Quantitative PCR high-resolution melting (qPCR-HRM) curve analysis, a new approach to simultaneously screen point mutations and large rearrangements: application to MLH1 germline mutations in Lynch syndrome.

PubMed

Rouleau, Etienne; Lefol, Cédrick; Bourdon, Violaine; Coulet, Florence; Noguchi, Tetsuro; Soubrier, Florent; Bièche, Ivan; Olschwang, Sylviane; Sobol, Hagay; Lidereau, Rosette

2009-06-01

Several techniques have been developed to screen mismatch repair (MMR) genes for deleterious mutations. Until now, two different techniques were required to screen for both point mutations and large rearrangements. For the first time, we propose a new approach, called "quantitative PCR (qPCR) high-resolution melting (HRM) curve analysis (qPCR-HRM)," which combines qPCR and HRM to obtain a rapid and cost-effective method suitable for testing a large series of samples. We designed PCR amplicons to scan the MLH1 gene using qPCR HRM. Seventy-six patients were fully scanned in replicate, including 14 wild-type patients and 62 patients with known mutations (57 point mutations and five rearrangements). To validate the detected mutations, we used sequencing and/or hybridization on a dedicated MLH1 array-comparative genomic hybridization (array-CGH). All point mutations and rearrangements detected by denaturing high-performance liquid chromatography (dHPLC)+multiplex ligation-dependent probe amplification (MLPA) were successfully detected by qPCR HRM. Three large rearrangements were characterized with the dedicated MLH1 array-CGH. One variant was detected with qPCR HRM in a wild-type patient and was located within the reverse primer. One variant was not detected with qPCR HRM or with dHPLC due to its proximity to a T-stretch. With qPCR HRM, prescreening for point mutations and large rearrangements are performed in one tube and in one step with a single machine, without the need for any automated sequencer in the prescreening process. In replicate, its reagent cost, sensitivity, and specificity are comparable to those of dHPLC+MLPA techniques. However, qPCR HRM outperformed the other techniques in terms of its rapidity and amount of data provided.

Printing 2-dimentional droplet array for single-cell reverse transcription quantitative PCR assay with a microfluidic robot.

PubMed

Zhu, Ying; Zhang, Yun-Xia; Liu, Wen-Wen; Ma, Yan; Fang, Qun; Yao, Bo

2015-04-01

This paper describes a nanoliter droplet array-based single-cell reverse transcription quantitative PCR (RT-qPCR) assay method for quantifying gene expression in individual cells. By sequentially printing nanoliter-scale droplets on microchip using a microfluidic robot, all liquid-handling operations including cell encapsulation, lysis, reverse transcription, and quantitative PCR with real-time fluorescence detection, can be automatically achieved. The inhibition effect of cell suspension buffer on RT-PCR assay was comprehensively studied to achieve high-sensitivity gene quantification. The present system was applied in the quantitative measurement of expression level of mir-122 in single Huh-7 cells. A wide distribution of mir-122 expression in single cells from 3061 copies/cell to 79998 copies/cell was observed, showing a high level of cell heterogeneity. With the advantages of full-automation in liquid-handling, simple system structure, and flexibility in achieving multi-step operations, the present method provides a novel liquid-handling mode for single cell gene expression analysis, and has significant potentials in transcriptional identification and rare cell analysis.

Printing 2-Dimentional Droplet Array for Single-Cell Reverse Transcription Quantitative PCR Assay with a Microfluidic Robot

PubMed Central

Zhu, Ying; Zhang, Yun-Xia; Liu, Wen-Wen; Ma, Yan; Fang, Qun; Yao, Bo

2015-01-01

This paper describes a nanoliter droplet array-based single-cell reverse transcription quantitative PCR (RT-qPCR) assay method for quantifying gene expression in individual cells. By sequentially printing nanoliter-scale droplets on microchip using a microfluidic robot, all liquid-handling operations including cell encapsulation, lysis, reverse transcription, and quantitative PCR with real-time fluorescence detection, can be automatically achieved. The inhibition effect of cell suspension buffer on RT-PCR assay was comprehensively studied to achieve high-sensitivity gene quantification. The present system was applied in the quantitative measurement of expression level of mir-122 in single Huh-7 cells. A wide distribution of mir-122 expression in single cells from 3061 copies/cell to 79998 copies/cell was observed, showing a high level of cell heterogeneity. With the advantages of full-automation in liquid-handling, simple system structure, and flexibility in achieving multi-step operations, the present method provides a novel liquid-handling mode for single cell gene expression analysis, and has significant potentials in transcriptional identification and rare cell analysis. PMID:25828383

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

USDA-ARS?s Scientific Manuscript database

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

A novel miniaturized PCR multi-reactor array fabricated using flip-chip bonding techniques

NASA Astrophysics Data System (ADS)

Zou, Zhi-Qing; Chen, Xiang; Jin, Qing-Hui; Yang, Meng-Su; Zhao, Jian-Long

2005-08-01

This paper describes a novel miniaturized multi-chamber array capable of high throughput polymerase chain reaction (PCR). The structure of the proposed device is verified by using finite element analysis (FEA) to optimize the thermal performance, and then implemented on a glass-silicon substrate using a standard MEMS process and post-processing. Thermal analysis simulation and verification of each reactor cell is equipped with integrated Pt temperature sensors and heaters at the bottom of the reaction chamber for real-time accurate temperature sensing and control. The micro-chambers are thermally separated from each other, and can be controlled independently. The multi-chip array was packaged on a printed circuit board (PCB) substrate using a conductive polymer flip-chip bonding technique, which enables effective heat dissipation and suppresses thermal crosstalk between the chambers. The designed system has successfully demonstrated a temperature fluctuation of ±0.5 °C during thermal multiplexing of up to 2 × 2 chambers, a full speed of 30 min for 30 cycle PCR, as well as the capability of controlling each chamber digitally and independently.

Customization and design of directed self-assembly using hybrid prepatterns

NASA Astrophysics Data System (ADS)

Cheng, Joy; Doerk, Gregory S.; Rettner, Charles T.; Singh, Gurpreet; Tjio, Melia; Truong, Hoa; Arellano, Noel; Balakrishnan, Srinivasan; Brink, Markus; Tsai, Hsinyu; Liu, Chi-Chun; Guillorn, Michael; Sanders, Daniel P.

2015-03-01

Diminishing error tolerance renders the customization of patterns created through directed self-assembly (DSA) extremely challenging at tighter pitch. A self-aligned customization scheme can be achieved using a hybrid prepattern comprising both organic and inorganic regions that serves as a guiding prepattern to direct the self-assembly of the block copolymers as well as a cut mask pattern for the DSA arrays aligned to it. In this paper, chemoepitaxy-based self-aligned customization is demonstrated using two types of organic-inorganic prepatterns. CHEETAH prepattern for "CHemoepitaxy Etch Trim using a self-Aligned Hardmask" of preferential hydrogen silsesquioxane (HSQ, inorganic resist), non-preferential organic underlayer is fabricated using electron beam lithography. Customized trench or hole arrays can be achieved through co-transfer of DSA-formed arrays and CHEETAH prepattern. Herein, we also introduce a tone-reversed version called reverse-CHEETAH (or rCHEETAH) in which customized line segments can be achieved through co-transfer of DSA-formed arrays formed on a prepattern wherein the inorganic HSQ regions are nonpreferential and the organic regions are PMMA preferential. Examples of two-dimensional self-aligned customization including 25nm pitch fin structures and an 8-bar "IBM" illustrate the versatility of this customization scheme using rCHEETAH.

Large-Scale Interaction Profiling of Protein Domains Through Proteomic Peptide-Phage Display Using Custom Peptidomes.

PubMed

Seo, Moon-Hyeong; Nim, Satra; Jeon, Jouhyun; Kim, Philip M

2017-01-01

Protein-protein interactions are essential to cellular functions and signaling pathways. We recently combined bioinformatics and custom oligonucleotide arrays to construct custom-made peptide-phage libraries for screening peptide-protein interactions, an approach we call proteomic peptide-phage display (ProP-PD). In this chapter, we describe protocols for phage display for the identification of natural peptide binders for a given protein. We finally describe deep sequencing for the analysis of the proteomic peptide-phage display.

Low-Latency Embedded Vision Processor (LLEVS)

DTIC Science & Technology

2016-03-01

26 3.2.3 Task 3 Projected Performance Analysis of FPGA- based Vision Processor ........... 31 3.2.3.1 Algorithms Latency Analysis ...Programmable Gate Array Custom Hardware for Real- Time Multiresolution Analysis . ............................................... 35...conduct data analysis for performance projections. The data acquired through measurements , simulation and estimation provide the requisite platform for

A novel lab-on-chip platform with integrated solid phase PCR and Supercritical Angle Fluorescence (SAF) microlens array for highly sensitive and multiplexed pathogen detection.

PubMed

Hung, Tran Quang; Chin, Wai Hoe; Sun, Yi; Wolff, Anders; Bang, Dang Duong

2017-04-15

Solid-phase PCR (SP-PCR) has become increasingly popular for molecular diagnosis and there have been a few attempts to incorporate SP-PCR into lab-on-a-chip (LOC) devices. However, their applicability for on-line diagnosis is hindered by the lack of sensitive and portable on-chip optical detection technology. In this paper, we addressed this challenge by combining the SP-PCR with super critical angle fluorescence (SAF) microlens array embedded in a microchip. We fabricated miniaturized SAF microlens array as part of a microfluidic chamber in thermoplastic material and performed multiplexed SP-PCR directly on top of the SAF microlens array. Attribute to the high fluorescence collection efficiency of the SAF microlens array, the SP-PCR assay on the LOC platform demonstrated a high sensitivity of 1.6 copies/µL, comparable to off-chip detection using conventional laser scanner. The combination of SP-PCR and SAF microlens array allows for on-chip highly sensitive and multiplexed pathogen detection with low-cost and compact optical components. The LOC platform would be widely used as a high-throughput biosensor to analyze food, clinical and environmental samples. Copyright © 2016 Elsevier B.V. All rights reserved.

[Phenotypic and genetic analysis of a patient presented with Tietz/Waardenburg type II a syndrome].

PubMed

Wang, Huanhuan; Tang, Lifang; Zhang, Jingmin; Hu, Qin; Chen, Yingwei; Xiao, Bing

2015-08-01

To determine the genetic cause for a patient featuring decreased pigmentation of the skin and iris, hearing loss and multiple congenital anomalies. Routine chromosomal banding was performed to analyze the karyotype of the patient and his parents. Single nucleotide polymorphism array (SNP array) was employed to identify cryptic chromosome aberrations, and quantitative real-time PCR was used to confirm the results. Karyotype analysis has revealed no obvious anomaly for the patient and his parents. SNP array analysis of the patient has demonstrated a 3.9 Mb deletion encompassing 3p13p14.1, which caused loss of entire MITF gene. The deletion was confirmed by quantitative real-time PCR. Clinical features of the patient have included severe bilateral hearing loss, decreased pigmentation of the skin and iris and multiple congenital anomalies. The patient, carrying a 3p13p14.1 deletion, has features of Tietz syndrome/Waardenburg syndrome type IIa. This case may provide additional data for the study of genotype-phenotype correlation of this disease.

A Novel High-Throughput Method for Molecular Detection of Human Pathogenic Viruses Using a Nanofluidic Real-Time PCR System

PubMed Central

Coudray-Meunier, Coralie; Fraisse, Audrey; Martin-Latil, Sandra; Delannoy, Sabine; Fach, Patrick; Perelle, Sylvie

2016-01-01

Human enteric viruses are recognized as the main causes of food- and waterborne diseases worldwide. Sensitive and quantitative detection of human enteric viruses is typically achieved through quantitative RT-PCR (RT-qPCR). A nanofluidic real-time PCR system was used to develop novel high-throughput methods for qualitative molecular detection (RT-qPCR array) and quantification of human pathogenic viruses by digital RT-PCR (RT-dPCR). The performance of high-throughput PCR methods was investigated for detecting 19 human pathogenic viruses and two main process controls used in food virology. The conventional real-time PCR system was compared to the RT-dPCR and RT-qPCR array. Based on the number of genome copies calculated by spectrophotometry, sensitivity was found to be slightly better with RT-qPCR than with RT-dPCR for 14 viruses by a factor range of from 0.3 to 1.6 log10. Conversely, sensitivity was better with RT-dPCR than with RT-qPCR for seven viruses by a factor range of from 0.10 to 1.40 log10. Interestingly, the number of genome copies determined by RT-dPCR was always from 1 to 2 log10 lower than the expected copy number calculated by RT-qPCR standard curve. The sensitivity of the RT-qPCR and RT-qPCR array assays was found to be similar for two viruses, and better with RT-qPCR than with RT-qPCR array for eighteen viruses by a factor range of from 0.7 to 3.0 log10. Conversely, sensitivity was only 0.30 log10 better with the RT-qPCR array than with conventional RT-qPCR assays for norovirus GIV detection. Finally, the RT-qPCR array and RT-dPCR assays were successfully used together to screen clinical samples and quantify pathogenic viruses. Additionally, this method made it possible to identify co-infection in clinical samples. In conclusion, given the rapidity and potential for large numbers of viral targets, this nanofluidic RT-qPCR assay should have a major impact on human pathogenic virus surveillance and outbreak investigations and is likely to be of benefit to public health. PMID:26824897

37 CFR 211.4 - Registration of claims of protection in mask works.

Code of Federal Regulations, 2010 CFR

2010-07-01

... adding metal-connection layers to unpersonalized gate arrays may separately register the entire unpersonalized gate array and the custom metallization layers. Applicants seeking to register separately entire unpersonalized gate arrays or custom metallization layers should make the nature of their claim clear at Space 8...

Implementation of Quality Management in Core Service Laboratories

PubMed Central

Creavalle, T.; Haque, K.; Raley, C.; Subleski, M.; Smith, M.W.; Hicks, B.

2010-01-01

CF-28 The Genetics and Genomics group of the Advanced Technology Program of SAIC-Frederick exists to bring innovative genomic expertise, tools and analysis to NCI and the scientific community. The Sequencing Facility (SF) provides next generation short read (Illumina) sequencing capacity to investigators using a streamlined production approach. The Laboratory of Molecular Technology (LMT) offers a wide range of genomics core services including microarray expression analysis, miRNA analysis, array comparative genome hybridization, long read (Roche) next generation sequencing, quantitative real time PCR, transgenic genotyping, Sanger sequencing, and clinical mutation detection services to investigators from across the NIH. As the technology supporting this genomic research becomes more complex, the need for basic quality processes within all aspects of the core service groups becomes critical. The Quality Management group works alongside members of these labs to establish or improve processes supporting operations control (equipment, reagent and materials management), process improvement (reengineering/optimization, automation, acceptance criteria for new technologies and tech transfer), and quality assurance and customer support (controlled documentation/SOPs, training, service deficiencies and continual improvement efforts). Implementation and expansion of quality programs within unregulated environments demonstrates SAIC-Frederick's dedication to providing the highest quality products and services to the NIH community.

Characterization of neuroendocrine tumors of the pancreas by real-time quantitative polymerase chain reaction. A methodological approach.

PubMed

Annaratone, Laura; Volante, Marco; Asioli, Sofia; Rangel, Nelson; Bussolati, Gianni

2013-06-01

The aim of this study was to assess the suitability of using real-time quantitative PCR (RT-qPCR) to characterize neuroendocrine (NE) tumors of the pancreas. For a series of tumors, we evaluated several genes of interest, and the data were matched with the "classical" immunohistochemical (IHC) features. In 21 cases, we extracted RNA from formalin-fixed paraffin-embedded (FFPE) blocks, and in nine cases, we also extracted RNA from fresh-frozen tissue. The RT-qPCR procedure was performed using two sets of customized arrays. The test using the first set, covering 96 genes of interest, was focused on assessing the feasibility of the procedure, and the results were used to select 18 genes indicative of NE differentiation, clinical behavior, and therapeutic responsiveness for use in the second set of arrays. Threshold cycle (Ct) values were used to calculate the fold-changes in gene expression using the 2-∆∆Ct method. Statistical procedures were used to analyze the results, which were matched with the IHC and follow-up data. Material from fresh-frozen samples performed better in terms of the level of amplification, but acceptable and concordant results were also obtained from FFPE samples. In addition, high concordance was observed between the mRNA and protein expression levels of somatostatin receptor type 2A (R = 0.52, p = 0.016). Genes associated with NE differentiation, as well as the gastrin-releasing peptide receptor and O-6-methylguanine-DNA methyltransferase genes, were underexpressed, whereas angiogenesis-associated markers (CDH13 and SLIT2) were overexpressed in tissues with malignant behavior. The RT-qPCR procedure is practical and feasible in economic terms for the characterization of NE tumors of the pancreas and can complement morphological and IHC-based evaluations. Thus, the results of the RT-qPCR procedure might offer an objective basis for therapeutic choices.

A novel genotoxin-specific qPCR array based on the metabolically competent human HepaRG™ cell line as a rapid and reliable tool for improved in vitro hazard assessment.

PubMed

Ates, Gamze; Mertens, Birgit; Heymans, Anja; Verschaeve, Luc; Milushev, Dimiter; Vanparys, Philippe; Roosens, Nancy H C; De Keersmaecker, Sigrid C J; Rogiers, Vera; Doktorova, Tatyana Y

2018-04-01

Although the value of the regulatory accepted batteries for in vitro genotoxicity testing is recognized, they result in a high number of false positives. This has a major impact on society and industries developing novel compounds for pharmaceutical, chemical, and consumer products, as afflicted compounds have to be (prematurely) abandoned or further tested on animals. Using the metabolically competent human HepaRG ™ cell line and toxicogenomics approaches, we have developed an upgraded, innovative, and proprietary gene classifier. This gene classifier is based on transcriptomic changes induced by 12 genotoxic and 12 non-genotoxic reference compounds tested at sub-cytotoxic concentrations, i.e., IC10 concentrations as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The resulting gene classifier was translated into an easy-to-handle qPCR array that, as shown by pathway analysis, covers several different cellular processes related to genotoxicity. To further assess the predictivity of the tool, a set of 5 known positive and 5 known negative test compounds for genotoxicity was evaluated. In addition, 2 compounds with debatable genotoxicity data were tested to explore how the qPCR array would classify these. With an accuracy of 100%, when equivocal results were considered positive, the results showed that combining HepaRG ™ cells with a genotoxin-specific qPCR array can improve (geno)toxicological hazard assessment. In addition, the developed qPCR array was able to provide additional information on compounds for which so far debatable genotoxicity data are available. The results indicate that the new in vitro tool can improve human safety assessment of chemicals in general by basing predictions on mechanistic toxicogenomics information.

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

PubMed Central

2013-01-01

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

Mus musculus-microRNA-449a ameliorates neuropathic pain by decreasing the level of KCNMA1 and TRPA1, and increasing the level of TPTE.

PubMed

Lu, Shan; Ma, Sichao; Wang, Yunyun; Huang, Tao; Zhu, Zhihua; Zhao, Guoqing

2017-07-01

Neuropathic pain is a nerve disorder characterized by the dysregulation of ion channels in dorsal root ganglion (DRG) neurons. MicroRNAs (miRs) may be associated with the molecular mechanisms underlying the altered levels of ion channels; however, the molecular mechanisms remain widely unknown. To investigate these mechanisms, the present study conducted a genomic analysis of miR between a unilateral spared nerve injury (SNI) model and sham control. Differentially expressed miRs between the SNI and sham groups were selected for transfection of DRG cells, a polymerase chain reaction (PCR) array analysis was subsequently performed. A total of three significantly differently expressed genes were selected from the results of the PCR array and further analyzed by reverse transcription‑quantitative PCR. Genomic analysis revealed that Mus musculus miR‑449a (mmu‑miR‑449a) was reduced in the SNI groups compared with the sham controls. The PCR array indicated that mmu‑miR‑449a‑transfection reduced the mRNA expression levels of transient receptor potential cation channel subfamily A member 1 (TRPA1), and calcium‑activated potassium channel subunit α‑1 (KCNMA1) and increased the level of transmembrane phosphatase with tension homology (TPTE) in the DRG cells (P<0.05). qRT‑PCR analysis further indicated that mmu‑miR‑449a transfection caused similar alterations in the mRNA expression levels of TRPA1, KCNMA1 and TPTE in DRG cells, respectively (P<0.05). Therefore, mmu‑miR‑449a may ameliorate neuropathic pain by decreasing the activity of the channel proteins TRPA1 and KCNMA1 and increasing the levels of TPTE. mmu‑miR‑449a may be a potential therapeutic molecule for the alleviation of neuropathic pain.

Demonstration of diet-induced decoupling of fatty acid and cholesterol synthesis by combining gene expression array and 2H2O quantification.

