov

    Multi-Tumor Tissue Microarrays A novel tool for high- throughput molecular profiling of tumor tissues Arrays Are Currently Available. Array Details To Order Slides Intramural Ordering Information: NCI/NIH personnel may directly contact Stephen M. Hewitt,

  9. Surface chemistries for antibody microarrays

    SciTech Connect

    Seurynck-Servoss, Shannon L.; Baird, Cheryl L.; Rodland, Karin D.; Zangar, Richard C.

    2007-05-01

    Enzyme-linked immunosorbent assay (ELISA) microarrays promise to be a powerful tool for the detection of disease biomarkers. The original technology for printing ELISA microarray chips and capturing antibodies on slides was derived from the DNA microarray field. However, due to the need to maintain antibody structure and function when immobilized, surface chemistries used for DNA microarrays are not always appropriate for ELISA microarrays. In order to identify better surface chemistries for antibody capture, a number of commercial companies and academic research groups have developed new slide types that could improve antibody function in microarray applications. In this review we compare and contrast the commercially available slide chemistries, as well as highlight some promising recent advances in the field.

  10. Tiling Microarray Analysis Tools

    SciTech Connect

    2005-05-04

    TiMAT is a package of 23 command line Java applications for use in the analysis of Affymetrix tiled genomic microarray data. TiMAT enables: 1) Rebuilding the genome annotation for entire tiled arrays (repeat filtering, chromosomal coordinate assignment). 2) Post processing of oligo intensity values (quantile normalization, median scaling, PMMM transformation), 3) Significance testing (Wilcoxon rank sum and signed rank tests, intensity difference and ratio tests) and Interval refinement (filtering based on multiple statistics, overlap comparisons), 4) Data visualization (detailed thumbnail/zoomed view with Interval Plots and data export to Affymetrix's Integrated Genome Browser) and Data reports (spreadsheet summaries and detailed profiles)

  11. Bio-microarray fabrication techniques--a review.

    PubMed

    Barbulovic-Nad, Irena; Lucente, Michael; Sun, Yu; Zhang, Mingjun; Wheeler, Aaron R; Bussmann, Markus

    2006-01-01

    Microarrays with biomolecules (e.g., DNA and proteins), cells, and tissues immobilized on solid substrates are important tools for biological research, including genomics, proteomics, and cell analysis. In this paper, the current state of microarray fabrication is reviewed. According to spot formation techniques, methods are categorized as "contact printing" and "non-contact printing." Contact printing is a widely used technology, comprising methods such as contact pin printing and microstamping. These methods have many advantages, including reproducibility of printed spots and facile maintenance, as well as drawbacks, including low-throughput fabrication of arrays. Non-contact printing techniques are newer and more varied, comprising photochemistry-based methods, laser writing, electrospray deposition, and inkjet technologies. These technologies emerged from other applications and have the potential to increase microarray fabrication throughput; however, there are several challenges in applying them to microarray fabrication, including interference from satellite drops and biomolecule denaturization. PMID:17095434

  12. Ecotoxicogenomics: Microarray interlaboratory comparability.

    PubMed

    Vidal-Dorsch, Doris E; Bay, Steven M; Moore, Shelly; Layton, Blythe; Mehinto, Alvine C; Vulpe, Chris D; Brown-Augustine, Marianna; Loguinov, Alex; Poynton, Helen; Garcia-Reyero, Natàlia; Perkins, Edward J; Escalon, Lynn; Denslow, Nancy D; Cristina, Colli-Dula R; Doan, Tri; Shukradas, Shweta; Bruno, Joy; Brown, Lorraine; Van Agglen, Graham; Jackman, Paula; Bauer, Megan

    2016-02-01

    Transcriptomic analysis can complement traditional ecotoxicology data by providing mechanistic insight, and by identifying sub-lethal organismal responses and contaminant classes underlying observed toxicity. Before transcriptomic information can be used in monitoring and risk assessment, it is necessary to determine its reproducibility and detect key steps impacting the reliable identification of differentially expressed genes. A custom 15K-probe microarray was used to conduct transcriptomics analyses across six laboratories with estuarine amphipods exposed to cyfluthrin-spiked or control sediments (10 days). Two sample types were generated, one consisted of total RNA extracts (Ex) from exposed and control samples (extracted by one laboratory) and the other consisted of exposed and control whole body amphipods (WB) from which each laboratory extracted RNA. Our findings indicate that gene expression microarray results are repeatable. Differentially expressed data had a higher degree of repeatability across all laboratories in samples with similar RNA quality (Ex) when compared to WB samples with more variable RNA quality. Despite such variability a subset of genes were consistently identified as differentially expressed across all laboratories and sample types. We found that the differences among the individual laboratory results can be attributed to several factors including RNA quality and technical expertise, but the overall results can be improved by following consistent protocols and with appropriate training. PMID:26363320

  13. The Genopolis Microarray Database

    PubMed Central

    Splendiani, Andrea; Brandizi, Marco; Even, Gael; Beretta, Ottavio; Pavelka, Norman; Pelizzola, Mattia; Mayhaus, Manuel; Foti, Maria; Mauri, Giancarlo; Ricciardi-Castagnoli, Paola

    2007-01-01

    Background Gene expression databases are key resources for microarray data management and analysis and the importance of a proper annotation of their content is well understood. Public repositories as well as microarray database systems that can be implemented by single laboratories exist. However, there is not yet a tool that can easily support a collaborative environment where different users with different rights of access to data can interact to define a common highly coherent content. The scope of the Genopolis database is to provide a resource that allows different groups performing microarray experiments related to a common subject to create a common coherent knowledge base and to analyse it. The Genopolis database has been implemented as a dedicated system for the scientific community studying dendritic and macrophage cells functions and host-parasite interactions. Results The Genopolis Database system allows the community to build an object based MIAME compliant annotation of their experiments and to store images, raw and processed data from the Affymetrix GeneChip® platform. It supports dynamical definition of controlled vocabularies and provides automated and supervised steps to control the coherence of data and annotations. It allows a precise control of the visibility of the database content to different sub groups in the community and facilitates exports of its content to public repositories. It provides an interactive users interface for data analysis: this allows users to visualize data matrices based on functional lists and sample characterization, and to navigate to other data matrices defined by similarity of expression values as well as functional characterizations of genes involved. A collaborative environment is also provided for the definition and sharing of functional annotation by users. Conclusion The Genopolis Database supports a community in building a common coherent knowledge base and analyse it. This fills a gap between a local database and a public repository, where the development of a common coherent annotation is important. In its current implementation, it provides a uniform coherently annotated dataset on dendritic cells and macrophage differentiation. PMID:17430566

  14. Living-cell microarrays.

    PubMed

    Yarmush, Martin L; King, Kevin R

    2009-01-01

    Living cells are remarkably complex. To unravel this complexity, living-cell assays have been developed that allow delivery of experimental stimuli and measurement of the resulting cellular responses. High-throughput adaptations of these assays, known as living-cell microarrays, which are based on microtiter plates, high-density spotting, microfabrication, and microfluidics technologies, are being developed for two general applications: (a) to screen large-scale chemical and genomic libraries and (b) to systematically investigate the local cellular microenvironment. These emerging experimental platforms offer exciting opportunities to rapidly identify genetic determinants of disease, to discover modulators of cellular function, and to probe the complex and dynamic relationships between cells and their local environment. PMID:19413510

  15. Tiling Microarray Analysis Tools

    Energy Science and Technology Software Center (ESTSC)

    2005-05-04

    TiMAT is a package of 23 command line Java applications for use in the analysis of Affymetrix tiled genomic microarray data. TiMAT enables: 1) Rebuilding the genome annotation for entire tiled arrays (repeat filtering, chromosomal coordinate assignment). 2) Post processing of oligo intensity values (quantile normalization, median scaling, PMMM transformation), 3) Significance testing (Wilcoxon rank sum and signed rank tests, intensity difference and ratio tests) and Interval refinement (filtering based on multiple statistics, overlap comparisons),more » 4) Data visualization (detailed thumbnail/zoomed view with Interval Plots and data export to Affymetrix's Integrated Genome Browser) and Data reports (spreadsheet summaries and detailed profiles)« less

  16. Application of ESTs in microarray analysis.

    PubMed

    Li, Weizhong; Olohan, Lisa; Williams, Daryl; Hughes, Margaret; Gracey, Andrew; Cossins, Andrew

    2009-01-01

    Microarray analyses provide information on the relative expression levels of large numbers of gene products (transcripts). As such they have been widely used to examine differences in gene expression across a variety of samples such as tissues and life-cycle stages. Due to a previous lack of sequence data, microarray analyses have typically centred on the study of well-characterised model organisms. However, the recent availability of large sets of expressed sequence tags (ESTs) generated for the purpose of gene discovery offers the opportunity to consider designing and applying microarray technology to a larger and more diverse set of species. Here we outline the array-design process involving the generation of an optimised set of oligoprobes from a minimally redundant but maximally representative list of sequences from raw EST data. We illustrate these principles by showing how we designed and fabricated a high-density oligoarray for the rainbow trout, a non-model species for which large numbers of ESTs, and a non-redundant assembly is available. This approach brings array technology within the reach of all investigators, even those with limited budgets. PMID:19277566

  17. MICROARRAY ANALYSIS IDENTIFIES GENES INVOLVED IN CROWN BUD DORMANCY IN LEAFY SPURGE (EUPHORBIA ESULA L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leafy spurge is a perennial rangeland weed that has become a model for weed genomics and cross-species research. Microarray analysis allows the simultaneous characterization of the expression from thousands of different genes from any given sampled tissue. We have used microarray analysis to follow ...

  18. Quantifying antibody binding on protein microarrays using microarray nonlinear calibration.

    PubMed

    Yu, Xiaobo; Wallstrom, Garrick; Magee, Dewey Mitchell; Qiu, Ji; Mendoza, D Eliseo A; Wang, Jie; Bian, Xiaofang; Graves, Morgan; LaBaer, Joshua

    2013-05-01

    We present a microarray nonlinear calibration (MiNC) method for quantifying antibody binding to the surface of protein microarrays that significantly increases the linear dynamic range and reduces assay variation compared with traditional approaches. A serological analysis of guinea pig Mycobacterium tuberculosis models showed that a larger number of putative antigen targets were identified with MiNC, which is consistent with the improved assay performance of protein microarrays. MiNC has the potential to be employed in biomedical research using multiplex antibody assays that need quantitation, including the discovery of antibody biomarkers, clinical diagnostics with multi-antibody signatures, and construction of immune mathematical models. PMID:23662896

  19. Basic Concepts of Microarrays and Potential Applications in Clinical Microbiology

    PubMed Central

    Miller, Melissa B.; Tang, Yi-Wei

    2009-01-01

    Summary: The introduction of in vitro nucleic acid amplification techniques, led by real-time PCR, into the clinical microbiology laboratory has transformed the laboratory detection of viruses and select bacterial pathogens. However, the progression of the molecular diagnostic revolution currently relies on the ability to efficiently and accurately offer multiplex detection and characterization for a variety of infectious disease pathogens. Microarray analysis has the capability to offer robust multiplex detection but has just started to enter the diagnostic microbiology laboratory. Multiple microarray platforms exist, including printed double-stranded DNA and oligonucleotide arrays, in situ-synthesized arrays, high-density bead arrays, electronic microarrays, and suspension bead arrays. One aim of this paper is to review microarray technology, highlighting technical differences between them and each platform's advantages and disadvantages. Although the use of microarrays to generate gene expression data has become routine, applications pertinent to clinical microbiology continue to rapidly expand. This review highlights uses of microarray technology that impact diagnostic microbiology, including the detection and identification of pathogens, determination of antimicrobial resistance, epidemiological strain typing, and analysis of microbial infections using host genomic expression and polymorphism profiles. PMID:19822891

  20. Microarray platform for omics analysis

    NASA Astrophysics Data System (ADS)

    Mecklenburg, Michael; Xie, Bin

    2001-09-01

    Microarray technology has revolutionized genetic analysis. However, limitations in genome analysis has lead to renewed interest in establishing 'omic' strategies. As we enter the post-genomic era, new microarray technologies are needed to address these new classes of 'omic' targets, such as proteins, as well as lipids and carbohydrates. We have developed a microarray platform that combines self- assembling monolayers with the biotin-streptavidin system to provide a robust, versatile immobilization scheme. A hydrophobic film is patterned on the surface creating an array of tension wells that eliminates evaporation effects thereby reducing the shear stress to which biomolecules are exposed to during immobilization. The streptavidin linker layer makes it possible to adapt and/or develop microarray based assays using virtually any class of biomolecules including: carbohydrates, peptides, antibodies, receptors, as well as them ore traditional DNA based arrays. Our microarray technology is designed to furnish seamless compatibility across the various 'omic' platforms by providing a common blueprint for fabricating and analyzing arrays. The prototype microarray uses a microscope slide footprint patterned with 2 by 96 flat wells. Data on the microarray platform will be presented.

  1. Wavelet and multi-fractal based analysis on DIC images in epithelium region to detect and diagnose the cancer progress among different grades of tissues

    NASA Astrophysics Data System (ADS)

    Mukhopadhyay, Sabyasachi; Das, Nandan K.; Pradhan, Asima; Ghosh, Nirmalya; Panigrahi, Prasanta K.

    2014-05-01

    DIC (Differential Interference Contrast Image) images of cervical pre-cancer tissues are taken from epithelium region, on which wavelet transform and multi-fractal analysis are applied. Discrete wavelet transform (DWT) through Daubechies basis are done for identifying fluctuations over polynomial trends for clear characterization and differentiation of tissues. A systematic investigation of denoised images is carried out through the continuous Morlet wavelet. The scalogram reveals the changes in coefficient peak values from grade-I to grade-III. Wavelet normalized energy plots are computed in order to show the difference of periodicity among different grades of cancerous tissues. Using the multi-fractal de-trended fluctuation analysis (MFDFA), it is observed that the values of Hurst exponent and width of singularity spectrum decrease as cancer progresses from grade-I to grade-III tissue.

  2. Validation of the Swine Protein-Annotated Oligonucleotide Microarray

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The specificity and utility of the Swine Protein-Annotated Oligonucleotide Microarray, or Pigoligoarray (www.pigoligoarray.org), has been evaluated by profiling the expression of transcripts from four porcine tissues. Tools for comparative analyses of expression on the Pigoligoarray were developed i...

  3. Progress in developing a living human tissue-engineered tri-leaflet heart valve assembled from tissue produced by the self-assembly approach.

    PubMed

    Dubé, Jean; Bourget, Jean-Michel; Gauvin, Robert; Lafrance, Hugues; Roberge, Charles J; Auger, François A; Germain, Lucie

    2014-08-01

    The aortic heart valve is constantly subjected to pulsatile flow and pressure gradients which, associated with cardiovascular risk factors and abnormal hemodynamics (i.e. altered wall shear stress), can cause stenosis and calcification of the leaflets and result in valve malfunction and impaired circulation. Available options for valve replacement include homograft, allogenic or xenogenic graft as well as the implantation of a mechanical valve. A tissue-engineered heart valve containing living autologous cells would represent an alternative option, particularly for pediatric patients, but still needs to be developed. The present study was designed to demonstrate the feasibility of using a living tissue sheet produced by the self-assembly method, to replace the bovine pericardium currently used for the reconstruction of a stented human heart valve. In this study, human fibroblasts were cultured in the presence of sodium ascorbate to produce tissue sheets. These sheets were superimposed to create a thick construct. Tissue pieces were cut from these constructs and assembled together on a stent, based on techniques used for commercially available replacement valves. Histology and transmission electron microscopy analysis showed that the fibroblasts were embedded in a dense extracellular matrix produced in vitro. The mechanical properties measured were consistent with the fact that the engineered tissue was resistant and could be cut, sutured and assembled on a wire frame typically used in bioprosthetic valve assembly. After a culture period in vitro, the construct was cohesive and did not disrupt or disassemble. The tissue engineered heart valve was stimulated in a pulsatile flow bioreactor and was able to sustain multiple duty cycles. This prototype of a tissue-engineered heart valve containing cells embedded in their own extracellular matrix and sewn on a wire frame has the potential to be strong enough to support physiological stress. The next step will be to test this valve extensively in a bioreactor and at a later date, in a large animal model in order to assess in vivo patency of the graft. PMID:24813743

  4. Microarray Analysis of Microbial Weathering

    NASA Astrophysics Data System (ADS)

    Olsson-Francis, K.; van Houdt, R.; Leys, N.; Mergeay, M.; Cockell, C. S.

    2010-04-01

    Microarray analysis of the heavy metal resistant bacterium, Cupriavidus metallidurans CH34 was used to investigate the genes involved in the weathering. The results demonstrated that large porin and membrane transporter genes were unregulated.

  5. Are glycan biosensors an alternative to glycan microarrays?

    PubMed Central

    Hushegyi, A.

    2016-01-01

    Complex carbohydrates (glycans) play an important role in nature and study of their interaction with proteins or intact cells can be useful for understanding many physiological and pathological processes. Such interactions have been successfully interrogated in a highly parallel way using glycan microarrays, but this technique has some limitations. Thus, in recent years glycan biosensors in numerous progressive configurations have been developed offering distinct advantages compared to glycan microarrays. Thus, in this review advances achieved in the field of label-free glycan biosensors are discussed.

  6. The bioinformatics of microarrays to study cancer: Advantages and disadvantages

    NASA Astrophysics Data System (ADS)

    Rodríguez-Segura, M. A.; Godina-Nava, J. J.; Villa-Treviño, S.

    2012-10-01

    Microarrays are devices designed to analyze simultaneous expression of thousands of genes. However, the process will adds noise into the information at each stage of the study. To analyze these thousands of data is necessary to use bioinformatics tools. The traditional analysis begins by normalizing data, but the obtained results are highly dependent on how it is conducted the study. It is shown the need to develop new strategies to analyze microarray. Liver tissue taken from an animal model in which is chemically induced cancer is used as an example.

  7. Screening of potential biomarkers for cholangiocarcinoma by integrated analysis of microarray data sets.

    PubMed

    Huang, Q-X; Cui, J-Y; Ma, H; Jia, X-M; Huang, F-L; Jiang, L-X

    2016-02-01

    Cholangiocarcinoma (CCA) continues to harbor a difficult prognosis and it is difficult to diagnose in its early stages. The molecular mechanisms of CCA oncogenesis and progression are poorly understood. This study aimed to identify candidate biomarkers for CCA. Integrated analysis of microarray data sets was performed to identify differentially expressed genes (DEGs) between CCA and normal tissues. Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were then performed to identify the functions of DEGs. Furthermore, the protein-protein interaction (PPI) network of DEGs was constructed. The expressions of DEGs were validated in human CCA tissues by qRT-PCR. A set of 712 DEGs were identified in CCA compared with normal tissues, including 306 upregulated and 406 downregulated DEGs. It can be shown from the KEGG pathway analysis that some pathways may have important roles in pathology of CCA, including peroxisome proliferator-activated receptor signaling pathway, bile secretion, cell cycle, fat digestion and absorption. PPI network indicated that the significant hub proteins were PKM, SPP1 and TPM1. The abnormally overexpression PKM, SPP1 and TPM1 were closely related to oncogenesis and progression of CCA. PKM, SPP1, TPM1, COL1A1 and COL1A2 may serve as candidate biomarkers for diagnosis and prognosis of CCA. PMID:26679756

  8. Quantitative proteomic analysis of paired colorectal cancer and non-tumorigenic tissues reveals signature proteins and perturbed pathways involved in CRC progression and metastasis.

    PubMed

    Sethi, Manveen K; Thaysen-Andersen, Morten; Kim, Hoguen; Park, Cheol Keun; Baker, Mark S; Packer, Nicolle H; Paik, Young-Ki; Hancock, William S; Fanayan, Susan

    2015-08-01

    Modern proteomics has proven instrumental in our understanding of the molecular deregulations associated with the development and progression of cancer. Herein, we profile membrane-enriched proteome of tumor and adjacent normal tissues from eight CRC patients using label-free nanoLC-MS/MS-based quantitative proteomics and advanced pathway analysis. Of the 948 identified proteins, 184 proteins were differentially expressed (P<0.05, fold change>1.5) between the tumor and non-tumor tissue (69 up-regulated and 115 down-regulated in tumor tissues). The CRC tumor and non-tumor tissues clustered tightly in separate groups using hierarchical cluster analysis of the differentially expressed proteins, indicating a strong CRC-association of this proteome subset. Specifically, cancer associated proteins such as FN1, TNC, DEFA1, ITGB2, MLEC, CDH17, EZR and pathways including actin cytoskeleton and RhoGDI signaling were deregulated. Stage-specific proteome signatures were identified including up-regulated ribosomal proteins and down-regulated annexin proteins in early stage CRC. Finally, EGFR(+) CRC tissues showed an EGFR-dependent down-regulation of cell adhesion molecules, relative to EGFR(-) tissues. Taken together, this study provides a detailed map of the altered proteome and associated protein pathways in CRC, which enhances our mechanistic understanding of CRC biology and opens avenues for a knowledge-driven search for candidate CRC protein markers. PMID:26054784

  9. A High-Fat Diet Containing Lard Accelerates Prostate Cancer Progression and Reduces Survival Rate in Mice: Possible Contribution of Adipose Tissue-Derived Cytokines

    PubMed Central

    Cho, Han Jin; Kwon, Gyoo Taik; Park, Heesook; Song, Hyerim; Lee, Ki Won; Kim, Jung-In; Park, Jung Han Yoon

    2015-01-01

    To examine the effects of high-fat diet (HFD) containing lard on prostate cancer development and progression and its underlying mechanisms, transgenic adenocarcinoma mouse prostate (TRAMP) and TRAMP-C2 allograft models, as well as in vitro culture models, were employed. In TRAMP mice, HFD feeding increased the incidence of poorly differentiated carcinoma and decreased that of prostatic intraepithelial neoplasia in the dorsolateral lobes of the prostate, which was accompanied by increased expression of proteins associated with proliferation and angiogenesis. HFD feeding also led to increased metastasis and decreased survival rate in TRAMP mice. In the allograft model, HFD increased solid tumor growth, the expression of proteins related to proliferation/angiogenesis, the number of lipid vacuoles in tumor tissues, and levels of several cytokines in serum and adipose tissue. In vitro results revealed that adipose tissue-conditioned media from HFD-fed mice stimulated the proliferation and migration of prostate cancer cells and angiogenesis compared to those from control-diet-fed mice. These results indicate that the increase of adipose tissue-derived soluble factors by HFD feeding plays a role in the growth and metastasis of prostate cancer via endocrine and paracrine mechanisms. These results provide evidence that a HFD containing lard increases prostate cancer development and progression, thereby reducing the survival rate. PMID:25912035

  10. A high-fat diet containing lard accelerates prostate cancer progression and reduces survival rate in mice: possible contribution of adipose tissue-derived cytokines.

    PubMed

    Cho, Han Jin; Kwon, Gyoo Taik; Park, Heesook; Song, Hyerim; Lee, Ki Won; Kim, Jung-In; Park, Jung Han Yoon

    2015-04-01

    To examine the effects of high-fat diet (HFD) containing lard on prostate cancer development and progression and its underlying mechanisms, transgenic adenocarcinoma mouse prostate (TRAMP) and TRAMP-C2 allograft models, as well as in vitro culture models, were employed. In TRAMP mice, HFD feeding increased the incidence of poorly differentiated carcinoma and decreased that of prostatic intraepithelial neoplasia in the dorsolateral lobes of the prostate, which was accompanied by increased expression of proteins associated with proliferation and angiogenesis. HFD feeding also led to increased metastasis and decreased survival rate in TRAMP mice. In the allograft model, HFD increased solid tumor growth, the expression of proteins related to proliferation/angiogenesis, the number of lipid vacuoles in tumor tissues, and levels of several cytokines in serum and adipose tissue. In vitro results revealed that adipose tissue-conditioned media from HFD-fed mice stimulated the proliferation and migration of prostate cancer cells and angiogenesis compared to those from control-diet-fed mice. These results indicate that the increase of adipose tissue-derived soluble factors by HFD feeding plays a role in the growth and metastasis of prostate cancer via endocrine and paracrine mechanisms. These results provide evidence that a HFD containing lard increases prostate cancer development and progression, thereby reducing the survival rate. PMID:25912035

  11. Tissue Array Research Program (TARP)

    Cancer.gov

    Objectives The TARP Lab’s objectives include development and distribution of multitumor tissue microarray slides and related technologies to cancer research investigators. This technology helps expedite discovery of the novel targets important in cancer t

  12. Karyotype versus Microarray Testing for Genetic Abnormalities after Stillbirth

    PubMed Central

    Reddy, Uma M.; Page, Grier P.; Saade, George R.; Silver, Robert M.; Thorsten, Vanessa R.; Parker, Corette B.; Pinar, Halit; Willinger, Marian; Stoll, Barbara J.; Heim-Hall, Josefine; Varner, Michael W.; Goldenberg, Robert L.; Bukowski, Radek; Wapner, Ronald J.; Drews-Botsch, Carolyn D.; O’Brien, Barbara M.; Dudley, Donald J.; Levy, Brynn

    2015-01-01

    Background Genetic abnormalities have been associated with 6 to 13% of stillbirths, but the true prevalence may be higher. Unlike karyotype analysis, microarray analysis does not require live cells, and it detects small deletions and duplications called copy-number variants. Methods The Stillbirth Collaborative Research Network conducted a population-based study of stillbirth in five geographic catchment areas. Standardized postmortem examinations and karyotype analyses were performed. A single-nucleotide polymorphism array was used to detect copy-number variants of at least 500 kb in placental or fetal tissue. Variants that were not identified in any of three databases of apparently unaffected persons were then classified into three groups: probably benign, clinical significance unknown, or pathogenic. We compared the results of karyotype and microarray analyses of samples obtained after delivery. Results In our analysis of samples from 532 stillbirths, microarray analysis yielded results more often than did karyotype analysis (87.4% vs. 70.5%, P<0.001) and provided better detection of genetic abnormalities (aneuploidy or pathogenic copy-number variants, 8.3% vs. 5.8%; P = 0.007). Microarray analysis also identified more genetic abnormalities among 443 antepartum stillbirths (8.8% vs. 6.5%, P = 0.02) and 67 stillbirths with congenital anomalies (29.9% vs. 19.4%, P = 0.008). As compared with karyotype analysis, microarray analysis provided a relative increase in the diagnosis of genetic abnormalities of 41.9% in all stillbirths, 34.5% in antepartum stillbirths, and 53.8% in stillbirths with anomalies. Conclusions Microarray analysis is more likely than karyotype analysis to provide a genetic diagnosis, primarily because of its success with nonviable tissue, and is especially valuable in analyses of stillbirths with congenital anomalies or in cases in which karyotype results cannot be obtained. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development.) PMID:23215556

  13. Comparing Bacterial DNA Microarray Fingerprints

    SciTech Connect

    Willse, Alan R.; Chandler, Darrell P.; White, Amanda M.; Protic, Miroslava; Daly, Don S.; Wunschel, Sharon C.

    2005-08-15

    Detecting subtle genetic differences between microorganisms is an important problem in molecular epidemiology and microbial forensics. In a typical investigation, gel electrophoresis is used to compare randomly amplified DNA fragments between microbial strains, where the patterns of DNA fragment sizes are proxies for a microbe's genotype. The limited genomic sample captured on a gel is often insufficient to discriminate nearly identical strains. This paper examines the application of microarray technology to DNA fingerprinting as a high-resolution alternative to gel-based methods. The so-called universal microarray, which uses short oligonucleotide probes that do not target specific genes or species, is intended to be applicable to all microorganisms because it does not require prior knowledge of genomic sequence. In principle, closely related strains can be distinguished if the number of probes on the microarray is sufficiently large, i.e., if the genome is sufficiently sampled. In practice, we confront noisy data, imperfectly matched hybridizations, and a high-dimensional inference problem. We describe the statistical problems of microarray fingerprinting, outline similarities with and differences from more conventional microarray applications, and illustrate the statistical fingerprinting problem for 10 closely related strains from three Bacillus species, and 3 strains from non-Bacillus species.

  14. Identification of differentially expressed genes in cutaneous squamous cell carcinoma by microarray expression profiling

    PubMed Central

    Nindl, Ingo; Dang, Chantip; Forschner, Tobias; Kuban, Ralf J; Meyer, Thomas; Sterry, Wolfram; Stockfleth, Eggert

    2006-01-01

    Background Carcinogenesis is a multi-step process indicated by several genes up- or down-regulated during tumor progression. This study examined and identified differentially expressed genes in cutaneous squamous cell carcinoma (SCC). Results Three different biopsies of 5 immunosuppressed organ-transplanted recipients each normal skin (all were pooled), actinic keratosis (AK) (two were pooled), and invasive SCC and additionally 5 normal skin tissues from immunocompetent patients were analyzed. Thus, total RNA of 15 specimens were used for hybridization with Affymetrix HG-U133A microarray technology containing 22,283 genes. Data analyses were performed by prediction analysis of microarrays using nearest shrunken centroids with the threshold 3.5 and ANOVA analysis was independently performed in order to identify differentially expressed genes (p < 0.05). Verification of 13 up- or down-regulated genes was performed by quantitative real-time reverse transcription (RT)-PCR and genes were additionally confirmed by sequencing. Broad coherent patterns in normal skin vs. AK and SCC were observed for 118 genes. Conclusion The majority of identified differentially expressed genes in cutaneous SCC were previously not described. PMID:16893473

  15. Three-dimensional lithographically-defined organotypic tissue arrays for quantitative analysis of morphogenesis and neoplastic progression

    SciTech Connect

    Nelson, Celeste M.; Inman, Jamie L.; Bissell, Mina J.

    2008-02-13

    Here we describe a simple micromolding method to construct three-dimensional arrays of organotypic epithelial tissue structures that approximate in vivo histology. An elastomeric stamp containing an array of posts of defined geometry and spacing is used to mold microscale cavities into the surface of type I collagen gels. Epithelial cells are seeded into the cavities and covered with a second layer of collagen. The cells reorganize into hollow tissues corresponding to the geometry of the cavities. Patterned tissue arrays can be produced in 3-4 h and will undergo morphogenesis over the following one to three days. The protocol can easily be adapted to study a variety of tissues and aspects of normal and neoplastic development.

  16. Development of a Highly Sensitive Glycan Microarray for Quantifying AFP-L3 for Early Prediction of Hepatitis B Virus–Related Hepatocellular Carcinoma

    PubMed Central

    Chou, Ruey-Hwang; Yen, Chia-Jui; Huang, Wei-Chien; Wu, Chung-Yi; Yu, Yung-Luen

    2014-01-01

    The α-fetoprotein fraction L3 (AFP-L3), which is synthesized by malignant cells and incorporates a fucosylated oligosaccharide, has been investigated as a diagnostic and prognostic marker for hepatocellular carcinoma (HCC). Quantification of AFP-L3 by conventional enzyme-linked immunosorbent assay (ELISA) has not always produced reliable results for serum samples with low AFP, and thus we evaluated the clinical utility of quantifying AFP-L3 using a new and highly sensitive glycan microarray assay. Sera from 9 patients with chronic hepatitis B and 32 patients with hepatitis B virus (HBV)-related HCC were tested for AFP-L3 level using the glycan microarray. Additionally, we compared receiver operator characteristic curves for the ELISA and glycan microarray methods for determination of the AFP-L3: AFP-L1 ratio in patient samples. This ratio was calculated for 8 HCC patients who underwent transarterial embolization therapy pre- or post-treatment with AFP-L3. Glycan microarrays showed that the AFP-L3 ratio of HBV-related HCC patients was significantly higher than that measured for chronic hepatitis B patients. Overall parameters for estimating AFP-L3% in HCC samples were as follows: sensitivity, 53.13%; specificity, 88.89%; and area under the curve, 0.75. The elevated AFP-L3% in the 8 patients with HBV-related HCC was strongly associated with HCC progression. Following one month of transarterial embolization therapy, the relative mean AFP-L3% decreased significantly. In addition, we compared Fut8 gene expression between paired tumor and non-tumor tissues from 24 patients with HBV-related HCC. The Fut8 mRNA expression was significantly increased in tumorous tissues in these patients than that in non-tumor tissue controls. Higher expression of Fut8 mRNA in tumorous tissues in these patients was associated with poor differentiation than well and moderate differentiation. Our results describe a new glycan microarray for the sensitive and rapid quantification of fucosylated AFP; this method is potentially applicable to screening changes in AFP-L3 level for assessment of HCC progression. PMID:24927126

  17. DNA microarrays: Types, Applications and their future

    PubMed Central

    Bumgarner, Roger

    2014-01-01

    This chapter provides an overview of DNA microarrays. Microarrays are a technology in which 1000’s of nucleic acids are bound to a surface and are used to measure the relative concentration of nucleic acid sequences in a mixture via hybridization and subsequent detection of the hybridization events. We first cover the history of microarrays and the antecedent technologies that led to their development. We then discuss the methods of manufacture of microarrays and the most common biological applications. The chapter ends with a brief discussion of the limitations of microarrays and discusses how microarrays are being rapidly replaced by DNA sequencing technologies. PMID:23288464

  18. Microarray expression profile analysis of aberrant long non-coding RNAs in esophageal squamous cell carcinoma.

    PubMed

    Yao, Juan; Huang, Jun-Xing; Lin, Mei; Wu, Zheng-Dong; Yu, Hong; Wang, Peng-Cheng; Ye, Jun; Chen, Ping; Wu, Jing; Zhao, Guo-Jun

    2016-06-01

    Increasing evidence indicates that long non-coding RNA (lncRNA) plays an important role in tumorigenesis. However, the function and regulatory mechanism of lncRNAs are still unclear in esophageal squamous cell carcinoma (ESCC). To address this challenge, we screened lncRNAs expression profiles in 3 pairs of ESCC and matched non-cancerous tissues by microarray assay and identified the relationship between lncRNAs expression in ESCC tissue and clinicopathological characteristics and prognosis of patients with ESCC. We found 182 lncRNAs that were significantly differently expressed in ESCC tissues versus the matched non-cancerous tissues. Gene ontology and pathway analysis results suggested that the primary biological processes of these genes were involved in extracellular matrix, immune responses, cell differentiation and cell proliferation. Through cis and trans analyzing, we found 4 lncRNAs (ENST00000480669, NONHSAT104436, NONHSAT126998 and NONHSAT112918) may play important roles in tumorigenesis of ESCC. The four lncRNAs were checked in 73 patients with ESCC. The results showed that they mainly related to tumor metastasis. Kaplan-Meier survival analysis showed that high expression of NONHSAT104436, NONHSAT126998 and low expression of ENST00000480669 were related to poor 3-year overall survival (P=0.003, 0.032 and 0.040, respectively). Multivariate analysis showed that NONHSAT104436 was an independent prognostic factor (P=0.017). Thus we concluded that, lncRNAs showed differently expression patterns in ESCC versus matched non-cancerous tissues, and aberrantly expressed lncRNA may play important roles in ESCC development and progression. Interestingly, the overexpression of NONHSAT104436 was tightly correlated with distant metastasis and, poor survival rate, which might indicate that NONHSAT104436 might play a very important part in ESCC tumor progression. PMID:27035335

  19. Microarray Developed on Plastic Substrates.

    PubMed

    Bañuls, María-José; Morais, Sergi B; Tortajada-Genaro, Luis A; Maquieira, Ángel

    2016-01-01

    There is a huge potential interest to use synthetic polymers as versatile solid supports for analytical microarraying. Chemical modification of polycarbonate (PC) for covalent immobilization of probes, micro-printing of protein or nucleic acid probes, development of indirect immunoassay, and development of hybridization protocols are described and discussed. PMID:26614067

  20. Microarray analysis: Uses and Limitations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The use of microarray technology has exploded in resent years. All areas of biological research have found application for this powerful platform. From human disease studies to microbial detection systems, a plethora of uses for this technology are currently in place with new uses being developed ...

  1. ANALYSIS OF DNA MICROARRAY DATA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Analysis of DNA microarrays involves the extraction of fluorescent intensity from raw image files generated by the scanner, storing the extracted data in a database, normalizing the data, conducting statistical analysis and finally querying the analyzed data to find biologically meaningful results. ...

  2. Microfluidic microarray systems and methods thereof

    DOEpatents

    West, Jay A. A.; Hukari, Kyle W.; Hux, Gary A.

    2009-04-28

    Disclosed are systems that include a manifold in fluid communication with a microfluidic chip having a microarray, an illuminator, and a detector in optical communication with the microarray. Methods for using these systems for biological detection are also disclosed.

  3. Image microarrays (IMA): Digital pathology's missing tool

    PubMed Central

    Hipp, Jason; Cheng, Jerome; Pantanowitz, Liron; Hewitt, Stephen; Yagi, Yukako; Monaco, James; Madabhushi, Anant; Rodriguez-canales, Jaime; Hanson, Jeffrey; Roy-Chowdhuri, Sinchita; Filie, Armando C.; Feldman, Michael D.; Tomaszewski, John E.; Shih, Natalie NC.; Brodsky, Victor; Giaccone, Giuseppe; Emmert-Buck, Michael R.; Balis, Ulysses J.

    2011-01-01

    Introduction: The increasing availability of whole slide imaging (WSI) data sets (digital slides) from glass slides offers new opportunities for the development of computer-aided diagnostic (CAD) algorithms. With the all-digital pathology workflow that these data sets will enable in the near future, literally millions of digital slides will be generated and stored. Consequently, the field in general and pathologists, specifically, will need tools to help extract actionable information from this new and vast collective repository. Methods: To address this limitation, we designed and implemented a tool (dCORE) to enable the systematic capture of image tiles with constrained size and resolution that contain desired histopathologic features. Results: In this communication, we describe a user-friendly tool that will enable pathologists to mine digital slides archives to create image microarrays (IMAs). IMAs are to digital slides as tissue microarrays (TMAs) are to cell blocks. Thus, a single digital slide could be transformed into an array of hundreds to thousands of high quality digital images, with each containing key diagnostic morphologies and appropriate controls. Current manual digital image cut-and-paste methods that allow for the creation of a grid of images (such as an IMA) of matching resolutions are tedious. Conclusion: The ability to create IMAs representing hundreds to thousands of vetted morphologic features has numerous applications in education, proficiency testing, consensus case review, and research. Lastly, in a manner analogous to the way conventional TMA technology has significantly accelerated in situ studies of tissue specimens use of IMAs has similar potential to significantly accelerate CAD algorithm development. PMID:22200030

  4. The Microarray Revolution: Perspectives from Educators

    ERIC Educational Resources Information Center

    Brewster, Jay L.; Beason, K. Beth; Eckdahl, Todd T.; Evans, Irene M.

    2004-01-01

    In recent years, microarray analysis has become a key experimental tool, enabling the analysis of genome-wide patterns of gene expression. This review approaches the microarray revolution with a focus upon four topics: 1) the early development of this technology and its application to cancer diagnostics; 2) a primer of microarray research,

  5. The Microarray Revolution: Perspectives from Educators

    ERIC Educational Resources Information Center

    Brewster, Jay L.; Beason, K. Beth; Eckdahl, Todd T.; Evans, Irene M.

    2004-01-01

    In recent years, microarray analysis has become a key experimental tool, enabling the analysis of genome-wide patterns of gene expression. This review approaches the microarray revolution with a focus upon four topics: 1) the early development of this technology and its application to cancer diagnostics; 2) a primer of microarray research,…

  6. Current Protocols in Chemical Biology Construction and Use of Glycan Microarrays

    PubMed Central

    Campbell, Christopher T.; Zhang, Yalong; Gildersleeve, Jeffrey C.

