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Sample records for progression tissue microarray

  1. 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

  2. PATMA: parser of archival tissue microarray.

    PubMed

    Roszkowiak, Lukasz; Lopez, Carlos

    2016-01-01

    Tissue microarrays are commonly used in modern pathology for cancer tissue evaluation, as it is a very potent technique. Tissue microarray slides are often scanned to perform computer-aided histopathological analysis of the tissue cores. For processing the image, splitting the whole virtual slide into images of individual cores is required. The only way to distinguish cores corresponding to specimens in the tissue microarray is through their arrangement. Unfortunately, distinguishing the correct order of cores is not a trivial task as they are not labelled directly on the slide. The main aim of this study was to create a procedure capable of automatically finding and extracting cores from archival images of the tissue microarrays. This software supports the work of scientists who want to perform further image processing on single cores. The proposed method is an efficient and fast procedure, working in fully automatic or semi-automatic mode. A total of 89% of punches were correctly extracted with automatic selection. With an addition of manual correction, it is possible to fully prepare the whole slide image for extraction in 2 min per tissue microarray. The proposed technique requires minimum skill and time to parse big array of cores from tissue microarray whole slide image into individual core images.

  3. PATMA: parser of archival tissue microarray

    PubMed Central

    2016-01-01

    Tissue microarrays are commonly used in modern pathology for cancer tissue evaluation, as it is a very potent technique. Tissue microarray slides are often scanned to perform computer-aided histopathological analysis of the tissue cores. For processing the image, splitting the whole virtual slide into images of individual cores is required. The only way to distinguish cores corresponding to specimens in the tissue microarray is through their arrangement. Unfortunately, distinguishing the correct order of cores is not a trivial task as they are not labelled directly on the slide. The main aim of this study was to create a procedure capable of automatically finding and extracting cores from archival images of the tissue microarrays. This software supports the work of scientists who want to perform further image processing on single cores. The proposed method is an efficient and fast procedure, working in fully automatic or semi-automatic mode. A total of 89% of punches were correctly extracted with automatic selection. With an addition of manual correction, it is possible to fully prepare the whole slide image for extraction in 2 min per tissue microarray. The proposed technique requires minimum skill and time to parse big array of cores from tissue microarray whole slide image into individual core images. PMID:27920955

  4. Tissue microarrays for early target evaluation.

    PubMed

    Simon, Ronald; Mirlacher, Martina; Sauter, Guido

    2004-09-01

    Early assessment of the probable biological importance of drug targets, the potential market size of a successful new drug, and possible treatment side effects are critical for risk management in drug development. A comprehensive molecular epidemiology analysis involving thousands of well-characterized human tissues will thus provide vital information for strategic decision-making. Tissue microarray (TMA) technology is ideally suited for such projects. The simultaneous analysis of thousands of tissues enables highly standardized, fast and affordable translational research studies of unprecedented scale.:

  5. Tissue Microarray Analysis Applied to Bone Diagenesis

    PubMed Central

    Mello, Rafael Barrios; Silva, Maria Regina Regis; Alves, Maria Teresa Seixas; Evison, Martin Paul; Guimarães, Marco Aurelio; Francisco, Rafaella Arrabaca; Astolphi, Rafael Dias; Iwamura, Edna Sadayo Miazato

    2017-01-01

    Taphonomic processes affecting bone post mortem are important in forensic, archaeological and palaeontological investigations. In this study, the application of tissue microarray (TMA) analysis to a sample of femoral bone specimens from 20 exhumed individuals of known period of burial and age at death is described. TMA allows multiplexing of subsamples, permitting standardized comparative analysis of adjacent sections in 3-D and of representative cross-sections of a large number of specimens. Standard hematoxylin and eosin, periodic acid-Schiff and silver methenamine, and picrosirius red staining, and CD31 and CD34 immunohistochemistry were applied to TMA sections. Osteocyte and osteocyte lacuna counts, percent bone matrix loss, and fungal spheroid element counts could be measured and collagen fibre bundles observed in all specimens. Decalcification with 7% nitric acid proceeded more rapidly than with 0.5 M EDTA and may offer better preservation of histological and cellular structure. No endothelial cells could be detected using CD31 and CD34 immunohistochemistry. Correlation between osteocytes per lacuna and age at death may reflect reported age-related responses to microdamage. Methodological limitations and caveats, and results of the TMA analysis of post mortem diagenesis in bone are discussed, and implications for DNA survival and recovery considered. PMID:28051148

  6. 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

  7. The tissue microarray OWL schema: An open-source tool for sharing tissue microarray data

    PubMed Central

    Kang, Hyunseok P.; Borromeo, Charles D.; Berman, Jules J.; Becich, Michael J.

    2010-01-01

    Background: Tissue microarrays (TMAs) are enormously useful tools for translational research, but incompatibilities in database systems between various researchers and institutions prevent the efficient sharing of data that could help realize their full potential. Resource Description Framework (RDF) provides a flexible method to represent knowledge in triples, which take the form Subject-Predicate-Object. All data resources are described using Uniform Resource Identifiers (URIs), which are global in scope. We present an OWL (Web Ontology Language) schema that expands upon the TMA data exchange specification to address this issue and assist in data sharing and integration. Methods: A minimal OWL schema was designed containing only concepts specific to TMA experiments. More general data elements were incorporated from predefined ontologies such as the NCI thesaurus. URIs were assigned using the Linked Data format. Results: We present examples of files utilizing the schema and conversion of XML data (similar to the TMA DES) to OWL. Conclusion: By utilizing predefined ontologies and global unique identifiers, this OWL schema provides a solution to the limitations of XML, which represents concepts defined in a localized setting. This will help increase the utilization of tissue resources, facilitating collaborative translational research efforts. PMID:20805954

  8. 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…

  9. Image quantification of high-throughput tissue microarray

    NASA Astrophysics Data System (ADS)

    Wu, Jiahua; Dong, Junyu; Zhou, Huiyu

    2006-03-01

    Tissue microarray (TMA) technology allows rapid visualization of molecular targets in thousands of tissue specimens at a time and provides valuable information on expression of proteins within tissues at a cellular and sub-cellular level. TMA technology overcomes the bottleneck of traditional tissue analysis and allows it to catch up with the rapid advances in lead discovery. Studies using TMA on immunohistochemistry (IHC) can produce a large amount of images for interpretation within a very short time. Manual interpretation does not allow accurate quantitative analysis of staining to be undertaken. Automatic image capture and analysis has been shown to be superior to manual interpretation. The aims of this work is to develop a truly high-throughput and fully automated image capture and analysis system. We develop a robust colour segmentation algorithm using hue-saturation-intensity (HSI) colour space to provide quantification of signal intensity and partitioning of staining on high-throughput TMA. Initial segmentation results and quantification data have been achieved on 16,000 TMA colour images over 23 different tissue types.

  10. Classification and immunohistochemical scoring of breast tissue microarray spots.

    PubMed

    Amaral, Telmo; McKenna, Stephen J; Robertson, Katherine; Thompson, Alastair

    2013-10-01

    Tissue microarrays (TMAs) facilitate the survey of very large numbers of tumors. However, the manual assessment of stained TMA sections constitutes a bottleneck in the pathologist's work flow. This paper presents a computational pipeline for automatically classifying and scoring breast cancer TMA spots that have been subjected to nuclear immunostaining. Spots are classified based on a bag of visual words approach. Immunohistochemical scoring is performed by computing spot features reflecting the proportion of epithelial nuclei that are stained and the strength of that staining. These are then mapped onto an ordinal scale used by pathologists. Multilayer perceptron classifiers are compared with latent topic models and support vector machines for spot classification, and with Gaussian process ordinal regression and linear models for scoring. Intraobserver variation is also reported. The use of posterior entropy to identify uncertain cases is demonstrated. Evaluation is performed using TMA images stained for progesterone receptor.

  11. Glucose transporter Glut-1 is detectable in peri-necrotic regions in many human tumor types but not normal tissues: Study using tissue microarrays.

    PubMed

    Airley, Rachel; Evans, Andrew; Mobasheri, Ali; Hewitt, Stephen M

    2010-05-20

    The hypoxic tumor microenvironment is associated with malignant progression and poor treatment response. The glucose transporter Glut-1 is a prognostic factor and putative hypoxia marker. So far, studies of Glut-1 in cancer have utilized conventional immunohistochemical analysis in a series of individual biopsy or surgical specimens. Tissue microarrays, however, provide a rapid, inexpensive means of profiling biomarker expression. To evaluate hypoxia markers, tissue cores must show the architectural features of hypoxia; i.e. viable tissue surrounding necrotic regions. Glut-1 may be a useful biomarker to validate tissue microarrays for use in studies of hypoxia-regulated genes in cancer. In this study, we carried out immunohistochemical detection of Glut-1 protein in many tumor and normal tissue types in a range of tissue microarrays. Glut-1 was frequently found in peri-necrotic regions, occurring in 9/34 lymphomas, 6/12 melanomas, and 5/16 glioblastomas; and in 43/54 lung, 22/84 colon, and 23/60 ovarian tumors. Expression was rare in breast (6/40) and prostate (1/57) tumors, and in normal tissue, was restricted to spleen, tongue, and CNS endothelium. In conclusion, tissue microarrays enable the observation of Glut-1 expression in peri-necrotic regions, which may be linked to hypoxia, and reflect previous studies showing differential Glut-1 expression across tumor types and non-malignant tissue.

  12. TMAinspiration: Decode Interdependencies in Multifactorial Tissue Microarray Data

    PubMed Central

    Boecker, Florian; Buerger, Horst; Mallela, Nikhil V.; Korsching, Eberhard

    2016-01-01

    There are no satisfying tools in tissue microarray (TMA) data analysis up to now to analyze the cooperative behavior of all measured markers in a multifactorial TMA approach. The developed tool TMAinspiration is not only offering an analysis option to close this gap but also offering an ecosystem consisting of quality control concepts and supporting scripts to make this approach a platform for informed practice and further research. The TMAinspiration method is specifically focusing on the demands of the TMA analysis by controlling errors and noise by a generalized regression scheme while at the same time avoiding to introduce a priori too many constraints into the analysis of the data. So, we are testing partitions of a proximity table to find an optimal support for a ranking scheme of molecular dependencies. The idea of combining several partitions to one ensemble, which is balancing the optimization process, is based on the main assumption that all these perspectives on the cellular network need to be self-consistent. Several application examples in breast cancer and one in squamous cell carcinoma demonstrate that this procedure is nicely confirming a priori knowledge on the expression characteristics of protein markers, while also integrating many new results discovered in the treasury of a bigger TMA experiment. The code and software are now freely available at: http://complex-systems.uni-muenster.de/tma_inspiration.html. PMID:27398021

  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.

  14. Large-scale meta-analysis of cancer microarray data identifies common transcriptional profiles of neoplastic transformation and progression

    PubMed Central

    Rhodes, Daniel R.; Yu, Jianjun; Shanker, K.; Deshpande, Nandan; Varambally, Radhika; Ghosh, Debashis; Barrette, Terrence; Pandey, Akhilesh; Chinnaiyan, Arul M.

    2004-01-01

    Many studies have used DNA microarrays to identify the gene expression signatures of human cancer, yet the critical features of these often unmanageably large signatures remain elusive. To address this, we developed a statistical method, comparative metaprofiling, which identifies and assesses the intersection of multiple gene expression signatures from a diverse collection of microarray data sets. We collected and analyzed 40 published cancer microarray data sets, comprising 38 million gene expression measurements from >3,700 cancer samples. From this, we characterized a common transcriptional profile that is universally activated in most cancer types relative to the normal tissues from which they arose, likely reflecting essential transcriptional features of neoplastic transformation. In addition, we characterized a transcriptional profile that is commonly activated in various types of undifferentiated cancer, suggesting common molecular mechanisms by which cancer cells progress and avoid differentiation. Finally, we validated these transcriptional profiles on independent data sets. PMID:15184677

  15. Expression of PPARγ and PTEN in human colorectal cancer: An immunohistochemical study using tissue microarray methodology.

    PubMed

    Lin, Mao Song; Huang, Jun Xing; Chen, Wei Chang; Zhang, Bao Feng; Fang, Jing; Zhou, Qiong; Hu, Ying; Gao, Heng Jun

    2011-11-01

    Although aberrations of peroxisome proliferator-activated receptor γ (PPARγ) and phosphatase and tensin homolog (PTEN) expression have been identified in several other cancer types, certain previous studies have revealed that PPARγ is abundant in normal and malignant tissue in the colon. The question of whether aberrant PTEN is involved in the initial stage or is a later event during colorectal carcinogenesis remains controversial. Relatively few studies have focused on the correlation of expression of PPARγ and PTEN in various tissues. In the present study, paraffin-embedded blocks from 139 patients with CRC, 18 adenomatous polyps and 50 paired paracancerous benign mucosas were selected and analysed in 4 tissue microarray (TMA) blocks comprising 104, 72, 130 and 54 cores, respectively. Expression of PPARγ and PTEN was examined using immunohistochemical staining on TMAs. There were no significant differences in the expression of PPARγ (P=0.055) and PTEN (P=0.100) between the colorectal cancers, adenomas and paracancerous mucosas. However, correlations of PPARγ expression with clinical stage (P=0.004) and PTEN expression with histological grade (P=0.006) and distant metastasis (P=0.015) were demonstrated in the CRC specimens. Although the differences in PPARγ and PTEN protein expression in human colorectal cancer may not be considered as early diagnostic markers, our results indicate that CRCs with a low expression or deletion of PTEN may progress towards invasion and even metastasis; thus, PTEN may have potential as a prognostic marker in human CRC.

  16. 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

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

    PubMed

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

    2015-11-10

    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.

  18. Gene expression profiling in gill tissues of White spot syndrome virus infected black tiger shrimp Penaeus monodon by DNA microarray.

    PubMed

    Shekhar, M S; Gomathi, A; Gopikrishna, G; Ponniah, A G

    2015-06-01

    White spot syndrome virus (WSSV) continues to be the most devastating viral pathogen infecting penaeid shrimp the world over. The genome of WSSV has been deciphered and characterized from three geographical isolates and significant progress has been made in developing various molecular diagnostic methods to detect the virus. However, the information on host immune gene response to WSSV pathogenesis is limited. Microarray analysis was carried out as an approach to analyse the gene expression in black tiger shrimp Penaeus monodon in response to WSSV infection. Gill tissues collected from the WSSV infected shrimp at 6, 24, 48 h and moribund stage were analysed for differential gene expression. Shrimp cDNAs of 40,059 unique sequences were considered for designing the microarray chip. The Cy3-labeled cRNA derived from healthy and WSSV-infected shrimp was subjected to hybridization with all the DNA spots in the microarray which revealed 8,633 and 11,147 as up- and down-regulated genes respectively at different time intervals post infection. The altered expression of these numerous genes represented diverse functions such as immune response, osmoregulation, apoptosis, nucleic acid binding, energy and metabolism, signal transduction, stress response and molting. The changes in gene expression profiles observed by microarray analysis provides molecular insights and framework of genes which are up- and down-regulated at different time intervals during WSSV infection in shrimp. The microarray data was validated by Real Time analysis of four differentially expressed genes involved in apoptosis (translationally controlled tumor protein, inhibitor of apoptosis protein, ubiquitin conjugated enzyme E2 and caspase) for gene expression levels. The role of apoptosis related genes in WSSV infected shrimp is discussed herein.

  19. COMPARATIVE ANALYSIS OF MICROARRAY DATA IDENTIFIES COMMON RESPONSES TO CALORIC RESTRICTION AMONG MOUSE TISSUES

    PubMed Central

    Swindell, William R.

    2009-01-01

    Caloric restriction has been extensively investigated as an intervention that both extends lifespan and delays age-related disease in mammals. In mice, much interest has centered on evaluating gene expression changes induced by caloric restriction (CR) in particular tissue types, but the overall systemic effect of CR among multiple tissues has been examined less extensively. This study presents a comparative analysis of microarray datasets that have collectively examined the effects of CR in ten different tissue types (liver, heart, muscle, hypothalamus, hippocampus, white adipose tissue, colon, kidney, lung, cochlea). Using novel methods for comparative analysis of microarray data, detailed comparisons of the effects of CR among tissues are provided, and 28 genes for which expression response to CR is most shared among tissues are identified. These genes characterize common responses to CR, which consist of both activation and inhibition of stress-response pathways. With respect to liver tissue, transcriptional effects of CR exhibited surprisingly little overlap with those of aging, and a variable degree of overlap with the potential CR-mimetic drug resveratrol. These analyses shed light on the systemic transcriptional activity associated with CR diets, and also illustrate new approaches for comparative analysis of microarray datasets in the context of aging biology. PMID:18155270

  20. Cell-cycle-associated markers and clinical outcome in human epithelial cancers: a tissue microarray study.

    PubMed

    Abdulkader, I; Sánchez, L; Cameselle-Teijeiro, J; Gude, F; Chávez, J E; López-López, R; Forteza, J; Fraga, M

    2005-12-01

    The development and progression of epithelial cancers are the result of an imbalance in signals promoting and inhibiting cellular proliferation and apoptosis. The aim of this study is to evaluate the expression of cell-cycle and apoptosis regulators and correlate them with clinical outcome in the most frequent carcinomas, in order to establish common prognostic biomarkers independent of cancer origin. Using tissue microarrays (TMAs), we have analysed the immuno-expression of Ki-67, Bcl-2, Bax, cyclin D1, cyclin D3, CDK1, CDK2, CDK6, p16, p21, and p27 in a series of 205 carcinomas of the large bowel, breast, lung and prostate (80, 73, 37 and 15 cases, respectively). By univariate analysis, positivity for p27, p16 and Bcl-2 was associated with better overall survival (P<0.0135, P<0.0442 and P<0.0001, respectively). The risk of mortality was 2.3-fold greater in patients without Bcl-2 expression. TMA immunohistochemical analysis identified a subset of epithelial cancers with overlapping alterations in cell-cycle checkpoints, apoptosis regulators and tumour suppressor pathways. We found that in most common epithelial cancers, regardless of origin, Bcl-2 appears to be the key biological factor influencing clinical behaviour.

  1. Tissue microarray (TMA) technology: miniaturized pathology archives for high-throughput in situ studies.

    PubMed

    Bubendorf, L; Nocito, A; Moch, H; Sauter, G

    2001-09-01

    Tissue microarray (TMA) technology allows a massive acceleration of studies correlating molecular in situ findings with clinico-pathological information. In this technique, cylindrical tissue samples are taken from up to 1000 different archival tissue blocks and subsequently placed into one empty 'recipient' paraffin block. Sections from TMA blocks can be used for all different types of in situ tissue analyses including immunohistochemistry and in situ hybridization. Multiple studies have demonstrated that findings obtained on TMAs are highly representative of their donor tissues, despite the small size of the individual specimens (diameter 0.6 mm). It is anticipated that TMAs will soon become a widely used tool for all types of tissue-based research. The availability of TMAs containing highly characterized tissues will enable every researcher to perform studies involving thousands of tumours rapidly. Therefore, TMAs will lead to a significant acceleration of the transition of basic research findings into clinical applications.

  2. 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

  3. 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.

  4. 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

  5. Deep learning for tissue microarray image-based outcome prediction in patients with colorectal cancer

    NASA Astrophysics Data System (ADS)

    Bychkov, Dmitrii; Turkki, Riku; Haglund, Caj; Linder, Nina; Lundin, Johan

    2016-03-01

    Recent advances in computer vision enable increasingly accurate automated pattern classification. In the current study we evaluate whether a convolutional neural network (CNN) can be trained to predict disease outcome in patients with colorectal cancer based on images of tumor tissue microarray samples. We compare the prognostic accuracy of CNN features extracted from the whole, unsegmented tissue microarray spot image, with that of CNN features extracted from the epithelial and non-epithelial compartments, respectively. The prognostic accuracy of visually assessed histologic grade is used as a reference. The image data set consists of digitized hematoxylin-eosin (H and E) stained tissue microarray samples obtained from 180 patients with colorectal cancer. The patient samples represent a variety of histological grades, have data available on a series of clinicopathological variables including long-term outcome and ground truth annotations performed by experts. The CNN features extracted from images of the epithelial tissue compartment significantly predicted outcome (hazard ratio (HR) 2.08; CI95% 1.04-4.16; area under the curve (AUC) 0.66) in a test set of 60 patients, as compared to the CNN features extracted from unsegmented images (HR 1.67; CI95% 0.84-3.31, AUC 0.57) and visually assessed histologic grade (HR 1.96; CI95% 0.99-3.88, AUC 0.61). As a conclusion, a deep-learning classifier can be trained to predict outcome of colorectal cancer based on images of H and E stained tissue microarray samples and the CNN features extracted from the epithelial compartment only resulted in a prognostic discrimination comparable to that of visually determined histologic grade.

  6. 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

    2015-09-09

    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.

  7. Comparative study of p63 and p53 expression in tissue microarrays of malignant melanomas.

    PubMed

    Brinck, Ulrich; Ruschenburg, Ilka; Di Como, Charles J; Buschmann, Nadine; Betke, Herbert; Stachura, Jerzy; Cordon-Cardo, Carlos; Korabiowska, Monika

    2002-12-01

    p63 is a known homologue of p53. In contrast to p53, however, p63 mutations are rarely seen in tumours. There have been several reports that p63 plays a regulatory role in the normal differentiation of cells, whereas its role in tumour biology must still be elucidated. The main aim of this study was to compare p63 and p53 expression in tissue microarrays of malignant melanomas and to establish any prognostic significance. p63 expression was found in 2 out of 59 tumours, both pT4. The p63 index did not exceed 30%. p53 expression was found in 27 out of 59 melanomas, with maximal expression in up to 80% of tumour cells. There were no correlations observed between the two markers. Multivariate analysis confirmed the prognostically independent role of p53. This study also confirmed that tissue microarrays can be used effectively for evaluation of the expression of certain tumour markers.

  8. Identification of Annexin A1 protein expression in human gastric adenocarcinoma using proteomics and tissue microarray

    PubMed Central

    Zhang, Zhi-Qiang; Li, Xiu-Juan; Liu, Gui-Tao; Xia, Yu; Zhang, Xiang-Yang; Wen, Hao

    2013-01-01

    AIM: To study the differential expression of Annexin A1 (ANXA1) protein in human gastric adenocarcinoma. This study was also designed to analyze the relationship between ANXA1 expression and the clinicopathological parameters of gastric carcinoma. METHODS: Purified gastric adenocarcinoma cells (GAC) and normal gastric epithelial cells (NGEC) were obtained from 15 patients with gastric cancer by laser capture microdissection. All of the peptide specimens were labeled as 18O/16O after trypsin digestion. Differential protein expressions were quantitatively identified between GAC and NGEC by nanoliter-reverse-phase liquid chromatography-mass/mass spectrometry (nano-RPLC-MS/MS). The expressions of ANXA1 in GAC and NGEC were verified by western blot analysis. The tissue microarray containing the expressed ANXA1 in 75 pairs of gastric carcinoma and paracarcinoma specimens was detected by immunohistochemistry (IHC). The relationship between ANXA1 expression and clinicopathological parametes of gastric carcinoma was analyzed. RESULTS: A total of 78 differential proteins were identified. Western blotting revealed that ANXA1 expression was significantly upregulated in GAC (2.17/1, P < 0.01). IHC results showed the correlations between ANXA1 protein expression and the clinicopathological parameters, including invasive depth (T stage), lymph node metastasis (N stage), distant metastasis (M stage) and tumour-lymph node metastasis stage (P < 0.01). However, the correlations between ANXA1 protein expression and the remaining clinicopathological parameters, including sex, age, histological differentiation and the size of tumour were not found (P > 0.05). CONCLUSION: The upregulated ANXA1 expression may be associated with carcinogenesis, progression, invasion and metastasis of GAC. This protein could be considered as a biomarker of clinical prognostic prediction and targeted therapy of GAC. PMID:24282368

  9. Identification of novel pancreatic adenocarcinoma cell-surface targets by gene expression profiling and tissue microarray

    PubMed Central

    Morse, David L.; Balagurunathan, Yoga; Hostetter, Galen; Trissal, Maria; Tafreshi, Narges K.; Burke, Nancy; Lloyd, Mark; Enkemann, Steven; Coppola, Domenico; Hruby, Victor; Gillies, Robert J.; Han, Haiyong

    2010-01-01

    Pancreatic cancer has a high mortality rate, which is generally related to the initial diagnosis coming at late stage disease combined with a lack of effective treatment options. Novel agents that selectively detect pancreatic cancer have potential for use in the molecular imaging of cancer, allowing for non-invasive determination of tumor therapeutic response and molecular characterization of the disease. Such agents may also be used for the targeted delivery of therapy to tumor cells while decreasing systemic effects. Using complementary assays of mRNA expression profiling to determine elevated expression in pancreatic cancer tissues relative to normal pancreas tissues, and validation of protein expression by immunohistochemistry on tissue microarray, we have identified cell-surface targets with potential for imaging and therapeutic agent development. Expression profiles of 2177 cell-surface genes for 28 pancreatic tumor specimens and 4 normal pancreas tissue samples were evaluated. Expression in normal tissues was evaluated using array data from 103 samples representing 28 organ sites as well as mining published data. One-hundred seventy unique targets were highly expressed in 2 or more of the pancreatic tumor specimens and were not expressed in the normal pancreas samples. Two targets (TLR2 and ABCC3) were further validated for protein expression by tissue microarray (TMA) based immunohistochemistry. These validated targets have potential for the development of diagnostic imaging and therapeutic agents for pancreatic cancer. PMID:20510208

  10. Loss of HITS (FAM107B) expression in cancers of multiple organs: tissue microarray analysis.

    PubMed

    Nakajima, Hideo; Koizumi, Keita; Tanaka, Takuji; Ishigaki, Yasuhito; Yoshitake, Yoshino; Yonekura, Hideto; Sakuma, Tsutomu; Fukushima, Toshihiro; Umehara, Hisanori; Ueno, Soichiro; Minamoto, Toshinari; Motoo, Yoshiharu

    2012-10-01

    Family with sequence similarity 107 (FAM107) proteins consist of two subtypes, FAM107A and FAM107B in mammals, possessing a conserved N-terminal domain of unknown function. Recently we found that FAM107B, an 18 kDa nuclear protein, is expressed in a broad range of tissues and is downregulated in gastrointestinal cancer. Because FAM107B expression is amplified by heat-shock stimulation, we designated it heat shock-inducible tumor small protein (HITS). Although data related to FAM107A as a candidate tumor suppressor have been accumulated, little biological information is available for HITS. In the present study, we examined HITS expression using immunohistochemistry with tissue microarrays and performed detailed statistical analyses. By screening a high-density multiple organ tumor and normal tissue microarray, HITS expression was decreased in tumor tissues of the breast, thyroid, testis and uterine cervix as well as the stomach and colon. Further analysis of tissue microarrays of individual organs showed that loss of HITS expression in cancer tissues was statistically significant and commonly observed in distinct organs in a histological type-specific manner. The HITS expression intensity was inversely correlated with the primary tumor size in breast and thyroid cancers. In addition, effects of tetracycline-inducible HITS expression on tumor growth were investigated in vivo. Forced expression of HITS inhibited tumor xenograft proliferation, compared with the mock-treated tumor xenograft model. These results show that loss of HITS expression is a common phenomenon observed in cancers of distinct organs and involved in tumor development and proliferation.

  11. Datamining Approach for Automation of Diagnosis of Breast Cancer in Immunohistochemically Stained Tissue Microarray Images

    PubMed Central

    Prasad, Keerthana; Zimmermann, Bernhard; Prabhu, Gopalakrishna; Pai, Muktha

    2010-01-01

    Cancer of the breast is the second most common human neoplasm, accounting for approximately one quarter of all cancers in females after cervical carcinoma. Estrogen receptor (ER), Progesteron receptor and human epidermal growth factor receptor (HER-2/neu) expressions play an important role in diagnosis and prognosis of breast carcinoma. Tissue microarray (TMA) technique is a high throughput technique which provides a standardized set of images which are uniformly stained, facilitating effective automation of the evaluation of the specimen images. TMA technique is widely used to evaluate hormone expression for diagnosis of breast cancer. If one considers the time taken for each of the steps in the tissue microarray process workflow, it can be observed that the maximum amount of time is taken by the analysis step. Hence, automated analysis will significantly reduce the overall time required to complete the study. Many tools are available for automated digital acquisition of images of the spots from the microarray slide. Each of these images needs to be evaluated by a pathologist to assign a score based on the staining intensity to represent the hormone expression, to classify them into negative or positive cases. Our work aims to develop a system for automated evaluation of sets of images generated through tissue microarray technique, representing the ER expression images and HER-2/neu expression images. Our study is based on the Tissue Microarray Database portal of Stanford university at http://tma.stanford.edu/cgi-bin/cx?n=her1, which has made huge number of images available to researchers. We used 171 images corresponding to ER expression and 214 images corresponding to HER-2/neu expression of breast carcinoma. Out of the 171 images corresponding to ER expression, 104 were negative and 67 were representing positive cases. Out of the 214 images corresponding to HER-2/neu expression, 112 were negative and 102 were representing positive cases. Our method has 92

  12. Datamining approach for automation of diagnosis of breast cancer in immunohistochemically stained tissue microarray images.

    PubMed

    Prasad, Keerthana; Zimmermann, Bernhard; Prabhu, Gopalakrishna; Pai, Muktha

    2010-05-28

    Cancer of the breast is the second most common human neoplasm, accounting for approximately one quarter of all cancers in females after cervical carcinoma. Estrogen receptor (ER), Progesteron receptor and human epidermal growth factor receptor (HER-2/neu) expressions play an important role in diagnosis and prognosis of breast carcinoma. Tissue microarray (TMA) technique is a high throughput technique which provides a standardized set of images which are uniformly stained, facilitating effective automation of the evaluation of the specimen images. TMA technique is widely used to evaluate hormone expression for diagnosis of breast cancer. If one considers the time taken for each of the steps in the tissue microarray process workflow, it can be observed that the maximum amount of time is taken by the analysis step. Hence, automated analysis will significantly reduce the overall time required to complete the study. Many tools are available for automated digital acquisition of images of the spots from the microarray slide. Each of these images needs to be evaluated by a pathologist to assign a score based on the staining intensity to represent the hormone expression, to classify them into negative or positive cases. Our work aims to develop a system for automated evaluation of sets of images generated through tissue microarray technique, representing the ER expression images and HER-2/neu expression images. Our study is based on the Tissue Microarray Database portal of Stanford university at http://tma.stanford.edu/cgi-bin/cx?n=her1, which has made huge number of images available to researchers. We used 171 images corresponding to ER expression and 214 images corresponding to HER-2/neu expression of breast carcinoma. Out of the 171 images corresponding to ER expression, 104 were negative and 67 were representing positive cases. Out of the 214 images corresponding to HER-2/neu expression, 112 were negative and 102 were representing positive cases. Our method has 92

  13. Rapid microarray-based genotyping of Chlamydia spp. strains from clinical tissue samples.

    PubMed

    Sachse, Konrad; Ruettger, Anke

    2015-01-01

    Pathogenic Chlamydia (C.) psittaci and C. trachomatis strains can be genotyped based on variations in the ompA genomic locus. In the present chapter, we describe rapid genotyping assays for both chlamydial agents using the ArrayStrip™ (AS) microarray platform. The test is targeting multiple discriminatory sites in the variable domains of the ompA gene by using 35 (C. psittaci) and 61 (C. trachomatis) oligonucleotide probes representing genotype-specific polymorphisms. In addition to discrimination among the established genotypes, this approach allows identification of atypical strains that were not accessible to typing using previously established techniques, such as PCR-RFLP or serotyping. The present DNA microarray assay can be conducted directly on clinical tissue samples and is suitable for tracing epidemiological chains and exploring the dissemination of particular genotypes. The procedure is easy to handle and economically affordable, and it allows genotyping of up to 32 clinical samples per day, thus lending itself for routine diagnosis as well.

  14. Applications of tissue microarray technology in immunohistochemistry: a study on c-kit expression in small cell lung cancer.

    PubMed

    Donati, Valentina; Faviana, Pinuccia; Dell'omodarme, Matteo; Prati, Maria Cristina; Camacci, Tiziano; De Ieso, Katia; Giannini, Riccardo; Lucchi, Marco; Mussi, Alfredo; Pingitore, Raffaele; Basolo, Fulvio; Fontanini, Gabriella

    2004-11-01

    Tissue microarray technology allows the immediate evaluation of molecular profiles of numerous different tissues, with savings of money and time. It was created for rapid, large-scale molecular studies, and the main concern regarding its possible broad acceptance is that the analysis of tissue microarrays instead of whole tissue sections may lead to false negative or positive results because of tissue heterogeneity. In the present study, we analyzed in 54 small cell lung cancers, by immunohistochemistry, the expression of the antigen c-kit, which seems to be important in these neoplasms' tumorigenesis, and compared the staining obtained on whole sections with that of the corresponding tissue microarrays. Although c-kit expression of the whole sections agreed with that of the corresponding biopsies in many cases, the correlation between whole sections and all the companion nonlost single cores or their mean value turned out to be highly significant only if the 36 double negatives (ie, both whole sections and companion tissue microarrays negative) were included (P <0.0001). In fact, if only cases positive to at least 1 of the tests (i.e. whole sections or corresponding tissue microarrays positive) were considered, the correlation was not significant (P=0.055). Tissue microarrays showed a good specificity (94.2% for all single cores and 92.3% for their mean value) but a rather poor sensitivity (respectively, 69.4% and 71.4%). Moreover, a high percentage (13.4%) of cores was lost, and this loss was not random. To sum up, in our experience, tissue microarray technology cannot be a substitute for whole sections in clinical diagnosis of individual cases.

  15. Decreased Expression of Inhibitor of Caspase-Activated DNase (ICAD) in Renal Cell Carcinoma – Tissue Microarray of Human Samples

    PubMed Central

    Rajandram, Retnagowri; Razack, Azad H. A.; Ng, Keng Lim

    2016-01-01

    Although primary localised tumours of renal cell carcinoma (RCC) can be treated relatively successfully with surgery, metastatic RCC has poor prognosis because of late diagnosis and resistance to therapies. In the present study, we were interested in profiling the protein expression of “inhibitor of caspase-activated DNase” (ICAD), an apoptosis inhibitor, in kidney cancer and its paired normal kidney. Immunohistochemistry with automated batch staining and morphometry using digital pathology were used to compare ICAD in 121 RCC specimens with their paired normal kidney tissue. Tissue microarray of formalin-fixed, paraffin-embedded archival tissue was used. Intensity and localisation of ICAD were compared between normal and cancer samples, and against grading within the cancers. The results demonstrated that, in this cohort, ICAD was highly expressed in the proximal tubular epithelium of normal kidney, and significantly decreased in clear cell RCC tissue (p < 0.05) as well as other subtypes of RCC (p < 0.01) compared with normal kidney. There was a tendency towards nuclear localisation of ICAD in clear cell RCC, but not in other subtypes of RCC. No significant association was found between ICAD intensity and grade of RCC. In summary, down-regulation of ICAD occurs in RCC. ICAD normally inhibits DNA fragmentation and apoptosis; thus, its down-regulation was unexpected in a cancer known for its resistance to apoptosis. However, these RCC samples were from primary, not metastatic, RCC sites, and down-regulated ICAD may be part of a progressive pathway that promotes RCC metastasis.

  16. 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.

  17. Validation of immature adipogenic status and identification of prognostic biomarkers in myxoid liposarcoma using tissue microarrays.

    PubMed

    Cheng, Hongwei; Dodge, Jim; Mehl, Erika; Liu, Shuzhen; Poulin, Neal; van de Rijn, Matt; Nielsen, Torsten O

    2009-09-01

    Myxoid liposarcoma displays variably aggressive behavior and responds poorly to available systemic therapies. Expression profiling followed by tissue microarray validation linked to patient outcome is a powerful approach for validating biological mechanisms and identifying prognostic biomarkers. We applied these techniques to independent series of primary myxoid liposarcomas in an effort to assess markers of adipose differentiation in myxoid liposarcoma and to identify prognostic markers that can be efficiently assessed by immunohistochemistry. Candidate genes were selected based on analysis of expression profiles from 9 primary myxoid/round liposarcomas and 45 other soft tissue tumors, and by reference to publicly available data sets. Protein products were validated on an adipose neoplasm tissue microarray, including 32 myxoid liposarcomas linked to patient outcome. Results were scored visually and correlated with clinical outcome by Kaplan-Meier and Cox regression analyses. In the study, by examining expression patterns of several lipogenic regulatory gene products, an immature adipogenic status was verified in myxoid liposarcomas. We also found that expression levels of the ret proto-oncogene, insulin-like growth factor 1 receptor, and insulin-like growth factor 2 correlate with poor metastasis-free survival, supporting a role for ERK/MAPK and PI3K/AKT pathways in clinically aggressive myxoid liposarcomas.

  18. Spectral Analysis of Breast Cancer on Tissue Microarrays: Seeing Beyond Morphology

    DTIC Science & Technology

    2004-05-01

    Release; Distribution Unlimited The views, opinions and/or findings contained in this report are those of the author( s ) and should not be construed as an...of Breast Cancer on Tissue Microarrays: DAMD17-02-1-0634 Seeing Beyond Morphology 6. AUTHOR( S ) David L. Rimm, M.D., Ph.D. 7. PERFORMING ORGANIZATION...NAME( S ) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION Yale University School of Medicine REPORT NUMBER New Haven, CT 06520-8047 E-Mail: david. rimm@yale

  19. Multi-Tissue Microarray Analysis Identifies a Molecular Signature of Regeneration

    PubMed Central

    Mercer, Sarah E.; Cheng, Chia-Ho; Atkinson, Donald L.; Krcmery, Jennifer; Guzman, Claudia E.; Kent, David T.; Zukor, Katherine; Marx, Kenneth A.; Odelberg, Shannon J.; Simon, Hans-Georg

    2012-01-01

    The inability to functionally repair tissues that are lost as a consequence of disease or injury remains a significant challenge for regenerative medicine. The molecular and cellular processes involved in complete restoration of tissue architecture and function are expected to be complex and remain largely unknown. Unlike humans, certain salamanders can completely regenerate injured tissues and lost appendages without scar formation. A parsimonious hypothesis would predict that all of these regenerative activities are regulated, at least in part, by a common set of genes. To test this hypothesis and identify genes that might control conserved regenerative processes, we performed a comprehensive microarray analysis of the early regenerative response in five regeneration-competent tissues from the newt Notophthalmus viridescens. Consistent with this hypothesis, we established a molecular signature for regeneration that consists of common genes or gene family members that exhibit dynamic differential regulation during regeneration in multiple tissue types. These genes include members of the matrix metalloproteinase family and its regulators, extracellular matrix components, genes involved in controlling cytoskeleton dynamics, and a variety of immune response factors. Gene Ontology term enrichment analysis validated and supported their functional activities in conserved regenerative processes. Surprisingly, dendrogram clustering and RadViz classification also revealed that each regenerative tissue had its own unique temporal expression profile, pointing to an inherent tissue-specific regenerative gene program. These new findings demand a reconsideration of how we conceptualize regenerative processes and how we devise new strategies for regenerative medicine. PMID:23300656

  20. Proteomic profiles of thyroid tumors by mass spectrometry-imaging on tissue microarrays.

    PubMed

    Galli, Manuel; Pagni, Fabio; De Sio, Gabriele; Smith, Andrew; Chinello, Clizia; Stella, Martina; L'Imperio, Vincenzo; Manzoni, Marco; Garancini, Mattia; Massimini, Diego; Mosele, Niccolò; Mauri, Giancarlo; Zoppis, Italo; Magni, Fulvio

    2016-12-09

    The current study proposes the successful use of a mass spectrometry-imaging technology that explores the composition of biomolecules and their spatial distribution directly on-tissue to differentially classify benign and malignant cases, as well as different histotypes. To identify new specific markers, we investigated with this technology a wide histological Tissue Microarray (TMA)-based thyroid lesion series. Results showed specific protein signatures for malignant and benign specimens and allowed to build clusters comprising several proteins with discriminant capabilities. Among them, FINC, ACTB1, LMNA, HSP7C and KAD1 were identified by LC-ESI-MS/MS and found up-expressed in malignant lesions. These findings represent the opening of further investigations for their translation into clinical practice, e.g. for setting up new immunohistochemical stainings, and for a better understanding of thyroid lesions.

  1. 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

  2. Protein Profiling Gastric Cancer and Neighboring Control Tissues Using High-Content Antibody Microarrays

    PubMed Central

    Sill, Martin; Schröder, Christoph; Shen, Ying; Marzoq, Aseel; Komel, Radovan; Hoheisel, Jörg D.; Nienhüser, Henrik; Schmidt, Thomas; Kastelic, Damjana

    2016-01-01

    In this study, protein profiling was performed on gastric cancer tissue samples in order to identify proteins that could be utilized for an effective diagnosis of this highly heterogeneous disease and as targets for therapeutic approaches. To this end, 16 pairs of postoperative gastric adenocarcinomas and adjacent non-cancerous control tissues were analyzed on microarrays that contain 813 antibodies targeting 724 proteins. Only 17 proteins were found to be differentially regulated, with much fewer molecules than the numbers usually identified in studies comparing tumor to healthy control tissues. Insulin-like growth factor-binding protein 7 (IGFBP7), S100 calcium binding protein A9 (S100A9), interleukin-10 (IL‐10) and mucin 6 (MUC6) exhibited the most profound variations. For an evaluation of the proteins’ capacity for discriminating gastric cancer, a Receiver Operating Characteristic curve analysis was performed, yielding an accuracy (area under the curve) value of 89.2% for distinguishing tumor from non-tumorous tissue. For confirmation, immunohistological analyses were done on tissue slices prepared from another cohort of patients with gastric cancer. The utility of the 17 marker proteins, and particularly the four molecules with the highest specificity for gastric adenocarcinoma, is discussed for them to act as candidates for diagnosis, even in serum, and targets for therapeutic approaches. PMID:27600085

  3. A standardized method for lectin microarray-based tissue glycome mapping

    PubMed Central

    Zou, Xia; Yoshida, Maki; Nagai-Okatani, Chiaki; Iwaki, Jun; Matsuda, Atsushi; Tan, Binbin; Hagiwara, Kozue; Sato, Takashi; Itakura, Yoko; Noro, Erika; Kaji, Hiroyuki; Toyoda, Masashi; Zhang, Yan; Narimatsu, Hisashi; Kuno, Atsushi

    2017-01-01

    The significance of glycomic profiling has been highlighted by recent findings that structural changes of glycans are observed in many diseases, including cancer. Therefore, glycomic profiling of the whole body (glycome mapping) under different physiopathological states may contribute to the discovery of reliable biomarkers with disease-specific alterations. To achieve this, standardization of high-throughput and in-depth analysis of tissue glycome mapping is needed. However, this is a great challenge due to the lack of analytical methodology for glycans on small amounts of endogenous glycoproteins. Here, we established a standardized method of lectin-assisted tissue glycome mapping. Formalin-fixed, paraffin-embedded tissue sections were prepared from brain, liver, kidney, spleen, and testis of two C57BL/6J mice. In total, 190 size-adjusted fragments with different morphology were serially collected from each tissue by laser microdissection and subjected to lectin microarray analysis. The results and subsequent histochemical analysis with selected lectins were highly consistent with previous reports of mass spectrometry-based N- and/or O-glycome analyses and histochemistry. This is the first report to look at both N- and O-glycome profiles of various regions within tissue sections of five different organs. This simple and reproducible mapping approach is also applicable to various disease model mice to facilitate disease-related biomarker discovery. PMID:28262709

  4. A standardized method for lectin microarray-based tissue glycome mapping.

    PubMed

    Zou, Xia; Yoshida, Maki; Nagai-Okatani, Chiaki; Iwaki, Jun; Matsuda, Atsushi; Tan, Binbin; Hagiwara, Kozue; Sato, Takashi; Itakura, Yoko; Noro, Erika; Kaji, Hiroyuki; Toyoda, Masashi; Zhang, Yan; Narimatsu, Hisashi; Kuno, Atsushi

    2017-03-06

    The significance of glycomic profiling has been highlighted by recent findings that structural changes of glycans are observed in many diseases, including cancer. Therefore, glycomic profiling of the whole body (glycome mapping) under different physiopathological states may contribute to the discovery of reliable biomarkers with disease-specific alterations. To achieve this, standardization of high-throughput and in-depth analysis of tissue glycome mapping is needed. However, this is a great challenge due to the lack of analytical methodology for glycans on small amounts of endogenous glycoproteins. Here, we established a standardized method of lectin-assisted tissue glycome mapping. Formalin-fixed, paraffin-embedded tissue sections were prepared from brain, liver, kidney, spleen, and testis of two C57BL/6J mice. In total, 190 size-adjusted fragments with different morphology were serially collected from each tissue by laser microdissection and subjected to lectin microarray analysis. The results and subsequent histochemical analysis with selected lectins were highly consistent with previous reports of mass spectrometry-based N- and/or O-glycome analyses and histochemistry. This is the first report to look at both N- and O-glycome profiles of various regions within tissue sections of five different organs. This simple and reproducible mapping approach is also applicable to various disease model mice to facilitate disease-related biomarker discovery.

  5. A curated collection of tissue microarray images and clinical outcome data of prostate cancer patients

    PubMed Central

    Zhong, Qing; Guo, Tiannan; Rechsteiner, Markus; Rüschoff, Jan H.; Rupp, Niels; Fankhauser, Christian; Saba, Karim; Mortezavi, Ashkan; Poyet, Cédric; Hermanns, Thomas; Zhu, Yi; Moch, Holger; Aebersold, Ruedi; Wild, Peter J.

    2017-01-01

    Microscopy image data of human cancers provide detailed phenotypes of spatially and morphologically intact tissues at single-cell resolution, thus complementing large-scale molecular analyses, e.g., next generation sequencing or proteomic profiling. Here we describe a high-resolution tissue microarray (TMA) image dataset from a cohort of 71 prostate tissue samples, which was hybridized with bright-field dual colour chromogenic and silver in situ hybridization probes for the tumour suppressor gene PTEN. These tissue samples were digitized and supplemented with expert annotations, clinical information, statistical models of PTEN genetic status, and computer source codes. For validation, we constructed an additional TMA dataset for 424 prostate tissues, hybridized with FISH probes for PTEN, and performed survival analysis on a subset of 339 radical prostatectomy specimens with overall, disease-specific and recurrence-free survival (maximum 167 months). For application, we further produced 6,036 image patches derived from two whole slides. Our curated collection of prostate cancer data sets provides reuse potential for both biomedical and computational studies. PMID:28291248

  6. 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.