PubMed

Jensen, Kristian K; Previs, Stephen F; Zhu, Lei; Herath, Kithsiri; Wang, Sheng-Ping; Bhat, Gowri; Hu, Guanghui; Miller, Paul L; McLaren, David G; Shin, Myung K; Vogt, Thomas F; Wang, Liangsu; Wong, Kenny K; Roddy, Thomas P; Johns, Douglas G; Hubbard, Brian K

2012-01-15

The liver is a crossroad for metabolism of lipid and carbohydrates, with acetyl-CoA serving as an important metabolic intermediate and a precursor for fatty acid and cholesterol biosynthesis pathways. A better understanding of the regulation of these pathways requires an experimental approach that provides both quantitative metabolic flux measurements and mechanistic insight. Under conditions of high carbohydrate availability, excess carbon is converted into free fatty acids and triglyceride for storage, but it is not clear how excessive carbohydrate availability affects cholesterol biosynthesis. To address this, C57BL/6J mice were fed either a low-fat, high-carbohydrate diet or a high-fat, carbohydrate-free diet. At the end of the dietary intervention, the two groups received (2)H(2)O to trace de novo fatty acid and cholesterol synthesis, and livers were collected for gene expression analysis. Expression of lipid and glucose metabolism genes was determined using a custom-designed pathway focused PCR-based gene expression array. The expression analysis showed downregulation of cholesterol biosynthesis genes and upregulation of fatty acid synthesis genes in mice receiving the high-carbohydrate diet compared with the carbohydrate-free diet. In support of these findings, (2)H(2)O tracer data showed that fatty acid synthesis was increased 10-fold and cholesterol synthesis was reduced by 1.6-fold in mice fed the respective diets. In conclusion, by applying gene expression analysis and tracer methodology, we show that fatty acid and cholesterol synthesis are differentially regulated when the carbohydrate intake in mice is altered.

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

PubMed Central

Zhu, Yuerong; Zhu, Yuelin; Xu, Wei

2008-01-01

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

Comprehensive GMO detection using real-time PCR array: single-laboratory validation.

PubMed

Mano, Junichi; Harada, Mioko; Takabatake, Reona; Furui, Satoshi; Kitta, Kazumi; Nakamura, Kosuke; Akiyama, Hiroshi; Teshima, Reiko; Noritake, Hiromichi; Hatano, Shuko; Futo, Satoshi; Minegishi, Yasutaka; Iizuka, Tayoshi

2012-01-01

We have developed a real-time PCR array method to comprehensively detect genetically modified (GM) organisms. In the method, genomic DNA extracted from an agricultural product is analyzed using various qualitative real-time PCR assays on a 96-well PCR plate, targeting for individual GM events, recombinant DNA (r-DNA) segments, taxon-specific DNAs, and donor organisms of the respective r-DNAs. In this article, we report the single-laboratory validation of both DNA extraction methods and component PCR assays constituting the real-time PCR array. We selected some DNA extraction methods for specified plant matrixes, i.e., maize flour, soybean flour, and ground canola seeds, then evaluated the DNA quantity, DNA fragmentation, and PCR inhibition of the resultant DNA extracts. For the component PCR assays, we evaluated the specificity and LOD. All DNA extraction methods and component PCR assays satisfied the criteria set on the basis of previous reports.

The database of chromosome imbalance regions and genes resided in lung cancer from Asian and Caucasian identified by array-comparative genomic hybridization

PubMed Central

2012-01-01

Background Cancer-related genes show racial differences. Therefore, identification and characterization of DNA copy number alteration regions in different racial groups helps to dissect the mechanism of tumorigenesis. Methods Array-comparative genomic hybridization (array-CGH) was analyzed for DNA copy number profile in 40 Asian and 20 Caucasian lung cancer patients. Three methods including MetaCore analysis for disease and pathway correlations, concordance analysis between array-CGH database and the expression array database, and literature search for copy number variation genes were performed to select novel lung cancer candidate genes. Four candidate oncogenes were validated for DNA copy number and mRNA and protein expression by quantitative polymerase chain reaction (qPCR), chromogenic in situ hybridization (CISH), reverse transcriptase-qPCR (RT-qPCR), and immunohistochemistry (IHC) in more patients. Results We identified 20 chromosomal imbalance regions harboring 459 genes for Caucasian and 17 regions containing 476 genes for Asian lung cancer patients. Seven common chromosomal imbalance regions harboring 117 genes, included gain on 3p13-14, 6p22.1, 9q21.13, 13q14.1, and 17p13.3; and loss on 3p22.2-22.3 and 13q13.3 were found both in Asian and Caucasian patients. Gene validation for four genes including ARHGAP19 (10q24.1) functioning in Rho activity control, FRAT2 (10q24.1) involved in Wnt signaling, PAFAH1B1 (17p13.3) functioning in motility control, and ZNF322A (6p22.1) involved in MAPK signaling was performed using qPCR and RT-qPCR. Mean gene dosage and mRNA expression level of the four candidate genes in tumor tissues were significantly higher than the corresponding normal tissues (P<0.001~P=0.06). In addition, CISH analysis of patients indicated that copy number amplification indeed occurred for ARHGAP19 and ZNF322A genes in lung cancer patients. IHC analysis of paraffin blocks from Asian Caucasian patients demonstrated that the frequency of PAFAH1B1 protein overexpression was 68% in Asian and 70% in Caucasian. Conclusions Our study provides an invaluable database revealing common and differential imbalance regions at specific chromosomes among Asian and Caucasian lung cancer patients. Four validation methods confirmed our database, which would help in further studies on the mechanism of lung tumorigenesis. PMID:22691236

Rapid identification of a novel complex I MT-ND3 m.10134C>A mutation in a Leigh syndrome patient.

PubMed

Miller, David K; Menezes, Minal J; Simons, Cas; Riley, Lisa G; Cooper, Sandra T; Grimmond, Sean M; Thorburn, David R; Christodoulou, John; Taft, Ryan J

2014-01-01

Leigh syndrome (LS) is a rare progressive multi-system neurodegenerative disorder, the genetics of which is frequently difficult to resolve. Rapid determination of the genetic etiology of LS in a 5-year-old girl facilitated inclusion in Edison Pharmaceutical's phase 2B clinical trial of EPI-743. SNP-arrays and high-coverage whole exome sequencing were performed on the proband, both parents and three unaffected siblings. Subsequent multi-tissue targeted high-depth mitochondrial sequencing was performed using custom long-range PCR amplicons. Tissue-specific mutant load was also assessed by qPCR. Complex I was interrogated by spectrophotometric enzyme assays and Western Blot. No putatively causal mutations were identified in nuclear-encoded genes. Analysis of low-coverage off-target mitochondrial reads revealed a previously unreported mitochondrial mutation in the proband in MT-ND3 (m.10134C>A, p.Q26K), a Complex I mitochondrial gene previously associated with LS. Targeted investigations demonstrated that this mutation was 1% heteroplasmic in the mother's blood and homoplasmic in the proband's blood, fibroblasts, liver and muscle. Enzyme assays revealed decreased Complex I activity. The identification of this novel LS MT-ND3 variant, the genomics of which was accomplished in less than 3.5 weeks, indicates that rapid genomic approaches may prove useful in time-sensitive cases with an unresolved genetic diagnosis.

Comparison of FilmArray and Quantitative Real-Time Reverse Transcriptase PCR for Detection of Zaire Ebolavirus from Contrived and Clinical Specimens

PubMed Central

Southern, Timothy R.; Racsa, Lori D.; Albariño, César G.; Fey, Paul D.; Hinrichs, Steven H.; Murphy, Caitlin N.; Herrera, Vicki L.; Sambol, Anthony R.; Hill, Charles E.; Ryan, Emily L.; Kraft, Colleen S.; Campbell, Shelley; Sealy, Tara K.; Schuh, Amy; Ritchie, James C.; Lyon, G. Marshall; Mehta, Aneesh K.; Varkey, Jay B.; Ribner, Bruce S.; Brantly, Kent P.; Ströher, Ute; Iwen, Peter C.

2015-01-01

Rapid, reliable, and easy-to-use diagnostic assays for detection of Zaire ebolavirus (ZEBOV) are urgently needed. The goal of this study was to examine the agreement among emergency use authorization (EUA) tests for the detection of ZEBOV nucleic acids, including the BioFire FilmArray BioThreat (BT) panel, the FilmArray BT-E panel, and the NP2 and VP40 quantitative real-time reverse transcriptase (qRT) PCR assays from the Centers for Disease Control and Prevention (CDC). Specimens used in this study included whole blood spiked with inactivated ZEBOV at known titers and whole-blood, plasma, and urine clinical specimens collected from persons diagnosed with Ebola virus disease (EVD). The agreement for FilmArray and qRT-PCR results using contrived whole-blood specimens was 100% (6/6 specimens) for each ZEBOV dilution from 4 × 107 to 4 × 102 50% tissue culture infective dose (TCID50)/ml, as well as the no-virus negative-control sample. The limit of detection for FilmArray and qRT-PCR assays with inactivated ZEBOV, based on duplicate positive results, was determined to be 4 × 102 TCID50/ml. Rates of agreement between FilmArray and qRT-PCR results for clinical specimens from patients with EVD were 85% (23/27 specimens) for whole-blood specimens, 90% (18/20 specimens) for whole-blood specimens tested by FilmArray testing and matched plasma specimens tested by qRT-PCR testing, and 85% (11/13 specimens) for urine specimens. Among 60 specimens, eight discordant results were noted, with ZEBOV nucleic acids being detected only by FilmArray testing in four specimens and only by qRT-PCR testing in the remaining four specimens. These findings demonstrate that the rapid and easy-to-use FilmArray panels are effective tests for evaluating patients with EVD. PMID:26157148

Customized versus Patient-Sized Cruciate-Retaining Total Knee Arthroplasty: An In Vivo Kinematics Study Using Mobile Fluoroscopy.

PubMed

Zeller, Ian M; Sharma, Adrija; Kurtz, William B; Anderle, Mathew R; Komistek, Richard D

2017-04-01

Historically, knee arthroplasties have been designed using average patient anatomy. Recent advances in imaging and manufacturing have facilitated the development of customized prostheses designed to fit the unique shape of individual patients. The purpose of this study is to determine if improving implant design through customized total knee arthroplasty (TKA) improves kinematic function. Using state-of-the-art mobile fluoroscopy, tibiofemoral kinematics were analyzed for 24 subjects with a customized individually made (CIM), cruciate-retaining TKA, and 14 subjects having an asymmetric condylar cruciate-retaining TKA. Subjects performed a weight-bearing deep knee bend and a rise from a seated position. Each patient was evaluated for weight-bearing range of motion, femorotibial translation, femorotibial axial rotation, and condylar liftoff occurrence. Subjects having a CIM TKA experienced greater weight-bearing knee flexion compared with the traditional posterior cruciate-retaining (PCR) TKA design. During flexion, the CIM TKA subjects consistently exhibited more posterior femoral rollback than the traditional PCR TKA subjects. The CIM TKA was found to have statistically greater axial rotation compared with the traditional PCR TKA (P = .05). Of note, only the CIM TKA patients experienced femoral internal rotation at full extension, as exhibited in a normal knee. Compared with the traditional PCR TKA, the CIM TKAs demonstrated minimal occurrences of paradoxical sliding and reverse rotation during flexion and extension. The CIM TKA subjects showed minimal liftoff and hence better stability in earlyflexion to midflexion compared with the traditional PCR subjects. The CIM TKA demonstrated kinematics more similar to a normal knee. Therefore, using customized implant technology through CIM TKA designs affords benefits including more normal motion compared with a traditional PCR TKA. Copyright © 2016 Elsevier Inc. All rights reserved.

Customizing microarrays for neuroscience drug discovery.

PubMed

Girgenti, Matthew J; Newton, Samuel S

2007-08-01

Microarray-based gene profiling has become the centerpiece of gene expression studies in the biological sciences. The ability to now interrogate the entire genome using a single chip demonstrates the progress in technology and instrumentation that has been made over the last two decades. Although this unbiased approach provides researchers with an immense quantity of data, obtaining meaningful insight is not possible without intensive data analysis and processing. Custom developed arrays have emerged as a viable and attractive alternative that can take advantage of this robust technology and tailor it to suit the needs and requirements of individual investigations. The ability to simplify data analysis, reduce noise and carefully optimize experimental conditions makes it a suitable tool that can be effectively utilized in neuroscience drug discovery efforts. Furthermore, incorporating recent advancements in fine focusing gene profiling to include specific cellular phenotypes can help resolve the complex cellular heterogeneity of the brain. This review surveys the use of microarray technology in neuroscience paying special attention to customized arrays and their potential in drug discovery. Novel applications of microarrays and ancillary techniques, such as laser microdissection, FAC sorting and RNA amplification, have also been discussed. The notion that a hypothesis-driven approach can be integrated into drug development programs is highlighted.

Microplate array diagonal gel electrophoresis (MADGE), CpG-PCR and temporal thermal ramp-MADGE (Melt-MADGE) for single nucleotide analyses in populations.

PubMed

Day, I N; O'Dell, S D; Spanakis, E; Weavind, G P

1999-02-01

Important requirements for molecular genetic epidemiological studies are economy, sample parallelism, convenience of setup and accessibility, goals inadequately met by existent approaches. We invented microplate array diagonal gel electrophoresis (MADGE) to gain simultaneously the advantages of simple setup, 96-well microplate compatibility, horizontal electrophoresis, and the resolution of polyacrylamide. At essentially no equipment cost (one simple plastic gel former), 10-100-fold savings on time for sample coding, liquid transfers, and data documentation, in addition to volume reductions and gel re-use, can be achieved. MADGE is compatible with ARMS, restriction analysis and other pattern analyses. CpG-PCR is a general PCR approach to CpG sites (10-20% of all human single base variation): both primers have 3' T, and are abutted to the CpG, forcing a TaqI restriction site if the CpG is intact. Typically, a 52 bp PCR product is then cut in half. CpG-PCR also illustrates that PAGE-MADGE readily permits analysis of 'ultrashort' PCRs. Melt-MADGE employs real-time-variable-temperature electrophoresis to examine duplex mobility during melting, achieving DGGE-like de novo, mutation scanning, but with the conveniences of arbitrary programmability, MADGE compatibility and short run time. This suite of methods enhances our capability to type or scan thousands of samples simultaneously, by 10-100-fold.

Comparison of the Idaho Technology FilmArray System to Real-Time PCR for Detection of Respiratory Pathogens in Children

PubMed Central

Pierce, Virginia M.; Elkan, Michael; Leet, Marilyn; McGowan, Karin L.

2012-01-01

The FilmArray Respiratory Panel (RP) multiplexed nucleic acid amplification test (Idaho Technology, Inc., Salt Lake City, UT) was compared to laboratory-developed real-time PCR assays for the detection of various respiratory viruses and certain bacterial pathogens. A total of 215 frozen archived pediatric respiratory specimens previously characterized as either negative or positive for one or more pathogens by real-time PCR were examined using the FilmArray RP system. Overall agreement between the FilmArray RP and corresponding real-time PCR assays for shared analytes was 98.6% (kappa = 0.92 [95% confidence interval (CI), 0.89 to 0.94]). The combined positive percent agreement was 89.4% (95% CI, 85.4 to 92.6); the negative percent agreement was 99.6% (95% CI, 99.2 to 99.8). The mean real-time PCR threshold cycle (CT) value for specimens with discordant results was 36.46 ± 4.54. Detection of coinfections and correct identification of influenza A virus subtypes were comparable to those of real-time PCR when using the FilmArray RP. The greatest comparative difference in sensitivity was observed for adenovirus; only 11 of 24 (45.8%; 95% CI, 27.9 to 64.9) clinical specimens positive for adenovirus by real-time PCR were also positive by the FilmArray RP. In addition, upon testing 20 characterized adenovirus serotypes prepared at high and low viral loads, the FilmArray RP did not detect serotypes 6 and 41 at either level and failed to detect serotypes 2, 20, 35, and 37 when viral loads were low. The FilmArray RP system is rapid and extremely user-friendly, with results available in just over 1 h with almost no labor involved. Its low throughput is a significant drawback for laboratories receiving large numbers of specimens, as only a single sample can be processed at a time with one instrument. PMID:22116144

Performance Analysis of a NASA Integrated Network Array

NASA Technical Reports Server (NTRS)

Nessel, James A.

2012-01-01

The Space Communications and Navigation (SCaN) Program is planning to integrate its individual networks into a unified network which will function as a single entity to provide services to user missions. This integrated network architecture is expected to provide SCaN customers with the capabilities to seamlessly use any of the available SCaN assets to support their missions to efficiently meet the collective needs of Agency missions. One potential optimal application of these assets, based on this envisioned architecture, is that of arraying across existing networks to significantly enhance data rates and/or link availabilities. As such, this document provides an analysis of the transmit and receive performance of a proposed SCaN inter-network antenna array. From the study, it is determined that a fully integrated internetwork array does not provide any significant advantage over an intra-network array, one in which the assets of an individual network are arrayed for enhanced performance. Therefore, it is the recommendation of this study that NASA proceed with an arraying concept, with a fundamental focus on a network-centric arraying.

Differential expression of THOC1 and ALY mRNP biogenesis/export factors in human cancers.

PubMed

Domínguez-Sánchez, María S; Sáez, Carmen; Japón, Miguel A; Aguilera, Andrés; Luna, Rosa

2011-02-17

One key step in gene expression is the biogenesis of mRNA ribonucleoparticle complexes (mRNPs). Formation of the mRNP requires the participation of a number of conserved factors such as the THO complex. THO interacts physically and functionally with the Sub2/UAP56 RNA-dependent ATPase, and the Yra1/REF1/ALY RNA-binding protein linking transcription, mRNA export and genome integrity. Given the link between genome instability and cancer, we have performed a comparative analysis of the expression patterns of THOC1, a THO complex subunit, and ALY in tumor samples. The mRNA levels were measured by quantitative real-time PCR and hybridization of a tumor tissue cDNA array; and the protein levels and distribution by immunostaining of a custom tissue array containing a set of paraffin-embedded samples of different tumor and normal tissues followed by statistical analysis. We show that the expression of two mRNP factors, THOC1 and ALY are altered in several tumor tissues. THOC1 mRNA and protein levels are up-regulated in ovarian and lung tumors and down-regulated in those of testis and skin, whereas ALY is altered in a wide variety of tumors. In contrast to THOC1, ALY protein is highly detected in normal proliferative cells, but poorly in high-grade cancers. These results suggest a differential connection between tumorogenesis and the expression levels of human THO and ALY. This study opens the possibility of defining mRNP biogenesis factors as putative players in cell proliferation that could contribute to tumor development.

Gene expression profile of mouse prostate tumors reveals dysregulations in major biological processes and identifies potential murine targets for preclinical development of human prostate cancer therapy.

PubMed

Haram, Kerstyn M; Peltier, Heidi J; Lu, Bin; Bhasin, Manoj; Otu, Hasan H; Choy, Bob; Regan, Meredith; Libermann, Towia A; Latham, Gary J; Sanda, Martin G; Arredouani, Mohamed S

2008-10-01

Translation of preclinical studies into effective human cancer therapy is hampered by the lack of defined molecular expression patterns in mouse models that correspond to the human counterpart. We sought to generate an open source TRAMP mouse microarray dataset and to use this array to identify differentially expressed genes from human prostate cancer (PCa) that have concordant expression in TRAMP tumors, and thereby represent lead targets for preclinical therapy development. We performed microarrays on total RNA extracted and amplified from eight TRAMP tumors and nine normal prostates. A subset of differentially expressed genes was validated by QRT-PCR. Differentially expressed TRAMP genes were analyzed for concordant expression in publicly available human prostate array datasets and a subset of resulting genes was analyzed by QRT-PCR. Cross-referencing differentially expressed TRAMP genes to public human prostate array datasets revealed 66 genes with concordant expression in mouse and human PCa; 56 between metastases and normal and 10 between primary tumor and normal tissues. Of these 10 genes, two, Sox4 and Tubb2a, were validated by QRT-PCR. Our analysis also revealed various dysregulations in major biologic pathways in the TRAMP prostates. We report a TRAMP microarray dataset of which a gene subset was validated by QRT-PCR with expression patterns consistent with previous gene-specific TRAMP studies. Concordance analysis between TRAMP and human PCa associated genes supports the utility of the model and suggests several novel molecular targets for preclinical therapy.

Custom oligonucleotide array-based CGH: a reliable diagnostic tool for detection of exonic copy-number changes in multiple targeted genes

PubMed Central

Vasson, Aurélie; Leroux, Céline; Orhant, Lucie; Boimard, Mathieu; Toussaint, Aurélie; Leroy, Chrystel; Commere, Virginie; Ghiotti, Tiffany; Deburgrave, Nathalie; Saillour, Yoann; Atlan, Isabelle; Fouveaut, Corinne; Beldjord, Cherif; Valleix, Sophie; Leturcq, France; Dodé, Catherine; Bienvenu, Thierry; Chelly, Jamel; Cossée, Mireille

2013-01-01

The frequency of disease-related large rearrangements (referred to as copy-number mutations, CNMs) varies among genes, and search for these mutations has an important place in diagnostic strategies. In recent years, CGH method using custom-designed high-density oligonucleotide-based arrays allowed the development of a powerful tool for detection of alterations at the level of exons and made it possible to provide flexibility through the possibility of modeling chips. The aim of our study was to test custom-designed oligonucleotide CGH array in a diagnostic laboratory setting that analyses several genes involved in various genetic diseases, and to compare it with conventional strategies. To this end, we designed a 12-plex CGH array (135k; 135 000 probes/subarray) (Roche Nimblegen) with exonic and intronic oligonucleotide probes covering 26 genes routinely analyzed in the laboratory. We tested control samples with known CNMs and patients for whom genetic causes underlying their disorders were unknown. The contribution of this technique is undeniable. Indeed, it appeared reproducible, reliable and sensitive enough to detect heterozygous single-exon deletions or duplications, complex rearrangements and somatic mosaicism. In addition, it improves reliability of CNM detection and allows determination of boundaries precisely enough to direct targeted sequencing of breakpoints. All of these points, associated with the possibility of a simultaneous analysis of several genes and scalability ‘homemade' make it a valuable tool as a new diagnostic approach of CNMs. PMID:23340513

High-performance single cell genetic analysis using microfluidic emulsion generator arrays.