    2011-01-01

    Glycosylation is an important post-translational modification that influences many biological processes critical for development, normal physiologic function, and diseases. Unfortunately, progress towards understanding the roles of glycans in biology has been slow due to the challenges of studying glycans and the proteins that interact with them. Glycan microarrays provide a high-throughput approach for the rapid analysis of carbohydrate-macromolecule interactions. Protocols detailed here are intended to help laboratories with basic familiarity of DNA or protein microarrays to begin printing and performing assays using glycan microarrays. Basic and advanced data processing are also detailed, along with strategies for improving reproducibility of data collected with glycan arrays. PMID:23836542

  7. DNA microarrays: sample quality control, array hybridization and scanning.

    PubMed

    Diaz, Elva; Barisone, Gustavo A

    2011-01-01

    Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested. PMID:21445042

  8. Recent progress in defining mechanisms and potential targets for prevention of normal tissue injury after radiation therapy

    SciTech Connect

    Anscher, Mitchell S. . E-mail: anscher@radonc.duke.edu; Chen, Liguang; Rabbani, Zahid; Kang Song; Larrier, Nicole; Huang Hong; Samulski, Thaddeus V.; Dewhirst, Mark W.; Brizel, David M.; Folz, Rodney J.; Vujaskovic, Zeljko

    2005-05-01

    The ability to optimize treatments for cancer on the basis of relative risks for normal tissue injury has important implications in oncology, because higher doses of radiation might, in some diseases, improve both local control and survival. To achieve this goal, a thorough understanding of the molecular mechanisms responsible for radiation-induced toxicity will be essential. Recent research has demonstrated that ionizing radiation triggers a series of genetic and molecular events, which might lead to chronic persistent alterations in the microenvironment and an aberrant wound-healing response. Disrupted epithelial-stromal cell communication might also be important. With the application of a better understanding of fundamental biology to clinical practice, new approaches to treating and preventing normal tissue injury can focus on correcting these disturbed molecular processes.

  9. Proton irradiation impacts age-driven modulations of cancer progression influenced by immune system transcriptome modifications from splenic tissue.

    PubMed

    Wage, Justin; Ma, Lili; Peluso, Michael; Lamont, Clare; Evens, Andrew M; Hahnfeldt, Philip; Hlatky, Lynn; Beheshti, Afshin

    2015-09-01

    Age plays a crucial role in the interplay between tumor and host, with additional impact due to irradiation. Proton irradiation of tumors induces biological modulations including inhibition of angiogenic and immune factors critical to 'hallmark' processes impacting tumor development. Proton irradiation has also provided promising results for proton therapy in cancer due to targeting advantages. Additionally, protons may contribute to the carcinogenesis risk from space travel (due to the high proportion of high-energy protons in space radiation). Through a systems biology approach, we investigated how host tissue (i.e. splenic tissue) of tumor-bearing mice was altered with age, with or without whole-body proton exposure. Transcriptome analysis was performed on splenic tissue from adolescent (68-day) versus old (736-day) C57BL/6 male mice injected with Lewis lung carcinoma cells with or without three fractionations of 0.5 Gy (1-GeV) proton irradiation. Global transcriptome analysis indicated that proton irradiation of adolescent hosts caused significant signaling changes within splenic tissues that support carcinogenesis within the mice, as compared with older subjects. Increases in cell cycling and immunosuppression in irradiated adolescent hosts with CDK2, MCM7, CD74 and RUVBL2 indicated these were the key genes involved in the regulatory changes in the host environment response (i.e. the spleen). Collectively, these results suggest that a significant biological component of proton irradiation is modulated by host age through promotion of carcinogenesis in adolescence and resistance to immunosuppression, carcinogenesis and genetic perturbation associated with advancing age. PMID:26253138

  10. Proton irradiation impacts age-driven modulations of cancer progression influenced by immune system transcriptome modifications from splenic tissue

    PubMed Central

    Wage, Justin; Ma, Lili; Peluso, Michael; Lamont, Clare; Evens, Andrew M.; Hahnfeldt, Philip; Hlatky, Lynn; Beheshti, Afshin

    2015-01-01

    Age plays a crucial role in the interplay between tumor and host, with additional impact due to irradiation. Proton irradiation of tumors induces biological modulations including inhibition of angiogenic and immune factors critical to ‘hallmark’ processes impacting tumor development. Proton irradiation has also provided promising results for proton therapy in cancer due to targeting advantages. Additionally, protons may contribute to the carcinogenesis risk from space travel (due to the high proportion of high-energy protons in space radiation). Through a systems biology approach, we investigated how host tissue (i.e. splenic tissue) of tumor-bearing mice was altered with age, with or without whole-body proton exposure. Transcriptome analysis was performed on splenic tissue from adolescent (68-day) versus old (736-day) C57BL/6 male mice injected with Lewis lung carcinoma cells with or without three fractionations of 0.5 Gy (1-GeV) proton irradiation. Global transcriptome analysis indicated that proton irradiation of adolescent hosts caused significant signaling changes within splenic tissues that support carcinogenesis within the mice, as compared with older subjects. Increases in cell cycling and immunosuppression in irradiated adolescent hosts with CDK2, MCM7, CD74 and RUVBL2 indicated these were the key genes involved in the regulatory changes in the host environment response (i.e. the spleen). Collectively, these results suggest that a significant biological component of proton irradiation is modulated by host age through promotion of carcinogenesis in adolescence and resistance to immunosuppression, carcinogenesis and genetic perturbation associated with advancing age. PMID:26253138

  11. Noninvasive near-infrared fluorescent protein-based imaging of tumor progression and metastases in deep organs and intraosseous tissues

    NASA Astrophysics Data System (ADS)

    Jiguet-Jiglaire, Carine; Cayol, Mylène; Mathieu, Sylvie; Jeanneau, Charlotte; Bouvier-Labit, Corinne; Ouafik, L.'houcine; El-Battari, Assou

    2014-01-01

    Whole-body imaging of experimental tumor growth is more feasible within the near-infrared (NIR) optical window because of the highest transparency of mammalian tissues within this wavelength spectrum, mainly due to improved tissue penetration and lower autofluorescence. We took advantage from the recently cloned infrared fluorescent protein (iRFP) together with a human immunodeficiency virus (HIV)-based lentiviral vector to produce virally transduced tumor cells that permanently express this protein. We then noninvasively explored metastatic spread as well as primary tumor growth in deep organs and behind bone barriers. Intrabone tumor growth was investigated through intracranial and intratibial injections of glioblastoma and osteosarcoma cells, respectively, and metastasis was assessed by tail vein injection of melanoma cells. We found that the emitted fluorescence is captured as sharp images regardless of the organ or tissue considered. Furthermore, by overlaying fluorescence spots with the white light, it was possible to afford whole-body images yet never observed before. This approach allowed us to continuously monitor the growth and dissemination of tumor cells with a small number of animals, minimal animal handling, and without the need for any additive. This iRFP-based system provides high-resolution readouts of tumorigenesis that should greatly facilitate preclinical trials with anticancer therapeutic molecules.

  12. The Current Status of DNA Microarrays

    NASA Astrophysics Data System (ADS)

    Shi, Leming; Perkins, Roger G.; Tong, Weida

    DNA microarray technology that allows simultaneous assay of thousands of genes in a single experiment has steadily advanced to become a mainstream method used in research, and has reached a stage that envisions its use in medical applications and personalized medicine. Many different strategies have been developed for manufacturing DNA microarrays. In this chapter, we discuss the manufacturing characteristics of seven microarray platforms that were used in a recently completed large study by the MicroArray Quality Control (MAQC) consortium, which evaluated the concordance of results across these platforms. The platforms can be grouped into three categories: (1) in situ synthesis of oligonucleotide probes on microarrays (Affymetrix GeneChip® arrays based on photolithography synthesis and Agilent's arrays based on inkjet synthesis); (2) spotting of presynthesized oligonucleotide probes on microarrays (GE Healthcare's CodeLink system, Applied Biosystems' Genome Survey Microarrays, and the custom microarrays printed with Operon's oligonucleotide set); and (3) deposition of presynthesized oligonucleotide probes on bead-based microarrays (Illumina's BeadChip microarrays). We conclude this chapter with our views on the challenges and opportunities toward acceptance of DNA microarray data in clinical and regulatory settings.

  13. The Current Status of DNA Microarrays

    NASA Astrophysics Data System (ADS)

    Shi, Leming; Perkins, Roger G.; Tong, Weida

    DNA microarray technology that allows simultaneous assay of thousands of genes in a single experiment has steadily advanced to become a mainstream method used in research, and has reached a stage that envisions its use in medical applications and personalized medicine. Many different strategies have been developed for manufacturing DNA microarrays. In this chapter, we discuss the manu facturing characteristics of seven microarray platforms that were used in a recently completed large study by the MicroArray Quality Control (MAQC) consortium, which evaluated the concordance of results across these platforms. The platforms can be grouped into three categories: (1) in situ synthesis of oligonucleotide probes on microarrays (Affymetrix GeneChip® arrays based on photolithography synthesis and Agilent's arrays based on inkjet synthesis); (2) spotting of presynthe-sized oligonucleotide probes on microarrays (GE Healthcare's CodeLink system, Applied Biosystems' Genome Survey Microarrays, and the custom microarrays printed with Operon's oligonucleotide set); and (3) deposition of presynthesized oligonucleotide probes on bead-based microarrays (Illumina's BeadChip microar-rays). We conclude this chapter with our views on the challenges and opportunities toward acceptance of DNA microarray data in clinical and regulatory settings.

  14. Evanescent-imaging-ellipsometry-based microarray reader.

    PubMed

    Venkatasubbarao, Srivatsa; Beaudry, Neil; Zhao, Yanming; Chipman, Russell

    2006-01-01

    We describe the development of a label-less ellipsometric imaging microarray reader. The ability of the ellipsometric microarray reader to measure binding of sample to microarray surface is verified using oligonucleotide complementary DNA (cDNA) microarrays. Polarized light illuminates the microarray surface through a glass substrate at an angle beyond the critical angle and changes in the polarization of totally internally reflected light resulting from binding events on the microarray surface are measured. This polarization change is used to measure the thickness of biomolecules bound to the microarray. A prototype ellipsometric imaging microarray reader is constructed and calibrated, and the performance is evaluated with cDNA microarrays. The microarray reader measures changes in refractive index changes as small as 0.0024 and thickness changes as small as 0.28 nm. The optimization of angle of incidence and substrate refractive index necessary to achieve high sensitivity is also described. This ellipsometric technique offers an attractive alternative to fluorescence-microarray readers in some genomic, proteomic, diagnostic, and sensing applications. PMID:16526905

  15. Fusion Transcript Discovery in Formalin-Fixed Paraffin-Embedded Human Breast Cancer Tissues Reveals a Link to Tumor Progression

    PubMed Central

    Ma, Yan; Ambannavar, Ranjana; Stephans, James; Jeong, Jennie; Dei Rossi, Andrew; Liu, Mei-Lan; Friedman, Adam J.; Londry, Jason J.; Abramson, Richard; Beasley, Ellen M.; Baker, Joffre; Levy, Samuel; Qu, Kunbin

    2014-01-01

    The identification of gene fusions promises to play an important role in personalized cancer treatment decisions. Many rare gene fusion events have been identified in fresh frozen solid tumors from common cancers employing next-generation sequencing technology. However the ability to detect transcripts from gene fusions in RNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor tissues, which exist in very large sample repositories for which disease outcome is known, is still limited due to the low complexity of FFPE libraries and the lack of appropriate bioinformatics methods. We sought to develop a bioinformatics method, named gFuse, to detect fusion transcripts in FFPE tumor tissues. An integrated, cohort based strategy has been used in gFuse to examine single-end 50 base pair (bp) reads generated from FFPE RNA-Sequencing (RNA-Seq) datasets employing two breast cancer cohorts of 136 and 76 patients. In total, 118 fusion events were detected transcriptome-wide at base-pair resolution across the 212 samples. We selected 77 candidate fusions based on their biological relevance to cancer and supported 61% of these using TaqMan assays. Direct sequencing of 19 of the fusion sequences identified by TaqMan confirmed them. Three unique fused gene pairs were recurrent across the 212 patients with 6, 3, 2 individuals harboring these fusions respectively. We show here that a high frequency of fusion transcripts detected at the whole transcriptome level correlates with poor outcome (P<0.0005) in human breast cancer patients. This study demonstrates the ability to detect fusion transcripts as biomarkers from archival FFPE tissues, and the potential prognostic value of the fusion transcripts detected. PMID:24727804

  16. Analysis of microarray experiments of gene expression profiling

    PubMed Central

    Tarca, Adi L.; Romero, Roberto; Draghici, Sorin

    2008-01-01

    The study of gene expression profiling of cells and tissue has become a major tool for discovery in medicine. Microarray experiments allow description of genome-wide expression changes in health and disease. The results of such experiments are expected to change the methods employed in the diagnosis and prognosis of disease in obstetrics and gynecology. Moreover, an unbiased and systematic study of gene expression profiling should allow the establishment of a new taxonomy of disease for obstetric and gynecologic syndromes. Thus, a new era is emerging in which reproductive processes and disorders could be characterized using molecular tools and fingerprinting. The design, analysis, and interpretation of microarray experiments require specialized knowledge that is not part of the standard curriculum of our discipline. This article describes the types of studies that can be conducted with microarray experiments (class comparison, class prediction, class discovery). We discuss key issues pertaining to experimental design, data preprocessing, and gene selection methods. Common types of data representation are illustrated. Potential pitfalls in the interpretation of microarray experiments, as well as the strengths and limitations of this technology, are highlighted. This article is intended to assist clinicians in appraising the quality of the scientific evidence now reported in the obstetric and gynecologic literature. PMID:16890548

  17. Microarray analysis in cardiac arrhythmias: a new perspective?

    PubMed

    Moric-Janiszewska, Ewa; Hibner, Grzegorz

    2013-07-01

    The opportunity to distinguish an accurate set of genes associated with multigenic diseases such as cardiomyopathies or cardiac arrhythmias was very limited before the genomic era. Numerous methods of measuring RNA abundance exist, including northern blotting, multiplex polymerase chain reaction (PCR), and quantitative real-time reverse transcriptase-PCR. However, these techniques might be used to assess the expression levels of only 10-50 genes at time. Today, DNA microarrays provide us with opportunity to simultaneously analyze tens of thousands of genes, giving a remarkable possibility to investigate the genomic contribution to cardiovascular diseases. A particular tissue at any stage of health or disease may be used to generate a genomic profile. Microarray techniques are already used in infectious diseases, oncology, and pharmacology to facilitate clinicians, risk-stratify patients, as well as to predict and assess therapeutic responses to drugs. In this paper, we describe recent advances in the use of various types of microarray technique in the diagnosis of arrhythmogenic heart disease. We also highlight other strategies and methods of differential gene typing comparing with pros and cons of microarray analysis. PMID:23614797

  18. Gene expression profiling of mouse embryos with microarrays

    PubMed Central

    Sharov, Alexei A.; Piao, Yulan; Ko, Minoru S. H.

    2011-01-01

    Global expression profiling by DNA microarrays provides a snapshot of cell and tissue status and becomes an essential tool in biological and medical sciences. Typical questions that can be addressed by microarray analysis in developmental biology include: (1) to find a set of genes expressed in a specific cell type; (2) to identify genes expressed commonly in multiple cell types; (3) to follow the time-course changes of gene expression patterns; (4) to demonstrate cell’s identity by showing similarities or differences among two or multiple cell types; (5) to find regulatory pathways and/or networks affected by gene manipulations, such as overexpression or repression of gene expression; (6) to find downstream target genes of transcription factors; (7) to find downstream target genes of cell signaling; (8) to examine the effects of environmental manipulation of cells on gene expression patterns; and (9) to find the effects of genetic manipulation in embryos and adults. Here we describe strategies for executing these experiments and monitoring changes of cell state with gene expression microarrays in application to mouse embryology. Both statistical assessment and interpretation of data are discussed. We also present a protocol for performing microarray analysis on a small amount of embryonic materials. PMID:20699157

  19. Extracellular Matrix, Nuclear and Chromatin Structure and GeneExpression in Normal Tissues and Malignant Tumors: A Work inProgress

    SciTech Connect

    Spencer, Virginia A.; Xu, Ren; Bissell, Mina J.

    2006-08-01

    Almost three decades ago, we presented a model where theextracellular matrix (ECM) was postulated to influence gene expressionand tissue-specificity through the action of ECM receptors and thecytoskeleton. This hypothesis implied that ECM molecules could signal tothe nucleus and that the unit of function in higher organisms was not thecell alone, but the cell plus its microenvironment. We now know that ECMinvokes changes in tissue and organ architecture and that tissue, cell,nuclear, and chromatin structure are changed profoundly as a result ofand during malignant progression. Whereas some evidence has beengenerated for a link between ECM-induced alterations in tissuearchitecture and changes in both nuclear and chromatin organization, themanner by which these changes actively induce or repress gene expressionin normal and malignant cells is a topic in need of further attention.Here, we will discuss some key findings that may provide insights intomechanisms through which ECM could influence gene transcription and howtumor cells acquire the ability to overcome these levels ofcontrol.

  20. Functional assessment of time course microarray data

    PubMed Central

    Nueda, María José; Sebastián, Patricia; Tarazona, Sonia; García-García, Francisco; Dopazo, Joaquín; Ferrer, Alberto; Conesa, Ana

    2009-01-01

    Motivation Time-course microarray experiments study the progress of gene expression along time across one or several experimental conditions. Most developed analysis methods focus on the clustering or the differential expression analysis of genes and do not integrate functional information. The assessment of the functional aspects of time-course transcriptomics data requires the use of approaches that exploit the activation dynamics of the functional categories to where genes are annotated. Methods We present three novel methodologies for the functional assessment of time-course microarray data. i) maSigFun derives from the maSigPro method, a regression-based strategy to model time-dependent expression patterns and identify genes with differences across series. maSigFun fits a regression model for groups of genes labeled by a functional class and selects those categories which have a significant model. ii) PCA-maSigFun fits a PCA model of each functional class-defined expression matrix to extract orthogonal patterns of expression change, which are then assessed for their fit to a time-dependent regression model. iii) ASCA-functional uses the ASCA model to rank genes according to their correlation to principal time expression patterns and assess functional enrichment on a GSA fashion. We used simulated and experimental datasets to study these novel approaches. Results were compared to alternative methodologies. Results Synthetic and experimental data showed that the different methods are able to capture different aspects of the relationship between genes, functions and co-expression that are biologically meaningful. The methods should not be considered as competitive but they provide different insights into the molecular and functional dynamic events taking place within the biological system under study. PMID:19534758

  1. Possible Role of GADD45γ Methylation in Diffuse Large B-Cell Lymphoma: Does It Affect the Progression and Tissue Involvement?

    PubMed Central

    Barış, İkbal Cansu; Caner, Vildan; Şen Türk, Nilay; Sarı, İsmail; Hacıoğlu, Sibel; Doğu, Mehmet Hilmi; Çetin, Ozan; Tepeli, Emre; Can, Özge; Bağcı, Gülseren; Keskin, Ali

    2015-01-01

    Objective: Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma among adults and is characterized by heterogeneous clinical, immunophenotypic, and genetic features. Different mechanisms deregulating cell cycle and apoptosis play a role in the pathogenesis of DLBCL. Growth arrest DNA damage-inducible 45 (GADD45γ) is an important gene family involved in these mechanisms. The aims of this study are to determine the frequency of GADD45γ methylation, to evaluate the correlation between GADD45γ methylation and protein expression, and to investigate the relation between methylation status and clinicopathologic parameters in DLBCL tissues and reactive lymphoid node tissues from patients with reactive lymphoid hyperplasia. Materials and Methods: Thirty-six tissue samples of DLBCL and 40 nonmalignant reactive lymphoid node tissues were analyzed in this study. Methylation-sensitive high-resolution melting analysis was used for the determination of GADD45γ methylation status. The GADD45γ protein expression was determined by immunohistochemistry. Results: GADD45γ methylation was frequent (50.0%) in DLBCL. It was also significantly higher in advanced-stage tumors compared with early-stage (p=0.041). In contrast, unmethylated GADD45γ was associated with nodal involvement as the primary anatomical site (p=0.040). Conclusion: The results of this study show that, in contrast to solid tumors, the frequency of GADD45γ methylation is higher and this epigenetic alteration of GADD45γ may be associated with progression in DLBCL. In addition, nodal involvement is more likely to be present in patients with unmethylated GADD45γ. PMID:25912017

  2. Lung cancer in uranium miners: A tissue resource and pilot study. Progress report, September 25, 1992--May 31, 1993

    SciTech Connect

    Samet, J.M.

    1993-05-01

    This project involves two related activities directed toward understanding respiratory carcinogenesis in radon-exposed former uranium miners. The first activity involves a continuation of the tissue resource of lung cancer cases from former underground uranium miners and comparison cases from non-miners. The second activity is a pilot study for a proposed longitudinal study of respiratory carcinogenesis in former uranium miners. The objectives are to facilitate the investigation of molecular changes in radon exposed lung cancer cases and to develop methods for prospectively studying clinical, cytologic, cytogenetic, and molecular changes in the multi-event process of respiratory carcinogenesis, and to assess the feasibility of recruiting former uranium miners into a longitudinal study that collects multiple biologic specimens.

  3. Progression from high insulin resistance to type 2 diabetes does not entail additional visceral adipose tissue inflammation.

    PubMed

    Barbarroja, Nuria; Lopez-Pedrera, Chary; Garrido-Sanchez, Lourdes; Mayas, Maria Dolores; Oliva-Olivera, Wilfredo; Bernal-Lopez, Maria Rosa; El Bekay, Rajaa; Tinahones, Francisco Jose

    2012-01-01

    Obesity is associated with a low-grade chronic inflammation state. As a consequence, adipose tissue expresses pro-inflammatory cytokines that propagate inflammatory responses systemically elsewhere, promoting whole-body insulin resistance and consequential islet β-cell exhaustation. Thus, insulin resistance is considered the early stage of type 2 diabetes. However, there is evidence of obese individuals that never develop diabetes indicating that the mechanisms governing the association between the increase of inflammatory factors and type 2 diabetes are much more complex and deserve further investigation. We studied for the first time the differences in insulin signalling and inflammatory pathways in blood and visceral adipose tissue (VAT) of 20 lean healthy donors and 40 equal morbidly obese (MO) patients classified in high insulin resistance (high IR) degree and diabetes state. We studied the changes in proinflammatory markers and lipid content from serum; macrophage infiltration, mRNA expression of inflammatory cytokines and transcription factors, activation of kinases involved in inflammation and expression of insulin signalling molecules in VAT. VAT comparison of these experimental groups revealed that type 2 diabetic-MO subjects exhibit the same pro-inflammatory profile than the high IR-MO patients, characterized by elevated levels of IL-1β, IL-6, TNFα, JNK1/2, ERK1/2, STAT3 and NFκB. Our work rules out the assumption that the inflammation should be increased in obese people with type 2 diabetes compared to high IR obese. These findings indicate that some mechanisms, other than systemic and VAT inflammation must be involved in the development of type 2 diabetes in obesity. PMID:23110196

  4. Progression from High Insulin Resistance to Type 2 Diabetes Does Not Entail Additional Visceral Adipose Tissue Inflammation

    PubMed Central

    Barbarroja, Nuria; Lopez-Pedrera, Chary; Garrido-Sanchez, Lourdes; Mayas, Maria Dolores; Oliva-Olivera, Wilfredo; Bernal-Lopez, Maria Rosa; El Bekay, Rajaa; Tinahones, Francisco Jose

    2012-01-01

    Obesity is associated with a low-grade chronic inflammation state. As a consequence, adipose tissue expresses pro-inflammatory cytokines that propagate inflammatory responses systemically elsewhere, promoting whole-body insulin resistance and consequential islet β-cell exhaustation. Thus, insulin resistance is considered the early stage of type 2 diabetes. However, there is evidence of obese individuals that never develop diabetes indicating that the mechanisms governing the association between the increase of inflammatory factors and type 2 diabetes are much more complex and deserve further investigation. We studied for the first time the differences in insulin signalling and inflammatory pathways in blood and visceral adipose tissue (VAT) of 20 lean healthy donors and 40 equal morbidly obese (MO) patients classified in high insulin resistance (high IR) degree and diabetes state. We studied the changes in proinflammatory markers and lipid content from serum; macrophage infiltration, mRNA expression of inflammatory cytokines and transcription factors, activation of kinases involved in inflammation and expression of insulin signalling molecules in VAT. VAT comparison of these experimental groups revealed that type 2 diabetic-MO subjects exhibit the same pro-inflammatory profile than the high IR-MO patients, characterized by elevated levels of IL-1β, IL-6, TNFα, JNK1/2, ERK1/2, STAT3 and NFκB. Our work rules out the assumption that the inflammation should be increased in obese people with type 2 diabetes compared to high IR obese. These findings indicate that some mechanisms, other than systemic and VAT inflammation must be involved in the development of type 2 diabetes in obesity. PMID:23110196

  5. Components of the endocannabinoid and dopamine systems are dysregulated in Huntington's disease: analysis of publicly available microarray datasets

    PubMed Central

    Laprairie, Robert B; Bagher, Amina M; Precious, Sophie V; Denovan-Wright, Eileen M

    2015-01-01

    The endocannabinoid system (ECS) and the dopaminergic system (DAS) are two major regulators of basal ganglia function. During Huntington's disease (HD) pathogenesis, the expression of genes in both the ECS and DAS is dysregulated. The purpose of this study was to determine the changes that were consistently observed in the ECS and DAS during HD progression in the central nervous system (CNS) and in the periphery in different models of HD and human HD tissue. To do this, we conducted a meta-analysis of differential gene expression in the ECS and DAS using publicly available microarray data. The consolidated data were summarized as observed changes in gene expression (OCGE) using a weighted sum for each gene. In addition, consolidated data were compared to previously published studies that were not available in the gene expression omnibus (GEO) database. The resulting data confirm gene expression changes observed using different approaches and provide novel insights into the consistency between changes observed in human tissue and various models, as well as disease stage- and tissue-specific transcriptional dysregulation in HD. The major implication of the systems-wide data presented here is that therapeutic strategies targeting the ECS or DAS must consider the dynamic changes in gene expression over time and in different body areas, which occur during HD progression and the interconnectedness of the two systems. PMID:25692022

  6. Gene expression profiling in peanut using high density oligonucleotide microarrays

    PubMed Central

    Payton, Paxton; Kottapalli, Kameswara Rao; Rowland, Diane; Faircloth, Wilson; Guo, Baozhu; Burow, Mark; Puppala, Naveen; Gallo, Maria

    2009-01-01

    Background Transcriptome expression analysis in peanut to date has been limited to a relatively small set of genes and only recently has a significant number of ESTs been released into the public domain. Utilization of these ESTs for oligonucleotide microarrays provides a means to investigate large-scale transcript responses to a variety of developmental and environmental signals, ultimately improving our understanding of plant biology. Results We have developed a high-density oligonucleotide microarray for peanut using 49,205 publicly available ESTs and tested the utility of this array for expression profiling in a variety of peanut tissues. To identify putatively tissue-specific genes and demonstrate the utility of this array for expression profiling in a variety of peanut tissues, we compared transcript levels in pod, peg, leaf, stem, and root tissues. Results from this experiment showed 108 putatively pod-specific/abundant genes, as well as transcripts whose expression was low or undetected in pod compared to peg, leaf, stem, or root. The transcripts significantly over-represented in pod include genes responsible for seed storage proteins and desiccation (e.g., late-embryogenesis abundant proteins, aquaporins, legumin B), oil production, and cellular defense. Additionally, almost half of the pod-abundant genes represent unknown genes allowing for the possibility of associating putative function to these previously uncharacterized genes. Conclusion The peanut oligonucleotide array represents the majority of publicly available peanut ESTs and can be used as a tool for expression profiling studies in diverse tissues. PMID:19523230

  7. Differential infection patterns of CD4+ T cells and lymphoid tissue viral burden distinguish progressive and nonprogressive lentiviral infections.

    PubMed

    Brenchley, Jason M; Vinton, Carol; Tabb, Brian; Hao, Xing Pei; Connick, Elizabeth; Paiardini, Mirko; Lifson, Jeffrey D; Silvestri, Guido; Estes, Jacob D

    2012-11-15

    Nonhuman primate natural hosts for simian immunodeficiency viruses (SIV) develop a nonresolving chronic infection but do not develop AIDS. Mechanisms to explain the nonprogressive nature of SIV infection in natural hosts that underlie maintained high levels of plasma viremia without apparent loss of target cells remain unclear. Here we used comprehensive approaches (ie, FACS sorting, quantitative RT-PCR, immunohistochemistry, and in situ hybridization) to study viral infection within subsets of peripheral blood and lymphoid tissue (LT) CD4(+) T cells in cohorts of chronically SIV-infected rhesus macaques (RMs), HIV-infected humans, and SIVsmm-infected sooty mangabeys (SMs). We find: (1) infection frequencies among CD4(+) T cells in chronically SIV-infected RMs are significantly higher than those in SIVsmm-infected SMs; (2) infected cells are found in distinct anatomic LT niches and different CD4(+) T-cell subsets in SIV-infected RMs and SMs, with infection patterns of RMs reflecting HIV infection in humans; (3) T(FH) cells are infected at higher frequencies in RMs and humans than in SMs; and (4) LT viral burden, including follicular dendritic cell deposition of virus, is increased in RMs and humans compared with SMs. These data provide insights into how natural hosts are able to maintain high levels of plasma viremia while avoiding development of immunodeficiency. PMID:22990012

  8. Differential infection patterns of CD4+ T cells and lymphoid tissue viral burden distinguish progressive and nonprogressive lentiviral infections

    PubMed Central

    Vinton, Carol; Tabb, Brian; Hao, Xing Pei; Connick, Elizabeth; Paiardini, Mirko; Lifson, Jeffrey D.; Silvestri, Guido

    2012-01-01

    Nonhuman primate natural hosts for simian immunodeficiency viruses (SIV) develop a nonresolving chronic infection but do not develop AIDS. Mechanisms to explain the nonprogressive nature of SIV infection in natural hosts that underlie maintained high levels of plasma viremia without apparent loss of target cells remain unclear. Here we used comprehensive approaches (ie, FACS sorting, quantitative RT-PCR, immunohistochemistry, and in situ hybridization) to study viral infection within subsets of peripheral blood and lymphoid tissue (LT) CD4+ T cells in cohorts of chronically SIV-infected rhesus macaques (RMs), HIV-infected humans, and SIVsmm-infected sooty mangabeys (SMs). We find: (1) infection frequencies among CD4+ T cells in chronically SIV-infected RMs are significantly higher than those in SIVsmm-infected SMs; (2) infected cells are found in distinct anatomic LT niches and different CD4+ T-cell subsets in SIV-infected RMs and SMs, with infection patterns of RMs reflecting HIV infection in humans; (3) TFH cells are infected at higher frequencies in RMs and humans than in SMs; and (4) LT viral burden, including follicular dendritic cell deposition of virus, is increased in RMs and humans compared with SMs. These data provide insights into how natural hosts are able to maintain high levels of plasma viremia while avoiding development of immunodeficiency. PMID:22990012

  9. Development and function of membrane systems in plant tissue. Annual technical progress report, 15 September 1981-15 August 1982

    SciTech Connect

    Hanson, J B

    1982-01-01

    Over the past 11 months we have continued investigation of ion transport mechanisms in corn roots and mitochondria. In mitochondria we find that only citrate and isocitrate are transported by the H/sup +//citrate symporter. However, the in vivo function of this carrier remains in doubt because citrate does not appear to be an effective substrate for corn mitochondria. Studies with roots have been directed to why various types of injury or shock all result in temporary blockage of the H/sup +/-efflux pump in the plasmamembrane. It appears this may be due to an injury-mediated Ca/sup 2 +/ influx into the tissue, which by raising free Ca/sup 2 +/ in the cytosal activates calmodulin (CaM). In turn, the Ca.CaM complex appears to activate protein kinase, phosphorylating membrane proteins. It is possible that one of these phosphorylated proteins is responsible for inactivation of the H/sup +/-ATPase. Future work is planned around the consequences of Ca/sup 2 +/ influx into the root cell subsequent to injury, investigating the recovery of the H/sup +/-ATPase and the initiation of the biosyntheses which lead to augmented ion transport.

  10. Self-assembling peptide nanofiber hydrogels in tissue engineering and regenerative medicine: Progress, design guidelines, and applications.

    PubMed

    Koutsopoulos, Sotirios

    2016-04-01

    Until the mid-1980s, mainly biologists were conducting peptide research. This changed with discoveries that opened new paths of research involving the use of peptides in bioengineering, biotechnology, biomedicine, nanotechnology, and bioelectronics. Peptide engineering and rational design of novel peptide sequences with unique and tailor-made properties further expanded the field. The discovery of short self-assembling peptides, which upon association form well-defined supramolecular architectures, created new and exciting areas of research. Depending on the amino acid sequence, the pH, and the type of the electrolyte in the medium, peptide self-assembly leads to the formation of nanofibers, which are further organized to form a hydrogel. In this review, the application of ionic complementary peptides which self-assemble to form nanofiber hydrogels for tissue engineering and regenerative medicine will be discussed through a selective presentation of the most important work performed during the last 25 years. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1002-1016, 2016. PMID:26707893

  11. Traumatic brain injury in young rats leads to progressive behavioral deficits coincident with altered tissue properties in adulthood.

    PubMed

    Ajao, David O; Pop, Viorela; Kamper, Joel E; Adami, Arash; Rudobeck, Emil; Huang, Lei; Vlkolinsky, Roman; Hartman, Richard E; Ashwal, Stephen; Obenaus, André; Badaut, Jérôme

    2012-07-20

    Traumatic brain injury (TBI) affects many infants and children, and results in enduring motor and cognitive impairments with accompanying changes in white matter tracts, yet few experimental studies in rodent juvenile models of TBI (jTBI) have examined the timeline and nature of these deficits, histologically and functionally. We used a single controlled cortical impact (CCI) injury to the parietal cortex of rats at post-natal day (P) 17 to evaluate behavioral alterations, injury volume, and morphological and molecular changes in gray and white matter, with accompanying measures of electrophysiological function. At 60 days post-injury (dpi), we found that jTBI animals displayed behavioral deficits in foot-fault and rotarod tests, along with a left turn bias throughout their early developmental stages and into adulthood. In addition, anxiety-like behaviors on the zero maze emerged in jTBI animals at 60 dpi. The final lesion constituted only ∼3% of brain volume, and morphological tissue changes were evaluated using MRI, as well as immunohistochemistry for neuronal nuclei (NeuN), myelin basic protein (MBP), neurofilament-200 (NF200), and oligodendrocytes (CNPase). White matter morphological changes were associated with a global increase in MBP immunostaining and reduced compound action potential amplitudes at 60 dpi. These results suggest that brain injury early in life can induce long-term white matter dysfunction, occurring in parallel with the delayed development and persistence of behavioral deficits, thus modeling clinical and longitudinal TBI observations. PMID:22697253

  12. Traumatic Brain Injury in Young Rats Leads to Progressive Behavioral Deficits Coincident with Altered Tissue Properties in Adulthood

    PubMed Central

    Ajao, David O.; Pop, Viorela; Kamper, Joel E.; Adami, Arash; Rudobeck, Emil; Huang, Lei; Vlkolinsky, Roman; Hartman, Richard E.; Ashwal, Stephen; Obenaus, André

    2012-01-01

    Abstract Traumatic brain injury (TBI) affects many infants and children, and results in enduring motor and cognitive impairments with accompanying changes in white matter tracts, yet few experimental studies in rodent juvenile models of TBI (jTBI) have examined the timeline and nature of these deficits, histologically and functionally. We used a single controlled cortical impact (CCI) injury to the parietal cortex of rats at post-natal day (P) 17 to evaluate behavioral alterations, injury volume, and morphological and molecular changes in gray and white matter, with accompanying measures of electrophysiological function. At 60 days post-injury (dpi), we found that jTBI animals displayed behavioral deficits in foot-fault and rotarod tests, along with a left turn bias throughout their early developmental stages and into adulthood. In addition, anxiety-like behaviors on the zero maze emerged in jTBI animals at 60 dpi. The final lesion constituted only ∼3% of brain volume, and morphological tissue changes were evaluated using MRI, as well as immunohistochemistry for neuronal nuclei (NeuN), myelin basic protein (MBP), neurofilament-200 (NF200), and oligodendrocytes (CNPase). White matter morphological changes were associated with a global increase in MBP immunostaining and reduced compound action potential amplitudes at 60 dpi. These results suggest that brain injury early in life can induce long-term white matter dysfunction, occurring in parallel with the delayed development and persistence of behavioral deficits, thus modeling clinical and longitudinal TBI observations. PMID:22697253

  13. Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray.

    PubMed

    Ramirez, Lisa S; Wang, Jun

    2016-01-01

    Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this report, we renovate the antibody microarray technology by using a novel approach for manufacturing and by introducing new features. The fabrication of our high-density antibody microarray is accomplished through perpendicularly oriented flow-patterning of single stranded DNAs and subsequent conversion mediated by DNA-antibody conjugates. This protocol outlines the critical steps in flow-patterning DNA, producing and purifying DNA-antibody conjugates, and assessing the quality of the fabricated microarray. The uniformity and sensitivity are comparable with conventional microarrays, while our microarray fabrication does not require the assistance of an array printer and can be performed in most research laboratories. The other major advantage is that the size of our microarray units is 10 times smaller than that of printed arrays, offering the unique capability of analyzing functional proteins from single cells when interfacing with generic microchip designs. This barcode technology can be widely employed in biomarker detection, cell signaling studies, tissue engineering, and a variety of clinical applications. PMID:26780370

  14. p53 and Beta-Catenin Expression in Gallbladder Tissues and Correlation with Tumor Progression in Gallbladder Cancer

    PubMed Central

    Ghosh, Mila; Sakhuja, Puja; Singh, Shivendra; Agarwal, Anil K.

    2013-01-01

    Background/Aim: The inactivation of the tumor suppressor gene and activation of the proto-oncogene are key steps in the development of human cancer. p53 and beta-catenin are examples of such genes, respectively. In the present study, our aim was to determine the role of these genes in the carcinogenesis of the gallbladder by immunohistochemistry. Patients and Methods: Sections from paraffin-embedded blocks of surgically resected specimens of gallbladder cancer (GBC) (80 cases), chronic cholecystitis (60 cases), and control gallbladders (10 cases) were stained with the monoclonal antibody p53, and polyclonal antibody beta-catenin. Results were scored semiquantitatively and statistical analysis performed. p53 expression was scored as percentage of the nuclei stained. Beta-catenin expression was scored as type of expression–membranous, cytoplasmic, and nuclear staining. Beta-catenin expression was correlated with tumor invasiveness, differentiation, and stage. Results: Over-expression of p53 was seen in 56.25% of GBC cases and was not seen in chronic cholecystitis or in control gallbladders. p53 expression in gallbladder cancer was significantly higher than in inflammatory or control gallbladders (P < 0.0001). p53 expression increased with increasing tumor grade (P = 0.039). Beta-catenin nuclear expression was seen in 75% cases of gallbladder cancer and in no case of chronic cholecystitis and control gallbladder. Beta-catenin nuclear expression increased with tumor depth invasiveness, and grade (P = 0.028 and P = 0.0152, respectively). Conclusion: p53 and beta-catenin nuclear expression is significantly higher in GBC. p53 expression correlates with increasing tumor grade while beta-catenin nuclear expression correlates with tumor grade and depth of invasion, thus suggesting a role for these genes in tumor progression of GBC. PMID:23319036

  15. Reverse phase protein microarrays advance to use in clinical trials

    PubMed Central

    Mueller, Claudius; Liotta, Lance A.; Espina, Virginia

    2010-01-01

    Individualizing cancer therapy for molecular targeted inhibitors requires a new class of molecular profiling technology that can map the functional state of the cancer cell signal pathways containing the drug targets. Reverse phase protein microarrays (RPMA) are a technology platform designed for quantitative, multiplexed analysis of specific phosphorylated, cleaved, or total (phosphorylated and non-phosphorylated) forms of cellular proteins from a limited amount of sample. This class of microarray can be used to interrogate tissue samples, cells, serum, or body fluids. RPMA were previously a research tool; now this technology has graduated to use in research clinical trials with clinical grade sensitivity and precision. In this review we describe the application of RPMA for multiplexed signal pathway analysis in therapeutic monitoring, biomarker discovery, and evaluation of pharmaceutical targets, and conclude with a summary of the technical aspects of RPMA construction and analysis. PMID:20974554

  16. Modeling Oncogenic Signaling in Colon Tumors by Multidirectional Analyses of Microarray Data Directed for Maximization of Analytical Reliability

    PubMed Central

    Rubel, Tymon; Paziewska, Agnieszka; Mikula, Michal; Jarosz, Dorota; Pachlewski, Jacek; Oledzki, Janusz; Ostrowsk, Jerzy

    2010-01-01

    Background Clinical progression of colorectal cancers (CRC) may occur in parallel with distinctive signaling alterations. We designed multidirectional analyses integrating microarray-based data with biostatistics and bioinformatics to elucidate the signaling and metabolic alterations underlying CRC development in the adenoma-carcinoma sequence. Methodology/Principal Findings Studies were performed on normal mucosa, adenoma, and carcinoma samples obtained during surgery or colonoscopy. Collections of cryostat sections prepared from the tissue samples were evaluated by a pathologist to control the relative cell type content. The measurements were done using Affymetrix GeneChip HG-U133plus2, and probe set data was generated using two normalization algorithms: MAS5.0 and GCRMA with least-variant set (LVS). The data was evaluated using pair-wise comparisons and data decomposition into singular value decomposition (SVD) modes. The method selected for the functional analysis used the Kolmogorov-Smirnov test. Expressional profiles obtained in 105 samples of whole tissue sections were used to establish oncogenic signaling alterations in progression of CRC, while those representing 40 microdissected specimens were used to select differences in KEGG pathways between epithelium and mucosa. Based on a consensus of the results obtained by two normalization algorithms, and two probe set sorting criteria, we identified 14 and 17 KEGG signaling and metabolic pathways that are significantly altered between normal and tumor samples and between benign and malignant tumors, respectively. Several of them were also selected from the raw microarray data of 2 recently published studies (GSE4183 and GSE8671). Conclusion/Significance Although the proposed strategy is computationally complex and labor–intensive, it may reduce the number of false results. PMID:20957034

  17. Microarray based analysis of gene regulation by microRNA in intervertebral disc degeneration.

    PubMed

    Hu, Peng; Feng, Bo; Wang, Guanglin; Ning, Bin; Jia, Tanghong

    2015-10-01

    The present study aimed to explore the underlying mechanism of the development of intervertebral disc degeneration (IDD) by bioinformatics based on microarray datasets. GSE 19943 and GSE 34095 datasets downloaded from Gene Expression Omnibus data were used to screen the differentially expressed genes (DEGs) in IDD. The correlation between microRNAs and target genes was investigated using different algorithms. The underlying molecular mechanisms of the target genes were then explored using Kyoto Encyclopedia of Genes and Genomes pathway and Gene Ontology function enrichment analysis. A total of 9 differentially expressed microRNAs, including 3 down‑ and 6 upregulated microRNAs and 850 DEGs were identified in tissue from patients with IDD. Two regulation networks of the target genes by microRNAs were constructed, including 33 upregulated microRNA‑target gene pairs and 4 downregulated microRNA‑target gene pairs. Certain target genes had been demonstrated to be involved in IDD progression via various pathways, including in the cell cycle and pathways in cancer. In addition, two important microRNAs (microRNA‑222 and microRNA‑589) were identified that were pivotal for the development of IDD, and their target genes, CDKNAB and SMAD4. In conclusion, a comprehensive miRNA‑target gene regulatory network was constructed, which was found to be important in IDD progression. PMID:26134418

  18. THE ABRF MARG MICROARRAY SURVEY 2005: TAKING THE PULSE ON THE MICROARRAY FIELD

    EPA Science Inventory

    Over the past several years microarray technology has evolved into a critical component of any discovery based program. Since 1999, the Association of Biomolecular Resource Facilities (ABRF) Microarray Research Group (MARG) has conducted biennial surveys designed to generate a pr...