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

    PubMed

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

    2012-05-31

    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

  8. Association between FOXM1 and hedgehog signaling pathway in human cervical carcinoma by tissue microarray analysis

    PubMed Central

    Chen, Hong; Wang, Jingjing; Yang, Hong; Chen, Dan; Li, Panpan

    2016-01-01

    Forkhead box M1 (FOXM1) and hedgehog (Hh) signaling pathway are implicated in the formation and development of human tumors, including cervical cancer. Previous studies have indicated that FOXM1 may be a downstream target gene of the Hh signaling pathway, but their association in cervical cancer is largely unknown. In the present study, the expression of FOXM1 and Hh signaling molecules was evaluated by immunohistochemical analysis in a tissue microarray that contained 70 cervical cancer tissues and 10 normal cervical tissues. In addition, the association of these molecules with clinicopathological parameters, and the association between FOXM1 and various molecules involved in the Hh signaling pathway was investigated. The results indicated that FOXM1 and Hh signaling molecules were overexpressed in cervical cancer tissues. The protein expression levels of FOXM1, glioma-associated oncogene 1 (GLI1) and smoothened (SMO) correlated with the clinical stage of the tumors, while the protein expression levels of Sonic Hh (SHh), patched 1 (PTCH1) and GLI1 correlated with the pathological grade of the tumors. The expression levels of GLI1 were lower in tissues without lymph node metastasis than in tissues with lymph node metastasis. In addition, FOXM1 expression correlated with GLI1, SHh and PTCH1 expression in cancer tissues. These findings confirmed the participation of FOXM1 and the Hh signaling pathway in cervical cancer. Furthermore, the finding that FOXM1 may be a downstream target gene of the Hh signaling pathway in cervical cancer provides a potential novel diagnostic and therapeutic target for cervical cancer. PMID:27698840

  9. Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas

    PubMed Central

    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

  10. Overview on Techniques to Construct Tissue Arrays with Special Emphasis on Tissue Microarrays

    PubMed Central

    Vogel, Ulrich

    2014-01-01

    With the advent of new histopathological staining techniques (histochemistry, immunohistochemistry, in situ hybridization) and the discovery of thousands of new genes, mRNA, and proteins by molecular biology, the need grew for a technique to compare many different cells or tissues on one slide in a cost effective manner and with the possibility to easily track the identity of each specimen: the tissue array (TA). Basically, a TA consists of at least two different specimens per slide. TAs differ in the kind of specimens, the number of specimens installed, the dimension of the specimens, the arrangement of the specimens, the embedding medium, the technique to prepare the specimens to be installed, and the technique to construct the TA itself. A TA can be constructed by arranging the tissue specimens in a mold and subsequently pouring the mold with the embedding medium of choice. In contrast, preformed so-called recipient blocks consisting of the embedding medium of choice have punched, drilled, or poured holes of different diameters and distances in which the cells or tissue biopsies will be deployed manually, semi-automatically, or automatically. The costs of constructing a TA differ from a few to thousands of Euros depending on the technique/equipment used. Remarkably high quality TAs can be also achieved by low cost techniques. PMID:27600339

  11. The extrinsic apoptotic signaling pathway in gastric adenocarcinomas assessed by tissue microarray.

    PubMed

    Gomes, Thiago S; Oshima, Celina T F; Segreto, Helena R C; Barrazueta, Luis M; Costa, Henrique O; Lima, Flavio O; Forones, Nora M; Ribeiro, Daniel A

    2011-10-15

    The purpose of this investigation was to analyze the immunoexpression of FasL, Fas, FADD, cleaved caspase 8, and cleaved caspase 3 in gastric cancer. Formalin-fixed and paraffin-embedded gastric adenocarcinoma tissues from 87 patients, including adjacent normal tissues, were included on tissue microarray by immunohistochemistry. The tumor and the adjacent normal tissues were positive for FasL in 66.7% and 90.6%, for Fas in 52.8% and 52.4%, for FADD in 67.4% and 82.3%, for cleaved caspase 8 in 27.9% and 37.7%, and for cleaved caspase 3 in 33.7% and 8.3%, respectively. FasL and the FADD from tumor were statistically different in relation to the histological type. Cleaved caspase 8 was statistically different in relation to clinical stage (p=0.031). The FADD from normal tissue was statistically different in relation to age (p=0.039), sex (p=0.055), clinical stage (p=0.019), and Fas was different in relation to tumor size (p=0.012). In the tumor, we observed a correlation between FasL and Fas, FasL and FADD, and FasL and cleaved caspase 3. In the adjacent normal tissue, a correlation was observed between FasL and Fas, FasL and FADD. There was no association of another marker with sex, age, clinical stage, and survival. Our results suggest that these proteins mediate the early extrinsic apoptotic pathway in gastric cancer and adjacent normal mucosa. FasL protein binds to Fas protein and subsequently binds to death receptor FADD signaling activation of the extrinsic apoptotic pathway. In this phase, there was inhibition of caspase 8 and, consequently, decreased apoptosis.

  12. TMA Navigator: Network inference, patient stratification and survival analysis with tissue microarray data.

    PubMed

    Lubbock, Alexander L R; Katz, Elad; Harrison, David J; Overton, Ian M

    2013-07-01

    Tissue microarrays (TMAs) allow multiplexed analysis of tissue samples and are frequently used to estimate biomarker protein expression in tumour biopsies. TMA Navigator (www.tmanavigator.org) is an open access web application for analysis of TMA data and related information, accommodating categorical, semi-continuous and continuous expression scores. Non-biological variation, or batch effects, can hinder data analysis and may be mitigated using the ComBat algorithm, which is incorporated with enhancements for automated application to TMA data. Unsupervised grouping of samples (patients) is provided according to Gaussian mixture modelling of marker scores, with cardinality selected by Bayesian information criterion regularization. Kaplan-Meier survival analysis is available, including comparison of groups identified by mixture modelling using the Mantel-Cox log-rank test. TMA Navigator also supports network inference approaches useful for TMA datasets, which often constitute comparatively few markers. Tissue and cell-type specific networks derived from TMA expression data offer insights into the molecular logic underlying pathophenotypes, towards more effective and personalized medicine. Output is interactive, and results may be exported for use with external programs. Private anonymous access is available, and user accounts may be generated for easier data management.

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

    PubMed

    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

  14. 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

  15. Association of molecular biomarkers expression with biochemical recurrence in prostate cancer through tissue microarray immunostaining.

    PubMed

    Ma, Ding; Zhou, Zhe; Yang, Bing; He, Qun; Zhang, Qian; Zhang, Xiang-Hua

    2015-10-01

    The aim of the present study was to investigate the prognostic role of metallothionein-2A (MT-2A), E-cadherin, interleukin-6 (IL-6), cyclin-E, proliferating cell nuclear antigen (PCNA) and B cell lymphoma (Bcl)-2 in the biochemical recurrence of prostate cancer (PCa) using tissue microarray immunostaining. Tissue specimens from 128 PCa patients who underwent radical prostatectomy were processed and transferred onto tissue microarrays. The clinicopathological parameters of PCa patients were also recorded. Following immunohistochemical examination of MT-2A, E-cadherin, IL-6, cyclin-E, PCNA and Bcl-2 expression in PCa specimens, association analysis of biomarkers expression with the biochemical recurrence of PCa was performed. The results revealed that the overall rate of biochemical recurrence was 30.5% (39/128) and the median biochemical recurrence-free time was 19 months (range, 6-35 months). The biochemical recurrence rates in low-, intermediate- and high-risk PCa classification were 14.8 (8/54), 38.7 (24/62) and 58.3% (7/12), respectively. Survival analysis demonstrated that a decreased biochemical recurrence-free survival rate was noted in PCa cases with positive MT-2A and cyclin E expression as well as those with negative E-cadherin expression (P=0.022, 0.028 and 0.011, respectively). Subsequent multivariate Cox analysis revealed that MT-2A [hazard ratio (HR)=2.01; 95% confidence interval (CI)=1.08-3.15; P=0.005], E-cadherin (HR=1.79; 95% CI=1.08-2.21; P=0.042) and cyclin E (HR=1.92; 95% CI=1.22-2.45; P=0.020) were independent predictors of the biochemical recurrence of PCa. In conclusion, the present study provided clinical evidence that evaluation of molecular biomarkers expression may improve clinical prognostic accuracy for the biochemical recurrence of PCa. Of note, the expression of MT-2A, cyclin E and E-cadherin may serve as independent predictors for biochemical recurrence of PCa.

  16. 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

  17. 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.

  18. Hyperspectral microscopic analysis of normal, benign and carcinoma microarray tissue sections

    NASA Astrophysics Data System (ADS)

    Maggioni, Mauro; Davis, Gustave L.; Warner, Frederick J.; Geshwind, Frank B.; Coppi, Andreas C.; DeVerse, Richard A.; Coifman, Ronald R.

    2006-02-01

    We apply a unique micro-optoelectromechanical tuned light source and new algorithms to the hyper-spectral microscopic analysis of human colon biopsies. The tuned light prototype (Plain Sight Systems Inc.) transmits any combination of light frequencies, range 440nm 700nm, trans-illuminating H and E stained tissue sections of normal (N), benign adenoma (B) and malignant carcinoma (M) colon biopsies, through a Nikon Biophot microscope. Hyper-spectral photomicrographs, randomly collected 400X magnication, are obtained with a CCD camera (Sensovation) from 59 different patient biopsies (20 N, 19 B, 20 M) mounted as a microarray on a single glass slide. The spectra of each pixel are normalized and analyzed to discriminate among tissue features: gland nuclei, gland cytoplasm and lamina propria/lumens. Spectral features permit the automatic extraction of 3298 nuclei with classification as N, B or M. When nuclei are extracted from each of the 59 biopsies the average classification among N, B and M nuclei is 97.1%; classification of the biopsies, based on the average nuclei classification, is 100%. However, when the nuclei are extracted from a subset of biopsies, and the prediction is made on nuclei in the remaining biopsies, there is a marked decrement in performance to 60% across the 3 classes. Similarly the biopsy classification drops to 54%. In spite of these classification differences, which we believe are due to instrument and biopsy normalization issues, hyper-spectral analysis has the potential to achieve diagnostic efficiency needed for objective microscopic diagnosis.

  19. A comparative performance study characterizing breast tissue microarrays using standard RGB and multispectral imaging

    NASA Astrophysics Data System (ADS)

    Qi, Xin; Cukierski, William; Foran, David J.

    2010-02-01

    The lack of clear consensus over the utility of multispectral imaging (MSI) for bright-field imaging prompted our team to investigate the benefit of using MSI on breast tissue microarrays (TMA). We have conducted performance studies to compare MSI with standard bright-field imaging in hematoxylin stained breast tissue. The methodology has three components. The first extracts a region of interest using adaptive thresholding and morphological processing. The second performs texture feature extraction from a local binary pattern within each spectral channel and compared to features of co-occurrence matrix and texture feature coding in third component. The third component performs feature selection and classification. For each spectrum, exhaustive feature selection was used to search for the combination of features that yields the best classification accuracy. AdaBoost with a linear perceptron least-square classifier was applied. The spectra carrying the greatest discriminatory power were automatically chosen and a majority vote was used to make the final classification. 92 breast TMA discs were included in the study. Sensitivity of 0.96 and specificity of 0.89 were achieved on the multispectral data, compared with sensitivity of 0.83 and specificity of 0.85 on RGB data. MSI consistently achieved better classification results than those obtained using standard RGB images. While the benefits of MSI for unmixing multi-stained specimens are well documented, this study demonstrated statistically significant improvements in the automated analysis of single stained bright-field images.

  20. Larger core size has superior technical and analytical accuracy in bladder tissue microarray.

    PubMed

    Eskaros, Adel Rh; Egloff, Shanna A Arnold; Boyd, Kelli L; Richardson, Joyce E; Hyndman, M Eric; Zijlstra, Andries

    2017-03-01

    The construction of tissue microarrays (TMAs) with cores from a large number of paraffin-embedded tissues (donors) into a single paraffin block (recipient) is an effective method of analyzing samples from many patient specimens simultaneously. For the TMA to be successful, the cores within it must capture the correct histologic areas from the donor blocks (technical accuracy) and maintain concordance with the tissue of origin (analytical accuracy). This can be particularly challenging for tissues with small histological features such as small islands of carcinoma in situ (CIS), thin layers of normal urothelial lining of the bladder, or cancers that exhibit intratumor heterogeneity. In an effort to create a comprehensive TMA of a bladder cancer patient cohort that accurately represents the tumor heterogeneity and captures the small features of normal and CIS, we determined how core size (0.6 vs 1.0 mm) impacted the technical and analytical accuracy of the TMA. The larger 1.0 mm core exhibited better technical accuracy for all tissue types at 80.9% (normal), 94.2% (tumor), and 71.4% (CIS) compared with 58.6%, 85.9%, and 63.8% for 0.6 mm cores. Although the 1.0 mm core provided better tissue capture, increasing the number of replicates from two to three allowed with the 0.6 mm core compensated for this reduced technical accuracy. However, quantitative image analysis of proliferation using both Ki67+ immunofluorescence counts and manual mitotic counts demonstrated that the 1.0 mm core size also exhibited significantly greater analytical accuracy (P=0.004 and 0.035, respectively, r(2)=0.979 and 0.669, respectively). Ultimately, our findings demonstrate that capturing two or more 1.0 mm cores for TMA construction provides superior technical and analytical accuracy over the smaller 0.6 mm cores, especially for tissues harboring small histological features or substantial heterogeneity.

  1. 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

  2. Research progress in liver tissue engineering.

    PubMed

    Zhang, Lei; Guan, Zheng; Ye, Jun-Song; Yin, Yan-Feng; Stoltz, Jean-François; de Isla, Natalia

    2017-01-01

    Liver transplantation is the definitive treatment for patients with end-stage liver diseases (ESLD). However, it is hampered by shortage of liver donor. Liver tissue engineering, aiming at fabricating new livers in vitro, provides a potential resolution for donor shortage. Three elements need to be considered in liver tissue engineering: seeding cell resources, scaffolds and bioreactors. Studies have shown potential cell sources as hepatocytes, hepatic cell line, mesenchymal stem cells and others. They need scaffolds with perfect biocompatiblity, suitable micro-structure and appropriate degradation rate, which are essential charateristics for cell attachment, proliferation and secretion in forming extracellular matrix. The most promising scaffolds in research include decellularized whole liver, collagens and biocompatible plastic. The development and function of cells in scaffold need a microenvironment which can provide them with oxygen, nutrition, growth factors, et al. Bioreactor is expected to fulfill these requirements by mimicking the living condition in vivo. Although there is great progress in these three domains, a large gap stays still between their researches and applications. Herein, we summarized the recent development in these three major fields which are indispensable in liver tissue engineering.

  3. 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.

  4. Application of new in situ hybridization probes for Ku70 and Ku80 in tissue microarrays of paraffin-embedded malignant melanomas: correlation with immunohistochemical analysis.

    PubMed

    Korabiowska, Monika; Bauer, Hanne; Quentin, Tomas; Stachura, Jerzy; Cordon-Cardo, Carlos; Brinck, Ulrich

    2004-02-01

    Ku70 and Ku80 proteins are responsible for the repair of DNA double-strand breaks and function as a regulatory subunit of the DNA-dependent protein kinase. In this study we analyzed expression of both genes in malignant melanoma tissue arrays applying in situ hybridization probes produced by our research group and using immunohistochemical analysis. Expression of both genes was down-regulated as melanoma progressed. In situ hybridization demonstrated more Ku70- and Ku80-positive cells than immunohistochemical methods, but the correlation between the two methods was highly significant (P <0.01). We conclude that the in situ hybridization assay for the detection of Ku70 and Ku80 expression used in this study is also suitable for tissue microarray analysis of paraffin-embedded melanoma samples. The laboratory procedure is much more complicated than the immunohistochemical method, however.

  5. Comparison of DNA ploidy status and DNA ploidy-related parameters in malignant melanoma tissue microarrays and full sections.

    PubMed

    Korabiowska, Monika; Cordon-Cardo, Carlos; Buschmann, Nadine; Stachura, Jerzy; Fischer, Gösta; Brinck, Ulrich

    2004-07-01

    A new high-throughput tissue-arraying technique, now frequently used in tumor pathology, requires standardization of methods of DNA analysis, previously applied in full histological sections. The main objectives of this study were to evaluate DNA ploidy status and DNA ploidy-related parameters using the CAS200 image analyzer in malignant melanoma tissue microarrays and to compare them with full histological sections. Comparison of DNA ploidy-related parameters, including percentage of diploid cells, percentage of aneuploid cells between 2c and 4c, percentage of tetraploid cells, percentage of aneuploid cells between 4c and 8c, percentage of octaploid cells, percentage of 16-ploid cells, and 5c exceeding rate, did not reveal any significant differences between malignant melanoma tissue microarrays and full sections. The DNA ploidy status according to Auer differed in 1 out of 59 cases investigated. Our study demonstrated that it is possible to evaluate DNA ploidy status and DNA ploidy-related parameters in tissue microarrays, which is of practical relevance to tumor pathology.

  6. 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

  7. Diagnostic utility of immunohistochemistry in distinguishing trichoepithelioma and basal cell carcinoma: evaluation using tissue microarray samples.

    PubMed

    Tebcherani, Antonio José; de Andrade, Heitor Franco; Sotto, Mirian N

    2012-10-01

    Trichoepithelioma is a benign neoplasm that shares both clinical and histological features with basal cell carcinoma. It is important to distinguish these neoplasms because they require different clinical behavior and therapeutic planning. Many studies have addressed the use of immunohistochemistry to improve the differential diagnosis of these tumors. These studies present conflicting results when addressing the same markers, probably owing to the small number of basaloid tumors that comprised their studies, which generally did not exceed 50 cases. We built a tissue microarray with 162 trichoepithelioma and 328 basal cell carcinoma biopsies and tested a panel of immune markers composed of CD34, CD10, epithelial membrane antigen, Bcl-2, cytokeratins 15 and 20 and D2-40. The results were analyzed using multiple linear and logistic regression models. This analysis revealed a model that could differentiate trichoepithelioma from basal cell carcinoma in 36% of the cases. The panel of immunohistochemical markers required to differentiate between these tumors was composed of CD10, cytokeratin 15, cytokeratin 20 and D2-40. The results obtained in this work were generated from a large number of biopsies and resulted in the confirmation of overlapping epithelial and stromal immunohistochemical profiles from these basaloid tumors. The results also corroborate the point of view that trichoepithelioma and basal cell carcinoma tumors represent two different points in the differentiation of a single cell type. Despite the use of panels of immune markers, histopathological criteria associated with clinical data certainly remain the best guideline for the differential diagnosis of trichoepithelioma and basal cell carcinoma.

  8. 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.

  9. 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

  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].

  11. A model for the design and construction of a resource for the validation of prognostic prostate cancer biomarkers: the Canary Prostate Cancer Tissue Microarray

    PubMed Central

    Hawley, Sarah; Fazli, Ladan; McKenney, Jesse K.; Simko, Jeff; Troyer, Dean; Nicolas, Marlo; Newcomb, Lisa F.; Cowan, Janet E.; Crouch, Luis; Ferrari, Michelle; Hernandez, Javier; Hurtado-Coll, Antonio; Kuchinsky, Kyle; Liew, Janet; Mendez-Meza, Rosario; Smith, Elizabeth; Tenggara, Imelda; Zhang, Xiaotun; Carroll, Peter R.; Chan, June M.; Gleave, Martin; Lance, Raymond; Lin, Daniel W.; Nelson, Peter S.; Thompson, Ian M.; Feng, Ziding; True, Lawrence D.; Brooks, James D.

    2012-01-01

    Tissue microarrays provide unique resources for rapid evaluation and validation of tissue biomarkers. The Canary Foundation Retrospective Prostate Tissue Microarray Resource used a rigorous statistical design, quota sampling, a variation of the case-cohort study, to select patients for inclusion in a multicenter, retrospective prostate cancer tissue microarray cohort. The study is designed to definitively validate tissue biomarkers of prostate cancer recurrence after radical prostatectomy. Tissue samples from over 1,000 participants treated for prostate cancer with radical prostatectomy between 1995 and 2004 were selected at six participating institutions in the United States and Canada. This design captured the heterogeneity of screening and clinical practices in the contemporary North American population. Standardized clinical data were collected in a centralized database. The project has been informative in several respects. The scale and complexity of assembling tissue microarrays (TMAs) with over 200 cases at each of six sites involved unanticipated levels of effort and time. Our statistical design promises to provide a model for outcome-based studies where tissue localization methods are applied to high-density tissue microarrays. PMID:23232570

  12. Expression and clinicopathological implication of DcR3 in lung cancer tissues: a tissue microarray study with 365 cases

    PubMed Central

    Zhang, Yu; Luo, Jie; He, Rongquan; Huang, Wenting; Li, Zuyun; Li, Ping; Dang, Yiwu; Chen, Gang; Li, Shikang

    2016-01-01

    Background Decoy receptor 3 (DcR3) has been reported to be involved in different cancers. However, few related researches have been accomplished on the role of DcR3 in lung cancer. Objective To explore the expression level and clinicopathological implication of DcR3 protein in lung cancer tissues. Materials and methods Immunohistochemistry was used to examine DcR3 protein expression in lung cancer (n=365) and normal lung tissues (n=26). The relationships between DcR3 expression and clinical parameters were further investigated. Furthermore, the diagnostic and clinicopathological value of DcR3 mRNA was analyzed based on The Cancer Genome Atlas database in lung cancer patients. Results Compared to normal lung tissues, DcR3 expression was significantly higher in lung cancer (P=0.007) tissues, including small-cell lung cancer (P=0.001) and non-small-cell lung cancer (P=0.008). In addition, DcR3 expression was related to tumor-node-metastasis (TNM) stage (P<0.001), tumor diameter (P=0.007), distant metastasis (P<0.001), and lymph node metastasis (P<0.001) in lung cancers. When concerning non-small-cell lung cancer, consistent correlations between DcR3 expression and TNM stage (P<0.001), tumor diameter (P=0.019), distant metastasis (P<0.001), and lymph node metastasis (P<0.001) were found. Simultaneously, in small-cell lung cancer, TNM stage (P=0.004) and lymph node metastasis (P=0.005) were also associated with DcR3 expression. Additionally, receiver operator characteristic curve revealed that the area under curve (AUC) of DcR3 was 0.637 (95% confidence interval [CI] 0.531–0.742) for lung cancer. Furthermore, DcR3 was overexpressed in both adenocarcinoma and squamous cell carcinoma tissues than in noncancerous lung tissues (all P<0.0001) based on the data from The Cancer Genome Atlas. AUC of DcR3 was 0.726 (95% CI 0.644–0.788) for lung adenocarcinoma patients and 0.647 (95% CI 0.566–0.728) for squamous cell carcinoma patients. DcR3 expression was also related to

  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.

  14. 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).

  15. Microarray analysis of CA1 pyramidal neurons in a mouse model of tauopathy reveals progressive synaptic dysfunction

    PubMed Central

    Alldred, Melissa J.; Duff, Karen E.; Ginsberg, Stephen D.

    2011-01-01

    The hTau mouse model of tauopathy was utilized to assess gene expression changes in vulnerable hippocampal CA1 neurons. CA1 pyramidal neurons were microaspirated via laser capture microdissection followed by RNA amplification in combination with custom-designed microarray analysis and qPCR validation in hTau mice and nontransgenic (ntg) littermates aged 11-14 months. Statistical analysis revealed ∼8% of all the genes on the array platform were dysregulated, with notable downregulation of several synaptic-related markers including synaptophysin (Syp), synaptojanin, and synaptobrevin, among others. Downregulation was also observed for select glutamate receptors (GluRs), Psd-95, TrkB, and several protein phosphatase subunits. In contrast, upregulation of tau isoforms and a calpain subunit were found. Microarray assessment of synaptic-related markers in a separate cohort of hTau mice at 7-8 months of age indicated only a few alterations compared to the 11-14 month cohort, suggesting progressive synaptic dysfunction occurs as tau accumulates in CA1 pyramidal neurons. An assessment of SYP and PSD-95 expression was performed in the hippocampal CA1 sector of hTau and ntg mice via confocal laser scanning microscopy along with hippocampal immunoblot analysis for protein-based validation of selected microarray observations. Results indicate significant decreases in SYP-immunoreactive and PSD-95-immunoreactive puncta as well as downregulation of SYP-immunoreactive and PSD-95-immunoreactive band intensity in hTau mice compared to age-matched ntg littermates. In summary, the high prevalence of downregulation of synaptic-related genes indicates that the moderately aged hTau mouse may be a model of tau-induced synaptodegeneration, and has profound effects on how we perceive progressive tau pathology affecting synaptic transmission in AD. PMID:22079237

  16. Alternations in genes expression of pathway signaling in esophageal tissue with atresia: results of expression microarray profiling.

    PubMed

    Smigiel, R; Lebioda, A; Blaszczyński, M; Korecka, K; Czauderna, P; Korlacki, W; Jakubiak, A; Bednarczyk, D; Maciejewski, H; Wizinska, P; Sasiadek, M M; Patkowski, D

    2015-04-01

    Esophageal atresia (EA) is a congenital defect of the esophagus involving the interruption of the esophagus with or without connection to the trachea (tracheoesophageal fistula [TEF]). EA/TEF may occur as an isolated anomaly, may be part of a complex of congenital defects (syndromic), or may develop within the context of a known syndrome or association. The molecular mechanisms underlying the development of EA are poorly understood. It is supposed that a combination of multigenic factors and epigenetic modification of genes play a role in its etiology. The aim of our work was to assess the human gene expression microarray study in esophageal tissue samples. Total RNA was extracted from 26 lower pouches of esophageal tissue collected during thoracoscopic EA repair in neonates with the isolated (IEA) and the syndromic form (SEA). We identified 787 downregulated and 841 upregulated transcripts between SEA and controls, and about 817 downregulated and 765 upregulated probes between IEA and controls. Fifty percent of these genes showed differential expression specific for either IEA or SEA. Functional pathway analysis revealed substantial enrichment for Wnt and Sonic hedgehog, as well as cytokine and chemokine signaling pathways. Moreover, we performed reverse transcription polymerase chain reaction study in a group of SHH and Wnt pathways genes with differential expression in microarray profiling to confirm the microarray expression results. We verified the altered expression in SFRP2 gene from the Wnt pathway as well as SHH, GLI1, GLI2, and GLI3 from the Sonic hedgehog pathway. The results suggest an important role of these pathways and genes for EA/TEF etiology.

  17. Detecting tissue-specific alternative splicing and disease-associated aberrant splicing of the PTCH gene with exon junction microarrays.

    PubMed

    Nagao, Kazuaki; Togawa, Naoyuki; Fujii, Katsunori; Uchikawa, Hideki; Kohno, Yoichi; Yamada, Masao; Miyashita, Toshiyuki

    2005-11-15

    Mutations in the human ortholog of Drosophila patched (PTCH) have been identified in patients with autosomal dominant nevoid basal cell carcinoma syndrome (NBCCS), characterized by minor developmental anomalies and an increased incidence of cancers such as medulloblastoma and basal cell carcinoma. We identified many isoforms of PTCH mRNA involving exons 1-5, exon 10 and a novel exon, 12b, generated by alternative splicing (AS), most of which have not been deposited in GenBank nor discussed earlier. To monitor splicing events of the PTCH gene, we designed oligonucleotide arrays on which exon probes and exon-exon junction probes as well as a couple of intron probes for the PTCH gene were placed in duplicate. Probe intensities were normalized on the basis of the total expression of PTCH and probe sensitivity. Tissue-specific regulation of AS identified with the microarrays closely correlated with the results obtained by RT-PCR. Of note, the novel exon, exon 12b, was specifically expressed in the brain and heart, especially in the cerebellum. Additionally, using these microarrays, we were able to detect disease-associated aberrant splicings of the PTCH gene in two patients with NBCCS. In both cases, cryptic splice donor sites located either in an exon or in an intron were activated because of the partial disruption of the consensus sequence for the authentic splice donor sites due to point mutations. Taken together, oligonucleotide microarrays containing exon junction probes are demonstrated to be a powerful tool to investigate tissue-specific regulation of AS and aberrant splicing taking place in genetic disorders.

  18. 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

  19. Large-scale atlas of microarray data reveals the distinct expression landscape of different tissues in Arabidopsis

    SciTech Connect

    He, Fei; Maslov, Sergei; Yoo, Shinjae; Wang, Daifeng; Kumari, Sunita; Gerstein, Mark; Ware, Doreen

    2016-05-25

    Here, transcriptome datasets from thousands of samples of the model plant Arabidopsis thaliana have been collectively generated by multiple individual labs. Although integration and meta-analysis of these samples has become routine in the plant research community, it is often hampered by the lack of metadata or differences in annotation styles by different labs. In this study, we carefully selected and integrated 6,057 Arabidopsis microarray expression samples from 304 experiments deposited to NCBI GEO. Metadata such as tissue type, growth condition, and developmental stage were manually curated for each sample. We then studied global expression landscape of the integrated dataset and found that samples of the same tissue tend to be more similar to each other than to samples of other tissues, even in different growth conditions or developmental stages. Root has the most distinct transcriptome compared to aerial tissues, but the transcriptome of cultured root is more similar to those of aerial tissues as the former samples lost their cellular identity. Using a simple computational classification method, we showed that the tissue type of a sample can be successfully predicted based on its expression profile, opening the door for automatic metadata extraction and facilitating re-use of plant transcriptome data. As a proof of principle we applied our automated annotation pipeline to 708 RNA-seq samples from public repositories and verified accuracy of our predictions with samples’ metadata provided by authors.

  20. Large-scale atlas of microarray data reveals the distinct expression landscape of different tissues in Arabidopsis

    DOE PAGES

    He, Fei; Maslov, Sergei; Yoo, Shinjae; ...

    2016-05-25

    Here, transcriptome datasets from thousands of samples of the model plant Arabidopsis thaliana have been collectively generated by multiple individual labs. Although integration and meta-analysis of these samples has become routine in the plant research community, it is often hampered by the lack of metadata or differences in annotation styles by different labs. In this study, we carefully selected and integrated 6,057 Arabidopsis microarray expression samples from 304 experiments deposited to NCBI GEO. Metadata such as tissue type, growth condition, and developmental stage were manually curated for each sample. We then studied global expression landscape of the integrated dataset andmore » found that samples of the same tissue tend to be more similar to each other than to samples of other tissues, even in different growth conditions or developmental stages. Root has the most distinct transcriptome compared to aerial tissues, but the transcriptome of cultured root is more similar to those of aerial tissues as the former samples lost their cellular identity. Using a simple computational classification method, we showed that the tissue type of a sample can be successfully predicted based on its expression profile, opening the door for automatic metadata extraction and facilitating re-use of plant transcriptome data. As a proof of principle we applied our automated annotation pipeline to 708 RNA-seq samples from public repositories and verified accuracy of our predictions with samples’ metadata provided by authors.« less

  1. 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.

  2. A 'waterfall' transfer-based workflow for improved quality of tissue microarray construction and processing in breast cancer research.

    PubMed

    Oberländer, M; Alkemade, H; Bünger, S; Ernst, F; Thorns, C; Braunschweig, T; Habermann, J K

    2014-07-01

    A major focus in cancer research is the identification of biomarkers for early diagnosis, therapy prediction and prognosis. Hereby, validation of target proteins on clinical samples is of high importance. Tissue microarrays (TMAs) represent an essential advancement for high-throughput analysis by assembling large numbers of tissue cores with high efficacy and comparability. However, limitations along TMA construction and processing exist. In our presented study, we had to overcome several obstacles in the construction and processing of high-density breast cancer TMAs to ensure good quality sections for further research. Exemplarily, 406 breast tissue cores from formalin-fixed and paraffin embedded samples of 245 patients were placed onto three recipient paraffin blocks. Sectioning was performed using a rotary microtome with a "waterfall" automated transfer system. Sections were stained by immunohistochemistry and immunofluorescence for nine proteins. The number and quality of cores after sectioning and staining was counted manually for each marker. In total, 97.1 % of all cores were available after sectioning, while further 96 % of the remaining cores were evaluable after staining. Thereby, normal tissue cores were more often lost compared to tumor tissue cores. Our workflow provides a robust method for manufacturing high-density breast cancer TMAs for subsequent IHC or IF staining without significant sample loss.

  3. 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

  4. Oligonucleotide microarray analysis reveals dysregulation of energy-related metabolism in insulin-sensitive tissues of type 2 diabetes patients.

    PubMed

    Wang, M; Wang, X C; Zhao, L; Zhang, Y; Yao, L L; Lin, Y; Peng, Y D; Hu, R M

    2014-06-17

    Impaired insulin action within skeletal muscle, adipose tissue, and the liver is an important characteristic of type 2 diabetes (T2D). In order to identify common underlying defects in insulin-sensitive tissues that may be involved in the pathogenesis of T2D, the gene expression profiles of skeletal muscle, visceral adipose tissue, and liver from autopsy donors with or without T2D were examined using oligonucleotide microarrays and quantitative reverse transcriptase-PCR. Compared with controls, 691 genes were commonly dysregulated in these three insulin-sensitive tissues of humans with T2D. These co-expressed genes were enriched within the mitochondrion, with suggested involvement in energy metabolic processes such as glycolysis and gluconeogenesis, fatty acid beta oxidative, tricarboxylic acid cycle, and electron transport. Genes related to energy metabolism were mostly downregulated in diabetic skeletal muscle and visceral adipose tissue, while they were upregulated in the diabetic liver. This observed dysregulation in energy-related metabolism may be the underlying factor leading to the molecular mechanisms responsible for the insulin resistance of patients with T2D.

  5. Study Progress on Tissue Culture of Maize Mature Embryo

    NASA Astrophysics Data System (ADS)

    Wang, Hongzhen; Cheng, Jun; Cheng, Yanping; Zhou, Xioafu

    It has been paid more and more attention on maize tissue culture as it is a basic work in maize genetic transformation, especially huge breakthrough has been made in maize tissue culture utilizing mature embryos as explants in the recent years. This paper reviewed the study progress on maize tissue culture and plant regeneration utilizing mature embryos as explants from callus induction, subculture, plant regeneration and browning reduction and so on.

  6. 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.

  7. Microarray gene expression profiles from mature gonad tissues of Atlantic bluefin tuna, Thunnus thynnus in the Gulf of Mexico

    PubMed Central

    2012-01-01

    Background Bluefin tunas are highly prized pelagic fish species representing a significant economic resource to fisheries throughout the world. Atlantic bluefin tuna (Thunnus thynnus) populations have significantly declined due to overexploitation. As a consequence of their value and population decline, T. thynnus has been the focus of considerable research effort concerning many aspects of their life history. However, in-depth understanding of T. thynnus reproductive biology is still lacking. Knowledge of reproductive physiology is a very important tool for determining effective fisheries and aquaculture management. Transcriptome techniques are proving powerful and provide novel insights into physiological processes. Construction of a microarray from T. thynnus ESTs sourced from reproductive tissues has provided an ideal platform to study the reproductive physiology of bluefin tunas. The aim of this investigation was to compare transcription profiles from the ovaries and testes of mature T. thynnus to establish sex specific variations underlying their reproductive physiology. Results Male and females T. thynnus gonad tissues were collected from the wild and histologically staged. Sub-samples of sexually mature tissues were also measured for their mRNA differential expression among the sexes using the custom microarray design BFT 4X44K. A total of 7068 ESTs were assessed for differential expression of which 1273 ESTs were significantly different (p<0.05) with >2 fold change in expression according to sex. Differential expression for 13 of these ESTs was validated with quantitative PCR. These include genes involved in egg envelope formation, hydration, and lipid transport/accumulation more highly expressed in ovaries compared with testis, while genes involved in meiosis, sperm motility and lipid metabolism were more highly expressed in testis compared with ovaries. Conclusions This investigation has furthered our knowledge of bluefin tunas reproductive biology by

  8. Performance of automated scoring of ER, PR, HER2, CK5/6 and EGFR in breast cancer tissue microarrays in the Breast Cancer Association Consortium

    PubMed Central

    Howat, William J; Blows, Fiona M; Provenzano, Elena; Brook, Mark N; Morris, Lorna; Gazinska, Patrycja; Johnson, Nicola; McDuffus, Leigh‐Anne; Miller, Jodi; Sawyer, Elinor J; Pinder, Sarah; van Deurzen, Carolien H M; Jones, Louise; Sironen, Reijo; Visscher, Daniel; Caldas, Carlos; Daley, Frances; Coulson, Penny; Broeks, Annegien; Sanders, Joyce; Wesseling, Jelle; Nevanlinna, Heli; Fagerholm, Rainer; Blomqvist, Carl; Heikkilä, Päivi; Ali, H Raza; Dawson, Sarah‐Jane; Figueroa, Jonine; Lissowska, Jolanta; Brinton, Louise; Mannermaa, Arto; Kataja, Vesa; Kosma, Veli‐Matti; Cox, Angela; Brock, Ian W; Cross, Simon S; Reed, Malcolm W; Couch, Fergus J; Olson, Janet E; Devillee, Peter; Mesker, Wilma E; Seyaneve, Caroline M; Hollestelle, Antoinette; Benitez, Javier; Perez, Jose Ignacio Arias; Menéndez, Primitiva; Bolla, Manjeet K; Easton, Douglas F; Schmidt, Marjanka K; Pharoah, Paul D; Sherman, Mark E

    2014-01-01

    Abstract Breast cancer risk factors and clinical outcomes vary by tumour marker expression. However, individual studies often lack the power required to assess these relationships, and large‐scale analyses are limited by the need for high throughput, standardized scoring methods. To address these limitations, we assessed whether automated image analysis of immunohistochemically stained tissue microarrays can permit rapid, standardized scoring of tumour markers from multiple studies. Tissue microarray sections prepared in nine studies containing 20 263 cores from 8267 breast cancers stained for two nuclear (oestrogen receptor, progesterone receptor), two membranous (human epidermal growth factor receptor 2 and epidermal growth factor receptor) and one cytoplasmic (cytokeratin 5/6) marker were scanned as digital images. Automated algorithms were used to score markers in tumour cells using the Ariol system. We compared automated scores against visual reads, and their associations with breast cancer survival. Approximately 65–70% of tissue microarray cores were satisfactory for scoring. Among satisfactory cores, agreement between dichotomous automated and visual scores was highest for oestrogen receptor (Kappa = 0.76), followed by human epidermal growth factor receptor 2 (Kappa = 0.69) and progesterone receptor (Kappa = 0.67). Automated quantitative scores for these markers were associated with hazard ratios for breast cancer mortality in a dose‐response manner. Considering visual scores of epidermal growth factor receptor or cytokeratin 5/6 as the reference, automated scoring achieved excellent negative predictive value (96–98%), but yielded many false positives (positive predictive value = 30–32%). For all markers, we observed substantial heterogeneity in automated scoring performance across tissue microarrays. Automated analysis is a potentially useful tool for large‐scale, quantitative scoring of immunohistochemically stained tissue

  9. 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

  10. Analysis of differentially expressed genes in placental tissues of preeclampsia patients using microarray combined with the Connectivity Map database.

    PubMed

    Song, Y; Liu, J; Huang, S; Zhang, L

    2013-12-01

    Preeclampsia (PE), which affects 2-7% of human pregnancies, causes significant maternal and neonatal morbidity and mortality. To better understand the pathophysiology of PE, the gene expression profiles of placental tissue from 5 controls and 5 PE patients were assessed using microarray. A total of 224 transcripts were significantly differentially expressed (>2-fold change and q value <0.05, SAM software). Gene Ontology (GO) enrichment analysis indicated that genes involved in hypoxia and oxidative and reductive processes were significantly changed. Three differentially expressed genes (DEGs) involved in these biological processes were further verified by quantitative real-time PCR. Finally, the potential therapeutic agents for PE were explored via the Connectivity Map database. In conclusion, the data obtained in this study might provide clues to better understand the pathophysiology of PE and to identify potential therapeutic agents for PE patients.

  11. Building quantitative prediction models for tissue residue of two explosives compounds in earthworms from microarray gene expression data.

    PubMed

    Gong, Ping; Loh, Po-Ru; Barker, Natalie D; Tucker, George; Wang, Nan; Zhang, Chenhua; Escalon, B Lynn; Berger, Bonnie; Perkins, Edward J

    2012-01-03

    Soil contamination near munitions plants and testing grounds is a serious environmental concern that can result in the formation of tissue chemical residue in exposed animals. Quantitative prediction of tissue residue still represents a challenging task despite long-term interest and pursuit, as tissue residue formation is the result of many dynamic processes including uptake, transformation, and assimilation. The availability of high-dimensional microarray gene expression data presents a new opportunity for computational predictive modeling of tissue residue from changes in expression profile. Here we analyzed a 240-sample data set with measurements of transcriptomic-wide gene expression and tissue residue of two chemicals, 2,4,6-trinitrotoluene (TNT) and 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX), in the earthworm Eisenia fetida. We applied two different computational approaches, LASSO (Least Absolute Shrinkage and Selection Operator) and RF (Random Forest), to identify predictor genes and built predictive models. Each approach was tested alone and in combination with a prior variable selection procedure that involved the Wilcoxon rank-sum test and HOPACH (Hierarchical Ordered Partitioning And Collapsing Hybrid). Model evaluation results suggest that LASSO was the best performer of minimum complexity on the TNT data set, whereas the combined Wilcoxon-HOPACH-RF approach achieved the highest prediction accuracy on the RDX data set. Our models separately identified two small sets of ca. 30 predictor genes for RDX and TNT. We have demonstrated that both LASSO and RF are powerful tools for quantitative prediction of tissue residue. They also leave more unknown than explained, however, allowing room for improvement with other computational methods and extension to mixture contamination scenarios.

  12. Current progress in 3D printing for cardiovascular tissue engineering.

    PubMed

    Mosadegh, Bobak; Xiong, Guanglei; Dunham, Simon; Min, James K

    2015-03-16

    3D printing is a technology that allows the fabrication of structures with arbitrary geometries and heterogeneous material properties. The application of this technology to biological structures that match the complexity of native tissue is of great interest to researchers. This mini-review highlights the current progress of 3D printing for fabricating artificial tissues of the cardiovascular system, specifically the myocardium, heart valves, and coronary arteries. In addition, how 3D printed sensors and actuators can play a role in tissue engineering is discussed. To date, all the work with building 3D cardiac tissues have been proof-of-principle demonstrations, and in most cases, yielded products less effective than other traditional tissue engineering strategies. However, this technology is in its infancy and therefore there is much promise that through collaboration between biologists, engineers and material scientists, 3D bioprinting can make a significant impact on the field of cardiovascular tissue engineering.

  13. Tenascin-C: a novel candidate marker for cancer stem cells in glioblastoma identified by tissue microarrays.

    PubMed

    Nie, Song; Gurrea, Mikel; Zhu, Jianhui; Thakolwiboon, Smathorn; Heth, Jason A; Muraszko, Karin M; Fan, Xing; Lubman, David M

    2015-02-06

    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.

  14. Inverse correlation between CD8+ inflammatory cells and E-cadherin expression in gallbladder cancer: Tissue microarray and imaging analysis

    PubMed Central

    Kai, Keita; Masuda, Masanori; Aishima, Shinichi

    2017-01-01

    AIM To investigated the association between the tumor cells’ expression of E-cadherin and the numbers of several types of inflammatory cells infiltrating into the invasive portion of gallbladder cancer (GBC). METHODS We analyzed 50 GBC cases for which a sufficient amount of tumor tissues for tissue microarray (TMA) had been saved. Three tissue cores (3.0 mm) of invasive lesion from each case were used for the TMA. The 4-μm cut sections on slides were immunostained using primary antibodies including E-cadherin for cancer cells, leukocyte common antigen for leukocyte, myeloperoxidase for neutrophils, CD3 for T cells, CD4 for helper T cells, CD8 for killer T cells, CD20 for B cells and CD68 for macrophages. The immunostained slides were digitally analyzed by imaging analysis software. RESULTS A significant inverse correlation between the number of infiltrating CD8+ cells at invasive areas and the expression of E-cadherin by cancer cells was observed (P = 0.0001), although the degree of this correlation was relatively weak (R = 0.32). The number of CD8+ cells and the cancer cells’ E-cadherin expression were also significantly correlated with tumor differentiation (well-differentiated vs poorly differentiated) (P = 0.0467 and P = 0.0294, respectively). Inverse correlation of T-stage and the number of CD8+ cell infiltration was observed with statistical significance in comparison of T2 and T3 cases (P = 0.0324). CONCLUSION Our findings indicate an inverse correlation of CD8+ T cell infiltration and cancer cells’ E-cadherin expression at invasive areas of GBC. Further analyses are essential to test these findings. PMID:28138440

  15. Validation of a low-cost modified technique for constructing tissue microarrays for canine mammary tumor analysis.

    PubMed

    Silva, Franciele Basso Fernandes; Leite, Juliana da Silva; de Mello, Marcela Freire Vallim; Ferreira, Ana Maria Reis

    2016-09-01

    Compared with conventional histological paraffin blocks, tissue microarray (TMA) represents a "high-throughput tool" that provides rapid results, a time- and cost-effective approach and simultaneous investigation of several tissue samples under the same conditions. Given the large number of cases of dogs affected with mammary tumors, the complexity of these tumors and their similarity with breast cancer in women, this study aimed to validate a low-cost modified method to construct TMAs for canine mammary tumor analysis using immunomarkers. Carcinoma cases were selected from canine mammary carcinomas in mixed tumors (CMT) because this tumor type is the most heterogeneous among the histopathological types of mammary tumors observed in female dogs. Through a histopathological examination, tumor representativity was compared between conventional sections and histological sections obtained from the TMA block; both were stained with hematoxylin and eosin. An immunohistochemistry analysis was performed to compare the percentages of immunoreactive cells obtained in whole tissue sections versus those obtained from sections from the TMA block. Streptavidin-biotin peroxidase complex and anti-PCNA, anti-vimentin and anti-pancytokeratin antibodies were used. Statistical analysis consisted of the nonparametric Friedman's test (p≤0.05) and descriptive statistical analysis. Histopathological analysis showed tumor representativity in all TMA cores selected for the study. There was no difference between the immunohistochemical analysis of mammary tumors using conventional histological sections or sections obtained from a single 1-mm-diameter TMA core, regardless of the marker used: PCNA (p=0.279), pancytokeratin (p=0.243) and vimentin (p=0.967). The results did not change even when the means of any number of cores were compared among each other and with the conventional histological section: PCNA (p=0.413), pancytokeratin (p=0.177) and vimentin (p=1.0). Therefore, this study

  16. 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.

  17. Comparison between whole mount tissue preparations and virtual tissue microarray samples for measuring Ki-67 and apoptosis indices in human bladder cancer

    PubMed Central

    Oshiro, Hisashi; Czerniak, Bogdan A.; Sakamaki, Kentaro; Tsuta, Koji; Bondaruk, Jolanta; Keyhani, Afsaneh; Dinney, Colin P.; Nagai, Takeshi; Kamat, Ashish M.

    2016-01-01

    Abstract Recent tissue microarray (TMA)-based studies have shown that cell proliferation- and apoptosis-related biomarkers are associated with clinical outcomes in patients with bladder urothelial carcinoma. However, little is known about the differences in these biomarker measurements between whole mount tissue preparations and TMAs. This study aimed to elucidate the discrepancy in the measurements of Ki-67 indices (KIs) and apoptosis indices (AIs) between whole mount tissue preparations and TMAs of bladder urothelial carcinoma samples. Whole mount tissue preparations for Ki-67 immunohistochemistry and terminal deoxynucleotidyl transferase dUTP nick end labeling were made from 30 patients who underwent transurethral resection of bladder urothelial carcinoma. Digital microscopy-assisted virtual TMAs, consisting of 3 small round areas (1 or 0.6 mm in diameter), were generated from the same whole mount tissue preparations. The measurement results in highly reactive areas of biomarkers were compared between the whole mount tissue preparation- and the TMA-based methods. Bland–Altman plot analysis, regression analysis, and Kendall τ were performed to investigate differences in the measurement results, systematic biases, and correlations between biomarkers. Although the Bland–Altman plot analysis demonstrated that almost all the plots were within the limits of agreement, fixed biases were detected in the 1- and 0.6-mm TMAs for the KI (0.181 and 0.222, respectively) and the AI (0.055 and 0.063, respectively). Proportional biases were also detected in the 1- and 0.6-mm TMAs for the AI (P < 0.001 and P < 0.001, respectively). Furthermore, positive correlations between KIs and AIs were observed in whole mount tissue preparations (r = 0.260, P = 0.044) and in the 1 mm TMAs (r = 0.375, P = 0.004); however, no such correlation was observed in the 0.6 mm TMAs. Our study suggests that the measurement results for certain biomarkers of bladder

  18. Comparison between whole mount tissue preparations and virtual tissue microarray samples for measuring Ki-67 and apoptosis indices in human bladder cancer: A cross-sectional study.

    PubMed

    Oshiro, Hisashi; Czerniak, Bogdan A; Sakamaki, Kentaro; Tsuta, Koji; Bondaruk, Jolanta; Keyhani, Afsaneh; Dinney, Colin P; Nagai, Takeshi; Kamat, Ashish M

    2016-08-01

    Recent tissue microarray (TMA)-based studies have shown that cell proliferation- and apoptosis-related biomarkers are associated with clinical outcomes in patients with bladder urothelial carcinoma. However, little is known about the differences in these biomarker measurements between whole mount tissue preparations and TMAs. This study aimed to elucidate the discrepancy in the measurements of Ki-67 indices (KIs) and apoptosis indices (AIs) between whole mount tissue preparations and TMAs of bladder urothelial carcinoma samples.Whole mount tissue preparations for Ki-67 immunohistochemistry and terminal deoxynucleotidyl transferase dUTP nick end labeling were made from 30 patients who underwent transurethral resection of bladder urothelial carcinoma. Digital microscopy-assisted virtual TMAs, consisting of 3 small round areas (1 or 0.6 mm in diameter), were generated from the same whole mount tissue preparations. The measurement results in highly reactive areas of biomarkers were compared between the whole mount tissue preparation- and the TMA-based methods. Bland-Altman plot analysis, regression analysis, and Kendall τ were performed to investigate differences in the measurement results, systematic biases, and correlations between biomarkers.Although the Bland-Altman plot analysis demonstrated that almost all the plots were within the limits of agreement, fixed biases were detected in the 1- and 0.6-mm TMAs for the KI (0.181 and 0.222, respectively) and the AI (0.055 and 0.063, respectively). Proportional biases were also detected in the 1- and 0.6-mm TMAs for the AI (P < 0.001 and P < 0.001, respectively). Furthermore, positive correlations between KIs and AIs were observed in whole mount tissue preparations (r = 0.260, P = 0.044) and in the 1 mm TMAs (r = 0.375, P = 0.004); however, no such correlation was observed in the 0.6 mm TMAs.Our study suggests that the measurement results for certain biomarkers of bladder urothelial

  19. High throughput tissue microarray analysis of FHIT expression in diffuse large cell B-cell lymphoma from Saudi Arabia.