PubMed

Zeng, Yong; Novak, Richard; Shuga, Joe; Smith, Martyn T; Mathies, Richard A

2010-04-15

High-throughput genetic and phenotypic analysis at the single cell level is critical to advance our understanding of the molecular mechanisms underlying cellular function and dysfunction. Here we describe a high-performance single cell genetic analysis (SCGA) technique that combines high-throughput microfluidic emulsion generation with single cell multiplex polymerase chain reaction (PCR). Microfabricated emulsion generator array (MEGA) devices containing 4, 32, and 96 channels are developed to confer a flexible capability of generating up to 3.4 x 10(6) nanoliter-volume droplets per hour. Hybrid glass-polydimethylsiloxane diaphragm micropumps integrated into the MEGA chips afford uniform droplet formation, controlled generation frequency, and effective transportation and encapsulation of primer functionalized microbeads and cells. A multiplex single cell PCR method is developed to detect and quantify both wild type and mutant/pathogenic cells. In this method, microbeads functionalized with multiple forward primers targeting specific genes from different cell types are used for solid-phase PCR in droplets. Following PCR, the droplets are lysed and the beads are pooled and rapidly analyzed by multicolor flow cytometry. Using Escherichia coli bacterial cells as a model, we show that this technique enables digital detection of pathogenic E. coli O157 cells in a high background of normal K12 cells, with a detection limit on the order of 1/10(5). This result demonstrates that multiplex SCGA is a promising tool for high-throughput quantitative digital analysis of genetic variation in complex populations.

High-Performance Single Cell Genetic Analysis Using Microfluidic Emulsion Generator Arrays

PubMed Central

Zeng, Yong; Novak, Richard; Shuga, Joe; Smith, Martyn T.; Mathies, Richard A.

2010-01-01

High-throughput genetic and phenotypic analysis at the single cell level is critical to advance our understanding of the molecular mechanisms underlying cellular function and dysfunction. Here we describe a high-performance single cell genetic analysis (SCGA) technique that combines high-throughput microfluidic emulsion generation with single cell multiplex PCR. Microfabricated emulsion generator array (MEGA) devices containing 4, 32 and 96 channels are developed to confer a flexible capability of generating up to 3.4 × 106 nanoliter-volume droplets per hour. Hybrid glass-polydimethylsiloxane diaphragm micropumps integrated into the MEGA chips afford uniform droplet formation, controlled generation frequency, and effective transportation and encapsulation of primer functionalized microbeads and cells. A multiplex single cell PCR method is developed to detect and quantify both wild type and mutant/pathogenic cells. In this method, microbeads functionalized with multiple forward primers targeting specific genes from different cell types are used for solid-phase PCR in droplets. Following PCR, the droplets are lysed, the beads are pooled and rapidly analyzed by multi-color flow cytometry. Using E. coli bacterial cells as a model, we show that this technique enables digital detection of pathogenic E. coli O157 cells in a high background of normal K12 cells, with a detection limit on the order of 1:105. This result demonstrates that multiplex SCGA is a promising tool for high-throughput quantitative digital analysis of genetic variation in complex populations. PMID:20192178

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

PubMed

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

2015-01-01

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

Adaptive Injection-locking Oscillator Array for RF Spectrum Analysis

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

Leung, Daniel

2011-04-19

A highly parallel radio frequency receiver using an array of injection-locking oscillators for on-chip, rapid estimation of signal amplitudes and frequencies is considered. The oscillators are tuned to different natural frequencies, and variable gain amplifiers are used to provide negative feedback to adapt the locking band-width with the input signal to yield a combined measure of input signal amplitude and frequency detuning. To further this effort, an array of 16 two-stage differential ring oscillators and 16 Gilbert-cell mixers is designed for 40-400 MHz operation. The injection-locking oscillator array is assembled on a custom printed-circuit board. Control and calibration is achievedmore » by on-board microcontroller.« less

The CRISPR-Associated Gene cas2 of Legionella pneumophila Is Required for Intracellular Infection of Amoebae

PubMed Central

Gunderson, Felizza F.; Cianciotto, Nicholas P.

2013-01-01

ABSTRACT Recent studies have shown that the clustered regularly interspaced palindromic repeats (CRISPR) array and its associated (cas) genes can play a key role in bacterial immunity against phage and plasmids. Upon analysis of the Legionella pneumophila strain 130b chromosome, we detected a subtype II-B CRISPR-Cas locus that contains cas9, cas1, cas2, cas4, and an array with 60 repeats and 58 unique spacers. Reverse transcription (RT)-PCR analysis demonstrated that the entire CRISPR-Cas locus is expressed during 130b extracellular growth in both rich and minimal media as well as during intracellular infection of macrophages and aquatic amoebae. Quantitative reverse transcription-PCR (RT-PCR) further showed that the levels of cas transcripts, especially those of cas1 and cas2, are elevated during intracellular growth relative to exponential-phase growth in broth. Mutants lacking components of the CRISPR-Cas locus were made and found to grow normally in broth and on agar media. cas9, cas1, cas4, and CRISPR array mutants also grew normally in macrophages and amoebae. However, cas2 mutants, although they grew typically in macrophages, were significantly impaired for infection of both Hartmannella and Acanthamoeba species. A complemented cas2 mutant infected the amoebae at wild-type levels, confirming that cas2 is required for intracellular infection of these host cells. PMID:23481601

Addressing fluorogenic real-time qPCR inhibition using the novel custom Excel file system 'FocusField2-6GallupqPCRSet-upTool-001' to attain consistently high fidelity qPCR reactions

PubMed Central

Ackermann, Mark R.

2006-01-01

The purpose of this manuscript is to discuss fluorogenic real-time quantitative polymerase chain reaction (qPCR) inhibition and to introduce/define a novel Microsoft Excel-based file system which provides a way to detect and avoid inhibition, and enables investigators to consistently design dynamically-sound, truly LOG-linear qPCR reactions very quickly. The qPCR problems this invention solves are universal to all qPCR reactions, and it performs all necessary qPCR set-up calculations in about 52 seconds (using a pentium 4 processor) for up to seven qPCR targets and seventy-two samples at a time – calculations that commonly take capable investigators days to finish. We have named this custom Excel-based file system "FocusField2-6GallupqPCRSet-upTool-001" (FF2-6-001 qPCR set-up tool), and are in the process of transforming it into professional qPCR set-up software to be made available in 2007. The current prototype is already fully functional. PMID:17033699

Custom ceramic microchannel-cooled array for high-power fiber-coupled application

NASA Astrophysics Data System (ADS)

Junghans, Jeremy; Feeler, Ryan; Stephens, Ed

2018-03-01

A low-SWaP (Size, Weight and Power) diode array has been developed for a high-power fiber-coupled application. High efficiency ( 65%) diodes enable high optical powers while minimizing thermal losses. A large amount of waste heat is still generated and must be extracted. Custom ceramic microchannel-coolers (MCCs) are used to dissipate the waste heat. The custom ceramic MCC was designed to accommodate long cavity length diodes and micro-lenses. The coolers provide similar thermal performance as copper MCCs however they are not susceptible to erosion and can be cooled with standard filtered water. The custom ceramic micro-channel cooled array was designed to be a form/fit replacement for an existing copperbased solution. Each array consisted of three-vertically stacked MCCs with 4 mm CL, 976 nm diodes and beamshaping micro-optics. The erosion and corrosion resistance of ceramic array is intended to mitigate the risk of copperbased MCC corrosion failures. Elimination of the water delivery requirements (pH, resistivity and dissolved oxygen control) further reduces the system SWaP while maintaining reliability. The arrays were fabricated and fully characterized. This work discusses the advantages of the ceramic MCC technology and describes the design parameters that were tailored for the fiber-coupled application. Additional configuration options (form/fit, micro-lensing, alternate coolants, etc.) and on-going design improvements are also discussed.

Leak Detection in Spacecraft Using a 64-Element Multiplexed Passive Array to Monitor Structure-Borne Noise

NASA Astrophysics Data System (ADS)

Holland, Stephen D.; Song, Jun-Ho; Chimenti, D. E.; Roberts, Ron

2006-03-01

We demonstrate an array sensor method intended to locate leaks in manned spacecraft using leak-generated, structure-borne ultrasonic noise. We have developed and tested a method for sensing and processing leak noise to reveal the leak location involving the use of a 64-element phased-array. Cross-correlations of ultrasonic noise waveforms from a leak into vacuum have been used with a phased-array analysis to find the direction from the sensor to the leak. This method measures the propagation of guided ultrasonic Lamb waves passing under the PZT array sensor in the spacecraft skin structure. This paper will describe the custom-designed array with integrated electronics, as well as the performance of the array in prototype applications. We show that this method can be used to successfully locate leaks to within a few millimeters on a 0.6-m square aluminum plate.

Sensitive single-stage PCR using custom-synthesized internal controls.

PubMed

Zimmermann, K; Rieger, M; Gross, P; Turecek, P L; Schwarz, H P

2000-04-01

A new approach for an internally controlled PCR was developed using a custom-synthesized oligonucleotide as the internal control. Three different PCR setups demonstrated the usefulness of this approach: (i) the addition of the respective internal control to samples containing ssDNA virus Parvo B19; (ii) the co-extraction of plasma samples and the respective internal control for the detection of the ssDNA virus TTV; and (iii) the addition of the respective internal control to a crude lysate of tail pieces for the genotyping of FVIII knockout mice, demonstrating that this approach is also applicable for dsDNA.

Genome-wide comparison of paired fresh frozen and formalin-fixed paraffin-embedded gliomas by custom BAC and oligonucleotide array comparative genomic hybridization: facilitating analysis of archival gliomas

PubMed Central

Mohapatra, Gayatry; Engler, David A.; Starbuck, Kristen D.; Kim, James C.; Bernay, Derek C.; Scangas, George A.; Rousseau, Audrey; Batchelor, Tracy T.; Betensky, Rebecca A.; Louis, David N.

2010-01-01

Molecular genetic analysis of cancer is rapidly evolving as a result of improvement in genomic technologies and the growing applicability of such analyses to clinical oncology. Array based comparative genomic hybridization (aCGH) is a powerful tool for detecting DNA copy number alterations (CNA), particularly in solid tumors, and has been applied to the study of malignant gliomas. In the clinical setting, however, gliomas are often sampled by small biopsies and thus formalin-fixed paraffin-embedded (FFPE) blocks are often the only tissue available for genetic analysis, especially for rare types of gliomas. Moreover, the biological basis for the marked intratumoral heterogeneity in gliomas is most readily addressed in FFPE material. Therefore, for gliomas, the ability to use DNA from FFPE tissue is essential for both clinical and research applications. In this study, we have constructed a custom bacterial artificial chromosome (BAC) array and show excellent sensitivity and specificity for detecting CNAs in a panel of paired frozen and FFPE glioma samples. Our study demonstrates a high concordance rate between CNAs detected in FFPE compared to frozen DNA. We have also developed a method of labeling DNA from FFPE tissue that allows efficient hybridization to oligonucleotide arrays. This labeling technique was applied to a panel of biphasic anaplastic oligoastrocytomas (AOA) to identify genetic changes unique to each component. Together, results from these studies suggest that BAC and oligonucleotide aCGH are sensitive tools for detecting CNAs in FFPE DNA, and can enable genome-wide analysis of rare, small and/or histologically heterogeneous gliomas. PMID:21080181

Using stereotactic brain atlases for small rodents and nonhuman primates for optrode array customization

NASA Astrophysics Data System (ADS)

Boutte, Ronald W.; Merlin, Sam; Griffiths, Brandon; Parry, Trent; Blair, Steve

2017-02-01

As the optogenetic field expands its need to target with high specificity only grows more crucial. This work will show a method for customizing soda-lime glass optrode arrays so that fine structures within the brains of small rodents and nonhuman primates can be optically interrogated below the outer cortical layer. An 8 × 6 array is customized for optrode length (400 μm ), optrode width (75 μm ), optrode pitch (400 μm ), backplane thickness (500 μm ), and overall form factor (3.45 mm × 2.65 mm ). The 400 μm long optrode is capable of illuminating the cortical Layer IV of rhesus macaque ( Macaca Fascicularis ) and the motor cortex of small mice ( Mus Musculus ).

1-Million droplet array with wide-field fluorescence imaging for digital PCR.

PubMed

Hatch, Andrew C; Fisher, Jeffrey S; Tovar, Armando R; Hsieh, Albert T; Lin, Robert; Pentoney, Stephen L; Yang, David L; Lee, Abraham P

2011-11-21

Digital droplet reactors are useful as chemical and biological containers to discretize reagents into picolitre or nanolitre volumes for analysis of single cells, organisms, or molecules. However, most DNA based assays require processing of samples on the order of tens of microlitres and contain as few as one to as many as millions of fragments to be detected. Presented in this work is a droplet microfluidic platform and fluorescence imaging setup designed to better meet the needs of the high-throughput and high-dynamic-range by integrating multiple high-throughput droplet processing schemes on the chip. The design is capable of generating over 1-million, monodisperse, 50 picolitre droplets in 2-7 minutes that then self-assemble into high density 3-dimensional sphere packing configurations in a large viewing chamber for visualization and analysis. This device then undergoes on-chip polymerase chain reaction (PCR) amplification and fluorescence detection to digitally quantify the sample's nucleic acid contents. Wide-field fluorescence images are captured using a low cost 21-megapixel digital camera and macro-lens with an 8-12 cm(2) field-of-view at 1× to 0.85× magnification, respectively. We demonstrate both end-point and real-time imaging ability to perform on-chip quantitative digital PCR analysis of the entire droplet array. Compared to previous work, this highly integrated design yields a 100-fold increase in the number of on-chip digitized reactors with simultaneous fluorescence imaging for digital PCR based assays.

Combined histochemical staining, RNA amplification, regional, and single cell cDNA analysis within the hippocampus.

PubMed

Ginsberg, Stephen D; Che, Shaoli

2004-08-01

The use of five histochemical stains (cresyl violet, thionin, hematoxylin & eosin, silver stain, and acridine orange) was evaluated in combination with an expression profiling paradigm that included regional and single cell analyses within the hippocampus of post-mortem human brains and adult mice. Adjacent serial sections of human and mouse hippocampus were labeled by histochemistry or neurofilament immunocytochemistry. These tissue sections were used as starting material for regional and single cell microdissection followed by a newly developed RNA amplification procedure (terminal continuation (TC) RNA amplification) and subsequent hybridization to custom-designed cDNA arrays. Results indicated equivalent levels of global hybridization signal intensity and relative expression levels for individual genes for hippocampi stained by cresyl violet, thionin, and hematoxylin & eosin, and neurofilament immunocytochemistry. Moreover, no significant differences existed between the Nissl stains and neurofilament immunocytochemistry for individual CA1 neurons obtained via laser capture microdissection. In contrast, a marked decrement was observed in adjacent hippocampal sections stained for silver stain and acridine orange, both at the level of the regional dissection and at the CA1 neuron population level. Observations made on the cDNA array platform were validated by real-time qPCR using primers directed against beta-actin and glyceraldehyde-3 phosphate dehydrogenase. Thus, this report demonstrated the utility of using specific Nissl stains, but not stains that bind RNA species directly, in both human and mouse brain tissues at the regional and cellular level for state-of-the-art molecular fingerprinting studies.

BEAT: Bioinformatics Exon Array Tool to store, analyze and visualize Affymetrix GeneChip Human Exon Array data from disease experiments

PubMed Central

2012-01-01

Background It is known from recent studies that more than 90% of human multi-exon genes are subject to Alternative Splicing (AS), a key molecular mechanism in which multiple transcripts may be generated from a single gene. It is widely recognized that a breakdown in AS mechanisms plays an important role in cellular differentiation and pathologies. Polymerase Chain Reactions, microarrays and sequencing technologies have been applied to the study of transcript diversity arising from alternative expression. Last generation Affymetrix GeneChip Human Exon 1.0 ST Arrays offer a more detailed view of the gene expression profile providing information on the AS patterns. The exon array technology, with more than five million data points, can detect approximately one million exons, and it allows performing analyses at both gene and exon level. In this paper we describe BEAT, an integrated user-friendly bioinformatics framework to store, analyze and visualize exon arrays datasets. It combines a data warehouse approach with some rigorous statistical methods for assessing the AS of genes involved in diseases. Meta statistics are proposed as a novel approach to explore the analysis results. BEAT is available at http://beat.ba.itb.cnr.it. Results BEAT is a web tool which allows uploading and analyzing exon array datasets using standard statistical methods and an easy-to-use graphical web front-end. BEAT has been tested on a dataset with 173 samples and tuned using new datasets of exon array experiments from 28 colorectal cancer and 26 renal cell cancer samples produced at the Medical Genetics Unit of IRCCS Casa Sollievo della Sofferenza. To highlight all possible AS events, alternative names, accession Ids, Gene Ontology terms and biochemical pathways annotations are integrated with exon and gene level expression plots. The user can customize the results choosing custom thresholds for the statistical parameters and exploiting the available clinical data of the samples for a multivariate AS analysis. Conclusions Despite exon array chips being widely used for transcriptomics studies, there is a lack of analysis tools offering advanced statistical features and requiring no programming knowledge. BEAT provides a user-friendly platform for a comprehensive study of AS events in human diseases, displaying the analysis results with easily interpretable and interactive tables and graphics. PMID:22536968

454 next generation-sequencing outperforms allele-specific PCR, Sanger sequencing, and pyrosequencing for routine KRAS mutation analysis of formalin-fixed, paraffin-embedded samples

PubMed Central

Altimari, Annalisa; de Biase, Dario; De Maglio, Giovanna; Gruppioni, Elisa; Capizzi, Elisa; Degiovanni, Alessio; D’Errico, Antonia; Pession, Annalisa; Pizzolitto, Stefano; Fiorentino, Michelangelo; Tallini, Giovanni

2013-01-01

Detection of KRAS mutations in archival pathology samples is critical for therapeutic appropriateness of anti-EGFR monoclonal antibodies in colorectal cancer. We compared the sensitivity, specificity, and accuracy of Sanger sequencing, ARMS-Scorpion (TheraScreen®) real-time polymerase chain reaction (PCR), pyrosequencing, chip array hybridization, and 454 next-generation sequencing to assess KRAS codon 12 and 13 mutations in 60 nonconsecutive selected cases of colorectal cancer. Twenty of the 60 cases were detected as wild-type KRAS by all methods with 100% specificity. Among the 40 mutated cases, 13 were discrepant with at least one method. The sensitivity was 85%, 90%, 93%, and 92%, and the accuracy was 90%, 93%, 95%, and 95% for Sanger sequencing, TheraScreen real-time PCR, pyrosequencing, and chip array hybridization, respectively. The main limitation of Sanger sequencing was its low analytical sensitivity, whereas TheraScreen real-time PCR, pyrosequencing, and chip array hybridization showed higher sensitivity but suffered from the limitations of predesigned assays. Concordance between the methods was k = 0.79 for Sanger sequencing and k > 0.85 for the other techniques. Tumor cell enrichment correlated significantly with the abundance of KRAS-mutated deoxyribonucleic acid (DNA), evaluated as ΔCt for TheraScreen real-time PCR (P = 0.03), percentage of mutation for pyrosequencing (P = 0.001), ratio for chip array hybridization (P = 0.003), and percentage of mutation for 454 next-generation sequencing (P = 0.004). Also, 454 next-generation sequencing showed the best cross correlation for quantification of mutation abundance compared with all the other methods (P < 0.001). Our comparison showed the superiority of next-generation sequencing over the other techniques in terms of sensitivity and specificity. Next-generation sequencing will replace Sanger sequencing as the reference technique for diagnostic detection of KRAS mutation in archival tumor tissues. PMID:23950653

Design and calibration of a six-axis MEMS sensor array for use in scoliosis correction surgery

NASA Astrophysics Data System (ADS)

Benfield, David; Yue, Shichao; Lou, Edmond; Moussa, Walied A.

2014-08-01

A six-axis sensor array has been developed to quantify the 3D force and moment loads applied in scoliosis correction surgery. Initially this device was developed to be applied during scoliosis correction surgery and augmented onto existing surgical instrumentation, however, use as a general load sensor is also feasible. The development has included the design, microfabrication, deployment and calibration of a sensor array. The sensor array consists of four membrane devices, each containing piezoresistive sensing elements, generating a total of 16 differential voltage outputs. The calibration procedure has made use of a custom built load application frame, which allows quantified forces and moments to be applied and compared to the outputs from the sensor array. Linear or non-linear calibration equations are generated to convert the voltage outputs from the sensor array back into 3D force and moment information for display or analysis.