  19. Protein-Binding Microarray Analysis of Tumor Suppressor AP2? Target Gene Specificity

    PubMed Central

    Schtz, Frdric; Grasso, Luigino; Egener-Kuhn, Tanja; Delaloye, Jean-Franois; Lehr, Hans-Anton; Vogel, Horst; Mermod, Nicolas

    2011-01-01

    Cheap and massively parallel methods to assess the DNA-binding specificity of transcription factors are actively sought, given their prominent regulatory role in cellular processes and diseases. Here we evaluated the use of protein-binding microarrays (PBM) to probe the association of the tumor suppressor AP2? with 6000 human genomic DNA regulatory sequences. We show that the PBM provides accurate relative binding affinities when compared to quantitative surface plasmon resonance assays. A PBM-based study of human healthy and breast tumor tissue extracts allowed the identification of previously unknown AP2? target genes and it revealed genes whose direct or indirect interactions with AP2? are affected in the diseased tissues. AP2? binding and regulation was confirmed experimentally in human carcinoma cells for novel target genes involved in tumor progression and resistance to chemotherapeutics, providing a molecular interpretation of AP2? role in cancer chemoresistance. Overall, we conclude that this approach provides quantitative and accurate assays of the specificity and activity of tumor suppressor and oncogenic proteins in clinical samples, interfacing genomic and proteomic assays. PMID:21876733

  20. Tumour class prediction and discovery by microarray-based DNA methylation analysis

    PubMed Central

    Adorján, Péter; Distler, Jürgen; Lipscher, Evelyne; Model, Fabian; Müller, Jürgen; Pelet, Cécile; Braun, Aron; Florl, Andrea R.; Gütig, David; Grabs, Gabi; Howe, André; Kursar, Mischo; Lesche, Ralf; Leu, Erik; Lewin, André; Maier, Sabine; Müller, Volker; Otto, Thomas; Scholz, Christian; Schulz, Wolfgang A.; Seifert, Hans-Helge; Schwope, Ina; Ziebarth, Heike; Berlin, Kurt; Piepenbrock, Christian; Olek, Alexander

    2002-01-01

    Aberrant DNA methylation of CpG sites is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation occurs in a tumour type-specific manner. However, large-scale analysis of candidate genes has so far been hampered by the lack of high throughput assays for methylation detection. We have developed the first microarray-based technique which allows genome-wide assessment of selected CpG dinucleotides as well as quantification of methylation at each site. Several hundred CpG sites were screened in 76 samples from four different human tumour types and corresponding healthy controls. Discriminative CpG dinucleotides were identified for different tissue type distinctions and used to predict the tumour class of as yet unknown samples with high accuracy using machine learning techniques. Some CpG dinucleotides correlate with progression to malignancy, whereas others are methylated in a tissue-specific manner independent of malignancy. Our results demonstrate that genome-wide analysis of methylation patterns combined with supervised and unsupervised machine learning techniques constitute a powerful novel tool to classify human cancers. PMID:11861926

  1. Protein Microarrays and Biomarkers of Infectious Disease

    PubMed Central

    Natesan, Mohan; Ulrich, Robert G.

    2010-01-01

    Protein microarrays are powerful tools that are widely used in systems biology research. For infectious diseases, proteome microarrays assembled from proteins of pathogens will play an increasingly important role in discovery of diagnostic markers, vaccines, and therapeutics. Distinct formats of protein microarrays have been developed for different applications, including abundance-based and function-based methods. Depending on the application, design issues should be considered, such as the need for multiplexing and label or label free detection methods. New developments, challenges, and future demands in infectious disease research will impact the application of protein microarrays for discovery and validation of biomarkers. PMID:21614200

  2. Collagenase and tissue plasminogen activator production in developing rat calvariae: normal progression despite fetal exposure to microgravity

    NASA Technical Reports Server (NTRS)

    Davis, B. A.; Sipe, B.; Gershan, L. A.; Fiacco, G. J.; Lorenz, T. C.; Jeffrey, J. J.; Partridge, N. C.

    1998-01-01

    Exposure to zero gravity has been shown to cause a decrease in bone formation. This implicates osteoblasts as the gravity-sensing cell in bone. Osteoblasts also are known to produce neutral proteinases, including collagenase and tissue plasminogen activator (tPA), which are thought to be important in bone development and remodeling. The present study investigated the effects of zero gravity on development of calvariae and their expression of collagenase and tPA. After in utero exposure to zero gravity for 9 days on the NASA STS-70 space shuttle mission, the calvariae of rat pups were examined by immunohistochemistry for the presence and location of these two proteinases. The ages of the pups were from gestational day 20 (G20) to postnatal (PN) day 35. Both collagenase and tPA were found to be present at all ages examined, with the greatest amount of both proteinases present in the PN14 rats. At later ages, high amounts were maintained for tPA but collagenase decreased substantially between ages PN21 to PN35. The location of collagenase was found to be associated with bone-lining cells, osteoblasts, osteocytes, and in the matrix along cement lines. In contrast, tPA was associated with endothelial cells lining the blood vessels entering bone. The presence and developmental expression of these two proteinases appeared to be unaffected by the exposure to zero gravity. The calvarial thickness of the pups was also examined; again the exposure to zero gravity showed little to no effect on the growth of the calvariae. Notably, from G20 to PN14, calvarial thickness increased dramatically, reaching a plateau after this age. It was apparent that elevated collagenase expression correlated with rapid bone growth in the period from G20 to PN14. To conclude, collagenase and tPA are present during the development of rat calvariae. Despite being produced by the same cell in vitro, i.e., the osteoblast, they are located in distinctly different places in bone in vivo. Their presence, developmental expression, and quantity do not seem to be affected by a brief exposure to zero gravity in utero.

  3. Micatu Tissue Arrayer | NCI Technology Transfer Center | TTC

    Cancer.gov

    An NCI researcher recognized a critical need to create a low-cost, easy-to-use tissue microarrayer (TMA), an instrument used by researchers and pathologists to accurately examine tissue samples from patients.

  4. Photoelectrochemical synthesis of DNA microarrays

    PubMed Central

    Chow, Brian Y.; Emig, Christopher J.; Jacobson, Joseph M.

    2009-01-01

    Optical addressing of semiconductor electrodes represents a powerful technology that enables the independent and parallel control of a very large number of electrical phenomena at the solid-electrolyte interface. To date, it has been used in a wide range of applications including electrophoretic manipulation, biomolecule sensing, and stimulating networks of neurons. Here, we have adapted this approach for the parallel addressing of redox reactions, and report the construction of a DNA microarray synthesis platform based on semiconductor photoelectrochemistry (PEC). An amorphous silicon photoconductor is activated by an optical projection system to create virtual electrodes capable of electrochemically generating protons; these PEC-generated protons then cleave the acid-labile dimethoxytrityl protecting groups of DNA phosphoramidite synthesis reagents with the requisite spatial selectivity to generate DNA microarrays. Furthermore, a thin-film porous glass dramatically increases the amount of DNA synthesized per chip by over an order of magnitude versus uncoated glass. This platform demonstrates that PEC can be used toward combinatorial bio-polymer and small molecule synthesis. PMID:19706433

  5. Yeast Proteomics and Protein Microarrays

    PubMed Central

    Chen, Rui; Snyder, Michael

    2010-01-01

    Our understanding of biological processes as well as human diseases has improved greatly thanks to studies on model organisms such as yeast. The power of scientific approaches with yeast lies in its relatively simple genome, its facile classical and molecular genetics, as well as the evolutionary conservation of many basic biological mechanisms. However, even in this simple model organism, systems biology studies, especially proteomic studies had been an intimidating task. During the past decade, powerful high-throughput technologies in proteomic research have been developed for yeast including protein microarray technology. The protein microarray technology allows the interrogation of protein-protein, protein-DNA, protein-small molecule interaction networks as well as post-translational modification networks in a large-scale, high-throughput manner. With this technology, many groundbreaking findings have been established in studies with the budding yeast Saccharomyces cerevisiae, most of which could have been unachievable with traditional approaches. Discovery of these networks has profound impact on explicating biological processes with a proteomic point of view, which may lead to a better understanding of normal biological phenomena as well as various human diseases. PMID:20728591

  6. 2008 Microarray Research Group (MARG Survey): Sensing the State of Microarray Technology

    EPA Science Inventory

    Over the past several years, the field of microarrays has grown and evolved drastically. In its continued efforts to track this evolution and transformation, the ABRF-MARG has once again conducted a survey of international microarray facilities and individual microarray users. Th...

  7. THE ABRF-MARG MICROARRAY SURVEY 2004: TAKING THE PULSE OF THE MICROARRAY FIELD

    EPA Science Inventory

    Over the past several years, the field of microarrays has grown and evolved drastically. In its continued efforts to track this evolution, the ABRF-MARG has once again conducted a survey of international microarray facilities and individual microarray users. The goal of the surve...

  8. 78 FR 18360 - Prospective Grant of Start-Up Exclusive License: Manual Device for Constructing Tissue Micro...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-26

    ... microarray construction having a block of embedding medium, a platform configured to retain the block, a... microarray. The field of use may be limited to the field of devices for construction of tissue...

  9. Ontology-Based Analysis of Microarray Data.

    PubMed

    Giuseppe, Agapito; Milano, Marianna

    2016-01-01

    The importance of semantic-based methods and algorithms for the analysis and management of biological data is growing for two main reasons. From a biological side, knowledge contained in ontologies is more and more accurate and complete, from a computational side, recent algorithms are using in a valuable way such knowledge. Here we focus on semantic-based management and analysis of protein interaction networks referring to all the approaches of analysis of protein-protein interaction data that uses knowledge encoded into biological ontologies.Semantic approaches for studying high-throughput data have been largely used in the past to mine genomic and expression data. Recently, the emergence of network approaches for investigating molecular machineries has stimulated in a parallel way the introduction of semantic-based techniques for analysis and management of network data. The application of these computational approaches to the study of microarray data can broad the application scenario of them and simultaneously can help the understanding of disease development and progress. PMID:25971913

  10. Alteration of liver glycopatterns during cirrhosis and tumor progression induced by HBV.

    PubMed

    Qin, Yannan; Zhong, Yaogang; Ma, Tianran; Wu, Fei; Wu, Haoxiang; Yu, Hanjie; Huang, Chen; Li, Zheng

    2016-04-01

    The incidence of hepatocellular carcinoma (HCC) is closely correlated with hepatitis B virus (HBV)-induced liver cirrhosis. Structural changes in the glycans of serum and tissue proteins are reliable indicators of liver damage. However, little is known about the alteration of liver glycopatterns during cirrhosis and tumor progression induced by HBV infection. This study compared the differential expression of liver glycopatterns in 7 sets of normal pericarcinomatous tissues (PCTs), cirrhotic, and tumor tissues from patients with liver cirrhosis and HCC induced by HBV using lectin microarrays. Fluorescence-based lectin histochemistry and lectin blotting were further utilized to validate and assess the expression and distribution of certain glycans in 9 sets of corresponding liver tissue sections. Eight lectins (e.g., Jacalin and AAL) revealed significant difference in cirrhotic tissues versus PCTs. Eleven lectins (e.g., EEL and SJA) showed significant alteration during cirrhotic and tumor progression. The expression of Galα1-3(Fucα1-2)Gal (EEL) and fucosyltransferase 1 was mainly increasing in the cytoplasm of hepatocytes during PCTs-cirrhotic-tumor tissues progression, while the expression of T antigen (ACA and PNA) was decreased sharply in cytoplasm of tumor hepatocytes. Understanding the precision alteration of liver glycopatterns related to the development of hepatitis, cirrhosis, and tumor induced by HBV infection may help elucidate the molecular mechanisms underlying the progression of chronic liver diseases and develop new antineoplastic therapeutic strategies. PMID:26833199

  11. Microarrays Made Simple: "DNA Chips" Paper Activity

    ERIC Educational Resources Information Center

    Barnard, Betsy

    2006-01-01

    DNA microarray technology is revolutionizing biological science. DNA microarrays (also called DNA chips) allow simultaneous screening of many genes for changes in expression between different cells. Now researchers can obtain information about genes in days or weeks that used to take months or years. The paper activity described in this article…

  12. BOS TAURUS 60MER OLIGONUCLEOTIDE MICROARRAY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bovine high-density long oligonucleotide microarrays were developed, tested and optimized. The bovine microarray with ~345,000 features (60mer oligonucleotides) representing 45,383 cattle unique sequences was designed and produced with Maskless Array Synthesizer technology. The 45,383 unique sequenc...

  13. Microarrays Made Simple: "DNA Chips" Paper Activity

    ERIC Educational Resources Information Center

    Barnard, Betsy

    2006-01-01

    DNA microarray technology is revolutionizing biological science. DNA microarrays (also called DNA chips) allow simultaneous screening of many genes for changes in expression between different cells. Now researchers can obtain information about genes in days or weeks that used to take months or years. The paper activity described in this article

  14. Protein-Based Microarray for the Detection of Pathogenic Bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microarrays have been used for gene expression and protein interaction studies, but recently, multianalyte diagnostic assays have employed the microarray platform. We developed a microarray immunoassay for bacteria, with biotinylated capture antibodies on streptavidin slides. To complete the fluor...

  15. Microarray Analysis for Saccharomyces cerevisiae

    PubMed Central

    Tighe, Scott; Hunter, Tim; Reed, Pat; Murray, Janet

    2011-01-01

    In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software. PMID:21505409

  16. DNA Microarrays for Identifying Fishes

    PubMed Central

    Nölte, M.; Weber, H.; Silkenbeumer, N.; Hjörleifsdottir, S.; Hreggvidsson, G. O.; Marteinsson, V.; Kappel, K.; Planes, S.; Tinti, F.; Magoulas, A.; Garcia Vazquez, E.; Turan, C.; Hervet, C.; Campo Falgueras, D.; Antoniou, A.; Landi, M.; Blohm, D.

    2008-01-01

    In many cases marine organisms and especially their diverse developmental stages are difficult to identify by morphological characters. DNA-based identification methods offer an analytically powerful addition or even an alternative. In this study, a DNA microarray has been developed to be able to investigate its potential as a tool for the identification of fish species from European seas based on mitochondrial 16S rDNA sequences. Eleven commercially important fish species were selected for a first prototype. Oligonucleotide probes were designed based on the 16S rDNA sequences obtained from 230 individuals of 27 fish species. In addition, more than 1200 sequences of 380 species served as sequence background against which the specificity of the probes was tested in silico. Single target hybridisations with Cy5-labelled, PCR-amplified 16S rDNA fragments from each of the 11 species on microarrays containing the complete set of probes confirmed their suitability. True-positive, fluorescence signals obtained were at least one order of magnitude stronger than false-positive cross-hybridisations. Single nontarget hybridisations resulted in cross-hybridisation signals at approximately 27% of the cases tested, but all of them were at least one order of magnitude lower than true-positive signals. This study demonstrates that the 16S rDNA gene is suitable for designing oligonucleotide probes, which can be used to differentiate 11 fish species. These data are a solid basis for the second step to create a “Fish Chip” for approximately 50 fish species relevant in marine environmental and fisheries research, as well as control of fisheries products. PMID:18270778

  17. Chaotic mixer improves microarray hybridization.

    PubMed

    McQuain, Mark K; Seale, Kevin; Peek, Joel; Fisher, Timothy S; Levy, Shawn; Stremler, Mark A; Haselton, Frederick R

    2004-02-15

    Hybridization is an important aspect of microarray experimental design which influences array signal levels and the repeatability of data within an array and across different arrays. Current methods typically require 24h and use target inefficiently. In these studies, we compare hybridization signals obtained in conventional static hybridization, which depends on diffusional target delivery, with signals obtained in a dynamic hybridization chamber, which employs a fluid mixer based on chaotic advection theory to deliver targets across a conventional glass slide array. Microarrays were printed with a pattern of 102 identical probe spots containing a 65-mer oligonucleotide capture probe. Hybridization of a 725-bp fluorescently labeled target was used to measure average target hybridization levels, local signal-to-noise ratios, and array hybridization uniformity. Dynamic hybridization for 1h with 1 or 10ng of target DNA increased hybridization signal intensities approximately threefold over a 24-h static hybridization. Similarly, a 10- or 60-min dynamic hybridization of 10ng of target DNA increased hybridization signal intensities fourfold over a 24h static hybridization. In time course studies, static hybridization reached a maximum within 8 to 12h using either 1 or 10ng of target. In time course studies using the dynamic hybridization chamber, hybridization using 1ng of target increased to a maximum at 4h and that using 10ng of target did not vary over the time points tested. In comparison to static hybridization, dynamic hybridization reduced the signal-to-noise ratios threefold and reduced spot-to-spot variation twofold. Therefore, we conclude that dynamic hybridization based on a chaotic mixer design improves both the speed of hybridization and the maximum level of hybridization while increasing signal-to-noise ratios and reducing spot-to-spot variation. PMID:14751256

  18. Chromatin immunoprecipitation and microarray-based analysis of protein location.

    PubMed

    Lee, Tong Ihn; Johnstone, Sarah E; Young, Richard A

    2006-01-01

    Genome-wide location analysis, also known as ChIP-Chip, combines chromatin immunoprecipitation and DNA microarray analysis to identify protein-DNA interactions that occur in living cells. Protein-DNA interactions are captured in vivo by chemical crosslinking. Cell lysis, DNA fragmentation and immunoaffinity purification of the desired protein will co-purify DNA fragments that are associated with that protein. The enriched DNA population is then labeled, combined with a differentially labeled reference sample and applied to DNA microarrays to detect enriched signals. Various computational and bioinformatic approaches are then applied to normalize the enriched and reference channels, to connect signals to the portions of the genome that are represented on the DNA microarrays, to provide confidence metrics and to generate maps of protein-genome occupancy. Here, we describe the experimental protocols that we use from crosslinking of cells to hybridization of labeled material, together with insights into the aspects of these protocols that influence the results. These protocols require approximately 1 week to complete once sufficient numbers of cells have been obtained, and have been used to produce robust, high-quality ChIP-chip results in many different cell and tissue types. PMID:17406303

  19. Microarrays as cancer keys: an array of possibilities.

    PubMed

    Mohr, Steve; Leikauf, George D; Keith, Grard; Rihn, Bertrand H

    2002-07-15

    Malignant transformation results from accumulation of genetic and epigenetic events. Functional studies of cancer will be crucial to our understanding of its complexity and polymorphism. There is no doubt that emerging genomic and proteomic technologies will facilitate such investigations. Microarray technology is a new and efficient approach to extract data of biomedical relevance for a wide range of applications. In cancer research, it will provide high-throughput and valuable insights into differences in an individual's tumor as compared with constitutional DNA, mRNA expression, and protein expression and activity. Across individuals, comparisons could provide tissue-specific disease signatures that provide diagnosis based on hundreds of informative genes. The resulting product should be a wealth of tumor-associated and tumor-specific biomarkers, which may help in cancer etiology, diagnosis, and therapy and ultimately lead to "molecular nosology" of cancers. This review highlights the recent developments in microarray technologies in cancer research, focuses on the results obtained so far, and describes the eventual use of microarray technology for clinical applications. PMID:12118031

  20. An oligonucleotide microarray for mouse imprinted genes profiling.

    PubMed

    Vig, A; Gallou-Kabani, C; Gross, M S; Fabre, A; Junien, C; Jais, J P

    2006-01-01

    Genomic imprinting is an epigenetic phenomenon unique to mammals that causes some genes to be expressed according to their parental origin. It results in developmental asymmetry in the function of the parental genomes. We describe here a method for the profiling of imprinted genes based on the development of a mouse imprinting microchip containing oligonucleotides corresponding to 493 genes, including most of the known imprinted genes (IG = 63), genes involved in epigenetic processes (EPI = 15), in metabolism (= 147), in obesity (= 10) and in neurotransmission (= 256) and housekeeping reference genes (= 2). This custom oligonucleotide microarray has been constructed to make data analysis and handling more manageable than pangenomic microarrays. As a proof of concept we present the differential expression of these 493 genes in different tissues (liver, placenta, embryo) of C57BL6/J mice fed different diets. Appropriate experimental strategies and statistical tools were defined at each step of the data analysis process with regard to the different sources of constraints. Data were confirmed by expression analyses based on quantitative real-time PCR. These oligochips should make it possible to increase our understanding of the involvement of imprinted genes in the timing of expression programs, tissue by tissue, stage by stage, in response to nutrients, lifestyles and other as yet unknown critical environmental factors in a variety of physiopathological situations, and in animals of different strains, ages and sexes. The use of oligonucleotides makes it possible to expand this microchip to include the increasing number of imprinted genes discovered. PMID:16575188

  1. Tissue Regeneration in the Chronically Inflamed Tumor Environment: Implications for Cell Fusion Driven Tumor Progression and Therapy Resistant Tumor Hybrid Cells.

    PubMed

    Dittmar, Thomas; Zänker, Kurt S

    2015-01-01

    The biological phenomenon of cell fusion in a cancer context is still a matter of controversial debates. Even though a plethora of in vitro and in vivo data have been published in the past decades the ultimate proof that tumor hybrid cells could originate in (human) cancers and could contribute to the progression of the disease is still missing, suggesting that the cell fusion hypothesis is rather fiction than fact. However, is the lack of this ultimate proof a valid argument against this hypothesis, particularly if one has to consider that appropriate markers do not (yet) exist, thus making it virtually impossible to identify a human tumor cell clearly as a tumor hybrid cell. In the present review, we will summarize the evidence supporting the cell fusion in cancer concept. Moreover, we will refine the cell fusion hypothesis by providing evidence that cell fusion is a potent inducer of aneuploidy, genomic instability and, most likely, even chromothripsis, suggesting that cell fusion, like mutations and aneuploidy, might be an inducer of a mutator phenotype. Finally, we will show that "accidental" tissue repair processes during cancer therapy could lead to the origin of therapy resistant cancer hybrid stem cells. PMID:26703575

  2. Tissue Regeneration in the Chronically Inflamed Tumor Environment: Implications for Cell Fusion Driven Tumor Progression and Therapy Resistant Tumor Hybrid Cells

    PubMed Central

    Dittmar, Thomas; Zänker, Kurt S.

    2015-01-01

    The biological phenomenon of cell fusion in a cancer context is still a matter of controversial debates. Even though a plethora of in vitro and in vivo data have been published in the past decades the ultimate proof that tumor hybrid cells could originate in (human) cancers and could contribute to the progression of the disease is still missing, suggesting that the cell fusion hypothesis is rather fiction than fact. However, is the lack of this ultimate proof a valid argument against this hypothesis, particularly if one has to consider that appropriate markers do not (yet) exist, thus making it virtually impossible to identify a human tumor cell clearly as a tumor hybrid cell. In the present review, we will summarize the evidence supporting the cell fusion in cancer concept. Moreover, we will refine the cell fusion hypothesis by providing evidence that cell fusion is a potent inducer of aneuploidy, genomic instability and, most likely, even chromothripsis, suggesting that cell fusion, like mutations and aneuploidy, might be an inducer of a mutator phenotype. Finally, we will show that “accidental” tissue repair processes during cancer therapy could lead to the origin of therapy resistant cancer hybrid stem cells. PMID:26703575

  3. Differentially profiling the low-expression transcriptomes of human hepatoma using a novel SSH/microarray approach

    PubMed Central

    Pan, Yi-Shin; Lee, Yun-Shien; Lee, Yung-Lin; Lee, Wei-Chen; Hsieh, Sen-Yung

    2006-01-01

    Background The main limitation in performing genome-wide gene-expression profiling is the assay of low-expression genes. Approaches with high throughput and high sensitivity for assaying low-expression transcripts are urgently needed for functional genomic studies. Combination of the suppressive subtractive hybridization (SSH) and cDNA microarray techniques using the subtracted cDNA clones as probes printed on chips has greatly improved the efficiency for fishing out the differentially expressed clones and has been used before. However, it remains tedious and inefficient sequencing works for identifying genes including the great number of redundancy in the subtracted amplicons, and sacrifices the original advantages of high sensitivity of SSH in profiling low-expression transcriptomes. Results We modified the previous combination of SSH and microarray methods by directly using the subtracted amplicons as targets to hybridize the pre-made cDNA microarrays (named as "SSH/microarray"). mRNA prepared from three pairs of hepatoma and non-hepatoma liver tissues was subjected to the SSH/microarray assays, as well as directly to regular cDNA microarray assays for comparison. As compared to the original SSH and microarray combination assays, the modified SSH/microarray assays allowed for much easier inspection of the subtraction efficiency and identification of genes in the subtracted amplicons without tedious and inefficient sequencing work. On the other hand, 5015 of the 9376 genes originally filtered out by the regular cDNA microarray assays because of low expression became analyzable by the SSH/microarray assays. Moreover, the SSH/microarray assays detected about ten times more (701 vs. 69) HCC differentially expressed genes (at least a two-fold difference and P < 0.01), particularly for those with rare transcripts, than did the regular cDNA microarray assays. The differential expression was validated in 9 randomly selected genes in 18 pairs of hepatoma/non-hepatoma liver tissues using quantitative RT-PCR. The SSH/microarray approaches resulted in identifying many differentially expressed genes implicated in the regulation of cell cycle, cell death, signal transduction and cell morphogenesis, suggesting the involvement of multi-biological processes in hepato-carcinogenesis. Conclusion The modified SSH/microarray approach is a simple but high-sensitive and high-efficient tool for differentially profiling the low-expression transcriptomes. It is most adequate for applying to functional genomic studies. PMID:16737534

  4. Annotating breast cancer microarray samples using ontologies

    PubMed Central

    Liu, Hongfang; Li, Xin; Yoon, Victoria; Clarke, Robert

    2008-01-01

    As the most common cancer among women, breast cancer results from the accumulation of mutations in essential genes. Recent advance in high-throughput gene expression microarray technology has inspired researchers to use the technology to assist breast cancer diagnosis, prognosis, and treatment prediction. However, the high dimensionality of microarray experiments and public access of data from many experiments have caused inconsistencies which initiated the development of controlled terminologies and ontologies for annotating microarray experiments, such as the standard microarray Gene Expression Data (MGED) ontology (MO). In this paper, we developed BCM-CO, an ontology tailored specifically for indexing clinical annotations of breast cancer microarray samples from the NCI Thesaurus. Our research showed that the coverage of NCI Thesaurus is very limited with respect to i) terms used by researchers to describe breast cancer histology (covering 22 out of 48 histology terms); ii) breast cancer cell lines (covering one out of 12 cell lines); and iii) classes corresponding to the breast cancer grading and staging. By incorporating a wider range of those terms into BCM-CO, we were able to indexed breast cancer microarray samples from GEO using BCM-CO and MGED ontology and developed a prototype system with web interface that allows the retrieval of microarray data based on the ontology annotations. PMID:18999108

  5. Seasonal dynamics of harmful algae in outer Oslofjorden monitored by microarray, qPCR, and microscopy.

    PubMed

    Dittami, Simon M; Hostyeva, Vladyslava; Egge, Elianne Sirnæs; Kegel, Jessica U; Eikrem, Wenche; Edvardsen, Bente

    2013-10-01

    Monitoring of marine microalgae is important to predict and manage harmful algal blooms. Microarray Detection of Toxic ALgae (MIDTAL) is an FP7-funded EU project aiming to establish a multi-species microarray as a tool to aid monitoring agencies. We tested the suitability of different prototype versions of the MIDTAL microarray for the monthly monitoring of a sampling station in outer Oslofjorden during a 1-year period. Microarray data from two different versions of the MIDTAL chip were compared to results from cell counts (several species) and quantitative real-time PCR (qPCR; only Pseudochattonella spp.). While results from generation 2.5 microarrays exhibited a high number of false positive signals, generation 3.3 microarray data generally correlated with microscopy and qPCR data, with three important limitations: (1) Pseudo-nitzschia cells were not reliably detected, possibly because cells were not sufficiently retained during filtration or lysed during the extraction, and because of low sensitivity of the probes; (2) in the case of samples with high concentrations of non-target species, the sensitivity of the arrays was decreased; (3) one occurrence of Alexandrium pseudogonyaulax was not detected due to a 1-bp mismatch with the genus probe represented on the microarray. In spite of these shortcomings our data demonstrate the overall progress made and the potential of the MIDTAL array. The case of Pseudochattonella - where two morphologically similar species impossible to separate by light microscopy were distinguished - in particular, underlines the added value of molecular methods such as microarrays in routine phytoplankton monitoring. PMID:23325054

  6. Metric learning for DNA microarray data analysis

    NASA Astrophysics Data System (ADS)

    Takeuchi, Ichiro; Nakagawa, Masao; Seto, Masao

    2009-12-01

    In many microarray studies, gene set selection is an important preliminary step for subsequent main task such as tumor classification, cancer subtype identification, etc. In this paper, we investigate the possibility of using metric learning as an alternative to gene set selection. We develop a simple metric learning algorithm aiming to use it for microarray data analysis. Exploiting a property of the algorithm, we introduce a novel approach for extending the metric learning to be adaptive. We apply the algorithm to previously studied microarray data on malignant lymphoma subtype identification.

  7. DNA Microarrays in Herbal Drug Research

    PubMed Central

    Chavan, Preeti; Joshi, Kalpana; Patwardhan, Bhushan

    2006-01-01

    Natural products are gaining increased applications in drug discovery and development. Being chemically diverse they are able to modulate several targets simultaneously in a complex system. Analysis of gene expression becomes necessary for better understanding of molecular mechanisms. Conventional strategies for expression profiling are optimized for single gene analysis. DNA microarrays serve as suitable high throughput tool for simultaneous analysis of multiple genes. Major practical applicability of DNA microarrays remains in DNA mutation and polymorphism analysis. This review highlights applications of DNA microarrays in pharmacodynamics, pharmacogenomics, toxicogenomics and quality control of herbal drugs and extracts. PMID:17173108

  8. Gene expression differences in adipose tissue associated with breast tumorigenesis

    PubMed Central

    Sturtz, Lori A; Deyarmin, Brenda; van Laar, Ryan; Yarina, William; Shriver, Craig D; Ellsworth, Rachel E

    2014-01-01

    Long thought to function only as an inert energy storage depot, the role of adipose tissue in breast tumorigenesis has been largely ignored. In light of increasing rates of obesity and use of breast conserving therapy and autologous fat grafting, improved understanding of the role of adipose tissue in tumor etiology is crucial. Thus, adipose tissue adjacent to and distant from invasive breast tumors (n = 20), or adjacent to non-malignant diagnoses (n = 20) was laser microdissected from post-menopausal women. Gene expression data were generated using microarrays and data analyzed to identify significant patterns of differential expression between adipose tissue groups at the individual gene and molecular pathway level. Pathway analysis revealed significant differences in immune response between non-malignant, distant, and tumor-adjacent adipose tissue, with the highest response in tumor-adjacent and lowest in non-malignant adipose tissue. Adipose tissue from invasive breasts exhibits increased expression of anti-inflammatory genes such as MARCO and VSIG4 while genes differentially expressed between tumor-adjacent and distant adipose tissue such as SPP1, RRM2, and MMP9, are associated with increased cellular proliferation, invasion, and angiogenesis. These data suggest that molecular profiles of adipose tissue differ depending on presence of or proximity to tumor cells. Heightened immunotolerance in adipose tissue from invasive breasts provides a microenvironment favorable to tumorigenesis. In addition, tumor-adjacent adipose tissue demonstrates expression of genes associated with tumor growth and progression. Thus, adipose tissue is not an inert component of the breast microenvironment but plays an active role in tumorigenesis. PMID:24719783

  9. Integrating Microarray Data and GRNs.

    PubMed

    Koumakis, L; Potamias, G; Tsiknakis, M; Zervakis, M; Moustakis, V

    2016-01-01

    With the completion of the Human Genome Project and the emergence of high-throughput technologies, a vast amount of molecular and biological data are being produced. Two of the most important and significant data sources come from microarray gene-expression experiments and respective databanks (e,g., Gene Expression Omnibus-GEO ( http://www.ncbi.nlm.nih.gov/geo )), and from molecular pathways and Gene Regulatory Networks (GRNs) stored and curated in public (e.g., Kyoto Encyclopedia of Genes and Genomes-KEGG ( http://www.genome.jp/kegg/pathway.html ), Reactome ( http://www.reactome.org/ReactomeGWT/entrypoint.html )) as well as in commercial repositories (e.g., Ingenuity IPA ( http://www.ingenuity.com/products/ipa )). The association of these two sources aims to give new insight in disease understanding and reveal new molecular targets in the treatment of specific phenotypes.Three major research lines and respective efforts that try to utilize and combine data from both of these sources could be identified, namely: (1) de novo reconstruction of GRNs, (2) identification of Gene-signatures, and (3) identification of differentially expressed GRN functional paths (i.e., sub-GRN paths that distinguish between different phenotypes). In this chapter, we give an overview of the existing methods that support the different types of gene-expression and GRN integration with a focus on methodologies that aim to identify phenotype-discriminant GRNs or subnetworks, and we also present our methodology. PMID:26134183

  10. Ultrasensitive DNA detection on microarrays

    NASA Astrophysics Data System (ADS)

    Jacak, Jaroslaw; Hesse, Jan; Hesch, Clemens; Kasper, Maria; Aberger, Fritz; Frischauf, Annemarie; Sonnleitner, Max; Freudenthaler, Guenter; Howorka, Stefan; Schuetz, Gerhard J.

    2005-03-01

    Genomic research is nowadays based on high throughput analytical techniques. Microarray assays are commonly used to determine DNA content of heterogeneous mixtures up to full genome scale. For low amounts of sample material this method, however, requires time consuming and error prone PCR based amplification steps. Here, we present an assay with the ability to characterize the cDNA content of a low number of cells using ultra-sensitive fluorescence microscopy. For detection, a newly developed chip reader was used. The instrument is based on a modified fluorescence microscope with single dye sensitivity. The highly sensitive CCD detector is operated in TDI mode, which allows avoiding overhead times for sample positioning and signal integration. This enabled the scanning of areas of 1x0.2cm2 within 50 seconds at a pixel size of 200nm. At this resolution, single dye molecules can be reliably detected with an average signal to background noise ratio of ~42. For DNA hybridization experiments, oligonucleotides were covalently linked to a newly developed aldehyde surface. Subsequently, fluorescence labeled complementary oligonucleotides were hybridized at various concentrations. Down to femto-molar oligonucleotide concentrations, specific signals were detected. At 10fM concentration signals of individual specifically hybridized oligonucleotide molecules were resolvable. This assay provides the conceptual basis for expression profiling of low amounts of sample material without signal amplification.

  11. AMIC@: All MIcroarray Clusterings @ once

    PubMed Central

    Geraci, Filippo; Pellegrini, Marco; Renda, M. Elena

    2008-01-01

    The AMIC@ Web Server offers a light-weight multi-method clustering engine for microarray gene-expression data. AMIC@ is a highly interactive tool that stresses user-friendliness and robustness by adopting AJAX technology, thus allowing an effective interleaved execution of different clustering algorithms and inspection of results. Among the salient features AMIC@ offers, there are: (i) automatic file format detection, (ii) suggestions on the number of clusters using a variant of the stability-based method of Tibshirani et al. (iii) intuitive visual inspection of the data via heatmaps and (iv) measurements of the clustering quality using cluster homogeneity. Large data sets can be processed efficiently by selecting algorithms (such as FPF-SB and k-Boost), specifically designed for this purpose. In case of very large data sets, the user can opt for a batch-mode use of the system by means of the Clustering wizard that runs all algorithms at once and delivers the results via email. AMIC@ is freely available and open to all users with no login requirement at the following URL http://bioalgo.iit.cnr.it/amica. PMID:18477631

  12. AMIC@: All MIcroarray Clusterings @ once.

    PubMed

    Geraci, Filippo; Pellegrini, Marco; Renda, M Elena

    2008-07-01

    The AMIC@ Web Server offers a light-weight multi-method clustering engine for microarray gene-expression data. AMIC@ is a highly interactive tool that stresses user-friendliness and robustness by adopting AJAX technology, thus allowing an effective interleaved execution of different clustering algorithms and inspection of results. Among the salient features AMIC@ offers, there are: (i) automatic file format detection, (ii) suggestions on the number of clusters using a variant of the stability-based method of Tibshirani et al. (iii) intuitive visual inspection of the data via heatmaps and (iv) measurements of the clustering quality using cluster homogeneity. Large data sets can be processed efficiently by selecting algorithms (such as FPF-SB and k-Boost), specifically designed for this purpose. In case of very large data sets, the user can opt for a batch-mode use of the system by means of the Clustering wizard that runs all algorithms at once and delivers the results via email. AMIC@ is freely available and open to all users with no login requirement at the following URL http://bioalgo.iit.cnr.it/amica. PMID:18477631

  13. Microarrays and toxicology: the advent of toxicogenomics.

    PubMed

    Nuwaysir, E F; Bittner, M; Trent, J; Barrett, J C; Afshari, C A

    1999-03-01

    The availability of genome-scale DNA sequence information and reagents has radically altered life-science research. This revolution has led to the development of a new scientific subdiscipline derived from a combination of the fields of toxicology and genomics. This subdiscipline, termed toxicogenomics, is concerned with the identification of potential human and environmental toxicants, and their putative mechanisms of action, through the use of genomics resources. One such resource is DNA microarrays or "chips," which allow the monitoring of the expression levels of thousands of genes simultaneously. Here we propose a general method by which gene expression, as measured by cDNA microarrays, can be used as a highly sensitive and informative marker for toxicity. Our purpose is to acquaint the reader with the development and current state of microarray technology and to present our view of the usefulness of microarrays to the field of toxicology. PMID:10204799

  14. SLIMarray: Lightweight software for microarray facility management

    PubMed Central

    Marzolf, Bruz; Troisch, Pamela

    2006-01-01

    Background Microarray core facilities are commonplace in biological research organizations, and need systems for accurately tracking various logistical aspects of their operation. Although these different needs could be handled separately, an integrated management system provides benefits in organization, automation and reduction in errors. Results We present SLIMarray (System for Lab Information Management of Microarrays), an open source, modular database web application capable of managing microarray inventories, sample processing and usage charges. The software allows modular configuration and is well suited for further development, providing users the flexibility to adapt it to their needs. SLIMarray Lite, a version of the software that is especially easy to install and run, is also available. Conclusion SLIMarray addresses the previously unmet need for free and open source software for managing the logistics of a microarray core facility. PMID:17147785

  15. Protein Microarrays: Novel Developments and Applications

    PubMed Central

    Berrade, Luis; Garcia, Angie E.