    PubMed

    Al Kuraya, Khawla; Siraj, Abdul Khalid; Bavi, Prashant; Al-Jomah, Naif; El-Solh, Hassan; Ezzat, Adnan; Al-Dayel, Fouad; Belgaumi, Asim; Al-Kofide, Amani; Sabbah, Rajeh; Sheikh, Salwa; Amr, Samir; Simon, Ronald; Sauter, Guido

    2006-08-01

    Recent studies have suggested a potential prognostic role of alterations of the fragile histidine triad (FHIT) gene in diffuse large B-cell lymphoma. To evaluate possible mechanisms of FHIT inactivation and to further clarify its potential prognostic relevance, we analyzed a set of 114 diffuse large B-cell lymphoma with clinical follow-up information. Tissue microarrays were analyzed by immunohistochemistry for protein expression, and corresponding DNA samples were analyzed for FHIT promotor hypermethlyation. Reduced or absent FHIT expression was found in 75 of 114 diffuse large B-cell lymphoma (66%), but was unrelated to clinical tumor stage or patient prognosis. FHIT promotor hypermethylation was observed in 29 of 93 (23%) interpretable diffuse large B-cell lymphoma. Hypermethylation was not significantly correlated to protein expression loss, which could be explained by competing mechanisms for FHIT inactivation in a substantial fraction of non FHIT hypermethylated diffuse large B-cell lymphoma. Hypermethylation was significantly associated with poor prognosis of diffuse large B-cell lymphoma patients and predominantly seen in nongerminal center diffuse large B-cell lymphoma (27%), but less frequent (13%) in germinal center diffuse large B-cell lymphoma. In summary, these data suggest that promotor hypermethylation is responsible for reduced FHIT expression in a substantial subset of diffuse large B-cell lymphoma, which is primarily composed of nongerminal center subtype with poor patient prognosis.

  20. Integrative proteomics and tissue microarray profiling indicate the association between overexpressed serum proteins and non-small cell lung cancer.

    PubMed

    Liu, Yansheng; Luo, Xiaoyang; Hu, Haichuan; Wang, Rui; Sun, Yihua; Zeng, Rong; Chen, Haiquan

    2012-01-01

    Lung cancer is the leading cause of cancer deaths worldwide. Clinically, the treatment of non-small cell lung cancer (NSCLC) can be improved by the early detection and risk screening among population. To meet this need, here we describe the application of extensive peptide level fractionation coupled with label free quantitative proteomics for the discovery of potential serum biomarkers for lung cancer, and the usage of Tissue microarray analysis (TMA) and Multiple reaction monitoring (MRM) assays for the following up validations in the verification phase. Using these state-of-art, currently available clinical proteomic approaches, in the discovery phase we confidently identified 647 serum proteins, and 101 proteins showed a statistically significant association with NSCLC in our 18 discovery samples. This serum proteomic dataset allowed us to discern the differential patterns and abnormal biological processes in the lung cancer blood. Of these proteins, Alpha-1B-glycoprotein (A1BG) and Leucine-rich alpha-2-glycoprotein (LRG1), two plasma glycoproteins with previously unknown function were selected as examples for which TMA and MRM verification were performed in a large sample set consisting about 100 patients. We revealed that A1BG and LRG1 were overexpressed in both the blood level and tumor sections, which can be referred to separate lung cancer patients from healthy cases.

  1. CD147 and Ki-67 overexpression confers poor prognosis in squamous cell carcinoma of oral tongue: A tissue microarray study

    PubMed Central

    Yu, Yau-Hua; Morales, Jose; Feng, Lei; Lee, J. Jack; El-Naggar, Adel K.; Vigneswaran, Nadarajah

    2015-01-01

    Squamous cell carcinoma of the oral tongue (SCCOT) exhibits high risk for recurrence and regional metastasis even after surgical resection. We assessed the clinicopathologic and prognostic significance of a group of functionally related biomarkers. We used a tissue microarray consisting SCCOT from 32 patients for this study. These patients were treated at the UT- M.D. Anderson Cancer Center from 1995 to 2008. Biomarker expression levels were examined by immunohistochemistry and graded semiquantitatively to determine their prognostic significance. CD147 and Tp63 expressions were significantly associated with a higher T-stage and Ki-67 labelling index as well as shorter overall survival (OS). Expression of Tp63 associated positively with poorly-differentiated histology. There was significant association of Tp63 with the expression levels of CD147 and Glut-1. Glut-1 overexpression was marginally associated with a higher T-stage. There was no prognostic significance of CD44v6 expression in SCCOT. SCCOT with CD147 overexpression in combination with high Ki-67 labelling index had poor OS. CD147 and Ki-67 overexpression is associated with aggressive disease with poor prognosis in SCCOT. PMID:25747176

  2. 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

  3. Overview of Protein Microarrays

    PubMed Central

    Reymond Sutandy, FX; Qian, Jiang; Chen, Chien-Sheng; Zhu, Heng

    2013-01-01

    Protein microarray is an emerging technology that provides a versatile platform for characterization of hundreds of thousands of proteins in a highly parallel and high-throughput way. Two major classes of protein microarrays are defined to describe their applications: analytical and functional protein microarrays. In addition, tissue or cell lysates can also be fractionated and spotted on a slide to form a reverse-phase protein microarray. While the fabrication technology is maturing, applications of protein microarrays, especially functional protein microarrays, have flourished during the past decade. Here, we will first review recent advances in the protein microarray technologies, and then present a series of examples to illustrate the applications of analytical and functional protein microarrays in both basic and clinical research. The research areas will include detection of various binding properties of proteins, study of protein posttranslational modifications, analysis of host-microbe interactions, profiling antibody specificity, and identification of biomarkers in autoimmune diseases. 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:23546620

  4. Hippocampus neuronal metabolic gene expression outperforms whole tissue data in accurately predicting Alzheimer's disease progression.

    PubMed

    Stempler, Shiri; Waldman, Yedael Y; Wolf, Lior; Ruppin, Eytan

    2012-09-01

    Numerous metabolic alterations are associated with the impairment of brain cells in Alzheimer's disease (AD). Here we use gene expression microarrays of both whole hippocampus tissue and hippocampal neurons of AD patients to investigate the ability of metabolic gene expression to predict AD progression and its cognitive decline. We find that the prediction accuracy of different AD stages is markedly higher when using neuronal expression data (0.9) than when using whole tissue expression (0.76). Furthermore, the metabolic genes' expression is shown to be as effective in predicting AD severity as the entire gene list. Remarkably, a regression model from hippocampal metabolic gene expression leads to a marked correlation of 0.57 with the Mini-Mental State Examination cognitive score. Notably, the expression of top predictive neuronal genes in AD is significantly higher than that of other metabolic genes in the brains of healthy subjects. All together, the analyses point to a subset of metabolic genes that is strongly associated with normal brain functioning and whose disruption plays a major role in AD.

  5. Using Ambystoma mexicanum (Mexican Axolotl) Embryos, Chemical Genetics, and Microarray Analysis to Identify Signaling Pathways Associated with Tissue Regeneration

    PubMed Central

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

    2015-01-01

    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 7 days 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

  6. Immunohistochemical expression of ARID1A in penile squamous cell carcinomas: a tissue microarray study of 112 cases.

    PubMed

    Faraj, Sheila F; Chaux, Alcides; Gonzalez-Roibon, Nilda; Munari, Enrico; Cubilla, Antonio L; Shih, Ie-Ming; Netto, George J

    2015-05-01

    ARID1A, a member of the chromatin remodeling genes family, has been suggested as a novel tumor suppressor gene in gynecologic malignancies. However, its role in penile cancer has yet to be determined. This study assesses the immunohistochemical expression of ARID1A in penile squamous cell carcinoma (SCC) and its association with pathologic features, human papillomavirus (HPV) status, and previously reported mammalian target of rapamycin pathway markers in the same cohort. Four tissue microarrays were constructed from 112 cases of formalin-fixed, paraffin-embedded penile SCC from Paraguay. Each tumor was sampled 3 to 12 times. ARID1A expression was evaluated by immunohistochemistry using a polyclonal rabbit anti-ARID1A (BAF250A) antibody. An H score was calculated in each spot as the sum of expression intensity (0-3+) by extent (0%-100%). Median H score per case was used for statistical analysis. ARID1A expression was observed in all cases, ranging from 3% to 100% of tumor cells (median, 95%). In 96 cases (86%), ARID1A expression was observed in 90% or more tumor cells. HPV DNA was detected in 20 (38%) of 52 analyzed samples. There was a significant trend of association between ARID1A and histologic grade. ARID1A expression was not associated with histologic subtype (P = .61) or HPV status (P = .18). ARID1A expression decreased with decreasing levels of PTEN expression (P = .01). ARID1A was expressed in penile SCC, in most cases at high levels. A significant trend of association was found between histologic grade and ARID1A expression, with lower ARID1A expression, lower histologic grades, and decreased PTEN expression.

  7. Immunohistochemical expression of ARID1A in penile squamous cell carcinomas: a tissue microarray study of 112 cases☆

    PubMed Central

    Faraj, Sheila F.; Chaux, Alcides; Gonzalez-Roibon, Nilda; Munari, Enrico; Cubilla, Antonio L.; Shih, Ie-Ming; Netto, George J.

    2015-01-01

    Summary ARID1A, a member of the chromatin remodeling genes family, has been suggested as a novel tumor suppressor gene in gynecologic malignancies. However, its role in penile cancer has yet to be determined. This study assesses the immunohistochemical expression of ARID1A in penile squamous cell carcinoma (SCC) and its association with pathologic features, human papillomavirus (HPV) status, and previously reported mammalian target of rapamycin pathway markers in the same cohort. Four tissue microarrays were constructed from 112 cases of formalin-fixed, paraffin-embedded penile SCC from Paraguay. Each tumor was sampled 3 to 12 times. ARID1A expression was evaluated by immunohistochemistry using a polyclonal rabbit anti-ARID1A (BAF250A) antibody. An H score was calculated in each spot as the sum of expression intensity (0-3+) by extent (0%-100%). Median H score per case was used for statistical analysis. ARID1A expression was observed in all cases, ranging from 3% to 100% of tumor cells (median, 95%). In 96 cases (86%), ARID1A expression was observed in 90% or more tumor cells. HPV DNA was detected in 20 (38%) of 52 analyzed samples. There was a significant trend of association between ARID1A and histologic grade. ARID1A expression was not associated with histologic subtype (P = .61) or HPV status (P = .18). ARID1A expression decreased with decreasing levels of PTEN expression (P = .01). ARID1A was expressed in penile SCC, in most cases at high levels. A significant trend of association was found between histologic grade and ARID1A expression, with lower ARID1A expression, lower histologic grades, and decreased PTEN expression. PMID:25776029

  8. 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.

  9. Best immunohistochemical panel in distinguishing adenocarcinoma from squamous cell carcinoma of lung: tissue microarray assay in resected lung cancer specimens.

    PubMed

    Kim, Mi Jin; Shin, Hyeong Chan; Shin, Kyeong Cheol; Ro, Jae Y

    2013-02-01

    The emergence of the targeted therapies for non-small cell lung carcinoma (NSCLC) has generated a need for accurate histologic subtyping of NSCLC. In this study, we assessed the utility of immunohistochemical markers that could be helpful in distinction between adenocarcinoma (ADC) and squamous cell carcinoma (SCC). We performed a battery of immunohistochemistry using tissue microarray for napsin-A, Thyroid transcription factor 1 (TTF-1), p63, cytokeratin (CK) 5/6, thrombomodulin (CD141), Epithelial-related antigen (MOC-31), carcinoembryonic antigen (CEA), Cyclooxygenase 2 (COX-2), high-molecular-weight CK (HMWCK), p27kip1 (p27), and Rb protein in 129 resected primary NSCLC with 81 ADCs and 48 SCCs and 10 metastatic ADC to the lung (primary in colon, 7 cases; stomach, 2 cases; vagina, 1 case). Cases of ADC and SCC were morphologically unequivocal and solid tumors with no definite squamous or glandular differentiation were excluded for this analysis. Napsin-A and TTF-1 were positive in 81% and 70% of ADC and in 0% and 2% of SCC, respectively, whereas P63 and CK5/6 were positive in 91% and 90% of SCC and in 9% and 4% of ADC, respectively (P < .001). CD141 stained significantly higher in SCC over ADC (positive in 2% of ADC and 46% of SCC. MOC-31, CEA, COX-2, HMWCK, p27, and Rb appeared to be not useful markers in distinction between ADC and SCC because of their low specificity. None of metastatic ADC to the lung showed positive for napsin-A and TTF-1. It was evident that combination of napsin-A, TTF-1, CK5/6, and p63 was the best immunohistochemical panel in differentiating ADC from SCC of the lung in this study. CD141 appeared to be a potential new marker for SCC with high specificity. Cyclooxygenase 2, MOC-31, CEA, HMWCK, p27, and Rb showed less specificity for differentiation ADC from SCC.

  10. Immunohistochemical expression of minichromosome maintenance complex protein 2 predicts biochemical recurrence in prostate cancer: a tissue microarray and digital imaging analysis-based study of 428 cases.

    PubMed

    Toubaji, Antoun; Sutcliffe, Siobhan; Chaux, Alcides; Lecksell, Kristen; Hicks, Jessica; De Marzo, Angelo M; Platz, Elizabeth A; Netto, George J

    2012-11-01

    Prostate cancer remains a major health problem in the United States. Established clinicopathologic parameters such as Gleason score, T stage, and prostate-specific antigen levels are currently the guiding tools for prognostication and disease management. The addition of biomarkers could increase the accuracy of these parameters for predicting disease progression, response to therapy, and survival. In this regard, the goal of this study was to evaluate minichromosome maintenance complex protein 2 and Ki-67 immunohistochemical expression as predictors of outcome in prostate cancer. For this purpose, 11 tissue microarrays were constructed using tumor and nontumor samples from 428 patients. Patients were divided into short-term (mean, 2.9 years) and long-term (mean, 14.1 years) follow-up groups. End points were biochemical recurrence for the short-term follow-up group and prostate cancer-related death for the long-term follow-up group. All men in the long-term follow-up group had biochemical recurrence at the time of recruitment. Expression of both markers was higher in tumor than in nontumor glands. Percentage of minichromosome maintenance complex protein 2 was associated with Gleason score in both groups. Percentage of Ki-67 was associated with Gleason score and pathologic stage only in the short-term follow-up group. Higher minichromosome maintenance complex protein 2 percentages were associated with biochemical recurrence in the short-term follow-up group. In the long-term follow-up group, neither minichromosome maintenance complex protein 2 nor Ki-67 levels predicted prostate cancer death. In conclusion, our results suggest that in patients treated by radical prostatectomy for clinically localized prostate cancer, immunohistochemistry for minichromosome maintenance complex protein 2 expression could be used to predict biochemical recurrence, independent of other known clinicopathologic factors.

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

    PubMed

    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.

  12. High‐throughput automated scoring of Ki67 in breast cancer tissue microarrays from the Breast Cancer Association Consortium

    PubMed Central

    Howat, William J; Daley, Frances; Zabaglo, Lila; McDuffus, Leigh‐Anne; Blows, Fiona; Coulson, Penny; Raza Ali, H; Benitez, Javier; Milne, Roger; Brenner, Herman; Stegmaier, Christa; Mannermaa, Arto; Chang‐Claude, Jenny; Rudolph, Anja; Sinn, Peter; Couch, Fergus J; Tollenaar, Rob A.E.M.; Devilee, Peter; Figueroa, Jonine; Sherman, Mark E; Lissowska, Jolanta; Hewitt, Stephen; Eccles, Diana; Hooning, Maartje J; Hollestelle, Antoinette; WM Martens, John; HM van Deurzen, Carolien; Investigators, kConFab; Bolla, Manjeet K; Wang, Qin; Jones, Michael; Schoemaker, Minouk; Broeks, Annegien; van Leeuwen, Flora E; Van't Veer, Laura; Swerdlow, Anthony J; Orr, Nick; Dowsett, Mitch; Easton, Douglas; Schmidt, Marjanka K; Pharoah, Paul D; Garcia‐Closas, Montserrat

    2016-01-01

    Abstract Automated methods are needed to facilitate high‐throughput and reproducible scoring of Ki67 and other markers in breast cancer tissue microarrays (TMAs) in large‐scale studies. To address this need, we developed an automated protocol for Ki67 scoring and evaluated its performance in studies from the Breast Cancer Association Consortium. We utilized 166 TMAs containing 16,953 tumour cores representing 9,059 breast cancer cases, from 13 studies, with information on other clinical and pathological characteristics. TMAs were stained for Ki67 using standard immunohistochemical procedures, and scanned and digitized using the Ariol system. An automated algorithm was developed for the scoring of Ki67, and scores were compared to computer assisted visual (CAV) scores in a subset of 15 TMAs in a training set. We also assessed the correlation between automated Ki67 scores and other clinical and pathological characteristics. Overall, we observed good discriminatory accuracy (AUC = 85%) and good agreement (kappa = 0.64) between the automated and CAV scoring methods in the training set. The performance of the automated method varied by TMA (kappa range= 0.37–0.87) and study (kappa range = 0.39–0.69). The automated method performed better in satisfactory cores (kappa = 0.68) than suboptimal (kappa = 0.51) cores (p‐value for comparison = 0.005); and among cores with higher total nuclei counted by the machine (4,000–4,500 cells: kappa = 0.78) than those with lower counts (50–500 cells: kappa = 0.41; p‐value = 0.010). Among the 9,059 cases in this study, the correlations between automated Ki67 and clinical and pathological characteristics were found to be in the expected directions. Our findings indicate that automated scoring of Ki67 can be an efficient method to obtain good quality data across large numbers of TMAs from multicentre studies. However, robust algorithm development and rigorous pre‐ and post

  13. High-throughput automated scoring of Ki67 in breast cancer tissue microarrays from the Breast Cancer Association Consortium.

    PubMed

    Abubakar, Mustapha; Howat, William J; Daley, Frances; Zabaglo, Lila; McDuffus, Leigh-Anne; Blows, Fiona; Coulson, Penny; Raza Ali, H; Benitez, Javier; Milne, Roger; Brenner, Herman; Stegmaier, Christa; Mannermaa, Arto; Chang-Claude, Jenny; Rudolph, Anja; Sinn, Peter; Couch, Fergus J; Tollenaar, Rob A E M; Devilee, Peter; Figueroa, Jonine; Sherman, Mark E; Lissowska, Jolanta; Hewitt, Stephen; Eccles, Diana; Hooning, Maartje J; Hollestelle, Antoinette; Wm Martens, John; Hm van Deurzen, Carolien; Investigators, kConFab; Bolla, Manjeet K; Wang, Qin; Jones, Michael; Schoemaker, Minouk; Broeks, Annegien; van Leeuwen, Flora E; Van't Veer, Laura; Swerdlow, Anthony J; Orr, Nick; Dowsett, Mitch; Easton, Douglas; Schmidt, Marjanka K; Pharoah, Paul D; Garcia-Closas, Montserrat

    2016-07-01

    Automated methods are needed to facilitate high-throughput and reproducible scoring of Ki67 and other markers in breast cancer tissue microarrays (TMAs) in large-scale studies. To address this need, we developed an automated protocol for Ki67 scoring and evaluated its performance in studies from the Breast Cancer Association Consortium. We utilized 166 TMAs containing 16,953 tumour cores representing 9,059 breast cancer cases, from 13 studies, with information on other clinical and pathological characteristics. TMAs were stained for Ki67 using standard immunohistochemical procedures, and scanned and digitized using the Ariol system. An automated algorithm was developed for the scoring of Ki67, and scores were compared to computer assisted visual (CAV) scores in a subset of 15 TMAs in a training set. We also assessed the correlation between automated Ki67 scores and other clinical and pathological characteristics. Overall, we observed good discriminatory accuracy (AUC = 85%) and good agreement (kappa = 0.64) between the automated and CAV scoring methods in the training set. The performance of the automated method varied by TMA (kappa range= 0.37-0.87) and study (kappa range = 0.39-0.69). The automated method performed better in satisfactory cores (kappa = 0.68) than suboptimal (kappa = 0.51) cores (p-value for comparison = 0.005); and among cores with higher total nuclei counted by the machine (4,000-4,500 cells: kappa = 0.78) than those with lower counts (50-500 cells: kappa = 0.41; p-value = 0.010). Among the 9,059 cases in this study, the correlations between automated Ki67 and clinical and pathological characteristics were found to be in the expected directions. Our findings indicate that automated scoring of Ki67 can be an efficient method to obtain good quality data across large numbers of TMAs from multicentre studies. However, robust algorithm development and rigorous pre- and post-analytical quality control procedures are

  14. 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.

  15. Application of in situ hybridization probes for MLH-1 and MSH-2 in tissue microarrays of paraffin-embedded malignant melanomas: correlation with immunohistochemistry and tumor stage.

    PubMed

    Korabiowska, Monika; Cordon-Cardo, Carlos; Jaenckel, Fredericke; Stachura, Jerzy; Fischer, Gösta; Brinck, Ulrich

    2004-12-01

    Defects in DNA mismatch-repair genes MLH1 and MSH2 reported primarily in hereditary nonpolyposis colorectal carcinoma are present in many sporadic tumors, including malignant melanomas. The main aim of this study was to investigate the expression of these genes in malignant melanomas in relation to tumor stage. An experiment was performed on paraffin-embedded tissue microarrays of malignant melanomas applying in situ hybridization with probes produced by our research group and immunohistochemical techniques. In situ hybridization demonstrated MLH1 expression in 45 of 59 melanomas and MSH2 expression in 51 of 59 melanomas. Immunohistochemistry detected MLH1 expression in 46 of 59 melanomas and MSH2 expression in 50 of 59 melanomas. Down-regulation of expression of both DNA mismatch repair genes in malignant melanomas was observed. The findings obtained by in situ hybridization and immunohistochemistry correlated significantly. Our study demonstrates the suitability of in situ hybridization with MLH1 and MSH2 probes for paraffin-embedded tissue. Tissue microarrays can be used successfully in both in situ hybridization and immunohistochemistry to analyze the expression of DNA mismatch-repair genes.

  16. 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.

  17. 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…

  18. 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

  19. Functional tissue engineering: Ten more years of progress.

    PubMed

    Guilak, Farshid; Baaijens, Frank P T

    2014-06-27

    "Functional tissue engineering" is a subset of the field of tissue engineering that was proposed by the United States National Committee on Biomechanics over a decade ago in order to place more emphasis on the roles of biomechanics and mechanobiology in tissue repair and regeneration. Over the past decade, there have been tremendous advances in this area, pointing out the critical role that biomechanical factors can play in the engineered repair of virtually all tissue and organ systems. In this special issue of the Journal of Biomechanics, we present a series of articles that address a broad array of the fundamental topics of functional tissue engineering, including: (1) measurement and modeling of the in vivo biomechanical environment and history in native and repair tissues; (2) further understanding of the biomechanical properties of native tissues across all geometric scales, in the context of repair or regeneration; (3) prioritization of specific biomechanical properties as design criteria; (4) development of biomaterials, scaffolds, and engineered tissues with prescribed biomechanical properties; (5) development of success criteria based on appropriate outcome measures; (6) investigation of the effects of mechanical factors on tissue repair in vivo; (7) investigation of the mechanisms by which physical factors may enhance tissue regeneration in vitro; and (8) development and validation of computational models of tissue growth and remodeling. These articles represent the tremendous expansion of this field in recent years, and emphasize the critical roles that biomechanics and mechanobiology play in controlling tissue repair and regeneration.

  20. 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.

  1. Feasibility of using tissue microarrays for the assessment of HER-2 gene amplification by fluorescence in situ hybridization in breast carcinoma.

    PubMed

    Bhargava, Rohit; Lal, Priti; Chen, Beiyun

    2004-12-01

    Tissue microarrays (TMAs) have been commonly used to study protein expression by immunohistochemistry (IHC). However, limited data exist on the validity of using TMAs to study gene amplification. In this study, we evaluated the feasibility of using breast carcinoma TMAs to study HER-2 gene amplification by fluorescence in situ hybridization (FISH). In addition, hormonal receptor status (ER and PR) and HER-2 protein overexpression by IHC were also studied, and results were compared with whole tissue sections. FISH for HER-2 was performed on formalin-fixed paraffin-embedded tissue from 114 invasive breast carcinomas both on whole tissue sections and on TMAs containing the same tumors. The TMA was created using 0.6-mm tissue cores with four sampled cores per tumor from the same tissue block used for whole section FISH. The PathVysion HER-2 probe kit was used for the FISH analysis. A ratio of HER-2:Chromosome17 > or =2.0 was interpreted as positive for gene amplification. The ER or PR was interpreted as positive when nuclear staining was detected in more than 10% of tumor cells. The HER-2 IHC (HercepTest; DAKO Corp, Carpinteria, CA) results were interpreted as 0, 1+, 2+, and 3+ according to standard criteria. The FISH results in the TMA and whole sections were concordant in 99 out of 101 successfully analyzed cases (99%). The FISH scores were consistent among the two to four cores in the majority of the cases. ER and PR results were concordant between whole sections and TMA cores in 97% (107/110) and 89% (97/109) cases, respectively. The overall concordance for HER-2 status by IHC between whole sections and TMA cores was 86% (94 out of 109 cases). TMAs are a reliable approach to study HER-2 gene amplification in a high throughput manner.

  2. Increased β‑catenin and c-myc expression predict aggressive growth of non-functioning pituitary adenomas: An assessment using a tissue microarray-based approach.

    PubMed

    Liu, Chunhui; Wu, Youtu; Yu, Shengyuan; Bai, Jiwei; Li, Chuzhong; Wu, Dan; Zhang, Yazhuo

    2017-04-01

    Non-functional pituitary adenomas (NFPAs) account for 80% of pituitary adenomas with the majority of these exhibiting recurrences post-surgery. Overexpression of β-catenin and c‑myc is common in numerous invasive tumors. The present study sought to investigate the correlation of β‑catenin and c‑myc expression levels with aggressive growth and recurrence of NFPAs, using immunohistochemical examination of tissue microarrays. Tissue microarrays comprised 212 NFPAs specimens and 10 healthy specimens as controls. NFPAs were categorized as non‑aggressive or aggressive. Immunohistochemical examination was performed to determine the expression of β‑catenin and c‑myc. Correlation of the expression levels of β‑catenin and c‑myc with clinicopathological parameters, including aggressiveness and recurrence, were assessed by univariate, multivariate and logistic regression analysis. Increased expression of β‑catenin and c‑myc was detected in the majority of aggressive NFPAs specimens (71.1 and 88.7%, respectively). There was a significant positive correlation between β‑catenin and c‑myc expression and aggressiveness [P=0.001, Odds Ratio (OR)=4.011; P<0.001, OR=30.833]. Only β‑catenin expression demonstrated a significant correlation with recurrence in NFPAs (P=0.021, OR=2.571). β‑catenin and c‑myc were demonstrated to be potential biomarkers for aggressive NFPAs and in the future, β-catenin may serve as a marker for aggressive behavior and recurrence in NFPAs.

  3. 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

  4. Distribution of the AQP4 water channel in normal human tissues: protein and tissue microarrays reveal expression in several new anatomical locations, including the prostate gland and seminal vesicles.

    PubMed

    Mobasheri, Ali; Marples, David; Young, Iain S; Floyd, Rachel V; Moskaluk, Christopher A; Frigeri, Antonio

    2007-01-01

    Aquaporins facilitate osmotically driven water movement across cell membranes. Aquaporin 4 (AQP4) is a major water channel in the central nervous system where it participates in cerebral water balance. AQP4 is also present in basolateral membranes of lower respiratory tract airway and renal collecting duct epithelial cells, gastric parietal cells and skeletal muscle cells. However, the distribution of AQP4 in many other tissues is still unknown. The aim of this study was to determine the expression and relative abundance of AQP4 in human Tissue MicroArrays (TMAs) and human protein microarrays by immunohistochemistry and chemiluminescence. In the central nervous system AQP4 was abundantly expressed in the cerebral cortex, cerebellar cortex (purkinje/granular layer), ependymal cell layer, hippocampus and spinal cord. Lower levels were detected in choroid plexus, white matter and meninges. In the musculoskeletal system AQP4 was highly expressed in the sarcolemma of skeletal muscle from the chest and neck. In the male genital system AQP4 was moderately expressed in seminiferous tubules, seminal vesicles, prostate and epidiymis. In the respiratory system AQP4 was moderately expressed in lung and bronchus. AQP expression was abundant in the kidney. In the gastrointestinal system AQP4 was moderately present in basolateral membranes of parietal cells at the base of gastric glands. AQP4 was also detected in salivary glands, adrenals, anterior pituitary, prostate and seminal vesicles. Human protein microarrays verified the TMA data. Our findings suggest that AQP4 is expressed more widely than previously thought in human organs and may be involved in prostatic and seminal fluid formation.

  5. Recent progress in interfacial tissue engineering approaches for osteochondral defects.

    PubMed

    Castro, Nathan J; Hacking, S Adam; Zhang, Lijie Grace

    2012-08-01

    This review provides a brief synopsis of the anatomy and physiology of the osteochondral interface, scaffold-based and non-scaffold based approaches for engineering both tissues independently as well as recent developments in the manufacture of gradient constructs. Novel manufacturing techniques and nanotechnology will be discussed with potential application in osteochondral interfacial tissue engineering.

  6. 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

  7. [Recent research progress of decellularization of native tissues].

    PubMed

    Dong, Jiaoming; Mo, Xiumei; Li, Yu; Chen, Dakai

    2012-10-01

    Biologic scaffold materials composed of extracellular matrix (ECM) are typically obtained in processes that involve decellularization of tissues or organs. Decellularized tissues and organs have been successfully used in a variety of tissue engineering/regenerative medicine applications. Preservation of the complex composition and three-dimensional ultrastructure of the ECM is highly desirable but it is recognized that all methods of decellularization result in disruption of the structure and potential loss of composition. The efficiency of cell removal from a tissue is dependent on the origin of the tissue and the physical, chemical, and enzymatic methods that are used. Each of these treatments affects the biochemical composition, tissue ultrastructure, and mechanical behavior of the remaining ECM scaffold, and all of the treatment methods affect the host response to the material as well. Tissue decellularization with preservation of ECM integrity and bioactivity can be optimized by making correct decisions regarding the agents and techniques utilized during processing. In this paper, the most commonly used decellularization methods are described, and consideration given to the effects of these methods upon the biologic scaffold material and recently described antigen removal strategy are presented.

  8. 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

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

    PubMed

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

    2012-01-01

    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.

  10. 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.

  11. 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

    Hassan, Hazem E; Myers, Alan L; Lee, Insong J; Chen, Hegang; Coop, Andrew; Eddington, Natalie D

    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.Dalpha, 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 x 10(-4)) and regulation of immune response (p = 5.090 x 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 (> or = 500 microM). Furthermore, brain uptake studies demonstrated that oxycodone-induced Abcg2 up-regulation resulted in a significant (p < 0.05) decrease (approximately 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

  12. Progress of National Multi-tissue Bank in Uruguay in the International Atomic Energy Agency (IAEA) Tissue Banking Programme.

    PubMed

    Alvarez, Inés; Del Carmen Saldías, Ma; Wodowoz, Olga; Pérez Campos, Héctor; Machin, Daniel; Silva, Walter; Sueta, Patricia; Pérez, Natalia; Acosta Md, Ma del Carmen

    2003-01-01

    The transplant law of 1971 based on informed consent, allows people to register their willingness to be a donor upon death. Since 1978 the governmental Institution, the National Bank of Organs and Tissues (BNOT), have been regulated the organ and tissue donation. Important progress was implemented in the BNOT and specially in the National Multi-tissue Bank (NMTB). Since 2001 with the participation in the IAEA Tissue Banking Programme, Quality System Management has been implemented in the NMTB. New bio-production for radiosterilized tissues for the first time and improved procedures were carried out. As a result an increased production of high-quality tissues was obtained and distributed for clinical use.

  13. 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.

  14. Gene therapy progress and prospects: in tissue engineering.

    PubMed

    Polak, J; Hench, L

    2005-12-01

    Tissue engineering (TE) has existed for several years as an area spanning many disciplines, including medicine and engineering. The use of stem cells as a biological basis for TE coupled with advances in materials science has opened up an entirely new chapter in medicine and holds the promise of major contributions to the repair, replacement and regeneration of damaged tissues and organs. In this article, we review the spectrum of stem cells and scaffolds being investigated for their potential applications in medicine.

  15. 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.

  16. Different detectability of cyclooxygenase-2 (COX-2) protein in standard paraffin sections and tissue microarrays of human melanomas and naevi - comparative study.

    PubMed

    Kuźbicki, Lukasz; Urban, Justyna; Chwirot, Barbara W

    2014-09-01

    Cyclooxygenase-2 (COX-2), overexpressed in many types of human cancer, may be a valuable marker for human melanoma. However, there are discrepancies between expression levels detected by different groups. The majority of the studies were carried out using standard paraffin sections. Tissue microarrays (TMAs) might enable analysis of COX-2 expression in numerous lesions. Our study assesses to what extent reprocessing of tissue samples used for preparing TMAs may influence reproducibility of data obtained for standard sections. The study included TMAs and standard histopathologic sections. COX-2 was detected by immunohistochemistry with two primary antibodies targeting different epitopes. COX-2 expression levels detected with both antibodies in standard sections were similar as in our previous study. Surprisingly, results obtained in TMAs were significantly different. While one of the antibodies yielded for TMAs results similar to standard sections, COX-2 expression levels found with the second antibody were very low and expression patterns strikingly different from those observed for standard sections and for both TMAs studied with the first antibody. Good performance of the antibodies found in standard sections of human skin and melanocytic lesions does not guarantee similar results in TMAs. The finding discloses a new aspect of immunohistochemical assays involving TMAs.

  17. Microarray expression profiles of genes in lung tissues of rats subjected to focal cerebral ischemia-induced lung injury following bone marrow-derived mesenchymal stem cell transplantation

    PubMed Central

    Hu, Yue; Xiong, Liu-Lin; Zhang, Piao; Wang, Ting-Hua

    2017-01-01

    Ischemia-induced stroke is the most common disease of the nervous system and is associated with a high mortality rate worldwide. Cerebral ischemia may lead to remote organ dysfunction, particular in the lungs, resulting in lung injury. Nowadays, bone marrow-derived mesenchymal stem cells (BMSCs) are widely studied in clinical trials as they may provide an effective solution to the treatment of neurological and cardiac diseases; however, the underlying molecular mechanisms remain unknown. In this study, a model of permanent focal cerebral ischemia-induced lung injury was successfully established and confirmed by neurological evaluation and lung injury scores. We demonstrated that the transplantation of BMSCs (passage 3) via the tail vein into the lung tissues attenuated lung injury. In order to elucidate the underlying molecular mechanisms, we analyzed the gene expression profiles in lung tissues from the rats with focal cerebral ischemia and transplanted with BMSCs using a Gene microarray. Moreover, the Gene Ontology database was employed to determine gene function. We found that the phosphoinositide 3-kinase (PI3K)-AKT signaling pathway, transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF) were downregulated in the BMSC transplantation groups, compared with the control group. These results suggested that BMSC transplantation may attenuate lung injury following focal cerebral ischemia and that this effect is associated with the downregulation of TGF-β, PDGF and the PI3K-AKT pathway. PMID:27922691

  18. Microarray expression profiles of genes in lung tissues of rats subjected to focal cerebral ischemia-induced lung injury following bone marrow-derived mesenchymal stem cell transplantation.

    PubMed

    Hu, Yue; Xiong, Liu-Lin; Zhang, Piao; Wang, Ting-Hua

    2017-01-01

    Ischemia-induced stroke is the most common disease of the nervous system and is associated with a high mortality rate worldwide. Cerebral ischemia may lead to remote organ dysfunction, particular in the lungs, resulting in lung injury. Nowadays, bone marrow-derived mesenchymal stem cells (BMSCs) are widely studied in clinical trials as they may provide an effective solution to the treatment of neurological and cardiac diseases; however, the underlying molecular mechanisms remain unknown. In this study, a model of permanent focal cerebral ischemia-induced lung injury was successfully established and confirmed by neurological evaluation and lung injury scores. We demonstrated that the transplantation of BMSCs (passage 3) via the tail vein into the lung tissues attenuated lung injury. In order to elucidate the underlying molecular mechanisms, we analyzed the gene expression profiles in lung tissues from the rats with focal cerebral ischemia and transplanted with BMSCs using a Gene microarray. Moreover, the Gene Ontology database was employed to determine gene function. We found that the phosphoinositide 3-kinase (PI3K)-AKT signaling pathway, transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF) were downregulated in the BMSC transplantation groups, compared with the control group. These results suggested that BMSC transplantation may attenuate lung injury following focal cerebral ischemia and that this effect is associated with the downregulation of TGF-β, PDGF and the PI3K-AKT pathway.

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

    PubMed

    Berthiaume, François; 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.

  20. Chromosome Microarray.

    PubMed

    Anderson, Sharon

    2016-01-01

    Over the last half century, knowledge about genetics, genetic testing, and its complexity has flourished. Completion of the Human Genome Project provided a foundation upon which the accuracy of genetics, genomics, and integration of bioinformatics knowledge and testing has grown exponentially. What is lagging, however, are efforts to reach and engage nurses about this rapidly changing field. The purpose of this article is to familiarize nurses with several frequently ordered genetic tests including chromosomes and fluorescence in situ hybridization followed by a comprehensive review of chromosome microarray. It shares the complexity of microarray including how testing is performed and results analyzed. A case report demonstrates how this technology is applied in clinical practice and reveals benefits and limitations of this scientific and bioinformatics genetic technology. Clinical implications for maternal-child nurses across practice levels are discussed.

  1. 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.

  2. Telomerase as a prognostic marker in breast cancer: high-throughput tissue microarray analysis of hTERT and hTR.

    PubMed

    Poremba, Christopher; Heine, Bernhard; Diallo, Raihanatou; Heinecke, Achim; Wai, Daniel; Schaefer, Karl-Ludwig; Braun, Yvonne; Schuck, Andreas; Lanvers, Claudia; Bànkfalvi, Agnes; Kneif, Sören; Torhorst, Joachim; Zuber, Markus; Köchli, Ossi R; Mross, Frank; Dieterich, Holger; Sauter, Guido; Stein, Harald; Fogt, Franz; Boecker, Werner

    2002-10-01

    Telomerase activity (TA) has been shown to correlate with poor clinical outcome in various tumour entities, indicating that tumours expressing this enzyme may be more aggressive and that TA may be a useful prognostic marker. For breast cancer, however, TA is a controversial prognostic marker; whereas some studies suggest an association between TA and disease outcome, others do not find this association. This study used tissue microarrays (breast carcinoma prognosis arrays) containing 611 samples (each 0.6 mm in diameter) from the tumour centre of paraffin-embedded breast carcinomas to analyse the catalytic subunit of telomerase, human telomerase reverse-transcriptase (hTERT), and the internal RNA component (hTR), which are the core components of the telomerase holoenzyme complex. hTERT protein expression was obtained by immunohistochemistry (human anti-telomerase antibody Ab-2, Calbiochem), and hTR RNA was measured by radioactive in situ hybridization. hTERT and hTR expression were determined semi-quantitatively and graded (scores 1-4). Clinical data, such as histological subtype, pT stage, tumour diameter, pN stage, BRE grade, tumour-specific survival (in months), patient's age and others, were available for statistical analysis. A statistically significant correlation was found between tumour-specific survival (overall survival) and hTERT expression (p < 0.0001) or hTR expression (p = 0.00110). Tumours with higher scores (scores 3, 4) for hTR and/or hTERT were associated with a worse prognosis. In multivariate analysis, hTERT expression was an independent prognostic factor. Previous studies, focusing on analysis of TA in smaller numbers of fresh-frozen breast carcinomas by the TRAP assay, gave controversial results with respect to TA as a prognostic marker. Using tissue microarrays from 611 breast carcinomas, this study has demonstrated that increased expression levels of the telomerase core components, hTERT and hTR, are associated with lower overall survival

  3. MALDI imaging mass spectrometry reveals multiple clinically relevant masses in colorectal cancer using large-scale tissue microarrays.

    PubMed

    Hinsch, Andrea; Buchholz, Malte; Odinga, Sinje; Borkowski, Carina; Koop, Christina; Izbicki, Jacob R; Wurlitzer, Marcus; Krech, Till; Wilczak, Waldemar; Steurer, Stefan; Jacobsen, Frank; Burandt, Eike-Christian; Stahl, Phillip; Simon, Ronald; Sauter, Guido; Schlüter, Hartmut

    2017-01-24

    For identification of clinically relevant masses to predict status, grade, relapse and prognosis of colorectal cancer we applied MALDI imaging mass spectrometry (IMS) to a tissue micro array (TMA) containing formalin-fixed and paraffin-embedded tissue samples from 349 patients. Analysis of our MALDI-IMS data revealed 27 different m/z signals associated with epithelial structures. Comparison of these signals showed significant association with status, grade and Ki-67 labeling index. 15 out of 27 IMS signals revealed a significant association with survival. For 7 signals (m/z 654, 776, 788, 904, 944, 975, and 1013) the absence, and for 8 signals (m/z 643, 678, 836, 886, 898, 1095, 1459, and 1477) the presence was associated with decreased life expectancy, including 5 masses (m/z 788, 836, 904, 944, and 1013) that provided prognostic information independently from the established prognosticators pT and pN. Combination of these 5 masses resulted in a 3-step classifier that provided prognostic information superior to univariate analysis. In addition, a total of 19 masses were associated with tumor stage, grade, metastasis, and cell proliferation. Our data demonstrate the suitability of combining IMS and large-scale TMAs to simultaneously identify and validate clinically useful molecular marker.

  4. Recent progresses in gene delivery-based bone tissue engineering.

    PubMed

    Lu, Chia-Hsin; Chang, Yu-Han; Lin, Shih-Yeh; Li, Kuei-Chang; Hu, Yu-Chen

    2013-12-01

    Gene therapy has converged with bone engineering over the past decade, by which a variety of therapeutic genes have been delivered to stimulate bone repair. These genes can be administered via in vivo or ex vivo approach using either viral or nonviral vectors. This article reviews the fundamental aspects and recent progresses in the gene therapy-based bone engineering, with emphasis on the new genes, viral vectors and gene delivery approaches.

  5. Comprehensive adipocytic and neurogenic tissue microarray analysis of NY-ESO-1 expression - a promising immunotherapy target in malignant peripheral nerve sheath tumor and liposarcoma

    PubMed Central

    Shurell, Elizabeth; Vergara-Lluri, Maria E.; Li, Yunfeng; Crompton, Joseph G.; Singh, Arun; Bernthal, Nicholas; Wu, Hong; Eilber, Fritz C.; Dry, Sarah M.

    2016-01-01

    Background Immunotherapy targeting cancer-testis antigen NY-ESO-1 shows promise for tumors with poor response to chemoradiation. Malignant peripheral nerve sheath tumors (MPNSTs) and liposarcomas (LPS) are chemoresistant and have few effective treatment options. Materials Methods Using a comprehensive tissue microarray (TMA) of both benign and malignant tumors in primary, recurrent, and metastatic samples, we examined NY-ESO-1 expression in peripheral nerve sheath tumor (PNST) and adipocytic tumors. The PNST TMA included 42 MPNSTs (spontaneous n = 26, NF1-associated n = 16), 35 neurofibromas (spontaneous n = 22, NF-1 associated n = 13), 11 schwannomas, and 18 normal nerves. The LPS TMA included 48 well-differentiated/dedifferentiated (WD/DD) LPS, 13 myxoid/round cell LPS, 3 pleomorphic LPS, 8 lipomas, 1 myelolipoma, and 3 normal adipocytic tissue samples. Stained in triplicate, NY-ESO-1 intensity and density were scored. Results NY-ESO-1 expression was exclusive to malignant tumors. 100% of myxoid/round cell LPS demonstrated NY-ESO-1 expression, while only 6% of WD/DD LPS showed protein expression, one of which was WD LPS. Of MPNST, 4/26 (15%) spontaneous and 2/16 (12%) NF1-associated MPNSTs demonstrated NY-ESO-1 expression. Strong NY-ESO-1 expression was observed in myxoid/round cell and dedifferentiated LPS, and MPNST in primary, neoadjuvant, and metastatic settings. Conclusions We found higher prevalence of NY-ESO-1 expression in MPNSTs than previously reported, highlighting a subset of MPNST patients who may benefit from immunotherapy. This study expands our understanding of NY-ESO-1 in WD/DD LPS and is the first demonstration of staining in a WD LPS and metastatic/recurrent myxoid/round cell LPS. These results suggest immunotherapy targeting NY-ESO-1 may benefit patients with aggressive tumors resistant to conventional therapy. PMID:27655679

  6. RUNX2 correlates with subtype-specific breast cancer in a human tissue microarray, and ectopic expression of Runx2 perturbs differentiation in the mouse mammary gland.

    PubMed

    McDonald, Laura; Ferrari, Nicola; Terry, Anne; Bell, Margaret; Mohammed, Zahra M; Orange, Clare; Jenkins, Alma; Muller, William J; Gusterson, Barry A; Neil, James C; Edwards, Joanne; Morris, Joanna S; Cameron, Ewan R; Blyth, Karen

    2014-05-01

    RUNX2, a master regulator of osteogenesis, is oncogenic in the lymphoid lineage; however, little is known about its role in epithelial cancers. Upregulation of RUNX2 in cell lines correlates with increased invasiveness and the capacity to form osteolytic disease in models of breast and prostate cancer. However, most studies have analysed the effects of this gene in a limited number of cell lines and its role in primary breast cancer has not been resolved. Using a human tumour tissue microarray, we show that high RUNX2 expression is significantly associated with oestrogen receptor (ER)/progesterone receptor (PR)/HER2-negative breast cancers and that patients with high RUNX2 expression have a poorer survival rate than those with negative or low expression. We confirm RUNX2 as a gene that has a potentially important functional role in triple-negative breast cancer. To investigate the role of this gene in breast cancer, we made a transgenic model in which Runx2 is specifically expressed in murine mammary epithelium under the control of the mouse mammary tumour virus (MMTV) promoter. We show that ectopic Runx2 perturbs normal development in pubertal and lactating animals, delaying ductal elongation and inhibiting lobular alveolar differentiation. We also show that the Runx2 transgene elicits age-related, pre-neoplastic changes in the mammary epithelium of older transgenic animals, suggesting that elevated RUNX2 expression renders such tissue more susceptible to oncogenic changes and providing further evidence that this gene might have an important, context-dependent role in breast cancer.

  7. 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

  8. 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...

  9. Cell-cycle and suppressor proteins expression in uterine cervix in HIV/HPV co-infection: comparative study by tissue micro-array (TMA)

    PubMed Central

    Nicol, Alcina F; Pires, Andréa Rodrigues Cordovil; de Souza, Simone R; Nuovo, Gerard J; Grinsztejn, Beatriz; Tristão, Aparecida; Russomano, Fabio B; Velasque, Luciane; e Silva, José R Lapa; Pirmez, Claude

    2008-01-01

    Background The oncoproteins of human papillomavirus (HPVs) directly effect cell-cycle control. We hypothesize that regulatory and cell cycle protein expression might be additionally modified in the cervix of HIV/HPV co-infected women. Methods We analyzed the expression of Rb, p27, VEGF and Elf-1 transcriptor factor by immunohistochemistry in 163 paraffin-embeded cervical samples using Tissue Micro-Array (TMA) and correlated this to HIV-1 and HPV infection. Results HIV/HPV co-infection was associated with a significant increase in expression (p < 0.001) of VEGF and p27 in both low and high grade CIN when compared to the cervices of women infected by HPV alone. Decreased Rb expression was evident with increased CIN grade in the cervices of women infected with HPV alone (p = 0.003 average of cells/mm2 in CIN I: 17.9, CIN II/III: 4.8, and tumor 3.9). Rb expression increased 3-fold for both low and high grade CIN with HPV/HIV-1 co-infection compared to HPV infection alone but did not reach statistical significance. There was a significant increase in Elf-1 expression in HPV+/HIV- women with CIN II/III and tumor (average of cells/mm2 in CIN I: 63.8; CIN II/III: 115.7 and tumor: 112.0, p = 0.005), in comparison to controls. Conclusion Co-infection of HPV and HIV leads to significant increase in the VEGF and p27 expression when compared to HPV+/HIV-negative infection that could facilitate viral persistence and invasive tumor development. PMID:18840277

  10. 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

  11. Up-regulation of cell cycle arrest protein BTG2 correlates with increased overall survival in breast cancer, as detected by immunohistochemistry using tissue microarray

    PubMed Central

    2010-01-01

    Background Previous studies have shown that the ADIPOR1, ADORA1, BTG2 and CD46 genes differ significantly between long-term survivors of breast cancer and deceased patients, both in levels of gene expression and DNA copy numbers. The aim of this study was to characterize the expression of the corresponding proteins in breast carcinoma and to determine their correlation with clinical outcome. Methods Protein expression was evaluated using immunohistochemistry in an independent breast cancer cohort of 144 samples represented on tissue microarrays. Fisher's exact test was used to analyze the differences in protein expression between dead and alive patients. We used Cox-regression multivariate analysis to assess whether the new markers predict the survival status of the patients better than the currently used markers. Results BTG2 expression was demonstrated in a significantly lower proportion of samples from dead patients compared to alive patients, both in overall expression (P = 0.026) and cell membrane specific expression (P = 0.013), whereas neither ADIPOR1, ADORA1 nor CD46 showed differential expression in the two survival groups. Furthermore, a multivariate analysis showed that a model containing BTG2 expression in combination with HER2 and Ki67 expression along with patient age performed better than a model containing the currently used prognostic markers (tumour size, nodal status, HER2 expression, hormone receptor status, histological grade, and patient age). Interestingly, BTG2 has previously been described as a tumour suppressor gene involved in cell cycle arrest and p53 signalling. Conclusions We conclude that high-level BTG2 protein expression correlates with prolonged survival in patients with breast carcinoma. PMID:20553615

  12. The epidermal growth factor receptor is frequently overexpressed in penile squamous cell carcinomas: a tissue microarray and digital image analysis study of 112 cases.