Operation's Concept for Array-Based Deep Space Network

NASA Technical Reports Server (NTRS)

Bagri, Durgadas S.; Statman, Joseph I.; Gatti, Mark S.

2005-01-01

The Array-based Deep Space Network (DSNArray) will be a part of more than 10(exp 3) times increase in the downlink/telemetry capability of the Deep space Network (DSN). The key function of the DSN-Array is to provide cost-effective, robust Telemetry, Tracking and Command (TT&C) services to the space missions of NASA and its international partners. It provides an expanded approach to the use of an array-based system. Instead of using the array as an element in the existing DSN, relying to a large extent on the DSN infrastructure, we explore a broader departure from the current DSN, using fewer elements of the existing DSN, and establishing a more modern Concept of Operations. This paper gives architecture of DSN-Array and its operation's philosophy. It also describes customer's view of operations, operations management and logistics - including maintenance philosophy, anomaly analysis and reporting.

Semicustom integrated circuits and the standard transistor array radix (STAR)

NASA Technical Reports Server (NTRS)

Edge, T. M.

1977-01-01

The development, application, pros and cons of the semicustom and custom approach to the integration of circuits are described. Improvements in terms of cost, reliability, secrecy, power, and size reduction are examined. Also presented is the standard transistor array radix, a semicustom approach to digital integrated circuits that offers the advantages of both custom and semicustom approaches to integration.

Localized transfection on arrays of magnetic beads coated with PCR products.

PubMed

Isalan, Mark; Santori, Maria Isabel; Gonzalez, Cayetano; Serrano, Luis

2005-02-01

High-throughput gene analysis would benefit from new approaches for delivering DNA or RNA into cells. Here we describe a simple system that allows any molecular biology laboratory to carry out multiple, parallel cell transfections on microscope coverslip arrays. By using magnetically defined positions and PCR product-coated paramagnetic beads, we achieved transfection in a variety of cell lines. Beads may be added to the cells at any time, allowing both spatial and temporal control of transfection. Because the beads may be coated with more than one gene construct, the method can be used to achieve cotransfection within single cells. Furthermore, PCR-generated mutants may be conveniently screened, bypassing cloning and plasmid purification steps. We illustrated the applicability of the method by screening combinatorial peptide libraries, fused to GFP, to identify previously unknown cellular localization motifs. In this way, we identified several localizing peptides, including structured localization signals based around the scaffold of a single C2H2 zinc finger.

Differential expression of THOC1 and ALY mRNP biogenesis/export factors in human cancers

PubMed Central

2011-01-01

Background One key step in gene expression is the biogenesis of mRNA ribonucleoparticle complexes (mRNPs). Formation of the mRNP requires the participation of a number of conserved factors such as the THO complex. THO interacts physically and functionally with the Sub2/UAP56 RNA-dependent ATPase, and the Yra1/REF1/ALY RNA-binding protein linking transcription, mRNA export and genome integrity. Given the link between genome instability and cancer, we have performed a comparative analysis of the expression patterns of THOC1, a THO complex subunit, and ALY in tumor samples. Methods The mRNA levels were measured by quantitative real-time PCR and hybridization of a tumor tissue cDNA array; and the protein levels and distribution by immunostaining of a custom tissue array containing a set of paraffin-embedded samples of different tumor and normal tissues followed by statistical analysis. Results We show that the expression of two mRNP factors, THOC1 and ALY are altered in several tumor tissues. THOC1 mRNA and protein levels are up-regulated in ovarian and lung tumors and down-regulated in those of testis and skin, whereas ALY is altered in a wide variety of tumors. In contrast to THOC1, ALY protein is highly detected in normal proliferative cells, but poorly in high-grade cancers. Conclusions These results suggest a differential connection between tumorogenesis and the expression levels of human THO and ALY. This study opens the possibility of defining mRNP biogenesis factors as putative players in cell proliferation that could contribute to tumor development. PMID:21329510

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2009-07-31

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

Type-Specific Detection of 30 Oncogenic Human Papillomaviruses by Genotyping both E6 and L1 Genes

PubMed Central

Peng, Junping; Gao, Lei; Guo, Junhua; Wang, Ting; Wang, Ling; Yao, Qing; Zhu, Haijun

2013-01-01

Human papillomavirus (HPV) is the principal cause of invasive cervical cancer and benign genital lesions. There are currently 30 HPV types linked to cervical cancer. HPV infection also leads to other types of cancer. We developed a 61-plex analysis of these 30 HPV types by examining two genes, E6 and L1, using MassARRAY matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (PCR-MS). Two hundred samples from homosexual males (HM) were screened by PCR-MS and MY09/MY11 primer set-mediated PCR (MY-PCR) followed by sequencing. One hundred thirty-five formalin-fixed, paraffin-embedded (FFPE) cervical cancer samples were also analyzed by PCR-MS, and results were compared to those of the commercially available GenoArray (GA) assay. One or more HPV types were identified in 64.5% (129/200) of the samples from HM. Comprising all 30 HPV types, PCR-MS detected 51.9% (67/129) of samples with multiple HPV types, whereas MY-PCR detected only one single HPV type in these samples. All PCR-MS results were confirmed by MY-PCR. In the cervical cancer samples, PCR-MS and GA detected 97% (131/135) and 90.4% (122/135) of HPV-positive samples, respectively. PCR-MS and GA results were fully concordant for 122 positive and 4 negative samples. The sequencing results for the 9 samples that tested negative by GA were completely concordant with the positive PCR-MS results. Multiple HPV types were identified in 25.2% (34/135) and 55.6% (75/135) of the cervical cancer samples by GA and PCR-MS, respectively, and results were confirmed by sequencing. The new assay allows the genotyping of >1,000 samples per day. It provides a good alternative to current methods, especially for large-scale investigations of multiple HPV infections and degraded FFPE samples. PMID:23152557

High-density fiber-optic DNA random microsphere array.

PubMed

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

2000-11-15

A high-density fiber-optic DNA microarray sensor was developed to monitor multiple DNA sequences in parallel. Microarrays were prepared by randomly distributing DNA probe-functionalized 3.1-microm-diameter microspheres in an array of wells etched in a 500-microm-diameter optical imaging fiber. Registration of the microspheres was performed using an optical encoding scheme and a custom-built imaging system. Hybridization was visualized using fluorescent-labeled DNA targets with a detection limit of 10 fM. Hybridization times of seconds are required for nanomolar target concentrations, and analysis is performed in minutes.

A girl with incomplete Prader-Willi syndrome and negative MS-PCR, found to have mosaic maternal UPD-15 at SNP array.

PubMed

Morandi, Anita; Bonnefond, Amélie; Lobbens, Stéphane; Carotenuto, Marco; Del Giudice, Emanuele Miraglia; Froguel, Philippe; Maffeis, Claudio

2015-11-01

The Prader-Willi syndrome (PWS) is caused by lack of expression of paternal allele of the 15q11.2-q13 region, due to deletions at paternal 15q11.2-q13 (<70%), maternal uniparental disomy of chromosome 15 (mat-UPD 15) (30%) or imprinting defects (1%). Hyperphagia, intellectual disabilities/behavioral disorders, neonatal hypotonia, and hypogonadism are cardinal features for PWS. Methylation sensitive PCR (MS-PCR) of the SNRPN locus, which assesses the presence of both the unmethylated (paternal) and the methylated (maternal) allele of 15q11.2-q13, is considered a sensitive reference technique for PWS diagnosis regardless of genetic subtype. We describe a 17-year-old girl with severe obesity, short stature, and intellectual disability, without hypogonadism and history of neonatal hypotonia, who was suspected to have an incomplete PWS. The MS-PCR showed a normal pattern with similar maternal and paternal electrophoretic bands. Afterwards, a SNP array showed the presence of iso-UPD 15, that is, UPD15 with two copies of the same chromosome 15, in about 50% of cells, suggesting a diagnosis of partial PWS due to mosaic maternal iso-UPD15 arisen as rescue of a post-fertilization error. A quantitative methylation analysis confirmed the presence of mosaic UPD15 in about 50% of cells. We propose that complete clinical criteria for PWS and MS-PCR should not be considered sensitive in suspecting and diagnosing partial PWS due to mosaic UPD15. In contrast, clinical suspicion based on less restrictive criteria followed by SNP array is a more powerful approach to diagnose atypical PWS due to UPD15 mosaicism. © 2015 Wiley Periodicals, Inc.

Riems influenza a typing array (RITA): An RT-qPCR-based low density array for subtyping avian and mammalian influenza a viruses.

PubMed

Hoffmann, Bernd; Hoffmann, Donata; Henritzi, Dinah; Beer, Martin; Harder, Timm C

2016-06-03

Rapid and sensitive diagnostic approaches are of the utmost importance for the detection of humans and animals infected by specific influenza virus subtype(s). Cascade-like diagnostics starting with the use of pan-influenza assays and subsequent subtyping devices are normally used. Here, we demonstrated a novel low density array combining 32 TaqMan(®) real-time RT-PCR systems in parallel for the specific detection of the haemagglutinin (HA) and neuraminidase (NA) subtypes of avian and porcine hosts. The sensitivity of the newly developed system was compared with that of the pan-influenza assay, and the specificity of all RT-qPCRs was examined using a broad panel of 404 different influenza A virus isolates representing 45 different subtypes. Furthermore, we analysed the performance of the RT-qPCR assays with diagnostic samples obtained from wild birds and swine. Due to the open format of the array, adaptations to detect newly emerging influenza A virus strains can easily be integrated. The RITA array represents a competitive, fast and sensitive subtyping tool that requires neither new machinery nor additional training of staff in a lab where RT-qPCR is already established.

Artificial Muscle (AM) Cilia Array for Underwater Systems

DTIC Science & Technology

2016-12-15

structures, including cilia-like structures. Specifically, a custom 3D printer was created that utilizes custom-made Nafion filament for 30 printing of custom... printing ) of IPMC material to create custom-shaped AM structures, including cilia-like structures. Various custom-shaped AM structures were fabricated via...integrating square cross-section IPMC actuators with a printed circuit board power delivery system. IV. Concise Accomplishments Performance

Genome wide analysis in a discordant monozygotic twin with caudal appendage and multiple congenital anomalies.

PubMed

Cogulu, O; Pariltay, E; Koroglu, O A; Aykut, A; Ozyurek, R; Levent, E; Kultursay, N; Ozkinay, F

2013-01-01

Caudal appendage is a rare dysmorphic feature of which etiologic mechanisms are not well understood. Here we report monozygotic (MZ) twin brothers who are discordant for the caudal appendage and multiple congenital anomalies. Twins were the product of a 33 weeks of gestation, monochorionic-diamniotic pregnancy. On admission the proband had micrognathia, beaked nose, hypospadias, caudal appendage and juxtaductal aorta coarctation. At birth, he was small for gestational age and he had transient hypothyroidism which was detected in the newborn period. Karyotype analysis showed 46,XY. Monozygosity was shown by 15 microsatellite markers plus amelogenin (AmpFlSTR Identifiler PCR Amplification Kit, Applied Biosystems). Genome-wide copy number analysis of the twins by DNA-DNA hybridization of whole genomic DNA (NimbleGen Human CGH 385K WG-T v2.0 array) showed a significant difference at two neighboring probes with Log2 ratio: 0.72088 which are located on chromosome 3p12.3. Further analysis by high resolution of chromosome 3 array (Roche NimbleGen Human HG18 CHR3 FT Median Probe Spacing 475 bp) and quantitative PCR analysis did not confirm the deletion.

Identification of a duplication within the GDF9 gene and novel candidate genes for primary ovarian insufficiency (POI) by a customized high-resolution array comparative genomic hybridization platform.

PubMed

Norling, A; Hirschberg, A L; Rodriguez-Wallberg, K A; Iwarsson, E; Wedell, A; Barbaro, M

2014-08-01

Can high-resolution array comparative genomic hybridization (CGH) analysis of DNA samples from women with primary ovarian insufficiency (POI) improve the diagnosis of the condition and identify novel candidate genes for POI? A mutation affecting the regulatory region of growth differentiation factor 9 (GDF9) was identified for the first time together with several novel candidate genes for POI. Most patients with POI do not receive a molecular diagnosis despite a significant genetic component in the pathogenesis. We performed a case-control study. Twenty-six patients were analyzed by array CGH for identification of copy number variants. Novel changes were investigated in 95 controls and in a separate population of 28 additional patients with POI. The experimental procedures were performed during a 1-year period. DNA samples from 26 patients with POI were analyzed by a customized 1M array-CGH platform with whole genome coverage and probe enrichment targeting 78 genes in sex development. By PCR amplification and sequencing, the breakpoint of an identified partial GDF9 gene duplication was characterized. A multiplex ligation-dependent probe amplification (MLPA) probe set for specific identification of deletions/duplications affecting GDF9 was developed. An MLPA probe set for the identification of additional cases or controls carrying novel candidate regions identified by array-CGH was developed. Sequencing of three candidate genes was performed. Eleven unique copy number changes were identified in a total of 11 patients, including a tandem duplication of 475 bp, containing part of the GDF9 gene promoter region. The duplicated region contains three NOBOX-binding elements and an E-box, important for GDF9 gene regulation. This aberration is likely causative of POI. Fifty-four patients were investigated for copy number changes within GDF9, but no additional cases were found. Ten aberrations constituting novel candidate regions were detected, including a second DNAH6 deletion in a patient with POI. Other identified candidate genes were TSPYL6, SMARCC1, CSPG5 and ZFR2. This is a descriptive study and no functional experiments were performed. The study illustrates the importance of analyzing small copy number changes in addition to sequence alterations in the genetic investigation of patients with POI. Also, promoter regions should be included in the investigation. The study was supported by grants from the Swedish Research council (project no 12198 to A.W. and project no 20324 to A.L.H.), Stockholm County Council (E.I., A.W. and K.R.W.), Foundation Frimurare Barnhuset (A.N., A.W. and M.B.), Karolinska Institutet (A.N., A.L.H., E.I., A.W. and M.B.), Novo Nordic Foundation (A.W.) and Svenska Läkaresällskapet (M.B.). The funding sources had no involvement in the design or analysis of the study. The authors have no competing interests to declare. Not applicable. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.

miRNome analysis using real-time PCR.

PubMed

Pontrelli, Paola; Accetturo, Matteo; Gesualdo, Loreto

2014-01-01

MicroRNAs (miRNAs) are short RNA molecules that regulate gene expression in eukaryotic organisms, thus influencing physiological mechanisms such as development, cell proliferation, cell death, and cell differentiation. The importance of the gene regulatory system operated by miRNAs is emerging as a central topic in the setting of several diseases included infectious disease and cancer. The different techniques used for the study of the entire "miRNome" give the opportunity to go better inside these novel mechanisms of gene expression regulation. In the following method we describe a protocol based on quantitative real-time PCR (qRT-PCR) with SYBR(®) green technology, to specifically analyze the expression levels of only those miRNAs that target genes involved in CTLs biogenesis and functions. Through an in silico approach, we designed a custom microRNA qPCR panel focused on those miRNAs relevant in regulation of CTLs-specific pathways. The panel we created was customized by EXIQON, since this company proposed a method based on the use of LNA enhanced primers, which guarantee increased affinity and specificity for each microRNA. The advantage of this protocol with respect to a whole miRNome analysis consists in the possibility to evidence weaker signals that otherwise would be secreted and remove the noise itself generated by other miRNAs not directly involved in the regulation of CTLs-specific pathways. This panel can be applicable in the study of CTLs behavior in pathological conditions such as infectious disease and cancer or can be used to characterize changes in patients' immune responsiveness after therapeutic intervention in order to understand the molecular mechanisms underlying these effects.

Forward-Looking Intracardiac Ultrasound Imaging Using a 1-D CMUT Array Integrated With Custom Front-End Electronics

PubMed Central

Nikoozadeh, Amin; Wygant, Ira O.; Lin, Der-Song; Oralkan, Ömer; Ergun, A. Sanlı; Stephens, Douglas N.; Thomenius, Kai E.; Dentinger, Aaron M.; Wildes, Douglas; Akopyan, Gina; Shivkumar, Kalyanam; Mahajan, Aman; Sahn, David J.; Khuri-Yakub, Butrus T.

2009-01-01

Minimally invasive catheter-based electrophysiological (EP) interventions are becoming a standard procedure in diagnosis and treatment of cardiac arrhythmias. As a result of technological advances that enable small feature sizes and a high level of integration, nonfluoroscopic intracardiac echocardiography (ICE) imaging catheters are attracting increasing attention. ICE catheters improve EP procedural guidance while reducing the undesirable use of fluoroscopy, which is currently the common catheter guidance method. Phased-array ICE catheters have been in use for several years now, although only for side-looking imaging. We are developing a forward-looking ICE catheter for improved visualization. In this effort, we fabricate a 24-element, fine-pitch 1-D array of capacitive micromachined ultrasonic transducers (CMUT), with a total footprint of 1.73 mm × 1.27 mm. We also design a custom integrated circuit (IC) composed of 24 identical blocks of transmit/receive circuitry, measuring 2.1 mm × 2.1 mm. The transmit circuitry is capable of delivering 25-V unipolar pulses, and the receive circuitry includes a transimpedance preamplifier followed by an output buffer. The CMUT array and the custom IC are designed to be mounted at the tip of a 10-Fr catheter for high-frame-rate forward-looking intracardiac imaging. Through-wafer vias incorporated in the CMUT array provide access to individual array elements from the back side of the array. We successfully flip-chip bond a CMUT array to the custom IC with 100% yield. We coat the device with a layer of polydimethylsiloxane (PDMS) to electrically isolate the device for imaging in water and tissue. The pulse-echo in water from a total plane reflector has a center frequency of 9.2 MHz with a 96% fractional bandwidth. Finally, we demonstrate the imaging capability of the integrated device on commercial phantoms and on a beating ex vivo rabbit heart (Langendorff model) using a commercial ultrasound imaging system. PMID:19126489

New customizable phased array UT instrument opens door for furthering research and better industrial implementation

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

Dao, Gavin; Ginzel, Robert

2014-02-18

Phased array UT as an inspection technique in itself continues to gain wide acceptance. However, there is much room for improvement in terms of implementation of Phased Array (PA) technology for every unique NDT application across several industries (e.g. oil and petroleum, nuclear and power generation, steel manufacturing, etc.). Having full control of the phased array instrument and customizing a software solution is necessary for more seamless and efficient inspections, from setting the PA parameters, collecting data and reporting, to the final analysis. NDT researchers and academics also need a flexible and open platform to be able to control variousmore » aspects of the phased array process. A high performance instrument with advanced PA features, faster data rates, a smaller form factor, and capability to adapt to specific applications, will be discussed.« less

ECL gate array with integrated PLL-based clock recovery and synthesis for high-speed data and telecom applications

NASA Astrophysics Data System (ADS)

Rosky, David S.; Coy, Bruce H.; Friedmann, Marc D.

1992-03-01

A 2500 gate mixed signal gate array has been developed that integrates custom PLL-based clock recovery and clock synthesis functions with 2500 gates of configurable logic cells to provide a single chip solution for 200 - 1244 MHz fiber based digital interface applications. By customizing the digital logic cells, any of the popular telecom and datacom standards may be implemented.

Three-color crystal digital PCR.

PubMed

Madic, J; Zocevic, A; Senlis, V; Fradet, E; Andre, B; Muller, S; Dangla, R; Droniou, M E

2016-12-01

Digital PCR is an exciting new field for molecular analysis, allowing unprecedented precision in the quantification of nucleic acids, as well as the fine discrimination of rare molecular events in complex samples. We here present a novel technology for digital PCR, Crystal Digital PCR™, which relies on the use of a single chip to partition samples into 2D droplet arrays, which are then subjected to thermal cycling and finally read using a three-color fluorescence scanning device. This novel technology thus allows three-color multiplexing, which entails a different approach to data analysis. In the present publication, we present this innovative workflow, which is both fast and user-friendly, and discuss associated data analysis issue, such as fluorescence spillover compensation and data representation. Lastly, we also present proof-of-concept of this three-color detection system, using a quadriplex assay for the detection of EGFR mutations L858R, L861Q and T790M.

Application of Nexus copy number software for CNV detection and analysis.

PubMed

Darvishi, Katayoon

2010-04-01

Among human structural genomic variation, copy number variants (CNVs) are the most frequently known component, comprised of gains/losses of DNA segments that are generally 1 kb in length or longer. Array-based comparative genomic hybridization (aCGH) has emerged as a powerful tool for detecting genomic copy number variants (CNVs). With the rapid increase in the density of array technology and with the adaptation of new high-throughput technology, a reliable and computationally scalable method for accurate mapping of recurring DNA copy number aberrations has become a main focus in research. Here we introduce Nexus Copy Number software, a platform-independent tool, to analyze the output files of all types of commercial and custom-made comparative genomic hybridization (CGH) and single-nucleotide polymorphism (SNP) arrays, such as those manufactured by Affymetrix, Agilent Technologies, Illumina, and Roche NimbleGen. It also supports data generated by various array image-analysis software tools such as GenePix, ImaGene, and BlueFuse. (c) 2010 by John Wiley & Sons, Inc.

High-throughput microfluidic single-cell digital polymerase chain reaction.