    2011-01-01

    Protein microarray technology possesses some of the greatest potential for providing direct information on protein function and potential drug targets. For example, functional protein microarrays are ideal tools suited for the mapping of biological pathways. They can be used to study most major types of interactions and enzymatic activities that take place in biochemical pathways and have been used for the analysis of simultaneous multiple biomolecular interactions involving protein-protein, protein-lipid, protein-DNA and protein-small molecule interactions. Because of this unique ability to analyze many kinds of molecular interactions en masse, the requirement of very small sample amount and the potential to be miniaturized and automated, protein microarrays are extremely well suited for protein profiling, drug discovery, drug target identification and clinical prognosis and diagnosis. The aim of this review is to summarize the most recent developments in the production, applications and analysis of protein microarrays. PMID:21116694

  16. Photopatterning of Hydrogel Microarrays in Closed Microchips.

    PubMed

    Gumuscu, Burcu; Bomer, Johan G; van den Berg, Albert; Eijkel, Jan C T

    2015-12-14

    To date, optical lithography has been extensively used for in situ patterning of hydrogel structures in a scale range from hundreds of microns to a few millimeters. The two main limitations which prevent smaller feature sizes of hydrogel structures are (1) the upper glass layer of a microchip maintains a large spacing (typically 525 μm) between the photomask and hydrogel precursor, leading to diffraction of UV light at the edges of mask patterns, (2) diffusion of free radicals and monomers results in irregular polymerization near the illumination interface. In this work, we present a simple approach to enable the use of optical lithography to fabricate hydrogel arrays with a minimum feature size of 4 μm inside closed microchips. To achieve this, we combined two different techniques. First, the upper glass layer of the microchip was thinned by mechanical polishing to reduce the spacing between the photomask and hydrogel precursor, and thereby the diffraction of UV light at the edges of mask patterns. The polishing process reduces the upper layer thickness from ∼525 to ∼100 μm, and the mean surface roughness from 20 to 3 nm. Second, we developed an intermittent illumination technique consisting of short illumination periods followed by relatively longer dark periods, which decrease the diffusion of monomers. Combination of these two methods allows for fabrication of 0.4 × 10(6) sub-10 μm sized hydrogel patterns over large areas (cm(2)) with high reproducibility (∼98.5% patterning success). The patterning method is tested with two different types of photopolymerizing hydrogels: polyacrylamide and polyethylene glycol diacrylate. This method enables in situ fabrication of well-defined hydrogel patterns and presents a simple approach to fabricate 3-D hydrogel matrices for biomolecule separation, biosensing, tissue engineering, and immobilized protein microarray applications. PMID:26558488

  17. Aggregation effect in microarray data analysis.

    PubMed

    Chen, Linlin; Almudevar, Anthony; Klebanov, Lev

    2013-01-01

    Inferring gene regulatory networks from microarray data has become a popular activity in recent years, resulting in an ever-increasing volume of publications. There are many pitfalls in network analysis that remain either unnoticed or scantily understood. A critical discussion of such pitfalls is long overdue. Here we discuss one feature of microarray data the investigators need to be aware of when embarking on a study of putative associations between elements of networks and pathways. PMID:23385538

  18. Contributions to Statistical Problems Related to Microarray Data

    ERIC Educational Resources Information Center

    Hong, Feng

    2009-01-01

    Microarray is a high throughput technology to measure the gene expression. Analysis of microarray data brings many interesting and challenging problems. This thesis consists three studies related to microarray data. First, we propose a Bayesian model for microarray data and use Bayes Factors to identify differentially expressed genes. Second, we…

  19. Differentially Expressed Proteins and Associated Histological and Disease Progression Changes in Cotyledon Tissue of a Resistant and Susceptible Genotype of Brassica napus Infected with Sclerotinia sclerotiorum

    PubMed Central

    Garg, Harsh; Li, Hua; Sivasithamparam, Krishnapillai; Barbetti, Martin J.

    2013-01-01

    Sclerotinia rot caused by Sclerotinia sclerotiorum is one of the most serious diseases of oilseed rape. To understand the resistance mechanisms in the Brassica napus to S. sclerotiorum, comparative disease progression, histological and proteomic studies were conducted of two B. napus genotypes (resistant cv. Charlton, susceptible cv. RQ001-02M2). At 72 and 96 h post inoculation (hpi), lesion size on cotyledons was significantly (P≤0.001) smaller in the resistant Charlton. Anatomical investigations revealed impeded fungal growth (at 24 hpi and onwards) and hyphal disintegration only on resistant Charlton. Temporal changes (12, 24, 48 and 72 hpi) in protein profile showed certain enzymes up-regulated only in resistant Charlton, such as those related to primary metabolic pathways, antioxidant defence, ethylene biosynthesis, pathogenesis related proteins, protein synthesis and protein folding, play a role in mediating defence responses against S. sclerotiorum. Similarly a eukaryotic translation initiation factor 5A enzyme with increased abundance in susceptible RQ001-02M2 and decreased levels in resistant Charlton has a role in increased susceptibility to this pathogen. This is the first time that the expression of these enzymes has been shown to be associated with mediating the defence response against S. sclerotinia in cotyledon tissue of a resistant cultivar of B. napus at a proteomics level. This study not only provides important new insights into the resistance mechanisms within B. napus against S. sclerotiorum, but opens the way for novel engineering of new B. napus varieties that over-express these key enzymes as a strategy to enhance resistance and better manage this devastating pathogen. PMID:23776450

  20. Integrative approaches for microarray data analysis.

    PubMed

    Waldron, Levi; Coller, Hilary A; Huttenhower, Curtis

    2012-01-01

    Microarrays were one of the first technologies of the genomic revolution to gain widespread adoption, rapidly expanding from a cottage industry to the source of thousands of experimental results. They were one of the first assays for which data repositories and metadata were standardized and researchers were required by many journals to make published data publicly available. Microarrays provide high-throughput insights into the biological functions of genes and gene products; however, they also present a "curse of dimensionality," whereby the availability of many gene expression measurements in few samples make it challenging to distinguish noise from true biological signal. All of these factors argue for integrative approaches to microarray data analysis, which combine data from multiple experiments to increase sample size, avoid laboratory-specific bias, and enable new biological insights not possible from a single experiment. Here, we discuss several approaches to integrative microarray analysis for a diverse range of applications, including biomarker discovery, gene function and interaction prediction, and regulatory network inference. We also show how, by integrating large microarray compendia with diverse genomic data types, more nuanced biological hypotheses can be explored computationally. This chapter provides overviews and brief descriptions of each of these approaches to microarray integration. PMID:22130880

  1. Evaluation of Surface Chemistries for Antibody Microarrays

    SciTech Connect

    Seurynck-Servoss, Shannon L.; White, Amanda M.; Baird, Cheryl L.; Rodland, Karin D.; Zangar, Richard C.

    2007-12-01

    Antibody microarrays are an emerging technology that promises to be a powerful tool for the detection of disease biomarkers. The current technology for protein microarrays has been primarily derived from DNA microarrays and is not fully characterized for use with proteins. For example, there are a myriad of surface chemistries that are commercially available for antibody microarrays, but no rigorous studies that compare these different surfaces. Therefore, we have used an enzyme-linked immunosorbent assay (ELISA) microarray platform to analyze 16 different commercially available slide types. Full standard curves were generated for 24 different assays. We found that this approach provides a rigorous and quantitative system for comparing the different slide types based on spot size and morphology, slide noise, spot background, lower limit of detection, and reproducibility. These studies demonstrate that the properties of the slide surface affect the activity of immobilized antibodies and the quality of data produced. Although many slide types can produce useful data, glass slides coated with poly-L-lysine or aminosilane, with or without activation with a crosslinker, consistently produce superior results in the ELISA microarray analyses we performed.

  2. The Impact of Photobleaching on Microarray Analysis

    PubMed Central

    von der Haar, Marcel; Preuß, John-Alexander; von der Haar, Kathrin; Lindner, Patrick; Scheper, Thomas; Stahl, Frank

    2015-01-01

    DNA-Microarrays have become a potent technology for high-throughput analysis of genetic regulation. However, the wide dynamic range of signal intensities of fluorophore-based microarrays exceeds the dynamic range of a single array scan by far, thus limiting the key benefit of microarray technology: parallelization. The implementation of multi-scan techniques represents a promising approach to overcome these limitations. These techniques are, in turn, limited by the fluorophores’ susceptibility to photobleaching when exposed to the scanner’s laser light. In this paper the photobleaching characteristics of cyanine-3 and cyanine-5 as part of solid state DNA microarrays are studied. The effects of initial fluorophore intensity as well as laser scanner dependent variables such as the photomultiplier tube’s voltage on bleaching and imaging are investigated. The resulting data is used to develop a model capable of simulating the expected degree of signal intensity reduction caused by photobleaching for each fluorophore individually, allowing for the removal of photobleaching-induced, systematic bias in multi-scan procedures. Single-scan applications also benefit as they rely on pre-scans to determine the optimal scanner settings. These findings constitute a step towards standardization of microarray experiments and analysis and may help to increase the lab-to-lab comparability of microarray experiment results. PMID:26378589

  3. Evaluation of Surface Chemistries for Antibody Microarrays

    PubMed Central

    Seurynck-Servoss, Shannon L.; White, Amanda M.; Baird, Cheryl L.; Rodland, Karin D.; Zangar, Richard C.

    2007-01-01

    Antibody microarrays are an emerging technology that promises to be a powerful tool for the detection of disease biomarkers. The current technology for protein microarrays has been primarily derived from DNA microarrays and is not fully characterized for use with proteins. For example, there are a myriad of surface chemistries that are commercially available for antibody microarrays, but no rigorous studies that compare these different surfaces. Therefore, we have used a sandwich enzyme-linked immunosorbent assay (ELISA) microarray platform to analyze 16 different commercially available slide types. Full standard curves were generated for 23 different assays. We found that this approach provides a rigorous and quantitative system for comparing the different slide types based on spot size and morphology, slide noise, spot background, lower limit of detection, and reproducibility. These studies demonstrate that the properties of the slide surface affect the activity of immobilized antibodies and the quality of data produced. Although many slide types produce useful data, glass slides coated with aldehyhyde silane, poly-L-lysine, or aminosilane, with or without activation with a crosslinker, consistently produce superior results in the sandwich ELISA microarray analyses we performed. PMID:17718996

  4. The Impact of Photobleaching on Microarray Analysis.

    PubMed

    von der Haar, Marcel; Preuß, John-Alexander; von der Haar, Kathrin; Lindner, Patrick; Scheper, Thomas; Stahl, Frank

    2015-01-01

    DNA-Microarrays have become a potent technology for high-throughput analysis of genetic regulation. However, the wide dynamic range of signal intensities of fluorophore-based microarrays exceeds the dynamic range of a single array scan by far, thus limiting the key benefit of microarray technology: parallelization. The implementation of multi-scan techniques represents a promising approach to overcome these limitations. These techniques are, in turn, limited by the fluorophores' susceptibility to photobleaching when exposed to the scanner's laser light. In this paper the photobleaching characteristics of cyanine-3 and cyanine-5 as part of solid state DNA microarrays are studied. The effects of initial fluorophore intensity as well as laser scanner dependent variables such as the photomultiplier tube's voltage on bleaching and imaging are investigated. The resulting data is used to develop a model capable of simulating the expected degree of signal intensity reduction caused by photobleaching for each fluorophore individually, allowing for the removal of photobleaching-induced, systematic bias in multi-scan procedures. Single-scan applications also benefit as they rely on pre-scans to determine the optimal scanner settings. These findings constitute a step towards standardization of microarray experiments and analysis and may help to increase the lab-to-lab comparability of microarray experiment results. PMID:26378589

  5. Assembly of ordered microsphere arrays: Platforms for microarrays

    NASA Astrophysics Data System (ADS)

    Xu, Wanling

    Microarrays are powerful tools in gene expression assessment, protein profiling, and protein function screening, as well as cell and tissue analysis. With thousands of small array spots assembled in an ordered array, these small devices makes it possible to screen for multiple targets in a fast, parallel, high-throughput manner. The well-developed technology of DNA microarrays, also called DNA chips, has proved successful in all kinds of biological experiments, including the human genome-sequencing project. The development of protein arrays has lagged behind that of DNA arrays mainly because of the greater complexity of proteins. Some parts of the microarray technology can be transplanted into the realm of protein arrays, while others cannot. The challenges from the complexity of protein targets demand more robust and powerful devices. Traditional planar arrays, in which proteins bind directly to a planar surface, have a drawback in that some proteins will be denatured or cluster together after immobilization. Microsphere-based microarrays represent a more advanced strategy. The functional proteins are first attached to microspheres; these microspheres are then immobilized in arrays on a planar surface. In this dissertation, two approaches to assembling arrays of microspheres will be discussed. The hydrodynamic approach uses surface micromachining and Deep Reactive Ion Etching techniques to form an array of channels through a silicon wafer. By drawing fluid containing the microspheres through the channels they become trapped in the channels and thereby immobilized. In the magnetic approach, permalloy films are deposited on a silicon substrate and subsequently patterned to form magnetic attachment sites. An external magnetic field is then applied and the magnetic microspheres then assemble on these sites. Both devices are able to immobilize microspheres in an ordered array, as opposed to coarsely grouping them in array spots. The assembled arrays are robust in that they ensure a resolution rate of almost 100%. In addition, different patterns of array spots with various spacings and diameters can be fabricated to satisfy different requirements. Moreover, the devices are easy to clean and reuse, and the experimental set-ups are relatively simple and portable. All these features make them good platforms for all kinds of microarrays.

  6. Tissue Array Research Program (TARP)

    Cancer.gov

    The following suggested protocols may be used when using the multi-tumor tissue microarray slides. H&E Protocol For Array Slides On Tape Section Slides The following suggested protocol was provided by David E. Kleiner M.D., Ph.D and Stephen M. Hewitt, M.D

  7. Alterations in vitamin D signaling pathway in gastric cancer progression: a study of vitamin D receptor expression in human normal, premalignant, and malignant gastric tissue

    PubMed Central

    Wen, Yanghui; Da, Mingxu; Zhang, Yongbin; Peng, Lingzhi; Yao, Jibin; Duan, Yaoxing

    2015-01-01

    Amount of studies in cells and animal models have proved vitamin D has multifarious antitumor effects. However, epidemiological studies showed inconsistent result on gastric cancer. The antitumor role is mainly mediated by the vitamin D receptor (VDR). Our hypothesis is that VDR may be abnormally (poorly) expressed in gastric cancer tissue. Present study is aimed at discovering and analyzing VDR expression in a series of human gastric tissues, including normal, premalignant, and malignant gastric tissue, and correlated VDR to the clinicopathological parameters of gastric cancer patients. VDR expression was detected by immunohistochemistry. The χ2 test was used to analyze the VDR expression as well as the relationship between VDR and the clinicopathological factors of gastric cancer patients. Compared with normal (82.61%) and premalignant tissues (73.64%), VDR was lower expressed in cancer tissues (57.61%), with a statistically significant difference (P = 0.001). Among cancer tissues, VDR was higher expressed in well and moderate differentiated tissues contrasted with tissues with poor differentiation, and higher expressed in small tumors (< 5 cm) compared with large tumors (≥ 5 cm), with a statistically significant difference respectively (P = 0.016, P = 0.009). A decline linear trend appeared when analyzing the statistical difference of VDR expression among normal, premalignant, and malignant gastric tissues. VDR expression has been on the decline from the premalignant stage, finally low expressed in gastric cancer tissues, especial in poorly differentiated tissues. VDR could be a potential prognostic factor for patients with gastric cancer. PMID:26722516

  8. Glycan Profiling of Plant Cell Wall Polymers using Microarrays

    PubMed Central

    Moller, Isabel E.; Pettolino, Filomena A.; Hart, Charlie; Lampugnani, Edwin R.; Willats, William G.T.; Bacic, Antony

    2012-01-01

    Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging. Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4. In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification. Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11. Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 & 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable. PMID:23271573

  9. Meta-Analysis of Public Microarray Datasets Reveals Voltage-Gated Calcium Gene Signatures in Clinical Cancer Patients

    PubMed Central

    Wang, Chih-Yang; Lai, Ming-Derg; Phan, Nam Nhut; Sun, Zhengda; Lin, Yen-Chang

    2015-01-01

    Voltage-gated calcium channels (VGCCs) are well documented to play roles in cell proliferation, migration, and apoptosis; however, whether VGCCs regulate the onset and progression of cancer is still under investigation. The VGCC family consists of five members, which are L-type, N-type, T-type, R-type and P/Q type. To date, no holistic approach has been used to screen VGCC family genes in different types of cancer. We analyzed the transcript expression of VGCCs in clinical cancer tissue samples by accessing ONCOMINE (www.oncomine.org), a web-based microarray database, to perform a systematic analysis. Every member of the VGCCs was examined across 21 different types of cancer by comparing mRNA expression in cancer to that in normal tissue. A previous study showed that altered expression of mRNA in cancer tissue may play an oncogenic role and promote tumor development; therefore, in the present findings, we focus only on the overexpression of VGCCs in different types of cancer. This bioinformatics analysis revealed that different subtypes of VGCCs (CACNA1C, CACNA1D, CACNA1B, CACNA1G, and CACNA1I) are implicated in the development and progression of diverse types of cancer and show dramatic up-regulation in breast cancer. CACNA1F only showed high expression in testis cancer, whereas CACNA1A, CACNA1C, and CACNA1D were highly expressed in most types of cancer. The current analysis revealed that specific VGCCs likely play essential roles in specific types of cancer. Collectively, we identified several VGCC targets and classified them according to different cancer subtypes for prospective studies on the underlying carcinogenic mechanisms. The present findings suggest that VGCCs are possible targets for prospective investigation in cancer treatment. PMID:26147197

  10. Storage and retrieval of microarray data and open source microarray database software.

    PubMed

    Sherlock, Gavin; Ball, Catherine A

    2005-07-01

    Microarray technology has been widely adopted by researchers who use both home-made microarrays and microarrays purchased from commercial vendors. Associated with the adoption of this technology has been a deluge of complex data, both from the microarrays themselves, and also in the form of associated meta data, such as gene annotation information, the properties and treatment of biological samples, and the data transformation and analysis steps taken downstream. In addition, standards for annotation and data exchange have been proposed, and are now being adopted by journals and funding agencies alike. The coupling of large quantities of complex data with extensive and complex standards require all but the most small-scale of microarray users to have access to a robust and scaleable database with various tools. In this review, we discuss some of the desirable properties of such a database, and look at the features of several freely available alternatives. PMID:15988049

  11. Visualization-based discovery and analysis of genomic aberrations in microarray data

    PubMed Central

    Myers, Chad L; Chen, Xing; Troyanskaya, Olga G

    2005-01-01

    Background Chromosomal copy number changes (aneuploidies) play a key role in cancer progression and molecular evolution. These copy number changes can be studied using microarray-based comparative genomic hybridization (array CGH) or gene expression microarrays. However, accurate identification of amplified or deleted regions requires a combination of visual and computational analysis of these microarray data. Results We have developed ChARMView, a visualization and analysis system for guided discovery of chromosomal abnormalities from microarray data. Our system facilitates manual or automated discovery of aneuploidies through dynamic visualization and integrated statistical analysis. ChARMView can be used with array CGH and gene expression microarray data, and multiple experiments can be viewed and analyzed simultaneously. Conclusion ChARMView is an effective and accurate visualization and analysis system for recognizing even small aneuploidies or subtle expression biases, identifying recurring aberrations in sets of experiments, and pinpointing functionally relevant copy number changes. ChARMView is freely available under the GNU GPL at . PMID:15953389

  12. Identifying pathogenic processes by integrating microarray data with prior knowledge

    PubMed Central

    2014-01-01

    Background It is of great importance to identify molecular processes and pathways that are involved in disease etiology. Although there has been an extensive use of various high-throughput methods for this task, pathogenic pathways are still not completely understood. Often the set of genes or proteins identified as altered in genome-wide screens show a poor overlap with canonical disease pathways. These findings are difficult to interpret, yet crucial in order to improve the understanding of the molecular processes underlying the disease progression. We present a novel method for identifying groups of connected molecules from a set of differentially expressed genes. These groups represent functional modules sharing common cellular function and involve signaling and regulatory events. Specifically, our method makes use of Bayesian statistics to identify groups of co-regulated genes based on the microarray data, where external information about molecular interactions and connections are used as priors in the group assignments. Markov chain Monte Carlo sampling is used to search for the most reliable grouping. Results Simulation results showed that the method improved the ability of identifying correct groups compared to traditional clustering, especially for small sample sizes. Applied to a microarray heart failure dataset the method found one large cluster with several genes important for the structure of the extracellular matrix and a smaller group with many genes involved in carbohydrate metabolism. The method was also applied to a microarray dataset on melanoma cancer patients with or without metastasis, where the main cluster was dominated by genes related to keratinocyte differentiation. Conclusion Our method found clusters overlapping with known pathogenic processes, but also pointed to new connections extending beyond the classical pathways. PMID:24758699

  13. An examination of the regulatory mechanism of Pxdn mutation-induced eye disorders using microarray analysis

    PubMed Central

    YANG, YANG; XING, YIQIAO; LIANG, CHAOQUN; HU, LIYA; XU, FEI; MEI, QI

    2016-01-01

    The present study aimed to identify biomarkers for peroxidasin (Pxdn) mutation-induced eye disorders and study the underlying mechanisms involved in this process. The microarray dataset GSE49704 was used, which encompasses 4 mouse samples from embryos with Pxdn mutation and 4 samples from normal tissues. After data preprocessing, the differentially expressed genes (DEGs) between Pxdn mutation and normal tissues were identified using the t-test in the limma package, followed by functional enrichment analysis. The protein-protein interaction (PPI) network was constructed based on the STRING database, and the transcriptional regulatory (TR) network was established using the GeneCodis database. Subsequently, the overlapping DEGs with high degrees in two networks were identified, as well as the sub-network extracted from the TR network. In total, 121 (75 upregulated and 46 downregulated) DEGs were identified, and these DEGs play important roles in biological processes (BPs), including neuron development and differentiation. A PPI network containing 25 nodes such as actin, alpha 1, skeletal muscle (Acta1) and troponin C type 2 (fast) (Tnnc2), and a TR network including 120 nodes were built. By comparing the two networks, seven crucial genes which overlapped were identified, including cyclin-dependent kinase inhibitor 1B (Cdkn1b), Acta1 and troponin T type 3 (Tnnt3). In the sub-network, Cdkn1b was predicted as the target of miRNAs such as mmu-miR-24 and transcription factors (TFs) including forkhead box O4 (FOXO4) and activating enhancer binding protein 4 (AP4). Thus, we suggest that seven crucial genes, including Cdkn1b, Acta1 and Tnnt3, play important roles in the progression of eye disorders such as glaucoma. We suggest that Cdkn1b exert its effects via the inhibition of proliferation and is mediated by mmu-miR-24 and targeted by the TFs FOXO4 and AP4. PMID:27121343

  14. PERFORMANCE CHARACTERISTICS OF 65-MER OLIGONUCLEOTIDE MICROARRAYS

    PubMed Central

    Lee, Myoyong; Xiang, Charlie C.; Trent, Jeffrey M.; Bittner, Michael L.

    2009-01-01

    Microarray fabrication using pre-synthesized long oligonucleotide is becoming increasingly important, but a study of large-scale array productions is not published yet. We addressed the issue of fabricating oligonucleotide microarrays by spotting commercial, pre-synthesized 65-mers with 5? amines representing 7500 murine genes. Amine-modified oligonucleotides were immobilized on glass slides having aldehyde groups via transient Schiff base formation followed by reduction to produce a covalent conjugate. When RNA derived from the same source was used for Cy3 and Cy5 labeling and hybridized to the same array, signal intensities spanning three orders of magnitude were observed, and the coefficient of variation between the two channels for all spots was 810%. To ascertain the reproducibility of ratio determination of these arrays, two triplicate hybridizations (with fluorochrome reversal) comparing RNAs from a fibroblast (NIH3T3) and a breast cancer (JC) cell line were carried out. The 95% confidence interval for all spots in the six hybridizations was 0.60 1.66. This level of reproducibility allows use of the full range of pattern finding and discriminant analysis typically applied to cDNA microarrays. Further comparative testing was carried out with oligonucleotide microarrays, cDNA microarrays and RT-PCR assays to examine the comparability of results across these different methodologies. PMID:17617369

  15. A comparison of two sets of microarray experiments to define allergic asthma expression pattern.

    PubMed

    Chamberland, Annie; Madore, Anne-Marie; Tremblay, Karine; Laviolette, Michel; Laprise, C

    2009-06-01

    Allergic asthma is a complex trait. Several approaches have been used to identify biomarkers involved in this disease. This study aimed at demonstrating the relevance and validity of microarrays in the definition of allergic asthma expression pattern. The authors compared the transcript expressions of bronchial biopsy of 2 different microarray experiments done 2 years apart, both including nonallergic healthy and allergic asthmatic subjects (n = 4 in each experiment). U95Av2 and U133A GeneChips detected respectively 89 and 40 differentially expressed genes. Fifty-five percent of the U133A genes were previously identified with the U95Av2 arrays. The immune signaling molecules and the proteolytic enzymes were the most preserved categories between the 2 experiments, because 3/4 of the genes identified by the U133A were also significant in the U95Av2 study for both categories. These results demonstrate the relevance of microarray experiments using bronchial tissues in allergic asthma. The comparison of these GeneChip studies suggests that earlier microarray results are as relevant as actual ones to target new genes of interest, particularly in function categories linked to the studied disease. Moreover, it demonstrates that microarrays are a valuable technology to target novel allergic asthma pathways as well as biomarkers. PMID:19842841

  16. Use of lectin microarray to differentiate gastric cancer from gastric ulcer

    PubMed Central

    Huang, Wei-Li; Li, Yang-Guang; Lv, Yong-Chen; Guan, Xiao-Hui; Ji, Hui-Fan; Chi, Bao-Rong

    2014-01-01

    AIM: To investigate the feasibility of lectin microarray for differentiating gastric cancer from gastric ulcer. METHODS: Twenty cases of human gastric cancer tissue and 20 cases of human gastric ulcer tissue were collected and processed. Protein was extracted from the frozen tissues and stored. The lectins were dissolved in buffer, and the sugar-binding specificities of lectins and the layout of the lectin microarray were summarized. The median of the effective data points for each lectin was globally normalized to the sum of medians of all effective data points for each lectin in one block. Formalin-fixed paraffin-embedded gastric cancer tissues and their corresponding gastric ulcer tissues were subjected to Ag retrieval. Biotinylated lectin was used as the primary antibody and HRP-streptavidin as the secondary antibody. The glycopatterns of glycoprotein in gastric cancer and gastric ulcer specimens were determined by lectin microarray, and then validated by lectin histochemistry. Data are presented as mean ± SD for the indicated number of independent experiments. RESULTS: The glycosylation level of gastric cancer was significantly higher than that in ulcer. In gastric cancer, most of the lectin binders showed positive signals and the intensity of the signals was stronger, whereas the opposite was the case for ulcers. Significant differences in the pathological score of the two lectins were apparent between ulcer and gastric cancer tissues using the same lectin. For MPL and VVA, all types of gastric cancer detected showed stronger staining and a higher positive rate in comparison with ulcer, especially in the case of signet ring cell carcinoma and intra-mucosal carcinoma. GalNAc bound to MPL showed a significant increase. A statistically significant association between MPL and gastric cancer was observed. As with MPL, there were significant differences in VVA staining between gastric cancer and ulcer. CONCLUSION: Lectin microarray can differentiate the different glycopatterns in gastric cancer and gastric ulcer, and the lectins MPL and VVA can be used as biomarkers. PMID:24833877

  17. Next station in microarray data analysis: GEPAS

    PubMed Central

    Montaner, David; Tárraga, Joaquín; Huerta-Cepas, Jaime; Burguet, Jordi; Vaquerizas, Juan M.; Conde, Lucía; Minguez, Pablo; Vera, Javier; Mukherjee, Sach; Valls, Joan; Pujana, Miguel A. G.; Alloza, Eva; Herrero, Javier; Al-Shahrour, Fátima; Dopazo, Joaquín

    2006-01-01

    The Gene Expression Profile Analysis Suite (GEPAS) has been running for more than four years. During this time it has evolved to keep pace with the new interests and trends in the still changing world of microarray data analysis. GEPAS has been designed to provide an intuitive although powerful web-based interface that offers diverse analysis options from the early step of preprocessing (normalization of Affymetrix and two-colour microarray experiments and other preprocessing options), to the final step of the functional annotation of the experiment (using Gene Ontology, pathways, PubMed abstracts etc.), and include different possibilities for clustering, gene selection, class prediction and array-comparative genomic hybridization management. GEPAS is extensively used by researchers of many countries and its records indicate an average usage rate of 400 experiments per day. The web-based pipeline for microarray gene expression data, GEPAS, is available at . PMID:16845056

  18. Designing microarray phantoms for hyperspectral imaging validation

    PubMed Central

    Clarke, Matthew L.; Lee, Ji Youn; Samarov, Daniel V.; Allen, David W.; Litorja, Maritoni; Nossal, Ralph; Hwang, Jeeseong

    2012-01-01

    The design and fabrication of custom-tailored microarrays for use as phantoms in the characterization of hyperspectral imaging systems is described. Corresponding analysis methods for biologically relevant samples are also discussed. An image-based phantom design was used to program a microarrayer robot to print prescribed mixtures of dyes onto microscope slides. The resulting arrays were imaged by a hyperspectral imaging microscope. The shape of the spots results in significant scattering signals, which can be used to test image analysis algorithms. Separation of the scattering signals allowed elucidation of individual dye spectra. In addition, spectral fitting of the absorbance spectra of complex dye mixtures was performed in order to determine local dye concentrations. Such microarray phantoms provide a robust testing platform for comparisons of hyperspectral imaging acquisition and analysis methods. PMID:22741076

  19. Analysis of High-Throughput ELISA Microarray Data

    SciTech Connect

    White, Amanda M.; Daly, Don S.; Zangar, Richard C.

    2011-02-23

    Our research group develops analytical methods and software for the high-throughput analysis of quantitative enzyme-linked immunosorbent assay (ELISA) microarrays. ELISA microarrays differ from DNA microarrays in several fundamental aspects and most algorithms for analysis of DNA microarray data are not applicable to ELISA microarrays. In this review, we provide an overview of the steps involved in ELISA microarray data analysis and how the statistically sound algorithms we have developed provide an integrated software suite to address the needs of each data-processing step. The algorithms discussed are available in a set of open-source software tools (http://www.pnl.gov/statistics/ProMAT).

  20. Integrating microarrays into disease-gene identification strategies.

    PubMed

    Dobrin, Seth E; Stephan, Dietrich A

    2003-05-01

    Positional cloning represents one of the most successful paradigm shifts in identifying the underlying patho-mechanisms in human disease. While traditional discovery tools focused on identifying defects at the tissue or cellular level, positional cloning identifies the damaged region of the genome as the preliminary step. While a large number of inherited single gene disorders have been mapped using this approach, a bottleneck still exists in combing through the genomic interval, often millions of nucleotides in length, to identify the nucleotide changes which result in a defective protein and subsequent disease. Along with the recent unravelling of the human genetic code, the development of massively parallel tools, such as microarrays, represent an equally important step forward in unraveling pathogenic genome dysfunctions. There are many emerging variants on microarray technology, such as expression arrays, exon arrays, array-based comparative genomic hybridization and sequencing arrays. Several of these platforms, if used properly, can accelerate the positional cloning process. The proper use of the platform is driven by knowledge of the underlying molecular defect being searched for and the operating characteristics of the array. The resultant insight forms the basis for improved molecular diagnostics and novel therapeutic targets. PMID:12779011

  1. Data-dependent kernel machines for microarray data classification.

    PubMed

    Xiong, Huilin; Zhang, Ya; Chen, Xue-Wen

    2007-01-01

    One important application of gene expression analysis is to classify tissue samples according to their gene expression levels. Gene expression data are typically characterized by high dimensionality and small sample size, which makes the classification task quite challenging. In this paper, we present a data-dependent kernel for microarray data classification. This kernel function is engineered so that the class separability of the training data is maximized. A bootstrapping-based resampling scheme is introduced to reduce the possible training bias. The effectiveness of this adaptive kernel for microarray data classification is illustrated with a k-Nearest Neighbor (KNN) classifier. Our experimental study shows that the data-dependent kernel leads to a significant improvement in the accuracy of KNN classifiers. Furthermore, this kernel-based KNN scheme has been demonstrated to be competitive to, if not better than, more sophisticated classifiers such as Support Vector Machines (SVMs) and the Uncorrelated Linear Discriminant Analysis (ULDA) for classifying gene expression data. PMID:17975270

  2. Comparing whole genomes using DNA microarrays.

    PubMed

    Gresham, David; Dunham, Maitreya J; Botstein, David

    2008-04-01

    The rapid accumulation of complete genomic sequences offers the opportunity to carry out an analysis of inter- and intra-individual genome variation within a species on a routine basis. Sequencing whole genomes requires resources that are currently beyond those of a single laboratory and therefore it is not a practical approach for resequencing hundreds of individual genomes. DNA microarrays present an alternative way to study differences between closely related genomes. Advances in microarray-based approaches have enabled the main forms of genomic variation (amplifications, deletions, insertions, rearrangements and base-pair changes) to be detected using techniques that are readily performed in individual laboratories using simple experimental approaches. PMID:18347592

  3. Microarrays - A Key Technology for Glycobiology

    NASA Astrophysics Data System (ADS)

    Liu, Yan; Feizi, Ten

    Carbohydrate chains of glycoproteins , glycolipids , and proteoglycans can mediate processes of biological and medical importance through their interactions with complementary proteins. The unraveling of these interactions is a priority therefore in biomedical sciences. Carbohydrate microarray technology is a new development at the frontiers of glycomics that has revolutionized the study of carbohydrate-protein interactions and the elucidation of their specificities in endogenous biological processes, immune defense mechanisms, and microbe-host interactions. In this chapter we briefly touch upon the principles of numerous platforms since the introduction of carbohydrate microarrays in 2002, and we highlight platforms that are beyond proof-of-concept, and have provided new biological information.

  4. Application of independent component analysis to microarrays

    PubMed Central

    Lee, Su-In; Batzoglou, Serafim

    2003-01-01

    We apply linear and nonlinear independent component analysis (ICA) to project microarray data into statistically independent components that correspond to putative biological processes, and to cluster genes according to over- or under-expression in each component. We test the statistical significance of enrichment of gene annotations within clusters. ICA outperforms other leading methods, such as principal component analysis, k-means clustering and the Plaid model, in constructing functionally coherent clusters on microarray datasets from Saccharomyces cerevisiae, Caenorhabditis elegans and human. PMID:14611662

  5. The use of microarrays in microbial ecology

    SciTech Connect

    Andersen, G.L.; He, Z.; DeSantis, T.Z.; Brodie, E.L.; Zhou, J.

    2009-09-15

    Microarrays have proven to be a useful and high-throughput method to provide targeted DNA sequence information for up to many thousands of specific genetic regions in a single test. A microarray consists of multiple DNA oligonucleotide probes that, under high stringency conditions, hybridize only to specific complementary nucleic acid sequences (targets). A fluorescent signal indicates the presence and, in many cases, the abundance of genetic regions of interest. In this chapter we will look at how microarrays are used in microbial ecology, especially with the recent increase in microbial community DNA sequence data. Of particular interest to microbial ecologists, phylogenetic microarrays are used for the analysis of phylotypes in a community and functional gene arrays are used for the analysis of functional genes, and, by inference, phylotypes in environmental samples. A phylogenetic microarray that has been developed by the Andersen laboratory, the PhyloChip, will be discussed as an example of a microarray that targets the known diversity within the 16S rRNA gene to determine microbial community composition. Using multiple, confirmatory probes to increase the confidence of detection and a mismatch probe for every perfect match probe to minimize the effect of cross-hybridization by non-target regions, the PhyloChip is able to simultaneously identify any of thousands of taxa present in an environmental sample. The PhyloChip is shown to reveal greater diversity within a community than rRNA gene sequencing due to the placement of the entire gene product on the microarray compared with the analysis of up to thousands of individual molecules by traditional sequencing methods. A functional gene array that has been developed by the Zhou laboratory, the GeoChip, will be discussed as an example of a microarray that dynamically identifies functional activities of multiple members within a community. The recent version of GeoChip contains more than 24,000 50mer oligonucleotide probes and covers more than 10,000 gene sequences in 150 gene categories involved in carbon, nitrogen, sulfur, and phosphorus cycling, metal resistance and reduction, and organic contaminant degradation. GeoChip can be used as a generic tool for microbial community analysis, and also link microbial community structure to ecosystem functioning. Examples of the application of both arrays in different environmental samples will be described in the two subsequent sections.

  6. Gene discovery and microarray analysis of cacao (Theobroma cacao L.) varieties.

    PubMed

    Jones, Paul G; Allaway, David; Gilmour, D Martin; Harris, Chris; Rankin, Debbie; Retzel, Ernest R; Jones, Chris A

    2002-12-01

    The cacao bean harvest from the relatively under developed tropical tree cacao (Theobroma cacao L.) is subject to high losses in potential production due to pests and diseases. To discover and understand the stability of putative natural resistance mechanisms in this commodity crop, essential for chocolate production, we undertook a gene-discovery program and demonstrated its use in gene-expression arrays. Sequencing and assembling bean and leaf cDNA library inserts produced a unique contig set of 1,380 members. High-quality annotation of this gene set using Blast and MetaFam produced annotation for 75% of the contigs and allowed us to identify the types of gene expressed in cacao beans and leaves. Microarrays were constructed using amplified inserts of the uni-gene set and challenged with bean and leaf RNA from five cacao varieties. The microarray performed well across the five randomly chosen cacao genotypes and did not show a bias towards either leaf or bean tissues. This demonstrates that the gene sequences are useful for microarray analysis across cacao genotypes and tissue types. The array results, when compared with real-time PCR results for selected genes, showed a correlation with differential gene-expression patterns. We intend that the resultant DNA sequences and molecular microarray platform will help the cacao community to understand the basis, likely stability and pathotype resistance range of candidate cacao plants. PMID:12447539

  7. Genome-Wide Microarray Expression and Genomic Alterations by Array-CGH Analysis in Neuroblastoma Stem-Like Cells

    PubMed Central

    Martínez-Soto, Soledad; Legarra, Sheila; Pata-Merci, Noémie; Guegan, Justine; Danglot, Giselle; Bernheim, Alain; Meléndez, Bárbara; Rey, Juan A.; Castresana, Javier S.