    PubMed

    Chaux, Alcides; Munari, Enrico; Katz, Betina; Sharma, Rajni; Lecksell, Kristen; Cubilla, Antonio L; Burnett, Arthur L; Netto, George J

    2013-12-01

    Disseminated penile cancer is usually treated with chemotherapy. However, response rates are far from acceptable. Recently, anti-epidermal growth factor receptor (EGFR) monoclonal antibodies have shown to be clinically useful in penile carcinomas. Nevertheless, only a few cases of penile carcinomas have been evaluated for EGFR expression. In this study, we assessed the immunohistochemical expression of EGFR in 112 patients with penile squamous cell carcinoma. We built 4 tissue microarrays and evaluated EGFR expression using a monoclonal mouse anti-EGFR antibody. For digital image analysis, we used the open-source software ImageJ version 1.47 (NIH, Bethesda, MD) along with the immunomembrane plug-in. Membranous EGFR expression was evaluated, taking into account staining completeness (0-10 points) and staining intensity (0-10 points) for a combined score (0-20 points). We classified the cases as follows: negative EGFR expression, 0 to 3 points; low EGFR expression, 4 to 8 points; and high EGFR expression, 9 to 20 points. The distribution of EGFR immunohistochemical expression was as follows: 13 cases (12%) were EGFR negative, 49 cases (44%) had low EGFR expression, and 50 cases (44%) had high EGFR expression. EGFR expression was not associated with histologic subtype (P = .47), histologic grade (P = .77), or human papillomavirus status (P = .14). In conclusion, immunohistochemical EGFR expression appears to be a common feature of penile carcinomas, independently of histologic subtype, histologic grade, and human papillomavirus presence. Whether or not EGFR expression is associated with EGFR gene mutation or if it can be used to predict response to therapy in patients with disseminated penile cancer should be evaluated in future studies.

  13. Comparison of in situ hybridization methods for the assessment of HER-2/neu gene amplification status in breast cancer using a tissue microarray

    PubMed Central

    Malicka-Durczak, Anna; Korski, Konstanty; Ibbs, Matthew

    2012-01-01

    Background This project compared HER-2/neu gene status in breast cancers, as demonstrated by FISH (fluorescent in situ hybridization) and CISH (chromogenic in situ hybridization) and using a tissue microarray (TMA). The study also aimed to show whether the TMA technique could be used in clinical diagnostics, rather than remain a scientific tool. Materials and methods A TMA was constructed using 121 breast cancer specimens, 6 cores from each specimen. Demonstration and assessment of HER-2/neu gene status was by FISH (Vysis Path) and CISH (DAKO Duo CISH). Results The 121 breast cancer specimens were divided into 3 groups by HER-2 status, as determined by immunohistochemistry. In the HER-2 negative group no amplification was observed in 36 out of 40 cases. 3 cases showed amplification by both methods and one by CISH alone. The equivocal HER-2 group showed no amplification in 30 out of 41 cases and amplification in 9 cases. One case was FISH negative CISH positive and one was discarded. In the HER-2 positive group, amplification was confirmed in 37 of the 40 cases by both methods. 3 cases were unsuitable for assessment. Conclusions This study indicated that CISH is a sensitive alternative to FISH in detecting HER2 gene amplification and may replace FISH in HER2 testing. Good agreement was observed between methods (98.5% – 119 out of 121 cases). Furthermore, as only 4 out of 121 cases were unsuitable for assessment (no signal or missing TMA cores) – it may be feasible to use TMA in diagnostics. PMID:24377000

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

    PubMed

    Tóth-Lipták, Judit; Piukovics, Klára; Borbényi, Zita; Demeter, Judit; Bagdi, Enikő; Krenács, László

    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.

  15. Microarrays in cancer research.

    PubMed

    Grant, Geraldine M; Fortney, Amanda; Gorreta, Francesco; Estep, Michael; Del Giacco, Luca; Van Meter, Amy; Christensen, Alan; Appalla, Lakshmi; Naouar, Chahla; Jamison, Curtis; Al-Timimi, Ali; Donovan, Jean; Cooper, James; Garrett, Carleton; Chandhoke, Vikas

    2004-01-01

    Microarray technology has presented the scientific community with a compelling approach that allows for simultaneous evaluation of all cellular processes at once. Cancer, being one of the most challenging diseases due to its polygenic nature, presents itself as a perfect candidate for evaluation by this approach. Several recent articles have provided significant insight into the strengths and limitations of microarrays. Nevertheless, there are strong indications that this approach will provide new molecular markers that could be used in diagnosis and prognosis of cancers. To achieve these goals it is essential that there is a seamless integration of clinical and molecular biological data that allows us to elucidate genes and pathways involved in various cancers. To this effect we are currently evaluating gene expression profiles in human brain, ovarian, breast and hematopoetic, lung, colorectal, head and neck and biliary tract cancers. To address the issues we have a joint team of scientists, doctors and computer scientists from two Virginia Universities and a major healthcare provider. The study has been divided into several focus groups that include; Tissue Bank Clinical & Pathology Laboratory Data, Chip Fabrication, QA/QC, Tissue Devitalization, Database Design and Data Analysis, using multiple microarray platforms. Currently over 300 consenting patients have been enrolled in the study with the largest number being that of breast cancer patients. Clinical data on each patient is being compiled into a secure and interactive relational database and integration of these data elements will be accomplished by a common programming interface. This clinical database contains several key parameters on each patient including demographic (risk factors, nutrition, co-morbidity, familial history), histopathology (non genetic predictors), tumor, treatment and follow-up information. Gene expression data derived from the tissue samples will be linked to this database, which

  16. 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.

  17. The prognostic implication of the expression of EGFR, p53, cyclin D1, Bcl-2 and p16 in primary locally advanced oral squamous cell carcinoma cases: a tissue microarray study.

    PubMed

    Solomon, Monica Charlotte; Vidyasagar, M S; Fernandes, Donald; Guddattu, Vasudev; Mathew, Mary; Shergill, Ankur Kaur; Carnelio, Sunitha; Chandrashekar, Chetana

    2016-12-01

    Oral squamous cell carcinomas comprise a heterogeneous tumor cell population with varied molecular characteristics, which makes prognostication of these tumors a complex and challenging issue. Thus, molecular profiling of these tumors is advantageous for an accurate prognostication and treatment planning. This is a retrospective study on a cohort of primary locally advanced oral squamous cell carcinomas (n = 178) of an Indian rural population. The expression of EGFR, p53, cyclin D1, Bcl-2 and p16 in a cohort of primary locally advanced oral squamous cell carcinomas was evaluated. A potential biomarker that can predict the tumor response to treatment was identified. Formalin-fixed paraffin-embedded tumor blocks of (n = 178) of histopathologically diagnosed cases of locally advanced oral squamous cell carcinomas were selected. Tissue microarray blocks were constructed with 2 cores of 2 mm diameter from each tumor block. Four-micron-thick sections were cut from these tissue microarray blocks. These tissue microarray sections were immunohistochemically stained for EGFR, p53, Bcl-2, cyclin D1 and p16. In this cohort, EGFR was the most frequently expressed 150/178 (84%) biomarker of the cases. Kaplan-Meier analysis showed a significant association (p = 0.038) between expression of p53 and a poor prognosis. A Poisson regression analysis showed that tumors that expressed p53 had a two times greater chance of recurrence (unadjusted IRR-95% CI 2.08 (1.03, 4.5), adjusted IRR-2.29 (1.08, 4.8) compared with the tumors that did not express this biomarker. Molecular profiling of oral squamous cell carcinomas will enable us to categorize our patients into more realistic risk groups. With biologically guided tumor characterization, personalized treatment protocols can be designed for individual patients, which will improve the quality of life of these patients.

  18. Adding the p16(INK4a) marker to the traditional 3-marker (ER/Vim/CEA) panel engenders no supplemental benefit in distinguishing between primary endocervical and endometrial adenocarcinomas in a tissue microarray study.

    PubMed

    Han, Chih-Ping; Lee, Ming-Yung; Kok, Lai-Fong; Ruan, Alexandra; Wu, Tina S; Cheng, Ya-Wen; Tyan, Yeu-Sheng; Lin, Ching-Yi

    2009-09-01

    Endocervical adenocarcinomas (ECAs) and endometrial adenocarcinomas (EMAs) are malignancies that affect the uterus; however, their biologic behaviors are quite different. This distinction has clinical significance, because the appropriate therapy may depend on the site of tumor origin. In this study, we not only compare the individual expression status of 4 immunomarkers [estrogen receptor (ER), vimentin (Vim), carcinoembryonic antigen (CEA), and p16], but also evaluate whether p16 adds value to the ER/Vim/CEA panel characteristics and performance in distinguishing between primary ECA and EMA. A tissue microarray (TMA) was constructed using paraffin-embedded, formalin-fixed tissues from 38 hysterectomy specimens, including 14 ECAs and 24 EMAs. Tissue microarray sections were immunostained with 4 antibodies, by the avidin-biotin complex method for antigen visualization. The staining intensity and area extent of the immunohistochemical reactions were evaluated using the semiquantitative scoring system. The 3 markers (ER, Vim, CEA) and their respective panel expressions showed statistically significant (P<0.05) frequency differences in ECA and EMA tumors. The p16 marker also revealed a significant frequency difference (P<0.05) between ECA and EMA, but did not demonstrate any supplementary benefit to the traditional 3-marker panel. In conclusion, when histomorphologic and clinical doubt exist as to the primary site of origin, we suggest that the conventional 3-marker (ER/Vim/CEA) panel is appropriate. Ancillary p16-marker testing does not add value to the 3-marker panel in distinguishing between primary ECA and EMA.

  19. Progress in reflectance confocal microscopy for imaging oral tissues in vivo

    NASA Astrophysics Data System (ADS)

    Peterson, Gary; Zanoni, Daniella K.; Migliacci, Jocelyn; Cordova, Miguel; Rajadhyaksha, Milind; Patel, Snehal

    2016-02-01

    We report progress in development and feasibility testing of reflectance confocal microscopy (RCM) for imaging in the oral cavity of humans. We adapted a small rigid relay telescope (120mm long x 14mm diameter) and a small water immersion objective lens (12mm diameter, NA 0.7) to a commercial handheld RCM scanner (Vivascope 3000, Caliber ID, Rochester NY). This scanner is designed for imaging skin but we adapted the front end (the objective lens and the stepper motor that axially translates) for intra-oral use. This adaption required a new approach to address the loss of the automated stepper motor for acquisition of images in depth. A helical spring-like cap (with a coverslip to contact tissue) was designed for approximately 150 um of travel. Additionally other methods for focusing optics were designed and evaluated. The relay telescope optics is being tested in a clinical setting. With the capture of video and "video-mosaicing", extended areas can be imaged. The feasibility of imaging oral tissues was initially investigated in volunteers. RCM imaging in buccal mucosa in vivo shows nuclear and cellular detail in the epithelium and epithelial junction, and connective tissue and blood flow in the underlying lamina propria. Similar detail, including filiform and fungiform papillae, can be seen on the tongue in vivo. Clinical testing during head and neck surgery is now in progress and patients are being imaged for both normal tissue and cancerous margins in lip and tongue mucosa.

  20. [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.

  1. 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

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

    PubMed

    Chakraborty, Debasis; 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 (S(3)VM) 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 S(3)VM 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.

  3. The effects of progressive anemia on jejunal mucosal and serosal tissue oxygenation in pigs.

    PubMed

    Haisjackl, M; Luz, G; Sparr, H; Germann, R; Salak, N; Friesenecker, B; Deusch, E; Meusburger, S; Hasibeder, W

    1997-03-01

    Anemia may promote intestinal hypoxia. We studied the effects of progressive isovolemic hemodilution on jejunal mucosal (Po2muc), and serosal tissue oxygen tension (Po2ser, Clark-type surface electrodes), mucosal microvascular hemoglobin oxygen saturation (Hbo2muc), and hematocrit (Hctmuc; tissue reflectance spectophotometry) in a jejunal segment. Twelve domestic pigs were anesthetized, paralyzed, and mechanically ventilated. Laparatomy was performed, arterial supply of a jejunal segment isolated, and constant pressure pump perfused. Seven animals were progressively hemodiluted to systemic hematocrits (Hctsys) of 20%, 15%, 10%, and 6%. Baseline for Po2muc, Po2ser and Hbo2muc was 23.5 +/- 2.1 mm Hg, 57.5 +/- 4 mm Hg, and 47.0% +/- 6.4% which were not different from the five controls. Despite a significant increase in jejunal blood flow, jejunal oxygen delivery decreased and oxygen extraction ratio increased significantly at Hctsys 10% and 6%. Po2ser decreased significantly below or at Hctsys of 15%, whereas Po2muc and Hbo2muc were maintained to Hctsys of 10%, but less than 10% Hbo2muc and mesenteric venous pH decreased significantly, implying that physiological limits of jejunal microvascular adaptation to severe anemia were reached. Decrease of Hctmuc was less pronounced than Hctsys. In conclusion, redistribution of jejunal blood flow and an increase in the ratio of mucosal to systemic hematocrit are the main mechanisms maintaining mucosal oxygen supply during progressive anemia.

  4. Current Progress in Tissue Engineering of Heart Valves: Multiscale Problems, Multiscale Solutions

    PubMed Central

    Cheung, Daniel Y; Duan, Bin; Butcher, Jonathan T.

    2016-01-01

    Introduction Heart valve disease is an increasingly prevalent and clinically serious condition. There are no clinically effective biological diagnostics or treatment strategies. The only recourse available is replacement with a prosthetic valve, but the inability of these devices to grow or respond biologically to their environments necessitates multiple resizing surgeries and life-long coagulation treatment, especially in children. Tissue engineering has a unique opportunity to impact heart valve disease by providing a living valve conduit, capable of growth and biological integration. Areas covered This review will cover current tissue engineering strategies in fabricating heart valves and their progress towards the clinic, including molded scaffolds using naturally-derived or synthetic polymers, decellularization, electrospinning, 3D bioprinting, hybrid techniques, and in vivo engineering. Expert opinion While much progress has been made to create functional living heart valves, a clinically viable product is not yet realized. The next leap in engineered living heart valves will require a deeper understanding of how the natural multi-scale structural and biological heterogeneity of the tissue ensures its efficient function. Related, improved fabrication strategies must be developed that can replicate this de novo complexity, which is likely instructive for appropriate cell differentiation and remodeling whether seeded with autologous stem cells in vitro or endogenously recruited cells. PMID:26027436

  5. Spinal osteoid osteoma progressed to osteoblastoma with paraspinal soft tissue mass: a unique presentation.

    PubMed

    Michaelides, Michalis; Pantziara, Maria; Petridou, Elia; Iacovou, Efrosini; Ioannides, Cleanthis

    2017-03-01

    Osteoid osteoma and osteoblastoma are rare benign bone-forming tumors with very similar histological features. They are nowadays considered as two distinct entities. Progression of an osteoid osteoma to osteoblastoma is considered very rare with only a few cases reported in the literature. Herein we describe a case of an osteoid osteoma of the thoracic spine in a 29-year-old woman that was initially treated conservatively and progressed to osteoblastoma 5 years following the initial diagnosis. Imaging revealed an increase in the size of the spinal lesion that was surrounded by extensive paraspinal abnormal soft tissue that raised suspicion for sarcomatous transformation. The final diagnosis was established by CT-guided biopsy of both the bone lesion and the paraspinal soft tissue, which excluded malignancy and revealed an osteoblastoma surrounded by plasma cell-rich chronic inflammation. The patient then underwent wide surgical excision of the lesion and paraspinal soft tissue component that confirmed the diagnosis. Follow-up with MRI over the next 12 months was unremarkable, with no signs of recurrence or spinal instability. This unique presentation of an osteoblastoma has not been previously described. This case also demonstrates the importance of follow-up of osteoid osteomas that are treated conservatively.

  6. Expression microarray analysis of papillary thyroid carcinoma and benign thyroid tissue: emphasis on the follicular variant and potential markers of malignancy

    PubMed Central

    Finn, S. P.; Smyth, P.; Cahill, S.; Streck, C.; O’Regan, E. M.; Flavin, R.; Sherlock, J.; Howells, D.; Henfrey, R.; Cullen, M.; Toner, M.; Timon, C.; O’Leary, J. J.

    2007-01-01

    The most common sub-variant of papillary thyroid carcinoma (PTC) is the so-called follicular variant (FVPTC), which is a particularly problematic lesion and can be challenging from a diagnostic viewpoint even in resected lesions. Although fine needle aspiration cytology is very useful in the diagnosis of PTC, its accuracy and utility would be greatly facilitated by the development of specific markers for PTC and its common variants. We used the recently developed Applied Biosystems 1700 microarray system to interrogate a series of 11 benign thyroid lesions and conditions and 14 samples of PTC (six with classic morphology and eight with follicular variant morphology). TaqMan® reverse transcriptase-polymerase chain reaction was used to validate the expression portfolios of 50 selected transcripts. Our data corroborates potential biomarkers previously identified in the literature, such as LGALS3, S100A11, LYN, BAX, and cluster of differentiation 44 (CD44). However, we have also identified numerous transcripts never previously implicated in thyroid carcinogenesis, and many of which are not represented on other microarray platforms. Diminished expression of metallothioneins featured strongly among these and suggests a possible role for this family as tumour suppressors in PTC. Fifteen transcripts were significantly associated with FVPTC morphology. Surprisingly, these genes were associated with an extremely narrow repertoire of functions, including the major histocompatibility complex and cathepsin families. PMID:17252232

  7. Structural and biochemical characterization of engineered tissue using FTIR spectroscopic imaging: melanoma progression as an example

    NASA Astrophysics Data System (ADS)

    Bhargava, Rohit; Kong, Rong

    2008-02-01

    Engineered tissue represents a convenient path to providing models for imaging and disease progression. The use of these models or phantoms is becoming increasingly prevalent. While structural characterization of these systems is well-documented, a combination of biochemical and structural knowledge is often helpful. Fourier transform infrared (FTIR) spectroscopic imaging is a rapidly emerging technique that combines the molecular selectivity of spectroscopy with the spatial specificity of optical microscopy. Here, we report on the application of FTIR spectroscopic for analysis of a melanoma model in engineered skin. We first characterize the biochemical properties, consistency and spectral changes in different layers of growing skin. Results provide simple indices for monitoring tissue consistency and reproducibility as a function of time. Second, we introduce malignant melanocytes to simulate tumor formation and growth. Both cellular changes associated with tumor formation and growth can be observed. FTIR images indicate holistic chemical changes during the tumor growth, allowing for the development of automated pathology protocols. FTIR imaging being non-destructive, further, samples remain entirely compatible with downstream tissue processing or staining. We specifically examined the correlation of structural changes, molecular content and reproducibility of the model systems. The development of analysis, integrating spectroscopy, imaging and computation will allow for quality control and standardization of both the structural and biochemical properties of tissue phantoms.

  8. Hypoxia-Inducible Factors: Mediators of Cancer Progression; Prognostic and Therapeutic Targets in Soft Tissue Sarcomas

    PubMed Central

    Sadri, Navid; Zhang, Paul J.

    2013-01-01

    Soft-tissue sarcomas remain aggressive tumors that result in death in greater than a third of patients due to either loco-regional recurrence or distant metastasis. Surgical resection remains the main choice of treatment for soft tissue sarcomas with pre- and/or post-operational radiation and neoadjuvant chemotherapy employed in more advanced stage disease. However, in recent decades, there has been little progress in the average five-year survival for the majority of patients with high-grade soft tissue sarcomas, highlighting the need for improved targeted therapeutic agents. Clinical and preclinical studies demonstrate that tumor hypoxia and up-regulation of hypoxia-inducible factors (HIFs) is associated with decreased survival, increased metastasis, and resistance to therapy in soft tissue sarcomas. HIF-mediated gene expression regulates many critical aspects of tumor biology, including cell survival, metabolic programming, angiogenesis, metastasis, and therapy resistance. In this review, we discuss HIFs and HIF-mediated genes as potential prognostic markers and therapeutic targets in sarcomas. Many pharmacological agents targeting hypoxia-related pathways are in development that may hold therapeutic potential for treating both primary and metastatic sarcomas that demonstrate increased HIF expression. PMID:24216979

  9. Monitoring Healing Progression and Characterizing the Mechanical Environment in Preclinical Models for Bone Tissue Engineering.

    PubMed

    Fountain, Stephanie; Windolf, Markus; Henkel, Jan; Tavakoli, Aramesh; Schuetz, Michael A; Hutmacher, Dietmar W; Epari, Devakara R

    2015-12-15

    The treatment of large segmental bone defects remains a significant clinical challenge. Due to limitations surrounding the use of bone grafts, tissue-engineered constructs for the repair of large bone defects could offer an alternative. Before translation of any newly developed tissue engineering (TE) approach to the clinic, efficacy of the treatment must be shown in a validated preclinical large animal model. Currently, biomechanical testing, histology, and microcomputed tomography are performed to assess the quality and quantity of the regenerated bone. However, in vivo monitoring of the progression of healing is seldom performed, which could reveal important information regarding time to restoration of mechanical function and acceleration of regeneration. Furthermore, since the mechanical environment is known to influence bone regeneration, and limb loading of the animals can poorly be controlled, characterizing activity and load history could provide the ability to explain variability in the acquired data sets and potentially outliers based on abnormal loading. Many approaches have been devised to monitor the progression of healing and characterize the mechanical environment in fracture healing studies. In this article, we review previous methods and share results of recent work of our group toward developing and implementing a comprehensive biomechanical monitoring system to study bone regeneration in preclinical TE studies.

  10. Microarray analysis in pulmonary hypertension

    PubMed Central

    Hoffmann, Julia; Wilhelm, Jochen; Olschewski, Andrea

    2016-01-01

    Microarrays are a powerful and effective tool that allows the detection of genome-wide gene expression differences between controls and disease conditions. They have been broadly applied to investigate the pathobiology of diverse forms of pulmonary hypertension, namely group 1, including patients with idiopathic pulmonary arterial hypertension, and group 3, including pulmonary hypertension associated with chronic lung diseases such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. To date, numerous human microarray studies have been conducted to analyse global (lung homogenate samples), compartment-specific (laser capture microdissection), cell type-specific (isolated primary cells) and circulating cell (peripheral blood) expression profiles. Combined, they provide important information on development, progression and the end-stage disease. In the future, system biology approaches, expression of noncoding RNAs that regulate coding RNAs, and direct comparison between animal models and human disease might be of importance. PMID:27076594

  11. DNA microarray technology in dermatology.

    PubMed

    Kunz, Manfred

    2008-03-01

    In recent years, DNA microarray technology has been used for the analysis of gene expression patterns in a variety of skin diseases, including malignant melanoma, psoriasis, lupus erythematosus, and systemic sclerosis. Many of the studies described herein confirmed earlier results on individual genes or functional groups of genes. However, a plethora of new candidate genes, gene patterns, and regulatory pathways have been identified. Major progresses were reached by the identification of a prognostic gene pattern in malignant melanoma, an immune signaling cluster in psoriasis, and a so-called interferon signature in systemic lupus erythematosus. In future, interference with genes or regulatory pathways with the use of different RNA interference technologies or targeted therapy may not only underscore the functional significance of microarray data but also may open interesting therapeutic perspectives. Large-scale gene expression analyses may also help to design more individualized treatment approaches of cutaneous diseases.

  12. Microarray analysis in pulmonary hypertension.

    PubMed

    Hoffmann, Julia; Wilhelm, Jochen; Olschewski, Andrea; Kwapiszewska, Grazyna

    2016-07-01

    Microarrays are a powerful and effective tool that allows the detection of genome-wide gene expression differences between controls and disease conditions. They have been broadly applied to investigate the pathobiology of diverse forms of pulmonary hypertension, namely group 1, including patients with idiopathic pulmonary arterial hypertension, and group 3, including pulmonary hypertension associated with chronic lung diseases such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. To date, numerous human microarray studies have been conducted to analyse global (lung homogenate samples), compartment-specific (laser capture microdissection), cell type-specific (isolated primary cells) and circulating cell (peripheral blood) expression profiles. Combined, they provide important information on development, progression and the end-stage disease. In the future, system biology approaches, expression of noncoding RNAs that regulate coding RNAs, and direct comparison between animal models and human disease might be of importance.

  13. 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...

  14. Annotating nonspecific SAGE tags with microarray data.

    PubMed

    Ge, Xijin; Jung, Yong-Chul; Wu, Qingfa; Kibbe, Warren A; Wang, San Ming

    2006-01-01

    SAGE (serial analysis of gene expression) detects transcripts by extracting short tags from the transcripts. Because of the limited length, many SAGE tags are shared by transcripts from different genes. Relying on sequence information in the general gene expression database has limited power to solve this problem due to the highly heterogeneous nature of the deposited sequences. Considering that the complexity of gene expression at a single tissue level should be much simpler than that in the general expression database, we reasoned that by restricting gene expression to tissue level, the accuracy of gene annotation for the nonspecific SAGE tags should be significantly improved. To test the idea, we developed a tissue-specific SAGE annotation database based on microarray data (). This database contains microarray expression information represented as UniGene clusters for 73 normal human tissues and 18 cancer tissues and cell lines. The nonspecific SAGE tag is first matched to the database by the same tissue type used by both SAGE and microarray analysis; then the multiple UniGene clusters assigned to the nonspecific SAGE tag are searched in the database under the matched tissue type. The UniGene cluster presented solely or at higher expression levels in the database is annotated to represent the specific gene for the nonspecific SAGE tags. The accuracy of gene annotation by this database was largely confirmed by experimental data. Our study shows that microarray data provide a useful source for annotating the nonspecific SAGE tags.

  15. Transient tissue priming via ROCK inhibition uncouples pancreatic cancer progression, sensitivity to chemotherapy, and metastasis.

    PubMed

    Vennin, Claire; Chin, Venessa T; Warren, Sean C; Lucas, Morghan C; Herrmann, David; Magenau, Astrid; Melenec, Pauline; Walters, Stacey N; Del Monte-Nieto, Gonzalo; Conway, James R W; Nobis, Max; Allam, Amr H; McCloy, Rachael A; Currey, Nicola; Pinese, Mark; Boulghourjian, Alice; Zaratzian, Anaiis; Adam, Arne A S; Heu, Celine; Nagrial, Adnan M; Chou, Angela; Steinmann, Angela; Drury, Alison; Froio, Danielle; Giry-Laterriere, Marc; Harris, Nathanial L E; Phan, Tri; Jain, Rohit; Weninger, Wolfgang; McGhee, Ewan J; Whan, Renee; Johns, Amber L; Samra, Jaswinder S; Chantrill, Lorraine; Gill, Anthony J; Kohonen-Corish, Maija; Harvey, Richard P; Biankin, Andrew V; Evans, T R Jeffry; Anderson, Kurt I; Grey, Shane T; Ormandy, Christopher J; Gallego-Ortega, David; Wang, Yingxiao; Samuel, Michael S; Sansom, Owen J; Burgess, Andrew; Cox, Thomas R; Morton, Jennifer P; Pajic, Marina; Timpson, Paul

    2017-04-05

    The emerging standard of care for patients with inoperable pancreatic cancer is a combination of cytotoxic drugs gemcitabine and Abraxane, but patient response remains moderate. Pancreatic cancer development and metastasis occur in complex settings, with reciprocal feedback from microenvironmental cues influencing both disease progression and drug response. Little is known about how sequential dual targeting of tumor tissue tension and vasculature before chemotherapy can affect tumor response. We used intravital imaging to assess how transient manipulation of the tumor tissue, or "priming," using the pharmaceutical Rho kinase inhibitor Fasudil affects response to chemotherapy. Intravital Förster resonance energy transfer imaging of a cyclin-dependent kinase 1 biosensor to monitor the efficacy of cytotoxic drugs revealed that priming improves pancreatic cancer response to gemcitabine/Abraxane at both primary and secondary sites. Transient priming also sensitized cells to shear stress and impaired colonization efficiency and fibrotic niche remodeling within the liver, three important features of cancer spread. Last, we demonstrate a graded response to priming in stratified patient-derived tumors, indicating that fine-tuned tissue manipulation before chemotherapy may offer opportunities in both primary and metastatic targeting of pancreatic cancer.

  16. 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

  17. Vitamin D Receptor Protein Expression in Tumor Tissue and Prostate Cancer Progression

    PubMed Central

    Hendrickson, Whitney K.; Flavin, Richard; Kasperzyk, Julie L.; Fiorentino, Michelangelo; Fang, Fang; Lis, Rosina; Fiore, Christopher; Penney, Kathryn L.; Ma, Jing; Kantoff, Philip W.; Stampfer, Meir J.; Loda, Massimo; Mucci, Lorelei A.; Giovannucci, Edward

    2011-01-01

    Purpose Data suggest that circulating 25-hydroxyvitamin D [25(OH)D] interacts with the vitamin D receptor (VDR) to decrease proliferation and increase apoptosis for some malignancies, although evidence for prostate cancer is less clear. How VDR expression in tumor tissue may influence prostate cancer progression has not been evaluated in large studies. Patients and Methods We examined protein expression of VDR in tumor tissue among 841 patients with prostate cancer in relation to risk of lethal prostate cancer within two prospective cohorts, the Physicians' Health Study and Health Professionals Follow-Up Study. We also examined the association of VDR expression with prediagnostic circulating 25(OH)D and 1,25-dihydroxyvitamin D levels and with two VDR single nucleotide polymorphisms, FokI and BsmI. Results Men whose tumors had high VDR expression had significantly lower prostate-specific antigen (PSA) at diagnosis (P for trend < .001), lower Gleason score (P for trend < .001), and less advanced tumor stage (P for trend < .001) and were more likely to have tumors harboring the TMPRSS2:ERG fusion (P for trend = .009). Compared with the lowest quartile, men whose tumors had the highest VDR expression had significantly reduced risk of lethal prostate cancer (hazard ratio [HR], 0.17; 95% CI, 0.07 to 0.41). This association was only slightly attenuated after adjustment for Gleason score and PSA at diagnosis (HR, 0.33; 95% CI, 0.13 to 0.83) or, additionally, for tumor stage (HR, 0.37; 95% CI, 0.14 to 0.94). Neither prediagnostic plasma vitamin D levels nor VDR polymorphisms were associated with VDR expression. Conclusion High VDR expression in prostate tumors is associated with a reduced risk of lethal cancer, suggesting a role of the vitamin D pathway in prostate cancer progression. PMID:21537045

  18. Parallel microarray profiling identifies ErbB4 as a determinant of cyst growth in ADPKD and a prognostic biomarker for disease progression.

    PubMed

    Streets, Andrew J; Magayr, Tajdida A; Huang, Linghong; Vergoz, Laura; Rossetti, Sandro; Simms, Roslyn J; Harris, Peter C; Peters, Dorien J M; Ong, Albert C M

    2017-04-01

    Autosomal dominant polycystic kidney disease (ADPKD) is the fourth most common cause of end-stage renal disease. The disease course can be highly variable and treatment options are limited. To identify new therapeutic targets and prognostic biomarkers of disease, we conducted parallel discovery microarray profiling in normal and diseased human PKD1 cystic kidney cells. A total of 1,515 genes and 5 miRNA were differentially expressed by more than twofold in PKD1 cells. Functional enrichment analysis identified 30 dysregulated signaling pathways including the epidermal growth factor (EGF) receptor pathway. In this paper, we report that the EGF/ErbB family receptor ErbB4 is a major factor driving cyst growth in ADPKD. Expression of ErbB4 in vivo was increased in human ADPKD and Pkd1 cystic kidneys, both transcriptionally and posttranscriptionally by mir-193b-3p. Ligand-induced activation of ErbB4 drives cystic proliferation and expansion suggesting a pathogenic role in cystogenesis. Our results implicate ErbB4 activation as functionally relevant in ADPKD, both as a marker of disease activity and as a new therapeutic target in this major kidney disease.

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

    PubMed Central

    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.

    2016-01-01

    Fibrosis compromises pancreatic ductal carcinoma (PDAC) treatment and contributes to patient mortality yet anti-stromal therapies are controversial. We found that human PDACs with impaired epithelial transforming growth factor β (TGF-β) signaling have elevated epithelial Stat3 activity and develop a stiffer, 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 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

  20. 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 Central

    Dragowska, Weislawa H.; Bally, Marcel B.

    2015-01-01

    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 (LCC6Luc) 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

  1. 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.

  2. Microarray in parasitic infections

    PubMed Central

    Sehgal, Rakesh; Misra, Shubham; Anand, Namrata; Sharma, Monika

    2012-01-01

    Modern biology and genomic sciences are rooted in parasitic disease research. Genome sequencing efforts have provided a wealth of new biological information that promises to have a major impact on our understanding of parasites. Microarrays provide one of the major high-throughput platforms by which this information can be exploited in the laboratory. Many excellent reviews and technique articles have recently been published on applying microarrays to organisms for which fully annotated genomes are at hand. However, many parasitologists work on organisms whose genomes have been only partially sequenced. This review is mainly focused on how to use microarray in these situations. PMID:23508469

  3. TMA for all: a new method for the construction of tissue microarrays without recipient paraffin block using custom-built needles

    PubMed Central

    Pires, Andréa Rodrigues Cordovil; Andreiuolo, Felipe da Matta; de Souza, Simone Rabello

    2006-01-01

    Background TMAs are becoming a useful tool for research and quality control methods, mostly for immunohistochemistry and in situ hybridization. Methods A new technique that allows building TMA blocks with more than 300 tissue cores without using a recipient paraffin block for the tissue cores and without using a commercial TMA builder instrument is described. This technique is based on the construction of TMA needles modifying conventional hypodermic needles to punch tissue cores from donor blocks, which are attached by double-side adhesive tape on a computer-generated paper grid used to align the cores on the block mould, which is filled with liquid paraffin. Results More than two hundred TMA blocks were constructed using this method, utilized in immunohistochemistry and histochemistry as positive and negative controls and also in research. Conclusion This technique has the following advantages: it is easy to reproduce, affordable, quick and creates uniform blocks with more than 300 cores aligned, adherent and easy to cut, with negligible losses during cutting and immunohistochemistry and in situ hybridization procedures. PMID:16869973

  4. Microarray analysis of microRNA expression in mouse fetus at 13.5 and 14.5 days post-coitum in ear and back skin tissues.

    PubMed

    Torres, Leda; Juárez, Ulises; García, Laura; Miranda-Ríos, Juan; Frias, Sara

    2016-09-01

    There is no information regarding the role of microRNAs in the development of the external ear in mammals. The purpose of this study was to determine the stage-specific expression of microRNA during external ear development in mice under normal conditions. GeneChip miRNA 3.0 arrays by Affymetrix were used to obtain miRNA expression profiles from mice fetal pinnae and back skin tissues at 13.5 days-post-coitum (dpc) and 14.5 dpc. Biological triplicates for each tissue were analyzed; one litter represents one biological replica, each litter had 16 fetuses on average. The results were analyzed with Affymetrix's Transcriptome Analysis Console software to identify differentially expressed miRNAs. The inquiry showed significant differential expression of 25 miRNAs at 13.5 dpc and 31 at 14.5 dpc, some of these miRNAs were predicted to target genes implicated in external ear development. One example is mmu-miR-10a whose low expression in pinnae is known to impact ear development by modulating Hoxa1 mRNA levels Garzon et al. (2006), Gavalas et al. (1998) [1], [2]. Other findings like the upregulation of mmu-miR-200c and mmu-miR-205 in the pinnae tissues of healthy mice are in agreement with what has been reported in human patients with microtia, in which down regulation of both miRNAs has been found Li et al. (2013) [3]. This study uncovered a spatiotemporal pattern of miRNA expression in the external ear, which results from continuous transcriptional changes during normal development of body structures. All microarray data are available at the Gene Expression Omnibus (GEO) at NCBI under accession number GSE64945.

  5. 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 1°C variation across a large (19×32cm) 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 2.95±0.03 % 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

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

    PubMed

    Pei, Tingting; Yu, Hongqiang; Wen, Chaoju; Guo, Tianqi; Zhou, Yanmin; Peng, Huimin

    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.

  7. 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

  8. Molecular diagnosis and prognosis with DNA microarrays.

    PubMed

    Wiltgen, Marco; Tilz, Gernot P

    2011-05-01

    Microarray analysis makes it possible to determine thousands of gene expression values simultaneously. Changes in gene expression, as a response to diseases, can be detected allowing a better understanding and differentiation of diseases at a molecular level. By comparing different kinds of tissue, for example healthy tissue and cancer tissue, the microarray analysis indicates induced gene activity, repressed gene activity or when there is no change in the gene activity level. Fundamental patterns in gene expression are extracted by several clustering and machine learning algorithms. Certain kinds of cancer can be divided into subtypes, with different clinical outcomes, by their specific gene expression patterns. This enables a better diagnosis and tailoring of individual patient treatments.

  9. Comparison of hepatocellular carcinoma miRNA expression profiling as evaluated by next generation sequencing and microarray.

    PubMed

    Murakami, Yoshiki; Tanahashi, Toshihito; Okada, Rina; Toyoda, Hidenori; Kumada, Takashi; Enomoto, Masaru; Tamori, Akihiro; Kawada, Norifumi; Taguchi, Y-h; Azuma, Takeshi

    2014-01-01

    MicroRNA (miRNA) expression profiling has proven useful in diagnosing and understanding the development and progression of several diseases. Microarray is the standard method for analyzing miRNA expression profiles; however, it has several disadvantages, including its limited detection of miRNAs. In recent years, advances in genome sequencing have led to the development of next-generation sequencing (NGS) technologies, which significantly advance genome sequencing speed and discovery. In this study, we compared the expression profiles obtained by next generation sequencing (NGS) with the profiles created using microarray to assess if NGS could produce a more accurate and complete miRNA profile. Total RNA from 14 hepatocellular carcinoma tumors (HCC) and 6 matched non-tumor control tissues were sequenced with Illumina MiSeq 50-bp single-end reads. Micro RNA expression profiles were estimated using miRDeep2 software. As a comparison, miRNA expression profiles for 11 out of 14 HCCs were also established by microarray (Agilent human microRNA microarray). The average total sequencing exceeded 2.2 million reads per sample and of those reads, approximately 57% mapped to the human genome. The average correlation for miRNA expression between microarray and NGS and subtraction were 0.613 and 0.587, respectively, while miRNA expression between technical replicates was 0.976. The diagnostic accuracy of HCC, p-value, and AUC were 90.0%, 7.22×10(-4), and 0.92, respectively. In summary, NGS created an miRNA expression profile that was reproducible and comparable to that produced by microarray. Moreover, NGS discovered novel miRNAs that were otherwise undetectable by microarray. We believe that miRNA expression profiling by NGS can be a useful diagnostic tool applicable to multiple fields of medicine.

  10. Multievidence microarray mining.

    PubMed

    Seifert, Martin; Scherf, Matthias; Epple, Anton; Werner, Thomas

    2005-10-01

    Microarray mining is a challenging task because of the superposition of several processes in the data. We believe that the combination of microarray data-based analyses (statistical significance analysis of gene expression) with array-independent analyses (literature-mining and promoter analysis) enables some of the problems of traditional array analysis to be overcome. As a proof-of-principle, we revisited publicly available microarray data derived from an experiment with platelet-derived growth factor (PDGF)-stimulated fibroblasts. Our strategy revealed results beyond the detection of the major metabolic pathway known to be linked to the PDGF response: we were able to identify the crosstalking regulatory networks underlying the metabolic pathway without using a priori knowledge about the experiment.

  11. DNA microarray technology. Introduction.

    PubMed

    Pollack, Jonathan R

    2009-01-01

    DNA microarray technology has revolutionized biological research by enabling genome-scale explorations. This chapter provides an overview of DNA microarray technology and its application to characterizing the physical genome, with a focus on cancer genomes. Specific areas discussed include investigations of DNA copy number alteration (and loss of heterozygosity), DNA methylation, DNA-protein (i.e., chromatin and transcription factor) interactions, DNA replication, and the integration of diverse genome-scale data types. Also provided is a perspective on recent advances and future directions in characterizing the physical genome.

  12. 3D Printing of Tissue Engineered Constructs for In Vitro Modeling of Disease Progression and Drug Screening.

    PubMed

    Vanderburgh, Joseph; Sterling, Julie A; Guelcher, Scott A

    2017-01-01

    2D cell culture and preclinical animal models have traditionally been implemented for investigating the underlying cellular mechanisms of human disease progression. However, the increasing significance of 3D vs. 2D cell culture has initiated a new era in cell culture research in which 3D in vitro models are emerging as a bridge between traditional 2D cell culture and in vivo animal models. Additive manufacturing (AM, also known as 3D printing), defined as the layer-by-layer fabrication of parts directed by digital information from a 3D computer-aided design file, offers the advantages of simultaneous rapid prototyping and biofunctionalization as well as the precise placement of cells and extracellular matrix with high resolution. In this review, we highlight recent advances in 3D printing of tissue engineered constructs that recapitulate the physical and cellular properties of the tissue microenvironment for investigating mechanisms of disease progression and for screening drugs.

  13. On-chip lectin microarray for glycoprofiling of different gastritis types and gastric cancer

    PubMed Central

    Roy, Bibhas; Chattopadhyay, Gautam; Mishra, Debasish; Das, Tamal; Chakraborty, Suman; Maiti, Tapas K.

    2014-01-01

    An on-chip lectin microarray based glycomic approach is employed to identify glyco markers for different gastritis and gastric cancer. Changes in protein glycosylation have impact on biological function and carcinogenesis. These altered glycosylation patterns in serum proteins and membrane proteins of tumor cells can be unique markers of cancer progression and hence have been exploited to diagnose various stages of cancer through lectin microarray technology. In the present work, we aimed to study the alteration of glycan structure itself in different stages of gastritis and gastric cancer thoroughly. In order to perform the study from both serum and tissue glycoproteins in an efficient and high-throughput manner, we indigenously developed and employed lectin microarray integrated on a microfluidic lab-on-a-chip platform. We analyzed serum and gastric biopsy samples from 8 normal, 15 chronic Type-B gastritis, 10 chronic Type-C gastritis, and 6 gastric adenocarcinoma patients and found that the glycoprofile obtained from tissue samples was more distinctive than that of the sera samples. We were able to establish signature glycoprofile for the three disease groups, that were absent in healthy normal individuals. In addition, our findings elucidated certain novel signature glycan expression in chronic gastritis and gastric cancer. In silico analysis showed that glycoprofile of chronic gastritis and gastric adenocarcinoma formed close clusters, confirming the previously hypothesized linkage between them. This signature can be explored further as gastric cancer marker to develop novel analytical tools and obtain in-depth understanding of the disease prognosis. PMID:24959308

  14. Protein Microarray Technology

    PubMed Central

    Hall, David A.; Ptacek, Jason

    2007-01-01

    Protein chips have emerged as a promising approach for a wide variety of applications including the identification of protein-protein interactions, protein-phospholipid interactions, small molecule targets, and substrates of proteins kinases. They can also be used for clinical diagnostics and monitoring disease states. This article reviews current methods in the generation and applications of protein microarrays. PMID:17126887

  15. [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.

  16. 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.

  17. [Progress in application of 3D bioprinting in cartilage regeneration and reconstruction for tissue engineering].

    PubMed

    Liao, Junlin; Wang, Shaohua; Chen, Jia; Xie, Hongju; Zhou, Jianda

    2017-02-28

    Three-dimensional (3D) bioprinting provides an advanced technology for tissue engineering and regenerative medicine because of its ability to produce the models or organs with higher precision and more suitable for human body. It has been successfully used to produce a variety of cartilage scaffold materials. In addition, 3D bioprinter can directly to print tissue and organs with live chondrocytes. In conclusion, 3D bioprinting may have broad prospect for cartilage regeneration and reconstruction in tissue engineering.

  18. Protein microarray applications: Autoantibody detection and posttranslational modification.

    PubMed

    Atak, Apurva; Mukherjee, Shuvolina; Jain, Rekha; Gupta, Shabarni; Singh, Vedita Anand; Gahoi, Nikita; K P, Manubhai; Srivastava, Sanjeeva

    2016-10-01

    The discovery of DNA microarrays was a major milestone in genomics; however, it could not adequately predict the structure or dynamics of underlying protein entities, which are the ultimate effector molecules in a cell. Protein microarrays allow simultaneous study of thousands of proteins/peptides, and various advancements in array technologies have made this platform suitable for several diagnostic and functional studies. Antibody arrays enable researchers to quantify the abundance of target proteins in biological fluids and assess PTMs by using the antibodies. Protein microarrays have been used to assess protein-protein interactions, protein-ligand interactions, and autoantibody profiling in various disease conditions. Here, we summarize different microarray platforms with focus on its biological and clinical applications in autoantibody profiling and PTM studies. We also enumerate the potential of tissue microarrays to validate findings from protein arrays as well as other approaches, highlighting their significance in proteomics.

  19. 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

  20. 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.

  1. 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.

  2. Ultrahigh density microarrays of solid samples.

    PubMed

    LeBaron, Matthew J; Crismon, Heidi R; Utama, Fransiscus E; Neilson, Lynn M; Sultan, Ahmed S; Johnson, Kevin J; Andersson, Eva C; Rui, Hallgeir

    2005-07-01

    We present a sectioning and bonding technology to make ultrahigh density microarrays of solid samples, cutting edge matrix assembly (CEMA). Maximized array density is achieved by a scaffold-free, self-supporting construction with rectangular array features that are incrementally scalable. This platform technology facilitates arrays of >10,000 tissue features on a standard glass slide, inclusion of unique sample identifiers for improved manual or automated tracking, and oriented arraying of stratified or polarized samples.

  3. Analyzing Microarray Data.

    PubMed

    Hung, Jui-Hung; Weng, Zhiping

    2017-03-01

    Because there is no widely used software for analyzing RNA-seq data that has a graphical user interface, this protocol provides an example of analyzing microarray data using Babelomics. This analysis entails performing quantile normalization and then detecting differentially expressed genes associated with the transgenesis of a human oncogene c-Myc in mice. Finally, hierarchical clustering is performed on the differentially expressed genes using the Cluster program, and the results are visualized using TreeView.

  4. Membrane-based microarrays

    NASA Astrophysics Data System (ADS)

    Dawson, Elliott P.; Hudson, James; Steward, John; Donnell, Philip A.; Chan, Wing W.; Taylor, Richard F.

    1999-11-01

    Microarrays represent a new approach to the rapid detection and identification of analytes. Studies to date have shown that the immobilization of receptor molecules (such as DNA, oligonucleotides, antibodies, enzymes and binding proteins) onto silicon and polymeric substrates can result in arrays able to detect hundreds of analytes in a single step. The formation of the receptor/analyte complex can, itself, lead to detection, or the complex can be interrogated through the use of fluorescent, chemiluminescent or radioactive probes and ligands.

  5. Real-time optical monitoring of permanent lesion progression in radiofrequency ablated cardiac tissue (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Singh-Moon, Rajinder P.; Hendon, Christine P.

    2016-02-01

    Despite considerable advances in guidance of radiofrequency ablation (RFA) therapies for atrial fibrillation, success rates have been hampered by an inability to intraoperatively characterize the extent of permanent injury. Insufficient lesions can elusively create transient conduction blockages that eventually reconduct. Prior studies suggest significantly greater met-myoglobin (Mmb) concentrations in the lesion core than those in the healthy myocardium and may serve as a marker for irreversible tissue damage. In this work, we present real-time monitoring of permanent injury through spectroscopic assessment of Mmb concentrations at the catheter tip. Atrial wedges (n=6) were excised from four fresh swine hearts and submerged under pulsatile flow of warm (37oC) phosphate buffered saline. A commercial RFA catheter inserted into a fiber optic sheath allowed for simultaneous measurement of tissue diffuse reflectance (DR) spectra (500-650nm) during application of RF energy. Optical measurements were continuously acquired before, during, and post-ablation, in addition to healthy neighboring tissue. Met-myoglobin, oxy-myoglobin, and deoxy-myoglobin concentrations were extracted from each spectrum using an inverse Monte Carlo method. Tissue injury was validated with Masson's trichrome and hematoxylin and eosin staining. Time courses revealed a rapid increase in tissue Mmb concentrations at the onset of RFA treatment and a gradual plateauing thereafter. Extracted Mmb concentrations were significantly greater post-ablation (p<0.0001) as compared to healthy tissue and correlated well with histological assessment of severe thermal tissue destruction. On going studies are aimed at integrating these findings with prior work on near infrared spectroscopic lesion depth assessment. These results support the use of spectroscopy-facilitated guidance of RFA therapies for real-time permanent injury estimation.