PubMed

White, A K; Heyries, K A; Doolin, C; Vaninsberghe, M; Hansen, C L

2013-08-06

Here we present an integrated microfluidic device for the high-throughput digital polymerase chain reaction (dPCR) analysis of single cells. This device allows for the parallel processing of single cells and executes all steps of analysis, including cell capture, washing, lysis, reverse transcription, and dPCR analysis. The cDNA from each single cell is distributed into a dedicated dPCR array consisting of 1020 chambers, each having a volume of 25 pL, using surface-tension-based sample partitioning. The high density of this dPCR format (118,900 chambers/cm(2)) allows the analysis of 200 single cells per run, for a total of 204,000 PCR reactions using a device footprint of 10 cm(2). Experiments using RNA dilutions show this device achieves shot-noise-limited performance in quantifying single molecules, with a dynamic range of 10(4). We performed over 1200 single-cell measurements, demonstrating the use of this platform in the absolute quantification of both high- and low-abundance mRNA transcripts, as well as micro-RNAs that are not easily measured using alternative hybridization methods. We further apply the specificity and sensitivity of single-cell dPCR to performing measurements of RNA editing events in single cells. High-throughput dPCR provides a new tool in the arsenal of single-cell analysis methods, with a unique combination of speed, precision, sensitivity, and specificity. We anticipate this approach will enable new studies where high-performance single-cell measurements are essential, including the analysis of transcriptional noise, allelic imbalance, and RNA processing.

Microwell Array Method for Rapid Generation of Uniform Agarose Droplets and Beads for Single Molecule Analysis.

PubMed

Li, Xingrui; Zhang, Dongfeng; Zhang, Huimin; Guan, Zhichao; Song, Yanling; Liu, Ruochen; Zhu, Zhi; Yang, Chaoyong

2018-02-20

Compartmentalization of aqueous samples in uniform emulsion droplets has proven to be a useful tool for many chemical, biological, and biomedical applications. Herein, we introduce an array-based emulsification method for rapid and easy generation of monodisperse agarose-in-oil droplets in a PDMS microwell array. The microwells are filled with agarose solution, and subsequent addition of hot oil results in immediate formation of agarose droplets due to the surface-tension of the liquid solution. Because droplet size is determined solely by the array unit dimensions, uniform droplets with preselectable diameters ranging from 20 to 100 μm can be produced with relative standard deviations less than 3.5%. The array-based droplet generation method was used to perform digital PCR for absolute DNA quantitation. The array-based droplet isolation and sol-gel switching property of agarose enable formation of stable beads by chilling the droplet array at -20 °C, thus, maintaining the monoclonality of each droplet and facilitating the selective retrieval of desired droplets. The monoclonality of droplets was demonstrated by DNA sequencing and FACS analysis, suggesting the robustness and flexibility of the approach for single molecule amplification and analysis. We believe our approach will lead to new possibilities for a great variety of applications, such as single-cell gene expression studies, aptamer selection, and oligonucleotide analysis.

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

PubMed

Jiang, Z; Gui, S; Zhang, Y

2011-05-01

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

Seven-panel solar wing deployment and on-orbit maneuvering analyses

NASA Astrophysics Data System (ADS)

Hwang, Earl

2005-05-01

BSS developed a new generation high power (~20kW) solar array to meet the customer demands. The high power solar array had the north and south solar wings of which designs were identical. Each side of the solar wing consists of three main conventional solar panels and the four-side panel swing-out new design. The fully deployed solar array surface area is 966 ft2. It was a quite challenging task to define the solar array's optimum design parameters and deployment scheme for such a huge solar array's successful deployment and on-orbit maneuvering. Hence, a deployable seven-flex-panel solar wing nonlinear math model and a fully deployed solar array/bus-payload math model were developed with the Dynamic Analysis and Design System (DADS) program codes utilizing the inherited and empirical data. Performing extensive parametric analyses with the math model, the optimum design parameters and the orbit maneuvering /deployment schemes were determined to meet all the design requirements, and for the successful solar wing deployment on-orbit.

Genome-wide comparison of paired fresh frozen and formalin-fixed paraffin-embedded gliomas by custom BAC and oligonucleotide array comparative genomic hybridization: facilitating analysis of archival gliomas.

PubMed

Mohapatra, Gayatry; Engler, David A; Starbuck, Kristen D; Kim, James C; Bernay, Derek C; Scangas, George A; Rousseau, Audrey; Batchelor, Tracy T; Betensky, Rebecca A; Louis, David N

2011-04-01

Array comparative genomic hybridization (aCGH) is a powerful tool for detecting DNA copy number alterations (CNA). Because diffuse malignant gliomas are often sampled by small biopsies, formalin-fixed paraffin-embedded (FFPE) blocks are often the only tissue available for genetic analysis; FFPE tissues are also needed to study the intratumoral heterogeneity that characterizes these neoplasms. In this paper, we present a combination of evaluations and technical advances that provide strong support for the ready use of oligonucleotide aCGH on FFPE diffuse gliomas. We first compared aCGH using bacterial artificial chromosome (BAC) arrays in 45 paired frozen and FFPE gliomas, and demonstrate a high concordance rate between FFPE and frozen DNA in an individual clone-level analysis of sensitivity and specificity, assuring that under certain array conditions, frozen and FFPE DNA can perform nearly identically. However, because oligonucleotide arrays offer advantages to BAC arrays in genomic coverage and practical availability, we next developed a method of labeling DNA from FFPE tissue that allows efficient hybridization to oligonucleotide arrays. To demonstrate utility in FFPE tissues, we applied this approach to biphasic anaplastic oligoastrocytomas and demonstrate CNA differences between DNA obtained from the two components. Therefore, BAC and oligonucleotide aCGH can be sensitive and specific tools for detecting CNAs in FFPE DNA, and novel labeling techniques enable the routine use of oligonucleotide arrays for FFPE DNA. In combination, these advances should facilitate genome-wide analysis of rare, small and/or histologically heterogeneous gliomas from FFPE tissues.

Isolation of human simple repeat loci by hybridization selection.

PubMed

Armour, J A; Neumann, R; Gobert, S; Jeffreys, A J

1994-04-01

We have isolated short tandem repeat arrays from the human genome, using a rapid method involving filter hybridization to enrich for tri- or tetranucleotide tandem repeats. About 30% of clones from the enriched library cross-hybridize with probes containing trimeric or tetrameric tandem arrays, facilitating the rapid isolation of large numbers of clones. In an initial analysis of 54 clones, 46 different tandem arrays were identified. Analysis of these tandem repeat loci by PCR showed that 24 were polymorphic in length; substantially higher levels of polymorphism were displayed by the tetrameric repeat loci isolated than by the trimeric repeats. Primary mapping of these loci by linkage analysis showed that they derive from 17 chromosomes, including the X chromosome. We anticipate the use of this strategy for the efficient isolation of tandem repeats from other sources of genomic DNA, including DNA from flow-sorted chromosomes, and from other species.

75 FR 55303 - Proposed Information Collection; Comment Request; Commercial Service Annual Customer Satisfaction...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-09-10

... Request; Commercial Service Annual Customer Satisfaction Survey AGENCY: International Trade Administration... assistance services. The CS uses an Annual Customer Satisfaction Survey to measure client's overall satisfaction with the full array of services and experiences they have had with the CS on an annual basis. The...

DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation.

PubMed

Nacheva, Elizabeth; Mokretar, Katya; Soenmez, Aynur; Pittman, Alan M; Grace, Colin; Valli, Roberto; Ejaz, Ayesha; Vattathil, Selina; Maserati, Emanuela; Houlden, Henry; Taanman, Jan-Willem; Schapira, Anthony H; Proukakis, Christos

2017-01-01

Potential bias introduced during DNA isolation is inadequately explored, although it could have significant impact on downstream analysis. To investigate this in human brain, we isolated DNA from cerebellum and frontal cortex using spin columns under different conditions, and salting-out. We first analysed DNA using array CGH, which revealed a striking wave pattern suggesting primarily GC-rich cerebellar losses, even against matched frontal cortex DNA, with a similar pattern on a SNP array. The aCGH changes varied with the isolation protocol. Droplet digital PCR of two genes also showed protocol-dependent losses. Whole genome sequencing showed GC-dependent variation in coverage with spin column isolation from cerebellum. We also extracted and sequenced DNA from substantia nigra using salting-out and phenol / chloroform. The mtDNA copy number, assessed by reads mapping to the mitochondrial genome, was higher in substantia nigra when using phenol / chloroform. We thus provide evidence for significant method-dependent bias in DNA isolation from human brain, as reported in rat tissues. This may contribute to array "waves", and could affect copy number determination, particularly if mosaicism is being sought, and sequencing coverage. Variations in isolation protocol may also affect apparent mtDNA abundance.

DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation

PubMed Central

Nacheva, Elizabeth; Mokretar, Katya; Soenmez, Aynur; Pittman, Alan M.; Grace, Colin; Valli, Roberto; Ejaz, Ayesha; Vattathil, Selina; Maserati, Emanuela; Houlden, Henry; Taanman, Jan-Willem; Schapira, Anthony H.

2017-01-01

Potential bias introduced during DNA isolation is inadequately explored, although it could have significant impact on downstream analysis. To investigate this in human brain, we isolated DNA from cerebellum and frontal cortex using spin columns under different conditions, and salting-out. We first analysed DNA using array CGH, which revealed a striking wave pattern suggesting primarily GC-rich cerebellar losses, even against matched frontal cortex DNA, with a similar pattern on a SNP array. The aCGH changes varied with the isolation protocol. Droplet digital PCR of two genes also showed protocol-dependent losses. Whole genome sequencing showed GC-dependent variation in coverage with spin column isolation from cerebellum. We also extracted and sequenced DNA from substantia nigra using salting-out and phenol / chloroform. The mtDNA copy number, assessed by reads mapping to the mitochondrial genome, was higher in substantia nigra when using phenol / chloroform. We thus provide evidence for significant method-dependent bias in DNA isolation from human brain, as reported in rat tissues. This may contribute to array “waves”, and could affect copy number determination, particularly if mosaicism is being sought, and sequencing coverage. Variations in isolation protocol may also affect apparent mtDNA abundance. PMID:28683077

FilmArray, an Automated Nested Multiplex PCR System for Multi-Pathogen Detection: Development and Application to Respiratory Tract Infection

PubMed Central

Poritz, Mark A.; Blaschke, Anne J.; Byington, Carrie L.; Meyers, Lindsay; Nilsson, Kody; Jones, David E.; Thatcher, Stephanie A.; Robbins, Thomas; Lingenfelter, Beth; Amiott, Elizabeth; Herbener, Amy; Daly, Judy; Dobrowolski, Steven F.; Teng, David H. -F.; Ririe, Kirk M.

2011-01-01

The ideal clinical diagnostic system should deliver rapid, sensitive, specific and reproducible results while minimizing the requirements for specialized laboratory facilities and skilled technicians. We describe an integrated diagnostic platform, the “FilmArray”, which fully automates the detection and identification of multiple organisms from a single sample in about one hour. An unprocessed biologic/clinical sample is subjected to nucleic acid purification, reverse transcription, a high-order nested multiplex polymerase chain reaction and amplicon melt curve analysis. Biochemical reactions are enclosed in a disposable pouch, minimizing the PCR contamination risk. FilmArray has the potential to detect greater than 100 different nucleic acid targets at one time. These features make the system well-suited for molecular detection of infectious agents. Validation of the FilmArray technology was achieved through development of a panel of assays capable of identifying 21 common viral and bacterial respiratory pathogens. Initial testing of the system using both cultured organisms and clinical nasal aspirates obtained from children demonstrated an analytical and clinical sensitivity and specificity comparable to existing diagnostic platforms. We demonstrate that automated identification of pathogens from their corresponding target amplicon(s) can be accomplished by analysis of the DNA melting curve of the amplicon. PMID:22039434

Digital Detection of Multiple Minority Mutants and Expression Levels of Multiple Colorectal Cancer-Related Genes Using Digital-PCR Coupled with Bead-Array

PubMed Central

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

2015-01-01

To simultaneously analyze mutations and expression levels of multiple genes on one detection platform, we proposed a method termed “multiplex ligation-dependent probe amplification–digital amplification coupled with hydrogel bead-array” (MLPA–DABA) and applied it to diagnose colorectal cancer (CRC). CRC cells and tissues were sampled to extract nucleic acid, perform MLPA with sequence-tagged probes, perform digital emulsion polymerase chain reaction (PCR), and produce a hydrogel bead-array to immobilize beads and form a single bead layer on the array. After hybridization with fluorescent probes, the number of colored beads, which reflects the abundance of expressed genes and the mutation rate, was counted for diagnosis. Only red or green beads occurred on the chips in the mixed samples, indicating the success of single-molecule PCR. When a one-source sample was analyzed using mixed MLPA probes, beads of only one color occurred, suggesting the high specificity of the method in analyzing CRC mutation and gene expression. In gene expression analysis of a CRC tissue from one CRC patient, the mutant percentage was 3.1%, and the expression levels of CRC-related genes were much higher than those of normal tissue. The highly sensitive MLPA–DABA succeeds in the relative quantification of mutations and gene expressions of exfoliated cells in stool samples of CRC patients on the same chip platform. MLPA–DABA coupled with hydrogel bead-array is a promising method in the non-invasive diagnosis of CRC. PMID:25880764

Alignment of sensor arrays in optical instruments using a geometric approach.

PubMed

Sawyer, Travis W

2018-02-01

Alignment of sensor arrays in optical instruments is critical to maximize the instrument's performance. While many commercial systems use standardized mounting threads for alignment, custom systems require specialized equipment and alignment procedures. These alignment procedures can be time-consuming, dependent on operator experience, and have low repeatability. Furthermore, each alignment solution must be considered on a case-by-case basis, leading to additional time and resource cost. Here I present a method to align a sensor array using geometric analysis. By imaging a grid pattern of dots, I show that it is possible to calculate the misalignment for a sensor in five degrees of freedom simultaneously. I first test the approach by simulating different cases of misalignment using Zemax before applying the method to experimentally acquired data of sensor misalignment for an echelle spectrograph. The results show that the algorithm effectively quantifies misalignment in five degrees of freedom for an F/5 imaging system, accurate to within ±0.87  deg in rotation and ±0.86  μm in translation. Furthermore, the results suggest that the method can also be applied to non-imaging systems with a small penalty to precision. This general approach can potentially improve the alignment of sensor arrays in custom instruments by offering an accurate, quantitative approach to calculating misalignment in five degrees of freedom simultaneously.

C%2B%2B tensor toolbox user manual.

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

Plantenga, Todd D.; Kolda, Tamara Gibson

2012-04-01

The C++ Tensor Toolbox is a software package for computing tensor decompositions. It is based on the Matlab Tensor Toolbox, and is particularly optimized for sparse data sets. This user manual briefly overviews tensor decomposition mathematics, software capabilities, and installation of the package. Tensors (also known as multidimensional arrays or N-way arrays) are used in a variety of applications ranging from chemometrics to network analysis. The Tensor Toolbox provides classes for manipulating dense, sparse, and structured tensors in C++. The Toolbox compiles into libraries and is intended for use with custom applications written by users.

Integrated Microfluidic Devices for Automated Microarray-Based Gene Expression and Genotyping Analysis

NASA Astrophysics Data System (ADS)

Liu, Robin H.; Lodes, Mike; Fuji, H. Sho; Danley, David; McShea, Andrew

Microarray assays typically involve multistage sample processing and fluidic handling, which are generally labor-intensive and time-consuming. Automation of these processes would improve robustness, reduce run-to-run and operator-to-operator variation, and reduce costs. In this chapter, a fully integrated and self-contained microfluidic biochip device that has been developed to automate the fluidic handling steps for microarray-based gene expression or genotyping analysis is presented. The device consists of a semiconductor-based CustomArray® chip with 12,000 features and a microfluidic cartridge. The CustomArray was manufactured using a semiconductor-based in situ synthesis technology. The micro-fluidic cartridge consists of microfluidic pumps, mixers, valves, fluid channels, and reagent storage chambers. Microarray hybridization and subsequent fluidic handling and reactions (including a number of washing and labeling steps) were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. Electrochemical micropumps were integrated in the cartridge to provide pumping of liquid solutions. A micromixing technique based on gas bubbling generated by electrochemical micropumps was developed. Low-cost check valves were implemented in the cartridge to prevent cross-talk of the stored reagents. Gene expression study of the human leukemia cell line (K562) and genotyping detection and sequencing of influenza A subtypes have been demonstrated using this integrated biochip platform. For gene expression assays, the microfluidic CustomArray device detected sample RNAs with a concentration as low as 0.375 pM. Detection was quantitative over more than three orders of magnitude. Experiment also showed that chip-to-chip variability was low indicating that the integrated microfluidic devices eliminate manual fluidic handling steps that can be a significant source of variability in genomic analysis. The genotyping results showed that the device identified influenza A hemagglutinin and neuraminidase subtypes and sequenced portions of both genes, demonstrating the potential of integrated microfluidic and microarray technology for multiple virus detection. The device provides a cost-effective solution to eliminate labor-intensive and time-consuming fluidic handling steps and allows microarray-based DNA analysis in a rapid and automated fashion.

Altered retinal microRNA expression profiles in early diabetic retinopathy: an in silico analysis.

PubMed

Xiong, Fen; Du, Xinhua; Hu, Jianyan; Li, Tingting; Du, Shanshan; Wu, Qiang

2014-07-01

MicroRNAs (miRNAs) - as negative regulators of target genes - are associated with various human diseases, but their precise role(s) in diabetic retinopathy (DR) remains to be elucidated. The aim of this study was to elucidate the involvement of miRNAs in early DR using in silico analysis to explore their gene expression patterns. We used the streptozotocin (STZ)-induced diabetic rat to investigate the roles of miRNAs in early DR. Retinal miRNA expression profiles from diabetic versus healthy control rats were examined by miRNA array analysis. Based on several bioinformatic systems, specifically, gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, we identified signatures of the potential pathological processes, gene functions, and signaling pathways that are influenced by dysregulated miRNAs. We used quantitative real-time polymerase chain reaction (qRT-PCR) to validate six (i.e. those with significant changes in expression levels) of the 17 miRNAs that were detected in the miRNA array. We also describe the significant role of the miRNA-gene network, which is based on the interactions between miRNAs and target genes. GO analysis of the 17 miRNAs detected in the miRNA array analysis revealed the most prevalent miRNAs to be those related to biological processes, olfactory bulb development and axonogenesis. These miRNAs also exert significant influence on additional pathways, including the mitogen-activated protein and calcium signaling pathways. Six of the seventeen miRNAs were chosen for qRT-PCR validation. With the exception of a slight difference in miRNA-350, our results are in close agreement with the differential expressions detected by array analysis. This study, which describes miRNA expression during the early developmental phases of DR, revealed extensive miRNA interactions. Based on both their target genes and signaling pathways, we suggest that miRNAs perform critical regulatory functions during the early stages of DR evolution.

The Ozobranchus leech is a candidate mechanical vector for the fibropapilloma-associated turtle herpesvirus found latently infecting skin tumors on Hawaiian green turtles (Chelonia mydas)

USGS Publications Warehouse

Greenblatt, R.J.; Work, Thierry M.; Balazs, G.; Sutton, C.A.; Casey, R.N.; Casey, J.W.

2004-01-01

Fibropapillomatosis (FP) of marine turtles is a neoplastic disease of ecological concern. A fibropapilloma-associated turtle herpesvirus (FPTHV) is consistently present, usually at loads exceeding one virus copy per tumor cell. DNA from an array of parasites of green turtles (Chelonia mydas) was examined with quantitative PCR (qPCR) to determine whether any carried viral loads are sufficient to implicate them as vectors for FPTHV. Marine leeches (Ozobranchus spp.) were found to carry high viral DNA loads; some samples approached 10 million copies per leech. Isopycnic sucrose density gradient/qPCR analysis confirmed that some of these copies were associated with particles of the density of enveloped viruses. The data implicate the marine leech Ozobranchus as a mechanical vector for FPTHV. Quantitative RT-PCR analysis of FPTHV gene expression indicated that most of the FPTHV copies in a fibropapilloma have restricted DNA polymerase expression, suggestive of latent infection.

Automated Forensic Animal Family Identification by Nested PCR and Melt Curve Analysis on an Off-the-Shelf Thermocycler Augmented with a Centrifugal Microfluidic Disk Segment.

PubMed

Keller, Mark; Naue, Jana; Zengerle, Roland; von Stetten, Felix; Schmidt, Ulrike

2015-01-01

Nested PCR remains a labor-intensive and error-prone biomolecular analysis. Laboratory workflow automation by precise control of minute liquid volumes in centrifugal microfluidic Lab-on-a-Chip systems holds great potential for such applications. However, the majority of these systems require costly custom-made processing devices. Our idea is to augment a standard laboratory device, here a centrifugal real-time PCR thermocycler, with inbuilt liquid handling capabilities for automation. We have developed a microfluidic disk segment enabling an automated nested real-time PCR assay for identification of common European animal groups adapted to forensic standards. For the first time we utilize a novel combination of fluidic elements, including pre-storage of reagents, to automate the assay at constant rotational frequency of an off-the-shelf thermocycler. It provides a universal duplex pre-amplification of short fragments of the mitochondrial 12S rRNA and cytochrome b genes, animal-group-specific main-amplifications, and melting curve analysis for differentiation. The system was characterized with respect to assay sensitivity, specificity, risk of cross-contamination, and detection of minor components in mixtures. 92.2% of the performed tests were recognized as fluidically failure-free sample handling and used for evaluation. Altogether, augmentation of the standard real-time thermocycler with a self-contained centrifugal microfluidic disk segment resulted in an accelerated and automated analysis reducing hands-on time, and circumventing the risk of contamination associated with regular nested PCR protocols.