    2014-01-01

    Neuroblastoma has a very diverse clinical behaviour: from spontaneous regression to a very aggressive malignant progression and resistance to chemotherapy. This heterogeneous clinical behaviour might be due to the existence of Cancer Stem Cells (CSC), a subpopulation within the tumor with stem-like cell properties: a significant proliferation capacity, a unique self-renewal capacity, and therefore, a higher ability to form new tumors. We enriched the CSC-like cell population content of two commercial neuroblastoma cell lines by the use of conditioned cell culture media for neurospheres, and compared genomic gains and losses and genome expression by array-CGH and microarray analysis, respectively (in CSC-like versus standard tumor cells culture). Despite the array-CGH did not show significant differences between standard and CSC-like in both analyzed cell lines, the microarray expression analysis highlighted some of the most relevant biological processes and molecular functions that might be responsible for the CSC-like phenotype. Some signalling pathways detected seem to be involved in self-renewal of normal tissues (Wnt, Notch, Hh and TGF-β) and contribute to CSC phenotype. We focused on the aberrant activation of TGF-β and Hh signalling pathways, confirming the inhibition of repressors of TGF-β pathway, as SMAD6 and SMAD7 by RT-qPCR. The analysis of the Sonic Hedgehog pathway showed overexpression of PTCH1, GLI1 and SMO. We found overexpression of CD133 and CD15 in SIMA neurospheres, confirming that this cell line was particularly enriched in stem-like cells. This work shows a cross-talk among different pathways in neuroblastoma and its importance in CSC-like cells. PMID:25392930

  8. Micro-Analyzer: automatic preprocessing of Affymetrix microarray data.

    PubMed

    Guzzi, Pietro Hiram; Cannataro, Mario

    2013-08-01

    A current trend in genomics is the investigation of the cell mechanism using different technologies, in order to explain the relationship among genes, molecular processes and diseases. For instance, the combined use of gene-expression arrays and genomic arrays has been demonstrated as an effective instrument in clinical practice. Consequently, in a single experiment different kind of microarrays may be used, resulting in the production of different types of binary data (images and textual raw data). The analysis of microarray data requires an initial preprocessing phase, that makes raw data suitable for use on existing analysis platforms, such as the TIGR M4 (TM4) Suite. An additional challenge to be faced by emerging data analysis platforms is the ability to treat in a combined way those different microarray formats coupled with clinical data. In fact, resulting integrated data may include both numerical and symbolic data (e.g. gene expression and SNPs regarding molecular data), as well as temporal data (e.g. the response to a drug, time to progression and survival rate), regarding clinical data. Raw data preprocessing is a crucial step in analysis but is often performed in a manual and error prone way using different software tools. Thus novel, platform independent, and possibly open source tools enabling the semi-automatic preprocessing and annotation of different microarray data are needed. The paper presents Micro-Analyzer (Microarray Analyzer), a cross-platform tool for the automatic normalization, summarization and annotation of Affymetrix gene expression and SNP binary data. It represents the evolution of the μ-CS tool, extending the preprocessing to SNP arrays that were not allowed in μ-CS. The Micro-Analyzer is provided as a Java standalone tool and enables users to read, preprocess and analyse binary microarray data (gene expression and SNPs) by invoking TM4 platform. It avoids: (i) the manual invocation of external tools (e.g. the Affymetrix Power Tools), (ii) the manual loading of preprocessing libraries, and (iii) the management of intermediate files, such as results and metadata. Micro-Analyzer users can directly manage Affymetrix binary data without worrying about locating and invoking the proper preprocessing tools and chip-specific libraries. Moreover, users of the Micro-Analyzer tool can load the preprocessed data directly into the well-known TM4 platform, extending in such a way also the TM4 capabilities. Consequently, Micro Analyzer offers the following advantages: (i) it reduces possible errors in the preprocessing and further analysis phases, e.g. due to the incorrect choice of parameters or due to the use of old libraries, (ii) it enables the combined and centralized pre-processing of different arrays, (iii) it may enhance the quality of further analysis by storing the workflow, i.e. information about the preprocessing steps, and (iv) finally Micro-Analzyer is freely available as a standalone application at the project web site http://sourceforge.net/projects/microanalyzer/. PMID:23731720

  9. Increased HOX C13 expression in metastatic melanoma progression

    PubMed Central

    2012-01-01

    Background The process of malignant transformation, progression and metastasis of melanoma is not completely understood. Recently, the microarray technology has been used to survey transcriptional differences that might provide insight into the metastatic process, but the validation of changing gene expression during metastatic transition period is poorly investigated. A large body of literature has been produced on the role of the HOX genes network in tumour evolution, suggesting the involvement of HOX genes in several types of human cancers. Deregulated paralogous group 13 HOX genes expression has been detected in melanoma, cervical cancer and odonthogenic tumors. Among these, Hox C13 is also involved in the expression control of the human keratin genes hHa5 and hHa2, and recently it was identified as a member of human DNA replication complexes. Methods In this study, to investigate HOX C13 expression in melanoma progression, we have compared its expression pattern between naevi, primary melanoma and metastasis. In addition HOXC13 profile pattern of expression has been evaluated in melanoma cell lines. Results Our results show the strong and progressive HOX C13 overexpression in metastatic melanoma tissues and cytological samples compared to nevi and primary melanoma tissues and cells. Conclusions The data presentated in the paper suggest a possible role of HOX C13 in metastatic melanoma switch. PMID:22583695

  10. Analysis of microRNA Microarrays in Cardiogenesis.

    PubMed

    Franco, Diego; Bonet, Fernando; Hernandez-Torres, Francisco; Lozano-Velasco, Estefania; Esteban, Francisco J; Aranega, Amelia E

    2016-01-01

    microRNAs are a subclass of noncoding RNAs which have been demonstrated to play pivotal roles in multiple cellular mechanisms. microRNAs are small RNA molecules of 22-24 nt in length capable of modulating protein translation and/or RNA stability by base-priming with complementary sequences of the mRNAs, normally at the 3'untranslated region. To date, over 2,000 microRNAs have been already identified in humans, and orthologous microRNAs have been also identified in distinct animals and plants ranging a wide vast of species. High-throughput analyses by microarrays have become a gold standard to analyze the changes on microRNA expression in normal and pathological cellular or tissue conditions. In this chapter, we provide insights into the usage of this uprising technology in the context of cardiac development and disease. PMID:25971912

  11. On integrating multi-experiment microarray data.

    PubMed

    Tsiliki, Georgia; Vlachakis, Dimitrios; Kossida, Sophia

    2014-05-28

    With the extensive use of microarray technology as a potential prognostic and diagnostic tool, the comparison and reproducibility of results obtained from the use of different platforms is of interest. The integration of those datasets can yield more informative results corresponding to numerous datasets and microarray platforms. We developed a novel integration technique for microarray gene-expression data derived by different studies for the purpose of a two-way Bayesian partition modelling which estimates co-expression profiles under subsets of genes and between biological samples or experimental conditions. The suggested methodology transforms disparate gene-expression data on a common probability scale to obtain inter-study-validated gene signatures. We evaluated the performance of our model using artificial data. Finally, we applied our model to six publicly available cancer gene-expression datasets and compared our results with well-known integrative microarray data methods. Our study shows that the suggested framework can relieve the limited sample size problem while reporting high accuracies by integrating multi-experiment data. PMID:24751870

  12. MICROARRAY DATA ANALYSIS USING MULTIPLE STATISTICAL MODELS

    EPA Science Inventory

    Microarray Data Analysis Using Multiple Statistical Models

    Wenjun Bao1, Judith E. Schmid1, Amber K. Goetz1, Ming Ouyang2, William J. Welsh2,Andrew I. Brooks3,4, ChiYi Chu3,Mitsunori Ogihara3,4, Yinhe Cheng5, David J. Dix1. 1National Health and Environmental Effects Researc...

  13. Shrinkage covariance matrix approach for microarray data

    NASA Astrophysics Data System (ADS)

    Karjanto, Suryaefiza; Aripin, Rasimah

    2013-04-01

    Microarray technology was developed for the purpose of monitoring the expression levels of thousands of genes. A microarray data set typically consists of tens of thousands of genes (variables) from just dozens of samples due to various constraints including the high cost of producing microarray chips. As a result, the widely used standard covariance estimator is not appropriate for this purpose. One such technique is the Hotelling's T2 statistic which is a multivariate test statistic for comparing means between two groups. It requires that the number of observations (n) exceeds the number of genes (p) in the set but in microarray studies it is common that n < p. This leads to a biased estimate of the covariance matrix. In this study, the Hotelling's T2 statistic with the shrinkage approach is proposed to estimate the covariance matrix for testing differential gene expression. The performance of this approach is then compared with other commonly used multivariate tests using a widely analysed diabetes data set as illustrations. The results across the methods are consistent, implying that this approach provides an alternative to existing techniques.

  14. Microarrays (DNA Chips) for the Classroom Laboratory

    ERIC Educational Resources Information Center

    Barnard, Betsy; Sussman, Michael; BonDurant, Sandra Splinter; Nienhuis, James; Krysan, Patrick

    2006-01-01

    We have developed and optimized the necessary laboratory materials to make DNA microarray technology accessible to all high school students at a fraction of both cost and data size. The primary component is a DNA chip/array that students "print" by hand and then analyze using research tools that have been adapted for classroom use. The primary…

  15. ANNOTATION OF THE AFFYMETRIX PORCINE GENOME MICROARRAY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Affymetrix Porcine Genome Microarray is minimally annotated. Less than 10% of the probe sets on this array are described with gene names, posing a challenge to biological interpretation of data. Lack of annotation is likely due to limited availability of full-length porcine cDNA sequence. Pr...

  16. Microarrays (DNA Chips) for the Classroom Laboratory

    ERIC Educational Resources Information Center

    Barnard, Betsy; Sussman, Michael; BonDurant, Sandra Splinter; Nienhuis, James; Krysan, Patrick

    2006-01-01

    We have developed and optimized the necessary laboratory materials to make DNA microarray technology accessible to all high school students at a fraction of both cost and data size. The primary component is a DNA chip/array that students "print" by hand and then analyze using research tools that have been adapted for classroom use. The primary

  17. DISC-BASED IMMUNOASSAY MICROARRAYS. (R825433)

    EPA Science Inventory

    Microarray technology as applied to areas that include genomics, diagnostics, environmental, and drug discovery, is an interesting research topic for which different chip-based devices have been developed. As an alternative, we have explored the principle of compact disc-based...

  18. On integrating multi-experiment microarray data

    PubMed Central

    Tsiliki, Georgia; Vlachakis, Dimitrios; Kossida, Sophia

    2014-01-01

    With the extensive use of microarray technology as a potential prognostic and diagnostic tool, the comparison and reproducibility of results obtained from the use of different platforms is of interest. The integration of those datasets can yield more informative results corresponding to numerous datasets and microarray platforms. We developed a novel integration technique for microarray gene-expression data derived by different studies for the purpose of a two-way Bayesian partition modelling which estimates co-expression profiles under subsets of genes and between biological samples or experimental conditions. The suggested methodology transforms disparate gene-expression data on a common probability scale to obtain inter-study-validated gene signatures. We evaluated the performance of our model using artificial data. Finally, we applied our model to six publicly available cancer gene-expression datasets and compared our results with well-known integrative microarray data methods. Our study shows that the suggested framework can relieve the limited sample size problem while reporting high accuracies by integrating multi-experiment data. PMID:24751870

  19. Diagnostic Oligonucleotide Microarray Fingerprinting of Bacillus Isolates

    SciTech Connect

    Chandler, Darrell P.; Alferov, Oleg; Chernov, Boris; Daly, Don S.; Golova, Julia; Perov, Alexander N.; Protic, Miroslava; Robison, Richard; Shipma, Matthew; White, Amanda M.; Willse, Alan R.

    2006-01-01

    A diagnostic, genome-independent microbial fingerprinting method using DNA oligonucleotide microarrays was used for high-resolution differentiation between closely related Bacillus strains, including two strains of Bacillus anthracis that are monomorphic (indistinguishable) via amplified fragment length polymorphism fingerprinting techniques. Replicated hybridizations on 391-probe nonamer arrays were used to construct a prototype fingerprint library for quantitative comparisons. Descriptive analysis of the fingerprints, including phylogenetic reconstruction, is consistent with previous taxonomic organization of the genus. Newly developed statistical analysis methods were used to quantitatively compare and objectively confirm apparent differences in microarray fingerprints with the statistical rigor required for microbial forensics and clinical diagnostics. These data suggest that a relatively simple fingerprinting microarray and statistical analysis method can differentiate between species in the Bacillus cereus complex, and between strains of B. anthracis. A synthetic DNA standard was used to understand underlying microarray and process-level variability, leading to specific recommendations for the development of a standard operating procedure and/or continued technology enhancements for microbial forensics and diagnostics.

  20. PRACTICAL STRATEGIES FOR PROCESSING AND ANALYZING SPOTTED OLIGONUCLEOTIDE MICROARRAY DATA

    EPA Science Inventory

    Thoughtful data analysis is as important as experimental design, biological sample quality, and appropriate experimental procedures for making microarrays a useful supplement to traditional toxicology. In the present study, spotted oligonucleotide microarrays were used to profile...

  1. Development and validation of a flax (Linum usitatissimum L.) gene expression oligo microarray

    PubMed Central

    2010-01-01

    Background Flax (Linum usitatissimum L.) has been cultivated for around 9,000 years and is therefore one of the oldest cultivated species. Today, flax is still grown for its oil (oil-flax or linseed cultivars) and its cellulose-rich fibres (fibre-flax cultivars) used for high-value linen garments and composite materials. Despite the wide industrial use of flax-derived products, and our actual understanding of the regulation of both wood fibre production and oil biosynthesis more information must be acquired in both domains. Recent advances in genomics are now providing opportunities to improve our fundamental knowledge of these complex processes. In this paper we report the development and validation of a high-density oligo microarray platform dedicated to gene expression analyses in flax. Results Nine different RNA samples obtained from flax inner- and outer-stems, seeds, leaves and roots were used to generate a collection of 1,066,481 ESTs by massive parallel pyrosequencing. Sequences were assembled into 59,626 unigenes and 48,021 sequences were selected for oligo design and high-density microarray (Nimblegen 385K) fabrication with eight, non-overlapping 25-mers oligos per unigene. 18 independent experiments were used to evaluate the hybridization quality, precision, specificity and accuracy and all results confirmed the high technical quality of our microarray platform. Cross-validation of microarray data was carried out using quantitative qRT-PCR. Nine target genes were selected on the basis of microarray results and reflected the whole range of fold change (both up-regulated and down-regulated genes in different samples). A statistically significant positive correlation was obtained comparing expression levels for each target gene across all biological replicates both in qRT-PCR and microarray results. Further experiments illustrated the capacity of our arrays to detect differential gene expression in a variety of flax tissues as well as between two contrasted flax varieties. Conclusion All results suggest that our high-density flax oligo-microarray platform can be used as a very sensitive tool for analyzing gene expression in a large variety of tissues as well as in different cultivars. Moreover, this highly reliable platform can also be used for the quantification of mRNA transcriptional profiling in different flax tissues. PMID:20964859

  2. Examining microarray slide quality for the EPA using SNL's hyperspectral microarray scanner.

    SciTech Connect

    Rohde, Rachel M.; Timlin, Jerilyn Ann

    2005-11-01

    This report summarizes research performed at Sandia National Laboratories (SNL) in collaboration with the Environmental Protection Agency (EPA) to assess microarray quality on arrays from two platforms of interest to the EPA. Custom microarrays from two novel, commercially produced array platforms were imaged with SNL's unique hyperspectral imaging technology and multivariate data analysis was performed to investigate sources of emission on the arrays. No extraneous sources of emission were evident in any of the array areas scanned. This led to the conclusions that either of these array platforms could produce high quality, reliable microarray data for the EPA toxicology programs. Hyperspectral imaging results are presented and recommendations for microarray analyses using these platforms are detailed within the report.

  3. Identifying Fishes through DNA Barcodes and Microarrays

    PubMed Central

    Kochzius, Marc; Seidel, Christian; Antoniou, Aglaia; Botla, Sandeep Kumar; Campo, Daniel; Cariani, Alessia; Vazquez, Eva Garcia; Hauschild, Janet; Hervet, Caroline; Hjörleifsdottir, Sigridur; Hreggvidsson, Gudmundur; Kappel, Kristina; Landi, Monica; Magoulas, Antonios; Marteinsson, Viggo; Nölte, Manfred; Planes, Serge; Tinti, Fausto; Turan, Cemal; Venugopal, Moleyur N.; Weber, Hannes; Blohm, Dietmar

    2010-01-01

    Background International fish trade reached an import value of 62.8 billion Euro in 2006, of which 44.6% are covered by the European Union. Species identification is a key problem throughout the life cycle of fishes: from eggs and larvae to adults in fisheries research and control, as well as processed fish products in consumer protection. Methodology/Principal Findings This study aims to evaluate the applicability of the three mitochondrial genes 16S rRNA (16S), cytochrome b (cyt b), and cytochrome oxidase subunit I (COI) for the identification of 50 European marine fish species by combining techniques of “DNA barcoding” and microarrays. In a DNA barcoding approach, neighbour Joining (NJ) phylogenetic trees of 369 16S, 212 cyt b, and 447 COI sequences indicated that cyt b and COI are suitable for unambiguous identification, whereas 16S failed to discriminate closely related flatfish and gurnard species. In course of probe design for DNA microarray development, each of the markers yielded a high number of potentially species-specific probes in silico, although many of them were rejected based on microarray hybridisation experiments. None of the markers provided probes to discriminate the sibling flatfish and gurnard species. However, since 16S-probes were less negatively influenced by the “position of label” effect and showed the lowest rejection rate and the highest mean signal intensity, 16S is more suitable for DNA microarray probe design than cty b and COI. The large portion of rejected COI-probes after hybridisation experiments (>90%) renders the DNA barcoding marker as rather unsuitable for this high-throughput technology. Conclusions/Significance Based on these data, a DNA microarray containing 64 functional oligonucleotide probes for the identification of 30 out of the 50 fish species investigated was developed. It represents the next step towards an automated and easy-to-handle method to identify fish, ichthyoplankton, and fish products. PMID:20838643

  4. Microarray analysis at single molecule resolution

    PubMed Central

    Mureşan, Leila; Jacak, Jarosław; Klement, Erich Peter; Hesse, Jan; Schütz, Gerhard J.

    2010-01-01

    Bioanalytical chip-based assays have been enormously improved in sensitivity in the recent years; detection of trace amounts of substances down to the level of individual fluorescent molecules has become state of the art technology. The impact of such detection methods, however, has yet not fully been exploited, mainly due to a lack in appropriate mathematical tools for robust data analysis. One particular example relates to the analysis of microarray data. While classical microarray analysis works at resolutions of two to 20 micrometers and quantifies the abundance of target molecules by determining average pixel intensities, a novel high resolution approach [1] directly visualizes individual bound molecules as diffraction limited peaks. The now possible quantification via counting is less susceptible to labeling artifacts and background noise. We have developed an approach for the analysis of high-resolution microarray images. It consists first of a single molecule detection step, based on undecimated wavelet transforms, and second, of a spot identification step via spatial statistics approach (corresponding to the segmentation step in the classical microarray analysis). The detection method was tested on simulated images with a concentration range of 0.001 to 0.5 molecules per square micron and signal-to-noise ratio (SNR) between 0.9 and 31.6. For SNR above 15 the false negatives relative error was below 15%. Separation of foreground/background proved reliable, in case foreground density exceeds background by a factor of 2. The method has also been applied to real data from high-resolution microarray measurements. PMID:20123580

  5. Design of a covalently bonded glycosphingolipid microarray.

    PubMed

    Arigi, Emma; Blixt, Ola; Buschard, Karsten; Clausen, Henrik; Levery, Steven B

    2012-01-01

    Glycosphingolipids (GSLs) are well known ubiquitous constituents of all eukaryotic cell membranes, yet their normal biological functions are not fully understood. As with other glycoconjugates and saccharides, solid phase display on microarrays potentially provides an effective platform for in vitro study of their functional interactions. However, with few exceptions, the most widely used microarray platforms display only the glycan moiety of GSLs, which not only ignores potential modulating effects of the lipid aglycone, but inherently limits the scope of application, excluding, for example, the major classes of plant and fungal GSLs. In this work, a prototype "universal" GSL-based covalent microarray has been designed, and preliminary evaluation of its potential utility in assaying protein-GSL binding interactions investigated. An essential step in development involved the enzymatic release of the fatty acyl moiety of the ceramide aglycone of selected mammalian GSLs with sphingolipid N-deacylase (SCDase). Derivatization of the free amino group of a typical lyso-GSL, lyso-G(M1), with a prototype linker assembled from succinimidyl-[(N-maleimidopropionamido)-diethyleneglycol] ester and 2-mercaptoethylamine, was also tested. Underivatized or linker-derivatized lyso-GSL were then immobilized on N-hydroxysuccinimide- or epoxide-activated glass microarray slides and probed with carbohydrate binding proteins of known or partially known specificities (i.e., cholera toxin B-chain; peanut agglutinin, a monoclonal antibody to sulfatide, Sulph 1; and a polyclonal antiserum reactive to asialo-G(M2)). Preliminary evaluation of the method indicated successful immobilization of the GSLs, and selective binding of test probes. The potential utility of this methodology for designing covalent microarrays that incorporate GSLs for serodiagnosis is discussed. PMID:22102144

  6. Methods in molecular cardiology: microarray technology

    PubMed Central

    van den Bosch, B.; Doevendans, P.A.; Lips, D.; Smeets, H.J.M.

    2003-01-01

    It has become more and more evident that changes in expression levels of genes can play an important role in cardiovascular diseases. Specific gene expression profiles may explain, for example, the pathophysiology of myocardial hypertrophy and pump failure and may provide clues for therapeutic interventions. Knowledge of gene expression patterns can also be applied for diagnostic and prognostic purposes, in which differences in gene activity can be used for classification. DNA microarray technology has become the method of choice to simultaneously study the expression of many different genes in a single assay. Each microarray contains many thousands of different DNA sequences attached to a glass slide. The amount of messenger RNA, which is a measure of gene activity, is compared for each gene on the microarray by labelling the mRNA with different fluorescently labelled nucleotides (Cy3 or Cy5) for the test and reference samples. After hybridisation to the microarray the relative amounts of a particular gene transcript in the two samples can be determined by measuring the signal intensities for the fluorescent groups (Cy3 and Cy5) and calculating signal ratios. This paper describes the development of in-house microarray technology, using commercially available cDNA collections. Several technical approaches will be compared and an overview of the pitfalls and possibilities will be presented. The technology will be explained in the context of our project to determine gene expression differences between normal, hypertrophic and failing heart. ImagesFigure 1Figure 2Figure 3Figure 4Figure 5Figure 6Figure 7Figure 9 PMID:25696214

  7. The development of common data elements for a multi-institute prostate cancer tissue bank: The Cooperative Prostate Cancer Tissue Resource (CPCTR) experience

    PubMed Central

    Patel, Ashokkumar A; Kajdacsy-Balla, André; Berman, Jules J; Bosland, Maarten; Datta, Milton W; Dhir, Rajiv; Gilbertson, John; Melamed, Jonathan; Orenstein, Jan; Tai, Kuei-Fang; Becich, Michael J

    2005-01-01

    Background The Cooperative Prostate Cancer Tissue Resource (CPCTR) is a consortium of four geographically dispersed institutions that are funded by the U.S. National Cancer Institute (NCI) to provide clinically annotated prostate cancer tissue samples to researchers. To facilitate this effort, it was critical to arrive at agreed upon common data elements (CDEs) that could be used to collect demographic, pathologic, treatment and clinical outcome data. Methods The CPCTR investigators convened a CDE curation subcommittee to develop and implement CDEs for the annotation of collected prostate tissues. The draft CDEs were refined and progressively annotated to make them ISO 11179 compliant. The CDEs were implemented in the CPCTR database and tested using software query tools developed by the investigators. Results By collaborative consensus the CPCTR CDE subcommittee developed 145 data elements to annotate the tissue samples collected. These included for each case: 1) demographic data, 2) clinical history, 3) pathology specimen level elements to describe the staging, grading and other characteristics of individual surgical pathology cases, 4) tissue block level annotation critical to managing a virtual inventory of cases and facilitating case selection, and 5) clinical outcome data including treatment, recurrence and vital status. These elements have been used successfully to respond to over 60 requests by end-users for tissue, including paraffin blocks from cases with 5 to 10 years of follow up, tissue microarrays (TMAs), as well as frozen tissue collected prospectively for genomic profiling and genetic studies. The CPCTR CDEs have been fully implemented in two major tissue banks and have been shared with dozens of other tissue banking efforts. Conclusion The freely available CDEs developed by the CPCTR are robust, based on "best practices" for tissue resources, and are ISO 11179 compliant. The process for CDE development described in this manuscript provides a framework model for other organ sites and has been used as a model for breast and melanoma tissue banking efforts. PMID:16111498

  8. A longitudinal study of MARS MRI scanning of soft-tissue lesions around metal-on-metal total hip arthroplasties and disease progression.

    PubMed

    Briant-Evans, T W; Lyle, N; Barbur, S; Hauptfleisch, J; Amess, R; Pearce, A R; Conn, K S; Stranks, G J; Britton, J M

    2015-10-01

    We investigated the changes seen on serial metal artefact reduction magnetic resonance imaging scans (MARS-MRI) of metal-on-metal total hip arthroplasties (MoM THAs). In total 155 THAs, in 35 male and 100 female patients (mean age 70.4 years, 42 to 91), underwent at least two MRI scans at a mean interval of 14.6 months (2.6 to 57.1), at a mean of 48.2 months (3.5 to 93.3) after primary hip surgery. Scans were graded using a modification of the Oxford classification. Progression of disease was defined as an increase in grade or a minimum 10% increase in fluid lesion volume at second scan. A total of 16 hips (30%) initially classified as 'normal' developed an abnormality on the second scan. Of those with 'isolated trochanteric fluid' 9 (47%) underwent disease progression, as did 7 (58%) of 'effusions'. A total of 54 (77%) of hips initially classified as showing adverse reactions to metal debris (ARMD) progressed, with higher rates of progression in higher grades. Disease progression was associated with high blood cobalt levels or an irregular pseudocapsule lining at the initial scan. There was no association with changes in functional scores. Adverse reactions to metal debris in MoM THAs may not be as benign as previous reports have suggested. Close radiological follow-up is recommended, particularly in high-risk groups. PMID:26430006

  9. Viral diagnosis in Indian livestock using customized microarray chips

    PubMed Central

    Yadav, Brijesh S; Pokhriyal, Mayank; Ratta, Barkha; Kumar, Ajay; Saxena, Meeta; Sharma, Bhaskar

    2015-01-01

    Viral diagnosis in Indian livestock using customized microarray chips is gaining momentum in recent years. Hence, it is possible to design customized microarray chip for viruses infecting livestock in India. Customized microarray chips identified Bovine herpes virus-1 (BHV-1), Canine Adeno Virus-1 (CAV-1), and Canine Parvo Virus-2 (CPV-2) in clinical samples. Microarray identified specific probes were further confirmed using RT-PCR in all clinical and known samples. Therefore, the application of microarray chips during viral disease outbreaks in Indian livestock is possible where conventional methods are unsuitable. It should be noted that customized application requires a detailed cost efficiency calculation. PMID:26912948

  10. Advancing translational research with next-generation protein microarrays.

    PubMed

    Yu, Xiaobo; Petritis, Brianne; LaBaer, Joshua

    2016-04-01

    Protein microarrays are a high-throughput technology used increasingly in translational research, seeking to apply basic science findings to enhance human health. In addition to assessing protein levels, posttranslational modifications, and signaling pathways in patient samples, protein microarrays have aided in the identification of potential protein biomarkers of disease and infection. In this perspective, the different types of full-length protein microarrays that are used in translational research are reviewed. Specific studies employing these microarrays are presented to highlight their potential in finding solutions to real clinical problems. Finally, the criteria that should be considered when developing next-generation protein microarrays are provided. PMID:26749402

  11. Optimised laser microdissection of the human ocular surface epithelial regions for microarray studies

    PubMed Central

    2013-01-01

    Background The most important challenge of performing insitu transcriptional profiling of the human ocular surface epithelial regions is obtaining samples in sufficient amounts, without contamination from adjacent tissue, as the region of interest is microscopic and closely apposed to other tissues regions. We have effectively collected ocular surface (OS) epithelial tissue samples from the Limbal Epithelial Crypt (LEC), limbus, cornea and conjunctiva of post-mortem cadaver eyes with laser microdissection (LMD) technique for gene expression studies with spotted oligonucleotide microarrays and Gene 1.0 ST arrays. Methods Human donor eyes (4 pairs for spotted oligonucleotide microarrays, 3 pairs for Gene 1.0 ST arrays) consented for research were included in this study with due ethical approval of the Nottingham Research Ethics Committee. Eye retrieval was performed within 36 hours of post-mortem period. The dissected corneoscleral buttons were immersed in OCT media and frozen in liquid nitrogen and stored at −80°C till further use. Microscopic tissue sections of interest were taken on PALM slides and stained with Toluidine Blue for laser microdissection with PALM microbeam systems. Optimisation of the laser microdissection technique was crucial for efficient and cost effective sample collection. Results The starting concentration of RNA as stipulated by the protocol of microarray platforms was taken as the cut-off concentration of RNA samples in our studies. The area of LMD tissue processed for spotted oligonucleotide microarray study ranged from 86,253 μm2 in LEC to 392,887 μm2 in LEC stroma. The RNA concentration of the LMD samples ranged from 22 to 92 pg/μl. The recommended starting concentration of the RNA samples used for Gene 1.0 ST arrays was 6 ng/5 μl. To achieve the desired RNA concentration the area of ocular surface epithelial tissue sample processed for the Gene 1.0 ST array experiments was approximately 100,0000 μm2 to 130,0000 μm2. RNA concentration of these samples ranged from 10.88 ng/12 μl to 25.8 ng/12 μl, with the RNA integrity numbers (RIN) for these samples from 3.3 to 7.9. RNA samples with RIN values below 2, that had failed to amplify satisfactorily were discarded. Conclusions The optimised protocol for sample collection and laser microdissection improved the RNA yield of the insitu ocular surface epithelial regions for effective microarray studies on spotted oligonucleotide and affymetrix platforms. PMID:24160452

  12. Lossless compression of microarray images using image-dependent finite-context models.

    PubMed

    Neves, António J R; Pinho, Armando J

    2009-02-01

    The use of microarray expression data in state-of-the-art biology has been well established. The widespread adoption of this technology, coupled with the significant volume of data generated per experiment, in the form of images, has led to significant challenges in storage and query retrieval. In this paper, we present a lossless bitplane-based method for efficient compression of microarray images. This method is based on arithmetic coding driven by image-dependent multibitplane finite-context models. It produces an embedded bitstream that allows progressive, lossy-to-lossless decoding. We compare the compression efficiency of the proposed method with three image compression standards (JPEG2000, JPEG-LS, and JBIG) and also with the two most recent specialized methods for microarray image coding. The proposed method gives better results for all images of the test sets and confirms the effectiveness of bitplane-based methods and finite-context modeling for the lossless compression of microarray images. PMID:19188108

  13. Microarray gene expression profiling and bioinformatics analysis of premature ovarian failure in a rat model.

    PubMed

    Li, Ji; Fan, Shengjun; Han, Dongwei; Xie, Jiaming; Kuang, Haixue; Ge, Pengling

    2014-12-01

    Premature ovarian failure (POF) remains one of the major gynecological problems worldwide which affected 1% of women. Even though tremendous achievements had been acquired as opposed to years past, molecular pathogenesis associated with POF is still unclear and needs to be well-defined. The aim of this study was to analyze the gene expression profiles in the POF rat model. To predict potential regulating factors, we firstly treated female Sprague Dawley (SD) rat with 4-vinylcyclohexene diepoxide (VCD). Total RNA from ovarian tissue was converted to cDNA and hybridized to mRNA Chip array. The differentially expressed genes (DEGs) were identified by two-sample t test and assessed using hierarchical clustering and Principal Component Analysis methods. Potential regulatory targets associated with these DEGs were constructed using BisoGenet in Cytoscape. Gene Ontology (GO) and functional enrichment analysis were performed using BiNGO and DAVID, respectively. As the results, 25 DEGs were found to be closely associated with POF initiation. Hierarchical clustering and Principal Component Analysis on the transcriptional profiles revealed an excellent separation of the vehicle and POF compartments. Pathway enrichment analysis based on the disease-gene interaction network analysis led to the identification of two core signaling pathways that were strongly affected during POF initiation and progression: immune response and cardiovascular disorders. In conclusion, we constructed a gene regulatory network associated with POF using the microarray gene expression profiling, and screened out some genes or transcription factors that may be used as potential molecular therapeutic targets for POF. PMID:25445499

  14. RNA-seq as a powerful tool for penaeid shrimp genetic progress

    PubMed Central

    Santos, Camilla A.; Blanck, Danielly V.; de Freitas, Patrcia D.

    2014-01-01

    The sequences of all different RNA transcripts present in a cell or tissue that are related to the gene expression and its functional control represent what it is called a transcriptome. The transcripts vary between cells, tissues, ontogenetic and environmental conditions, and the knowledge that can be gained through them is of a solid relevance for genetic applications in aquaculture. Some of the techniques used in transcriptome studies, such as microarrays, are being replaced for next-generation sequencing approaches. RNA-seq emerges as a new possibility for the transcriptome complexity analysis as well as for the candidate genes and polymorphisms identification of penaeid species. Thus, it may also help to understand the determination of complex traits mechanisms and genetic improvement of stocks. In this review, it is first introduced an overview of transcriptome analysis by RNA-seq, followed by a discussion of how this approach may be applied in genetic progress within penaeid stocks. PMID:25221571

  15. PMD: A Resource for Archiving and Analyzing Protein Microarray data

    PubMed Central

    Xu, Zhaowei; Huang, Likun; Zhang, Hainan; Li, Yang; Guo, Shujuan; Wang, Nan; Wang, Shi-hua; Chen, Ziqing; Wang, Jingfang; Tao, Sheng-ce

    2016-01-01

    Protein microarray is a powerful technology for both basic research and clinical study. However, because there is no database specifically tailored for protein microarray, the majority of the valuable original protein microarray data is still not publically accessible. To address this issue, we constructed Protein Microarray Database (PMD), which is specifically designed for archiving and analyzing protein microarray data. In PMD, users can easily browse and search the entire database by experimental name, protein microarray type, and sample information. Additionally, PMD integrates several data analysis tools and provides an automated data analysis pipeline for users. With just one click, users can obtain a comprehensive analysis report for their protein microarray data. The report includes preliminary data analysis, such as data normalization, candidate identification, and an in-depth bioinformatics analysis of the candidates, which include functional annotation, pathway analysis, and protein-protein interaction network analysis. PMD is now freely available at www.proteinmicroarray.cn. PMID:26813635

  16. Tissues from the irradiated dog/mouse archive

    SciTech Connect

    Gayle Woloschak

    2007-04-01

    The purpose of this project is to organize the databases/information and organize and move the tissues from the long-term dog (4,000 dogs) and mouse (over 30,000 mice) radiation experiments done at Argonne National Laboratory during the 1970's and 80's to Northwestern University. These studies were done with the intention of understanding the effects of exposure to radiation at a variety of different doses, dose-rates, and radiation qualities on end-points such as life-shortening, carcinogenesis, cause of death, shifts in disease incidence and other biological parameters. Organ and tissue samples from these animals including cancers, metastases and other significant degenerative and inflammatory lesions and those in a regular protocol of normal tissues were preserved in paraffin blocks, tissue impressions and sections and represent a great resource for the radiation biology community. These collections are particularly significant since these experiments are not likely to be repeated because of the extreme cost of monies and time for such large-scale animal studies. The long-term goal is to make these tissues and databases available to the wider scientific community so that questions such as tissue sensitivity, early and late effects, low dose and protracted dose responses of normal and tumor tissues, etc. can be examined and defined. Recent advances in biology particularly at the subcellular and molecular level now permit microarray-based gene expression array analyses from paraffin-embedded tissues (where RNA samples are significantly degraded), synchrotron-based studies of metal and other elemental distribution patterns in tissues, PCR-based analyses for mutation detection, and other similar approaches that were not available when the long¬ term animal studies were designed and initiated. Understanding the basis and progression of radiation damage should also permit rational approaches to prevention and mitigation of those damages. Therefore, as stated earlier, these tissues and their related documentation, represent a significant resource for future studies. For this project, we propose to accomplish the following objectives: (1) inventory and organize the tissues, blood smears, wet-tissues and paper-¬based information that is available in the tissue bank at Argonne National Laboratory; (2) convert the existing Oracle database of the mouse studies to MS Access( the dog data is already in this format which is far more user friendly and widely used in business and research) , (3) move the remaining samples and documentation from dogs that had been transferred from ANL to New Mexico (in Dr. F. Hahn's care) to Northwestern University and add these to the inventory; (4) move the tissues and Access database at Argonne National Laboratory to Northwestern University.

  17. Cellular Signal Transduction Pathways by Leptin in Colorectal Cancer Tissue: Preliminary Results

    PubMed Central

    Nowakowska-Zajdel, Ewa; Mazurek, Urszula; Stachowicz, Malgorzata; Niedworok, Elzbieta; Fatyga, Edyta; Muc-Wierzgoń, Małgorzata

    2011-01-01

    The aim of the study was to analyse genes typing with the use of the oligonucleotide microarray technique (HG-U133A, Affymetrix) differentiating colorectal cancer tissues from tissues assessed histopathologically as healthy ones among a panel of 91 mRNA of genes encoding proteins involved in activation of cellular signal transduction pathways by leptin. Frozen tumor specimens from 11 colon cancer patients in various stages of clinical progression of the disease in an I–IV stage scale according to the TNM staging were used in molecular tests. Among the genes participating in the cascade of signal transfer in cell activated by leptin, the following ones: AKT1, STAT3, MCL1 were qualified as differentiating stage I and II and VEGFC, CCNDI the encoding genes respectively as differentiating III and IV stage neoplasm. It is necessary to extend studies of analysis of cellular signal transduction pathways by leptin in colorectal cancer initiation and transformation processes. PMID:22363883

  18. What are microarrays teaching us about sleep?

    PubMed Central

    Mackiewicz, Miroslaw; Zimmerman, John E.; Shockley, Keith R.; Churchill, Gary A.; Pack, Allan I.

    2010-01-01

    Many fundamental questions about sleep remain unanswered. The presence of sleep across phyla suggests that it must serve a basic cellular and/or molecular function. Microarray studies, performed in several model systems, have identified classes of genes that are sleep-state regulated. This has led to the following concepts: first, a function of sleep is to maintain synaptic homeostasis; second, sleep is a stage of macromolecule biosynthesis; third, extending wakefulness leads to downregulation of several important metabolic pathways; and, fourth, extending wakefulness leads to endoplasmic reticulum stress. In human studies, microarrays are being applied to the identification of biomarkers for sleepiness and for the common debilitating condition of obstructive sleep apnea. PMID:19162550

  19. Glycan microarrays for decoding the glycome

    PubMed Central

    Rillahan, Cory D.; Paulson, James C.