  6. Rapid progression to pulmonary arterial hypertension crisis associated with mixed connective tissue disease in an 11-year-old girl.

    PubMed

    Okura, Yuka; Takezaki, Shunichiro; Yamazaki, Yasuhiro; Yamada, Masafumi; Kobayashi, Ichiro; Ariga, Tadashi

    2013-09-01

    Mixed connective tissue disease (MCTD) is rare in pediatric rheumatic diseases. Pulmonary arterial hypertension (PAH) associated with MCTD usually progresses gradually and is difficult to note at the asymptomatic phase. We report a 11-year-old girl with MCTD complicated with rapidly progressive PAH. Although PAH was not detected by echocardiogram or chest CT scan at the initial examination, it became clear in 1 year and suddenly came to cardiac arrest during an invasive procedure. She was successfully treated with extracorporeal assist and both vasodilative and immunosuppressive medication. A combination of echocardiogram and plasma BNP levels could be a useful marker for the follow-up of such cases. PAH could develop early in the course of pediatric MCTD and needs attention to unexpected acute exacerbation, especially under emotional stress.

  7. [Research progress on application of carbon nanotubes in bone tissue engineering scaffold].

    PubMed

    Yao, Mengzhu; Sheng, Xiaoxia; Lin, Jun; Gao, Jianqing

    2016-03-01

    Carbon nanotubes possess excellent mechanical and electrical properties and demonstrate broad application prospects in medical fields. Carbon nanotubes are composed of inorganic materials, natural biodegradable polymer or synthetic biodegradable polymer. The composite bone tissue engineering scaffolds are constructed by particle-hole method, lyophilization, microsphere aggregation method, electrostatic spinning or three-dimensional printing. Composite scaffolds overcome the shortcomings of single material and have good biocompatibility, osteoconduction and osteoinduction. With the study of surface chemistry, toxicology, and biocompatibility, a degradable "human-friendly" carbon nanotubes composite bone tissue scaffold will be available; and under the drive of new fabrication techniques, the clinical application of carbon nanotubes composite bone tissue engineering scaffolds will be better developed.

  8. [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.

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

    PubMed

    Gao, Peng; Jiang, Dapeng; Li, Zhaozhu

    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.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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.

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

    PubMed Central

    Weissman, Irving

    2015-01-01

    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

  16. Mixed connective tissue disease presenting with progressive scleroderma symptoms in a 10-year-old girl.

    PubMed

    Latuśkiewicz-Potemska, Joanna; Zygmunt, Agnieszka; Biernacka-Zielińska, Małgorzata; Stańczyk, Jerzy; Smolewska, Elżbieta

    2013-10-01

    Mixed connective tissue disease (MCTD) is a systemic inflammatory disease affecting connective tissue with the underlying autoimmunological mechanism. The core of MCTD is an appearance of symptoms of several other inflammatory diseases of connective tissue - systemic lupus erythematosus, systemic scleroderma, poly- or dermatomyositis, rheumatoid arthritis at the same time, accompanied by a high level of anti-ribonucleoprotein antibodies (anti-U1RNP). The disease was described more than 40 years ago by Sharp et al. During recent years, many efforts to better understand clinical and serological features of MCTD have been made. Diagnosis of MCTD can be difficult. Obligatory international diagnostic criteria are required to be fulfilled. Several versions of such criteria have been proposed, but the most widely used one was described by Kasukawa. There is no consensus about treatment - a choice of drugs depends on symptoms. We present a case of a 10-year-old girl with sclerodactyly and trophic damages of fingers accompanied by symptoms of Raynaud's phenomenon. After an almost 2-year course of the disease, a diagnosis of MCTD has been established.

  17. Aberrant Expression Profile of Long Noncoding RNA in Human Sinonasal Squamous Cell Carcinoma by Microarray Analysis

    PubMed Central

    Meng, Ling-zhao; Sun, Jing-wu; Yang, Fan

    2016-01-01

    Objectives. This study aimed to identify aberrantly expressed long noncoding RNAs (lncRNAs) profile of sinonasal squamous cell carcinoma (SSCC) and explore their potential functions. Methods. We investigated lncRNA and mRNA expression in SSCC and paired adjacent noncancerous tissues obtained from 6 patients with microarrays. Gene ontology (GO) analysis and pathway analysis were utilized to investigate the gene function. Gene signal-network and lncRNA-mRNA network were depicted. Quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to validate 5 lncRNAs in a second set of paired SSCC and adjacent noncancerous tissues obtained from 22 additional patients. Results. We identified significantly differentially expressed lncRNAs (n = 3146) and mRNAs (n = 2208) in SSCC relative to noncancerous tissues. The GO annotation indicated that there are some core gene products that may be attributed to the progress of SSCC. The pathway analysis identified many pathways associated with cancer. The results of lncRNA-mRNA network and gene signal-network implied some core lncRNAs/mRNAs might play important roles in SSCC pathogenesis. The results of qRT-PCR showed that all of the 5 lncRNAs were differentially expressed and consistent with the microarray results. Conclusion. Our study is the first screening and analysis of lncRNAs expression profile in SSCC and may offer new insights into pathogenesis of this disease. PMID:28044124

  18. 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

  19. Magnetic Analysis of Post-mortem Hippocampal Tissue from Alzheimer's Patients: Changes with Progression of the Disease.

    NASA Astrophysics Data System (ADS)

    Fuller, M.; Zinin, P.; Favia, J.; Tatsumi, L.; Kletetschka, G.; Adachi, T.

    2007-12-01

    Increases of iron in the human brain with age have been observed and may be accompanied by the development of neurodegenerative diseases, such as Alzheimer's. We have measured the magnetic characteristics of several sets of slides of hippocampal tissue from deceased Alzheimer patients. The slides were made available by the Harvard Brain Bank. The pathology of the tissue was classified in the Braak stages I to VI used to describe the progression of the disease. In general, the slides from patients with higher Braak stages and development of fibrillary tangles and plaques had greater magnetic moments than did those with Braak stage II. However, the peak values were at stage IV and V. To mitigate errors due to the inevitable differences in masses of the tissue on individual slides and their precise location in the hippocampus, ratios of magnetic properties were also observed. Ratios of Anhysteretic Remanent Magnetizaton (ARM) to Isothermal Remanent Magnetization (IRM) were obtained and showed a decrease from Stage II to the more advanced stages, with the minimum values at stages IV and V. The acquisition and demagnetization of IRM are consistent with the presence of magnetite, but also indicate a magnetically harder phase.

  20. Progress in the tissue engineering and stem cell industry "are we there yet?".

    PubMed

    Jaklenec, Ana; Stamp, Andrea; Deweerd, Elizabeth; Sherwin, Angela; Langer, Robert

    2012-06-01

    This report presents a detailed update to our 2008 publication on the tissue engineering (TE) and stem cell industry. Data are reported through mid 2011 showing an almost three-fold growth in commercial sales over the past 4 years. In addition, the number of companies selling products or offering services has increased over two-fold to 106, and they are generating a remarkable $3.5 billion in sales. Overall, the TE and stem cell sector is spending $3.6 billion and employing almost 14,000 employees. These data suggest the TE and stem cell industry has stabilized and is on a path pointing toward continued success.

  1. 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

  2. Identification of tumor-associated autoantigens for the diagnosis of colorectal cancer in serum using high density protein microarrays.

    PubMed

    Babel, Ingrid; Barderas, Rodrigo; Díaz-Uriarte, Ramón; Martínez-Torrecuadrada, Jorge Luis; Sánchez-Carbayo, Marta; Casal, J Ignacio

    2009-10-01

    There is a mounting evidence of the existence of autoantibodies associated to cancer progression. Antibodies are the target of choice for serum screening because of their stability and suitability for sensitive immunoassays. By using commercial protein microarrays containing 8000 human proteins, we examined 20 sera from colorectal cancer (CRC) patients and healthy subjects to identify autoantibody patterns and associated antigens. Forty-three proteins were differentially recognized by tumoral and reference sera (p value <0.04) in the protein microarrays. Five immunoreactive antigens, PIM1, MAPKAPK3, STK4, SRC, and FGFR4, showed the highest prevalence in cancer samples, whereas ACVR2B was more abundant in normal sera. Three of them, PIM1, MAPKAPK3, and ACVR2B, were used for further validation. A significant increase in the expression level of these antigens on CRC cell lines and colonic mucosa was confirmed by immunoblotting and immunohistochemistry on tissue microarrays. A diagnostic ELISA based on the combination of MAPKAPK3 and ACVR2B proteins yielded specificity and sensitivity values of 73.9 and 83.3% (area under the curve, 0.85), respectively, for CRC discrimination after using an independent sample set containing 94 sera representative of different stages of progression and control subjects. In summary, these studies confirmed the presence of specific autoantibodies for CRC and revealed new individual markers of disease (PIM1, MAPKAPK3, and ACVR2B) with the potential to diagnose CRC with higher specificity and sensitivity than previously reported serum biomarkers.

  3. Estrogen Receptor (ER)α-regulated Lipocalin 2 Expression in Adipose Tissue Links Obesity with Breast Cancer Progression*

    PubMed Central

    Drew, Brian G.; Hamidi, Habib; Zhou, Zhenqi; Villanueva, Claudio J.; Krum, Susan A.; Calkin, Anna C.; Parks, Brian W.; Ribas, Vicent; Kalajian, Nareg Y.; Phun, Jennifer; Daraei, Pedram; Christofk, Heather R.; Hewitt, Sylvia C.; Korach, Kenneth S.; Tontonoz, Peter; Lusis, Aldons J.; Slamon, Dennis J.; Hurvitz, Sara A.; Hevener, Andrea L.

    2015-01-01

    Obesity is associated with increased breast cancer (BrCA) incidence. Considering that inactivation of estrogen receptor (ER)α promotes obesity and metabolic dysfunction in women and female mice, understanding the mechanisms and tissue-specific sites of ERα action to combat metabolic-related disease, including BrCA, is of clinical importance. To study the role of ERα in adipose tissue we generated fat-specific ERα knock-out (FERKO) mice. Herein we show that ERα deletion increased adipocyte size, fat pad weight, and tissue expression and circulating levels of the secreted glycoprotein, lipocalin 2 (Lcn2), an adipokine previously associated with BrCA development. Chromatin immunoprecipitation and luciferase reporter studies showed that ERα binds the Lcn2 promoter to repress its expression. Because adipocytes constitute an important cell type of the breast microenvironment, we examined the impact of adipocyte ERα deletion on cancer cell behavior. Conditioned medium from ERα-null adipocytes and medium containing pure Lcn2 increased proliferation and migration of a subset of BrCA cells in culture. The proliferative and promigratory effects of ERα-deficient adipocyte-conditioned medium on BrCA cells was reversed by Lcn2 deletion. BrCA cell responsiveness to exogenous Lcn2 was heightened in cell types where endogenous Lcn2 expression was minimal, but components of the Lcn2 signaling pathway were enriched, i.e. SLC22A17 and 3-hydroxybutyrate dehydrogenase (BDH2). In breast tumor biopsies from women diagnosed with BrCA we found that BDH2 expression was positively associated with adiposity and circulating Lcn2 levels. Collectively these data suggest that reduction of ERα expression in adipose tissue promotes adiposity and is linked with the progression and severity of BrCA via increased adipocyte-specific Lcn2 production and enhanced tumor cell Lcn2 sensitivity. PMID:25468909

  4. Recent progresses in understanding of water interacting with biomolecules, and inside living cells and tissues

    NASA Astrophysics Data System (ADS)

    Ford, R. C.; Li, J.

    Recent inelastic and quasi-elastic neutron scattering measurements of water in cell preparations has provided information on the interfacial (or bound) water molecules. The experiments show that the interfacial water molecules can be readily distinguished from those in the bulk (bulk water), especially using inelastic neutron scattering data over the 20-130 meV range. Studies of intact biological systems - whole cells and tissues - demonstrated the feasibility of using these methods to assess the degree of interfacial water and their potential for monitoring physiological changes. Here we also describe the effect of heat shock and osmotic stress on yeast and E. coli cells, and show that the interfacial water content increases with elevated osmolarity and heat shock, and decreases under hypoosmotic conditions.

  5. 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.

  6. Optimized T7 amplification system for microarray analysis.

    PubMed

    Pabón, C; Modrusan, Z; Ruvolo, M V; Coleman, I M; Daniel, S; Yue, H; Arnold, L J

    2001-10-01

    Glass cDNA microarray technologies offer a highly parallel approach for profiling expressed gene sequences in disease-relevant tissues. However, standard hybridization and detection protocols are insufficient for milligram quantities of tissue, such as those derived from needle biopsies. Amplification systems utilizing T7 RNA polymerase can provide multiple cRNA copies from mRNA transcripts, permitting microarray studies with reduced sample inputs. Here, we describe an optimized T7-based amplification system for microarray analysis that yields between 200- and 700-fold amplification. This system was evaluated with both mRNA and total RNA samples and provided microarray sensitivity and precision that are comparable to our standard production process without amplification. The size distributions of amplified cRNA ranged from 200 bp to 4 kb and were similar to original mRNA profiles. These amplified cRNA samples were fluorescently labeled by reverse transcription and hybridized to microarrays comprising approximately 10,000 cDNA targets using a dual-channel format. Replicate hybridization experiments were conducted with the same and different tissues in each channel to assess the sensitivity and precision of differential expression ratios. Statistical analysis of differential expression ratios showed the lower limit of detection to be about 2-fold within and between amplified data sets, and about 3-fold when comparing amplified data to unamplified data (99.5% confidence).

  7. Sporophytic ovule tissues modulate the initiation and progression of apomixis in Hieracium.

    PubMed

    Tucker, Matthew R; Okada, Takashi; Johnson, Susan D; Takaiwa, Fumio; Koltunow, Anna M G

    2012-05-01

    Apomixis in Hieracium subgenus Pilosella initiates in ovules when sporophytic cells termed aposporous initial (AI) cells enlarge near sexual cells undergoing meiosis. AI cells displace the sexual structures and divide by mitosis to form unreduced embryo sac(s) without meiosis (apomeiosis) that initiate fertilization-independent embryo and endosperm development. In some Hieracium subgenus Pilosella species, these events are controlled by the dominant LOSS OF APOMEIOSIS (LOA) and LOSS OF PARTHENOGENESIS (LOP) loci. In H. praealtum and H. piloselloides, which both contain the same core LOA locus, the timing and frequency of AI cell formation is altered in derived mutants exhibiting abnormal funiculus growth and in transgenic plants expressing rolB which alters cellular sensitivity to auxin. The impact on apomictic and sexual reproduction was examined here when a chimeric RNAse gene was targeted to the funiculus and basal portions of the ovule, and also when polar auxin transport was inhibited during ovule development following N-1-naphthylphthalamic acid (NPA) application. Both treatments led to ovule deformity in the funiculus and distal parts of the ovule and LOA-dependent alterations in the timing, position, and frequency of AI cell formation. In the case of NPA treatment, this correlated with increased expression of DR5:GFP in the ovule, which marks the accumulation of the plant hormone auxin. Our results show that sporophytic information potentiated by funiculus growth and polar auxin transport influences ovule development, the initiation of apomixis, and the progression of embryo sac development in Hieracium. Signals associated with ovule pattern formation and auxin distribution or perception may influence the capacity of sporophytic ovule cells to respond to LOA.

  8. 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

  9. Plasma levels of the tissue inhibitor matrix metalloproteinase-3 as a potential biomarker in oral cancer progression

    PubMed Central

    Su, Chun-Wen; Su, Bo-Feng; Chiang, Whei-Ling; Yang, Shun-Fa; Chen, Mu-Kuan; Lin, Chiao-Wen

    2017-01-01

    Oral cancer is the most common malignancy with poor prognosis and is the fourth most common cancer in men in Taiwan. The tissue inhibitor of metalloproteinase-3 (TIMP3) acts as a tumor suppressor gene by inhibiting the growth, angiogenesis, migration, and invasion of cancer cells. However, few studies have examined the association of plasma TIMP3 levels with oral squamous cell carcinoma (OSCC), and the role of plasma TIMP3 levels in OSCC progression is still unclear. We measured the plasma TIMP3 levels of 450 OSCC patients and 64 healthy controls by using a commercial enzyme-linked immunosorbent assay. We also analyzed TIMP3 mRNA levels of 328 OSCC patients and 32 normal tissues from The Cancer Genome Atlas (TCGA) dataset. Our results revealed that plasma TIMP3 levels were significantly lower in patients with OSCC than in healthy controls (p < 0.001). Moreover, plasma TIMP3 levels in patients with OSCC were significantly associated with the tumor stage and tumor status but not with the lymph node status, metastasis, and cell differentiation. To verify our findings, we also examined TCGA bioinformatics database and discovered similar results for the association with the pathological stage of OSCC. In conclusion, our results suggest that plasma TIMP3 is a potential biomarker for predicting the tumor stage and T status in patients with OSCC. PMID:28138307

  10. Plant-pathogen interactions: what microarray tells about it?

    PubMed

    Lodha, T D; Basak, J

    2012-01-01

    Plant defense responses are mediated by elementary regulatory proteins that affect expression of thousands of genes. Over the last decade, microarray technology has played a key role in deciphering the underlying networks of gene regulation in plants that lead to a wide variety of defence responses. Microarray is an important tool to quantify and profile the expression of thousands of genes simultaneously, with two main aims: (1) gene discovery and (2) global expression profiling. Several microarray technologies are currently in use; most include a glass slide platform with spotted cDNA or oligonucleotides. Till date, microarray technology has been used in the identification of regulatory genes, end-point defence genes, to understand the signal transduction processes underlying disease resistance and its intimate links to other physiological pathways. Microarray technology can be used for in-depth, simultaneous profiling of host/pathogen genes as the disease progresses from infection to resistance/susceptibility at different developmental stages of the host, which can be done in different environments, for clearer understanding of the processes involved. A thorough knowledge of plant disease resistance using successful combination of microarray and other high throughput techniques, as well as biochemical, genetic, and cell biological experiments is needed for practical application to secure and stabilize yield of many crop plants. This review starts with a brief introduction to microarray technology, followed by the basics of plant-pathogen interaction, the use of DNA microarrays over the last decade to unravel the mysteries of plant-pathogen interaction, and ends with the future prospects of this technology.

  11. Microarray simulator as educational tool.

    PubMed

    Ruusuvuori, Pekka; Nykter, Matti; Mäkiraatikka, Eeva; Lehmussola, Antti; Korpelainen, Tomi; Erkkilä, Timo; Yli-Harja, Olli

    2007-01-01

    As many real-world applications, microarray measurements are inapplicable for large-scale teaching purposes due to their laborious preparation process and expense. Fortunately, many phases of the array preparation process can be efficiently demonstrated by using a software simulator tool. Here we propose the use of microarray simulator as an aiding tool in teaching of computational biology. Three case studies on educational use of the simulator are presented, which demonstrate the effect of gene knock-out, synthetic time series, and effect of noise sources. We conclude that the simulator, used for teaching the principles of microarray measurement technology, proved to be a useful tool in education.

  12. 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...

  13. Expression profile of long non-coding RNAs in colorectal cancer: A microarray analysis.

    PubMed

    Luo, Jia; Xu, Luning; Jiang, Yigui; Zhuo, Dexiang; Zhang, Shengjun; Wu, Lianhui; Xu, Huadong; Huang, Yue

    2016-04-01

    Colorectal cancer (CRC) is one of the most prevalent malignant tumors and the second cause of cancer-related mortality worldwide. Due to increased morbidity and mortality rates, there is an urgent need to understand the pathogenesis of CRC, discover strategies that can improve diagnosis, and ultimately identify therapies targeting this disease. Over the past several years, research into tumor progression mechanisms has been devoted to identifying and understanding various coding and non-coding regions of the genome and how these genetic variants may affect tumorigenesis and progression. Recently, long non-coding RNAs (lncRNAs), which are non‑protein coding transcripts longer than 200 nucleotides, have emerged as a key aspect in tumor pathogenesis. In the present study, we examined the lncRNA and mRNA expression profiles in 4 patients with colon adenocarcinoma, with paired adjacent normal tissues as controls. Microarray data showed that a total of 3,523 lncRNAs and 2,515 mRNAs were consistently differentially expressed in the CRC tissues compared to adjacent normal tissues. Upon comparison of the differentially expressed transcripts between the groups, we identified 22 pathways which were related to the upregulated transcripts and 24 pathways that corresponded to the downregulated transcripts. Gene ontology analysis revealed that the upregulated transcripts were predominantly enriched in DNA metabolic processes, and the downregulated transcripts were predominantly enriched in organic hydroxyl compound metabolic processes. Coding-non-coding gene co-expression analysis showed that these differentially expressed lncRNAs were closely correlated with 'Wnt signaling pathway' components, whose aberrant activation plays a central role in CRC, indicating that a functional correlation exists between them. In conclusion, the results of the microarray and informatic analysis strongly suggest that lncRNA dysregulation is involved in the complicated process of CRC development

  14. Chemistry of Natural Glycan Microarray

    PubMed Central

    Song, Xuezheng; Heimburg-Molinaro, Jamie; Cummings, Richard D.; Smith, David F.

    2014-01-01

    Glycan microarrays have become indispensable tools for studying protein-glycan interactions. Along with chemo-enzymatic synthesis, glycans isolated from natural sources have played important roles in array development and will continue to be a major source of glycans. N- and O-glycans from glycoproteins, and glycans from glycosphingolipids can be released from corresponding glycoconjugates with relatively mature methods, although isolation of large numbers and quantities of glycans are still very challenging. Glycosylphosphatidylinositol (GPI)-anchors and glycosaminoglycans (GAGs) are less represented on current glycan microarrays. Glycan microarray development has been greatly facilitated by bifunctional fluorescent linkers, which can be applied in a “Shotgun Glycomics” approach to incorporate isolated natural glycans. Glycan presentation on microarrays may affect glycan binding by GBPs, often through multivalent recognition by the GBP. PMID:24487062

  15. M@IA: a modular open-source application for microarray workflow and integrative datamining.

    PubMed

    Le Béchec, Antony; Zindy, Pierre; Sierocinski, Thomas; Petritis, Dimitri; Bihouée, Audrey; Le Meur, Nolwenn; Léger, Jean; Théret, Nathalie

    2008-01-01

    Microarray technology is a widely used approach to gene expression analysis. Many tools for microarray management and data analysis have been developed, and recently new methods have been proposed for deciphering biological pathways by integrating microarray data with other data sources. However, to improve microarray analysis and provide meaningful gene interaction networks, integrated software solutions are still needed. Therefore, we developed M@IA, an environment for DNA microarray data analysis allowing gene network reconstruction. M@IA is a microarray integrated application which includes all of the steps of a microarray study, from MIAME-compliant raw data storage and processing gene expression analysis. Furthermore, M@IA allows automatic gene annotation based on ontology, metabolic/signalling pathways, protein interaction, miRNA and transcriptional factor associations, as well as integrative analysis of gene interaction networks. Statistical and graphical methods facilitate analysis, yielding new hypotheses on gene expression data. To illustrate our approach, we applied M@IA modules to microarray data taken from an experiment on liver tissue. We integrated differentially expressed genes with additional biological information, thus identifying new molecular interaction networks that are associated with fibrogenesis. M@IA is a new application for microarray management and data analysis, offering functional insights into microarray data by the combination of gene expression data and biological knowledge annotation based on interactive graphs. M@IA is an interactive multi-user interface based on a flexible modular architecture and it is freely available for academic users at http://maia.genouest.org.

  16. 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. PMID:27231487

  17. 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.

  18. Cardiac tissue-restricted deletion of plakoglobin results in progressive cardiomyopathy and activation of {beta}-catenin signaling.

    PubMed

    Li, Jifen; Swope, David; Raess, Natalia; Cheng, Lan; Muller, Eliane J; Radice, Glenn L

    2011-03-01

    Mutations in the plakoglobin (JUP) gene have been identified in arrhythmogenic right ventricular cardiomyopathy (ARVC) patients. However, the mechanisms underlying plakoglobin dysfunction involved in the pathogenesis of ARVC remain poorly understood. Plakoglobin is a component of both desmosomes and adherens junctions located at the intercalated disc (ICD) of cardiomyocytes, where it functions to link cadherins to the cytoskeleton. In addition, plakoglobin functions as a signaling protein via its ability to modulate the Wnt/β-catenin signaling pathway. To investigate the role of plakoglobin in ARVC, we generated an inducible cardiorestricted knockout (CKO) of the plakoglobin gene in mice. Plakoglobin CKO mice exhibited progressive loss of cardiac myocytes, extensive inflammatory infiltration, fibrous tissue replacement, and cardiac dysfunction similar to those of ARVC patients. Desmosomal proteins from the ICD were decreased, consistent with altered desmosome ultrastructure in plakoglobin CKO hearts. Despite gap junction remodeling, plakoglobin CKO hearts were refractory to induced arrhythmias. Ablation of plakoglobin caused increase β-catenin stabilization associated with activated AKT and inhibition of glycogen synthase kinase 3β. Finally, β-catenin/TCF transcriptional activity may contribute to the cardiac hypertrophy response in plakoglobin CKO mice. This novel model of ARVC demonstrates for the first time how plakoglobin affects β-catenin activity in the heart and its implications for disease pathogenesis.

  19. Microarray Technologies in Fungal Diagnostics.

    PubMed

    Rupp, Steffen

    2017-01-01

    Microarray technologies have been a major research tool in the last decades. In addition they have been introduced into several fields of diagnostics including diagnostics of infectious diseases. Microarrays are highly parallelized assay systems that initially were developed for multiparametric nucleic acid detection. From there on they rapidly developed towards a tool for the detection of all kind of biological compounds (DNA, RNA, proteins, cells, nucleic acids, carbohydrates, etc.) or their modifications (methylation, phosphorylation, etc.). The combination of closed-tube systems and lab on chip devices with microarrays further enabled a higher automation degree with a reduced contamination risk. Microarray-based diagnostic applications currently complement and may in the future replace classical methods in clinical microbiology like blood cultures, resistance determination, microscopic and metabolic analyses as well as biochemical or immunohistochemical assays. In addition, novel diagnostic markers appear, like noncoding RNAs and miRNAs providing additional room for novel nucleic acid based biomarkers. Here I focus an microarray technologies in diagnostics and as research tools, based on nucleic acid-based arrays.

  20. 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.

  1. Microarray analysis on the lncRNA expression profile in male hepatocelluar carcinoma patients with chronic hepatitis B virus infection

    PubMed Central

    Niu, Jianjun; Lin, Yong; Liu, Pingguo; Yu, Yiwen; Su, Chenghao; Wang, Xiaomin

    2016-01-01

    Long non-coding RNAs are involved with development and progression of cancer, and the advance of microarray technology allows the researchers to investigate the complete expression profile of lncRNA in various kinds of sample. We enrolled 5 male primary HCC cases with chronic HBV infection and the HCC and normal tissues have been obtained during the resection surgery. After total RNA extraction, the lncRNA microarray analysis was conducted to determine the lncRNA and mRNA expression signals. 612 lncRNAs and 1,064 mRNAs were significantly up-regulated in HCC tissue while 656 lncRNAs and 1,532 mRNAs were down-regulated in HCC tissues. Compared with normal tissues, XLOC_007433 (fold change: 12.80) and AC144449.1 (fold change: 27.20) were the most over- and under-expressed lncRNAs in HCC tissues. As for the mRNA, THBS4 (fold change:41.13) and CXCL14 (fold change: 58.03) were the most over- and under-expressed mRNAs in HCC tissues when comparing with their normal counterparts. In total, 4,552 pairs of lncRNA-mRNA were identified and the co-expression network was constructed. Moreover, the gene ontology enrichment analysis showed that the significantly different transcript between HCC and normal tissues were mainly associated with response to wounding, inflammatory response, protein hetrodimerization activity, response to stress which involved with biological process and molecular function. The pathway analysis suggested that the most significant pathways consisted of alcoholism, regulatory RNA pathways and RNA polymerase transcription. Several novel differentially expressed lncRNAs and mRNAs were identified in the present study. PMID:27769059

  2. A lectin-based cell microarray approach to analyze the mammalian granulosa cell surface glycosylation profile.

    PubMed

    Accogli, Gianluca; Desantis, Salvatore; Martino, Nicola Antonio; Dell'Aquila, Maria Elena; Gemeiner, Peter; Katrlík, Jaroslav

    2016-10-01

    The high complexity of glycome, the repertoire of glycans expressed in a cell or in an organism, is difficult to analyze and the use of new technologies has accelerated the progress of glycomics analysis. In the last decade, the microarray approaches, and in particular glycan and lectin microarrays, have provided new insights into evaluation of cell glycosylation status. Here we present a cell microarray method based on cell printing on microarray slides for the analysis of the glycosylation pattern of the cell glycocalyx. In order to demonstrate the reliability of the developed method, the glycome profiles of equine native uncultured mural granulosa cells (uGCs) and in vitro cultured mural granulosa cells (cGCs) were determined and compared. The method consists in the isolation of GCs, cell printing into arrays on microarray slide, incubation with a panel of biotinylated lectins, reaction with fluorescent streptavidin and signal intensity detection by a microarray scanner. Cell microarray technology revealed that glycocalyx of both uGCs and cGCs contains N-glycans, sialic acid terminating glycans, N-acetylglucosamine and O-glycans. The comparison of uGCs and cGCs glycan signals indicated an increase in the expression of sialic acids, N-acetylglucosamine, and N-glycans in cGCs. Glycan profiles determined by cell microarray agreed with those revealed by lectin histochemistry. The described cell microarray method represents a simple and sensitive procedure to analyze cell surface glycome in mammalian cells.

  3. 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

  4. 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

  5. Development of a highly sensitive glycan microarray for quantifying AFP-L3 for early prediction of hepatitis B virus-related hepatocellular carcinoma.

    PubMed

    Wu, Chen-Shiou; Lee, Teng-Yu; 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

  6. Microfluidic microarray systems and methods thereof

    SciTech Connect

    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.

  7. 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

  8. 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,…

  9. Metastasis Initiating Cells in Primary Prostate Cancer Tissues From Transurethral Resection of the Prostate (TURP) Predicts Castration-Resistant Progression and Survival of Prostate Cancer Patients

    PubMed Central

    Li, Qinlong; Li, Quanlin; Nuccio, Jill; Liu, Chunyan; Duan, Peng; Wang, Ruoxiang; Jones, Lawrence W.; Chung, Leland W. K.; Zhau, Haiyen E.

    2016-01-01

    BACKGROUND We previouslyreported that the activation of RANK and c-Met signaling components in both experimental mouse models and human prostate cancer (PC) specimens predicts bone metastatic potential and PC patient survival. This study addresses whether a population of metastasis-initiating cells (MICs) known to express a stronger RANKL, phosphorylated c-Met (p-c-Met), and neuropilin-1 (NRP1) signaling network than bystander or dormant cells (BDCs) can be detected in PC tissues from patients subjected to transurethral resection of the prostate (TURP) for urinary obstruction prior to the diagnosis of PC with or without prior hormonal manipulation, and whether the relative abundance of MICs over BDCs could predict castration-resistant progression and PC patient survival. METHODS We employed a multiplexed quantum-dot labeling (mQDL) protocol to detect and quantify MICs and BDCs at the single cell level in TURP tissues obtained from 44 PC patients with documented overall survival and castration resistance status. RESULTS PC tissues with a higher number of MICs and an activated RANK signaling network, including increased expression of RANKL, p-c-Met, and NRP1 compared to BDCs, were found to correlate with the development of castration resistance and overall survival. CONCLUSIONS The assessment of PC cells with MIC and BDC phenotypes in primary PC tissues from hormone-naïve patients can predict the progression to castration resistance and the overall survival of PC patients. PMID:25990623

  10. Disc-based microarrays: principles and analytical applications.

    PubMed

    Morais, Sergi; Puchades, Rosa; Maquieira, Ángel

    2016-07-01

    The idea of using disk drives to monitor molecular biorecognition events on regular optical discs has received considerable attention during the last decade. CDs, DVDs, Blu-ray discs and other new optical discs are universal and versatile supports with the potential for development of protein and DNA microarrays. Besides, standard disk drives incorporated in personal computers can be used as compact and affordable optical reading devices. Consequently, a CD technology, resulting from the audio-video industry, has been used to develop analytical applications in health care, environmental monitoring, food safety and quality assurance. The review presents and critically evaluates the current state of the art of disc-based microarrays with illustrative examples, including past, current and future developments. Special mention is made of the analytical developments that use either chemically activated or raw standard CDs where proteins, oligonucleotides, peptides, haptens or other biological probes are immobilized. The discs are also used to perform the assays and must maintain their readability with standard optical drives. The concept and principle of evolving disc-based microarrays and the evolution of disk drives as optical detectors are also described. The review concludes with the most relevant uses ordered chronologically to provide an overview of the progress of CD technology applications in the life sciences. Also, it provides a selection of important references to the current literature. Graphical Abstract High density disc-based microarrays.

  11. 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.

  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 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.

  13. 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

  14. 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.

  15. Tumor-Derived Tissue Factor Aberrantly Activates Complement and Facilitates Lung Tumor Progression via Recruitment of Myeloid-Derived Suppressor Cells

    PubMed Central

    Han, Xiao; Zha, Haoran; Yang, Fei; Guo, Bo; Zhu, Bo

    2017-01-01

    The initiator of extrinsic coagulation, tissue factor (TF), and its non-coagulant isoform alternatively spliced TF (asTF) are closely associated with tumor development. In the tumor microenvironment, the role of TF-induced coagulation in tumor progression remains to be fully elucidated. Using TF-knockdown lung tumor cells, we showed that TF is the dominant component of procoagulant activity but is dispensable in the cellular biology of tumor cells. In a xenograft model, using immunohistochemical analysis and flow cytometry analysis of the tumor microenvironment, we demonstrated that TF-induced fibrin deposition, which is correlated with complement activation and myeloid-derived suppressor cell (MDSC) recruitment, is positively associated with tumor progression. C5aR antagonism blunted the effect of TF on tumor progression and decreased MDSC recruitment. In conclusion, our data suggested that in tumor microenvironment, TF-induced coagulation activated the complement system and subsequently recruited myeloid-derived suppressor cells to promote tumor growth, which brings new insights into the coagulation-induced complement activation within the tumor microenvironment during tumor progression. PMID:28106852

  16. Tumor-Derived Tissue Factor Aberrantly Activates Complement and Facilitates Lung Tumor Progression via Recruitment of Myeloid-Derived Suppressor Cells.

    PubMed

    Han, Xiao; Zha, Haoran; Yang, Fei; Guo, Bo; Zhu, Bo

    2017-01-19

    The initiator of extrinsic coagulation, tissue factor (TF), and its non-coagulant isoform alternatively spliced TF (asTF) are closely associated with tumor development. In the tumor microenvironment, the role of TF-induced coagulation in tumor progression remains to be fully elucidated. Using TF-knockdown lung tumor cells, we showed that TF is the dominant component of procoagulant activity but is dispensable in the cellular biology of tumor cells. In a xenograft model, using immunohistochemical analysis and flow cytometry analysis of the tumor microenvironment, we demonstrated that TF-induced fibrin deposition, which is correlated with complement activation and myeloid-derived suppressor cell (MDSC) recruitment, is positively associated with tumor progression. C5aR antagonism blunted the effect of TF on tumor progression and decreased MDSC recruitment. In conclusion, our data suggested that in tumor microenvironment, TF-induced coagulation activated the complement system and subsequently recruited myeloid-derived suppressor cells to promote tumor growth, which brings new insights into the coagulation-induced complement activation within the tumor microenvironment during tumor progression.

  17. Immunohistochemistry Study of P53 and C-erbB-2 Expression in Trophoblastic Tissue and Their Predictive Values in Diagnosing Malignant Progression of Simple Molar Pregnancy

    PubMed Central

    Hasanzadeh, Malihe; Sharifi, Norrie; Farazestanian, Marjaneh; Nazemian, Seyed Saman; Madani Sani, Faezeh

    2016-01-01

    Background Finding a tumor marker to predict the aggressive behavior of molar pregnancy in early stages has yet been a topic for studies. Objectives In this survey we planned to study patients with molar pregnancy to 1) assess the p53 and c-erbB-2 expression in trophoblastic tissue, 2) to study the relationship between their expression intensity and progression of a molar pregnancy to gestational trophoblastic neoplasia, and 3) to determine a cut off value for the amount of p53 and c-erbB-2 expression which might correlate with aggressive behavior of molar pregnancy. Patients and Methods In a prospective cross sectional study by using a high accuracy technique EnVision Tm system for immunohistochemistry staining of molar pregnancy samples, we evaluated p53 and c-erbB-2 expression in cytotrophoblast and syncytiotrophoblast and the correlation of their expression with progression of molar pregnancy to gestational trophoblastic neoplasia (GTN). Normal prostatic tissue and Breast cancer tissue were used as positive controls. Results We studied 28 patients with simple molar pregnancy (SMP) and 30 with GTN. Cytotrophobalst had significantly higher expression of p53 and c-erbB-2 and syncytiotrophoblast had greater expression of p53 in GTN group as compared to SMP group. The cut off values for percentage of p53 positive immunostained cytotrophoblast and syncytiotrophoblast were 5.5% and 2.5%. In c-erbB-2 positive membranous stained cytotrophoblast the cut off was 12.5%. Conclusions Our data suggests that over expression of p53 and c-erbB-2 is associated with malignant progression of molar pregnancy. We encountered that high expression of p53 and c-erbB-2 in trophoblastic cells could predict gestational trophoblastic neoplasia during the early stages. PMID:27703642

  18. 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

  19. Recommendations for the use of microarrays in prenatal diagnosis.

    PubMed

    Suela, Javier; López-Expósito, Isabel; Querejeta, María Eugenia; Martorell, Rosa; Cuatrecasas, Esther; Armengol, Lluis; Antolín, Eugenia; Domínguez Garrido, Elena; Trujillo-Tiebas, María José; Rosell, Jordi; García Planells, Javier; Cigudosa, Juan Cruz

    2017-04-07

    Microarray technology, recently implemented in international prenatal diagnosis systems, has become one of the main techniques in this field in terms of detection rate and objectivity of the results. This guideline attempts to provide background information on this technology, including technical and diagnostic aspects to be considered. Specifically, this guideline defines: the different prenatal sample types to be used, as well as their characteristics (chorionic villi samples, amniotic fluid, fetal cord blood or miscarriage tissue material); variant reporting policies (including variants of uncertain significance) to be considered in informed consents and prenatal microarray reports; microarray limitations inherent to the technique and which must be taken into account when recommending microarray testing for diagnosis; a detailed clinical algorithm recommending the use of microarray testing and its introduction into routine clinical practice within the context of other genetic tests, including pregnancies in families with a genetic history or specific syndrome suspicion, first trimester increased nuchal translucency or second trimester heart malformation and ultrasound findings not related to a known or specific syndrome. This guideline has been coordinated by the Spanish Association for Prenatal Diagnosis (AEDP, «Asociación Española de Diagnóstico Prenatal»), the Spanish Human Genetics Association (AEGH, «Asociación Española de Genética Humana») and the Spanish Society of Clinical Genetics and Dysmorphology (SEGCyD, «Sociedad Española de Genética Clínica y Dismorfología»).

  20. Surface characterization of carbohydrate microarrays.

    PubMed

    Scurr, David J; Horlacher, Tim; Oberli, Matthias A; Werz, Daniel B; Kroeck, Lenz; Bufali, Simone; Seeberger, Peter H; Shard, Alexander G; Alexander, Morgan R

    2010-11-16

    Carbohydrate microarrays are essential tools to determine the biological function of glycans. Here, we analyze a glycan array by time-of-flight secondary ion mass spectrometry (ToF-SIMS) to gain a better understanding of the physicochemical properties of the individual spots and to improve carbohydrate microarray quality. The carbohydrate microarray is prepared by piezo printing of thiol-terminated sugars onto a maleimide functionalized glass slide. The hyperspectral ToF-SIMS imaging data are analyzed by multivariate curve resolution (MCR) to discern secondary ions from regions of the array containing saccharide, linker, salts from the printing buffer, and the background linker chemistry. Analysis of secondary ions from the linker common to all of the sugar molecules employed reveals a relatively uniform distribution of the sugars within the spots formed from solutions with saccharide concentration of 0.4 mM and less, whereas a doughnut shape is often formed at higher-concentration solutions. A detailed analysis of individual spots reveals that in the larger spots the phosphate buffered saline (PBS) salts are heterogeneously distributed, apparently resulting in saccharide concentrated at the rim of the spots. A model of spot formation from the evaporating sessile drop is proposed to explain these observations. Saccharide spot diameters increase with saccharide concentration due to a reduction in surface tension of the saccharide solution compared to PBS. The multivariate analytical partial least squares (PLS) technique identifies ions from the sugars that in the complex ToF-SIMS spectra correlate with the binding of galectin proteins.

  1. Diagnostic challenges for multiplexed protein microarrays.

    PubMed

    Master, Stephen R; Bierl, Charlene; Kricka, Larry J

    2006-11-01

    Multiplexed protein analysis using planar microarrays or microbeads is growing in popularity for simultaneous assays of antibodies, cytokines, allergens, drugs and hormones. However, this new assay format presents several new operational issues for the clinical laboratory, such as the quality control of protein-microarray-based assays, the release of unrequested test data and the use of diagnostic algorithms to transform microarray data into diagnostic results.

  2. Automated Microarray Image Analysis Toolbox for MATLAB

    SciTech Connect

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

    2005-09-01

    The Automated Microarray Image Analysis (AMIA) Toolbox for MATLAB is a flexible, open-source microarray image analysis tool that allows the user to customize analysis of sets of microarray images. This tool provides several methods of identifying and quantify spot statistics, as well as extensive diagnostic statistics and images to identify poor data quality or processing. The open nature of this software allows researchers to understand the algorithms used to provide intensity estimates and to modify them easily if desired.

  3. Independent component analysis of Alzheimer's DNA microarray gene expression data

    PubMed Central

    Kong, Wei; Mou, Xiaoyang; Liu, Qingzhong; Chen, Zhongxue; Vanderburg, Charles R; Rogers, Jack T; Huang, Xudong

    2009-01-01

    Background Gene microarray technology is an effective tool to investigate the simultaneous activity of multiple cellular pathways from hundreds to thousands of genes. However, because data in the colossal amounts generated by DNA microarray technology are usually complex, noisy, high-dimensional, and often hindered by low statistical power, their exploitation is difficult. To overcome these problems, two kinds of unsupervised analysis methods for microarray data: principal component analysis (PCA) and independent component analysis (ICA) have been developed to accomplish the task. PCA projects the data into a new space spanned by the principal components that are mutually orthonormal to each other. The constraint of mutual orthogonality and second-order statistics technique within PCA algorithms, however, may not be applied to the biological systems studied. Extracting and characterizing the most informative features of the biological signals, however, require higher-order statistics. Results ICA is one of the unsupervised algorithms that can extract higher-order statistical structures from data and has been applied to DNA microarray gene expression data analysis. We performed FastICA method on DNA microarray gene expression data from Alzheimer's disease (AD) hippocampal tissue samples and consequential gene clustering. Experimental results showed that the ICA method can improve the clustering results of AD samples and identify significant genes. More than 50 significant genes with high expression levels in severe AD were extracted, representing immunity-related protein, metal-related protein, membrane protein, lipoprotein, neuropeptide, cytoskeleton protein, cellular binding protein, and ribosomal protein. Within the aforementioned categories, our method also found 37 significant genes with low expression levels. Moreover, it is worth noting that some oncogenes and phosphorylation-related proteins are expressed in low levels. In comparison to the PCA and support

  4. 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...

  5. Recent progress in the understanding of tissue culture-induced genome level changes in plants and potential applications.

    PubMed

    Neelakandan, Anjanasree K; Wang, Kan

    2012-04-01

    In vitro cell and tissue-based systems have tremendous potential in fundamental research and for commercial applications such as clonal propagation, genetic engineering and production of valuable metabolites. Since the invention of plant cell and tissue culture techniques more than half a century ago, scientists have been trying to understand the morphological, physiological, biochemical and molecular changes associated with tissue culture responses. Establishment of de novo developmental cell fate in vitro is governed by factors such as genetic make-up, stress and plant growth regulators. In vitro culture is believed to destabilize the genetic and epigenetic program of intact plant tissue and can lead to chromosomal and DNA sequence variations, methylation changes, transposon activation, and generation of somaclonal variants. In this review, we discuss the current status of understanding the genomic and epigenomic changes that take place under in vitro conditions. It is hoped that a precise and comprehensive knowledge of the molecular basis of these variations and acquisition of developmental cell fate would help to devise strategies to improve the totipotency and embryogenic capability in recalcitrant species and genotypes, and to address bottlenecks associated with clonal propagation.

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

    PubMed

    Ramirez, Lisa S; Wang, Jun

    2016-01-06

    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.

  7. Semiconductor quantum dots for multiplexed bio-detection on solid-state microarrays.

    PubMed

    Rousserie, Gilles; Sukhanova, Alyona; Even-Desrumeaux, Klervi; Fleury, Fabrice; Chames, Patrick; Baty, Daniel; Oleinikov, Vladimir; Pluot, Michel; Cohen, Jacques H M; Nabiev, Igor

    2010-04-01

    Understanding cellular systems requires identification and analysis of their multiple components and determination of how they act together and are regulated. Microarray technology is one of the few tools that is able to solve such problems. It is based on high-throughput recognition of a target to the probe and has the potential to simultaneously measure the presence of numerous molecules in multiplexed tests, all contained in a small drop of test fluid. Microarrays allow the parallel analysis of genomic or proteomic content in healthy versus disease-affected or altered tissues or cells. The signal read-out from the microarrays is done with organic dyes which often suffer of photobleaching, low brightness and background fluorescence. Recent data show that the use of fluorescent nanocrystals named "quantum dots" (QDs) allows to push these limits away. QDs are sufficiently bright to be detected as individual particles, extremely resistant against photobleaching and provide unique possibilities for multiplexing, thus supplying the microarray technology with a novel read-out option enabling the sensitivity of detection to reach the single-molecule level. This paper reviews QDs applications to microarray-based detection and demonstrates how the combination of microarray and QDs technologies may increase sensitivity and highly parallel capacities of multiplexed microarrays. Such a combination should provide the breakthrough results in drug discovery, cancer diagnosis and establish new therapeutic approaches through the identification of binding target molecules and better understanding of cell signalling pathways.

  8. Living Cell Microarrays: An Overview of Concepts

    PubMed Central

    Jonczyk, Rebecca; Kurth, Tracy; Lavrentieva, Antonina; Walter, Johanna-Gabriela; Scheper, Thomas; Stahl, Frank

    2016-01-01

    Living cell microarrays are a highly efficient cellular screening system. Due to the low number of cells required per spot, cell microarrays enable the use of primary and stem cells and provide resolution close to the single-cell level. Apart from a variety of conventional static designs, microfluidic microarray systems have also been established. An alternative format is a microarray consisting of three-dimensional cell constructs ranging from cell spheroids to cells encapsulated in hydrogel. These systems provide an in vivo-like microenvironment and are preferably used for the investigation of cellular physiology, cytotoxicity, and drug screening. Thus, many different high-tech microarray platforms are currently available. Disadvantages of many systems include their high cost, the requirement of specialized equipment for their manufacture, and the poor comparability of results between different platforms. In this article, we provide an overview of static, microfluidic, and 3D cell microarrays. In addition, we describe a simple method for the printing of living cell microarrays on modified microscope glass slides using standard DNA microarray equipment available in most laboratories. Applications in research and diagnostics are discussed, e.g., the selective and sensitive detection of biomarkers. Finally, we highlight current limitations and the future prospects of living cell microarrays. PMID:27600077

  9. Clustering Short Time-Series Microarray

    NASA Astrophysics Data System (ADS)

    Ping, Loh Wei; Hasan, Yahya Abu

    2008-01-01

    Most microarray analyses are carried out on static gene expressions. However, the dynamical study of microarrays has lately gained more attention. Most researches on time-series microarray emphasize on the bioscience and medical aspects but few from the numerical aspect. This study attempts to analyze short time-series microarray mathematically using STEM clustering tool which formally preprocess data followed by clustering. We next introduce the Circular Mould Distance (CMD) algorithm with combinations of both preprocessing and clustering analysis. Both methods are subsequently compared in terms of efficiencies.

  10. Tissue damage-associated "danger signals" influence T-cell responses that promote the progression of preneoplasia to cancer.