78 FR 69018 - Improving the Resiliency of Mobile Wireless Communications Networks; Reliability and Continuity...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-11-18

... offer their customers an increasing array of ``smartphones'' and data-centric devices, such as tablets... basis to provide service to their customers? 38. Additionally, the proposal would allow providers to... customers with comparable levels of service within that county? 40. Second, will consumers find this metric...

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

PubMed Central

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

2001-01-01

Mammalian nutritional status affects the homeostatic balance of multiple physiological processes and their associated gene expression. Although DNA array analysis can monitor large numbers of genes, there are no reports of expression profiling of a micronutrient deficiency in an intact animal system. In this report, we have tested the feasibility of using cDNA arrays to compare the global changes in expression of genes of known function that occur in the early stages of rodent zinc deficiency. The gene-modulating effects of this deficiency were demonstrated by real-time quantitative PCR measurements of altered mRNA levels for metallothionein 1, zinc transporter 2, and uroguanylin, all of which have been previously documented as zinc-regulated genes. As a result of the low level of inherent noise within this model system and application of a recently reported statistical tool for statistical analysis of microarrays [Tusher, V.G., Tibshirani, R. & Chu, G. (2001) Proc. Natl. Acad. Sci. USA 98, 5116–5121], we demonstrate the ability to reproducibly identify the modest changes in mRNA abundance produced by this single micronutrient deficiency. Among the genes identified by this array profile are intestinal genes that influence signaling pathways, growth, transcription, redox, and energy utilization. Additionally, the influence of dietary zinc supply on the expression of some of these genes was confirmed by real-time quantitative PCR. Overall, these data support the effectiveness of cDNA array expression profiling to investigate the pleiotropic effects of specific nutrients and may provide an approach to establishing markers for assessment of nutritional status. PMID:11717422

Xp22.3 genomic deletions involving the CDKL5 gene in girls with early onset epileptic encephalopathy.

PubMed

Mei, Davide; Marini, Carla; Novara, Francesca; Bernardina, Bernardo D; Granata, Tiziana; Fontana, Elena; Parrini, Elena; Ferrari, Anna R; Murgia, Alessandra; Zuffardi, Orsetta; Guerrini, Renzo

2010-04-01

Mutations of the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) cause an X-linked encephalopathy with early onset intractable epilepsy, including infantile spasms and other seizure types, and a Rett syndrome (RTT)-like phenotype. Very limited information is available on the frequency and phenotypic spectrum associated with CDKL5 deletions/duplications. We investigated the role of CDKL5 deletions/duplications in causing early onset intractable epilepsy of unknown etiology in girls. We studied 49 girls with early onset intractable epilepsy, with or without infantile spasms, and developmental impairment, for whom no etiologic factors were obvious after clinical examination, brain magnetic resonance imaging (MRI) and expanded screening for inborn errors of metabolism. We performed CDKL5 gene mutation analysis in all and multiplex ligation dependent probe amplification assay (MLPA) in those who were mutation negative. Custom Array-comparative genomic hybridization (CGH), breakpoint polymerase chain reaction (PCR) analysis, and X-inactivation studies were performed in patients in whom MLPA uncovered a genomic alteration. We found CDKL5 mutations in 8.2% (4 of 49) of patients and genomic deletions in 8.2% (4 of 49). Overall, abnormalities of the CDKL5 gene accounted for 16.3% (8 of 49) of patients. CDKL5 gene deletions are an under-ascertained cause of early onset intractable epilepsy in girls. Genetic testing of CDKL5, including both mutation and deletion/duplication analysis, should be considered in this clinical subgroup.

Detection of siRNA Mediated Target mRNA Cleavage Activities in Human Cells by a Novel Stem-Loop Array RT-PCR Analysis

DTIC Science & Technology

2016-09-07

sequences of the target mRNA, and a double stranded stem at the 5′ end that forms a stem -loop to function as a forceps to stabilize the secondary...E-mjournal homepage: www.elsevier.com/locate/bbrepDetection of siRNA-mediated target mRNA cleavage activities in human cells by a novel stem -loop...challenges for the accurate and efficient detection and verification of cleavage sites on target mRNAs. Here we used a sensitive stem -loop array reverse

Ultrasensitive Label-free Electronic Chip for DNA Analysis Using Carbon Nanotube Nanoelectrode Arrays

NASA Technical Reports Server (NTRS)

Li, Jun; Koehne, Jessica; Chen, Hua; Cassell, Alan; Ng, Hou Tee; Ye, Qi; Han, Jie; Meyyappan, M.

2004-01-01

There is a strong need for faster, cheaper, and simpler methods for nucleic acid analysis in today s clinical tests. Nanotechnologies can potentially provide solutions to these requirements by integrating nanomaterials with biofunctionalities. Dramatic improvement in the sensitivity and multiplexing can be achieved through the high-degree miniaturization. Here, we present our study in the development of an ultrasensitive label-free electronic chip for DNA/RNA analysis based on carbon nanotube nanoelectrode arrays. A reliable nanoelectrode array based on vertically aligned multi-walled carbon nanotubes (MWNTs) embedded in a SiO2 matrix is fabricated using a bottom-up approach. Characteristic nanoelectrode behavior is observed with a low-density MWNT nanoelectrode array in measuring both the bulk and surface immobilized redox species. The open-end of MWNTs are found to present similar properties as graphite edge-plane electrodes, with a wide potential window, flexible chemical functionalities, and good biocompatibility. A BRCA1 related oligonucleotide probe with 18 bases is covalently functionalized at the open ends of the MWNTs and specifically hybridized with an oligonucleotide target as well as a PCR amplicon. The guanine bases in the target molecules are employed as the signal moieties for the electrochemical measurements. Ru(bpy)3(2+) mediator is used to further amplify the guanine oxidation signal. This technique has been employed for direct electrochemical detection of label-free PCR amplicon through specific hybridization with the BRCAl probe. The detection limit is estimated to be less than approximately 1000 DNA molecules, approaching the limit of the sensitivity by laser-based fluorescence techniques in DNA microarray. This system provides a general electronic platform for rapid molecular diagnostics in applications requiring ultrahigh sensitivity, high-degree of miniaturization, simple sample preparation, and low- cost operation.

The microRNA Expression Profile in Donation after Cardiac Death (DCD) Livers and Its Ability to Identify Primary Non Function.

PubMed

Khorsandi, Shirin Elizabeth; Quaglia, Alberto; Salehi, Siamak; Jassem, Wayel; Vilca-Melendez, Hector; Prachalias, Andreas; Srinivasan, Parthi; Heaton, Nigel

2015-01-01

Donation after cardiac death (DCD) livers are marginal organs for transplant and their use is associated with a higher risk of primary non function (PNF) or early graft dysfunction (EGD). The aim was to determine if microRNA (miRNA) was able to discriminate between DCD livers of varying clinical outcome. DCD groups were categorized as PNF retransplanted within a week (n=7), good functional outcome (n=7) peak aspartate transaminase (AST) ≤ 1000 IU/L and EGD (n=9) peak AST ≥ 2500 IU/L. miRNA was extracted from archival formalin fixed post-perfusion tru-cut liver biopsies. High throughput expression analysis was performed using miRNA arrays. Bioinformatics for expression data analysis was performed and validated with real time quantitative PCR (RT-qPCR). The function of miRNA of interest was investigated using computational biology prediction algorithms. From the array analysis 16 miRNAs were identified as significantly different (p<0.05). On RT-qPCR miR-155 and miR-940 had the highest expression across all three DCD clinical groups. Only one miRNA, miR-22, was validated with marginal significance, to have differential expression between the three groups (p=0.049). From computational biology miR-22 was predicted to affect signalling pathways that impact protein turnover, metabolism and apoptosis/cell cycle. In conclusion, microRNA expression patterns have a low diagnostic potential clinically in discriminating DCD liver quality and outcome.

Detecting and Genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays

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

Call, Douglas R.; Brockman, Fred J.; Chandler, Darrell P.

2000-12-01

Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification.more » The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFU ml-1 (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products and the system is amenable to automation.« less

Detecting and genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays

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

Call, Douglas R.; Brockman, Fred J.; Chandler, Darrell P.

2001-07-05

Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification.more » The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFUs ml-1 (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products and the system is amenable to automation.« less

Motion camera based on a custom vision sensor and an FPGA architecture

NASA Astrophysics Data System (ADS)

Arias-Estrada, Miguel

1998-09-01

A digital camera for custom focal plane arrays was developed. The camera allows the test and development of analog or mixed-mode arrays for focal plane processing. The camera is used with a custom sensor for motion detection to implement a motion computation system. The custom focal plane sensor detects moving edges at the pixel level using analog VLSI techniques. The sensor communicates motion events using the event-address protocol associated to a temporal reference. In a second stage, a coprocessing architecture based on a field programmable gate array (FPGA) computes the time-of-travel between adjacent pixels. The FPGA allows rapid prototyping and flexible architecture development. Furthermore, the FPGA interfaces the sensor to a compact PC computer which is used for high level control and data communication to the local network. The camera could be used in applications such as self-guided vehicles, mobile robotics and smart surveillance systems. The programmability of the FPGA allows the exploration of further signal processing like spatial edge detection or image segmentation tasks. The article details the motion algorithm, the sensor architecture, the use of the event- address protocol for velocity vector computation and the FPGA architecture used in the motion camera system.

Analysis of Stress Distributions Under Lightweight Wheeled Vehicles

DTIC Science & Technology

2013-10-09

For a balanced analysis it is important at examine the full scale error ε f . Sinkage error, although large in a relative sense is typically on the...director of the Edgerton Center at MIT, to Thuan Doan, and to Meccanotecnica Riesi SRL for collaborating on manufacturing the custom sensing array...a pulling/braking force at the vehicle axle. Fx = T −Rc (33) The importance of drawbar force is obvious, since a positive drawbar force implies that

LGI1 microdeletion in autosomal dominant lateral temporal epilepsy

PubMed Central

Fanciulli, M.; Santulli, L.; Errichiello, L.; Barozzi, C.; Tomasi, L.; Rigon, L.; Cubeddu, T.; de Falco, A.; Rampazzo, A.; Michelucci, R.; Uzzau, S.; Striano, S.; de Falco, F.A.; Striano, P.

2012-01-01

Objectives: To characterize clinically and genetically a family with autosomal dominant lateral temporal epilepsy (ADLTE) negative to LGI1 exon sequencing test. Methods: All participants were personally interviewed and underwent neurologic examination. Most affected subjects underwent EEG and neuroradiologic examinations (CT/MRI). Available family members were genotyped with the HumanOmni1-Quad v1.0 single nucleotide polymorphism (SNP) array beadchip and copy number variations (CNVs) were analyzed in each subject. LGI1 gene dosage was performed by real-time quantitative PCR (qPCR). Results: The family had 8 affected members (2 deceased) over 3 generations. All of them showed GTC seizures, with focal onset in 6 and unknown onset in 2. Four patients had focal seizures with auditory features. EEG showed only minor sharp abnormalities in 3 patients and MRI was unremarkable in all the patients examined. Three family members presented major depression and anxiety symptoms. Routine LGI1 exon sequencing revealed no point mutation. High-density SNP array CNV analysis identified a genomic microdeletion about 81 kb in size encompassing the first 4 exons of LGI1 in all available affected members and in 2 nonaffected carriers, which was confirmed by qPCR analysis. Conclusions: This is the first microdeletion affecting LGI1 identified in ADLTE. Families with ADLTE in which no point mutations are revealed by direct exon sequencing should be screened for possible genomic deletion mutations by CNV analysis or other appropriate methods. Overall, CNV analysis of multiplex families may be useful for identifying microdeletions in novel disease genes. PMID:22496201

Promoter methylation assay of SASH1 gene in hepatocellular carcinoma.

PubMed

Peng, Liu; Wei, He; Liren, Li

2014-01-01

To analyse the relationship between the expression of SASH1 and its methylation level in human hepatocellular carcinoma. Expression levels of SASH1 were examined with real-time PCR (RT-PCR) in tissues and cells, and methylation analysis was performed with MassArray. The expression levels of SASH1 were strongly reduced in liver cancer tissues compared with adjacent normal tissues. Quantitative methylation analysis by MassArray revealed different CpG sites in SASH1 promoter shared similar methylation pattern between liver cancer tissues and adjacent normal tissues and the CpG sites of significant difference in methylation level were found as follows: CpG_3, CpG_17, CpG_21.22, CpG_25, CpG_26.27, CpG_28, CpG_34.35.36 and CpG_51.52. Moreover, 5-aza-2'-deoxycytidine treatment of Hep-G2 cell line caused significant elevation of SASH1 mRNA. Based on these data, we propose that increase of DNA methylation degree in the promoter region of SASH1 gene, particularly CpG_26.27 sites, possibly repressed SASH1 expression in liver cancer.

Promoter methylation assay of SASH1 gene in breast cancer.

PubMed

Sheyu, Lin; Hui, Liu; Junyu, Zhang; Jiawei, Xu; Honglian, Wang; Qing, Sang; Hengwei, Zhang; Xuhui, Guo; Qinghe, Xing; Lin, He

2013-01-01

To analyze the relationship between the expression of SASH1 and its methylation level of SASH1 gene promoter in human breast cancer. Expression levels of SASH1 were examined in breast cancer tissues and adjacent normal tissues with immunohistochemistry and with real time PCR (RT-PCR) methylation analysis was performed with MassArray. Immunohistochemistry showed that SASH1 expression was strongly reduced in breast cancer compared with adjacent normal tissues. Quantitative methylation analysis by MassArray revealed that CpG sites in SASH1 promoter shared similar methylation pattern in tumor tissue and adjacent normal tissue. The CpG sites with significant difference in methylation level were CpG_26.27 and CpG_54.55. Moreover, 5-aza-2'-deoxycytidine (5-Aza-dc) treatment of tumor cell line MDA-MB-231 caused significant elevation of SASH1 mRNA. Based on these data, we propose that increase of DNA methylation level in the promoter region of gene SASH1, particularly CpG_26.27 or CpG_54.55 sites, possibly repressed SASH1 expression in breast cancer.

Comparison of detection methods for HPV status as a prognostic marker for loco-regional control after radiochemotherapy in patients with HNSCC.

PubMed

Linge, Annett; Schötz, Ulrike; Löck, Steffen; Lohaus, Fabian; von Neubeck, Cläre; Gudziol, Volker; Nowak, Alexander; Tinhofer, Inge; Budach, Volker; Sak, Ali; Stuschke, Martin; Balermpas, Panagiotis; Rödel, Claus; Bunea, Hatice; Grosu, Anca-Ligia; Abdollahi, Amir; Debus, Jürgen; Ganswindt, Ute; Lauber, Kirsten; Pigorsch, Steffi; Combs, Stephanie E; Mönnich, David; Zips, Daniel; Baretton, Gustavo B; Buchholz, Frank; Krause, Mechthild; Belka, Claus; Baumann, Michael

2018-04-01

To compare six HPV detection methods in pre-treatment FFPE tumour samples from patients with locally advanced head and neck squamous cell carcinoma (HNSCC) who received postoperative (N = 175) or primary (N = 90) radiochemotherapy. HPV analyses included detection of (i) HPV16 E6/E7 RNA, (ii) HPV16 DNA (PCR-based arrays, A-PCR), (iii) HPV DNA (GP5+/GP6+ qPCR, (GP-PCR)), (iv) p16 (immunohistochemistry, p16 IHC), (v) combining p16 IHC and the A-PCR result and (vi) combining p16 IHC and the GP-PCR result. Differences between HPV positive and negative subgroups were evaluated for the primary endpoint loco-regional control (LRC) using Cox regression. Correlation between the HPV detection methods was high (chi-squared test, p < 0.001). While p16 IHC analysis resulted in several false positive classifications, A-PCR, GP-PCR and the combination of p16 IHC and A-PCR or GP-PCR led to results comparable to RNA analysis. In both cohorts, Cox regression analyses revealed significantly prolonged LRC for patients with HPV positive tumours irrespective of the detection method. The most stringent classification was obtained by detection of HPV16 RNA, or combining p16 IHC with A-PCR or GP-PCR. This approach revealed the lowest rate of recurrence in patients with tumours classified as HPV positive and therefore appears most suited for patient stratification in HPV-based clinical studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression Profiling in Bemisia tabaci under Insecticide Treatment: Indicating the Necessity for Custom Reference Gene Selection

PubMed Central

Zhou, Xuguo; Gao, Xiwu

2014-01-01

Finding a suitable reference gene is the key for qRT-PCR analysis. However, none of the reference gene discovered thus far can be utilized universally under various biotic and abiotic experimental conditions. In this study, we further examine the stability of candidate reference genes under a single abiotic factor, insecticide treatment. After being exposed to eight commercially available insecticides, which belong to five different classes, the expression profiles of eight housekeeping genes in the sweetpotato whitefly, Bemisia tabaci, one of the most invasive and destructive pests in the world, were investigated using qRT-PCR analysis. In summary, elongation factor 1α (EF1α), α-tubulin (TUB1α) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were identified as the most stable reference genes under the insecticide treatment. The initial assessment of candidate reference genes was further validated with the expression of two target genes, a P450 (Cyp6cm1) and a glutathione S-transferase (GST). However, ranking of reference genes varied substantially among intra- and inter-classes of insecticides. These combined data strongly suggested the necessity of conducting custom reference gene selection designed for each and every experimental condition, even when examining the same abiotic or biotic factor. PMID:24498122

The optics inside an automated single molecule array analyzer

NASA Astrophysics Data System (ADS)

McGuigan, William; Fournier, David R.; Watson, Gary W.; Walling, Les; Gigante, Bill; Duffy, David C.; Rissin, David M.; Kan, Cheuk W.; Meyer, Raymond E.; Piech, Tomasz; Fishburn, Matthew W.

2014-02-01

Quanterix and Stratec Biomedical have developed an instrument that enables the automated measurement of multiple proteins at concentration ~1000 times lower than existing immunoassays. The instrument is based on Quanterix's proprietary Single Molecule Array technology (Simoa™ ) that facilitates the detection and quantification of biomarkers previously difficult to measure, thus opening up new applications in life science research and in-vitro diagnostics. Simoa is based on trapping individual beads in arrays of femtoliter-sized wells that, when imaged with sufficient resolution, allows for counting of single molecules associated with each bead. When used to capture and detect proteins, this approach is known as digital ELISA (Enzyme-linked immunosorbent assay). The platform developed is a merger of many science and engineering disciplines. This paper concentrates on the optical technologies that have enabled the development of a fully-automated single molecule analyzer. At the core of the system is a custom, wide field-of-view, fluorescence microscope that images arrays of microwells containing single molecules bound to magnetic beads. A consumable disc containing 24 microstructure arrays was developed previously in collaboration with Sony DADC. The system cadence requirements, array dimensions, and requirement to detect single molecules presented significant optical challenges. Specifically, the wide field-of-view needed to image the entire array resulted in the need for a custom objective lens. Additionally, cost considerations for the system required a custom solution that leveraged the image processing capabilities. This paper will discuss the design considerations and resultant optical architecture that has enabled the development of an automated digital ELISA platform.

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

PubMed

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

2011-06-07

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

Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation.

PubMed

Sharma, Sunita; Sapkota, Dipak; Xue, Ying; Sun, Yang; Finne-Wistrand, Anna; Bruland, Ove; Mustafa, Kamal

2016-01-01

Selection of appropriate osteoinductive growth factors, suitable delivery method and proper supportive scaffold are critical for a successful outcome in bone tissue engineering using bone marrow stromal cells (BMSC). This study examined the molecular and functional effect of a combination of adenoviral mediated expression of bone morphogenetic protein-2 (BMP2) in BMSC and recently developed and characterized, biodegradable Poly(L-lactide-co-є-caprolactone){poly(LLA-co-CL)}scaffolds in osteogenic molecular changes and ectopic bone formation by using in vitro and in vivo approaches. Pathway-focused custom PCR array, validation using TaqMan based quantitative RT-PCR (qRT-PCR) and ALP staining showed significant up-regulation of several osteogenic and angiogenic molecules, including ALPL and RUNX2 in ad-BMP2 BMSC group grown in poly(LLA-co-CL) scaffolds both at 3 and 14 days. Micro CT and histological analyses of the subcutaneously implanted scaffolds in NOD/SCID mice revealed significantly increased radiopaque areas, percentage bone volume and formation of vital bone in ad-BMP2 scaffolds as compared to the control groups both at 2 and 8 weeks. The increased bone formation in the ad-BMP2 group in vivo was paralleled at the molecular level with concomitant over-expression of a number of osteogenic and angiogenic genes including ALPL, RUNX2, SPP1, ANGPT1. The increased bone formation in ad-BMP2 explants was not found to be associated with enhanced endochondral activity as evidenced by qRT-PCR (SOX9 and FGF2) and Safranin O staining. Taken together, combination of adenoviral mediated BMP-2 expression in BMSC grown in the newly developed poly(LLA-co-CL) scaffolds induced expression of osteogenic markers and enhanced bone formation in vivo.