    2011-01-01

    In the last decade glycan microarrays have revolutionized the analysis of the specificity of glycan binding proteins, providing information that simultaneously illuminates the biology mediated by them and decodes the information content of the glycome. Numerous methods have emerged for arraying glycans in a ‘chip’ format, and glycan libraries have been assembled that address the diversity of the human glycome. Such arrays have been successfully used for analysis of glycan binding proteins that mediate mammalian biology, host-pathogen interactions, immune recognition of glycans relevant to vaccine production and cancer antigens. This review covers the development of glycan microarrays and applications that have provided insights into the roles of mammalian and microbial glycan binding proteins. PMID:21469953

  20. Immobilization Techniques for Microarray: Challenges and Applications

    PubMed Central

    Nimse, Satish Balasaheb; Song, Keumsoo; Sonawane, Mukesh Digambar; Sayyed, Danishmalik Rafiq; Kim, Taisun

    2014-01-01

    The highly programmable positioning of molecules (biomolecules, nanoparticles, nanobeads, nanocomposites materials) on surfaces has potential applications in the fields of biosensors, biomolecular electronics, and nanodevices. However, the conventional techniques including self-assembled monolayers fail to position the molecules on the nanometer scale to produce highly organized monolayers on the surface. The present article elaborates different techniques for the immobilization of the biomolecules on the surface to produce microarrays and their diagnostic applications. The advantages and the drawbacks of various methods are compared. This article also sheds light on the applications of the different technologies for the detection and discrimination of viral/bacterial genotypes and the detection of the biomarkers. A brief survey with 115 references covering the last 10 years on the biological applications of microarrays in various fields is also provided. PMID:25429408

  1. Statistical Considerations for Analysis of Microarray Experiments

    PubMed Central

    Owzar, Kouros; Barry, William T.; Jung, Sin-Ho

    2014-01-01

    Microarray technologies enable the simultaneous interrogation of expressions from thousands of genes from a biospecimen sample taken from a patient. This large set of expressions generate a genetic profile of the patient that may be used to identify potential prognostic or predictive genes or genetic models for clinical outcomes. The aim of this article is to provide a broad overview of some of the major statistical considerations for the design and analysis of microarrays experiments conducted as correlative science studies to clinical trials. An emphasis will be placed on how the lack of understanding and improper use of statistical concepts and methods will lead to noise discovery and misinterpretation of experimental results. PMID:22212230

  2. High Frequency of Chlamydia trachomatis Mixed Infections Detected by Microarray Assay in South American Samples

    PubMed Central

    Gallo Vaulet, Lucía; Entrocassi, Carolina; Portu, Ana I.; Castro, Erica; Di Bartolomeo, Susana; Ruettger, Anke; Sachse, Konrad; Rodriguez Fermepin, Marcelo

    2016-01-01

    Chlamydia trachomatis is one of the most common sexually transmitted infections worldwide. Based on sequence variation in the ompA gene encoding the major outer membrane protein, the genotyping scheme distinguishes 17 recognized genotypes, i.e. A, B, Ba, C, D, Da, E, F, G, H, I, Ia, J, K, L1, L2, and L3. Genotyping is an important tool for epidemiological tracking of C. trachomatis infections, including the revelation of transmission pathways and association with tissue tropism and pathogenicity. Moreover, genotyping can be useful for clinicians to establish the correct treatment when LGV strains are detected. Recently a microarray assay was described that offers several advantages, such as rapidity, ease of standardization and detection of mixed infections. The aim of this study was to evaluate the performance of the DNA microarray-based assay for C. trachomatis genotyping of clinical samples already typed by PCR-RFLP from South America. The agreement between both typing techniques was 90.05% and the overall genotype distribution obtained with both techniques was similar. Detection of mixed-genotype infections was significantly higher using the microarray assay (8.4% of cases) compared to PCR-RFLP (0.5%). Among 178 samples, the microarray assay identified 10 ompA genotypes, i.e. D, Da, E, F, G, H, I, J, K and L2. The most predominant type was genotype E, followed by D and F. PMID:27082962

  3. High Frequency of Chlamydia trachomatis Mixed Infections Detected by Microarray Assay in South American Samples.

    PubMed

    Gallo Vaulet, Lucía; Entrocassi, Carolina; Portu, Ana I; Castro, Erica; Di Bartolomeo, Susana; Ruettger, Anke; Sachse, Konrad; Rodriguez Fermepin, Marcelo

    2016-01-01

    Chlamydia trachomatis is one of the most common sexually transmitted infections worldwide. Based on sequence variation in the ompA gene encoding the major outer membrane protein, the genotyping scheme distinguishes 17 recognized genotypes, i.e. A, B, Ba, C, D, Da, E, F, G, H, I, Ia, J, K, L1, L2, and L3. Genotyping is an important tool for epidemiological tracking of C. trachomatis infections, including the revelation of transmission pathways and association with tissue tropism and pathogenicity. Moreover, genotyping can be useful for clinicians to establish the correct treatment when LGV strains are detected. Recently a microarray assay was described that offers several advantages, such as rapidity, ease of standardization and detection of mixed infections. The aim of this study was to evaluate the performance of the DNA microarray-based assay for C. trachomatis genotyping of clinical samples already typed by PCR-RFLP from South America. The agreement between both typing techniques was 90.05% and the overall genotype distribution obtained with both techniques was similar. Detection of mixed-genotype infections was significantly higher using the microarray assay (8.4% of cases) compared to PCR-RFLP (0.5%). Among 178 samples, the microarray assay identified 10 ompA genotypes, i.e. D, Da, E, F, G, H, I, J, K and L2. The most predominant type was genotype E, followed by D and F. PMID:27082962

  4. High-Throughput Nano-Biofilm Microarray for Antifungal Drug Discovery

    PubMed Central

    Srinivasan, Anand; Leung, Kai P.; Lopez-Ribot, Jose L.; Ramasubramanian, Anand K.

    2013-01-01

    ABSTRACT Micro- and nanoscale technologies have radically transformed biological research from genomics to tissue engineering, with the relative exception of microbial cell culture, which is still largely performed in microtiter plates and petri dishes. Here, we present nanoscale culture of the opportunistic fungal pathogen Candida albicans on a microarray platform. The microarray consists of 1,200 individual cultures of 30 nl of C. albicans biofilms (“nano-biofilms”) encapsulated in an inert alginate matrix. We demonstrate that these nano-biofilms are similar to conventional macroscopic biofilms in their morphological, architectural, growth, and phenotypic characteristics. We also demonstrate that the nano-biofilm microarray is a robust and efficient tool for accelerating the drug discovery process: (i) combinatorial screening against a collection of 28 antifungal compounds in the presence of immunosuppressant FK506 (tacrolimus) identified six drugs that showed synergistic antifungal activity, and (ii) screening against the NCI challenge set small-molecule library identified three heretofore-unknown hits. This cell-based microarray platform allows for miniaturization of microbial cell culture and is fully compatible with other high-throughput screening technologies. PMID:23800397

  5. Sensitive and high resolution subcutaneous fluorescence in vivo imaging using upconversion nanoparticles and microarrays.

    PubMed

    Li, Xin; Li, Zhuoqi; Gan, Wupeng; Wang, Tongzhou; Zhao, Songmin; Lu, Ying; Cheng, Jing; Huang, Guoliang

    2013-07-01

    A sensitive and high resolution small animal in vivo imaging system using upconversion nanoparticles (UNPs) and microarrays was developed. The fluorescence tomography using UNPs could achieve higher precision than that using ordinary fluorophores, which was theoretically explained by the finite element method (FEM). Given the autofluorescence-insensitive property of UNPs, a high subcutaneous detection sensitivity of 0.93 × 10(-4) wt% could be achieved with a UNP volume of ∼10 μL in tissue phantoms. Furthermore, UNP fluorophore microarrays (25, 50 and 100 μm arrays) embedded under mouse skin were prepared for subcutaneous in vivo detection. An optical clearing method was applied to enhance the skin transparency and improve the spatial resolution. The results demonstrated that the optimized system could achieve a spatial resolution of 50 μm for in vivo detection of subcutaneous UNP microarrays. Taken together, we conclude that the proposed system and UNP microarrays could achieve sensitive, high resolution subcutaneous in vivo detection, and have great potential for high throughput detection of tumors and other diseases. PMID:23687650

  6. Microarray-based identification of differentially expressed genes in extramammary Pagets disease

    PubMed Central

    Lin, Jin-Ran; Liang, Jun; Zhang, Qiao-An; Huang, Qiong; Wang, Shang-Shang; Qin, Hai-Hong; Chen, Lian-Jun; Xu, Jin-Hua

    2015-01-01

    Extramammary Pagets disease (EMPD) is a rare cutaneous malignancy accounting for approximately 1-2% of vulvar cancers. The rarity of this disease has caused difficulties in characterization and the molecular mechanism underlying EMPD development remains largely unclear. Here we used microarray analysis to identify differentially expressed genes in EMPD of the scrotum comparing with normal epithelium from healthy donors. Agilent single-channel microarray was used to compare the gene expression between 6 EMPD specimens and 6 normal scrotum epithelium samples. A total of 799 up-regulated genes and 723 down-regulated genes were identified in EMPD tissues. Real-time PCR was conducted to verify the differential expression of some representative genes, including ERBB4, TCF3, PAPSS2, PIK3R3, PRLR, SULT1A1, TCF7L1, and CREB3L4. Generally, the real-time PCR results were consistent with microarray data, and the expression of ERBB4, PRLR, TCF3, PIK3R3, SULT1A1, and TCF7L1 was significantly overexpressed in EMPD (P<0.05). Moreover, the overexpression of PRLR in EMPD, a receptor for the anterior pituitary hormone prolactin (PRL), was confirmed by immunohistochemistry. These data demonstrate that the differentially expressed genes from the microarray-based identification are tightly associated with EMPD occurrence. PMID:26221264

  7. Protein Microarray Analysis in Patients With Asthma*

    PubMed Central

    Kim, Hyo-Bin; Kim, Chang-Keun; Iijima, Koji; Kobayashi, Takao; Kita, Hirohito

    2010-01-01

    Background Microarray technology offers a new opportunity to gain insight into global gene and protein expression profiles in asthma. To identify novel factors produced in the asthmatic airway, we analyzed sputum samples by using a membrane-based human cytokine microarray technology in patients with bronchial asthma (BA). Methods Induced sputum was obtained from 28 BA subjects, 20 nonasthmatic atopic control (AC) subjects, and 38 nonasthmatic nonatopic normal control (NC) subjects. The microarray samples of subjects were randomly selected from nine BA subjects, three AC subjects, and six NC subjects. Sputum supernatants were analyzed using a custom human cytokine array (RayBio Custom Human Cytokine Array; RayBiotech; Norcross, GA) designed to analyze 79 specific cytokines simultaneously. The levels of growth-regulated oncogene (GRO)-α, eotaxin-2, and pulmonary and activation-regulated chemokine (PARC)/CCL18 were measured by sandwich enzyme-linked immunosorbent assays (ELISAs), and eosinophil-derived neurotoxin (EDN) was measured by radioimmunoassay. Results By microarray, the signal intensities for GRO-α, eotaxin-2, and PARC were significantly higher in BA subjects than in AC and NC subjects (p = 0.036, p = 0.042, and p = 0.033, respectively). By ELISA, the sputum PARC protein levels were significantly higher in BA subjects than in AC and NC subjects (p < 0.0001). Furthermore, PARC levels correlated significantly with sputum eosinophil percentages (r = 0.570, p < 0.0001) and the levels of EDN(r = 0.633, p < 0.0001), the regulated upon activation, normal T cell expressed and secreted cytokine (r = 0.440, p < 0.001), interleukin-4 (r = 0.415, p < 0.01), and interferon-γ (r = 0.491, p < 0.001). Conclusions By a nonbiased screening approach, a chemokine, PARC, is elevated in sputum specimens from patients with asthma. PARC may play important roles in development of airway eosinophilic inflammation in asthma. PMID:19017877

  8. A review of software for microarray genotyping

    PubMed Central

    2011-01-01

    The focus of this review is software for the genotyping of microarray single nucleotide polymorphisms, in particular software for Affymetrix and Illumina arrays. Different statistical principles and ideas have been applied to the construction of genotyping algorithms -- for example, likelihood versus Bayesian modelling, and whether to genotype one or all arrays at a time. The release of new arrays is generally followed by new, or updated, algorithms. PMID:21712191

  9. High-Throughput Enzyme Kinetics Using Microarrays

    SciTech Connect

    Guoxin Lu; Edward S. Yeung

    2007-11-01

    We report a microanalytical method to study enzyme kinetics. The technique involves immobilizing horseradish peroxidase on a poly-L-lysine (PLL)- coated glass slide in a microarray format, followed by applying substrate solution onto the enzyme microarray. Enzyme molecules are immobilized on the PLL-coated glass slide through electrostatic interactions, and no further modification of the enzyme or glass slide is needed. In situ detection of the products generated on the enzyme spots is made possible by monitoring the light intensity of each spot using a scientific-grade charged-coupled device (CCD). Reactions of substrate solutions of various types and concentrations can be carried out sequentially on one enzyme microarray. To account for the loss of enzyme from washing in between runs, a standard substrate solution is used for calibration. Substantially reduced amounts of substrate solution are consumed for each reaction on each enzyme spot. The Michaelis constant K{sub m} obtained by using this method is comparable to the result for homogeneous solutions. Absorbance detection allows universal monitoring, and no chemical modification of the substrate is needed. High-throughput studies of native enzyme kinetics for multiple enzymes are therefore possible in a simple, rapid, and low-cost manner.

  10. Calibration of microarray gene-expression data.

    PubMed

    Binder, Hans; Preibisch, Stephan; Berger, Hilmar

    2010-01-01

    Calibration of microarray measurements aims at removing systematic biases from the probe-level data to get expression estimates that linearly correlate with the transcript abundance in the studied samples. The improvement of calibration methods is an essential prerequisite for estimating absolute expression levels, which, in turn, are required for quantitative analyses of transcriptional regulation, for example, in the context of gene profiling of diseases. We address hybridization on microarrays as a reaction process in a complex environment and express the measured intensities as a function of the input quantities of the experiment. Popular calibration methods such as MAS5, dChip, RMA, gcRMA, vsn, and PLIER are briefly reviewed and assessed in light of the hybridization model and of previous benchmark studies. We present our hook method, a new calibration approach that is based on a graphical summary of the actual hybridization characteristics of a particular microarray. Although single-chip related, hook performs as well as the multi-chip-related gcRMA, presently one of the best state-of-the-art methods for estimating expression values. The hook method, in addition, provides a set of chip summary characteristics that evaluate the performance of a given hybridization. The algorithm of the method is briefly described and its performance is exemplified. PMID:19882273

  11. G protein-coupled receptor (GPCR) microarrays

    NASA Astrophysics Data System (ADS)

    Fang, Ye; Frutos, Anthony G.; Lahiri, Joydeep

    2002-06-01

    G protein-coupled receptors (GPCRs) are the largest family of cell surface proteins involved in transmitting extracellular signals to the interior of the cell. These membrane-spanning proteins constitute one of the most important families of drug targets. Despite their importance, the power and utility of microarray technology has not been extended to GPCRs or other membrane proteins because of issues due to immobilization - these proteins typically need to be embedded in membrane environment to maintain their native conformations. This paper describes the fabrication of GPCR microarrays by conventional robotic pin-printing and demonstrates straightforward assays for screening of ligands on these arrays. GPCRs, obtained as membrane preparations form cell lines over-expressing particular GPCRs, were arrayed using a quill-pin printer. The arrays were incubated with solutions of labeled cognate ligands and unlabeled compounds, and imaged using a fluorescence scanner. The assays conducted were designed to test: (i) the specificity of ligand binding among different families of GPCRs; (ii) the selectivity of ligand binding and inhibition among different members of a GPCR family; (iii) the affinity of ligand binding. The results showed highly selective binding of ligands to arrays of receptors, with affinities similar to those reported in the literature and obtained suing other techniques. This demonstration of membrane-protein arrays and associated assays overcomes a fundamental limitation in protein microchip technology - the lack of practical microarray based methods for membrane proteins.

  12. Integrating data from heterogeneous DNA microarray platforms.

    PubMed

    Valente, Eduardo; Rocha, Miguel

    2015-01-01

    DNA microarrays are one of the most used technologies for gene expression measurement. However, there are several distinct microarray platforms, from different manufacturers, each with its own measurement protocol, resulting in data that can hardly be compared or directly integrated. Data integration from multiple sources aims to improve the assertiveness of statistical tests, reducing the data dimensionality problem. The integration of heterogeneous DNA microarray platforms comprehends a set of tasks that range from the re-annotation of the features used on gene expression, to data normalization and batch effect elimination. In this work, a complete methodology for gene expression data integration and application is proposed, which comprehends a transcript-based re-annotation process and several methods for batch effect attenuation. The integrated data will be used to select the best feature set and learning algorithm for a brain tumor classification case study. The integration will consider data from heterogeneous Agilent and Affymetrix platforms, collected from public gene expression databases, such as The Cancer Genome Atlas and Gene Expression Omnibus. PMID:26673932

  13. Inverse Langmuir method for oligonucleotide microarray analysis

    PubMed Central

    Mulders, Geert CWM; Barkema, Gerard T; Carlon, Enrico

    2009-01-01

    Background An algorithm for the analysis of Affymetrix Genechips is presented. This algorithm, referred to as the Inverse Langmuir Method (ILM), estimates the binding of transcripts to complementary probes using DNA/RNA hybridization free energies, and the hybridization between partially complementary transcripts in solution using RNA/RNA free energies. The balance between these two competing reactions allows for the translation of background-subtracted intensities into transcript concentrations. Results To validate the ILM, it is applied to publicly available microarray data from a multi-lab comparison study. Here, microarray experiments are performed on samples which deviate only in few genes. The log2 fold change between these two samples, as obtained from RT-PCR experiments, agrees well with the log2 fold change as obtained with the ILM, indicating that the ILM determines changes in the expression level accurately. We also show that the ILM allows for the identification of outlying probes, as it yields independent concentration estimates per probe. Conclusion The ILM is robust and offers an interesting alternative to purely statistical algorithms for microarray data analysis. PMID:19232092

  14. Chicken sperm transcriptome profiling by microarray analysis.

    PubMed

    Singh, R P; Shafeeque, C M; Sharma, S K; Singh, R; Mohan, J; Sastry, K V H; Saxena, V K; Azeez, P A

    2016-03-01

    It has been confirmed that mammalian sperm contain thousands of functional RNAs, and some of them have vital roles in fertilization and early embryonic development. Therefore, we attempted to characterize transcriptome of the sperm of fertile chickens using microarray analysis. Spermatozoal RNA was pooled from 10 fertile males and used for RNA preparation. Prior to performing the microarray, RNA quality was assessed using a bioanalyzer, and gDNA and somatic cell RNA contamination was assessed by CD4 and PTPRC gene amplification. The chicken sperm transcriptome was cross-examined by analysing sperm and testes RNA on a 4 44K chicken array, and results were verified by RT-PCR. Microarray analysis identified 21?639 predominantly nuclear-encoded transcripts in chicken sperm. The majority (66.55%) of the sperm transcripts were shared with the testes, while surprisingly, 33.45% transcripts were detected (raw signal intensity greater than 50) only in the sperm and not in the testes. The greatest proportion of up-regulated transcripts were responsible for signal transduction (63.20%) followed by embryonic development (56.76%) and cell structure (56.25%). Of the 20 most abundant transcripts, 18 remain uncharacterized, whereas the least abundant genes were mostly associated with the ribosome. These findings lay a foundation for more detailed investigations on sperm RNAs in chickens to identify sperm-based biomarkers for fertility. PMID:26868024

  15. The role of APE/Ref-1 signaling pathway in hepatocellular carcinoma progression

    PubMed Central

    YANG, ZHEN; YANG, SUN; MISNER, BOBBYE J.; LIU-SMITH, FENG; MEYSKENS, FRANK L.

    2014-01-01

    Hepatocellular carcinoma (HCC) is responsible for a third of the estimated cancer-caused deaths worldwide. To deeply understand the mechanisms controlling HCC progression is of primary importance to develop new approaches for treatment. Apurinic/apyrimidinic endonuclease-1/redox effector factor 1 (APE/Ref-1) has been uncovered elevated in various types of cancer, including HCC. Additionally, HCC progression is always correlated with elevated copper (Cu). Our previous data demonstrated that Cu treatment initiated APE/Ref-1 expression and its downstream targets. Therefore, we hypothesized that APE/Ref-1 may be involved in HCC progression through mediating the effect of Cu to its signaling cascades. Following different treatments, human HCC cell line (Hep3B) and immortalized non-malignant hepatocyte cell line (THLE3) were analyzed to explore the role of APE/Ref-1 signaling pathway. Unstained human tissue microarrays (TMA) were subjected to IHC analysis to study the relationship between APE/Ref-1 expression and clinic features. APE/Ref-1 was upregulated in HCC cells consistent with the strong expression of APE/Ref-1 in HCC tissue microarray. Greater cytoplasmic accumulation of APE/Ref-1 was found in poorly differentiated and more aggressive tumors. Also we provide evidence to show that APE/Ref-1 signaling pathway stimulates cellular proliferation, enhances antiapoptosis, and facilitates metastasis through experimental knockdown of APE/Ref-1 using siRNA in Hep3B cells or overexpressing APE/Ref-1 in THLE3 cells. These results define a novel role of APE/Ref-1 in HCC progression as being an important mediating and potentiating molecule, and also provide a basis for further investigations utilizing appropriate APE/Ref-1 inhibitors in combination with chemo-drugs for HCC treatment. PMID:25109342

  16. Pleiotropic effects and compensation mechanisms determine tissue specificity in mitochondrial myopathy and sideroblastic anemia (MLASA).

    PubMed

    Bykhovskaya, Yelena; Mengesha, Emebet; Fischel-Ghodsian, Nathan

    2007-06-01

    The tissue specificity of mitochondrial diseases is poorly understood. Recently, tissue-specific quantitative differences of the components of the mitochondrial translation system have been found to correlate with disease presentation in fatal hepatopathy caused by mutations in mitochondrial translation factor EFG1. MLASA is an autosomal recessive inherited progressive oxidative phosphorylation disorder that affects muscle and erythroid cells. The disease is caused by the homozygous point mutation C656T (R116W) in the catalytic domain of the pseudouridylate synthase 1 (PUS1) gene, which leads to a complete lack of pseudouridylation at the expected sites in mitochondrial and cytoplasmic tRNAs. Despite the presence of these altered tRNAs, most tissues are unaffected, and even in muscle and erythroid cells the disease phenotype only slowly emerges over the course of years. In order to elucidate intracellular pathways through which the homozygous mutation leads to tissue-restricted phenotype, we performed microarray expression analysis of EBV-transformed lymphoblasts from MLASA patients, heterozygous parents, and controls using human Beadchip microarray with 47,296 transcripts. Genes coding for proteins involved in DNA transcription and its regulation, and metal binding proteins, demonstrated major differences in expression between patients and all other individuals with normal phenotype. Genes coding for ribosomal proteins differed significantly between individual with at least one copy of the mutated PUS1 gene and controls. These findings indicate that the lack of tRNA pseudouridylation can be overcome by compensatory changes in levels of ribosomal proteins, and that the disease phenotype in affected tissues is likely due to pleiotropic effects of PUS1p on non-tRNA molecules involved in DNA transcription and iron metabolism. Similar combinations of mechanisms may play a role in the tissue specificity of other mitochondrial disorders. PMID:17374500

  17. Pleiotropic Effects and Compensation Mechanisms Determine Tissue Specificity in Mitochondrial Myopathy and Sideroblastic Anemia (MLASA)

    PubMed Central

    Bykhovskaya, Yelena; Mengesha, Emebet; Fischel-Ghodsian, Nathan

    2007-01-01

    The tissue specificity of mitochondrial diseases is poorly understood. Recently, tissue-specific quantitative differences of the components of the mitochondrial translation system have been found to correlate with disease presentation in fatal hepatopathy caused by mutations in mitochondrial translation factor EFG1. MLASA is an autosomal recessive inherited progressive oxidative phosphorylation disorder that affects muscle and erythroid cells. The disease is caused by the homozygous point mutation C656T (R116W) in the catalytic domain of the pseudouridylate synthase 1 (PUS1) gene, which leads to a complete lack of pseudouridylation at the expected sites in mitochondrial and cytoplasmic tRNAs. Despite the presence of these altered tRNAs, most tissues are unaffected, and even in muscle and erythroid cells the disease phenotype only slowly emerges over the course of years. In order to elucidate intracellular pathways through which the homozygous mutation leads to tissue-restricted phenotype, we performed microarray expression analysis of EBV-transformed lymphoblasts from MLASA patients, heterozygous parents, and controls using human Beadchip microarray with 47,296 transcripts. Genes coding for proteins involved in DNA transcription and its regulation, and metal binding proteins, demonstrated major differences in expression between patients and all other individuals with normal phenotype. Genes coding for ribosomal proteins differed significantly between individual with at least one copy of the mutated PUS1 gene and controls. These findings indicate that the lack of tRNA pseudouridylation can be overcome by compensatory changes in levels of ribosomal proteins, and that the disease phenotype in affected tissues is likely due to pleiotropic effects of PUS1p on non-tRNA molecules involved in DNA transcription and iron metabolism. Similar combinations of mechanisms may play a role in the tissue specificity of other mitochondrial disorders. PMID:17374500

  18. Symptomatic and asymptomatic benign prostatic hyperplasia: Molecular differentiation by using microarrays

    PubMed Central

    Prakash, Kulkarni; Pirozzi, Gregorio; Elashoff, Michael; Munger, William; Waga, Iwao; Dhir, Rajiv; Kakehi, Yoshiyuki; Getzenberg, Robert H.

    2002-01-01

    Benign prostatic hyperplasia (BPH) is a disease of unknown etiology that significantly affects the quality of life in aging men. Histologic BPH may present itself either as symptomatic or asymptomatic in nature. To elucidate the molecular differences underlying BPH, gene expression profiles from the prostate transition zone tissue have been analyzed by using microarrays. A set of 511 differentially expressed genes distinguished symptomatic and asymptomatic BPH. This genetic signature separates BPH from normal tissue but does not seem to change with age. These data could provide novel approaches for alleviating symptoms and hyperplasia in BPH. PMID:12032329

  19. Symptomatic and asymptomatic benign prostatic hyperplasia: Molecular differentiation by using microarrays

    NASA Astrophysics Data System (ADS)

    Prakash, Kulkarni; Pirozzi, Gregorio; Elashoff, Michael; Munger, William; Waga, Iwao; Dhir, Rajiv; Kakehi, Yoshiyuki; Getzenberg, Robert H.

    2002-05-01

    Benign prostatic hyperplasia (BPH) is a disease of unknown etiology that significantly affects the quality of life in aging men. Histologic BPH may present itself either as symptomatic or asymptomatic in nature. To elucidate the molecular differences underlying BPH, gene expression profiles from the prostate transition zone tissue have been analyzed by using microarrays. A set of 511 differentially expressed genes distinguished symptomatic and asymptomatic BPH. This genetic signature separates BPH from normal tissue but does not seem to change with age. These data could provide novel approaches for alleviating symptoms and hyperplasia in BPH.

  20. Development of a microarray chip for gene expression in rabbit ocular research

    PubMed Central

    Liu, Li; Timmers, Adrian; Esson, Douglas W.; Shiroma, Lineu; Meyers, Craig; Berceli, Scott; Tao, Ming; Wistow, Graeme; Schultz, Gregory S.; Sherwood, Mark B.

    2007-01-01

    Purpose To develop a microarray for the rabbit that can be used for ocular gene expression research. Methods Messenger RNA was isolated from anterior segment tissues (cornea, conjunctiva, and iris) and posterior segment tissues (lens, retina, and sclera) of rabbit eyes and used to create two independent cDNA libraries through the NEIBank project. Clones from each of these libraries were sequenced from both the 5' and 3' ends. These sequences and those from the National Center for Biotechnology Information (NCBI) taxonomy database for rabbit were combined and electronically assembled into a set of unique nonoverlapping continuous sequences (contigs). For each contig, a homology search was performed using BLASTX and BLASTN against both the NCBI NR and NT databases to provide gene annotation. Unique contigs were sent to Agilent Technologies, where 60 base oligonucleotide probes were designed and synthesized, in situ, on two different arrays in an 8 array x 1900 element format. Glaucoma filtration surgery was performed on one eye of six rabbits. After 14 days, tissue was harvested from the conjunctiva and Tenon's capsule of both the surgically treated and untreated control eyes. Total RNA from each sample was labeled with cyanine dyes and hybridized to our custom microarrays. Results Of the 3,154 total probes present on the two arrays, 2,522 had a signal value above the background. The expression of 315 genes was significantly altered by glaucoma filtration surgery. Genes whose expression was altered included proteins associated with inflammatory response, defense response, and proteins involved in synthesis of the extracellular matrix. Conclusions The results of this rabbit microarray study are consistent with those from other wound healing studies, indicating that this array can provide valid information on broad patterns of gene expression. This is the first microarray available for rabbit studies and is a valuable tool that can be used to study molecular events in the eye. PMID:17293780

  1. Expression of p53, p21(CIP1/WAF1) and eIF4E in the adjacent tissues of oral squamous cell carcinoma: establishing the molecular boundary and a cancer progression model.

    PubMed

    Li, Yi; Li, Bo; Xu, Bo; Han, Bo; Xia, Hui; Chen, Qian-Ming; Li, Long-Jiang

    2015-09-01

    The present study evaluated the expression of key molecules and the status of DNA in both oral squamous cell carcinoma (OSCC) and adjacent tissues to establish a molecular surgical boundary and provide a cancer progression model. Biopsy samples from 50 OSCC patients were divided into T (cancer), P1 (0-0.5 cm), P2 (0.5-1 cm), P3 (1-1.5 cm) and P4 (1.5-2 cm) groups based on the distances from the visible boundary of the primary focus. Twenty samples of normal mucosa were used as controls. We used immunohistochemical staining and flow cytometry to evaluate p53, p21(CIP1/WAF1), eIF4E and Ki-67 expression and to determine DNA status, respectively. Sub-mucosal invasion was present in the P1 and P2 groups as determined by haematoxylin and eosin staining. Mutant p53 expression decreased gradually from cancerous to normal mucosae, whereas p21(CIP1/WAF1) expression displayed an opposite trend. eIF4E expression decreased from cancerous to normal mucosae. Ki-67 expression, the heteroploidy ratio, S-phase fraction and proliferative index decreased gradually with the distance from the tumour centre. Based on these results, we suggest that the resection boundary in OSCC surgery should be beyond 2 cm from the tumour. Additionally, the adjacent tissues of the primary focus could be used as a model for assessing cancer progression. PMID:25835715

  2. Expression of p53, p21 CIP1/WAF1 and eIF4E in the adjacent tissues of oral squamous cell carcinoma: establishing the molecular boundary and a cancer progression model

    PubMed Central

    Li, Yi; Li, Bo; Xu, Bo; Han, Bo; Xia, Hui; Chen, Qian-Ming; Li, Long-Jiang

    2015-01-01

    The present study evaluated the expression of key molecules and the status of DNA in both oral squamous cell carcinoma (OSCC) and adjacent tissues to establish a molecular surgical boundary and provide a cancer progression model. Biopsy samples from 50 OSCC patients were divided into T (cancer), P1 (0–0.5 cm), P2 (0.5–1 cm), P3 (1–1.5 cm) and P4 (1.5–2 cm) groups based on the distances from the visible boundary of the primary focus. Twenty samples of normal mucosa were used as controls. We used immunohistochemical staining and flow cytometry to evaluate p53, p21 CIP1/WAF1 , eIF4E and Ki-67 expression and to determine DNA status, respectively. Sub-mucosal invasion was present in the P1 and P2 groups as determined by haematoxylin and eosin staining. Mutant p53 expression decreased gradually from cancerous to normal mucosae, whereas p21 CIP1/WAF1 expression displayed an opposite trend. eIF4E expression decreased from cancerous to normal mucosae. Ki-67 expression, the heteroploidy ratio, S-phase fraction and proliferative index decreased gradually with the distance from the tumour centre. Based on these results, we suggest that the resection boundary in OSCC surgery should be beyond 2 cm from the tumour. Additionally, the adjacent tissues of the primary focus could be used as a model for assessing cancer progression. PMID:25835715

  3. DNA Microarray for Detection of Gastrointestinal Viruses

    PubMed Central

    Martnez, Miguel A.; Soto-del Ro, Mara de los Dolores; Gutirrez, Rosa Mara; Chiu, Charles Y.; Greninger, Alexander L.; Contreras, Juan Francisco; Lpez, Susana; Arias, Carlos F.

    2014-01-01

    Gastroenteritis is a clinical illness of humans and other animals that is characterized by vomiting and diarrhea and caused by a variety of pathogens, including viruses. An increasing number of viral species have been associated with gastroenteritis or have been found in stool samples as new molecular tools have been developed. In this work, a DNA microarray capable in theory of parallel detection of more than 100 viral species was developed and tested. Initial validation was done with 10 different virus species, and an additional 5 species were validated using clinical samples. Detection limits of 1 103 virus particles of Human adenovirus C (HAdV), Human astrovirus (HAstV), and group A Rotavirus (RV-A) were established. Furthermore, when exogenous RNA was added, the limit for RV-A detection decreased by one log. In a small group of clinical samples from children with gastroenteritis (n = 76), the microarray detected at least one viral species in 92% of the samples. Single infection was identified in 63 samples (83%), and coinfection with more than one virus was identified in 7 samples (9%). The most abundant virus species were RV-A (58%), followed by Anellovirus (15.8%), HAstV (6.6%), HAdV (5.3%), Norwalk virus (6.6%), Human enterovirus (HEV) (9.2%), Human parechovirus (1.3%), Sapporo virus (1.3%), and Human bocavirus (1.3%). To further test the specificity and sensitivity of the microarray, the results were verified by reverse transcription-PCR (RT-PCR) detection of 5 gastrointestinal viruses. The RT-PCR assay detected a virus in 59 samples (78%). The microarray showed good performance for detection of RV-A, HAstV, and calicivirus, while the sensitivity for HAdV and HEV was low. Furthermore, some discrepancies in detection of mixed infections were observed and were addressed by reverse transcription-quantitative PCR (RT-qPCR) of the viruses involved. It was observed that differences in the amount of genetic material favored the detection of the most abundant virus. The microarray described in this work should help in understanding the etiology of gastroenteritis in humans and animals. PMID:25355758

  4. Array-based analysis on tobacco plastid transcripts: preparation of a genomic microarray containing all genes and all intergenic regions.

    PubMed

    Nakamura, Takahiro; Furuhashi, Yumiko; Hasegawa, Keiko; Hashimoto, Hiroshi; Watanabe, Kazufumi; Obokata, Junichi; Sugita, Mamoru; Sugiura, Masahiro

    2003-08-01

    The plastid genome of higher plants includes about 120 genes. We adopted genomic array technologies to the tobacco plastid genome. A microarray was constructed, consisting of 220 DNA fragments that cover the whole genome sequence. Each DNA fragment corresponds to a single known gene or an intergenic region. We evaluated reliability of this microarray by comparing the plastid RNA level in light- or dark-grown tobacco seedlings. The transcripts encoding photosynthetic subunits increased significantly in light-grown tissues as expected. Furthermore, we found unexpected signals in several intergenic regions, suggesting the existence of novel transcripts in tobacco plastids. PMID:12941879

  5. A cell spot microarray method for production of high density siRNA transfection microarrays

    PubMed Central

    2011-01-01

    Background High-throughput RNAi screening is widely applied in biological research, but remains expensive, infrastructure-intensive and conversion of many assays to HTS applications in microplate format is not feasible. Results Here, we describe the optimization of a miniaturized cell spot microarray (CSMA) method, which facilitates utilization of the transfection microarray technique for disparate RNAi analyses. To promote rapid adaptation of the method, the concept has been tested with a panel of 92 adherent cell types, including primary human cells. We demonstrate the method in the systematic screening of 492 GPCR coding genes for impact on growth and survival of cultured human prostate cancer cells. Conclusions The CSMA method facilitates reproducible preparation of highly parallel cell microarrays for large-scale gene knockdown analyses. This will be critical towards expanding the cell based functional genetic screens to include more RNAi constructs, allow combinatorial RNAi analyses, multi-parametric phenotypic readouts or comparative analysis of many different cell types. PMID:21443765

  6. Evaluation of toxicity of the mycotoxin citrinin using yeast ORF DNA microarray and Oligo DNA microarray

    PubMed Central

    Iwahashi, Hitoshi; Kitagawa, Emiko; Suzuki, Yoshiteru; Ueda, Youji; Ishizawa, Yo-hei; Nobumasa, Hitoshi; Kuboki, Yoshihide; Hosoda, Hiroshi; Iwahashi, Yumiko

    2007-01-01

    Background Mycotoxins are fungal secondary metabolites commonly present in feed and food, and are widely regarded as hazardous contaminants. Citrinin, one of the very well known mycotoxins that was first isolated from Penicillium citrinum, is produced by more than 10 kinds of fungi, and is possibly spread all over the world. However, the information on the action mechanism of the toxin is limited. Thus, we investigated the citrinin-induced genomic response for evaluating its toxicity. Results Citrinin inhibited growth of yeast cells at a concentration higher than 100 ppm. We monitored the citrinin-induced mRNA expression profiles in yeast using the ORF DNA microarray and Oligo DNA microarray, and the expression profiles were compared with those of the other stress-inducing agents. Results obtained from both microarray experiments clustered together, but were different from those of the mycotoxin patulin. The oxidative stress response genes – AADs, FLR1, OYE3, GRE2, and MET17 – were significantly induced. In the functional category, expression of genes involved in "metabolism", "cell rescue, defense and virulence", and "energy" were significantly activated. In the category of "metabolism", genes involved in the glutathione synthesis pathway were activated, and in the category of "cell rescue, defense and virulence", the ABC transporter genes were induced. To alleviate the induced stress, these cells might pump out the citrinin after modification with glutathione. While, the citrinin treatment did not induce the genes involved in the DNA repair. Conclusion Results from both microarray studies suggest that citrinin treatment induced oxidative stress in yeast cells. The genotoxicity was less severe than the patulin, suggesting that citrinin is less toxic than patulin. The reproducibility of the expression profiles was much better with the Oligo DNA microarray. However, the Oligo DNA microarray did not completely overcome cross hybridization. PMID:17408496

  7. An insight into normal and pathological pregnancies using large-scale microarrays: lessons from microarrays.

    PubMed

    Chaouat, Gérard; Rodde, Nathalie; Petitbarat, Marie; Bulla, Roberta; Rahmati, Mona; Dubanchet, Sylvie; Zourbas, Sandrine; Bataillon, Isabelle; Coqué, Nathalie; Hennuy, Benoit; Martal, Jacques; Munaut, Carine; Aubert, Julie; Sérazin, Valérie; Steffen, Thiel; Jensenius, Jens Christian; Foidart, Jean Michel; Sandra, Olivier; Tedesco, Francesco; Lédée, Nathalie

    2011-05-01

    In the introduction, we briefly recall old but classic evidence that there is no tolerance to paternal alloantigens in a first pregnancy. Therefore, we performed small- and large-scale microarrays in CBA × DBA/2 and CBA × BALB/c combinations, recently described as a murine model for preeclampsia. Our results are in line with other data suggesting a very early deregulation of local immune vascular events rather than a break of immune tolerance. Other data presented at the Tioman 2010 Preeclampsia Workshop supporting this hypothesis are briefly summarised, as well as indications and caveats from a recent human microarray on implantation failure and recurrent pregnancy loss. PMID:21329986

  8. Detection of Pathogenic Vibrio spp. in Shellfish by Using Multiplex PCR and DNA Microarrays

    PubMed Central

    Panicker, Gitika; Call, Douglas R.; Krug, Melissa J.; Bej, Asim K.