    PubMed

    He, Ying; Zha, Jikun; Wang, Yamin; Liu, Wenhua; Yang, Xuanming; Yu, Ping

    2013-01-15

    T-cell responses may be shaped by sterile "danger signals" that are constituted by damage-associated molecular patterns (DAMP). However, whether and what type of adaptive immune responses are triggered in vivo by DAMPs induced by tumor progression are not well characterized. In this study, we report that the production of HMGB1, an established DAMP released by dying cells, was critical for tumor progression in an established mouse model of prostate cancer. HMGB1 was required for the activation and intratumoral accumulation of T cells that expressed cytokine lymphotoxinα(1)β(2) (LT) on their surface. Intriguingly, these tumor-activated T cells recruited macrophages to the lesion and were essential to promote the preneoplasia to invasive carcinoma in an LTβ receptor (LTβR)-dependent manner. Taken together, our findings suggest that the release of HMGB1 as an endogenous danger signal is important for priming an adaptive immune response that promotes malignant progression, with implications for cancer prevention and therapy.

  11. Microarray analysis of diet-induced alterations in gene expression in the ACI rat prostate.

    PubMed

    Reyes, Niradiz; Iatropoulos, Michael; Mittelman, Abraham; Geliebter, Jan

    2002-08-01

    The natural history of prostate cancer is a multistage process that involves the transition from normal tissue to subclinical cancer, with progression to carcinoma in situ and eventually metastatic disease. Evidence suggests that a high-fat diet plays a critical role in the biology and progression of the disease. ACI rats were maintained for two generations on high beef fat or control diets for 18 months. Affymetrix microarrays were used to analyze the mRNA expression levels in the dorsolateral prostates of rats on the different diets. Approximately 4752 genes and expressed sequence tag (EST) were expressed in the prostates of rats on either diet. Twenty-seven genes were upregulated and 28 genes downregulated in the high beef fat diet. Data analysis indicated that a high beef fat diet affects the expression of genes involved in inflammation, glucose and fatty acid metabolism, androgen metabolism, potential tumor suppression and protein kinase activity, as well as intracellular and extracellular matrix molecules, growth factors and androgen responsive genes. Results from these and future studies will lead to a better understanding of the effect of diet on gene expression in the prostate and facilitate the rational design and assessment of potential dietary programs for prostate cancer prevention.

  12. 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

  13. 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...

  14. 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...

  15. Construction and analysis of tissue microarrays in the era of digital pathology: a pilot study targeting CDX1 and CDX2 in a colon cancer cohort of 612 patients

    PubMed Central

    Nolte, Sarah; Zlobec, Inti; Lugli, Alessandro; Hohenberger, Werner; Croner, Roland; Merkel, Susanne; Hartmann, Arndt; Geppert, Carol I

    2017-01-01

    Abstract CDX1 and CDX2 are possibly predictive biomarkers in colorectal cancer. We combined digitally‐guided (next generation) TMA construction (ngTMA) and the utility of digital image analysis (DIA) to assess accuracy, tumour heterogeneity and the selective impact of different combined intensity‐percentage levels on prognosis.CDX1 and CDX2 immunohistochemistry was performed on ngTMAs covering normal tissue, tumour centre and invasive front. The percentages of all epithelial cells per staining intensity per core were analysed digitally. Beyond classical prognosis analysis following REMARK guidelines, we investigated pre‐analytical conditions, three different types of heterogeneity (mosaic‐like, targeted and haphazard) and influences on cohort segregation and patient selection. The ngTMA‐DIA approach produced robust biomarker data with infrequent core loss and excellent on‐target punching. The detailed assessment of tumour heterogeneity could – except for a certain diffuse mosaic‐like heterogeneity – exclude differences between the invasive front and tumour centre, as well as detect haphazard clonal heterogeneous elements. Moreover, lower CDX1 and CDX2 counts correlated with mucinous histology, higher TNM stage, higher tumour grade and worse survival (p < 0.01, all). Different protein expression intensity levels shared comparable prognostic power and a great overlap in patient selection. The combination of ngTMA with DIA enhances accuracy and controls for biomarker analysis. Beyond the confirmation of CDX1 and CDX2 as prognostically relevant markers in CRC, this study highlights the greater robustness of CDX2 in comparison to CDX1. For the assessment of CDX2 protein loss, cut‐points as percentage data of complete protein loss can be deduced as a recommendation. PMID:28138402

  16. 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.

  17. Analysis of Protein Glycosylation and Phosphorylation Using Liquid Phase Separation, Protein Microarray Technology, and Mass Spectrometry

    PubMed Central

    Zhao, Jia; Patwa, Tasneem H.; Pal, Manoj; Qiu, Weilian; Lubman, David M.

    2010-01-01

    Summary Protein glycosylation and phosphorylation are very common posttranslational modifications. The alteration of these modifications in cancer cells is closely related to the onset and progression of cancer and other disease states. In this protocol, strategies for monitoring the changes in protein glycosylation and phosphorylation in serum or tissue cells on a global scale and specifically characterizing these alterations are included. The technique is based on lectin affinity enrichment for glycoproteins, all liquid-phase two-dimensional fractionation, protein microarray, and mass spectrometry technology. Proteins are separated based on pI in the first dimension using chromatofocusing (CF) or liquid isoelectric focusing (IEF) followed by the second-dimension separation using nonporous silica RP-HPLC. Five lectins with different binding specificities to glycan structures are used for screening glycosylation patterns in human serum through a biotin–streptavidin system. Fluorescent phosphodyes and phosphospecific antibodies are employed to detect specific phosphorylated proteins in cell lines or human tissues. The purified proteins of interest are identified by peptide sequencing. Their modifications including glycosylation and phosphorylation could be further characterized by mass-spectrometry-based approaches. These strategies can be used in biological samples for large-scale glycoproteome/phosphoproteome screening as well as for individual protein modification analysis. PMID:19241043

  18. Microarray analysis in caudal medulla of cattle orally challenged with bovine spongiform encephalopathy.

    PubMed

    Almeida, L M; Basu, U; Williams, J L; Moore, S S; Guan, L L

    2011-10-25

    Bovine spongiform encephalopathy (BSE) is a fatal disorder in cattle characterized by progressive neurodegeneration of the central nervous system. We investigated the molecular mechanisms involved in neurodegeneration during prion infection through the identification of genes that are differentially expressed (DE) between experimentally infected and non-challenged cattle. Gene expression of caudal medulla from control and orally infected animals was compared by microarray analysis using 24,000 bovine oligonucleotides representing 16,846 different genes to identify DE genes associated with BSE disease. In total, 182 DE genes were identified between normal and BSE-infected tissues (>2.0-fold change, P < 0.01); 81 DE genes had gene ontology functions, which included synapse function, calcium ion regulation, immune and inflammatory response, apoptosis, and cytoskeleton organization; 13 of these genes were found to be involved in 26 different Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The expression of five DE genes associated with synapse function (tachykinin, synuclein, neuropeptide Y, cocaine, amphetamine-responsive transcript, and synaptosomal-associated protein 25 kDa) and three DE genes associated with calcium ion regulation (parvalbumin, visinin-like, and cadherin) was further validated in the medulla tissue of cattle at different infection times (6, 12, 42, and 45 months post-infection) by qRT-PCR. These data will contribute to a better understanding of the molecular mechanisms of neuropathology in bovine species.

  19. 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.

  20. Progress in theoretical, experimental, and computational investigations in turbid tissue phantoms and human teeth using laser infrared photothermal radiometry

    NASA Astrophysics Data System (ADS)

    Mandelis, Andreas

    2002-03-01

    This paper reviews and describes the state-of-the-art in the development of frequency-domain infrared photothermal radiometry (FD-PTR) for biomedical and dental applications. The emphasis is placed on the measurement of the optical and thermal properties of tissue-like materials using FD-PTR. A rigorous three-dimensional thermal-wave formulation with three-dimensional diffuse and coherent photon-density-wave sources is presented, and is applied to data from model tissue phantoms and dental enamel samples. The combined theoretical, experimental and computational methodology shows good promise with regard to its analytical ability to measure optical properties of turbid media uniquely, as compared to PPTR, which exhibits uniqueness problems. From data sets obtained with calibrated test phantoms, the reduced optical scattering and absorption coefficients were found to be within 20% and 10%, respectively, from the independently derived values using Mie scattering theory and spectrophotometric measurements. Furthermore, the state-of-the-art and recent developments in applications of laser infrared FD-PTR to dental caries research is described, with examples and histological studies from carious dental tissue. The correlation of PTR signals with modulated dental luminescence is discussed as a very promising potential quantitative methodology for the clinical diagnosis of sub-surface incipient dental caries. The application of the turbid-medium thermal-wave model to the measurement of the optical absorption and scattering coefficients of enamel is also presented.

  1. Polyanionic collagen membranes for guided tissue regeneration: Effect of progressive glutaraldehyde cross-linking on biocompatibility and degradation.

    PubMed

    Veríssimo, D M; Leitão, R F C; Ribeiro, R A; Figueiró, S D; Sombra, A S B; Góes, J C; Brito, G A C

    2010-10-01

    The ultimate goal of periodontal therapy is to control periodontal tissue inflammation and to produce predictable regeneration of that part of the periodontium which has been lost as a result of periodontal disease. In guided tissue regeneration membranes function as mechanical barriers, excluding the epithelium and gingival corium from the root surface and allowing regeneration by periodontal ligament cells. This report aims to study the effect of glutaraldehyde (GA) cross-linking on mineralized polyanionic collagen (PAC) membranes by conducting a histological evaluation of the tissue response (biocompatibility) and by assessing the biodegradation of subcutaneous membrane implants in rats. We studied six different samples: a PAC, a PAC mineralized by alternate soaking processes for either 25 or 75 cycles (PAC 25 and PAC 75, respectively) and these films cross-linked by GA. Inflammatory infiltrate, cytokine dosage, fibrosis capsule thickness, metalloproteinase immunohistochemistry and membrane biodegradation after 1, 7, 15 and 30 days were measured. The inflammatory response was found to be more intense in membranes without cross-linking, while the fibrosis capsules became thicker in cross-linked membranes after 30 days. The membranes without cross-linking suffered intense biodegradation, while the membranes with cross-linking remained intact after 30 days. The cross-linking with GA reduced the inflammatory response and prevented degradation of the membranes over the entire course of the observation period. These membranes are thus an attractive option when the production of new bone depends on the prolonged presence of a mechanical barrier.

  2. 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.

  3. 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…

  4. 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.

  5. Progressive changes in composition of lymphocytes in lung tissues from patients with non-small-cell lung cancer

    PubMed Central

    del Mar Valenzuela-Membrives, María; Perea-García, Francisco; Sanchez-Palencia, Abel; Ruiz-Cabello, Francisco; Gómez-Morales, Mercedes; Miranda-León, María Teresa; Galindo-Angel, Inmaculada; Fárez-Vidal, María Esther

    2016-01-01

    Immune cell infiltration is a common feature of many human solid tumors. Innate and adaptative immune systems contribute to tumor immunosurveillance. We investigated whether tumors evade immune surveillance by inducing states of tolerance and/or through the inability of some immune subpopulations to effectively penetrate tumor nests. Immunohistochemistry and flow cytometry analysis were used to study the composition and distribution of immune subpopulations in samples of peripheral blood, tumor tissue (TT), adjacent tumor tissue (ATT), distant non-tumor tissue (DNTT), cancer nests, cancer stroma, and invasive margin in 61 non-small-cell lung cancer (NSCLC) patients. A significantly higher percentage of T and B cells and significantly lower percentage of NK cells were detected in TT than in DNTT. Memory T cells (CD4+CD45RO+, CD8+CD45RO+) and activated T cells (CD8+DR+) were more prevalent in TT. Alongside this immune activation, the percentage of T cells with immunosuppressive activity was higher in TT than in DNTT. B- cells were practically non-existent in tumor nests and were preferentially located in the invasive margin. The dominant NK cell phenotype in peripheral blood and DNTT was the cytotoxic phenotype (CD56+ CD16+), while the presence of these cells was significantly decreased in ATT and further decreased in TT. Finally, the immunologic response differed between adenocarcinoma and squamous cell carcinoma and according to the tumor differentiation grade. These findings on the infiltration of innate and adaptative immune cells into tumors contribute to a more complete picture of the immune reaction in NSCLC. PMID:27689405

  6. Genome-wide copy number aberrations and HER2 and FGFR1 alterations in primary breast cancer by molecular inversion probe microarray.

    PubMed

    Chen, Hui; Singh, Rajesh R; Lu, Xinyan; Huo, Lei; Yao, Hui; Aldape, Kenneth; Abraham, Ronald; Virani, Shumaila; Mehrotra, Meenakshi; Mishra, Bal Mukund; Bousamra, Alex; Albarracin, Constance; Wu, Yun; Roy-Chowdhuri, Sinchita; Shamanna, Rashmi Kanagal; Routbort, Mark J; Medeiros, L Jeffrey; Patel, Keyur P; Broaddus, Russell; Sahin, Aysegul; Luthra, Rajyalakshmi

    2017-01-24

    Breast cancer remains the second leading cause of cancer-related death in women despite stratification based on standard hormonal receptor (HR) and HER2 testing. Additional prognostic markers are needed to improve breast cancer treatment. Chromothripsis, a catastrophic genome rearrangement, has been described recently in various cancer genomes and affects cancer progression and prognosis. However, little is known about chromothripsis in breast cancer. To identify novel prognostic biomarkers in breast cancer, we used molecular inversion probe (MIP) microarray to explore genome-wide copy number aberrations (CNA) and breast cancer-related gene alterations in DNA extracted from formalin-fixed paraffin-embedded tissue. We examined 42 primary breast cancers with known HR and HER2 status assessed via immunohistochemistry and FISH and analyzed MIP microarray results for correlation with standard tests and survival outcomes. Global genome-wide CNA ranged from 0.2% to 65.7%. Chromothripsis-like patterns were observed in 23/38 (61%) cases and were more prevalent in cases with ≥10% CNA (20/26, 77%) than in cases with <10% CNA (3/12, 25%; p<0.01). Most frequently involved chromosomal segment was 17q12-q21, the HER2 locus. Chromothripsis-like patterns involving 17q12 were observed in 8/19 (42%) of HER2-amplified tumors but not in any of the tumors without HER2 amplification (0/19; p<0.01). HER2 amplification detected by MIP microarray was 95% concordant with conventional testing (39/41). Interestingly, 21% of patients (9/42) had fibroblast growth factor receptor 1 (FGFR1)amplification and had a 460% higher risk for mortality than those without FGFR1 amplification (p<0.01). In summary, MIP microarray provided a robust assessment of genomic CNA of breast cancer.

  7. Analysis of DNA microarray expression data.

    PubMed

    Simon, Richard

    2009-06-01

    DNA microarrays are powerful tools for studying biological mechanisms and for developing prognostic and predictive classifiers for identifying the patients who require treatment and are best candidates for specific treatments. Because microarrays produce so much data from each specimen, they offer great opportunities for discovery and great dangers or producing misleading claims. Microarray based studies require clear objectives for selecting cases and appropriate analysis methods. Effective analysis of microarray data, where the number of measured variables is orders of magnitude greater than the number of cases, requires specialized statistical methods which have recently been developed. Recent literature reviews indicate that serious problems of analysis exist a substantial proportion of publications. This manuscript attempts to provide a non-technical summary of the key principles of statistical design and analysis for studies that utilize microarray expression profiling.

  8. Microarray Applications in Microbial Ecology Research.

    SciTech Connect

    Gentry, T.; Schadt, C.; Zhou, J.

    2006-04-06

    Microarray technology has the unparalleled potential tosimultaneously determine the dynamics and/or activities of most, if notall, of the microbial populations in complex environments such as soilsand sediments. Researchers have developed several types of arrays thatcharacterize the microbial populations in these samples based on theirphylogenetic relatedness or functional genomic content. Several recentstudies have used these microarrays to investigate ecological issues;however, most have only analyzed a limited number of samples withrelatively few experiments utilizing the full high-throughput potentialof microarray analysis. This is due in part to the unique analyticalchallenges that these samples present with regard to sensitivity,specificity, quantitation, and data analysis. This review discussesspecific applications of microarrays to microbial ecology research alongwith some of the latest studies addressing the difficulties encounteredduring analysis of complex microbial communities within environmentalsamples. With continued development, microarray technology may ultimatelyachieve its potential for comprehensive, high-throughput characterizationof microbial populations in near real-time.

  9. In control: systematic assessment of microarray performance.

    PubMed

    van Bakel, Harm; Holstege, Frank C P

    2004-10-01

    Expression profiling using DNA microarrays is a powerful technique that is widely used in the life sciences. How reliable are microarray-derived measurements? The assessment of performance is challenging because of the complicated nature of microarray experiments and the many different technology platforms. There is a mounting call for standards to be introduced, and this review addresses some of the issues that are involved. Two important characteristics of performance are accuracy and precision. The assessment of these factors can be either for the purpose of technology optimization or for the evaluation of individual microarray hybridizations. Microarray performance has been evaluated by at least four approaches in the past. Here, we argue that external RNA controls offer the most versatile system for determining performance and describe how such standards could be implemented. Other uses of external controls are discussed, along with the importance of probe sequence availability and the quantification of labelled material.

  10. 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.

  11. Fecal source tracking in water using a mitochondrial DNA microarray.

    PubMed

    Vuong, Nguyet-Minh; Villemur, Richard; Payment, Pierre; Brousseau, Roland; Topp, Edward; Masson, Luke

    2013-01-01

    A mitochondrial-based microarray (mitoArray) was developed for rapid identification of the presence of 28 animals and one family (cervidae) potentially implicated in fecal pollution in mixed activity watersheds. Oligonucleotide probes for genus or subfamily-level identification were targeted within the 12S rRNA - Val tRNA - 16S rRNA region in the mitochondrial genome. This region, called MI-50, was selected based on three criteria: 1) the ability to be amplified by universal primers 2) these universal primer sequences are present in most commercial and domestic animals of interest in source tracking, and 3) that sufficient sequence variation exists within this region to meet the minimal requirements for microarray probe discrimination. To quantify the overall level of mitochondrial DNA (mtDNA) in samples, a quantitative-PCR (Q-PCR) universal primer pair was also developed. Probe validation was performed using DNA extracted from animal tissues and, for many cases, animal-specific fecal samples. To reduce the amplification of potentially interfering fish mtDNA sequences during the MI-50 enrichment step, a clamping PCR method was designed using a fish-specific peptide nucleic acid. DNA extracted from 19 water samples were subjected to both array and independent PCR analyses. Our results confirm that the mitochondrial microarray approach method could accurately detect the dominant animals present in water samples emphasizing the potential for this methodology in the parallel scanning of a large variety of animals normally monitored in fecal source tracking.

  12. New 3-D microarray platform based on macroporous polymer monoliths.

    PubMed

    Rober, M; Walter, J; Vlakh, E; Stahl, F; Kasper, C; Tennikova, T

    2009-06-30

    Polymer macroporous monoliths are widely used as efficient sorbents in different, mostly dynamic, interphase processes. In this paper, monolithic materials strongly bound to the inert glass surface are suggested as operative matrices at the development of three-dimensional (3-D) microarrays. For this purpose, several rigid macroporous copolymers differed by reactivity and hydrophobic-hydrophilic properties were synthesized and tested: (1) glycidyl methacrylate-co-ethylene dimethacrylate (poly(GMA-co-EDMA)), (2) glycidyl methacrylate-co-glycerol dimethacrylate (poly(GMA-co-GDMA)), (3) N-hydroxyphthalimide ester of acrylic acid-co-glycidyl methacrylate-co-ethylene dimethacrylate (poly(HPIEAA-co-GMA-co-EDMA)), (4) 2-cyanoethyl methacrylate-co-ethylene dimethacrylate (poly(CEMA-co-EDMA)), and (5) 2-cyanoethyl methacrylate-co-2-hydroxyethyl methacrylate-co-ethylene dimethacrylate (poly(CEMA-co-HEMA-co-EDMA)). The constructed devices were used as platforms for protein microarrays construction and model mouse IgG-goat anti-mouse IgG affinity pair was used to demonstrate the potential of developed test-systems, as well as to optimize microanalytical conditions. The offered microarray platforms were applied to detect the bone tissue marker osteopontin directly in cell culture medium.

  13. Glycan profiling of endometrial cancers using lectin microarray.

    PubMed

    Nishijima, Yoshihiro; Toyoda, Masashi; Yamazaki-Inoue, Mayu; Sugiyama, Taro; Miyazawa, Masaki; Muramatsu, Toshinari; Nakamura, Kyoko; Narimatsu, Hisashi; Umezawa, Akihiro; Mikami, Mikio

    2012-10-01

    Cell surface glycans change during the process of malignant transformation. To characterize and distinguish endometrial cancer and endometrium, we performed glycan profiling using an emerging modern technology, lectin microarray analysis. The three cell lines, two from endometrial cancers [well-differentiated type (G1) and poorly differentiated type (G3)] and one from normal endometrium, were successfully categorized into three independent groups by 45 lectins. Furthermore, in cancer cells, a clear difference between G1 and G3 type was observed for the glycans recognized with six lectins, Ulex europaeus agglutinin I (UEA-I), Sambucus sieboldiana agglutinin (SSA), Sambucus nigra agglutinin (SNA), Trichosanthes japonica agglutinin I (TJA-I), Amaranthus caudatus agglutinin (ACA), and Bauhinia purpurea lectin (BPL). The lectin microarray analysis using G3 type tissues demonstrated that stage I and stage III or IV were distinguished depending on signal pattern of three lectins, Dolichos biflorus agglutinin (DBA), BPL, and ACA. In addition, the analysis of the glycans on the ovarian cancer cells showed that only anticancer drug-sensitive cell lines had almost no activities to specific three lectins. Glycan profiling by the lectin microarray may be used to assess the characteristics of tumors and potentially to predict the success of chemotherapy treatment.

  14. 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.

  15. 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

  16. 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.

  17. Regulated necrosis-related molecule mRNA expression in humans and mice and in murine acute tissue injury and systemic autoimmunity leading to progressive organ damage, and progressive fibrosis

    PubMed Central

    Honarpisheh, Mohsen; Desai, Jyaysi; Marschner, Julian A.; Weidenbusch, Marc; Lech, Maciej; Vielhauer, Volker; Anders, Hans-Joachim; Mulay, Shrikant R.

    2016-01-01

    The species-specific, as well as organ-specific expression of regulated necrosis (RN)-related molecules, is not known. We determined the expression levels of tumour necrosis factor receptor-1 (TNFR1), receptor activated protein kinase (RIPK)1, RIPK3, mixed lineage kinase domain-like (MLKL), CASP8, Fas-associated protein with death domain (FADD), cellular inhibitor of apoptosis protein (CIAP)1, CIAP2, glutathione peroxidase-4 (GPX4), cyclophilin D (CYPD), CASP1, NLRP3 and poly(ADP-ribose) polymerase-1 (PARP1) in human and mouse solid organs. We observed significant differences in expression of these molecules between human and mice. In addition, we characterized their expression profiles in acute as well as persistent tissue injury and chronic tissue remodelling using acute and chronic kidney injury models. We observed that the degree and pattern of induction of RN-related molecules were highly dependent on the trigger and disease pathogenesis. Furthermore, we studied their expression patterns in mice with lupus-like systemic autoimmunity, which revealed that the expression of MLKL, GPX4 and PARP1 significantly increased in the spleen along disease progression and CASP1, RIPK1, RIPK3 and CYPD were higher at the earlier stages but were significantly decreased in the later stages. In contrast, in the kidney, the expression of genes involved in pyroptosis, e.g. NLRP3 and CASP1 were significantly increased and TNFR1, RIPK1, RIPK3, CIAP1/2 and GPX4 were significantly decreased along the progression of lupus nephritis (LN). Thus, the organ- and species-specific expression of RN-related molecules should be considered during designing experiments, interpreting the results as well as extrapolating the conclusions from one species or organ to another species or organ respectively. PMID:27811014

  18. Microarray-integrated optoelectrofluidic immunoassay system.

    PubMed

    Han, Dongsik; Park, Je-Kyun

    2016-05-01

    A microarray-based analytical platform has been utilized as a powerful tool in biological assay fields. However, an analyte depletion problem due to the slow mass transport based on molecular diffusion causes low reaction efficiency, resulting in a limitation for practical applications. This paper presents a novel method to improve the efficiency of microarray-based immunoassay via an optically induced electrokinetic phenomenon by integrating an optoelectrofluidic device with a conventional glass slide-based microarray format. A sample droplet was loaded between the microarray slide and the optoelectrofluidic device on which a photoconductive layer was deposited. Under the application of an AC voltage, optically induced AC electroosmotic flows caused by a microarray-patterned light actively enhanced the mass transport of target molecules at the multiple assay spots of the microarray simultaneously, which reduced tedious reaction time from more than 30 min to 10 min. Based on this enhancing effect, a heterogeneous immunoassay with a tiny volume of sample (5 μl) was successfully performed in the microarray-integrated optoelectrofluidic system using immunoglobulin G (IgG) and anti-IgG, resulting in improved efficiency compared to the static environment. Furthermore, the application of multiplex assays was also demonstrated by multiple protein detection.

  19. MARS: Microarray analysis, retrieval, and storage system

    PubMed Central

    Maurer, Michael; Molidor, Robert; Sturn, Alexander; Hartler, Juergen; Hackl, Hubert; Stocker, Gernot; Prokesch, Andreas; Scheideler, Marcel; Trajanoski, Zlatko

    2005-01-01

    Background Microarray analysis has become a widely used technique for the study of gene-expression patterns on a genomic scale. As more and more laboratories are adopting microarray technology, there is a need for powerful and easy to use microarray databases facilitating array fabrication, labeling, hybridization, and data analysis. The wealth of data generated by this high throughput approach renders adequate database and analysis tools crucial for the pursuit of insights into the transcriptomic behavior of cells. Results MARS (Microarray Analysis and Retrieval System) provides a comprehensive MIAME supportive suite for storing, retrieving, and analyzing multi color microarray data. The system comprises a laboratory information management system (LIMS), a quality control management, as well as a sophisticated user management system. MARS is fully integrated into an analytical pipeline of microarray image analysis, normalization, gene expression clustering, and mapping of gene expression data onto biological pathways. The incorporation of ontologies and the use of MAGE-ML enables an export of studies stored in MARS to public repositories and other databases accepting these documents. Conclusion We have developed an integrated system tailored to serve the specific needs of microarray based research projects using a unique fusion of Web based and standalone applications connected to the latest J2EE application server technology. The presented system is freely available for academic and non-profit institutions. More information can be found at . PMID:15836795

  20. Tissue angiotensin II during progression or ventricular hypertrophy to heart failure in hypertensive rats; differential effects on PKC epsilon and PKC beta.

    PubMed

    Inagaki, Koichi; Iwanaga, Yoshitaka; Sarai, Nobuaki; Onozawa, Yoko; Takenaka, Hiroyuki; Mochly-Rosen, Daria; Kihara, Yasuki

    2002-10-01

    The protein kinase C (PKC) family has been implicated as second messengers in mechanosensitive modulation of cardiac hypertrophy. However, little information is available on the role of expression and activation of specific cardiac PKC isozymes during development of left ventricular hypertrophy (LVH) and failure (LVF). Dahl salt-sensitive rats fed an 8% salt diet developed systemic hypertension and concentric LVH at 11 weeks of age that is followed by left ventricle (LV) dilatation and global hypokinesis at 17 weeks. Among several PKC isozymes expressed in the LV myocardium, only PKC epsilon showed a 94% increase at the LVH stage. At the LVF stage, however, PKC epsilon returned to the control level, whereas PKC beta I and beta II increased by 158% and 155%, respectively. Hearts were studied at each stage using the Langendorff set-up, and a LV balloon was inflated to achieve an equivalent diastolic wall stress. Following mechanical stretch, PKC epsilon was significantly activated in LVH myocardium in which tissue angiotensin II levels were increased by 59%. Pre-treatment with valsartan, an AT(1)-receptor blocker, abolished the stretch-mediated PKC epsilon activation. Mechanical stretch no longer induced PKC epsilon activation in LVF. Chronic administration of valsartan blunted the progression of LVF and inhibited the increase in PKC beta. Mechanosensitive PKC epsilon activation is augmented and therefore may contribute to the development of compensatory hypertrophy. This effect was dependent on activation of tissue angiotensin II. However, this compensatory mechanism becomes inactive in LVF, where PKC beta may participate in the progression to cardiac dysfunction and LV remodeling.

  1. Systematic review of accuracy of prenatal diagnosis for abnormal chromosome diseases by microarray technology.

    PubMed

    Xu, H B; Yang, H; Liu, G; Chen, H

    2014-10-31

    The accuracy of prenatal diagnosis for abnormal chromosome diseases by chromosome microarray technology and karyotyping were compared. A literature search was carried out in the MEDLINE database with the keywords "chromosome" and "karyotype" and "genetic testing" and "prenatal diagnosis" and "oligonucleotide array sequence". The studies obtained were filtered by using the QUADAS tool, and studies conforming to the quality standard were fully analyzed. There was one paper conforming to the QUADAS standards including 4406 gravidas with adaptability syndromes of prenatal diagnosis including elderly parturient women, abnormal structure by type-B ultrasound, and other abnormalities. Microarray technology yielded successful diagnoses in 4340 cases (98.8%), and there was no need for tissue culture in 87.9% of the samples. All aneuploids and non-parallel translocations in 4282 cases of non-chimera identified by karyotyping could be detected using microarray analysis technology, whereas parallel translocations and fetal triploids could not be detected by microarray analysis technology. In the samples with normal karyotyping results, type-B ultrasound showed that 6% of chromosomal deficiencies or chromosome duplications could be detected by microarray technology, and the same abnormal chromosomes were detected in 1.7% of elderly parturient women and samples with positive serology screening results. In the prenatal diagnosis test, compared with karyotyping, microarray technology could identify the extra cell genetic information with clinical significance, aneuploids, and non-parallel translocations; however, its disadvantage is that it could not identify parallel translocations and triploids.

  2. Microarray Data Processing Techniques for Genome-Scale Network Inference from Large Public Repositories.

    PubMed

    Chockalingam, Sriram; Aluru, Maneesha; Aluru, Srinivas

    2016-09-19

    Pre-processing of microarray data is a well-studied problem. Furthermore, all popular platforms come with their own recommended best practices for differential analysis of genes. However, for genome-scale network inference using microarray data collected from large public repositories, these methods filter out a considerable number of genes. This is primarily due to the effects of aggregating a diverse array of experiments with different technical and biological scenarios. Here we introduce a pre-processing pipeline suitable for inferring genome-scale gene networks from large microarray datasets. We show that partitioning of the available microarray datasets according to biological relevance into tissue- and process-specific categories significantly extends the limits of downstream network construction. We demonstrate the effectiveness of our pre-processing pipeline by inferring genome-scale networks for the model plant Arabidopsis thaliana using two different construction methods and a collection of 11,760 Affymetrix ATH1 microarray chips. Our pre-processing pipeline and the datasets used in this paper are made available at http://alurulab.cc.gatech.edu/microarray-pp.

  3. 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.

  4. 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

  5. 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

  6. A newly developed DNA microarray is useful to assess induction of cytochromes p450 in the cynomolgus monkey.

    PubMed

    Ise, Ryota; Uehara, Shotaro; Akiyama, Hideo; Kondo, Satoshi; Iwasaki, Kazuhide; Nagata, Ryoichi; Nobumasa, Hitoshi; Yamazaki, Hiroshi; Uno, Yasuhiro

    2011-06-01

    Cytochromes P450 (P450s or CYPs) are a gene family of highly homologous genes and include the CYP1-4 family, which is relevant to drug metabolism. In the cynomolgus monkey (which is frequently used in drug metabolism studies), numerous CYPs (mfCYPs) have been identified in the CYP1-4 family. DNA microarrays are useful for high-throughput screening assays; however, there is a potential problem with cross-hybridization of highly homologous genes in the gene family. This problem might be solved with the use of low-density DNA microarrays, with which specific validation can be performed for the genes on the microarray. We have developed a DNA microarray for the 20 mfCYPs and have evaluated and validated its specificity and usefulness. First, in both DNA microarray and quantitative polymerase chain reaction (qPCR) analyses, hepatic expression of each mfCYP correlated well, and similar tissue expression patterns were observed for five representative mfCYPs, confirming the specificity of the DNA microarray. Second, the usefulness of this DNA microarray was validated by induction analysis of mfCYPs in primary hepatocytes, which successfully detected known responders, but also novel responders (mfCYP2C43, mfCYP2C75, and mfCYP3A5 for rifampicin), as confirmed by qPCR analysis. This DNA microarray can thus be utilized for high-throughput assays during drug development.

  7. Urinary monocyte chemoattractant protein-1 (MCP-1) and connective tissue growth factor (CCN2) as prognostic markers for progression of diabetic nephropathy.

    PubMed

    Tam, Frederick W K; Riser, Bruce L; Meeran, Karim; Rambow, JoAnn; Pusey, Charles D; Frankel, Andrew H

    2009-07-01

    Profibrotic growth factors and inflammatory chemokines have been implicated in the pathogenesis of diabetic nephropathy (DN). However, measurement of urinary monocyte chemoattractant protein-1 (MCP-1) and connective tissue growth factor (CCN2) as prognostic markers has not previously been reported, and neither have two such molecules in urine been examined in a single study of DN. In this prospective observational study, 43 adult diabetic patients were studied, 40 were followed up for 6years. Urinary MCP-1/creatinine ratios were found to be significantly higher in patients with macroalbuminuria (3.3- and 2.1-fold higher (p<0.01) than normoalbuminuric and microalbuminuric patients, respectively). CCN2 exhibited a pattern different from that of urinary MCP-1. Urinary CCN2/creatinine ratios were greatly elevated in both microalbuminuric and macroalbuminuric patients (125- and 74-fold higher than normoalbuminuric patients, respectively, p<0.01 and p<0.05, respectively). Further, urinary CCN2, but not MCP-1, correlated with progression of microalbuminuria (R=0.49, p<0.05). In contrast, MCP-1, but not CCN2, correlated with the rate of eGFR decline for all patients (R=0.61, p<0.0001), reflective of its predictive value in patients with macroalbuminuria, but not for patients with microalbuminuria or normoalbuminuria. In conclusion, increased urinary CCN2 is associated with the early progression of DN, whereas MCP-1 is associated with later stage disease.

  8. Mutational analysis using oligonucleotide microarrays

    PubMed Central

    Hacia, J.; Collins, F.

    1999-01-01

    The development of inexpensive high throughput methods to identify individual DNA sequence differences is important to the future growth of medical genetics. This has become increasingly apparent as epidemiologists, pathologists, and clinical geneticists focus more attention on the molecular basis of complex multifactorial diseases. Such undertakings will rely upon genetic maps based upon newly discovered, common, single nucleotide polymorphisms. Furthermore, candidate gene approaches used in identifying disease associated genes necessitate screening large sequence blocks for changes tracking with the disease state. Even after such genes are isolated, large scale mutational analyses will often be needed for risk assessment studies to define the likely medical consequences of carrying a mutated gene.
This review concentrates on the use of oligonucleotide arrays for hybridisation based comparative sequence analysis. Technological advances within the past decade have made it possible to apply this technology to many different aspects of medical genetics. These applications range from the detection and scoring of single nucleotide polymorphisms to mutational analysis of large genes. Although we discuss published scientific reports, unpublished work from the private sector12 could also significantly affect the future of this technology.


Keywords: mutational analysis; oligonucleotide microarrays; DNA chips PMID:10528850

  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.

  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.

  11. 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

  12. 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

  13. 3D Biomaterial Microarrays for Regenerative Medicine: Current State-of-the-Art, Emerging Directions and Future Trends.

    PubMed

    Gaharwar, Akhilesh K; Arpanaei, Ayyoob; Andresen, Thomas L; Dolatshahi-Pirouz, Alireza

    2016-01-27

    Three dimensional (3D) biomaterial microarrays hold enormous promise for regenerative medicine because of their ability to accelerate the design and fabrication of biomimetic materials. Such tissue-like biomaterials can provide an appropriate microenvironment for stimulating and controlling stem cell differentiation into tissue-specific lineages. The use of 3D biomaterial microarrays can, if optimized correctly, result in a more than 1000-fold reduction in biomaterials and cells consumption when engineering optimal materials combinations, which makes these miniaturized systems very attractive for tissue engineering and drug screening applications.

  14. 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.

  15. 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…

  16. 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

  17. 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

  18. A Combinatorial Protein Microarray for Probing Materials Interaction with Pancreatic Islet Cell Populations

    PubMed Central

    Delalat, Bahman; Rojas-Canales, Darling M.; Rasi Ghaemi, Soraya; Waibel, Michaela; Harding, Frances J.; Penko, Daniella; Drogemuller, Christopher J.; Loudovaris, Thomas; Coates, Patrick T. H.; Voelcker, Nicolas H.

    2016-01-01

    Pancreatic islet transplantation has become a recognized therapy for insulin-dependent diabetes mellitus. During isolation from pancreatic tissue, the islet microenvironment is disrupted. The extracellular matrix (ECM) within this space not only provides structural support, but also actively signals to regulate islet survival and function. In addition, the ECM is responsible for growth factor presentation and sequestration. By designing biomaterials that recapture elements of the native islet environment, losses in islet function and number can potentially be reduced. Cell microarrays are a high throughput screening tool able to recreate a multitude of cellular niches on a single chip. Here, we present a screening methodology for identifying components that might promote islet survival. Automated fluorescence microscopy is used to rapidly identify islet derived cell interaction with ECM proteins and immobilized growth factors printed on arrays. MIN6 mouse insulinoma cells, mouse islets and, finally, human islets are progressively screened. We demonstrate the capability of the platform to identify ECM and growth factor protein candidates that support islet viability and function and reveal synergies in cell response. PMID:27600088

  19. High-throughput cellular microarray platforms: applications in drug discovery, toxicology and stem cell research

    PubMed Central

    Fernandes, Tiago G.; Diogo, Maria Margarida; Clark, Douglas S.; Dordick, Jonathan S.; Cabral, Joaquim M.S.

    2017-01-01

    Cellular microarrays are powerful experimental tools for high-throughput screening of large numbers of test samples. Miniaturization increases assay throughput while reducing reagent consumption and the number of cells required, making these systems attractive for a wide range of assays in drug discovery, toxicology, stem cell research and potentially therapy. Here, we provide an overview of the emerging technologies that can be used to generate cellular microarrays, and we highlight recent significant advances in the field. This emerging and multidisciplinary approach offers new opportunities for the design and control of stem cells in tissue engineering and cellular therapies and promises to expedite drug discovery in the biotechnology and pharmaceutical industries. PMID:19398140

  20. Systematic Expression Profiling of the Mouse Transcriptome Using RIKEN cDNA Microarrays

    PubMed Central

    Bono, Hidemasa; Yagi, Ken; Kasukawa, Takeya; Nikaido, Itoshi; Tominaga, Naoko; Miki, Rika; Mizuno, Yosuke; Tomaru, Yasuhiro; Goto, Hitoshi; Nitanda, Hiroyuki; Shimizu, Daisuke; Makino, Hirochika; Morita, Tomoyuki; Fujiyama, Junshin; Sakai, Takehito; Shimoji, Takashi; Hume, David A.; Hayashizaki, Yoshihide; Okazaki, Yasushi

    2003-01-01

    The number of known mRNA transcripts in the mouse has been greatly expanded by the RIKEN Mouse Gene Encyclopedia project. Validation of their reproducible expression in a tissue is an important contribution to the study of functional genomics. In this report, we determine the expression profile of 57,931 clones on 20 mouse tissues using cDNA microarrays. Of these 57,931 clones, 22,928 clones correspond to the FANTOM2 clone set. The set represents 20,234 transcriptional units (TUs) out of 33,409 TUs in the FANTOM2 set. We identified 7206 separate clones that satisfied stringent criteria for tissue-specific expression. Gene Ontology terms were assigned for these 7206 clones, and the proportion of `molecular function' ontology for each tissue-specific clone was examined. These data will provide insights into the function of each tissue. Tissue-specific gene expression profiles obtained using our cDNA microarrays were also compared with the data extracted from the GNF Expression Atlas based on Affymetrix microarrays. One major outcome of the RIKEN transcriptome analysis is the identification of numerous nonprotein-coding mRNAs. The expression profile was also used to obtain evidence of expression for putative noncoding RNAs. In addition, 1926 clones (70%) of 2768 clones that were categorized as “unknown EST,” and 1969 (58%) clones of 3388 clones that were categorized as “unclassifiable” were also shown to be reproducibly expressed. PMID:12819129

  1. Microarrays: molecular allergology and nanotechnology for personalised medicine (I).

    PubMed

    Lucas, J M

    2010-01-01

    The diagnosis of antibody-mediated allergic disorders is based on the clinical findings and the detection of allergen-specific IgE based on in vitro and in vivo techniques, together with allergen provocation tests. In vitro diagnostic techniques have progressed enormously following the introduction of the advances made in proteomics and nanotechnology--offering tools for the diagnosis and investigation of allergy at molecular level. The most advanced developments are the microarray techniques, which in genomics allowed rapid description of the human genetic code, and which now have been applied to proteomics, broadening the field for research and clinical use. Together with these technological advances, the characterisation of most of the different proteins generating specific IgE and which conform each natural allergen, as well as their purification or genetic engineering-based synthesis, have been crucial elements--offering the possibility of identifying disease-causing allergens at molecular level, establishing a component-resolved diagnosis (CRD), using them to study the natural course of the disease, and applying them to improvements in specific immunotherapy. Microarrays of allergic components offer results relating to hundreds of these allergenic components in a single test, and use a small amount of serum that can be obtained from capillary blood. The availability of new molecules will allow the development of panels including new allergenic components and sources, which will require evaluation for clinical use. The present study reviews these new developments, component-resolved diagnosis, and the development of microarray techniques as a critical element for furthering our knowledge of allergic disease.

  2. Validation of affinity reagents using antigen microarrays.

    PubMed

    Sjöberg, Ronald; Sundberg, Mårten; Gundberg, Anna; Sivertsson, Asa; Schwenk, Jochen M; Uhlén, Mathias; Nilsson, Peter

    2012-06-15

    There is a need for standardised validation of affinity reagents to determine their binding selectivity and specificity. This is of particular importance for systematic efforts that aim to cover the human proteome with different types of binding reagents. One such international program is the SH2-consortium, which was formed to generate a complete set of renewable affinity reagents to the SH2-domain containing human proteins. Here, we describe a microarray strategy to validate various affinity reagents, such as recombinant single-chain antibodies, mouse monoclonal antibodies and antigen-purified polyclonal antibodies using a highly multiplexed approach. An SH2-specific antigen microarray was designed and generated, containing more than 6000 spots displayed by 14 identical subarrays each with 406 antigens, where 105 of them represented SH2-domain containing proteins. Approximately 400 different affinity reagents of various types were analysed on these antigen microarrays carrying antigens of different types. The microarrays revealed not only very detailed specificity profiles for all the binders, but also showed that overlapping target sequences of spotted antigens were detected by off-target interactions. The presented study illustrates the feasibility of using antigen microarrays for integrative, high-throughput validation of various types of binders and antigens.

  3. Probe-Level Analysis of Expression Microarrays Characterizes Isoform-Specific Degradation during Mouse Oocyte Maturation

    PubMed Central

    Salisbury, Jesse; Hutchison, Keith W.; Wigglesworth, Karen; Eppig, John J.; Graber, Joel H.

    2009-01-01

    Background Gene expression microarrays have provided many insights into changes in gene expression patterns between different tissue types, developmental stages, and disease states. Analyses of these data focused primarily measuring the relative abundance of transcripts of a gene, while treating most or all transcript isoforms as equivalent. Differences in the selection between transcript isoforms can, however, represent critical changes to either the protein product or the posttranscriptional regulation of the transcript. Novel analyses on existing microarray data provide fresh insights and new interpretations into transcriptome-wide changes in expression. Methodology A probe-level analysis of existing gene expression arrays revealed differences in mRNA processing, primarily affecting the 3′-untranslated region. Working with the example of microarrays drawn from a transcriptionally silent period of mouse oocyte development, probe-level analysis (implemented here as rmodel) identified genes whose transcript isoforms have differing stabilities. Comparison of micorarrays measuring cDNA generated from oligo-dT and random primers revealed further differences in the polyadenylation status of some transcripts. Additional analysis provided evidence for sequence-targeted cleavage, including putative targeting sequences, as one mechanism of degradation for several hundred transcripts in the maturing oocyte. Conclusions The capability of probe-level analysis to elicit novel findings from existing expression microarray data was demonstrated. The characterization of differences in stability between transcript isoforms in maturing mouse oocytes provided some mechanistic details of degradation. Similar analysis of existing archives of expression microarray data will likely provide similar discoveries. PMID:19834616

  4. In Situ-Synthesized Novel Microarray Optimized for Mouse Stem Cell and Early Developmental Expression Profiling

    PubMed Central

    Carter, Mark G.; Hamatani, Toshio; Sharov, Alexei A.; Carmack, Condie E.; Qian, Yong; Aiba, Kazuhiro; Ko, Naomi T.; Dudekula, Dawood B.; Brzoska, Pius M.; Hwang, S. Stuart; Ko, Minoru S.H.

    2003-01-01

    Applications of microarray technologies to mouse embryology/genetics have been limited, due to the nonavailability of microarrays containing large numbers of embryonic genes and the gap between microgram quantities of RNA required by typical microarray methods and the miniscule amounts of tissue available to researchers. To overcome these problems, we have developed a microarray platform containing in situ-synthesized 60-mer oligonucleotide probes representing approximately 22,000 unique mouse transcripts, assembled primarily from sequences of stem cell and embryo cDNA libraries. We have optimized RNA labeling protocols and experimental designs to use as little as 2 ng total RNA reliably and reproducibly. At least 98% of the probes contained in the microarray correspond to clones in our publicly available collections, making cDNAs readily available for further experimentation on genes of interest. These characteristics, combined with the ability to profile very small samples, make this system a resource for stem cell and embryogenomics research. [Supplemental material is available online at www.genome.org and at the NIA Mouse cDNA Project Web site, http://lgsun.grc.nia.nih.gov/cDNA/cDNA.html.] PMID:12727912

  5. Generation of a non-small cell lung cancer transcriptome microarray

    PubMed Central

    Tanney, Austin; Oliver, Gavin R; Farztdinov, Vadim; Kennedy, Richard D; Mulligan, Jude M; Fulton, Ciaran E; Farragher, Susan M; Field, John K; Johnston, Patrick G; Harkin, D Paul; Proutski, Vitali; Mulligan, Karl A

    2008-01-01

    Background Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality worldwide. At present no reliable biomarkers are available to guide the management of this condition. Microarray technology may allow appropriate biomarkers to be identified but present platforms are lacking disease focus and are thus likely to miss potentially vital information contained in patient tissue samples. Methods A combination of large-scale in-house sequencing, gene expression profiling and public sequence and gene expression data mining were used to characterise the transcriptome of NSCLC and the data used to generate a disease-focused microarray – the Lung Cancer DSA research tool. Results Built on the Affymetrix GeneChip platform, the Lung Cancer DSA research tool allows for interrogation of ~60,000 transcripts relevant to Lung Cancer, tens of thousands of which are unavailable on leading commercial microarrays. Conclusion We have developed the first high-density disease specific transcriptome microarray. We present the array design process and the results of experiments carried out to demonstrate the array's utility. This approach serves as a template for the development of other disease transcriptome microarrays, including non-neoplastic diseases. PMID:18513400

  6. Profiling of differentially expressed genes in human gingival epithelial cells and fibroblasts by DNA microarray.

    PubMed

    Abiko, Yoshimitsu; Hiratsuka, Koichi; Kiyama-Kishikawa, Michiko; Tsushima, Katsumasa; Ohta, Mitsuhiro; Sasahara, Hiroshige

    2004-03-01

    Gingival epithelial cells and fibroblasts play important roles and have a harmonious relationship under normal and disease conditions, but the precise differences between theses cells remain unknown. To study the differences in gene expression between human gingival epithelial cells (HGE) and human gingival fibroblasts (HGF), mRNA was recovered from primary cultured cells and analyzed using cDNA microarray technology. The cDNA retro-transcribed from equal quantities of mRNA was labeled with the fluorescent dyes Cy5 and Cy3. The mixed probes were then hybridized with 7276 genes on the DNA microarray, after which fluorescence signals were scanned and further analyzed using GeneSpring software. Of the 7276 genes screened, 469 showed expression levels that were more than 2-fold greater in HGE than in HGF, while 293 showed expression levels that were more than 2-fold greater in HGF than in HGE. To confirm the reliability of the microarray results, keratin K5 and desmocolin, and vimentin and gp130, which showed higher mRNA levels in HGE and HGF, respectively, were selected and their mRNA levels were further analyzed by RT-PCR. The results of RT-PCR correlated well with those of microarray analysis. The present findings using a DNA microarray to detect differences in the gene expression profiles of HGE and HGF may be beneficial for genetic diagnosis of periodontal tissue metabolism and periodontal diseases.