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

PubMed

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

2018-06-15

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

Proof of Concept Study to Assess Fetal Gene Expression in Amniotic Fluid by NanoArray PCR

PubMed Central

Massingham, Lauren J.; Johnson, Kirby L.; Bianchi, Diana W.; Pei, Shermin; Peter, Inga; Cowan, Janet M.; Tantravahi, Umadevi; Morrison, Tom B.

2011-01-01

Microarray analysis of cell-free RNA in amniotic fluid (AF) supernatant has revealed differential fetal gene expression as a function of gestational age and karyotype. Once informative genes are identified, research moves to a more focused platform such as quantitative reverse transcriptase-PCR. Standardized NanoArray PCR (SNAP) is a recently developed gene profiling technology that enables the measurement of transcripts from samples containing reduced quantities or degraded nucleic acids. We used a previously developed SNAP gene panel as proof of concept to determine whether fetal functional gene expression could be ascertained from AF supernatant. RNA was extracted and converted to cDNA from 19 AF supernatant samples of euploid fetuses between 15 to 20 weeks of gestation, and transcript abundance of 21 genes was measured. Statistically significant differences in expression, as a function of advancing gestational age, were observed for 5 of 21 genes. ANXA5, GUSB, and PPIA showed decreasing gene expression over time, whereas CASC3 and ZNF264 showed increasing gene expression over time. Statistically significantly increased expression of MTOR and STAT2 was seen in female compared with male fetuses. This study demonstrates the feasibility of focused fetal gene expression analysis using SNAP technology. In the future, this technique could be optimized to examine specific genes instrumental in fetal organ system function, which could be a useful addition to prenatal care. PMID:21827969

High-performance integrated pick-up circuit for SPAD arrays in time-correlated single photon counting

NASA Astrophysics Data System (ADS)

Acconcia, Giulia; Cominelli, Alessandro; Peronio, Pietro; Rech, Ivan; Ghioni, Massimo

2017-05-01

The analysis of optical signals by means of Single Photon Avalanche Diodes (SPADs) has been subject to a widespread interest in recent years. The development of multichannel high-performance Time Correlated Single Photon Counting (TCSPC) acquisition systems has undergone a fast trend. Concerning the detector performance, best in class results have been obtained resorting to custom technologies leading also to a strong dependence of the detector timing jitter from the threshold used to determine the onset of the photogenerated current flow. In this scenario, the avalanche current pick-up circuit plays a key role in determining the timing performance of the TCSPC acquisition system, especially with a large array of SPAD detectors because of electrical crosstalk issues. We developed a new current pick-up circuit based on a transimpedance amplifier structure able to extract the timing information from a 50-μm-diameter custom technology SPAD with a state-of-art timing jitter as low as 32ps and suitable to be exploited with SPAD arrays. In this paper we discuss the key features of this structure and we present a new version of the pick-up circuit that also provides quenching capabilities in order to minimize the number of interconnections required, an aspect that becomes more and more crucial in densely integrated systems.

'FloraArray' for screening of specific DNA probes representing the characteristics of a certain microbial community.

PubMed

Yokoi, Takahide; Kaku, Yoshiko; Suzuki, Hiroyuki; Ohta, Masayuki; Ikuta, Hajime; Isaka, Kazuichi; Sumino, Tatsuo; Wagatsuma, Masako

2007-08-01

To investigate uncharacterized microbial communities, a custom DNA microarray named 'FloraArray' was developed for screening specific probes that would represent the characteristics of a microbial community. The array was prepared by spotting 2000 plasmid DNAs from a genomic shotgun library of a sludge sample on a DNA microarray. By comparative hybridization of the array with two different samples of genomic DNA, one from the activated sludge and the other from a nonactivated sludge sample of an anaerobic ammonium oxidation (anammox) bacterial community, specific spots were visualized as a definite fluctuating profile in an MA (differential intensity ratio vs. spot intensity) plot. About 300 spots of the array accounted for the candidate probes to represent anammox reaction of the activated sludge. After sequence analysis of the probes and examination of the results of blastn searches against the reported anammox reference sequence, complete matches were found for 161 probes (58.3%) and >90% matches were found for 242 probes (87.1%). These results demonstrate that 'FloraArray' could be a useful tool for screening specific DNA molecules of unknown microbial communities.

Biomechanical Characterization of Cardiomyocyte Using PDMS Pillar with Microgrooves

PubMed Central

Oyunbaatar, Nomin-Erdene; Lee, Deok-Hyu; Patil, Swati J.; Kim, Eung-Sam; Lee, Dong-Weon

2016-01-01

This paper describes the surface-patterned polydimethylsiloxane (PDMS) pillar arrays for enhancing cell alignment and contraction force in cardiomyocytes. The PDMS micropillar (μpillar) arrays with microgrooves (μgrooves) were fabricated using a unique micro-mold made using SU-8 double layer processes. The spring constant of the μpillar arrays was experimentally confirmed using atomic force microscopy (AFM). After culturing cardiac cells on the two different types of μpillar arrays, with and without grooves on the top of μpillar, the characteristics of the cardiomyocytes were analyzed using a custom-made image analysis system. The alignment of the cardiomyocytes on the μgrooves of the μpillars was clearly observed using a DAPI staining process. The mechanical force generated by the contraction force of the cardiomyocytes was derived from the displacement of the μpillar arrays. The contraction force of the cardiomyocytes aligned on the μgrooves was 20% higher than that of the μpillar arrays without μgrooves. The experimental results prove that applied geometrical stimulus is an effective method for aligning and improving the contraction force of cardiomyocytes. PMID:27517924

Method for replicating an array of nucleic acid probes

DOEpatents

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

1998-01-01

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

Exercising calf muscle T₂∗ changes correlate with pH, PCr recovery and maximum oxidative phosphorylation.

PubMed

Schmid, Albrecht Ingo; Schewzow, Kiril; Fiedler, Georg Bernd; Goluch, Sigrun; Laistler, Elmar; Wolzt, Michael; Moser, Ewald; Meyerspeer, Martin

2014-05-01

Skeletal muscle metabolism is impaired in disorders like diabetes mellitus or peripheral vascular disease. The skeletal muscle echo planar imaging (EPI) signal (S(EPI) ) and its relation to energy metabolism are still debated. Localised ³¹P MRS and S(EPI) data from gastrocnemius medialis of 19 healthy subjects were combined in one scanning session to study direct relationships between phosphocreatine (PCr), pH kinetics and parameters of T₂∗ time courses. Dynamic spectroscopy (semi-LASER) and EPI were performed immediately before, during and after 5 min of plantar flexions. Data were acquired in a 7 T MR scanner equipped with a custom-built ergometer and a dedicated ³¹P/¹H radio frequency (RF) coil array. Using a form-fitted multi-channel ³¹P/¹H coil array resulted in high signal-to-noise ratio (SNR). PCr and pH in the gastrocnemius medialis muscle were quantified from each ³¹P spectrum, acquired every 6 s. During exercise, SEPI (t) was found to be a linear function of tissue pH(t) (cross-correlation r = -0.85 ± 0.07). Strong Pearson's correlations were observed between post exercise time-to-peak (TTP) of SEPI and (a) the time constant of PCr recovery τPCr recovery (r = 0.89, p < 10⁻⁶), (b) maximum oxidative phosphorylation using the linear model, Q(max, lin) (r = 0.65, p = 0.002), the adenosine-diphosphate-driven model, Q(max,ADP) (r = 0.73, p = 0.0002) and (c) end exercise pH (r = 0.60, p = 0.005). Based on combined accurately localised ³¹P MRS and T₂∗ weighted MRI, both with high temporal resolution, strong correlations of the skeletal muscle SEPI during exercise and tissue pH time courses and of post exercise SEPI and parameters of energy metabolism were observed. In conclusion, a tight coupling between skeletal muscle metabolic activity and tissue T₂∗ signal weighting, probably induced by osmotically driven water shift, exists and can be measured non-invasively, using NMR at 7 T. © 2014 The Authors. NMR in Biomedicine published by John Wiley & Sons, Ltd.

A 4×8-Gbps VCSEL array driver ASIC and integration with a custom array transmitter module for the LHC front-end transmission

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

Guo, Di; Liu, Chonghan; Chen, Jinghong

This paper describes the design, fabrication and experiment results of a 4×8-Gbps Vertical-Cavity Surface-Emitting Laser (VCSEL) array driver ASIC with the adjustable active-shunt peaking technique and the novel balanced output structure under the Silicon-on-Sapphire (SOS) process, and a custom array optical transmitter module, featuring a compact size of 10 mm×15 mm×5.3 mm. Both the array driver ASIC and the module have been fully tested after integration as a complete parallel transmitter. Optical eye diagram of each channel passes the eye mask at 8 Gbps/ch with adjacent channel working simultaneously with a power consumption of 150 mW/ch. As a result, themore » optical transmission of Bit-Error Rate (BER) less than 10E-12 is achieved at an aggregated data rate of 4×8-Gbps.« less

A 4×8-Gbps VCSEL array driver ASIC and integration with a custom array transmitter module for the LHC front-end transmission

DOE PAGES

Guo, Di; Liu, Chonghan; Chen, Jinghong; ...

2016-03-21

This paper describes the design, fabrication and experiment results of a 4×8-Gbps Vertical-Cavity Surface-Emitting Laser (VCSEL) array driver ASIC with the adjustable active-shunt peaking technique and the novel balanced output structure under the Silicon-on-Sapphire (SOS) process, and a custom array optical transmitter module, featuring a compact size of 10 mm×15 mm×5.3 mm. Both the array driver ASIC and the module have been fully tested after integration as a complete parallel transmitter. Optical eye diagram of each channel passes the eye mask at 8 Gbps/ch with adjacent channel working simultaneously with a power consumption of 150 mW/ch. As a result, themore » optical transmission of Bit-Error Rate (BER) less than 10E-12 is achieved at an aggregated data rate of 4×8-Gbps.« less

Automated Forensic Animal Family Identification by Nested PCR and Melt Curve Analysis on an Off-the-Shelf Thermocycler Augmented with a Centrifugal Microfluidic Disk Segment

PubMed Central

Zengerle, Roland; von Stetten, Felix; Schmidt, Ulrike

2015-01-01

Nested PCR remains a labor-intensive and error-prone biomolecular analysis. Laboratory workflow automation by precise control of minute liquid volumes in centrifugal microfluidic Lab-on-a-Chip systems holds great potential for such applications. However, the majority of these systems require costly custom-made processing devices. Our idea is to augment a standard laboratory device, here a centrifugal real-time PCR thermocycler, with inbuilt liquid handling capabilities for automation. We have developed a microfluidic disk segment enabling an automated nested real-time PCR assay for identification of common European animal groups adapted to forensic standards. For the first time we utilize a novel combination of fluidic elements, including pre-storage of reagents, to automate the assay at constant rotational frequency of an off-the-shelf thermocycler. It provides a universal duplex pre-amplification of short fragments of the mitochondrial 12S rRNA and cytochrome b genes, animal-group-specific main-amplifications, and melting curve analysis for differentiation. The system was characterized with respect to assay sensitivity, specificity, risk of cross-contamination, and detection of minor components in mixtures. 92.2% of the performed tests were recognized as fluidically failure-free sample handling and used for evaluation. Altogether, augmentation of the standard real-time thermocycler with a self-contained centrifugal microfluidic disk segment resulted in an accelerated and automated analysis reducing hands-on time, and circumventing the risk of contamination associated with regular nested PCR protocols. PMID:26147196

Customized Molecular Phenotyping by Quantitative Gene Expression and Pattern Recognition Analysis

PubMed Central

Akilesh, Shreeram; Shaffer, Daniel J.; Roopenian, Derry

2003-01-01

Description of the molecular phenotypes of pathobiological processes in vivo is a pressing need in genomic biology. We have implemented a high-throughput real-time PCR strategy to establish quantitative expression profiles of a customized set of target genes. It enables rapid, reproducible data acquisition from limited quantities of RNA, permitting serial sampling of mouse blood during disease progression. We developed an easy to use statistical algorithm—Global Pattern Recognition—to readily identify genes whose expression has changed significantly from healthy baseline profiles. This approach provides unique molecular signatures for rheumatoid arthritis, systemic lupus erythematosus, and graft versus host disease, and can also be applied to defining the molecular phenotype of a variety of other normal and pathological processes. PMID:12840047

Automated methods for multiplexed pathogen detection.

PubMed

Straub, Timothy M; Dockendorff, Brian P; Quiñonez-Díaz, Maria D; Valdez, Catherine O; Shutthanandan, Janani I; Tarasevich, Barbara J; Grate, Jay W; Bruckner-Lea, Cynthia J

2005-09-01

Detection of pathogenic microorganisms in environmental samples is a difficult process. Concentration of the organisms of interest also co-concentrates inhibitors of many end-point detection methods, notably, nucleic acid methods. In addition, sensitive, highly multiplexed pathogen detection continues to be problematic. The primary function of the BEADS (Biodetection Enabling Analyte Delivery System) platform is the automated concentration and purification of target analytes from interfering substances, often present in these samples, via a renewable surface column. In one version of BEADS, automated immunomagnetic separation (IMS) is used to separate cells from their samples. Captured cells are transferred to a flow-through thermal cycler where PCR, using labeled primers, is performed. PCR products are then detected by hybridization to a DNA suspension array. In another version of BEADS, cell lysis is performed, and community RNA is purified and directly labeled. Multiplexed detection is accomplished by direct hybridization of the RNA to a planar microarray. The integrated IMS/PCR version of BEADS can successfully purify and amplify 10 E. coli O157:H7 cells from river water samples. Multiplexed PCR assays for the simultaneous detection of E. coli O157:H7, Salmonella, and Shigella on bead suspension arrays was demonstrated for the detection of as few as 100 cells for each organism. Results for the RNA version of BEADS are also showing promising results. Automation yields highly purified RNA, suitable for multiplexed detection on microarrays, with microarray detection specificity equivalent to PCR. Both versions of the BEADS platform show great promise for automated pathogen detection from environmental samples. Highly multiplexed pathogen detection using PCR continues to be problematic, but may be required for trace detection in large volume samples. The RNA approach solves the issues of highly multiplexed PCR and provides "live vs. dead" capabilities. However, sensitivity of the method will need to be improved for RNA analysis to replace PCR.

Automated Methods for Multiplexed Pathogen Detection

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

Straub, Tim M.; Dockendorff, Brian P.; Quinonez-Diaz, Maria D.

2005-09-01

Detection of pathogenic microorganisms in environmental samples is a difficult process. Concentration of the organisms of interest also co-concentrates inhibitors of many end-point detection methods, notably, nucleic acid methods. In addition, sensitive, highly multiplexed pathogen detection continues to be problematic. The primary function of the BEADS (Biodetection Enabling Analyte Delivery System) platform is the automated concentration and purification of target analytes from interfering substances, often present in these samples, via a renewable surface column. In one version of BEADS, automated immunomagnetic separation (IMS) is used to separate cells from their samples. Captured cells are transferred to a flow-through thermal cyclermore » where PCR, using labeled primers, is performed. PCR products are then detected by hybridization to a DNA suspension array. In another version of BEADS, cell lysis is performed, and community RNA is purified and directly labeled. Multiplexed detection is accomplished by direct hybridization of the RNA to a planar microarray. The integrated IMS/PCR version of BEADS can successfully purify and amplify 10 E. coli O157:H7 cells from river water samples. Multiplexed PCR assays for the simultaneous detection of E. coli O157:H7, Salmonella, and Shigella on bead suspension arrays was demonstrated for the detection of as few as 100 cells for each organism. Results for the RNA version of BEADS are also showing promising results. Automation yields highly purified RNA, suitable for multiplexed detection on microarrays, with microarray detection specificity equivalent to PCR. Both versions of the BEADS platform show great promise for automated pathogen detection from environmental samples. Highly multiplexed pathogen detection using PCR continues to be problematic, but may be required for trace detection in large volume samples. The RNA approach solves the issues of highly multiplexed PCR and provides ''live vs. dead'' capabilities. However, sensitivity of the method will need to be improved for RNA analysis to replace PCR.« less

Characterization of Custom-Designed Charge-Coupled Devices for Applications to Gas and Aerosol Monitoring Sensorcraft Instrument

NASA Technical Reports Server (NTRS)

Refaat, Tamer F.; Abedin, M. Nurul; Farnsworth, Glenn R.; Garcia, Christopher S.; Zawodny, Joseph M.

2005-01-01

Custom-designed charge-coupled devices (CCD) for Gas and Aerosols Monitoring Sensorcraft instrument were developed. These custom-designed CCD devices are linear arrays with pixel format of 512x1 elements and pixel size of 10x200 sq m. These devices were characterized at NASA Langley Research Center to achieve a full well capacity as high as 6,000,000 e-. This met the aircraft flight mission requirements in terms of signal-to-noise performance and maximum dynamic range. Characterization and analysis of the electrical and optical properties of the CCDs were carried out at room temperature. This includes measurements of photon transfer curves, gain coefficient histograms, read noise, and spectral response. Test results obtained on these devices successfully demonstrated the objectives of the aircraft flight mission. In this paper, we describe the characterization results and also discuss their applications to future mission.

Zoom‐in comparative genomic hybridisation arrays for the characterisation of variable breakpoint contiguous gene syndromes

PubMed Central

Johnston, Jennifer J; Walker, Robert L; Davis, Sean; Facio, Flavia; Turner, Joyce T; Bick, David P; Daentl, Donna L; Ellison, Jay W; Meltzer, Paul S; Biesecker, Leslie G

2007-01-01

Contiguous gene syndromes cause disorders via haploinsufficiency for adjacent genes. Some contiguous gene syndromes (CGS) have stereotypical breakpoints, but others have variable breakpoints. In CGS that have variable breakpoints, the extent of the deletions may be correlated with severity. The Greig cephalopolysyndactyly contiguous gene syndrome (GCPS‐CGS) is a multiple malformation syndrome caused by haploinsufficiency of GLI3 and adjacent genes. In addition, non‐CGS GCPS can be caused by deletions or duplications in GLI3. Although fluorescence in situ hybridisation (FISH) can identify large deletion mutations in patients with GCPS or GCPS‐CGS, it is not practical for identification of small intragenic deletions or insertions, and it is difficult to accurately characterise the extent of the large deletions using this technique. We have designed a custom comparative genomic hybridisation (CGH) array that allows identification of deletions and duplications at kilobase resolution in the vicinity of GLI3. The array averages one probe every 730 bp for a total of about 14 000 probes over 10 Mb. We have analysed 16 individuals with known or suspected deletions or duplications. In 15 of 16 individuals (14 deletions and 1 duplication), the array confirmed the prior results. In the remaining patient, the normal CGH array result was correct, and the prior assessment was a false positive quantitative polymerase chain reaction result. We conclude that high‐density CGH array analysis is more sensitive than FISH analysis for detecting deletions and provides clinically useful results on the extent of the deletion. We suggest that high‐density CGH array analysis should replace FISH analysis for assessment of deletions and duplications in patients with contiguous gene syndromes caused by variable deletions. PMID:17098889

Direct elicitation of template concentration from quantification cycle (Cq) distributions in digital PCR.

PubMed

Mojtahedi, Mitra; Fouquier d'Hérouël, Aymeric; Huang, Sui

2014-01-01

Digital PCR (dPCR) exploits limiting dilution of a template into an array of PCR reactions. From this array the number of reactions that contain at least one (as opposed to zero) initial template is determined, allowing inferring the original template concentration. Here we present a novel protocol to efficiently infer the concentration of a sample and its optimal dilution for dPCR from few targeted qPCR assays. By taking advantage of the real-time amplification feature of qPCR as opposed to relying on endpoint PCR assessment as in standard dPCR prior knowledge of template concentration is not necessary. This eliminates the need for serial dilutions in a separate titration and reduces the number of necessary reactions. We describe the theory underlying our approach and discuss experimental moments that contribute to uncertainty. We present data from a controlled experiment where the initial template concentration is known as proof of principle and apply our method on directly monitoring transcript level change during cell differentiation as well as gauging amplicon numbers in cDNA samples after pre-amplification. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Method for replicating an array of nucleic acid probes

DOEpatents

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

1998-08-18

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

Transcriptome discovery in non-model wild fish species for the development of quantitative transcript abundance assays

USGS Publications Warehouse

Hahn, Cassidy M.; Iwanowicz, Luke R.; Cornman, Robert S.; Mazik, Patricia M.; Blazer, Vicki S.