    2004-01-01

    This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50°C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 102 to 103 CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers. PMID:15574946

  9. Hippo transducer TAZ promotes epithelial mesenchymal transition and supports pancreatic cancer progression

    PubMed Central

    Xie, Dacheng; Cui, Jiujie; Xia, Tian; Jia, Zhiliang; Wang, Liang; Wei, Wenfei; Zhu, Anna; Gao, Yong; Xie, Keping; Quan, Ming

    2015-01-01

    Transcriptional co-activator with PDZ binding motif (TAZ) is a transducer of the Hippo pathway and promotes cancer development and progression. In the present study, we sought to determine the roles and underlying mechanisms of elevated expression and activation of TAZ in pancreatic cancer development and progression. The mechanistic role of TAZ and Hippo signaling in promotion of pancreatic cancer development and progression was examined using cell culture, molecular biology, and mouse models. The relevance of our experimental and mechanistic findings was validated using human pancreatic tumor specimens. We found that TAZ expression was markedly higher in pancreatic tumors than in normal pancreatic tissue. Further analysis of the correlation of TAZ expression with tissue microarray clinicopathologic parameters revealed that this expression was positively associated with tumor differentiation. Also, TAZ expression was higher in pancreatic cancer cell lines than in pancreatic ductal epithelial cells. TAZ activation in pancreatic cancer cells promoted their proliferation, migration, invasion, and epithelial-mesenchymal transition. Further mechanistic studies demonstrated that aberrant expression and activation of TAZ in pancreatic cancer cells resulted from suppression of the expression of Merlin, a positive regulator upstream of the Hippo pathway, and that the oncogenic function of TAZ in pancreatic cancer cells was mediated by TEA/ATTS domain transcription factors. Therefore, TAZ functioned as an oncogene and promoted pancreatic cancer epithelial-mesenchymal transition and progression. TAZ thus may be a target for effective therapeutic strategies for pancreatic cancer. PMID:26416426

  10. High-throughput allogeneic antibody detection using protein microarrays.

    PubMed

    Paul, Jed; Sahaf, Bita; Perloff, Spenser; Schoenrock, Kelsi; Wu, Fang; Nakasone, Hideki; Coller, John; Miklos, David

    2016-05-01

    Enzyme-linked immunosorbent assays (ELISAs) have traditionally been used to detect alloantibodies in patient plasma samples post hematopoietic cell transplantation (HCT); however, protein microarrays have the potential to be multiplexed, more sensitive, and higher throughput than ELISAs. Here, we describe the development of a novel and sensitive microarray method for detection of allogeneic antibodies against minor histocompatibility antigens encoded on the Y chromosome, called HY antigens. Six microarray surfaces were tested for their ability to bind recombinant protein and peptide HY antigens. Significant allogeneic immune responses were determined in male patients with female donors by considering normal male donor responses as baseline. HY microarray results were also compared with our previous ELISA results. Our overall goal was to maximize antibody detection for both recombinant protein and peptide epitopes. For detection of HY antigens, the Epoxy (Schott) protein microarray surface was both most sensitive and reliable and has become the standard surface in our microarray platform. PMID:26902899

  11. Identification of significant features in DNA microarray data

    PubMed Central

    Bair, Eric

    2013-01-01

    DNA microarrays are a relatively new technology that can simultaneously measure the expression level of thousands of genes. They have become an important tool for a wide variety of biological experiments. One of the most common goals of DNA microarray experiments is to identify genes associated with biological processes of interest. Conventional statistical tests often produce poor results when applied to microarray data owing to small sample sizes, noisy data, and correlation among the expression levels of the genes. Thus, novel statistical methods are needed to identify significant genes in DNA microarray experiments. This article discusses the challenges inherent in DNA microarray analysis and describes a series of statistical techniques that can be used to overcome these challenges. The problem of multiple hypothesis testing and its relation to microarray studies are also considered, along with several possible solutions. PMID:24244802

  12. Microarray gene expression analysis of the human airway in patients exposed to sulfur mustard.

    PubMed

    Najafi, Ali; Masoudi-Nejad, Ali; Imani Fooladi, Abbas Ali; Ghanei, Mostafa; Nourani, Mohamad Reza

    2014-08-01

    There is much data about the acute effects of sulfur mustard gas on humans, animals and cells. But less is known regarding the molecular basics of chronic complications in humans. Basically, mustard gas, as an alkylating agent, causes several chronic problems in the eyes, skin and more importantly in the pulmonary system which is the main cause of death. Although recent proteomic research has been carried out on bronchoalveolar lavage (BAL) and serum, but high-throughput transcriptomics have not yet been applied to chronic airway remodeling. This is the first cDNA-microarray report on the chronic human mustard lung disease, 25 years after exposure during the Iran-Iraq war. Microarray transcriptional profiling indicated that a total of 122 genes were significantly dysregulated in tissues located in the airway of patients. These genes are associated with the extracellular matrix components, apoptosis, stress response, inflammation and mucus secretion. PMID:24823320

  13. Microarray Technology and Its Applications for Detecting Plasma microRNA Biomarkers in Digestive Tract Cancers.

    PubMed

    Konishi, Hirotaka; Ichikawa, Daisuke; Arita, Tomohiro; Otsuji, Eigo

    2016-01-01

    Many cancers are known to be regulated by microRNAs (miRNAs), and the relationships between tissue miRNA expression levels and the amounts of miRNA circulating in the plasma (or plasma miRNA) have been examined in many types of cancers, including digestive tract cancers. The role of plasma miRNAs has yet to be elucidated in detail; therefore a comprehensive analysis of plasma miRNAs using microarrays should assist in establishing the utility of liquid biopsy or companion diagnosis. We here described the 3D-Gene(®) miRNA microarray (TORAY) currently used in our laboratory and introduced a trial application in digestive tract cancer diagnosis. PMID:26614071

  14. Combined mass quantitation and phenotyping of intact extracellular vesicles by a microarray platform.

    PubMed

    Gagni, Paola; Cretich, Marina; Benussi, Luisa; Tonoli, Elisa; Ciani, Miriam; Ghidoni, Roberta; Santini, Benedetta; Galbiati, Elisabetta; Prosperi, Davide; Chiari, Marcella

    2016-01-01

    The interest towards extracellular vesicles (EVs) has grown exponentially over the last few years; being involved in intercellular communication and serving as reservoirs for biomarkers for tumors, they have a great potential for liquid biopsy development, possibly replacing many costly and invasive tissue biopsies. Here we propose, for the first time, the use of a Si/SiO2 interferometric, microarray platform for multiparametric intact EVs analysis combining label-free EVs mass quantitation and high sensitivity fluorescence based phenotyping. Label free interferometric measurement allows to quantify the amount of vesicles captured by printed antibodies while, on the same chip, EVs are also detected by fluorescence in a sandwich immunoassay. The proposed method simultaneously detects, quantify and phenotype intact EVs in a microarray format. PMID:26703266

  15. Protein Microarrays--Without a Trace

    SciTech Connect

    Camarero, J A

    2007-04-05

    Many experimental approaches in biology and biophysics, as well as applications in diagnosis and drug discovery, require proteins to be immobilized on solid supports. Protein microarrays, for example, provide a high-throughput format to study biomolecular interactions. The technique employed for protein immobilization is a key to the success of these applications. Recent biochemical developments are allowing, for the first time, the selective and traceless immobilization of proteins generated by cell-free systems without the need for purification and/or reconcentration prior to the immobilization step.

  16. Optimal gene expression analysis by microarrays.

    PubMed

    Miller, Lance D; Long, Philip M; Wong, Limsoon; Mukherjee, Sayan; McShane, Lisa M; Liu, Edison T

    2002-11-01

    DNA microarrays make possible the rapid and comprehensive assessment of the transcriptional activity of a cell, and as such have proven valuable in assessing the molecular contributors to biological processes and in the classification of human cancers. The major challenge in using this technology is the analysis of its massive data output, which requires computational means for interpretation and a heightened need for quality data. The optimal analysis requires an accounting and control of the many sources of variance within the system, an understanding of the limitations of the statistical approaches, and the ability to make sense of the results through intelligent database interrogation. PMID:12450790

  17. Prenatal chromosomal microarray for the Catholic physician

    PubMed Central

    Bringman, Jay J.

    2014-01-01

    Prenatal chromosomal microarray (CMA) is a test that is used to diagnose certain genetic problems in the fetus. While the test has been used in the pediatric setting for several years, it is now being introduced for use in the prenatal setting. The test offers great hope for detection of certain genetic defects in the fetus so that early intervention can be performed to improve the outcome for that individual. As with many biotechnical advances, CMA comes with certain bioethical issues that need to be addressed prior to its implementation. This paper is intended to provide guidance to all those that provide counseling regarding genetic testing options during pregnancy. PMID:24899750

  18. ProMAT: protein microarray analysis tool

    SciTech Connect

    White, Amanda M.; Daly, Don S.; Varnum, Susan M.; Anderson, Kevin K.; Bollinger, Nikki; Zangar, Richard C.

    2006-04-04

    Summary: ProMAT is a software tool for statistically analyzing data from ELISA microarray experiments. The software estimates standard curves, sample protein concentrations and their uncertainties for multiple assays. ProMAT generates a set of comprehensive figures for assessing results and diagnosing process quality. The tool is available for Windows or Mac, and is distributed as open-source Java and R code. Availability: ProMAT is available at http://www.pnl.gov/statistics/ProMAT. ProMAT requires Java version 1.5.0 and R version 1.9.1 (or more recent versions) which are distributed with the tool.

  19. α-SMA-Cre-mediated excision of PDK1 reveals an essential role of PDK1 in regulating morphology of cardiomyocyte and tumor progression in tissue microenvironment.

    PubMed

    Qian, X-J; Li, X-L; Xu, X; Wang, X; Feng, Q-T; Yang, C-J

    2015-04-01

    The phosphoinositide-3 kinase (PI3K) - phosphoinositide-dependent protein kinase 1 (PDK1)-Akt/protein kinase B (PKB) cascade plays a critical role in cardiovascular development and tumor genesis. But the role of PDK1 in the microenvironment of heart and tumor remains unknown. To clarify the effects of PDK1 on tissue microenvironment in vivo, here, we created α-SMA-Cre-mediated excision of PDK1 mice. And the mice were injected subcutaneously with Lewis lung carcinoma (LLC) cells. We found PDK1-deficient mice had post-natal praecox dilated cardiomyopathy, decelerated tumor growth and severe tumor metastasis. Histopathological analysis revealed abnormality of vascular microenvironment in heart and primary tumor. In conclusion, PDK1 plays a pivotal role in regulating cardiac function and tumor metastasis by interfering with microenvironment. PMID:25601552

  20. Oral tocotrienols are transported to human tissues and delay the progression of the model for end-stage liver disease score in patients.

    PubMed

    Patel, Viren; Rink, Cameron; Gordillo, Gayle M; Khanna, Savita; Gnyawali, Urmila; Roy, Sashwati; Shneker, Bassel; Ganesh, Kasturi; Phillips, Gary; More, J Layne; Sarkar, Atom; Kirkpatrick, Robert; Elkhammas, Elmahdi A; Klatte, Emily; Miller, Michael; Firstenberg, Michael S; Chiocca, E Antonio; Nesaretnam, Kalanithi; Sen, Chandan K

    2012-03-01

    The natural vitamin E family is composed of 8 members equally divided into 2 classes: tocopherols (TCP) and tocotrienols (TE). A growing body of evidence suggests TE possess potent biological activity not shared by TCP. The primary objective of this work was to determine the concentrations of TE (200 mg mixed TE, b.i.d.) and TCP [200 mg α-TCP, b.i.d.)] in vital tissues and organs of adults receiving oral supplementation. Eighty participants were studied. Skin and blood vitamin E concentrations were determined from healthy participants following 12 wk of oral supplementation of TE or TCP. Vital organ vitamin E levels were determined by HPLC in adipose, brain, cardiac muscle, and liver of surgical patients following oral TE or TCP supplementation (mean duration, 20 wk; range, 1-96 wk). Oral supplementation of TE significantly increased the TE tissue concentrations in blood, skin, adipose, brain, cardiac muscle, and liver over time. α-TE was delivered to human brain at a concentration reported to be neuroprotective in experimental models of stroke. In prospective liver transplantation patients, oral TE lowered the model for end-stage liver disease (MELD) score in 50% of patients supplemented, whereas only 20% of TCP-supplemented patients demonstrated a reduction in MELD score. This work provides, to our knowledge, the first evidence demonstrating that orally supplemented TE are transported to vital organs of adult humans. The findings of this study, in the context of the current literature, lay the foundation for Phase II clinical trials testing the efficacy of TE against stroke and end-stage liver disease in humans. PMID:22298568

  1. An examination of the regulatory mechanism of Pxdn mutation-induced eye disorders using microarray analysis.

    PubMed

    Yang, Yang; Xing, Yiqiao; Liang, Chaoqun; Hu, Liya; Xu, Fei; Mei, Qi

    2016-06-01

    The present study aimed to identify biomarkers for peroxidasin (Pxdn) mutation-induced eye disorders and study the underlying mechanisms involved in this process. The microarray dataset GSE49704 was used, which encompasses 4 mouse samples from embryos with Pxdn mutation and 4 samples from normal tissues. After data preprocessing, the differentially expressed genes (DEGs) between Pxdn mutation and normal tissues were identified using the t-test in the limma package, followed by functional enrichment analysis. The protein-protein interaction (PPI) network was constructed based on the STRING database, and the transcriptional regulatory (TR) network was established using the GeneCodis database. Subsequently, the overlapping DEGs with high degrees in two networks were identified, as well as the sub-network extracted from the TR network. In total, 121 (75 upregulated and 46 downregulated) DEGs were identified, and these DEGs play important roles in biological processes (BPs), including neuron development and differentiation. A PPI network containing 25 nodes such as actin, alpha 1, skeletal muscle (Acta1) and troponin C type 2 (fast) (Tnnc2), and a TR network including 120 nodes were built. By comparing the two networks, seven crucial genes which overlapped were identified, including cyclin‑dependent kinase inhibitor 1B (Cdkn1b), Acta1 and troponin T type 3 (Tnnt3). In the sub-network, Cdkn1b was predicted as the target of miRNAs such as mmu-miR-24 and transcription factors (TFs) including forkhead box O4 (FOXO4) and activating enhancer binding protein 4 (AP4). Thus, we suggest that seven crucial genes, including Cdkn1b, Acta1 and Tnnt3, play important roles in the progression of eye disorders such as glaucoma. We suggest that Cdkn1b exert its effects via the inhibition of proliferation and is mediated by mmu-miR-24 and targeted by the TFs FOXO4 and AP4. PMID:27121343

  2. Circular RNA: a novel biomarker for progressive laryngeal cancer

    PubMed Central

    Xuan, Lijia; Qu, Lingmei; Zhou, Han; Wang, Peng; Yu, Haoyang; Wu, Tianyi; Wang, Xin; Li, Qiuying; Tian, Linli; Liu, Ming; Sun, Yanan

    2016-01-01

    Circular RNAs (circRNAs), a class of endogenous RNAs, are characterized by covalently closed continuous loop without 5’ to 3’ polarity and polyadenylated tail. Recent studies indicated that circRNAs might play an important role in cancer. However, the function of circRNA in laryngeal squamous cell cancer tissues (LSCC) is still unknown. In this study, we investigated the expression of circRNAs in 4 paired LSCC tissues and adjacent non-tumor tissues by microarray analysis. Results showed significant upregulation (n = 302) of or downregulation (n = 396) of 698 circRNAs in LSCC tissues. We further detected hsa_circRNA_100855 as the most upregulated circRNA and hsa_circRNA_104912 as the most downregulated circRNA using qRT-PCR methods. Results showed that hsa_circRNA_100855 level was significantly higher in LSCC than in the corresponding adjacent non-neoplastic tissues. Patients with T3-4 stage, neck nodal metastasis or advanced clinical stage had higher hsa_circRNA_100855 expression. The hsa_circRNA_104912 level was significantly lower in LSCC than in corresponding adjacent non-neoplastic tissues. Patients with T3-4 stage, neck nodal metastasis, poor differentiation or advanced clinical stage had a lower hsa_circRNA_104912 expression. Overall, our data suggest that circRNAs play an important role in the tumorigenesis of LSCC and may serve as novel and stable biomarkers for the diagnosis and progress of LSCC.

  3. Microarray comparative genomic hybridization analysis of tubular breast carcinoma shows recurrent loss of the CDH13 locus on 16q.

    PubMed

    Riener, Marc-Oliver; Nikolopoulos, Elisabeth; Herr, Alexander; Wild, Peter Johannes; Hausmann, Michael; Wiech, Thorsten; Orlowska-Volk, Marzenna; Lassmann, Silke; Walch, Axel; Werner, Martin

    2008-11-01

    Tubular breast carcinoma is a highly differentiated carcinoma with an excellent prognosis. Distinct genetic alterations in tubular breast carcinoma cells have been described, especially broad genetic losses on the q-arm of chromosome 16. These are more common in lobular breast carcinoma and low-grade ductal carcinoma in situ than in ductal breast carcinoma and high-grade ductal carcinoma in situ. To further delineate the molecular changes involved in tubular breast carcinoma more precisely, we examined 23 formalin-fixed and paraffin wax-embedded tissue samples (21 of tubular breast carcinoma and 2 of nonneoplastic breast epithelium) by microarray-based comparative genomic hybridization focusing on 287 genomic target clones of oncogenes and tumor suppressor genes. The results obtained from all nonneoplastic tissue samples of breast epithelium indicate no DNA copy number changes. In the tubular breast carcinoma samples, the highest frequencies for DNA sequence copy number losses were detected for CDH13 (in 86% of the samples) and MSH2, KCNK12 (in 52% of the samples). The highest frequencies of DNA sequence copy number gains were detected for HRAS and D13S319XYZ (each in 62% of the samples). Using principal component analysis, 3 subgroups of tubular breast carcinomas showing relative genetic changes were identified. For validation, the most frequent DNA copy number loss for CDH13 (18/21) was confirmed using fluorescence in situ hybridization in 4 of 5 tubular breast carcinomas analyzed. The newly identified genes with considerable copy number changes may include so far unknown candidate genes for the development and progression of tubular breast carcinoma, such as CDH13. The study provides the starting point for further delineating their detailed influence on the pathogenesis of tubular breast carcinoma. PMID:18656243

  4. CGcgh: a tool for molecular karyotyping using DNA microarray-based comparative genomic hybridization (array-CGH).

    PubMed

    Lee, Yun-Shien; Chao, Angel; Chao, An-Shine; Chang, Shuenn-Dyh; Chen, Chun-Houh; Wu, Wei-Ming; Wang, Tzu-Hao; Wang, Hsin-Shih

    2008-11-01

    Microarray-based comparative genomic hybridization (array-CGH) is a technique by which variations in copy numbers between two genomes can be analyzed using DNA microarrays. Array CGH has been used to survey chromosomal amplifications and deletions in fetal aneuploidies or cancer tissues. Herein we report a user-friendly, MATLAB-based, array CGH analyzing program, Chang Gung comparative genomic hybridization (CGcgh), as a standalone PC version. The analyzed chromosomal data are displayed in a graphic interface, and CGcgh allows users to launch a corresponding G-banding ideogram. The abnormal DNA copy numbers (gains and losses) can be identified automatically using a user defined window size (default value is 50 probes) and sequential student t-tests with sliding windows along with chromosomes. CGcgh has been tested in multiple karyotype-confirmed human samples, including five published cases and trisomies 13, 18, 21 and X from our laboratories, and 18 cases of which microarray data are available publicly. CGcgh can be used to detect the copy number changes in small genomic regions, which are commonly encountered by clinical geneticists. CGcgh works well for the data from cDNA microarray, spotted oligonucleotide microarrays, and Affymetrix Human Mapping Arrays (10K, 100K, 500K Array Sets). The program can be freely downloaded from http://www.mcu.edu.tw/department/biotec/en%5Fpage/CGcgh/ . PMID:18712492

  5. Refractive index change detection based on porous silicon microarray

    NASA Astrophysics Data System (ADS)

    Chen, Weirong; Jia, Zhenhong; Li, Peng; Lv, Guodong; Lv, Xiaoyi

    2016-05-01

    By combining photolithography with the electrochemical anodization method, a microarray device of porous silicon (PS) photonic crystal was fabricated on the crystalline silicon substrate. The optical properties of the microarray were analyzed with the transfer matrix method. The relationship between refractive index and reflectivity of each array element of the microarray at 633 nm was also studied, and the array surface reflectivity changes were observed through digital imaging. By means of the reflectivity measurement method, reflectivity changes below 10-3 can be observed based on PS microarray. The results of this study can be applied to the detection of biosensor arrays.

  6. Versatile protein microarray based on carbohydrate-binding modules.

    PubMed

    Ofir, Keren; Berdichevsky, Yevgeny; Benhar, Itai; Azriel-Rosenfeld, Ronit; Lamed, Raphael; Barak, Yoav; Bayer, Edward A; Morag, Ely

    2005-05-01

    Non-DNA microarrays, such as protein, peptide and small molecule microarrays, can potentially revolutionize the high-throughput screening tools currently used in basic and pharmaceutical research. However, fundamental obstacles remain that limit their rapid and widespread implementation as an alternative bioanalytical approach. These include the prerequisite for numerous proteins in active and purified form, ineffectual immobilization strategies and inadequate means for quality control of the considerable numbers of multiple reagents. This study describes a simple yet efficient strategy for the production of non-DNA microarrays, based on the tenacious affinity of a carbohydrate-binding module (CBM) for its three-dimensional substrate, i.e., cellulose. Various microarray formats are described, e.g., conventional and single-chain antibody microarrays and peptide microarrays for serodiagnosis of human immunodeficiency virus patients. CBM-based microarray technology overcomes many of the previous obstacles that have hindered fabrication of non-DNA microarrays and provides a technically simple but effective alternative to conventional microarray technology. PMID:15825150

  7. Laser Capture Microdissection of Embryonic Cells and Preparation of RNA for Microarray Assays

    PubMed Central

    Redmond, Latasha C.; Pang, Christopher J.; Dumur, Catherine; Haar, Jack L.; Lloyd, Joyce A.

    2014-01-01

    In order to compare the global gene expression profiles of different embryonic cell types, it is first necessary to isolate the specific cells of interest. The purpose of this chapter is to provide a step-by-step protocol to perform laser capture microdissection (LCM) on embryo samples and obtain sufficient amounts of high-quality RNA for microarray hybridizations. Using the LCM/microarray strategy on mouse embryo samples has some challenges, because the cells of interest are available in limited quantities. The first step in the protocol is to obtain embryonic tissue, and immediately cryoprotect and freeze it in a cryomold containing Optimal Cutting Temperature freezing media (Sakura Finetek), using a dry ice–isopentane bath. The tissue is then cryosectioned, and the microscope slides are processed to fix, stain, and dehydrate the cells. LCM is employed to isolate specific cell types from the slides, identified under the microscope by virtue of their morphology. Detailed protocols are provided for using the currently available ArcturusXT LCM instrument and CapSure® LCM Caps, to which the selected cells adhere upon laser capture. To maintain RNA integrity, upon removing a slide from the final processing step, or attaching the first cells on the LCM cap, LCM is completed within 20 min. The cells are then immediately recovered from the LCM cap using a denaturing solution that stabilizes RNA integrity. RNA is prepared using standard methods, modified for working with small samples. To ensure the validity of the microarray data, the quality of the RNA is assessed using the Agilent bioanalyzer. Only RNA that is of sufficient integrity and quantity is used to perform microarray assays. This chapter provides guidance regarding troubleshooting and optimization to obtain high-quality RNA from cells of limited availability, obtained from embryo samples by LCM. PMID:24318813

  8. Isolation and characterization of the major form of human MUC18 cDNA gene and correlation of MUC18 over-expression in prostate cancer cell lines and tissues with malignant progression.

    PubMed

    Wu, G J; Wu, M W; Wang, S W; Liu, Z; Qu, P; Peng, Q; Yang, H; Varma, V A; Sun, Q C; Petros, J A; Lim, S D; Amin, M B

    2001-11-14

    Ectopical expression of huMUC18, a cell adhesion molecule in the immunoglobulin gene superfamily, causes a non-metastatic human melanoma cell line to become metastatic in a nude mouse system. To determine if MUC18 expression correlates with the development and malignant progression of prostate cancer, we investigated differential expression of human MUC18 (huMUC18) in normal prostate epithelial cells, prostate cancer cell lines, and prostatic normal and cancer tissues. We cloned and characterized the human MUC18 (huMUC18) cDNA gene from three human prostate cancer cell lines and three human melanoma cell lines. The cDNA sequences from the six human cancer cell lines were identical except differences in one to five nucleotides. The deduced amino acid sequences of the longest ORF were 646 amino acids that were identical in these cDNAs except for one to three amino acid residues. The amino acid sequences of all our huMUC18 cDNA genes are similar to that cloned by other group (GenBank access #M28882) except differences in the same seven amino acids. We conclude that huMUC18 cDNA gene reported here represents the gene product from a major allele. The MUC18 mRNA and protein was expressed in three metastatic prostate cancer cell lines (TSU-PR1, DU145, and PC-3), but not in one non-metastatic prostate cancer cell line (LNCaP.FGC). The expression of huMUC18 in these four cell lines is positively related to their extent of in vitro motility and invasiveness and in vivo metastasis in nude mice. HuMUC18 protein was also expressed at high levels in extracts prepared from tissue sample sections containing high grade prostatic intraepithelial neoplasia (PIN), but weakly expressed in extracts prepared from cultured primary normal prostatic epithelial cells and the normal prostate gland. Immunohistochemical analysis showed that huMUC18 was expressed at higher levels in the epithelial cells of high-grade PIN and prostatic carcinomas, and in cells of a perineural invasion, a lymph node, and a lung metastases compared to that in normal or benign hyperplastic epithelium (BPH). We therefore conclude that MUC18 expression is increased during prostate cancer initiation (high grade PIN) and progression to carcinoma, and in metastatic cell lines and metastatic carcinoma. Increased expression of MUC18 is implicated to play an important role in developing and malignant progression of human prostate cancer. Furthermore, the lacking of predominant cytoplasmic membrane expression of MUC18 appeared to correlate with malignant progression of prostate cancer. PMID:11722842

  9. Analysis of gene expression profiles as a tool to uncover tumor markers of liver cancer progression in a rat model

    PubMed Central

    VÁSQUEZ-GARZÓN, VERÓNICA R.; BELTRÁN-RAMÍREZ, OLGA; SALCIDO-NEYOY, MARTHA E.; CERVANTE-ANAYA, NANCY; VILLA-TREVIÑO, SAÚL

    2015-01-01

    Establishing a transcriptomic profile of human hepatocellular liver cancer (HCC) progression is a complex undertaking. A rat model of HCC was employed to develop a transcriptomic profile. Using three interventions, preneoplastic lesions appeared after 30 days and they progressed to HCC by 9 months. Preneoplastic and cancer lesions were characterized for transcriptomic analysis, and RNA from total liver homogenates was obtained at 1, 7, 11 and 16 days after the initiation treatment. RNA from dissected persistent preneoplastic lesions, adjacent tissue or cancer tissue was used for 30 days, and 5, 9, 12 and 18 months. The GeneChip® Rat Exon 1.0 ST arrays, Partek software and an Affymetrix console were employed for these analyses. LGALS3BP was differentially expressed at each time point, from the initial period, through the preneoplastic evolution period and until the end of cancer progression period. Twelve differentially expressed genes common to the preneoplastic evolution and to the cancer progression period were detected, which included ABCC3. Validation of the microarrays was confirmed by reverse transcription-quantitative polymerase chain reaction of six genes, including LGALS3BP and ABCC3. Of note, the proteins of these two genes are associated with the multidrug response complex, and evasion of immune surveillance and negative regulation of T cell proliferation. This model is useful for identifying candidate genes, and to validate them with regards to determining their relevance in rat HCC progression. PMID:25798242

  10. Visualization of microarray results to assist interpretation.

    PubMed

    Papatheodorou, Irene; Sergot, Marek; Randall, Marty; Stewart, Graham R; Robertson, Brian D

    2004-01-01

    Whole genome microarrays allow assessment of the profile of genes expressed under particular experimental conditions, including external stimuli such as pH or temperature, and internal changes brought about by deleting or over-expressing a gene. Such experiments produce large data sets, for which sophisticated analysis software is available. What is lacking are tools for analysing data sets from different experiments, in order to test and generate hypotheses about the links between regulatory networks. We describe here a method for presenting results from different experiments as a directed graph constructed using an automated graph drawing program xneato, enhanced by a logic program designed to cluster data and aid in the generation of hypotheses about possible gene interactions. A web-based front-end to the system has been constructed to explore and manipulate the graphical displays produced. Results of microarray experiments on Mycobacterium tuberculosis were used to develop and evaluate the visualization tool and initiate the development of an inference system for gene interactions based on such data. The GeneGraph project can be accessed at: zebrafish.doc.ic.ac.uk PMID:15207497

  11. Randomized probe selection algorithm for microarray design.

    PubMed

    Gasieniec, Leszek; Li, Cindy Y; Sant, Paul; Wong, Prudence W H

    2007-10-01

    DNA microarray technology, originally developed to measure the level of gene expression, has become one of the most widely used tools in genomic study. The crux of microarray design lies in how to select a unique probe that distinguishes a given genomic sequence from other sequences. Due to its significance, probe selection attracts a lot of attention. Various probe selection algorithms have been developed in recent years. Good probe selection algorithms should produce a small number of candidate probes. Efficiency is also crucial because the data involved are usually huge. Most existing algorithms are usually not sufficiently selective and quite a large number of probes are returned. We propose a new direction to tackle the problem and give an efficient algorithm based on randomization to select a small set of probes and demonstrate that such a small set of probes is sufficient to distinguish each sequence from all the other sequences. Based on the algorithm, we have developed probe selection software RandPS, which runs efficiently in practice. The software is available on our website (http://www.csc.liv.ac.uk/ approximately cindy/RandPS/RandPS.htm). We test our algorithm via experiments on different genomes (Escherichia coli, Saccharamyces cerevisiae, etc.) and our algorithm is able to output unique probes for most of the genes efficiently. The other genes can be identified by a combination of at most two probes. PMID:17628606

  12. Laser direct writing of biomolecule microarrays

    NASA Astrophysics Data System (ADS)

    Serra, P.; Fernández-Pradas, J. M.; Berthet, F. X.; Colina, M.; Elvira, J.; Morenza, J. L.

    Protein-based biosensors are highly efficient tools for protein detection and identification. The production of these devices requires the manipulation of tiny amounts of protein solutions in conditions preserving their biological properties. In this work, laser induced forward transfer (LIFT) was used for spotting an array of a purified bacterial antigen in order to check the viability of this technique for the production of protein microarrays. A pulsed Nd:YAG laser beam (355 nm wavelength, 10 ns pulse duration) was used to transfer droplets of a solution containing the Treponema pallidum 17 kDa protein antigen on a glass slide. Optical microscopy showed that a regular array of micrometric droplets could be precisely and uniformly spotted onto a solid substrate. Subsequently, it was proved that LIFT deposition of a T. pallidum 17 kDa antigen onto nylon-coated glass slides preserves its antigenic reactivity and diagnostic properties. These results support that LIFT is suitable for the production of protein microarrays and pave the way for future diagnostics applications.

  13. Stroma-Derived Connective Tissue Growth Factor Maintains Cell Cycle Progression and Repopulation Activity of Hematopoietic Stem Cells In Vitro

    PubMed Central

    Istvánffy, Rouzanna; Vilne, Baiba; Schreck, Christina; Ruf, Franziska; Pagel, Charlotta; Grziwok, Sandra; Henkel, Lynette; Prazeres da Costa, Olivia; Berndt, Johannes; Stümpflen, Volker; Götze, Katharina S.; Schiemann, Matthias; Peschel, Christian; Mewes, Hans-Werner; Oostendorp, Robert A.J.

    2015-01-01

    Summary Hematopoietic stem cells (HSCs) are preserved in co-cultures with UG26-1B6 stromal cells or their conditioned medium. We performed a genome-wide study of gene expression changes of UG26-1B6 stromal cells in contact with Lineage− SCA-1+ KIT+ (LSK) cells. This analysis identified connective tissue growth factor (CTGF) to be upregulated in response to LSK cells. We found that co-culture of HSCs on CTGF knockdown stroma (shCtgf) shows impaired engraftment and long-term quality. Further experiments demonstrated that CD34− CD48− CD150+ LSK (CD34− SLAM) cell numbers from shCtgf co-cultures increase in G0 and senescence and show delayed time to first cell division. To understand this observation, a CTGF signaling network model was assembled, which was experimentally validated. In co-culture experiments of CD34− SLAM cells with shCtgf stromal cells, we found that SMAD2/3-dependent signaling was activated, with increasing p27Kip1 expression and downregulating cyclin D1. Our data support the view that LSK cells modulate gene expression in the niche to maintain repopulating HSC activity. PMID:26527384

  14. Tissue Mechanics and Fibrosis

    PubMed Central

    Wells, Rebecca G.

    2013-01-01

    Mechanical forces are essential to the development and progression of fibrosis, and are likely to be as important as soluble factors. These forces regulate the phenotype and proliferation of myofibroblasts and other cells in damaged tissues, the activation of growth factors, the structure and mechanics of the matrix, and, potentially, tissue patterning. Better understanding of the variety and magnitude of forces, the characteristics of those forces in biological tissues, and their impact on fibrosis in multiple tissues is needed and may lead to identification of important new therapeutic targets. PMID:23434892

  15. Systems-wide chicken DNA microarrays, gene expression profiling, and discovery of functional genes.

    PubMed

    Cogburn, L A; Wang, X; Carre, W; Rejto, L; Porter, T E; Aggrey, S E; Simon, J

    2003-06-01

    The goal of our current consortium project is to launch a new era--functional genomics of poultry--by providing genomic resources [expressed sequence tags (EST) and DNA microarrays] and by examining global gene expression in target tissues of chickens. DNA microarray analysis has been a fruitful strategy for the identification of functional genes in several model organisms (i.e., human, rodents, fruit fly, etc.). We have constructed and normalized five tissue-specific or multiple-tissue chicken cDNA libraries [liver, fat, breast, and leg muscle/epiphyseal growth plate, pituitary/hypothalamus/pineal, and reproductive tract (oviduct/ovary/testes)] for high-throughput DNA sequencing of EST. DNA sequence clustering was used to build contigs of overlapping sequence and to identify unique, non-redundant EST clones (unigenes), which permitted printing of systems-wide chicken DNA microarrays. One of the most promising genetic resources for gene exploration and functional gene mapping is provided by two sets of experimental lines of broiler-type chickens developed at INRA, France, by divergent selection for extremes in growth traits (fast-growing versus slow-growing; fatness versus leanness at a similar growth rate). We are using DNA microarrays for global gene expression profiling to identify candidate genes and to map growth, metabolic, and regulatory pathways that control important production traits. Candidate genes will be used for functional gene mapping and QTL analysis of F2 progeny from intercrosses made between divergent genetic lines (fat x lean lines; fast-growing x slow-growing lines). Using our first chicken liver microarray, we have already identified several interesting differentially expressed genes in commercial broilers and in divergently selected broiler lines. Many of these candidate genes are involved in the lipogenic pathway and are controlled in part by the thyrotropic axis. Thus, genome-wide transcriptional profiling is a powerful tool used to visualize the cascade of genetic circuits that govern complex biological responses. Global gene expression profiling and QTL scans should enable us to functionally map the genetic pathways that control growth, development, and metabolism of chickens. This emerging technology will have broad applications for poultry breeding programs (i.e., use of molecular markers) and for future production systems (i.e., the health and welfare of birds and the quality of poultry products). PMID:12817449

  16. Adenoviral low density lipoprotein receptor attenuates progression of atherosclerosis and decreases tissue cholesterol levels in a murine model of familial hypercholesterolemia.

    PubMed

    Jacobs, Frank; Van Craeyveld, Eline; Feng, Yingmei; Snoeys, Jan; De Geest, Bart

    2008-12-01

    Familial hypercholesterolemia is an autosomal codominant disease characterized by high concentrations of pro-atherogenic lipoproteins and premature atherosclerosis secondary to low density lipoprotein receptor (LDLr) deficiency. In the current study, the effects of gene transfer with 5 x 10(10) particles of E1E3E4-deleted adenoviral vectors expressing the LDLr (AdLDLr) or VLDLr (AdVLDLr) under control of the hepatocyte-specific human alpha(1)-antitrypsin promoter and 4 copies of the human apo E enhancer in C57BL/6 LDLr(-/-) mice were investigated. Evaluation was performed in both sexes and in mice fed either standard chow or an atherogenic diet containing 0.2% cholesterol and 10% coconut oil. Compared to control mice, AdLDLr and AdVLDLr persistently decreased plasma non-HDL cholesterol in both sexes and on both diets. Six months after LDLr gene transfer in mice fed the atherogenic diet, average intimal area was 2.5-fold (p<0.01) and 3.2-fold (p<0.001) lower in male and female mice, respectively, compared to controls. In mice fed standard chow, intimal area was reduced 22-fold (p<0.001) and 21-fold (p<0.001) after LDLr gene transfer in male and female mice, respectively. We show that non-HDL lipoproteins are more atherogenic in female mice, independent of sex differences of plasma HDL cholesterol levels, and that saturated fat does not have an effect on atherosclerosis independent of plasma cholesterol levels. Finally, quantification of tissue cholesterol levels indicates that AdLDLr does not induce cholesterol accumulation in the liver and reduces cholesterol content in the myocardium, quadriceps muscle and kidney. In conclusion, hepatocyte-specific LDLr gene transfer significantly improves cholesterol homeostasis in LDLr(-/-) mice. PMID:18378244

  17. SpOT the Correct Tissue Every Time in Multi-Tissue Blocks

    PubMed Central

    Coffey, Anna H.; Berry, Deborah L.; Johnson, Michael D.

    2015-01-01

    Multi-tissue paraffin blocks provide high throughput analysis with increased efficiency, experimental uniformity, and reduced time and cost. Tissue microarrays make up the majority of multi-tissue paraffin blocks, but increasingly, researchers are using non-arrayed blocks containing larger tissues from multiple individuals which can provide many of the advantages of tissue microarrays without substantial investment in planning and equipment. A critical component of any multi-tissue analysis is the orientation method used to identify each individual tissue. Although methods exist to maintain proper orientation and identification of tissues in multi-tissue blocks, most are not well-suited to non-arrayed blocks, may consume valuable space within an array and/or are difficult to produce in the standard histology laboratory. The Specimen Orientation Tag (SpOT) is a simple, low cost orientation tool that is clearly visible in paraffin blocks and all tissue sections for reliable specimen identification in arrayed and non-arrayed layouts. The SpOT provides advantages over existing orientation methods for non-arrayed blocks as it does not require any direct modification to the tissue and allows for flexibility in the arrangement of tissue pieces. PMID:26067587

  18. Microarray analysis of metallothioneins in human diseases--A review.

    PubMed

    Krizkova, Sona; Kepinska, Marta; Emri, Gabriella; Rodrigo, Miguel Angel Merlos; Tmejova, Katerina; Nerudova, Danuse; Kizek, Rene; Adam, Vojtech

    2016-01-01

    Metallothioneins (MTs), low molecular mass cysteine-rich proteins, which are able to bind up to 20 monovalent and up to 7 divalent heavy metal ions are widely studied due to their functions in detoxification of metals, scavenging free radicals and cells protection against the oxidative stress. It was found that the loss of the protective effects of MT leads to an escalation of pathogenic processes and carcinogenesis. The most extensive area is MTs expression for oncological applications, where the information about gene patterns is helpful for the identification biological function, resistance to drugs and creating the correct chemotherapy. In other medical applications the effect of oxidative stress to cell lines exposed to heavy metals and hydrogen peroxide is studied as well as influence of drugs and cytokines on MTs expression and MTs expression in the adipose tissue. The precise detection of low metallothionein concentrations and its isoforms is necessary to understand the connection between quantity and isoforms of MTs to size, localization and type of cancer. This information is necessary for well-timed therapy and increase the chance to survival. Microarray chips appear as good possibility for finding all information about expression of MTs genes and isoforms not only in cancer, but also in other diseases, especially diabetes, obesity, cardiovascular diseases, ageing, osteoporosis, psychiatric disorders and as the effects of toxic drugs and pollutants, which is discussed in this review. PMID:26454339

  19. Human protein atlas and the use of microarray technologies.

    PubMed

    Hober, S; Uhlén, M

    2008-02-01

    Currently one of the most challenging tasks in biological and medical research is to explore and understand the function of all proteins encoded by the genome of an organism. A systematic approach based on the genome sequences is feasible because the full genome of many organisms presently is available and many more are underway. For the production of expression atlases different strategies are used. Early attempts to acquire information about protein expression levels have focused on the analysis of mRNA levels within different tissues and cell types. Recently, novel strategies to focus directly on protein levels have been developed. To assess global protein expression in a systematic and high-throughput manner, methods based on design of specific affinity ligands to recognize the proteins have been presented. By subsequently using these affinity molecules for detection of the corresponding proteins in a wide range of platforms, important information can be gained. This article focuses on strategies to profile protein levels and in particular the human protein atlas initiative and the use of microarray technologies. PMID:18187316

  20. Microarray Analysis of Whole Genome Expression of Intracellular Mycobacterium tuberculosis

    PubMed Central

    Waddell, Simon J.; Butcher, Philip D.

    2011-01-01

    Analysis of the changing mRNA expression profile of Mycobacterium tuberculosis though the course of infection promises to advance our understanding of how mycobacteria are able to survive the host immune response. The difficulties of sample extraction from distinct mycobacterial populations, and of measuring mRNA expression profiles of multiple genes has limited the impact of gene expression studies on our interpretation of this dynamic infection process. The development of whole genome microarray technology together with advances in sample collection have allowed the expression pattern of the whole M. tuberculosis genome to be compared across a number of different in vitro conditions, murine and human tissue culture models and in vivo infection samples. This review attempts to produce a summative model of the M. tuberculosis response to infection derived from or reflected in these gene expression datasets. The mycobacterial response to the intracellular environment is characterised by the utilisation of lipids as a carbon source and the switch from aerobic/microaerophilic to anaerobic respiratory pathways. Other genes induced in the macrophage phagosome include those likely to be involved in the maintenance of the cell wall and genes related to DNA damage, heat shock, iron sequestration and nutrient limitation. The comparison of transcriptional data from in vitro models of infection with complex in vivo samples, together with the use of bacterial RNA amplification strategies to sample defined populations of bacilli, should allow us to make conclusions about M. tuberculosis physiology and host microenvironments during natural infection. PMID:17504113

  1. Experimental Approaches to Microarray Analysis of Tumor Samples

    ERIC Educational Resources Information Center

    Furge, Laura Lowe; Winter, Michael B.; Meyers, Jacob I.; Furge, Kyle A.