  7. The role of DNA microarrays in the evaluation of fetal death.

    PubMed

    Reddy, Uma M; Page, Grier P; Saade, George R

    2012-04-01

    Fetal death occurs in 15% of clinically recognized pregnancies. Cytogenetic abnormalities are present in 50% of spontaneous abortions (fetal deaths < 20 weeks) whereas the rate is 6% to 13% for stillbirths (fetal deaths ≥ 20 weeks). Microarray has been demonstrated to increase the diagnosis of genetic abnormalities by providing coverage of the entire genome at a higher density, detecting as small as 50 to 100 kb deletions or duplications, known as copy number changes. Microarray is particularly suited for evaluation of fetal death because DNA can still be analyzed in macerated fetuses and nonviable tissue, two situations where culturing and karyotyping is known to have low yield. Microarray has already proven successful in providing additional genetic information beyond karyotype in spontaneous abortion. The few studies on the use of microarray in stillbirth evaluation have been promising, demonstrating an increase in the diagnosis of clinically relevant genetic abnormalities when compared with karyotype. As the cost and technology improve, microarray may ultimately become the first line screen for genetic abnormalities in stillbirth. The accurate diagnosis of a genetic abnormality as the cause for fetal death may provide closure for families, prevent unnecessary treatments, and enable clinicians to more accurately counsel and manage subsequent pregnancies.

  8. Posttranslational Modification Assays on Functional Protein Microarrays.

    PubMed

    Neiswinger, Johnathan; Uzoma, Ijeoma; Cox, Eric; Rho, HeeSool; Jeong, Jun Seop; Zhu, Heng

    2016-10-03

    Protein microarray technology provides a straightforward yet powerful strategy for identifying substrates of posttranslational modifications (PTMs) and studying the specificity of the enzymes that catalyze these reactions. Protein microarray assays can be designed for individual enzymes or a mixture to establish connections between enzymes and substrates. Assays for four well-known PTMs-phosphorylation, acetylation, ubiquitylation, and SUMOylation-have been developed and are described here for use on functional protein microarrays. Phosphorylation and acetylation require a single enzyme and are easily adapted for use on an array. The ubiquitylation and SUMOylation cascades are very similar, and the combination of the E1, E2, and E3 enzymes plus ubiquitin or SUMO protein and ATP is sufficient for in vitro modification of many substrates.

  9. Identification of Novel Cholesteatoma-Related Gene Expression Signatures Using Full-Genome Microarrays

    PubMed Central

    Klenke, Christin; Janowski, Sebastian; Borck, Daniela; Widera, Darius; Ebmeyer, Jörg; Kalinowski, Jörn; Leichtle, Anke; Hofestädt, Ralf; Upile, Tahwinder; Kaltschmidt, Christian; Kaltschmidt, Barbara; Sudhoff, Holger

    2012-01-01

    Background Cholesteatoma is a gradually expanding destructive epithelial lesion within the middle ear. It can cause extensive local tissue destruction in the temporal bone and can initially lead to the development of conductive hearing loss via ossicular erosion. As the disease progresses, sensorineural hearing loss, vertigo or facial palsy may occur. Cholesteatoma may promote the spread of infection through the tegmen of the middle ear and cause meningitis or intracranial infections with abscess formation. It must, therefore, be considered as a potentially life-threatening middle ear disease. Methods and Findings In this study, we investigated differentially expressed genes in human cholesteatomas in comparison to regular auditory canal skin using Whole Human Genome Microarrays containing 19,596 human genes. In addition to already described up-regulated mRNAs in cholesteatoma, such as MMP9, DEFB2 and KRT19, we identified 3558 new cholesteatoma-related transcripts. 811 genes appear to be significantly differentially up-regulated in cholesteatoma. 334 genes were down-regulated more than 2-fold. Significantly regulated genes with protein metabolism activity include matrix metalloproteinases as well as PI3, SERPINB3 and SERPINB4. Genes like SPP1, KRT6B, PRPH, SPRR1B and LAMC2 are known as genes with cell growth and/or maintenance activity. Transport activity genes and signal transduction genes are LCN2, GJB2 and CEACAM6. Three cell communication genes were identified; one CDH19 and two from the S100 family. Conclusions This study demonstrates that the expression profile of cholesteatoma is similar to a metastatic tumour and chronically inflamed tissue. Based on the investigated profiles we present novel protein-protein interaction and signal transduction networks, which include cholesteatoma-regulated transcripts and may be of great value for drug targeting and therapy development. PMID:23285167

  10. 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

  11. 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

  12. Overview of DNA microarrays: types, applications, and their future.

    PubMed

    Bumgarner, Roger

    2013-01-01

    This unit provides an overview of DNA microarrays. Microarrays are a technology in which thousands 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. This overview first discusses the history of microarrays and the antecedent technologies that led to their development. This is followed by discussion of the methods of manufacture of microarrays and the most common biological applications. The unit ends with a brief description of the limitations of microarrays and discusses how microarrays are being rapidly replaced by DNA sequencing technologies.

  13. 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).

  14. 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

  15. Sharpin promotes hepatocellular carcinoma progression via transactivation of Versican expression

    PubMed Central

    Tanaka, Y; Tateishi, K; Nakatsuka, T; Kudo, Y; Takahashi, R; Miyabayashi, K; Yamamoto, K; Asaoka, Y; Ijichi, H; Tateishi, R; Shibahara, J; Fukayama, M; Ishizawa, T; Hasegawa, K; Kokudo, N; Koike, K

    2016-01-01

    Sharpin (Shank-associated RH domain-interacting protein, also known as SIPL1) is a multifunctional molecule that participates in various biological settings, including nuclear factor-κB signaling activation and tumor suppressor gene inhibition. Sharpin is upregulated in various types of cancers, including hepatocellular carcinoma (HCC), and is implicated in tumor progression. However, the exact roles of Sharpin in tumorigenesis and tumor progression remain largely unknown. Here we report novel mechanisms of HCC progression through Sharpin overexpression. In our study, Sharpin was upregulated in human HCC tissues. Increased Sharpin expression enhanced hepatoma cell invasion, whereas decrease in Sharpin expression by RNA interference inhibited invasion. Microarray analysis identified that Versican, a chondroitin sulfate proteoglycan that plays crucial roles in tumor progression and invasion, was also upregulated in Sharpin-expressing stable cells. Versican expression increased in the majority of HCC tissues and knocking down of Versican greatly attenuated hepatoma cell invasion. Sharpin expression resulted in a significant induction of Versican transcription synergistically with Wnt/β-catenin pathway activation. Furthermore, Sharpin-overexpressing cells had high tumorigenic properties in vivo. These results demonstrate that Sharpin promotes Versican expression synergistically with the Wnt/β-catenin pathway, potentially contributing to HCC development. A Sharpin/Versican axis could be an attractive therapeutic target for this currently untreatable cancer. PMID:27941932

  16. 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

  17. 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

  18. 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

    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.

  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. Progressive lung cancer determined by expression profiling and transcriptional regulation.

    PubMed

    Han, Namshik; Dol, Zulkifli; Vasieva, Olga; Hyde, Russell; Liloglou, Triantafillos; Raji, Olaide; Brambilla, Elisabeth; Brambilla, Christian; Martinet, Yves; Sozzi, Gabriella; Risch, Angela; Montuenga, Luis M; Brass, Andy; Field, John K

    2012-07-01

    Clinically, our ability to predict disease outcome for patients with early stage lung cancer is currently poor. To address this issue, tumour specimens were collected at surgery from non-small cell lung cancer (NSCLC) patients as part of the European Early Lung Cancer (EUELC) consortium. The patients were followed-up for three years post-surgery and patients who suffered progressive disease (PD, tumour recurrence, metastasis or a second primary) or remained disease-free (DF) during follow-up were identified. RNA from both tumour and adjacent-normal lung tissue was extracted from patients and subjected to microarray expression profiling. These samples included 36 adenocarcinomas and 23 squamous cell carcinomas from both PD and DF patients. The microarray data was subject to a series of systematic bioinformatics analyses at gene, network and transcription factor levels. The focus of these analyses was 2-fold: firstly to determine whether there were specific biomarkers capable of differentiating between PD and DF patients, and secondly, to identify molecular networks which may contribute to the progressive tumour phenotype. The experimental design and analyses performed permitted the clear differentiation between PD and DF patients using a set of biomarkers implicated in neuroendocrine signalling and allowed the inference of a set of transcription factors whose activity may differ according to disease outcome. Potential links between the biomarkers, the transcription factors and the genes p21/CDKN1A and Myc, which have previously been implicated in NSCLC development, were revealed by a combination of pathway analysis and microarray meta-analysis. These findings suggest that neuroendocrine-related genes, potentially driven through p21/CDKN1A and Myc, are closely linked to whether or not a NSCLC patient will have poor clinical outcome.

  1. 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...

  2. Raman-based microarray readout: a review.

    PubMed

    Haisch, Christoph

    2016-07-01

    For a quarter of a century, microarrays have been part of the routine analytical toolbox. Label-based fluorescence detection is still the commonest optical readout strategy. Since the 1990s, a continuously increasing number of label-based as well as label-free experiments on Raman-based microarray readout concepts have been reported. This review summarizes the possible concepts and methods and their advantages and challenges. A common label-based strategy is based on the binding of selective receptors as well as Raman reporter molecules to plasmonic nanoparticles in a sandwich immunoassay, which results in surface-enhanced Raman scattering signals of the reporter molecule. Alternatively, capture of the analytes can be performed by receptors on a microarray surface. Addition of plasmonic nanoparticles again leads to a surface-enhanced Raman scattering signal, not of a label but directly of the analyte. This approach is mostly proposed for bacteria and cell detection. However, although many promising readout strategies have been discussed in numerous publications, rarely have any of them made the step from proof of concept to a practical application, let alone routine use. Graphical Abstract Possible realization of a SERS (Surface-Enhanced Raman Scattering) system for microarray readout.

  3. Analytical Protein Microarrays: Advancements Towards Clinical Applications

    PubMed Central

    Sauer, Ursula

    2017-01-01

    Protein microarrays represent a powerful technology with the potential to serve as tools for the detection of a broad range of analytes in numerous applications such as diagnostics, drug development, food safety, and environmental monitoring. Key features of analytical protein microarrays include high throughput and relatively low costs due to minimal reagent consumption, multiplexing, fast kinetics and hence measurements, and the possibility of functional integration. So far, especially fundamental studies in molecular and cell biology have been conducted using protein microarrays, while the potential for clinical, notably point-of-care applications is not yet fully utilized. The question arises what features have to be implemented and what improvements have to be made in order to fully exploit the technology. In the past we have identified various obstacles that have to be overcome in order to promote protein microarray technology in the diagnostic field. Issues that need significant improvement to make the technology more attractive for the diagnostic market are for instance: too low sensitivity and deficiency in reproducibility, inadequate analysis time, lack of high-quality antibodies and validated reagents, lack of automation and portable instruments, and cost of instruments necessary for chip production and read-out. The scope of the paper at hand is to review approaches to solve these problems. PMID:28146048

  4. Analytical Protein Microarrays: Advancements Towards Clinical Applications.

    PubMed

    Sauer, Ursula

    2017-01-29

    Protein microarrays represent a powerful technology with the potential to serve as tools for the detection of a broad range of analytes in numerous applications such as diagnostics, drug development, food safety, and environmental monitoring. Key features of analytical protein microarrays include high throughput and relatively low costs due to minimal reagent consumption, multiplexing, fast kinetics and hence measurements, and the possibility of functional integration. So far, especially fundamental studies in molecular and cell biology have been conducted using protein microarrays, while the potential for clinical, notably point-of-care applications is not yet fully utilized. The question arises what features have to be implemented and what improvements have to be made in order to fully exploit the technology. In the past we have identified various obstacles that have to be overcome in order to promote protein microarray technology in the diagnostic field. Issues that need significant improvement to make the technology more attractive for the diagnostic market are for instance: too low sensitivity and deficiency in reproducibility, inadequate analysis time, lack of high-quality antibodies and validated reagents, lack of automation and portable instruments, and cost of instruments necessary for chip production and read-out. The scope of the paper at hand is to review approaches to solve these problems.

  5. 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…

  6. 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.

  7. 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...

  8. 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.

  9. A Method of Microarray Data Storage Using Array Data Type

    PubMed Central

    Tsoi, Lam C.; Zheng, W. Jim

    2009-01-01

    A well-designed microarray database can provide valuable information on gene expression levels. However, designing an efficient microarray database with minimum space usage is not an easy task since designers need to integrate the microarray data with the information of genes, probe annotation, and the descriptions of each microarray experiment. Developing better methods to store microarray data can greatly improve the efficiency and usefulness of such data. A new schema is proposed to store microarray data by using array data type in an object-relational database management system – PostgreSQL. The implemented database can store all the microarray data from the same chip in an array data structure. The variable length array data type in PostgreSQL can store microarray data from same chip. The implementation of our schema can help to increase the data retrieval and space efficiency. PMID:17392028

  10. 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...

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. 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

    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 χ(2) 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.

  16. Use of microarray technology to profile gene expression patterns important for reproduction in cattle.

    PubMed

    Evans, A C O; Forde, N; O'Gorman, G M; Zielak, A E; Lonergan, P; Fair, T

    2008-07-01

    Fertility in cattle is a major component of many agricultural enterprises and there is pressure to devise methods to improve this. A number of approaches are ongoing, one of which is to better understand the cellular and molecular events of the development of reproductive tissues and to use these as targets for developing new strategies. Microarray technologies now allow us the potential to determine the transcriptional profile of expressed genes in a given tissue. This review focuses on the types of microarrays available for studies in cattle and concludes that genes associated with one or more of the cellular processes of cell survival/death, intracellular signalling, transcription and translation, cell division and proliferation and cellular metabolism are the main transcriptional pathways that control the development of ovarian follicles, oocytes, early embryos and the uterine endometrium about the time of the establishment of pregnancy.

  17. The microarray explorer tool for data mining of cDNA microarrays: application for the mammary gland.

    PubMed

    Lemkin, P F; Thornwall, G C; Walton, K D; Hennighausen, L

    2000-11-15

    The Microarray Explorer (MAExplorer) is a versatile Java-based data mining bioinformatic tool for analyzing quantitative cDNA expression profiles across multiple microarray platforms and DNA labeling systems. It may be run as either a stand-alone application or as a Web browser applet over the Internet. With this program it is possible to (i) analyze the expression of individual genes, (ii) analyze the expression of gene families and clusters, (iii) compare expression patterns and (iv) directly access other genomic databases for clones of interest. Data may be downloaded as required from a Web server or in the case of the stand-alone version, reside on the user's computer. Analyses are performed in real-time and may be viewed and directly manipulated in images, reports, scatter plots, histograms, expression profile plots and cluster analyses plots. A key feature is the clone data filter for constraining a working set of clones to those passing a variety of user-specified logical and statistical tests. Reports may be generated with hypertext Web access to UniGene, GenBank and other Internet databases for sets of clones found to be of interest. Users may save their explorations on the Web server or local computer and later recall or share them with other scientists in this groupware Web environment. The emphasis on direct manipulation of clones and sets of clones in graphics and tables provides a high level of interaction with the data, making it easier for investigators to test ideas when looking for patterns. We have used the MAExplorer to profile gene expression patterns of 1500 duplicated genes isolated from mouse mammary tissue. We have identified genes that are preferentially expressed during pregnancy and during lactation. One gene we identified, carbonic anhydrase III, is highly expressed in mammary tissue from virgin and pregnant mice and in gene knock-out mice with underdeveloped mammary epithelium. Other genes, which include those encoding milk proteins

  18. Ribosomal RNA depletion or exclusion has negligible effect on the detection of viruses in a pan viral microarray.

    PubMed

    McGowan, Sarah; Nunez-Garcia, Javier; Steinbach, Falko; La Rocca, Anna; Blake, Damer; Dastjerdi, Akbar

    2014-10-01

    Pan viral DNA microarrays, which can detect known, novel and multiple viral infections, are major laboratory assets contributing to the control of infectious diseases. The large quantity of ribosomal RNA (rRNA) found in tissue samples is thought to be a major factor contributing to the comparatively lower sensitivity of detecting RNA viruses, as a sequence-independent PCR is used to amplify unknown samples for microarray analysis. This study aimed to determine whether depletion or exclusion of rRNA can improve microarray detection and simplify its analysis. Therefore, two different rRNA depletion and exclusion protocols, RiboMinus™ technology and non-rRNA binding hexanucleotides, were applied to the microarray sample processing and the outcome was compared with those of the sequence-independent amplification protocol. This study concludes that the two procedures, described to deplete or exclude rRNA, have negligible effect on the microarrays detection and analysis and might only in combination with further techniques result in a significant enhancement of sensitivity. Currently, existing protocols of random amplification and background adjustment are pertinent for the purpose of sample processing for microarray analysis.

  19. Transcript copy number estimation using a mouse whole-genome oligonucleotide microarray

    PubMed Central

    Carter, Mark G; Sharov, Alexei A; VanBuren, Vincent; Dudekula, Dawood B; Carmack, Condie E; Nelson, Charlie; Ko, Minoru SH

    2005-01-01

    The ability to quantitatively measure the expression of all genes in a given tissue or cell with a single assay is an exciting promise of gene-expression profiling technology. An in situ-synthesized 60-mer oligonucleotide microarray designed to detect transcripts from all mouse genes was validated, as well as a set of exogenous RNA controls derived from the yeast genome (made freely available without restriction), which allow quantitative estimation of absolute endogenous transcript abundance. PMID:15998450

  20. DNA microarray analysis of the liver of mice treated with cobalt chloride.

    PubMed

    Matsumoto, Kanako; Fujishiro, Hitomi; Satoh, Masahiko; Himeno, Seiichiro

    2010-12-01

    To investigate the in vivo effects of cobalt chloride on gene expression at early time points, DNA microarray analysis was performed on the liver of mice injected subcutaneously with cobalt chloride. The liver tissue samples were taken 0.5, 1, and 3 hr after injection. Of the 14 genes up-regulated at 0.5 hr after injection, 7 are related to immunological responses, and 4 of the 7 were found to be involved in the activation of interferon.

  1. Integrated analysis of microarray data and gene function information.

    PubMed

    Cui, Yan; Zhou, Mi; Wong, Wing Hung

    2004-01-01

    Microarray data should be interpreted in the context of existing biological knowledge. Here we present integrated analysis of microarray data and gene function classification data using homogeneity analysis. Homogeneity analysis is a graphical multivariate statistical method for analyzing categorical data. It converts categorical data into graphical display. By simultaneously quantifying the microarray-derived gene groups and gene function categories, it captures the complex relations between biological information derived from microarray data and the existing knowledge about the gene function. Thus, homogeneity analysis provides a mathematical framework for integrating the analysis of microarray data and the existing biological knowledge.

  2. 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

  3. Viral diagnosis in Indian livestock using customized microarray chips.

    PubMed

    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.

  4. Transcriptional profiling and biochemical analysis of mechanically induced cartilaginous tissues

    PubMed Central

    Salisbury Palomares, Kristy T.; Gerstenfeld, Louis C.; Wigner, Nathan A.; Lenburg, Marc E.; Einhorn, Thomas A.; Morgan, Elise F.

    2010-01-01

    Objective In order to characterize patterns of molecular expression that lead to cartilage formation in vivo in a post-natal setting, mRNA expression profiling was carried out across the timecourse of mechanically induced chondrogenesis. Methods Retired breeder Sprague-Dawley rats underwent production of a non-critical-size, transverse femoral osteotomy. Experimental animals (n=45) were subjected to bending stimulation (60° cyclic motion in the sagittal plane for 15 minutes/day) of the osteotomy gap beginning on post-operative day (POD) 10. Control animals (n=32) experienced continuous rigid fixation. mRNA isolated on POD 10, 17, 24, and 38 was analyzed using a microarray containing 608 genes involved in skeletal development, tissue differentiation, fracture healing, and mechanotransduction. The glycosaminoglycan (GAG) content of the stimulated tissues was compared to native articular cartilage as a means of assessing the progression of chondrogenic development of the tissues. Results The majority of the 100 genes that were differentially expressed were upregulated in response to mechanical stimulation. Many of these genes are associated with articular cartilage development and maintenance, diarthroidal joint development, cell adhesion, extracellular matrix synthesis, signal transduction, and skeletal development. Quantitative real-time PCR results were consistent with the microarray findings. The GAG content of the stimulated tissues increased over time and was no different from that of articular cartilage at POD 38. Conclusions The mechanical stimulation caused upregulation of genes principally involved in joint cavity morphogenesis and critical to articular cartilage function. Further study of this type of stimulation may identify key signaling events required for post-natal, hyaline cartilage formation. PMID:20131271

  5. Technical Advances of the Recombinant Antibody Microarray Technology Platform for Clinical Immunoproteomics

    PubMed Central

    Delfani, Payam; Dexlin Mellby, Linda; Nordström, Malin; Holmér, Andreas; Ohlsson, Mattias; Borrebaeck, Carl A. K.; Wingren, Christer

    2016-01-01

    In the quest for deciphering disease-associated biomarkers, high-performing tools for multiplexed protein expression profiling of crude clinical samples will be crucial. Affinity proteomics, mainly represented by antibody-based microarrays, have during recent years been established as a proteomic tool providing unique opportunities for parallelized protein expression profiling. But despite the progress, several main technical features and assay procedures remains to be (fully) resolved. Among these issues, the handling of protein microarray data, i.e. the biostatistics parts, is one of the key features to solve. In this study, we have therefore further optimized, validated, and standardized our in-house designed recombinant antibody microarray technology platform. To this end, we addressed the main remaining technical issues (e.g. antibody quality, array production, sample labelling, and selected assay conditions) and most importantly key biostatistics subjects (e.g. array data pre-processing and biomarker panel condensation). This represents one of the first antibody array studies in which these key biostatistics subjects have been studied in detail. Here, we thus present the next generation of the recombinant antibody microarray technology platform designed for clinical immunoproteomics. PMID:27414037

  6. Respiratory Tularemia: Francisella Tularensis and Microarray Probe Designing

    PubMed Central

    Ranjbar, Reza; Behzadi, Payam; Mammina, Caterina

    2016-01-01

    Background: Francisella tularensis (F. tularensis) is the etiological microorganism for tularemia. There are different forms of tularemia such as respiratory tularemia. Respiratory tularemia is the most severe form of tularemia with a high rate of mortality; if not treated. Therefore, traditional microbiological tools and Polymerase Chain Reaction (PCR) are not useful for a rapid, reliable, accurate, sensitive and specific diagnosis. But, DNA microarray technology does. DNA microarray technology needs to appropriate microarray probe designing. Objective: The main goal of this original article was to design suitable long oligo microarray probes for detection and identification of F. tularensis. Method: For performing this research, the complete genomes of F. tularensis subsp. tularensis FSC198, F. tularensis subsp. holarctica LVS, F. tularensis subsp. mediasiatica, F. tularensis subsp. novicida (F. novicida U112), and F. philomiragia subsp. philomiragia ATCC 25017 were studied via NCBI BLAST tool, GView and PanSeq Servers and finally the microarray probes were produced and processed via AlleleID 7.7 software and Oligoanalyzer tool, respectively. Results: In this in silico investigation, a number of long oligo microarray probes were designed for detecting and identifying F. tularensis. Among these probes, 15 probes were recognized as the best candidates for microarray chip designing. Conclusion: Calibrated microarray probes reduce the biasis of DNA microarray technology as an advanced, rapid, accurate and cost-effective molecular diagnostic tool with high specificity and sensitivity. Professional microarray probe designing provides us with much more facility and flexibility regarding preparation of a microarray diagnostic chip. PMID:28077973

  7. Detecting outlier samples in microarray data.

    PubMed

    Shieh, Albert D; Hung, Yeung Sam

    2009-01-01

    In this paper, we address the problem of detecting outlier samples with highly different expression patterns in microarray data. Although outliers are not common, they appear even in widely used benchmark data sets and can negatively affect microarray data analysis. It is important to identify outliers in order to explore underlying experimental or biological problems and remove erroneous data. We propose an outlier detection method based on principal component analysis (PCA) and robust estimation of Mahalanobis distances that is fully automatic. We demonstrate that our outlier detection method identifies biologically significant outliers with high accuracy and that outlier removal improves the prediction accuracy of classifiers. Our outlier detection method is closely related to existing robust PCA methods, so we compare our outlier detection method to a prominent robust PCA method.

  8. Plasmonically amplified fluorescence bioassay with microarray format

    NASA Astrophysics Data System (ADS)

    Gogalic, S.; Hageneder, S.; Ctortecka, C.; Bauch, M.; Khan, I.; Preininger, Claudia; Sauer, U.; Dostalek, J.

    2015-05-01

    Plasmonic amplification of fluorescence signal in bioassays with microarray detection format is reported. A crossed relief diffraction grating was designed to couple an excitation laser beam to surface plasmons at the wavelength overlapping with the absorption and emission bands of fluorophore Dy647 that was used as a label. The surface of periodically corrugated sensor chip was coated with surface plasmon-supporting gold layer and a thin SU8 polymer film carrying epoxy groups. These groups were employed for the covalent immobilization of capture antibodies at arrays of spots. The plasmonic amplification of fluorescence signal on the developed microarray chip was tested by using interleukin 8 sandwich immunoassay. The readout was performed ex situ after drying the chip by using a commercial scanner with high numerical aperture collecting lens. Obtained results reveal the enhancement of fluorescence signal by a factor of 5 when compared to a regular glass chip.

  9. 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

  10. PMD: A Resource for Archiving and Analyzing Protein Microarray data.

    PubMed

    Xu, Zhaowei; Huang, Likun; Zhang, Hainan; Li, Yang; Guo, Shujuan; Wang, Nan; Wang, Shi-Hua; Chen, Ziqing; Wang, Jingfang; Tao, Sheng-Ce

    2016-01-27

    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.

  11. 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

  12. Metadata management and semantics in microarray repositories.

    PubMed

    Kocabaş, F; Can, T; Baykal, N

    2011-12-01

    The number of microarray and other high-throughput experiments on primary repositories keeps increasing as do the size and complexity of the results in response to biomedical investigations. Initiatives have been started on standardization of content, object model, exchange format and ontology. However, there are backlogs and inability to exchange data between microarray repositories, which indicate that there is a great need for a standard format and data management. We have introduced a metadata framework that includes a metadata card and semantic nets that make experimental results visible, understandable and usable. These are encoded in syntax encoding schemes and represented in RDF (Resource Description Frame-word), can be integrated with other metadata cards and semantic nets, and can be exchanged, shared and queried. We demonstrated the performance and potential benefits through a case study on a selected microarray repository. We concluded that the backlogs can be reduced and that exchange of information and asking of knowledge discovery questions can become possible with the use of this metadata framework.

  13. Methods for fabricating microarrays of motile bacteria.

    PubMed

    Rozhok, Sergey; Shen, Clifton K-F; Littler, Pey-Lih H; Fan, Zhifang; Liu, Chang; Mirkin, Chad A; Holz, Richard C

    2005-04-01

    Motile bacterial cell microarrays were fabricated by attaching Escherichia coli K-12 cells onto predesigned 16-mercaptohexadecanoic acid patterned microarrays, which were covalently functionalized with E. coli antibodies or poly-L-lysine. By utilizing 11-mercaptoundecyl-penta(ethylene glycol) or 11-mercapto-1-undecanol as passivating molecules, nonspecific binding of E. coli was significantly reduced. Microcontact printing and dip-pen nanolithography were used to prepare microarrays for bacterial adhesion, which was studied by optical fluorescence and atomic force microscopy. These data indicate that single motile E. coli can be attached to predesigned line or dot features and binding can occur via the cell body or the flagella of bacteria. Adherent bacteria are viable (remain alive and motile after adhesion to patterned surface features) for more than four hours. Individual motile bacterial cells can be placed onto predesigned surface features that are at least 1.3 microm in diameter or larger. The importance of controlling the adhesion of single bacterial cell to a surface is discussed with regard to biomotor design.

  14. A New Distribution Family for Microarray Data.

    PubMed

    Kelmansky, Diana Mabel; Ricci, Lila

    2017-02-10

    The traditional approach with microarray data has been to apply transformations that approximately normalize them, with the drawback of losing the original scale. The alternative stand point taken here is to search for models that fit the data, characterized by the presence of negative values, preserving their scale; one advantage of this strategy is that it facilitates a direct interpretation of the results. A new family of distributions named gpower-normal indexed by p∈R is introduced and it is proven that these variables become normal or truncated normal when a suitable gpower transformation is applied. Expressions are given for moments and quantiles, in terms of the truncated normal density. This new family can be used to model asymmetric data that include non-positive values, as required for microarray analysis. Moreover, it has been proven that the gpower-normal family is a special case of pseudo-dispersion models, inheriting all the good properties of these models, such as asymptotic normality for small variances. A combined maximum likelihood method is proposed to estimate the model parameters, and it is applied to microarray and contamination data. Rcodes are available from the authors upon request.

  15. 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.

  16. 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

  17. Analysis of Microarray Data on Gene Expression and Methylation to Identify Long Non-coding RNAs in Non-small Cell Lung Cancer

    PubMed Central

    Feng, Nannan; Ching, Travers; Wang, Yu; Liu, Ben; Lin, Hongyan; Shi, Oumin; Zhang, Xiaohong; Zheng, Min; Zheng, Xin; Gao, Ming; Zheng, Zhi-jie; Yu, Herbert; Garmire, Lana; Qian, Biyun

    2016-01-01

    To identify what long non-coding RNAs (lncRNAs) are involved in non-small cell lung cancer (NSCLC), we analyzed microarray data on gene expression and methylation. Gene expression chip and HumanMethylation450BeadChip were used to interrogate genome-wide expression and methylation in tumor samples. Differential expression and methylation were analyzed through comparing tumors with adjacent non-tumor tissues. LncRNAs expressed differentially and correlated with coding genes and DNA methylation were validated in additional tumor samples using RT-qPCR and pyrosequencing. In vitro experiments were performed to evaluate lncRNA’s effects on tumor cells. We identified 8,500 lncRNAs expressed differentially between tumor and non-tumor tissues, of which 1,504 were correlated with mRNA expression. Two of the lncRNAs, LOC146880 and ENST00000439577, were positively correlated with expression of two cancer-related genes, KPNA2 and RCC2, respectively. High expression of LOC146880 and ENST00000439577 were also associated with poor survival. Analysis of lncRNA expression in relation to DNA methylation showed that LOC146880 expression was down-regulated by DNA methylation in its promoter. Lowering the expression of LOC146880 or ENST00000439577 in tumor cells could inhibit cell proliferation, invasion and migration. Analysis of microarray data on gene expression and methylation allows us to identify two lncRNAs, LOC146880 and ENST00000439577, which may promote the progression of NSCLC. PMID:27849024

  18. DNA Microarray for Detection of Gastrointestinal Viruses

    PubMed Central

    Martínez, Miguel A.; Soto-del Río, María de los Dolores; Gutiérrez, Rosa María; Chiu, Charles Y.; Greninger, Alexander L.; Contreras, Juan Francisco; López, 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

  19. DNA microarray for detection of gastrointestinal viruses.

    PubMed

    Martínez, Miguel A; Soto-Del Río, María de Los Dolores; Gutiérrez, Rosa María; Chiu, Charles Y; Greninger, Alexander L; Contreras, Juan Francisco; López, Susana; Arias, Carlos F; Isa, Pavel

    2015-01-01

    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 × 10(3) 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

  20. 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.

  1. 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

  2. Evaluation of gene importance in microarray data based upon probability of selection

    PubMed Central

    Fu, Li M; Fu-Liu, Casey S

    2005-01-01

    Background Microarray devices permit a genome-scale evaluation of gene function. This technology has catalyzed biomedical research and development in recent years. As many important diseases can be traced down to the gene level, a long-standing research problem is to identify specific gene expression patterns linking to metabolic characteristics that contribute to disease development and progression. The microarray approach offers an expedited solution to this problem. However, it has posed a challenging issue to recognize disease-related genes expression patterns embedded in the microarray data. In selecting a small set of biologically significant genes for classifier design, the nature of high data dimensionality inherent in this problem creates substantial amount of uncertainty. Results Here we present a model for probability analysis of selected genes in order to determine their importance. Our contribution is that we show how to derive the P value of each selected gene in multiple gene selection trials based on different combinations of data samples and how to conduct a reliability analysis accordingly. The importance of a gene is indicated by its associated P value in that a smaller value implies higher information content from information theory. On the microarray data concerning the subtype classification of small round blue cell tumors, we demonstrate that the method is capable of finding the smallest set of genes (19 genes) with optimal classification performance, compared with results reported in the literature. Conclusion In classifier design based on microarray data, the probability value derived from gene selection based on multiple combinations of data samples enables an effective mechanism for reducing the tendency of fitting local data particularities. PMID:15784140

  3. Chronic Trypanosoma cruzi infection potentiates adipose tissue macrophage polarization toward an anti-inflammatory M2 phenotype and contributes to diabetes progression in a diet-induced obesity model

    PubMed Central

    Cabalén, María E.; Cabral, María F.; Sanmarco, Liliana M.; Andrada, Marta C.; Onofrio, Luisina I.; Ponce, Nicolás E.; Aoki, María P.; Gea, Susana; Cano, Roxana C.

    2016-01-01

    Chronic obesity and Chagas disease (caused by the protozoan Trypanosoma cruzi) represent serious public health concerns. The interrelation between parasite infection, adipose tissue, immune system and metabolism in an obesogenic context, has not been entirely explored. A novel diet-induced obesity model (DIO) was developed in C57BL/6 wild type mice to examine the effect of chronic infection (DIO+I) on metabolic parameters and on obesity-related disorders. Dyslipidemia, hyperleptinemia, and cardiac/hepatic steatosis were strongly developed in DIO mice. Strikingly, although these metabolic alterations were collectively improved by infection, plasmatic apoB100 levels remain significantly increased in DIO+I, suggesting the presence of pro-atherogenic small and dense LDL particles. Moreover, acute insulin resistance followed by chronic hyperglycemia with hypoinsulinemia was found, evidencing an infection-related-diabetes progression. These lipid and glucose metabolic changes seemed to be highly dependent on TLR4 expression since TLR4−/− mice were protected from obesity and its complications. Notably, chronic infection promoted a strong increase in MCP-1 producing macrophages with a M2 (F4/80+CD11c-CD206+) phenotype associated to oxidative stress in visceral adipose tissue of DIO+I mice. Importantly, infection reduced lipid content but intensified inflammatory infiltrates in target tissues. Thus, parasite persistence in an obesogenic environment and the resulting host immunometabolic dysregulation may contribute to diabetes/atherosclerosis progression. PMID:26921251

  4. Chronic Trypanosoma cruzi infection potentiates adipose tissue macrophage polarization toward an anti-inflammatory M2 phenotype and contributes to diabetes progression in a diet-induced obesity model.

    PubMed

    Cabalén, María E; Cabral, María F; Sanmarco, Liliana M; Andrada, Marta C; Onofrio, Luisina I; Ponce, Nicolás E; Aoki, María P; Gea, Susana; Cano, Roxana C

    2016-03-22

    Chronic obesity and Chagas disease (caused by the protozoan Trypanosoma cruzi) represent serious public health concerns. The interrelation between parasite infection, adipose tissue, immune system and metabolism in an obesogenic context, has not been entirely explored. A novel diet-induced obesity model (DIO) was developed in C57BL/6 wild type mice to examine the effect of chronic infection (DIO+I) on metabolic parameters and on obesity-related disorders. Dyslipidemia, hyperleptinemia, and cardiac/hepatic steatosis were strongly developed in DIO mice. Strikingly, although these metabolic alterations were collectively improved by infection, plasmatic apoB100 levels remain significantly increased in DIO+I, suggesting the presence of pro-atherogenic small and dense LDL particles. Moreover, acute insulin resistance followed by chronic hyperglycemia with hypoinsulinemia was found, evidencing an infection-related-diabetes progression. These lipid and glucose metabolic changes seemed to be highly dependent on TLR4 expression since TLR4-/- mice were protected from obesity and its complications. Notably, chronic infection promoted a strong increase in MCP-1 producing macrophages with a M2 (F4/80+CD11c-CD206+) phenotype associated to oxidative stress in visceral adipose tissue of DIO+I mice. Importantly, infection reduced lipid content but intensified inflammatory infiltrates in target tissues. Thus, parasite persistence in an obesogenic environment and the resulting host immunometabolic dysregulation may contribute to diabetes/atherosclerosis progression.

  5. Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays.

    PubMed

    Panicker, Gitika; Call, Douglas R; Krug, Melissa J; Bej, Asim K

    2004-12-01

    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 degrees 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 10(2) to 10(3) 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.

  6. An algorithm for finding biologically significant features in microarray data based on a priori manifold learning.

    PubMed

    Hira, Zena M; Trigeorgis, George; Gillies, Duncan F

    2014-01-01

    Microarray databases are a large source of genetic data, which, upon proper analysis, could enhance our understanding of biology and medicine. Many microarray experiments have been designed to investigate the genetic mechanisms of cancer, and analytical approaches have been applied in order to classify different types of cancer or distinguish between cancerous and non-cancerous tissue. However, microarrays are high-dimensional datasets with high levels of noise and this causes problems when using machine learning methods. A popular approach to this problem is to search for a set of features that will simplify the structure and to some degree remove the noise from the data. The most widely used approach to feature extraction is principal component analysis (PCA) which assumes a multivariate Gaussian model of the data. More recently, non-linear methods have been investigated. Among these, manifold learning algorithms, for example Isomap, aim to project the data from a higher dimensional space onto a lower dimension one. We have proposed a priori manifold learning for finding a manifold in which a representative set of microarray data is fused with relevant data taken from the KEGG pathway database. Once the manifold has been constructed the raw microarray data is projected onto it and clustering and classification can take place. In contrast to earlier fusion based methods, the prior knowledge from the KEGG databases is not used in, and does not bias the classification process--it merely acts as an aid to find the best space in which to search the data. In our experiments we have found that using our new manifold method gives better classification results than using either PCA or conventional Isomap.

  7. Genetics Home Reference: progressive osseous heteroplasia

    MedlinePlus

    ... and muscle tissue. Bone that forms outside the skeleton is called heterotopic or ectopic bone. In progressive ... preventing bony tissue from being produced outside the skeleton. The GNAS gene mutations that cause progressive osseous ...

  8. Microarray Technology Applied to Human Allergic Disease

    PubMed Central

    Hamilton, Robert G.

    2017-01-01

    IgE antibodies serve as the gatekeeper for the release of mediators from sensitized (IgE positive) mast cells and basophils following a relevant allergen exposure which can lead to an immediate-type hypersensitivity (allergic) reaction. Purified recombinant and native allergens were combined in the 1990s with state of the art chip technology to establish the first microarray-based IgE antibody assay. Triplicate spots to over 100 allergenic molecules are immobilized on an amine-activated glass slide to form a single panel multi-allergosorbent assay. Human antibodies, typically of the IgE and IgG isotypes, specific for one or many allergens bind to their respective allergen(s) on the chip. Following removal of unbound serum proteins, bound IgE antibody is detected with a fluorophore-labeled anti-human IgE reagent. The fluorescent profile from the completed slide provides a sensitization profile of an allergic patient which can identify IgE antibodies that bind to structurally similar (cross-reactive) allergen families versus molecules that are unique to a single allergen specificity. Despite its ability to rapidly analyze many IgE antibody specificities in a single simple assay format, the chip-based microarray remains less analytically sensitive and quantitative than its singleplex assay counterpart (ImmunoCAP, Immulite). Microgram per mL quantities of allergen-specific IgG antibody can also complete with nanogram per mL quantities of specific IgE for limited allergen binding sites on the chip. Microarray assays, while not used in clinical immunology laboratories for routine patient IgE antibody testing, will remain an excellent research tool for defining sensitization profiles of populations in epidemiological studies. PMID:28134842

  9. Development of a microarray for identification of pathogenic Clostridium species

    PubMed Central

    Janvilisri, Tavan; Scaria, Joy; Gleed, Robin; Fubini, Susan; Bonkosky, Michelle M.; Gröhn, Yrjö T.; Chang, Yung-Fu

    2009-01-01

    In recent years, Clostridium species have rapidly reemerged as human and animal pathogens. The detection and identification of pathogenic Clostridium species is therefore critical for clinical diagnosis and antimicrobial therapy. Traditional diagnostic techniques for clostridia are laborious, time-consuming and may adversely affect the therapeutic outcome. In this study, we developed an oligonucleotide diagnostic microarray for pathogenic Clostridium species. The microarray specificity was tested against 65 Clostridium isolates. The applicability of this microarray in a clinical setting was assessed with the use of mock stool samples. The microarray was successful in discriminating at least four species with the limit of detection as low as 104 CFU/ml. In addition, the pattern of virulence and antibiotic resistance genes of tested strains were determined through the microarrays. This approach demonstrates the high-throughput detection and identification of Clostridium species and provides advantages over traditional methods. Microarray-based techniques are promising applications for clinical diagnosis and epidemiological investigations. PMID:19879710

  10. Application of DNA microarray technology to gerontological studies.

    PubMed

    Masuda, Kiyoshi; Kuwano, Yuki; Nishida, Kensei; Rokutan, Kazuhito

    2013-01-01

    Gene expression patterns change dramatically in aging and age-related events. The DNA microarray is now recognized as a useful device in molecular biology and widely used to identify the molecular mechanisms of aging and the biological effects of drugs for therapeutic purpose in age-related diseases. Recently, numerous technological advantages have led to the evolution of DNA microarrays and microarray-based techniques, revealing the genomic modification and all transcriptional activity. Here, we show the step-by-step methods currently used in our lab to handling the oligonucleotide microarray and miRNA microarray. Moreover, we introduce the protocols of ribonucleoprotein [RNP] immunoprecipitation followed by microarray analysis (RIP-chip) which reveal the target mRNA of age-related RNA-binding proteins.

  11. 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.

  12. 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.

  13. 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.

  14. Chemiluminescence microarrays in analytical chemistry: a critical review.

    PubMed

    Seidel, Michael; Niessner, Reinhard

    2014-09-01

    Multi-analyte immunoassays on microarrays and on multiplex DNA microarrays have been described for quantitative analysis of small organic molecules (e.g., antibiotics, drugs of abuse, small molecule toxins), proteins (e.g., antibodies or protein toxins), and microorganisms, viruses, and eukaryotic cells. In analytical chemistry, multi-analyte detection by use of analytical microarrays has become an innovative research topic because of the possibility of generating several sets of quantitative data for different analyte classes in a short time. Chemiluminescence (CL) microarrays are powerful tools for rapid multiplex analysis of complex matrices. A wide range of applications for CL microarrays is described in the literature dealing with analytical microarrays. The motivation for this review is to summarize the current state of CL-based analytical microarrays. Combining analysis of different compound classes on CL microarrays reduces analysis time, cost of reagents, and use of laboratory space. Applications are discussed, with examples from food safety, water safety, environmental monitoring, diagnostics, forensics, toxicology, and biosecurity. The potential and limitations of research on multiplex analysis by use of CL microarrays are discussed in this review.

  15. Studying cellular processes and detecting disease with protein microarrays

    SciTech Connect

    Zangar, Richard C.; Varnum, Susan M.; Bollinger, Nikki

    2005-10-31

    Protein microarrays are a rapidly developing analytic tool with diverse applications in biomedical research. These applications include profiling of disease markers or autoimmune responses, understanding molecular pathways, protein modifications and protein activities. One factor that is driving this expanding usage is the wide variety of experimental formats that protein microarrays can take. In this review, we provide a short, conceptual overview of the different approaches for protein microarray. We then examine some of the most significant applications of these microarrays to date, with an emphasis on how global protein analyses can be used to facilitate biomedical research.

  16. Re-Annotator: Annotation Pipeline for Microarray Probe Sequences.

    PubMed

    Arloth, Janine; Bader, Daniel M; Röh, Simone; Altmann, Andre

    2015-01-01

    Microarray technologies are established approaches for high throughput gene expression, methylation and genotyping analysis. An accurate mapping of the array probes is essential to generate reliable biological findings. However, manufacturers of the microarray platforms typically provide incomplete and outdated annotation tables, which often rely on older genome and transcriptome versions that differ substantially from up-to-date sequence databases. Here, we present the Re-Annotator, a re-annotation pipeline for microarray probe sequences. It is primarily designed for gene expression microarrays but can also be adapted to other types of microarrays. The Re-Annotator uses a custom-built mRNA reference database to identify the positions of gene expression array probe sequences. We applied Re-Annotator to the Illumina Human-HT12 v4 microarray platform and found that about one quarter (25%) of the probes differed from the manufacturer's annotation. In further computational experiments on experimental gene expression data, we compared Re-Annotator to another probe re-annotation tool, ReMOAT, and found that Re-Annotator provided an improved re-annotation of microarray probes. A thorough re-annotation of probe information is crucial to any microarray analysis. The Re-Annotator pipeline is freely available at http://sourceforge.net/projects/reannotator along with re-annotated files for Illumina microarrays HumanHT-12 v3/v4 and MouseRef-8 v2.

  17. Application of DNA microarrays in occupational health research.

    PubMed

    Koizumi, Shinji

    2004-01-01

    The profiling of gene expression patterns with DNA microarrays is recently being widely used not only in basic molecular biological studies but also in the practical fields. In clinical application, for example, this technique is expected to be quite useful in making a correct diagnosis. In the pharmacological area, the microarray analysis can be applied to drug discovery and individualized drug treatment. Although not so popular as these examples, DNA microarrays could also be a powerful tool in studies relevant to occupational health. This review will describe the outline of gene expression profiling with DNA microarrays and prospects in occupational health research.

  18. 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.

  19. 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

  20. Zeptosens' protein microarrays: a novel high performance microarray platform for low abundance protein analysis.

    PubMed

    Pawlak, Michael; Schick, Eginhard; Bopp, Martin A; Schneider, Michael J; Oroszlan, Peter; Ehrat, Markus

    2002-04-01

    Protein microarrays are considered an enabling technology, which will significantly expand the scope of current protein expression and protein interaction analysis. Current technologies, such as two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry, allowing the identification of biologically relevant proteins, have a high resolving power, but also considerable limitations. As was demonstrated by Gygi et al. (Proc. Nat. Acad. Sci. USA 2000,97, 9390-9395), most spots in 2-DE, observed from whole cell extracts, are from high abundance proteins, whereas low abundance proteins, such as signaling molecules or kinases, are only poorly represented. Protein microarrays are expected to significantly expedite the discovery of new markers and targets of pharmaceutical interest, and to have the potential for high-throughput applications. Key factors to reach this goal are: high read-out sensitivity for quantification also of low abundance proteins, functional analysis of proteins, short assay analysis times, ease of handling and the ability to integrate a variety of different targets and new assays. Zeptosens has developed a revolutionary new bioanalytical system based on the proprietary planar waveguide technology which allows us to perform multiplexed, quantitative biomolecular interaction analysis with highest sensitivity in a microarray format upon utilizing the specific advantages of the evanescent field fluorescence detection. The analytical system, comprising an ultrasensitive fluorescence reader and microarray chips with integrated microfluidics, enables the user to generate a multitude of high fidelity data in applications such as protein expression profiling or investigating protein-protein interactions. In this paper, the important factors for developing high performance protein microarray systems, especially for targeting low abundant messengers of relevant biological information, will be discussed and the performance of the system will

  1. Manufacturing DNA microarrays from unpurified PCR products

    PubMed Central

    Diehl, Frank; Beckmann, Boris; Kellner, Nadine; Hauser, Nicole C.; Diehl, Susanne; Hoheisel, Jörg D.

    2002-01-01

    For the production of DNA microarrays from PCR products, purification of the the DNA fragments prior to spotting is a major expense in cost and time. Also, a considerable amount of material is lost during this process and contamination might occur. Here, a protocol is presented that permits the manufacture of microarrays from unpurified PCR products on aminated surfaces such as glass slides coated with the widely used poly(l-lysine) or aminosilane. The presence of primer molecules in the PCR sample does not increase the non-specific signal upon hybridisation. Overall, signal intensity on arrays made of unpurified PCR products is 94% of the intensity obtained with the respective purified molecules. This slight loss in signal, however, is offset by a reduced variation in the amount of DNA present at the individual spot positions across an array, apart from the considerable savings in time and cost. In addition, a larger number of arrays can be made from one batch of amplification products. PMID:12177307

  2. Inferring genetic networks from microarray data.

    SciTech Connect

    May, Elebeoba Eni; Davidson, George S.; Martin, Shawn Bryan; Werner-Washburne, Margaret C.; Faulon, Jean-Loup Michel

    2004-06-01

    In theory, it should be possible to infer realistic genetic networks from time series microarray data. In practice, however, network discovery has proved problematic. The three major challenges are: (1) inferring the network; (2) estimating the stability of the inferred network; and (3) making the network visually accessible to the user. Here we describe a method, tested on publicly available time series microarray data, which addresses these concerns. The inference of genetic networks from genome-wide experimental data is an important biological problem which has received much attention. Approaches to this problem have typically included application of clustering algorithms [6]; the use of Boolean networks [12, 1, 10]; the use of Bayesian networks [8, 11]; and the use of continuous models [21, 14, 19]. Overviews of the problem and general approaches to network inference can be found in [4, 3]. Our approach to network inference is similar to earlier methods in that we use both clustering and Boolean network inference. However, we have attempted to extend the process to better serve the end-user, the biologist. In particular, we have incorporated a system to assess the reliability of our network, and we have developed tools which allow interactive visualization of the proposed network.