    2008-01-01

    Comprehensive measurement of gene expression using high-density nucleic acid arrays (i.e. microarrays) has become an important tool for investigating the molecular differences in clinical and research samples. Consequently, inclusion of discussion in biochemistry, molecular biology, or other appropriate courses of microarray technologies has

  2. Application of Microarray Technology to Investigate Salmonella and Antimicrobial Resistance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microarrays have been developed for the study of various aspects of Salmonella, which is a model system for investigating pathogenesis. Microarrays were used to analyze the gene expression of Salmonella in various environments that mimic the host environment and these studies have helped to elucidat...

  3. Experimental Approaches to Microarray Analysis of Tumor Samples

    ERIC Educational Resources Information Center

    Furge, Laura Lowe; Winter, Michael B.; Meyers, Jacob I.; Furge, Kyle A.

    2008-01-01

    Comprehensive measurement of gene expression using high-density nucleic acid arrays (i.e. microarrays) has become an important tool for investigating the molecular differences in clinical and research samples. Consequently, inclusion of discussion in biochemistry, molecular biology, or other appropriate courses of microarray technologies has…

  4. The Importance of Normalization on Large and Heterogeneous Microarray Datasets

    EPA Science Inventory

    DNA microarray technology is a powerful functional genomics tool increasingly used for investigating global gene expression in environmental studies. Microarrays can also be used in identifying biological networks, as they give insight on the complex gene-to-gene interactions, ne...

  5. Applications of microarray technology in breast cancer research

    PubMed Central

    Cooper, Colin S

    2001-01-01

    Microarrays provide a versatile platform for utilizing information from the Human Genome Project to benefit human health. This article reviews the ways in which microarray technology may be used in breast cancer research. Its diverse applications include monitoring chromosome gains and losses, tumour classification, drug discovery and development, DNA resequencing, mutation detection and investigating the mechanism of tumour development. PMID:11305951

  6. Insights into Osteoarthritis Progression Revealed by Analyses of Both Knee Tibiofemoral Compartments

    PubMed Central

    Chou, Ching-Heng; Lee, Ming Ta Michael; Song, I-Wen; Lu, Liang-Suei; Shen, Hsain-Chung; Lee, Chian-Her; Wu, Jer-Yuarn; Chen, Yuan-Tsong; Kraus, Virginia Byers; Wu, Chia-Chun

    2016-01-01

    Objective To identify disease relevant genes and pathways associated with knee Osteoarthritis (OA) progression in human subjects using medial and lateral compartment dominant OA knee tissue. Design Gene expression of knee cartilage was comprehensively assessed for three regions of interest from human medial dominant OA (n=10) and non-OA (n=6) specimens. Histology and gene expression were compared for the regions with minimal degeneration, moderate degeneration and significant degeneration. Agilent whole-genome microarray was performed and data were analyzed using Agilent GeneSpring GX11.5. Significant differentially regulated genes were further investigated by Ingenuity Pathway Analysis (IPA) to identify functional categories. To confirm their association with disease severity as opposed to site within the knee, 30 differentially expressed genes, identified by microarray, were analyzed by quantitative reverse-transcription polymerase chain reaction on additional medial (n=16) and lateral (n=10) compartment dominant knee OA samples. Results A total of 767 genes were differentially expressed ≥two-fold (P ≤0.05) in lesion compared to relatively intact regions. Analysis of these data by IPA predicted biological functions related to an imbalance of anabolism and catabolism of cartilage matrix components. Up-regulated expression of IL11, POSTN, TNFAIP6, and down-regulated expression of CHRDL2, MATN4, SPOCK3, VIT, PDE3B were significantly associated with OA progression and validated in both medial and lateral compartment dominant OA samples. Conclusions Our study provides a strategy for identifying targets whose modification may have the potential to ameliorate pathological alternations and progression of disease in cartilage and to serve as biomarkers for identifying individuals susceptible to progression. PMID:25575966

  7. APPLICATION OF PHYLOGENETIC MICROARRAYS TO INTERROGATION OF HUMAN MICROBIOTA

    PubMed Central

    Paliy, Oleg; Agans, Richard

    2011-01-01

    Human-associated microbiota is recognized to play vital roles in maintaining host health, and it is implicated in many disease states. While the initial surge in the profiling of these microbial communities was achieved with Sanger and next generation sequencing, many oligonucleotide microarrays have also been developed recently for this purpose. Containing probes complementary to small ribosomal subunit RNA gene sequences of community members, such phylogenetic arrays provide direct quantitative comparisons of microbiota composition among samples and between sample groups. Some of the developed microarrays including PhyloChip, Microbiota Array, and HITChip can simultaneously measure the presence and abundance of hundreds and thousands of phylotypes in a single sample. This review describes the currently available phylogenetic microarrays that can be used to analyse human microbiota, delineates the approaches for the optimization of microarray use, and provides examples of recent findings based on microarray interrogation of human-associated microbial communities. PMID:22092522

  8. DNA microarrays--techniques and applications in microbial systems.

    PubMed

    Majtán, T; Bukovská, G; Timko, J

    2004-01-01

    Genome projects produce a huge amount of sequence information. As a result, the focus of genomics research is turning toward deduction of functional information about newly discovered genes. Thus structural genomics paves the way for a new discipline called functional genomics by providing the information required for microarray manufacture. Microarray technology is the result of automation and miniaturization in the detection of differential gene expression. By using this technology one can make a parallel analysis of RNA abundance and DNA homology for thousands of genes in a single experiment. Over the past several years, this unique technology has been used to explore hundreds transcriptional patterns and genome differences for a variety of microbial species. Applications of microarrays extend beyond the boundaries of basic biology into diagnostics, environmental monitoring, pharmacology, toxicology and biotechnology. We describe comprehensive nature of DNA microarray technology with emphasis on fabrication of DNA microarrays and application of this technology in biological environment with primary accent on microbial systems. PMID:15881400

  9. An ultralow background substrate for protein microarray technology.

    PubMed

    Feng, Hui; Zhang, Qingyang; Ma, Hongwei; Zheng, Bo

    2015-08-21

    We herein report an ultralow background substrate for protein microarrays. Conventional protein microarray substrates often suffer from non-specific protein adsorption and inhomogeneous spot morphology. Consequently, surface treatment and a suitable printing solution are required to improve the microarray performance. In the current work, we improved the situation by developing a new microarray substrate based on a fluorinated ethylene propylene (FEP) membrane. A polydopamine microspot array was fabricated on the FEP membrane, with proteins conjugated to the FEP surface through polydopamine. Uniform microspots were obtained on FEP without the application of a special printing solution. The modified FEP membrane demonstrated ultralow background signal and was applied in protein and peptide microarray analysis. PMID:26134063

  10. cDNA Microarray Screening in Food Safety

    PubMed Central

    ROY, SASHWATI; SEN, CHANDAN K

    2009-01-01

    The cDNA microarray technology and related bioinformatics tools presents a wide range of novel application opportunities. The technology may be productively applied to address food safety. In this mini-review article, we present an update highlighting the late breaking discoveries that demonstrate the vitality of cDNA microarray technology as a tool to analyze food safety with reference to microbial pathogens and genetically modified foods. In order to bring the microarray technology to mainstream food safety, it is important to develop robust user-friendly tools that may be applied in a field setting. In addition, there needs to be a standardized process for regulatory agencies to interpret and act upon microarray-based data. The cDNA microarray approach is an emergent technology in diagnostics. Its values lie in being able to provide complimentary molecular insight when employed in addition to traditional tests for food safety, as part of a more comprehensive battery of tests. PMID:16466843

  11. A Comparative Study of Normalization Methods Used in Statistical Analysis of Oligonucleotide Microarray Data

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Normalization methods used in the statistical analysis of oligonucleotide microarray data were evaluated. The oligonucleotide microarray is considered an efficient analytical tool for analyzing thousands of genes simultaneously in a single experiment. However, systematic variation in microarray, ori...

  12. Giant Magnetoresistive Sensors for DNA Microarray

    PubMed Central

    Xu, Liang; Yu, Heng; Han, Shu-Jen; Osterfeld, Sebastian; White, Robert L.; Pourmand, Nader; Wang, Shan X.

    2009-01-01

    Giant magnetoresistive (GMR) sensors are developed for a DNA microarray. Compared with the conventional fluorescent sensors, GMR sensors are cheaper, more sensitive, can generate fully electronic signals, and can be easily integrated with electronics and microfluidics. The GMR sensor used in this work has a bottom spin valve structure with an MR ratio of 12%. The single-strand target DNA detected has a length of 20 bases. Assays with DNA concentrations down to 10 pM were performed, with a dynamic range of 3 logs. A double modulation technique was used in signal detection to reduce the 1/f noise in the sensor while circumventing electromagnetic interference. The logarithmic relationship between the magnetic signal and the target DNA concentration can be described by the Temkin isotherm. Furthermore, GMR sensors integrated with microfluidics has great potential of improving the sensitivity to 1 pM or below, and the total assay time can be reduced to less than 1 hour. PMID:20824116

  13. Role of lectin microarrays in cancer diagnosis.

    PubMed

    Syed, Parvez; Gidwani, Kamlesh; Kekki, Henna; Leivo, Janne; Pettersson, Kim; Lamminmäki, Urpo

    2016-04-01

    The majority of cell differentiation associated tumor markers reported to date are either glycoproteins or glycolipids. Despite there being a large number of glycoproteins reported as candidate markers for various cancers, only a handful are approved by the US Food and Drug Administration. Lectins, which bind to the glycan part of the glycoproteins, can be exploited to identify aberrant glycosylation patterns, which in turn would help in enhancing the specificity of cancer diagnosis. Although conventional techniques such as HPLC and MS have been instrumental in performing the glycomic analyses, these techniques lack multiplexity. Lectin microarrays have proved to be useful in studying multiple lectin-glycan interactions in a single experiment and, with the advances made in the field, hold a promise of enabling glycomic profiling of cancers in a fast and efficient manner. PMID:26841254

  14. Chapter 9 - Methylation Analysis by Microarray

    PubMed Central

    Deatherage, Daniel E.; Potter, Dustin; Yan, Pearlly S.; Huang, Tim H.-M.; Lin, Shili

    2010-01-01

    Differential Methylation Hybridization (DMH) is a high-throughput DNA methylation screening tool that utilizes methylation-sensitive restriction enzymes to profile methylated fragments by hybridizing them to a CpG island microarray. This array contains probes spanning all the 27,800 islands annotated in the UCSC Genome Browser. Herein we describe a DMH protocol with clearly identified quality control points. In this manner, samples that are unlikely to provide good read-outs for differential methylation profiles between the test and the control samples will be identified and repeated with appropriate modifications. The step-by-step laboratory DMH protocol is described. In addition, we provide descriptions regarding DMH data analysis, including image quantification, background correction, and statistical procedures for both exploratory analysis and more formal inferences. Issues regarding quality control are addressed as well. PMID:19488875

  15. Digital microarray analysis for digital artifact genomics

    NASA Astrophysics Data System (ADS)

    Jaenisch, Holger; Handley, James; Williams, Deborah

    2013-06-01

    We implement a Spatial Voting (SV) based analogy of microarray analysis for digital gene marker identification in malware code sections. We examine a famous set of malware formally analyzed by Mandiant and code named Advanced Persistent Threat (APT1). APT1 is a Chinese organization formed with specific intent to infiltrate and exploit US resources. Manidant provided a detailed behavior and sting analysis report for the 288 malware samples available. We performed an independent analysis using a new alternative to the traditional dynamic analysis and static analysis we call Spatial Analysis (SA). We perform unsupervised SA on the APT1 originating malware code sections and report our findings. We also show the results of SA performed on some members of the families associated by Manidant. We conclude that SV based SA is a practical fast alternative to dynamics analysis and static analysis.

  16. Lipid Microarray Biosensor for Biotoxin Detection.

    SciTech Connect

    Singh, Anup K.; Throckmorton, Daniel J.; Moran-Mirabal, Jose C.; Edel, Joshua B.; Meyer, Grant D.; Craighead, Harold G.

    2006-05-01

    We present the use of micron-sized lipid domains, patterned onto planar substrates and within microfluidic channels, to assay the binding of bacterial toxins via total internal reflection fluorescence microscopy (TIRFM). The lipid domains were patterned using a polymer lift-off technique and consisted of ganglioside-populated DSPC:cholesterol supported lipid bilayers (SLBs). Lipid patterns were formed on the substrates by vesicle fusion followed by polymer lift-off, which revealed micron-sized SLBs containing either ganglioside GT1b or GM1. The ganglioside-populated SLB arrays were then exposed to either Cholera toxin subunit B (CTB) or Tetanus toxin fragment C (TTC). Binding was assayed on planar substrates by TIRFM down to 1 nM concentration for CTB and 100 nM for TTC. Apparent binding constants extracted from three different models applied to the binding curves suggest that binding of a protein to a lipid-based receptor is strongly affected by the lipid composition of the SLB and by the substrate on which the bilayer is formed. Patterning of SLBs inside microfluidic channels also allowed the preparation of lipid domains with different compositions on a single device. Arrays within microfluidic channels were used to achieve segregation and selective binding from a binary mixture of the toxin fragments in one device. The binding and segregation within the microfluidic channels was assayed with epifluorescence as proof of concept. We propose that the method used for patterning the lipid microarrays on planar substrates and within microfluidic channels can be easily adapted to proteins or nucleic acids and can be used for biosensor applications and cell stimulation assays under different flow conditions. KEYWORDS. Microarray, ganglioside, polymer lift-off, cholera toxin, tetanus toxin, TIRFM, binding constant.4

  17. Molecular mechanisms of osteoarthritis using gene microarrays.

    PubMed

    Cui, Shuo; Zhang, Xinying; Hai, Sen; Lu, Hong; Chen, Yongcai; Li, Chao; Tong, Pengfei; Lu, Fei; Yuan, Zhengjiang

    2015-01-01

    This study aimed to investigate the molecular mechanisms of osteoarthritis (OA) by microarray analysis. Three gene expression datasets GSE1919, 19664 and 55235 were downloaded from the Gene Expression Omnibus, and data of OA samples and healthy controls were used. After data preprocessing, differential expression analysis between the OA group and controls was performed using LIMMA (Linear Models for Microarray Data) package and genes with |log2FC (fold change)|>1 and P<0.05 were screened as DEGs (differentially expressed genes). The screened DEGs were then subject to functional annotation and pathway enrichment analysis using DAVID (Database for Annotation Visualization and Integrated Discovery). Next, gene-set enrichment analysis was performed using Enrichment map Cytoscape plug-in, followed by detecting sub-networks using clusterONE. Finally, risk subpathways were screened using iSubpathwayMiner package. A total of 141 DEGs were screened, including 52 up-regulated ones and 89 down-regulated ones. These DEGs were enriched in 48 GO terms that were mainly related to locomotory behavior, taxis, adhesion, and 11 pathways that were related to cytokine-cytokine receptor interaction, ECM-receptor interaction, focal adhesion, as well as several signaling pathways. The enrichment map enriched gene-sets mainly related to cell death and apoptosis, and extracellular components. The risk pathways up-regulated DEGs were exclusively related to arachidonic acid metabolism and glycosphingolipid biosynthesis, and the top two risk pathways were tyrosine metabolism for the down-regulated ones. From this study we conclude that genes involved in cell death and apoptosis, as well as cell-extracellular matrix interaction, may be essential for OA pathogenesis by altering multiple signaling pathways. PMID:25468726

  18. Bystander effect: biological endpoints and microarray analysis.

    PubMed

    Chaudhry, M Ahmad

    2006-05-11

    In cell populations exposed to ionizing radiation, the biological effects occur in a much larger proportion of cells than are estimated to be traversed by radiation. It has been suggested that irradiated cells are capable of providing signals to the neighboring unirradiated cells resulting in damage to these cells. This phenomenon is termed the bystander effect. The bystander effect induces persistent, long-term, transmissible changes that result in delayed death and neoplastic transformation. Because the bystander effect is relevant to carcinogenesis, it could have significant implications for risk estimation for radiation exposure. The nature of the bystander effect signal and how it impacts the unirradiated cells remains to be elucidated. Examination of the changes in gene expression could provide clues to understanding the bystander effect and could define the signaling pathways involved in sustaining damage to these cells. The microarray technology serves as a tool to gain insight into the molecular pathways leading to bystander effect. Using medium from irradiated normal human diploid lung fibroblasts as a model system we examined gene expression alterations in bystander cells. The microarray data revealed that the radiation-induced gene expression profile in irradiated cells is different from unirradiated bystander cells suggesting that the pathways leading to biological effects in the bystander cells are different from the directly irradiated cells. The genes known to be responsive to ionizing radiation were observed in irradiated cells. Several genes were upregulated in cells receiving media from irradiated cells. Surprisingly no genes were found to be downregulated in these cells. A number of genes belonging to extracellular signaling, growth factors and several receptors were identified in bystander cells. Interestingly 15 genes involved in the cell communication processes were found to be upregulated. The induction of receptors and the cell communication processes in bystander cells receiving media from irradiated cells supports the active involvement of these processes in inducing bystander effect. PMID:16414093

  19. On the Statics for Micro-Array Data Analysis

    NASA Astrophysics Data System (ADS)

    Urushibara, Tomoko; Akasaka, Shizu; Ito, Makiko; Suzuki, Tomonori; Miyazaki, Satoru

    2010-01-01

    Recently after human genome sequence has been determined almost perfectly, more and more researchers have been studying genes in detail. Therefore, we are sure that accumulated gene information for human will be getting more important in the near future to develop customized medicine and to make gene interactions clear. Among plenty of information, micro array might be one of the most important analysis method for genes because it is the technique that can get big amount of the gene expressions data from one time experiment and also can be used for DNA isolation. To get the novel knowledge from micro array data, we need to enrich statistical tools for its data analysis. So far, many mathematical theories and definition have been proposing. However, many of those proposals are tested with strict conditions or customized to data for specific species. In this paper, we reviewed existing typical statistical methods for micro array analysis and discussed the repeatability of the analysis, construction the guideline with more general procedure. First we analyzed the micro array data for TG rats, with statistical methods of family-wise error rate (FWER) control approach and False Discovery Rate (FDR) control approach. As existing report, no significantly different gene could be detected with FWER control approach. On the other hand, we could find several genes significantly with FDR control approach even q=0.5. To find out the reliability of FDR control approach with micro array conditions, we have analyzed 2 more pieces of data from Gene Expression Omnibus (GEO) public database on the web site with SAM in addition to FWER and FDR control approaches. We could find a certain number of significantly different genes with BH method and SAM in the case of q=0.05. However, we have to note that the number and kinds of detected genes are different when we compare our result with the one from the published paper. Even if the same approach is used to analyze the same micro array data, we might get a different result because the distinct definition for micro array data has not been set yet. It means that from the same data we will get different results depending on researchers. We are afraid that this problem will have a big effect on developing new medicines and to progress the next step, like a 2nd screening. So, we suggest that we should have certain guidelines to analyze Micro-Array data validly with statistic method and it will surely be helpful for Micro-Array analysis for medical studies in the future.

  20. Obesity and prostate cancer: gene expression signature of human periprostatic adipose tissue

    PubMed Central

    2012-01-01

    Background Periprostatic (PP) adipose tissue surrounds the prostate, an organ with a high predisposition to become malignant. Frequently, growing prostatic tumor cells extend beyond the prostatic organ towards this fat depot. This study aimed to determine the genome-wide expression of genes in PP adipose tissue in obesity/overweight (OB/OW) and prostate cancer patients. Methods Differentially expressed genes in human PP adipose tissue were identified using microarrays. Analyses were conducted according to the donors' body mass index characteristics (OB/OW versus lean) and prostate disease (extra prostatic cancer versus organ confined prostate cancer versus benign prostatic hyperplasia). Selected genes with altered expression were validated by real-time PCR. Ingenuity Pathway Analysis (IPA) was used to investigate gene ontology, canonical pathways and functional networks. Results In the PP adipose tissue of OB/OW subjects, we found altered expression of genes encoding molecules involved in adipogenic/anti-lipolytic, proliferative/anti-apoptotic, and mild immunoinflammatory processes (for example, FADS1, down-regulated, and LEP and ANGPT1, both up-regulated). Conversely, in the PP adipose tissue of subjects with prostate cancer, altered genes were related to adipose tissue cellular activity (increased cell proliferation/differentiation, cell cycle activation and anti-apoptosis), whereas a downward impact on immunity and inflammation was also observed, mostly related to the complement (down-regulation of CFH). Interestingly, we found that the microRNA MIRLET7A2 was overexpressed in the PP adipose tissue of prostate cancer patients. Conclusions Obesity and excess adiposity modified the expression of PP adipose tissue genes to ultimately foster fat mass growth. In patients with prostate cancer the expression profile of PP adipose tissue accounted for hypercellularity and reduced immunosurveillance. Both findings may be liable to promote a favorable environment for prostate cancer progression. PMID:23009291

  1. Microarray sampling-platform fabrication using bubble-jet technology for a biochip system.

    PubMed

    Allain, L R; Askari, M; Stokes, D L; Vo-Dinh, T

    2001-09-01

    The fabrication of microarrays containing PCR-amplified genomic DNA extracts from mice tumors on a Zetaprobe membrane using a modified thermal ink-jet printer is described. A simple and cost-effective procedure for the fabrication of microarrays containing biological samples using a modified bubble-jet printing system is presented. Because of their mass-produced design, ink-jet printers are a much cheaper alternative to conventional spotting techniques. The usefulness of the biochip microarray platform is illustrated by the detection of human fragile histidine triad (FHIT), a tumor suppressor gene. Subcutaneous carcinomas were induced with MKN/FHIT and MKN/E4 cell lines in immunodeficient mice. Several weeks into their development, the tumors from both groups of mice were removed and subjected to DNA extraction by lysis of tissue samples. The extracted DNA samples were amplified by PCR (30 cycles) using the primers corresponding to nucleotides 2 to 18 of the FHIT sequence. The resulting solution was transferred to the individual reservoirs of a three-color cartridge from a conventional thermal ink-jet printer (HP 694C), and arrays were printed on to a Zetaprobe membrane. After spotting, these membranes were used in a hybridization assay, using fluorescent probes, and detected with a biochip. PMID:11678184

  2. Microarray Profiling of Lymphocytes in Internal Diseases With an Altered Immune Response: Potential and Methodology

    PubMed Central

    Gladkevich, Anatoliy; Nelemans, S. Adriaan; Kauffman, Henk F.; Korf, Jakob

    2005-01-01

    Recently it has become possible to investigate expression of all human genes with microarray technique. The authors provide arguments to consider peripheral white blood cells and in particular lymphocytes as a model for the investigation of pathophysiology of asthma, RA, and SLE diseases in which inflammation is a major component. Lymphocytes are an alternative to tissue biopsies that are most often difficult to collect systematically. Lymphocytes express more than 75% of the human genome, and, being an important part of the immune system, they play a central role in the pathogenesis of asthma, RA, and SLE. Here we review alterations of gene expression in lymphocytes and methodological aspects of the microarray technique in these diseases. Lymphocytic genes may become activated because of a general nonspecific versus disease-specific mechanism. The authors suppose that in these diseases microarray profiles of gene expression in lymphocytes can be disease specific, rather than inflammation specific. Some potentials and pitfalls of the array technologies are discussed. Optimal clinical designs aimed to identify disease-specific genes are proposed. Lymphocytes can be explored for research, diagnostic, and possible treatment purposes in these diseases, but their precise value should be clarified in future investigation. PMID:16489251

  3. VAMPIRE microarray suite: a web-based platform for the interpretation of gene expression data

    PubMed Central

    Hsiao, Albert; Ideker, Trey; Olefsky, Jerrold M.; Subramaniam, Shankar

    2005-01-01

    Microarrays are invaluable high-throughput tools used to snapshot the gene expression profiles of cells and tissues. Among the most basic and fundamental questions asked of microarray data is whether individual genes are significantly activated or repressed by a particular stimulus. We have previously presented two Bayesian statistical methods for this level of analysis, collectively known as variance-modeled posterior inference with regional exponentials (VAMPIRE). These methods each require a sophisticated modeling step followed by integration of a posterior probability density. We present here a publicly available, web-based platform that allows users to easily load data, associate related samples and identify differentially expressed features using the VAMPIRE statistical framework. In addition, this suite of tools seamlessly integrates a novel gene annotation tool, known as GOby, which identifies statistically overrepresented gene groups. Unlike other tools in this genre, GOby can localize enrichment while respecting the hierarchical structure of annotation systems like Gene Ontology (GO). By identifying statistically significant enrichment of GO terms, Kyoto Encyclopedia of Genes and Genomes pathways, and TRANSFAC transcription factor binding sites, users can gain substantial insight into the physiological significance of sets of differentially expressed genes. The VAMPIRE microarray suite can be accessed at . PMID:15980550

  4. Histopathology of melanosis coli and determination of its associated genes by comparative analysis of expression microarrays.

    PubMed

    Li, Xiao-Αn; Zhou, Yan; Zhou, Shu-Χian; Liu, Hai-Rong; Xu, Jin-Mei; Gao, Long; Yu, Xian-Jing; Li, Xiao-Hui

    2015-10-01

    Melanosis coli (MC) refers to the condition characterized by abnormal brown or black pigmentation deposits on the colonic mucosa. However, the histopathological findings and genes associated with the pathogenesis of melanosis coli remain to be fully elucidated. The present study aimed to examine the histopathological features and differentially expressed genes of MC. This involved performing hematoxylin and eosin staining, specific staining and immunohistochemistry on tissues sections, which were isolated from patients diagnosed with MC. DNA expression microarray analysis, western blotting and immunofluorescence assays were performed to analyze the differentially expressed genes of melanosis coli. The results demonstrated that the pigment deposits in MC consisted of lipofuscin. A TUNEL assay revealed that a substantial number of apoptotic cells were present within the macrophages and superficial lamina propria of the colonic epithelium. Expression microarray analysis revealed that the significantly downregulated genes were CYP3A4, CYP3A7, UGT2B11 and UGT2B15 in melanosis coli. Western blotting and immunofluorescence assays indicated that the expression of CYP3A4 in the normal tissue was higher than in the MC tissue. The results of the present study provided a comprehensive description of the histopathological characteristics and pathogenesis of MC and for the first time, to the best of our knowledge, demonstrated that the cytochrome P450‑associated genes were significantly downregulated in melanosis coli. This novel information can be used to assist in further investigations of melanosis coli. PMID:26238215

  5. Histopathology of melanosis coli and determination of its associated genes by comparative analysisof expression microarrays

    PubMed Central

    LI, XIAO-AN; ZHOU, YAN; ZHOU, SHU-XIAN; LIU, HAI-RONG; XU, JIN-MEI; GAO, LONG; YU, XIAN-JING; LI, XIAO-HUI

    2015-01-01

    Melanosis coli (MC) refers to the condition characterized by abnormal brown or black pigmentation deposits on the colonic mucosa. However, the histopathological findings and genes associated with the pathogenesis of melanosis coli remain to be fully elucidated. The present study aimed to examine the histopathological features and differentially expressed genes of MC. This involved performing hematoxylin and eosin staining, specific staining and immunohistochemistry on tissues sections, which were isolated from patients diagnosed with MC. DNA expression microarray analysis, western blotting and immunofluorescence assays were performed to analyze the differentially expressed genes of melanosis coli. The results demonstrated that the pigment deposits in MC consisted of lipofuscin. A TUNEL assay revealed that a substantial number of apoptotic cells were present within the macrophages and superficial lamina propria of the colonic epithelium. Expression microarray analysis revealed that the significantly downregulated genes were CYP3A4, CYP3A7, UGT2B11 and UGT2B15 in melanosis coli. Western blotting and immunofluorescence assays indicated that the expression of CYP3A4 in the normal tissue was higher than in the MC tissue. The results of the present study provided a comprehensive description of the histopathological characteristics and pathogenesis of MC and for the first time, to the best of our knowledge, demonstrated that the cytochrome P450-associated genes were significantly downregulated in melanosis coli. This novel information can be used to assist in further investigations of melanosis coli. PMID:26238215

  6. Design and evaluation of Actichip, a thematic microarray for the study of the actin cytoskeleton

    PubMed Central

    Muller, Jean; Mehlen, André; Vetter, Guillaume; Yatskou, Mikalai; Muller, Arnaud; Chalmel, Frédéric; Poch, Olivier; Friederich, Evelyne; Vallar, Laurent

    2007-01-01

    Background The actin cytoskeleton plays a crucial role in supporting and regulating numerous cellular processes. Mutations or alterations in the expression levels affecting the actin cytoskeleton system or related regulatory mechanisms are often associated with complex diseases such as cancer. Understanding how qualitative or quantitative changes in expression of the set of actin cytoskeleton genes are integrated to control actin dynamics and organisation is currently a challenge and should provide insights in identifying potential targets for drug discovery. Here we report the development of a dedicated microarray, the Actichip, containing 60-mer oligonucleotide probes for 327 genes selected for transcriptome analysis of the human actin cytoskeleton. Results Genomic data and sequence analysis features were retrieved from GenBank and stored in an integrative database called Actinome. From these data, probes were designed using a home-made program (CADO4MI) allowing sequence refinement and improved probe specificity by combining the complementary information recovered from the UniGene and RefSeq databases. Actichip performance was analysed by hybridisation with RNAs extracted from epithelial MCF-7 cells and human skeletal muscle. Using thoroughly standardised procedures, we obtained microarray images with excellent quality resulting in high data reproducibility. Actichip displayed a large dynamic range extending over three logs with a limit of sensitivity between one and ten copies of transcript per cell. The array allowed accurate detection of small changes in gene expression and reliable classification of samples based on the expression profiles of tissue-specific genes. When compared to two other oligonucleotide microarray platforms, Actichip showed similar sensitivity and concordant expression ratios. Moreover, Actichip was able to discriminate the highly similar actin isoforms whereas the two other platforms did not. Conclusion Our data demonstrate that Actichip is a powerful alternative to commercial high density microarrays for cytoskeleton gene profiling in normal or pathological samples. Actichip is available upon request. PMID:17727702

  7. ARACNe-based inference, using curated microarray data, of Arabidopsis thaliana root transcriptional regulatory networks

    PubMed Central

    2014-01-01

    Background Uncovering the complex transcriptional regulatory networks (TRNs) that underlie plant and animal development remains a challenge. However, a vast amount of data from public microarray experiments is available, which can be subject to inference algorithms in order to recover reliable TRN architectures. Results In this study we present a simple bioinformatics methodology that uses public, carefully curated microarray data and the mutual information algorithm ARACNe in order to obtain a database of transcriptional interactions. We used data from Arabidopsis thaliana root samples to show that the transcriptional regulatory networks derived from this database successfully recover previously identified root transcriptional modules and to propose new transcription factors for the SHORT ROOT/SCARECROW and PLETHORA pathways. We further show that these networks are a powerful tool to integrate and analyze high-throughput expression data, as exemplified by our analysis of a SHORT ROOT induction time-course microarray dataset, and are a reliable source for the prediction of novel root gene functions. In particular, we used our database to predict novel genes involved in root secondary cell-wall synthesis and identified the MADS-box TF XAL1/AGL12 as an unexpected participant in this process. Conclusions This study demonstrates that network inference using carefully curated microarray data yields reliable TRN architectures. In contrast to previous efforts to obtain root TRNs, that have focused on particular functional modules or tissues, our root transcriptional interactions provide an overview of the transcriptional pathways present in Arabidopsis thaliana roots and will likely yield a plethora of novel hypotheses to be tested experimentally. PMID:24739361

  8. Home-built integrated microarray system (IMAS). A three-laser confocal fluorescence scanner coupled with a microarray printer.

    PubMed

    Tragoulias, Sotirios S; Obeid, Pierre J; Tataridis, Ioannis E; Christopoulos, Theodore K

    2008-03-01

    Microarray technology covers the urgent need to exploit the accumulated genetic information from large-scale sequencing projects and facilitate investigations on a genome-wide scale. Although most applications focus on DNA microarrays, the technology has expanded to microarrays of proteins, peptides, carbohydrates, and small molecules aiming either at detection/quantification of biomolecules or investigation of biomolecular interactions in a massively parallel manner. Microarray experiments require two specialized instruments: An arrayer (or printer), for construction of microarrays, and a readout instrument (scanner). We have designed, constructed, and characterized the first integrated microarray system (IMAS) that combines the functions of a microarrayer and a three-laser confocal fluorescence scanner into a single instrument and provides excellent flexibility for the researcher. The three-axis robotic system that moves the printing head carrying multiple pins for arraying is also used for moving the microarray slide in front of a stationary optical system during scanning. Since the translation stages are the most expensive and crucial components of microarray printers and scanners, the proposed design reduces considerably the cost of the instrument and enhances remarkably its operative flexibility. Experiments were carried out at resolutions of 2.5, 5, 10, and 20 microm. The scanner detects 0.128 nmol L(-1) carboxyfluorescein (spots with diameters of 70 microm) corresponding to 1.8 molecules microm(-2). The linear range extends over 3.5 orders of magnitude (R(2) = 0.997) and the dynamic range covers almost five orders of magnitude. DNA microarray model experiments were carried out, including staining with SYBR Green I and hybridization with oligonucleotides labeled with the fluorescent dyes Alexa 488, Alexa 594, and Alexa 633. PMID:18228007

  9. Microarrays for identifying binding sites and probing structure of RNAs

    PubMed Central

    Kierzek, Ryszard; Turner, Douglas H.; Kierzek, Elzbieta

    2015-01-01

    Oligonucleotide microarrays are widely used in various biological studies. In this review, application of oligonucleotide microarrays for identifying binding sites and probing structure of RNAs is described. Deep sequencing allows fast determination of DNA and RNA sequence. High-throughput methods for determination of secondary structures of RNAs have also been developed. Those methods, however, do not reveal binding sites for oligonucleotides. In contrast, microarrays directly determine binding sites while also providing structural insights. Microarray mapping can be used over a wide range of experimental conditions, including temperature, pH, various cations at different concentrations and the presence of other molecules. Moreover, it is possible to make universal microarrays suitable for investigations of many different RNAs, and readout of results is rapid. Thus, microarrays are used to provide insight into oligonucleotide sequences potentially able to interfere with biological function. Better understanding of structure–function relationships of RNA can be facilitated by using microarrays to find RNA regions capable to bind oligonucleotides. That information is extremely important to design optimal sequences for antisense oligonucleotides and siRNA because both bind to single-stranded regions of target RNAs. PMID:25505162

  10. A perspective on microarrays: current applications, pitfalls, and potential uses

    PubMed Central

    Jaluria, Pratik; Konstantopoulos, Konstantinos; Betenbaugh, Michael; Shiloach, Joseph

    2007-01-01

    With advances in robotics, computational capabilities, and the fabrication of high quality glass slides coinciding with increased genomic information being available on public databases, microarray technology is increasingly being used in laboratories around the world. In fact, fields as varied as: toxicology, evolutionary biology, drug development and production, disease characterization, diagnostics development, cellular physiology and stress responses, and forensics have benefiting from its use. However, for many researchers not familiar with microarrays, current articles and reviews often address neither the fundamental principles behind the technology nor the proper designing of experiments. Although, microarray technology is relatively simple, conceptually, its practice does require careful planning and detailed understanding of the limitations inherently present. Without these considerations, it can be exceedingly difficult to ascertain valuable information from microarray data. Therefore, this text aims to outline key features in microarray technology, paying particular attention to current applications as outlined in recent publications, experimental design, statistical methods, and potential uses. Furthermore, this review is not meant to be comprehensive, but rather substantive; highlighting important concepts and detailing steps necessary to conduct and interpret microarray experiments. Collectively, the information included in this text will highlight the versatility of microarray technology and provide a glimpse of what the future may hold. PMID:17254338

  11. Microarray Gene Expression Analysis to Evaluate Cell Type Specific Expression of Targets Relevant for Immunotherapy of Hematological Malignancies

    PubMed Central

    Honders, M. W.; Kremer, A. N.; van Kooten, C.; Out, C.; Hiemstra, P. S.; de Boer, H. C.; Jager, M. J.; Schmelzer, E.; Vries, R. G.; Al Hinai, A. S.; Kroes, W. G.; Monajemi, R.; Goeman, J. J.; Böhringer, S.; Marijt, W. A. F.; Falkenburg, J. H. F.; Griffioen, M.

    2016-01-01

    Cellular immunotherapy has proven to be effective in the treatment of hematological cancers by donor lymphocyte infusion after allogeneic hematopoietic stem cell transplantation and more recently by targeted therapy with chimeric antigen or T-cell receptor-engineered T cells. However, dependent on the tissue distribution of the antigens that are targeted, anti-tumor responses can be accompanied by undesired side effects. Therefore, detailed tissue distribution analysis is essential to estimate potential efficacy and toxicity of candidate targets for immunotherapy of hematological malignancies. We performed microarray gene expression analysis of hematological malignancies of different origins, healthy hematopoietic cells and various non-hematopoietic cell types from organs that are often targeted in detrimental immune responses after allogeneic stem cell transplantation leading to graft-versus-host disease. Non-hematopoietic cells were also cultured in the presence of IFN-γ to analyze gene expression under inflammatory circumstances. Gene expression was investigated by Illumina HT12.0 microarrays and quality control analysis was performed to confirm the cell-type origin and exclude contamination of non-hematopoietic cell samples with peripheral blood cells. Microarray data were validated by quantitative RT-PCR showing strong correlations between both platforms. Detailed gene expression profiles were generated for various minor histocompatibility antigens and B-cell surface antigens to illustrate the value of the microarray dataset to estimate efficacy and toxicity of candidate targets for immunotherapy. In conclusion, our microarray database provides a relevant platform to analyze and select candidate antigens with hematopoietic (lineage)-restricted expression as potential targets for immunotherapy of hematological cancers. PMID:27171398

  12. Role of carbohydrate response element-binding protein (ChREBP) in generating an aerobic metabolic phenotype and in breast cancer progression

    PubMed Central

    Airley, R E; McHugh, P; Evans, A R; Harris, B; Winchester, L; Buffa, F M; Al-Tameemi, W; Leek, R; Harris, A L

    2014-01-01

    Background: The lipogenic transcription factor carbohydrate response element-binding protein (ChREBP) may play a key role in malignant progression of breast cancer by allowing metabolic adaptations to take place in response to changes in oxygenation. Methods: Immunohistochemical analysis of ChREBP was carried out in human breast tumour tissue microarrays representative of malignant progression from normal breast through to metastatic cancer. The ChREBP protein and mRNA expressions were then analysed in a series of breast cancers for correlative analysis with common and breast-specific hypoxia signatures, and survival. Results: In invasive ductal carcinoma, ChREBP correlated significantly with mean ‘downregulated' hypoxia scores (r=0.3, P<0.015, n=67) and in two distinct breast progression arrays, ChREBP protein also increased with malignant progression (P<0.001). However, bioinformatic analysis of a large data set (2136 cases) revealed an apparent reversal in the relationship between ChREBP mRNA level and clinical outcome – not only being significantly correlated with increased survival (log rank P<0.001), but also downregulated in malignant tissue compared with adjacent normal tissue. Conclusion: The ChREBP expression may be reflective of an aerobic metabolic phenotype that may conflict with hypoxia-induced signalling but provide a mechanism for growth at the oxygenated edge of the tumours. PMID:24366300

  13. Investigating the biochemical progression of liver disease through fibrosis, cirrhosis, dysplasia, and hepatocellular carcinoma using Fourier transform infrared spectroscopic imaging

    NASA Astrophysics Data System (ADS)

    Sreedhar, Hari; Pant, Mamta; Ronquillo, Nemencio R.; Davidson, Bennett; Nguyen, Peter; Chennuri, Rohini; Choi, Jacqueline; Herrera, Joaquin A.; Hinojosa, Ana C.; Jin, Ming; Kajdacsy-Balla, Andre; Guzman, Grace; Walsh, Michael J.

    2014-03-01

    Hepatocellular carcinoma (HCC) is the most common form of primary hepatic carcinoma. HCC ranks the fourth most prevalent malignant tumor and the third leading cause of cancer related death in the world. Hepatocellular carcinoma develops in the context of chronic liver disease and its evolution is characterize