  3. Solution processed organic microarray with inverted structure

    NASA Astrophysics Data System (ADS)

    Toglia, Patrick; Lewis, Jason; Lafalce, Evan; Jiang, Xiaomei

    2011-03-01

    We have fabricated inverted organic microarray using a novel solution-based technique. The array consists of 60 small (1 square mm) solar cells on a one inch by one inch glass substrate. The device utilizes photoactive materials such as a blend of poly(3-hexylthiophene) (P3HT) and [6,6]-phenyl-C61-butyric acid methyl ester (PCBM). Manipulation of active layer nanomorphology has been done by choice of solvents and annealing conditions. Detailed analysis of device physics including current voltage characteristics, external quantum efficiency and carrier recombinations will be presented and complimented by AFM images and glazing angle XRD of the active layer under different processing conditions. The procedure described here has the full potential for use in future fabrication of microarrays with single cell as small as 0.01 square mm for application in DC power supplies for electrostatic Microelectromechanical systems (MEMS) devices. This work was supported by New Energy Technology Inc. and Florida High Tech Corridor Matching Fund (FHT 09-18).

  4. Clickable Polymeric Coating for Glycan Microarrays.

    PubMed

    Zilio, Caterina; Sola, Laura; Cretich, Marina; Bernardi, Anna; Chiari, Marcella

    2017-01-01

    The interaction of carbohydrates with a variety of biological targets, including antibodies, proteins, viruses, and cells are of utmost importance in many aspects of biology. Glycan microarrays are increasingly used to determine the binding specificity of glycan-binding proteins. In this study, a novel microarray support is reported for the fabrication of glycan arrays that combines the higher sensitivity of a layered Si-SiO2 surface with a novel polymeric coating easily modifiable by subsequent click reaction. The alkyne-containing copolymer, adsorbed from an aqueous solution, produces a coating by a single step procedure and serves as a soft, tridimensional support for the oriented immobilization of carbohydrates via azide/alkyne Cu (I) catalyzed "click" reaction. The advantages of a functional 3D polymer coating making use of a click chemistry immobilization are combined with the high fluorescence sensitivity and superior signal-to-noise ratio of a Si-SiO2 substrate. The proposed approach enables the attachment of complex sugars on a silicon oxide surface by a method that does not require skilled personnel and chemistry laboratories.

  5. Laser capture microdissection of embryonic cells and preparation of RNA for microarray assays.

    PubMed

    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.

  6. A biofidelic computational model of the female pelvic system to understand effect of bladder fill and progressive vaginal tissue stiffening due to prolapse on anterior vaginal wall.

    PubMed

    Chanda, Arnab; Unnikrishnan, Vinu; Richter, Holly E; Lockhart, Mark E

    2016-11-01

    Treatment of anterior vaginal prolapse (AVP), suffered by over 500,000 women in the USA, is a challenge in urogynecology because of the poorly understood mechanics of AVP. Recently, computational modeling combined with finite element method has been used to model AVP through the study of pelvic floor muscle and connective tissue impairments on the anterior vaginal wall (AVW). Also, the effects of pelvic organ displacements on the AVW were studied numerically. In our current work, an MRI-based full-scale biofidelic computational model of the female pelvic system composed of the urinary bladder, vaginal canal, and the uterus was developed, and a novel finite element method framework was employed to simulate vaginal tissue stiffening and also bladder filling due to expansion for the first time. A mesh convergence study was conducted to choose a computationally efficient mesh, and a non-linear hyperelastic Yeoh's material model was adopted for the study. The AVW displacements, mechanical stresses, and strains were estimated at varying degrees of bladder fills and vaginal tissue stiffening. Both bladder filling and vaginal stiffening were found to increase the stress concentration on the AVW with varying trends, which have been discussed in detail in the paper. To our knowledge, this study is the first to estimate the individual and combined effects of bladder filling and vaginal tissue stiffening due to prolapse on the AVW. Copyright © 2016 John Wiley & Sons, Ltd.

  7. New insights about host response to smallpox using microarray data

    PubMed Central

    Esteves, Gustavo H; Simoes, Ana CQ; Souza, Estevao; Dias, Rodrigo A; Ospina, Raydonal; Venancio, Thiago M

    2007-01-01

    Background Smallpox is a lethal disease that was endemic in many parts of the world until eradicated by massive immunization. Due to its lethality, there are serious concerns about its use as a bioweapon. Here we analyze publicly available microarray data to further understand survival of smallpox infected macaques, using systems biology approaches. Our goal is to improve the knowledge about the progression of this disease. Results We used KEGG pathways annotations to define groups of genes (or modules), and subsequently compared them to macaque survival times. This technique provided additional insights about the host response to this disease, such as increased expression of the cytokines and ECM receptors in the individuals with higher survival times. These results could indicate that these gene groups could influence an effective response from the host to smallpox. Conclusion Macaques with higher survival times clearly express some specific pathways previously unidentified using regular gene-by-gene approaches. Our work also shows how third party analysis of public datasets can be important to support new hypotheses to relevant biological problems. PMID:17718913

  8. Overexpression of Tissue VEGF-A May Portend an Increased Likelihood of Progression in a Phase II Trial of Bevacizumab and Erlotinib in Resistant Ovarian Cancer

    PubMed Central

    Chambers, Setsuko K.; Clouser, Mary C.; Baker, Amanda F.; Roe, Denise J.; Cui, Haiyan; Brewer, Molly A.; Hatch, Kenneth D.; Gordon, Michael S.; Janicek, Mike F.; Isaacs, Jeffrey D.; Gordon, Alan N.; Nagle, Raymond B.; Wright, Heather M.; Cohen, Janice L.; Alberts, David S.

    2010-01-01

    Purpose This phase II trial evaluated bevacizumab plus erlotinib in platinum-resistant ovarian cancer; exploratory biomarker analyses, including that of tumor vascular endothelial growth factor (VEGF-A), were also performed. Experimental Design Forty heavily pretreated patients received erlotinib 150 mg/day orally and bevacizumab 10 mg/kg intravenously every 2 weeks until disease progression. Primary end points were objective response rate and response duration; secondary end points included progression-free survival (PFS), toxicity, and correlations between angiogenic protein levels, toxicity, and efficacy. Results Grade 3 toxicities included skin rash (n=6), diarrhea (n=5), fatigue (n=4), and hypertension (n=3). Grade 4 toxicities were myocardial infarction (n=1) and nasal septal perforation (n=1). Only 1 grade 3 fistula and 1 grade 2 bowel perforation were observed. Nine (23.1%) of 39 evaluable patients had a response (median duration 36.1+ weeks, 1 complete response), and 10 (25.6%) patients achieved stable disease, for a disease control rate of 49%. Median PFS was 4 months, and 6-month PFS was 30.8%. Biomarker analyses identified an association between tumor cell VEGF-A expression and progression (P=0.03); for every 100 unit increase in the VEGF-A score, there was a 3.7-fold increase in the odds of progression (95% CI: 1.1–16.6). Conclusions Bevacizumab plus erlotinib in heavily pretreated ovarian cancer patients was clinically active and well tolerated. Erlotinib did not appear to contribute to efficacy. Our study raises the intriguing possibility that high levels of tumor cell VEGF-A, capable of both autocrine and paracrine interactions, are associated with resistance to bevacizumab, emphasizing the complexity of the tumor microenvironment. PMID:21041183

  9. Testing an aflatoxin B1 gene signature in rat archival tissues.

    PubMed

    Merrick, B Alex; Auerbach, Scott S; Stockton, Patricia S; Foley, Julie F; Malarkey, David E; Sills, Robert C; Irwin, Richard D; Tice, Raymond R

    2012-05-21

    Archival tissues from laboratory studies represent a unique opportunity to explore the relationship between genomic changes and agent-induced disease. In this study, we evaluated the applicability of qPCR for detecting genomic changes in formalin-fixed, paraffin-embedded (FFPE) tissues by determining if a subset of 14 genes from a 90-gene signature derived from microarray data and associated with eventual tumor development could be detected in archival liver, kidney, and lung of rats exposed to aflatoxin B1 (AFB1) for 90 days in feed at 1 ppm. These tissues originated from the same rats used in the microarray study. The 14 genes evaluated were Adam8, Cdh13, Ddit4l, Mybl2, Akr7a3, Akr7a2, Fhit, Wwox, Abcb1b, Abcc3, Cxcl1, Gsta5, Grin2c, and the C8orf46 homologue. The qPCR FFPE liver results were compared to the original liver microarray data and to qPCR results using RNA from fresh frozen liver. Archival liver paraffin blocks yielded 30 to 50 μg of degraded RNA that ranged in size from 0.1 to 4 kB. qPCR results from FFPE and fresh frozen liver samples were positively correlated (p ≤ 0.05) by regression analysis and showed good agreement in direction and proportion of change with microarray data for 11 of 14 genes. All 14 transcripts could be amplified from FFPE kidney RNA except the glutamate receptor gene Grin2c; however, only Abcb1b was significantly upregulated from control. Abundant constitutive transcripts, S18 and β-actin, could be amplified from lung FFPE samples, but the narrow RNA size range (25-500 bp length) prevented consistent detection of target transcripts. Overall, a discrete gene signature derived from prior transcript profiling and representing cell cycle progression, DNA damage response, and xenosensor and detoxication pathways was successfully applied to archival liver and kidney by qPCR and indicated that gene expression changes in response to subchronic AFB1 exposure occurred predominantly in the liver, the primary target for AFB1-induced

  10. 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

  11. 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…

  12. Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

    PubMed Central

    Linger, Yvonne; Kukhtin, Alexander; Golova, Julia; Perov, Alexander; Qu, Peter; Knickerbocker, Christopher; Cooney, Christopher G.; Chandler, Darrell P.

    2014-01-01

    Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice. PMID:24796567

  13. Demonstrating a multi-drug resistant Mycobacterium tuberculosis amplification microarray.

    PubMed

    Linger, Yvonne; Kukhtin, Alexander; Golova, Julia; Perov, Alexander; Qu, Peter; Knickerbocker, Christopher; Cooney, Christopher G; Chandler, Darrell P

    2014-04-25

    Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

  14. 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...

  15. Screening and characterization of plant cell walls using carbohydrate microarrays.

    PubMed

    Sørensen, Iben; Willats, William G T

    2011-01-01

    Plant cells are surrounded by cell walls built largely from complex carbohydrates. The primary walls of growing plant cells consist of interdependent networks of three polysaccharide classes: cellulose, cross-linking glycans (also known as hemicelluloses), and pectins. Cellulose microfibrils are tethered together by cross-linking glycans, and this assembly forms the major load-bearing component of primary walls, which is infiltrated with pectic polymers. In the secondary walls of woody tissues, pectins are much reduced and walls are reinforced with the phenolic polymer lignin. Plant cell walls are essential for plant life and also have numerous industrial applications, ranging from wood to nutraceuticals. Enhancing our knowledge of cell wall biology and the effective use of cell wall materials is dependent to a large extent on being able to analyse their fine structures. We have developed a suite of techniques based on microarrays probed with monoclonal antibodies with specificity for cell wall components, and here we present practical protocols for this type of analysis.

  16. Microarray analysis of Xenopus endoderm expressing Ptf1a

    PubMed Central

    Bilogan, Cassandra K.; Horb, Marko E.

    2012-01-01

    Pancreas specific transcription factor 1a (Ptf1a), a bHLH transcription factor, has two temporally distinct functions during pancreas development; initially it is required for early specification of the entire pancreas, while later it is required for proper differentiation and maintenance of only acinar cells. The importance of Ptf1a function was revealed by the fact that loss of Ptf1a leads to pancreas agenesis in humans. While Ptf1a is one of the most important pancreatic transcription factors, little is known about the differences between the regulatory networks it controls during initial specification of the pancreas as opposed to acinar cell development, and to date no comprehensive analysis of its downstream targets has been published. In this paper, we use Xenopus embryos to identify putative downstream targets of Ptf1a. We isolated anterior endoderm tissue overexpressing Ptf1a at two early stages, NF32 and NF36, and compared their gene expression profiles using microarrays. Our results revealed that Ptf1a regulates genes with a wide variety of functions, providing insight into the complexity of the regulatory network required for pancreas specification. PMID:22815262

  17. 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.

  18. 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

  19. Uses of Dendrimers for DNA Microarrays

    PubMed Central

    Caminade, Anne-Marie; Padié, Clément; Laurent, Régis; Maraval, Alexandrine; Majoral, Jean-Pierre

    2006-01-01

    Biosensors such as DNA microarrays and microchips are gaining an increasing importance in medicinal, forensic, and environmental analyses. Such devices are based on the detection of supramolecular interactions called hybridizations that occur between complementary oligonucleotides, one linked to a solid surface (the probe), and the other one to be analyzed (the target). This paper focuses on the improvements that hyperbranched and perfectly defined nanomolecules called dendrimers can provide to this methodology. Two main uses of dendrimers for such purpose have been described up to now; either the dendrimer is used as linker between the solid surface and the probe oligonucleotide, or the dendrimer is used as a multilabeled entity linked to the target oligonucleotide. In the first case the dendrimer generally induces a higher loading of probes and an easier hybridization, due to moving away the solid phase. In the second case the high number of localized labels (generally fluorescent) induces an increased sensitivity, allowing the detection of small quantities of biological entities.

  20. Meta-analysis of incomplete microarray studies.

    PubMed

    Zollinger, Alix; Davison, Anthony C; Goldstein, Darlene R

    2015-10-01

    Meta-analysis of microarray studies to produce an overall gene list is relatively straightforward when complete data are available. When some studies lack information-providing only a ranked list of genes, for example-it is common to reduce all studies to ranked lists prior to combining them. Since this entails a loss of information, we consider a hierarchical Bayes approach to meta-analysis using different types of information from different studies: the full data matrix, summary statistics, or ranks. The model uses an informative prior for the parameter of interest to aid the detection of differentially expressed genes. Simulations show that the new approach can give substantial power gains compared with classical meta-analysis and list aggregation methods. A meta-analysis of 11 published studies with different data types identifies genes known to be involved in ovarian cancer and shows significant enrichment.

  1. Software and tools for microarray data analysis.

    PubMed

    Mehta, Jai Prakash; Rani, Sweta

    2011-01-01

    A typical microarray experiment results in series of images, depending on the experimental design and number of samples. Software analyses the images to obtain the intensity at each spot and quantify the expression for each transcript. This is followed by normalization, and then various data analysis techniques are applied on the data. The whole analysis pipeline requires a large number of software to accurately handle the massive amount of data. Fortunately, there are large number of freely available and commercial software to churn the massive amount of data to manageable sets of differentially expressed genes, functions, and pathways. This chapter describes the software and tools which can be used to analyze the gene expression data right from the image analysis to gene list, ontology, and pathways.

  2. DNA microarray-based mutation discovery and genotyping.

    PubMed

    Gresham, David

    2011-01-01

    DNA microarrays provide an efficient means of identifying single-nucleotide polymorphisms (SNPs) in DNA samples and characterizing their frequencies in individual and mixed samples. We have studied the parameters that determine the sensitivity of DNA probes to SNPs and found that the melting temperature (T (m)) of the probe is the primary determinant of probe sensitivity. An isothermal-melting temperature DNA microarray design, in which the T (m) of all probes is tightly distributed, can be implemented by varying the length of DNA probes within a single DNA microarray. I describe guidelines for designing isothermal-melting temperature DNA microarrays and protocols for labeling and hybridizing DNA samples to DNA microarrays for SNP discovery, genotyping, and quantitative determination of allele frequencies in mixed samples.

  3. Fluorescence detection in (sub-)nanoliter microarrays

    NASA Astrophysics Data System (ADS)

    van den Doel, L. Richard; Vellekoop, Michael J.; Sarro, Pasqualina M.; Picioreanu, S.; Moerman, R.; Frank, J.; van Dedem, G. W. K.; Hjelt, Kari H.; van Vliet, Lucas J.; Young, Ian T.

    1999-06-01

    The goal of our TU Delft interfaculty research program is to develop intelligent molecular diagnostic systems (IMDS) that can analyze liquid samples that contain a variety of biochemical compounds such as those associated with fermentation processes. One specific project within the IMDS program focuses on photon sensors. In order to analyze the liquid samples we use dedicated microarrays. At this stage, these are basically miniaturized micro titre plates. Typical dimensions of a vial are 200 X 200 X 20 micrometer3. These dimensions may be varied and the shape of the vials can be modified with a result that the volume of the vials varies from 0.5 to 1.6 nl. For all experiments, we have used vials with the shape of a truncated pyramid. These vials are fabricated in silicon by a wet etching process. For testing purposes the vials are filled with rhodamine solutions of various concentrations. To avoid evaporation glycerol-water (1:1, v/v) with a viscosity of 8.3 times the viscosity of water is used as solvent. We aim at wide field-of-view imaging at the expense of absolute sensitivity: the field-of-view increases quadratically with decreasing magnification. Small magnification, however, implies low Numerical Aperture (NA). The ability of a microscope objective to collect photons is proportional to the square of the NA. To image the entire microarray we have used an epi-illumination fluorescence microscope equipped with a low magnification (2.5 X/0.075) objective and a scientific CCD camera to integrate the photons emitted from the fluorescing particles in the solutions in the vials. From these experiments we found that for this setup the detection limit is on the order of micromolar concentrations of fluorescing particles. This translates to 108 molecules per vial.

  4. 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

  5. Microarray analysis reveals altered circulating microRNA expression in mice infected with Coxsackievirus B3

    PubMed Central

    Sun, Chaoyu; Tong, Lei; Zhao, Wenran; Wang, Yan; Meng, Yuan; Lin, Lexun; Liu, Bingchen; Zhai, Yujia; Zhong, Zhaohua; Li, Xueqi

    2016-01-01

    Coxsackievirus B3 (CVB3) is a common causative agent in the development of inflammatory cardiomyopathy. However, whether the expression of peripheral blood microRNAs (miRNAs) is altered in this process is unknown. The present study investigated changes to miRNA expression in the peripheral blood of CVB3-infected mice. Utilizing miRNA microarray technology, differential miRNA expression was examined between normal and CVB3-infected mice. The present results suggest that specific miRNAs were differentially expressed in the peripheral blood of mice infected with CVB3, varying with infection duration. Using miRNA microarray analysis, a total of 96 and 89 differentially expressed miRNAs were identified in the peripheral blood of mice infected with CVB3 for 3 and 6 days, respectively. Quantitative polymerase chain reaction was used to validate differentially expressed miRNAs, revealing a consistency of these results with the miRNA microarray analysis results. The biological functions of the differentially expressed miRNAs were then predicted by bioinformatics analysis. The potential biological roles of differentially expressed miRNAs included hypertrophic cardiomyopathy, dilated cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy. These results may provide important insights into the mechanisms responsible for the progression of CVB3 infection. PMID:27698715

  6. 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

    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.

  7. The expression pattern of long non-coding RNA PVT1 in tumor tissues and in extracellular vesicles of colorectal cancer correlates with cancer progression.

    PubMed

    Guo, Kai; Yao, Jie; Yu, Qiang; Li, Zijian; Huang, Hu; Cheng, Jianguo; Wang, Zhigang; Zhu, Yunfeng

    2017-04-01

    The plasmacytoma variant translocation 1 gene (PVT1) is a large non-coding locus at adjacent of c-Myc, and long non-coding RNA PVT1 is now recognized as a cancerous gene co-amplified with c-Myc in various cancers. But the expression and functional role of PVT1 in colorectal cancer are still unelucidated. In addition, all the reported long non-coding RNAs so far are discovered in either cells or tissues, but no research about long non-coding RNAs detection in extracellular vesicles has been reported yet. In the present study, we firstly investigated the expression of PVT1 in colorectal cancer specimens and its correlation with the expression of c-Myc and other related genes by real-time polymerase chain reaction. Then, we isolated the extracellular vesicles from colorectal cancer cells culturing medium by differential centrifugation and detected the PVT1 expression in extracellular vesicles by using real-time polymerase chain reaction. The PVT1 targeting siRNA was transfected into SW480 and SW620 cells, and 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay and flow cytometry were used to evaluate the cell proliferation and apoptosis. The results showed that the PVT1 expression in tumor tissues was higher than that in normal tissues, which was significantly correlated with the expression of c-Myc and three c-Myc regulating genes FUBP1, EZH2, and NPM1 and also correlated with the expression of two other PVT1-associated transcript factors nuclear factor-κB and myocyte-specific enhancer factor 2A. Here, we reported for the first time that PVT1 as a long non-coding RNA was successfully detected in extracellular vesicles excluded from SW620 and SW480 cells, and the expression level of PVT1 was higher in extracellular vesicles from the more aggressive cell SW620 than from SW480. The results also showed that by down-regulating the PVT1 expression, the c-Myc expression was suppressed, the cell proliferation was inhibited, and

  8. Immune and inflammatory gene signature in rat cerebrum in subarachnoid hemorrhage with microarray analysis.

    PubMed

    Lee, Chu-I; Chou, An-Kuo; Lin, Ching-Chih; Chou, Chia-Hua; Loh, Joon-Khim; Lieu, Ann-Shung; Wang, Chih-Jen; Huang, Chi-Ying F; Howng, Shen-Long; Hong, Yi-Ren

    2012-01-01

    Cerebral vasospasm following subarachnoid hemorrhage (SAH) has been studied in terms of a contraction of the major cerebral arteries, but the effect of cerebrum tissue in SAH is not yet well understood. To gain insight into the biology of SAH-expressing cerebrum, we employed oligonucleotide microarrays to characterize the gene expression profiles of cerebrum tissue at the early stage of SAH. Functional gene expression in the cerebrum was analyzed 2 h following stage 1-hemorrhage in Sprague-Dawley rats. mRNA was investigated by performing microarray and quantitative real-time PCR analyses, and protein expression was determined by Western blot analysis. In this study, 18 upregulated and 18 downregulated genes displayed at least a 1.5-fold change. Five genes were verified by real-time PCR, including three upregulated genes [prostaglandin E synthase (PGES), CD14 antigen, and tissue inhibitor of metalloproteinase 1 (TIMP1)] as well as two downregulated genes [KRAB-zinc finger protein-2 (KZF-2) and γ-aminobutyric acid B receptor 1 (GABA B receptor)]. Notably, there were functional implications for the three upregulated genes involved in the inflammatory SAH process. However, the mechanisms leading to decreased KZF-2 and GABA B receptor expression in SAH have never been characterized. We conclude that oligonucleotide microarrays have the potential for use as a method to identify candidate genes associated with SAH and to provide novel investigational targets, including genes involved in the immune and inflammatory response. Furthermore, understanding the regulation of MMP9/TIMP1 during the early stages of SAH may elucidate the pathophysiological mechanisms in SAH rats.

  9. 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

  10. Identification of a Novel Coronavirus from a Beluga Whale by Using a Panviral Microarray ▿ †

    PubMed Central

    Mihindukulasuriya, Kathie A.; Wu, Guang; St. Leger, Judy; Nordhausen, Robert W.; Wang, David

    2008-01-01

    The emergence of viruses such as severe acute respiratory syndrome coronavirus and Nipah virus has underscored the role of animal reservoirs in human disease and the need for reservoir surveillance. Here, we used a panviral DNA microarray to investigate the death of a captive beluga whale in an aquatic park. A highly divergent coronavirus, tentatively named coronavirus SW1, was identified in liver tissue from the deceased whale. Subsequently, the entire genome of SW1 was sequenced, yielding a genome of 31,686 nucleotides. Phylogenetic analysis revealed SW1 to be a novel virus distantly related to but most similar to group III coronaviruses. PMID:18353961

  11. Leptospiral Outer Membrane Protein Microarray, a Novel Approach to Identification of Host Ligand-Binding Proteins

    PubMed Central

    Matsunaga, James; Haake, David A.

    2012-01-01

    Leptospirosis is a zoonosis with worldwide distribution caused by pathogenic spirochetes belonging to the genus Leptospira. The leptospiral life cycle involves transmission via freshwater and colonization of the renal tubules of their reservoir hosts. Infection requires adherence to cell surfaces and extracellular matrix components of host tissues. These host-pathogen interactions involve outer membrane proteins (OMPs) expressed on the bacterial surface. In this study, we developed an Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 OMP microarray containing all predicted lipoproteins and transmembrane OMPs. A total of 401 leptospiral genes or their fragments were transcribed and translated in vitro and printed on nitrocellulose-coated glass slides. We investigated the potential of this protein microarray to screen for interactions between leptospiral OMPs and fibronectin (Fn). This approach resulted in the identification of the recently described fibronectin-binding protein, LIC10258 (MFn8, Lsa66), and 14 novel Fn-binding proteins, denoted Microarray Fn-binding proteins (MFns). We confirmed Fn binding of purified recombinant LIC11612 (MFn1), LIC10714 (MFn2), LIC11051 (MFn6), LIC11436 (MFn7), LIC10258 (MFn8, Lsa66), and LIC10537 (MFn9) by far-Western blot assays. Moreover, we obtained specific antibodies to MFn1, MFn7, MFn8 (Lsa66), and MFn9 and demonstrated that MFn1, MFn7, and MFn9 are expressed and surface exposed under in vitro growth conditions. Further, we demonstrated that MFn1, MFn4 (LIC12631, Sph2), and MFn7 enable leptospires to bind fibronectin when expressed in the saprophyte, Leptospira biflexa. Protein microarrays are valuable tools for high-throughput identification of novel host ligand-binding proteins that have the potential to play key roles in the virulence mechanisms of pathogens. PMID:22961849

  12. 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

  13. Systematic gene microarray analysis of the lncRNA expression profiles in human uterine cervix carcinoma.

    PubMed

    Chen, Jie; Fu, Ziyi; Ji, Chenbo; Gu, Pingqing; Xu, Pengfei; Yu, Ningzhu; Kan, Yansheng; Wu, Xiaowei; Shen, Rong; Shen, Yan

    2015-05-01

    The human uterine cervix carcinoma is one of the most well-known malignancy reproductive system cancers, which threatens women health globally. However, the mechanisms of the oncogenesis and development process of cervix carcinoma are not yet fully understood. Long non-coding RNAs (lncRNAs) have been proved to play key roles in various biological processes, especially development of cancer. The function and mechanism of lncRNAs on cervix carcinoma is still rarely reported. We selected 3 cervix cancer and normal cervix tissues separately, then performed lncRNA microarray to detect the differentially expressed lncRNAs. Subsequently, we explored the potential function of these dysregulated lncRNAs through online bioinformatics databases. Finally, quantity real-time PCR was carried out to confirm the expression levels of these dysregulated lncRNAs in cervix cancer and normal tissues. We uncovered the profiles of differentially expressed lncRNAs between normal and cervix carcinoma tissues by using the microarray techniques, and found 1622 upregulated and 3026 downregulated lncRNAs (fold-change>2.0) in cervix carcinoma compared to the normal cervical tissue. Furthermore, we found HOXA11-AS might participate in cervix carcinogenesis by regulating HOXA11, which is involved in regulating biological processes of cervix cancer. This study afforded expression profiles of lncRNAs between cervix carcinoma tissue and normal cervical tissue, which could provide database for further research about the function and mechanism of key-lncRNAs in cervix carcinoma, and might be helpful to explore potential diagnosis factors and therapeutic targets for cervix carcinoma.

  14. The application of protein microarray assays in psychoneuroimmunology.

    PubMed

    Ayling, K; Bowden, T; Tighe, P; Todd, I; Dilnot, E M; Negm, O H; Fairclough, L; Vedhara, K

    2017-01-01

    Protein microarrays are miniaturized multiplex assays that exhibit many advantages over the commonly used enzyme-linked immunosorbent assay (ELISA). This article aims to introduce protein microarrays to readers of Brain, Behavior, and Immunity and demonstrate its utility and validity for use in psychoneuroimmunological research. As part of an ongoing investigation of psychological and behavioral influences on influenza vaccination responses, we optimized a novel protein microarray to quantify influenza-specific antibody levels in human sera. Reproducibility was assessed by calculating intra- and inter-assay coefficients of variance on serially diluted human IgG concentrations. A random selection of samples was analyzed by microarray and ELISA to establish validity of the assay. For IgG concentrations, intra-assay and inter-assay precision profiles demonstrated a mean coefficient of variance of 6.7% and 11.5% respectively. Significant correlations were observed between microarray and ELISA for all antigens, demonstrating the microarray is a valid alternative to ELISA. Protein microarrays are a highly robust, novel assay method that could be of significant benefit for researchers working in psychoneuroimmunology. They offer high throughput, fewer resources per analyte and can examine concurrent neuro-immune-endocrine mechanisms.

  15. Imaging combined autoimmune and infectious disease microarrays

    NASA Astrophysics Data System (ADS)

    Ewart, Tom; Raha, Sandeep; Kus, Dorothy; Tarnopolsky, Mark

    2006-09-01

    Bacterial and viral pathogens are implicated in many severe autoimmune diseases, acting through such mechanisms as molecular mimicry, and superantigen activation of T-cells. For example, Helicobacter pylori, well known cause of stomach ulcers and cancers, is also identified in ischaemic heart disease (mimicry of heat shock protein 65), autoimmune pancreatitis, systemic sclerosis, autoimmune thyroiditis (HLA DRB1*0301 allele susceptibility), and Crohn's disease. Successful antibiotic eradication of H.pylori often accompanies their remission. Yet current diagnostic devices, and test-limiting cost containment, impede recognition of the linkage, delaying both diagnosis and therapeutic intervention until the chronic debilitating stage. We designed a 15 minute low cost 39 antigen microarray assay, combining autoimmune, viral and bacterial antigens1. This enables point-of-care serodiagnosis and cost-effective narrowly targeted concurrent antibiotic and monoclonal anti-T-cell and anti-cytokine immunotherapy. Arrays of 26 pathogen and 13 autoimmune antigens with IgG and IgM dilution series were printed in triplicate on epoxysilane covalent binding slides with Teflon well masks. Sera diluted 1:20 were incubated 10 minutes, washed off, anti-IgG-Cy3 (green) and anti-IgM-Dy647 (red) were incubated for 5 minutes, washed off and the slide was read in an ArrayWoRx(e) scanning CCD imager (Applied Precision, Issaquah, WA). As a preliminary model for the combined infectious disease-autoimmune diagnostic microarray we surveyed 98 unidentified, outdated sera that were discarded after Hepatitis B antibody testing. In these, significant IgG or IgM autoantibody levels were found: dsDNA 5, ssDNA 11, Ro 2, RNP 7, SSB 4, gliadin 2, thyroglobulin 13 cases. Since control sera showed no autoantibodies, the high frequency of anti-DNA and anti-thyroglobulin antibodies found in infected sera lend increased support for linkage of infection to subsequent autoimmune disease. Expansion of the antigen

  16. Protein Microarrays with Novel Microfluidic Methods: Current Advances.

    PubMed

    Dixit, Chandra K; Aguirre, Gerson R

    2014-07-01

    Microfluidic-based micromosaic technology has allowed the pattering of recognition elements in restricted micrometer scale areas with high precision. This controlled patterning enabled the development of highly multiplexed arrays multiple analyte detection. This arraying technology was first introduced in the beginning of 2001 and holds tremendous potential to revolutionize microarray development and analyte detection. Later, several microfluidic methods were developed for microarray application. In this review we discuss these novel methods and approaches which leverage the property of microfluidic technologies to significantly improve various physical aspects of microarray technology, such as enhanced imprinting homogeneity, stability of the immobilized biomolecules, decreasing assay times, and reduction of the costs and of the bulky instrumentation.

  17. Deciphering the glycosaminoglycan code with the help of microarrays.

    PubMed

    de Paz, Jose L; Seeberger, Peter H

    2008-07-01

    Carbohydrate microarrays have become a powerful tool to elucidate the biological role of complex sugars. Microarrays are particularly useful for the study of glycosaminoglycans (GAGs), a key class of carbohydrates. The high-throughput chip format enables rapid screening of large numbers of potential GAG sequences produced via a complex biosynthesis while consuming very little sample. Here, we briefly highlight the most recent advances involving GAG microarrays built with synthetic or naturally derived oligosaccharides. These chips are powerful tools for characterizing GAG-protein interactions and determining structure-activity relationships for specific sequences. Thereby, they contribute to decoding the information contained in specific GAG sequences.

  18. Microarray Gene Expression Analysis to Evaluate Cell Type Specific Expression of Targets Relevant for Immunotherapy of Hematological Malignancies.

    PubMed

    Pont, M J; 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.

  19. miR-450b-5p induced by oncogenic KRAS is required for colorectal cancer progression

    PubMed Central

    Deng, Dan-ling; Jiao, Hong-Li; Li, Ting-Ting; Wang, Shu-Yang; Wang, Yong-Xia; Xiao, Zhi-Yuan; Wei, Wen-ting; Chen, Yan-Ru; Qiu, Jun-Feng; Yang, Run-Wei; Lin, Jie; Liang, Li; Liao, Wen-Ting; Ding, Yan-Qing

    2016-01-01

    The development and progression of CRC are regarded as a complicated network and progressive event including genetic and/or epigenetic alterations. Recent researches revealed that MicroRNAs are biomarkers and regulators of CRC progression. Analyses of published microarray datasets revealed that miR-450b-5p was highly up-regulated in CRC tissues. In addition, high expression of miR-450b-5p was significantly associated with KRAS mutation. However, the role of miR-450b-5p in the progression of CRC remains unknown. Here, we sought to validate the expression of miR-450b-5p in CRC tissues and investigate the role and underlying mechanism of miR-450b-5p in the progression of CRC. The results revealed that miR-450b-5p was up-regulated in CRC tissues, high expression level of miR-450b-5p was positively associated with poor differentiation, advanced TNM classification and poor prognosis. Moreover, miR-450b-5p was especially high in KRAS-mutated cell lines and could be up-regulated by KRAS/AP-1 signaling. Functional validation revealed that overexpression of miR-450b-5p promoted cell proliferation and tumor growth while inhibited apoptosis of CRC cells. Furthermore, we demonstrated that miR-450b-5p directly bound the 3′-UTRs of SFRP2 and SIAH1, and activated Wnt/β-Catenin signaling. In conclusion, miR-450b-5p induced by oncogenic KRAS is required for colorectal cancer progression. Collectively, our work helped to understand the precise role of miR-450b-5p in the progression of CRC, and might promote the development of new therapeutic strategies against CRC. PMID:27494869

  20. Microarrays (DNA chips) for the classroom laboratory.

    PubMed

    Barnard, Betsy; Sussman, Michael; Bondurant, Sandra Splinter; Nienhuis, James; Krysan, Patrick

    2006-09-01

    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 adaptation is the use of a simulated cDNA target. The low density DNA array we discuss here was used to demonstrate differential expression of several Arabidopsis thaliana genes related to photosynthesis and photomorphogenesis. The methods we present here can be used with any biological organism whose sequence is known. Furthermore, these methods can be adapted to exhibit a variety of differential gene expression patterns under different experimental conditions. The materials and tools we discuss have been applied in classrooms at West High School in Madison, WI. We have also shared these materials with high school teachers attending professional development courses at the University of Wisconsin-Madison.

  1. Cell microarrays on photochemically modified polytetrafluoroethylene.

    PubMed

    Mikulikova, Regina; Moritz, Sieglinde; Gumpenberger, Thomas; Olbrich, Michael; Romanin, Christoph; Bacakova, Lucie; Svorcik, Vaclav; Heitz, Johannes

    2005-09-01

    We studied the adhesion, proliferation, and viability of human umbilical vein endothelial cells (HUVEC) and human embryonic kidney cells (HEK) on modified spots at polytetrafluoroethylene (PTFE) surfaces. The viability of the cells was assessed using an aqueous non-radioactive cell proliferation assay. Round spots with a diameter of 100 microm were modified by exposure to the ultraviolet (UV) light of a Xe(2)(*)-excimer lamp at a wavelength of 172 nm in an ammonia atmosphere employing a contact mask. The spots were arranged in a quadratic pattern with 300 microm center-to-center spot distances. With optimized degree of modification, the cells adhered to the modified spots with a high degree of selectivity (70-90%). The adhered cells on the spots proliferated. This resulted in a significant increase in the number of adhering HUVECS or HEK cells after seeding and in the formation of confluent cell clusters after 3-4 days. With higher start seeding density, these clusters were not only confined to the modified spots but extended several micrometer to the neighborhood. The high potential of the cell microarrays for gene analysis in living cells was demonstrated with HEK cells transfected by yellow fluorescent protein (YFP).

  2. Antibody microarrays for native toxin detection.

    PubMed

    Rucker, Victor C; Havenstrite, Karen L; Herr, Amy E

    2005-04-15

    We have developed antibody-based microarray techniques for the multiplexed detection of cholera toxin beta-subunit, diphtheria toxin, anthrax lethal factor and protective antigen, Staphylococcus aureus enterotoxin B, and tetanus toxin C fragment in spiked samples. Two detection schemes were investigated: (i) a direct assay in which fluorescently labeled toxins were captured directly by the antibody array and (ii) a competition assay that employed unlabeled toxins as reporters for the quantification of native toxin in solution. In the direct assay, fluorescence measured at each array element is correlated with labeled toxin concentration to yield baseline binding information (Langmuir isotherms and affinity constants). Extending from the direct assay, the competition assay yields information on the presence, identity, and concentration of toxins. A significant advantage of the competition assay over reported profiling assays is the minimal sample preparation required prior to analysis because the competition assay obviates the need to fluorescently label native proteins in the sample of interest. Sigmoidal calibration curves and detection limits were established for both assay formats. Although the sensitivity of the direct assay is superior to that of the competition assay, detection limits for unmodified toxins in the competition assay are comparable to values reported previously for sandwich-format immunoassays of antibodies arrayed on planar substrates. As a demonstration of the potential of the competition assay for unlabeled toxin detection, we conclude with a straightforward multiplexed assay for the differentiation and identification of both native S. aureus enterotoxin B and tetanus toxin C fragment in spiked dilute serum samples.

  3. Microarray analysis of DNA replication timing.

    PubMed

    Karnani, Neerja; Taylor, Christopher M; Dutta, Anindya

    2009-01-01

    Although all of the DNA in an eukaryotic cell replicates during the S-phase of cell cycle, there is a significant difference in the actual time in S-phase when a given chromosomal segment replicates. Methods are described here for generation of high-resolution temporal maps of DNA replication in synchronized human cells. This method does not require amplification of DNA before microarray hybridization and so avoids errors introduced during PCR. A major advantage of using this procedure is that it facilitates finer dissection of replication time in S-phase. Also, it helps delineate chromosomal regions that undergo biallelic or asynchronous replication, which otherwise are difficult to detect at a genome-wide scale by existing methods. The continuous TR50 (time of completion of 50% replication) maps of replication across chromosomal segments identify regions that undergo acute transitions in replication timing. These transition zones can play a significant role in identifying insulators that separate chromosomal domains with different chromatin modifications.

  4. Transcriptome Analysis of Zebrafish Embryogenesis Using Microarrays

    PubMed Central

    Mathavan, Sinnakaruppan; Lee, Serene G. P; Mak, Alicia; Miller, Lance D; Murthy, Karuturi Radha Krishna; Govindarajan, Kunde R; Tong, Yan; Wu, Yi Lian; Lam, Siew Hong; Yang, Henry; Ruan, Yijun; Korzh, Vladimir; Gong, Zhiyuan; Liu, Edison T; Lufkin, Thomas

    2005-01-01

    Zebrafish (Danio rerio) is a well-recognized model for the study of vertebrate developmental genetics, yet at the same time little is known about the transcriptional events that underlie zebrafish embryogenesis. Here we have employed microarray analysis to study the temporal activity of developmentally regulated genes during zebrafish embryogenesis. Transcriptome analysis at 12 different embryonic time points covering five different developmental stages (maternal, blastula, gastrula, segmentation, and pharyngula) revealed a highly dynamic transcriptional profile. Hierarchical clustering, stage-specific clustering, and algorithms to detect onset and peak of gene expression revealed clearly demarcated transcript clusters with maximum gene activity at distinct developmental stages as well as co-regulated expression of gene groups involved in dedicated functions such as organogenesis. Our study also revealed a previously unidentified cohort of genes that are transcribed prior to the mid-blastula transition, a time point earlier than when the zygotic genome was traditionally thought to become active. Here we provide, for the first time to our knowledge, a comprehensive list of developmentally regulated zebrafish genes and their expression profiles during embryogenesis, including novel information on the temporal expression of several thousand previously uncharacterized genes. The expression data generated from this study are accessible to all interested scientists from our institute resource database (http://giscompute.gis.a-star.edu.sg/~govind/zebrafish/data_download.html). PMID:16132083

  5. Advances in allergen-microarray technology for diagnosis and monitoring of allergy: the MeDALL allergen-chip.

    PubMed

    Lupinek, Christian; Wollmann, Eva; Baar, Alexandra; Banerjee, Srinita; Breiteneder, Heimo; Broecker, Barbara M; Bublin, Merima; Curin, Mirela; Flicker, Sabine; Garmatiuk, Tetiana; Hochwallner, Heidrun; Mittermann, Irene; Pahr, Sandra; Resch, Yvonne; Roux, Kenneth H; Srinivasan, Bharani; Stentzel, Sebastian; Vrtala, Susanne; Willison, Leanna N; Wickman, Magnus; Lødrup-Carlsen, Karin C; Antó, Josep Maria; Bousquet, Jean; Bachert, Claus; Ebner, Daniel; Schlederer, Thomas; Harwanegg, Christian; Valenta, Rudolf

    2014-03-01

    Allergy diagnosis based on purified allergen molecules provides detailed information regarding the individual sensitization profile of allergic patients, allows monitoring of the development of allergic disease and of the effect of therapies on the immune response to individual allergen molecules. Allergen microarrays contain a large variety of allergen molecules and thus allow the simultaneous detection of allergic patients' antibody reactivity profiles towards each of the allergen molecules with only minute amounts of serum. In this article we summarize recent progress in the field of allergen microarray technology and introduce the MeDALL allergen-chip which has been developed for the specific and sensitive monitoring of IgE and IgG reactivity profiles towards more than 170 allergen molecules in sera collected in European birth cohorts. MeDALL is a European research program in which allergen microarray technology is used for the monitoring of the development of allergic disease in childhood, to draw a geographic map of the recognition of clinically relevant allergens in different populations and to establish reactivity profiles which are associated with and predict certain disease manifestations. We describe technical advances of the MeDALL allergen-chip regarding specificity, sensitivity and its ability to deliver test results which are close to in vivo reactivity. In addition, the usefulness and numerous advantages of allergen microarrays for allergy research, refined allergy diagnosis, monitoring of disease, of the effects of therapies, for improving the prescription of specific immunotherapy and for prevention are discussed.

  6. A New Way to Introduce Microarray Technology in a Lecture/Laboratory Setting by Studying the Evolution of This Modern Technology

    ERIC Educational Resources Information Center

    Rowland-Goldsmith, Melissa

    2009-01-01

    DNA microarray is an ordered grid containing known sequences of DNA, which represent many of the genes in a particular organism. Each DNA sequence is unique to a specific gene. This technology enables the researcher to screen many genes from cells or tissue grown in different conditions. We developed an undergraduate lecture and laboratory…

  7. Solitary Bone Plasmacytoma Progressing into Retroperitoneal Plasma Cell Myeloma with No Related End Organ or Tissue Impairment: A Case Report and Review of the Literature

    PubMed Central

    Tikku, Gargi; Jain, Monica; Mridha, Asit; Grover, Rajesh

    2014-01-01

    Solitary bone plasmacytomas and plasma cell myeloma are clonal proliferations of plasma cells. Many patients with solitary bone plasmacytomas develop plasma cell myeloma on follow-up. We present a case of a 70-year-old man who presented with fracture and a lytic lesion in the subtrochanteric region of the left femur and was assigned a diagnosis of solitary bone plasmacytoma. He received local curative radiotherapy. However, 4 months later his serum M protein and β2-microglobulin levels increased to 2.31 g/dL and 5.965 mg/L, respectively. He complained of abdominal fullness and constipation. Ultrasound and non-contrast CT imaging revealed multiple retroperitoneal masses. Colonoscopic examination was normal. Biopsy of the a retroperitoneal mass confirmed it to be a plasmacytoma. Repeat hemogram, blood urea, serum creatinine, skeletal survey, and bone marrow examination revealed no abnormalities. This is an unusual presentation of plasma cell myeloma, which manifested as multiple huge extramedullary retroperitoneal masses and arose from a solitary bone plasmacytoma, without related end organ or tissue impairment and bone marrow plasmacytosis. The patient succumbed to his disease 8 months after the appearance of the retroperitoneal masses. This case highlights the importance of close monitoring of patients diagnosed with solitary bone plasmacytoma with increased serum M protein and serum β2-microglobulin levels, so that early therapy can be instituted to prevent conversion to plasma cell myeloma. PMID:25330522

  8. Solitary bone plasmacytoma progressing into retroperitoneal plasma cell myeloma with no related end organ or tissue impairment: a case report and review of the literature.

    PubMed

    Tikku, Gargi; Jain, Monica; Mridha, Asit; Grover, Rajesh

    2014-09-05

    Solitary bone plasmacytomas and plasma cell myeloma are clonal proliferations of plasma cells. Many patients with solitary bone plasmacytomas develop plasma cell myeloma on follow-up. We present a case of a 70-year-old man who presented with fracture and a lytic lesion in the subtrochanteric region of the left femur and was assigned a diagnosis of solitary bone plasmacytoma. He received local curative radiotherapy. However, 4 months later his serum M protein and β2-microglobulin levels increased to 2.31 g/dL and 5.965 mg/L, respectively. He complained of abdominal fullness and constipation. Ultrasound and non-contrast CT imaging revealed multiple retroperitoneal masses. Colonoscopic examination was normal. Biopsy of the a retroperitoneal mass confirmed it to be a plasmacytoma. Repeat hemogram, blood urea, serum creatinine, skeletal survey, and bone marrow examination revealed no abnormalities. This is an unusual presentation of plasma cell myeloma, which manifested as multiple huge extramedullary retroperitoneal masses and arose from a solitary bone plasmacytoma, without related end organ or tissue impairment and bone marrow plasmacytosis. The patient succumbed to his disease 8 months after the appearance of the retroperitoneal masses. This case highlights the importance of close monitoring of patients diagnosed with solitary bone plasmacytoma with increased serum M protein and serum β2-microglobulin levels, so that early therapy can be instituted to prevent conversion to plasma cell myeloma.

  9. Evaluation of Hepatic Tissue Blood Flow Using Xenon Computed Tomography with Fibrosis Progression in Nonalcoholic Fatty Liver Disease: Comparison with Chronic Hepatitis C

    PubMed Central

    Shigefuku, Ryuta; Takahashi, Hideaki; Kato, Masaki; Yoshida, Yoshihito; Suetani, Keigo; Noguchi, Yohei; Hatsugai, Moriaki; Nakahara, Kazunari; Ikeda, Hiroki; Kobayashi, Minoru; Matsunaga, Kotaro; Matsumoto, Nobuyuki; Okuse, Chiaki; Itoh, Fumio; Maeyama, Shiro; Sase, Shigeru; Suzuki, Michihiro

    2014-01-01

    Aims The present study evaluated the utility of xenon computed tomography (Xe-CT) as a noninvasive diagnostic procedure for the measurement of hepatic tissue blood flow (TBF) in patients with nonalcoholic fatty liver disease (NAFLD) or chronic hepatitis C (CH-C). Methods Xe-CT was performed in 93 patients with NAFLD and in 109 patients with CH-C. Subjects were classified into one of three groups, based on fibrosis stage: group 1, no bridging fibrosis; group 2, bridging fibrosis; and group 3, liver cirrhosis. Correlations between hepatic TBFs in each fibrosis stage were examined. Results In group 1, portal venous TBF (PVTBF), hepatic arterial (HATBF), and total hepatic TBF (THTBF) were significantly lower in patients with in nonalcoholic steatohepatitis (NASH) than in those with CH-C (p < 0.001, p < 0.05, p < 0.001, respectively). In group 2, PVTBF and THTBF were significantly lower in patients with in NASH than in those with CH-C (p < 0.001, p < 0.05, respectively). In group 3, hepatic TBFs were not significantly different when comparing patients with NASH and those with CH-C. Conclusions PVTBF decreased due to fat infiltration. Therefore, hemodynamic changes occur relatively earlier in NAFLD than in CH-C. Patients with NASH should be monitored carefully for portal hypertensive complications in the early fibrosis stage. PMID:24424317

  10. Cell-Based Microarrays for In Vitro Toxicology.

    PubMed

    Wegener, Joachim

    2015-01-01

    DNA/RNA and protein