2016-01-01

Environmental studies increasingly identify the presence of both contaminants of emerging concern (CECs) and legacy contaminants in aquatic environments; however, the biological effects of these compounds on resident fishes remain largely unknown. High throughput methodologies were employed to establish partial transcriptomes for three wild-caught, non-model fish species; smallmouth bass (Micropterus dolomieu), white sucker (Catostomus commersonii) and brown bullhead (Ameiurus nebulosus). Sequences from these transcriptome databases were utilized in the development of a custom nCounter CodeSet that allowed for direct multiplexed measurement of 50 transcript abundance endpoints in liver tissue. Sequence information was also utilized in the development of quantitative real-time PCR (qPCR) primers. Cross-species hybridization allowed the smallmouth bass nCounter CodeSet to be used for quantitative transcript abundance analysis of an additional non-model species, largemouth bass (Micropterus salmoides). We validated the nCounter analysis data system with qPCR for a subset of genes and confirmed concordant results. Changes in transcript abundance biomarkers between sexes and seasons were evaluated to provide baseline data on transcript modulation for each species of interest.

Comparison of the FilmArray Respiratory Panel and Prodesse Real-Time PCR Assays for Detection of Respiratory Pathogens ▿ †

PubMed Central

Loeffelholz, M. J.; Pong, D. L.; Pyles, R. B.; Xiong, Y.; Miller, A. L.; Bufton, K. K.; Chonmaitree, T.

2011-01-01

We compared the diagnostic performance and overall respiratory pathogen detection rate of the premarket version of the FilmArray Respiratory Panel (RP) multiplex PCR assay (Idaho Technology, Inc., Salt Lake City, UT) with those of the Food and Drug Administration (FDA)-cleared Prodesse ProFlu+, ProFAST+, ProParaflu+, Pro hMPV+, and ProAdeno+ real-time PCR assays (Gen-Probe, San Diego, CA). The assays were performed on a panel of 192 nasopharyngeal-secretion specimens collected from 81 children under 1 year of age with upper respiratory tract symptoms. To resolve discordant results and confirm pathogens detected only by the larger FilmArray panel, we performed laboratory-developed real-time PCR assays. Among viruses detectable by both commercial assays (adenovirus, human metapneumovirus, influenza A virus, influenza B virus, parainfluenza viruses 1 to 3, and respiratory syncytial virus), the FilmArray and Prodesse assays showed good overall agreement (181/192 [94.3%]; kappa = 0.87; 95% CI, 0.79 to 0.94). FilmArray RP detected more parainfluenza viruses 1 and 3 than ProParaflu+ (18 versus 13) while ProAdeno+ detected more adenoviruses (11 versus 6), but these differences were not statistically significant. Additionally, FilmArray RP detected 138 pathogens (confirmed as true positives) not included in the Prodesse assays (rhinovirus [RV]/enterovirus [EV], 118; bocavirus, 8; coronavirus, 7; parainfluenza virus 4, 4; Mycoplasma pneumoniae, 1). FilmArray RP was cleared by the FDA following the completion of this study. The FDA-cleared version includes the following targets: adenovirus, coronaviruses HKU1 and NL63, human metapneumovirus (hMPV), influenza A virus (to type level only), influenza A H1 seasonal virus, influenza A H3 seasonal virus, influenza A virus H1-2009, influenza B virus, parainfluenza viruses 1 to 4, respiratory syncytial virus (RSV), and RV/EV (no differentiation). The larger panel in the FilmArray RP assay allowed the detection of additional respiratory pathogens compared to the Prodesse assays. In this population of young children with upper respiratory tract infection, RV/EV accounted for the majority of the additional pathogens detected by FilmArray RP. PMID:21998418

Bacterial discrimination by means of a universal array approach mediated by LDR (ligase detection reaction)

PubMed Central

Busti, Elena; Bordoni, Roberta; Castiglioni, Bianca; Monciardini, Paolo; Sosio, Margherita; Donadio, Stefano; Consolandi, Clarissa; Rossi Bernardi, Luigi; Battaglia, Cristina; De Bellis, Gianluca

2002-01-01

Background PCR amplification of bacterial 16S rRNA genes provides the most comprehensive and flexible means of sampling bacterial communities. Sequence analysis of these cloned fragments can provide a qualitative and quantitative insight of the microbial population under scrutiny although this approach is not suited to large-scale screenings. Other methods, such as denaturing gradient gel electrophoresis, heteroduplex or terminal restriction fragment analysis are rapid and therefore amenable to field-scale experiments. A very recent addition to these analytical tools is represented by microarray technology. Results Here we present our results using a Universal DNA Microarray approach as an analytical tool for bacterial discrimination. The proposed procedure is based on the properties of the DNA ligation reaction and requires the design of two probes specific for each target sequence. One oligo carries a fluorescent label and the other a unique sequence (cZipCode or complementary ZipCode) which identifies a ligation product. Ligated fragments, obtained in presence of a proper template (a PCR amplified fragment of the 16s rRNA gene) contain either the fluorescent label or the unique sequence and therefore are addressed to the location on the microarray where the ZipCode sequence has been spotted. Such an array is therefore "Universal" being unrelated to a specific molecular analysis. Here we present the design of probes specific for some groups of bacteria and their application to bacterial diagnostics. Conclusions The combined use of selective probes, ligation reaction and the Universal Array approach yielded an analytical procedure with a good power of discrimination among bacteria. PMID:12243651

Single step generation of protein arrays from DNA by cell-free expression and in situ immobilisation (PISA method).

PubMed

He, M; Taussig, M J

2001-08-01

We describe a format for production of protein arrays termed 'protein in situ array' (PISA). A PISA is rapidly generated in one step directly from PCR-generated DNA fragments by cell-free protein expression and in situ immobilisation at a surface. The template for expression is DNA encoding individual proteins or domains, which is produced by PCR using primers designed from information in DNA databases. Coupled transcription and translation is carried out on a surface to which the tagged protein adheres as soon as it is synthesised. Because proteins generated by cell-free synthesis are usually soluble and functional, this method can overcome problems of insolubility or degradation associated with bacterial expression of recombinant proteins. Moreover, the use of PCR-generated DNA enables rapid production of proteins or domains based on genome information alone and will be particularly useful where cloned material is not available. Here we show that human single-chain antibody fragments (three domain, V(H)/K form) and an enzyme (luciferase) can be functionally arrayed by the PISA method.

The Microbial Detection Array Combined with Random Phi29-Amplification Used as a Diagnostic Tool for Virus Detection in Clinical Samples

PubMed Central

Erlandsson, Lena; Rosenstierne, Maiken W.; McLoughlin, Kevin; Jaing, Crystal; Fomsgaard, Anders

2011-01-01

A common technique used for sensitive and specific diagnostic virus detection in clinical samples is PCR that can identify one or several viruses in one assay. However, a diagnostic microarray containing probes for all human pathogens could replace hundreds of individual PCR-reactions and remove the need for a clear clinical hypothesis regarding a suspected pathogen. We have established such a diagnostic platform for random amplification and subsequent microarray identification of viral pathogens in clinical samples. We show that Phi29 polymerase-amplification of a diverse set of clinical samples generates enough viral material for successful identification by the Microbial Detection Array, demonstrating the potential of the microarray technique for broad-spectrum pathogen detection. We conclude that this method detects both DNA and RNA virus, present in the same sample, as well as differentiates between different virus subtypes. We propose this assay for diagnostic analysis of viruses in clinical samples. PMID:21853040

Optimization of multiplexed PCR on an integrated microfluidic forensic platform for rapid DNA analysis.

PubMed

Estes, Matthew D; Yang, Jianing; Duane, Brett; Smith, Stan; Brooks, Carla; Nordquist, Alan; Zenhausern, Frederic

2012-12-07

This study reports the design, prototyping, and assay development of multiplexed polymerase chain reaction (PCR) on a plastic microfluidic device. Amplification of 17 DNA loci is carried out directly on-chip as part of a system for continuous workflow processing from sample preparation (SP) to capillary electrophoresis (CE). For enhanced performance of on-chip PCR amplification, improved control systems have been developed making use of customized Peltier assemblies, valve actuators, software, and amplification chemistry protocols. Multiple enhancements to the microfluidic chip design have been enacted to improve the reliability of sample delivery through the various on-chip modules. This work has been enabled by the encapsulation of PCR reagents into a solid phase material through an optimized Solid Phase Encapsulating Assay Mix (SPEAM) bead-based hydrogel fabrication process. SPEAM bead technology is reliably coupled with precise microfluidic metering and dispensing for efficient amplification and subsequent DNA short tandem repeat (STR) fragment analysis. This provides a means of on-chip reagent storage suitable for microfluidic automation, with the long shelf-life necessary for point-of-care (POC) or field deployable applications. This paper reports the first high quality 17-plex forensic STR amplification from a reference sample in a microfluidic chip with preloaded solid phase reagents, that is designed for integration with up and downstream processing.

High-resolution mapping and sequence analysis of 597 cDNA clones transcribed from the 1 Mb region in human chromosome 4q16.3 containing Huntington disease gene

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

Hadano, S.; Ishida, Y.; Tomiyasu, H.

1994-09-01

To complete a transcription map of the 1 Mb region in human chromosome 4p16.3 containing the Huntington disease (HD) gene, the isolation of cDNA clones are being performed throughout. Our method relies on a direct screening of the cDNA libraries probed with single copy microclones from 3 YAC clones spanning 1 Mbp of the HD gene region. AC-DNAs were isolated by a preparative pulsed-field gel electrophoresis, amplified by both a single unique primer (SUP)-PCR and a linker ligation PCR, and 6 microclone-DNA libraries were generated. Then, 8,640 microclones from these libraries were independently amplified by PCR, and arrayed onto themore » membranes. 800-900 microclones that were not cross-hybridized with total human and yeast genomic DNA, TAC vector DNA, and ribosomal cDNA on a dot hybridization (putatively carrying single copy sequences) were pooled to make 9 probe pools. A total of {approximately}1.8x10{sup 7} plaques from the human brain cDNA libraries was screened with 9 pool-probes, and then 672 positive cDNA clones were obtained. So far, 597 cDNA clones were defined and arrayed onto a map of the 1 Mbp of the HD gene region by hybridization with HD region-specific cosmid contigs and YAC clones. Further characterization including a DNA sequencing and Northern blot analysis is currently underway.« less

Preliminary Concept of Operations for the Deep Space Array-Based Network

NASA Astrophysics Data System (ADS)

Bagri, D. S.; Statman, J. I.

2004-05-01

The Deep Space Array-Based Network (DSAN) will be an array-based system, part of a greater than 1000 times increase in the downlink/telemetry capability of the Deep Space Network. The key function of the DSAN is provision of cost-effective, robust telemetry, tracking, and command services to the space missions of NASA and its international partners. This article presents an expanded approach to the use of an array-based system. Instead of using the array as an element in the existing Deep Space Network (DSN), relying to a large extent on the DSN infrastructure, we explore a broader departure from the current DSN, using fewer elements of the existing DSN, and establishing a more modern concept of operations. For example, the DSAN will have a single 24 x 7 monitor and control (M&C) facility, while the DSN has four 24 x 7 M&C facilities. The article gives the architecture of the DSAN and its operations philosophy. It also briefly describes the customer's view of operations, operations management, logistics, anomaly analysis, and reporting.

Detection of Respiratory Viruses in Sputum from Adults by Use of Automated Multiplex PCR

PubMed Central

Walsh, Edward E.; Formica, Maria A.; Falsey, Ann R.

2014-01-01

Respiratory tract infections (RTI) frequently cause hospital admissions among adults. Diagnostic viral reverse transcriptase PCR (RT-PCR) of nose and throat swabs (NTS) is useful for patient care by informing antiviral use and appropriate isolation. However, automated RT-PCR systems are not amenable to utilizing sputum due to its viscosity. We evaluated a simple method of processing sputum samples in a fully automated respiratory viral panel RT-PCR assay (FilmArray). Archived sputum and NTS samples collected in 2008-2012 from hospitalized adults with RTI were evaluated. A subset of sputum samples positive for 10 common viruses by a uniplex RT-PCR was selected. A sterile cotton-tip swab was dunked in sputum, swirled in 700 μL of sterile water (dunk and swirl method) and tested by the FilmArray assay. Quantitative RT-PCR was performed on “dunked” sputum and NTS samples for influenza A (Flu A), respiratory syncytial virus (RSV), coronavirus OC43 (OC43), and human metapneumovirus (HMPV). Viruses were identified in 31% of 965 illnesses using a uniplex RT-PCR. The sputum sample was the only sample positive for 105 subjects, including 35% (22/64) of influenza cases and significantly increased the diagnostic yield of NTS alone (302/965 [31%] versus 197/965 [20%]; P = 0.0001). Of 108 sputum samples evaluated by the FilmArray assay using the dunk and swirl method, 99 (92%) were positive. Quantitative RT-PCR revealed higher mean viral loads in dunked sputum samples compared to NTS samples for Flu A, RSV, and HMPV (P = 0.0001, P = 0.006, and P = 0.011, respectively). The dunk and swirl method is a simple and practical method for reliably processing sputum samples in a fully automated PCR system. The higher viral loads in sputa may increase detection over NTS testing alone. PMID:25056335

Wafer-level radiometric performance testing of uncooled microbolometer arrays

NASA Astrophysics Data System (ADS)

Dufour, Denis G.; Topart, Patrice; Tremblay, Bruno; Julien, Christian; Martin, Louis; Vachon, Carl

2014-03-01

A turn-key semi-automated test system was constructed to perform on-wafer testing of microbolometer arrays. The system allows for testing of several performance characteristics of ROIC-fabricated microbolometer arrays including NETD, SiTF, ROIC functionality, noise and matrix operability, both before and after microbolometer fabrication. The system accepts wafers up to 8 inches in diameter and performs automated wafer die mapping using a microscope camera. Once wafer mapping is completed, a custom-designed quick insertion 8-12 μm AR-coated Germanium viewport is placed and the chamber is pumped down to below 10-5 Torr, allowing for the evaluation of package-level focal plane array (FPA) performance. The probe card is electrically connected to an INO IRXCAM camera core, a versatile system that can be adapted to many types of ROICs using custom-built interface printed circuit boards (PCBs). We currently have the capability for testing 384x288, 35 μm pixel size and 160x120, 52 μm pixel size FPAs. For accurate NETD measurements, the system is designed to provide an F/1 view of two rail-mounted blackbodies seen through the Germanium window by the die under test. A master control computer automates the alignment of the probe card to the dies, the positioning of the blackbodies, FPA image frame acquisition using IRXCAM, as well as data analysis and storage. Radiometric measurement precision has been validated by packaging dies measured by the automated probing system and re-measuring the SiTF and Noise using INO's pre-existing benchtop system.

"Mini-array" transcriptional analysis of the Listeria monocytogenes lecithinase operon as a class project: A student investigative molecular biology laboratory experience*.

PubMed

Christensen, Douglas; Jovic, Marko

2006-05-01

This report describes a molecular biotechnology-based laboratory curriculum developed to accompany an undergraduate genetics course. During the course of a semester, students researched the pathogen, developed a research question, designed experiments, and performed transcriptional analysis of a set of genes that confer virulence to the food-borne pathogen, Listeria monocytogenes. Gene fragments were amplified via PCR and utilized in "mini-arrays," a dot-blot-based format suitable for the simultaneous transcriptional analysis of multiple genes. The project provides exposure to a wide range of molecular techniques and can be easily modified for variations in class size. Data are generated at various steps of the process, allowing for student interpretation, troubleshooting, and assessment opportunities. Copyright © 2006 International Union of Biochemistry and Molecular Biology, Inc.

Rapid Identification of Shiga Toxin-Producing Escherichia coli O Serogroups from Fresh Produce and Raw Milk Enrichment Cultures by Luminex Bead-Based Suspension Array.

PubMed

Kase, Julie A; Maounounen-Laasri, Anna; Lin, Andrew

2016-09-01

The U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM) Chapter 4a describes a Luminex microbead-based suspension array used to screen colonies for 11 clinically relevant Shiga toxin-producing Escherichia coli (STEC) serogroups: O26, O45, O91, O103, O104, O111, O113, O121, O128, O145, and O157. We evaluated the usefulness of this method to identify STEC-positive enrichment samples before agar plating. Twelve E. coli strains were added to three types of fresh produce (bagged baby spinach, alfalfa sprouts, and cilantro) at levels near the detection limit of the test. A subset of these strains (six O serogroups) was similarly evaluated in raw milk. For comparison, portions of each of the 168 enrichment cultures were analyzed for serogroup by a real-time PCR assay and a Bio-Plex 200 assay with the bead-based suspensions. No false-positive results were obtained. Of the 112 samples with a reported cycle threshold (C T ) value, 101 undiluted, diluted, or extracted enrichment cultures also produced ratios above 5.0 in the Bio-Plex assay. When PCR C T values approached or were greater than 35, Bio-Plex detection became less reliable. Using undiluted or extracted enrichment cultures resulted in a significantly larger number of positive results. With the same enrichment material prepared for real-time PCR analysis as described in the BAM Chapter 4a, the STEC microbead-based suspension array can accurately screen food enrichment cultures.

Differential expression of extracellular matrix constituents and cell adhesion molecules between malignant pleural mesothelioma and mesothelial hyperplasia.

PubMed

Alì, Greta; Borrelli, Nicla; Riccardo, Giannini; Proietti, Agnese; Pelliccioni, Serena; Niccoli, Cristina; Boldrini, Laura; Lucchi, Marco; Mussi, Alfredo; Fontanini, Gabriella

2013-11-01

Malignant pleural mesothelioma (MPM) is a highly aggressive neoplasm associated with asbestos exposure. Currently, the molecular mechanisms that induce MPM development are still unknown. The purpose of this study was to identify new molecular biomarkers for mesothelial carcinogenesis. We analyzed a panel of 84 genes involved in extracellular matrix remodeling and cell adhesion by polymerase chain reaction (PCR) array in 15 samples of epithelioid mesothelioma and 10 samples of reactive mesothelial hyperplasia (MH; 3 of 25 samples were inadequate for mRNA analysis). To validate the differentially expressed genes identified by PCR array, we analyzed 27 more samples by immunohistochemistry, in addition to the 25 samples already studied. Twenty-five genes were differentially expressed in MPM and MH by PCR array. Of these we studied matrix metalloproteinase 7 (MMP7), MMP14, CD44, and integrin, alpha3 expression by immunohistochemistry in 26 epithelioid MPM and 26 MH samples from the entire series of 52 cases. We observed higher MMP14 and integrin, alpha3 expression in MPM samples compared with MH samples (p = 0.000002 and p = 0.000002, respectively). Conversely, CD44 expression was low in most (57.7%) mesothelioma samples but only in 11.5% of the MH samples (p = 0.0013). As regards MMP7, we did not observe differential expression between MH and MPM samples. We have extensively studied genes involved in cell adhesion and extracellular matrix remodeling in MPM and MH samples, gaining new insight into the pathophysiology of mesothelioma. Moreover, our data suggest that these factors could be potential biomarkers for MPM.

Developing a gate-array capability at a research and development laboratory

NASA Astrophysics Data System (ADS)

Balch, J. W.; Current, K. W.; Magnuson, W. G., Jr.; Pocha, M. D.

1983-03-01

Experiences in developing a gate array capability for low volume applications in a research and development (R and D) laboratory are described. By purchasing unfinished wafers and doing the customization steps in-house. Turnaround time was shortened to as little as one week and the direct costs reduced to as low as $5K per design. Designs generally require fast turnaround (a few weeks to a few months) and very low volumes (1 to 25). Design costs must be kept at a minimum. After reviewing available commercial gate array design and fabrication services, it was determined that objectives would best be met by using existing internal integrated circuit fabrication facilities, the COMPUTERVISION interactive graphics layout system, and extensive computational capabilities. The reasons and the approach taken for; selection for a particular gate array wafer, adapting a particular logic simulation program, and how layout aids were enhanced are discussed. Testing of the customized chips is described. The content, schedule, and results of the internal gate array course recently completed are discussed. Finally, problem areas and near term plans are presented.

A Simple PCR Method for Rapid Genotype Analysis of Mycobacterium ulcerans

PubMed Central

Stinear, Timothy; Davies, John K.; Jenkin, Grant A.; Portaels, Françoise; Ross, Bruce C.; OppEdIsano, Frances; Purcell, Maria; Hayman, John A.; Johnson, Paul D. R.

2000-01-01

Two high-copy-number insertion sequences, IS2404 and IS2606, were recently identified in Mycobacterium ulcerans and were shown by Southern hybridization to possess restriction fragment length polymorphism between strains from different geographic origins. We have designed a simple genotyping method that captures these differences by PCR amplification of the region between adjacent copies of IS2404 and IS2606. We have called this system 2426 PCR. The method is rapid, reproducible, sensitive, and specific for M. ulcerans, and it has confirmed previous studies suggesting a clonal population structure of M. ulcerans within a geographic region. M. ulcerans isolates from Australia, Papua New Guinea, Malaysia, Surinam, Mexico, Japan, China, and several countries in Africa were easily differentiated based on an array of 4 to 14 PCR products ranging in size from 200 to 900 bp. Numerical analysis of the banding patterns suggested a close evolutionary link between M. ulcerans isolates from Africa and southeast Asia. The application of 2426 PCR to total DNA, extracted directly from M. ulcerans-infected tissue specimens without culture, demonstrated the sensitivity and specificity of this method and confirmed for the first time that both animal and human isolates from areas of endemicity in southeast Australia have the same genotype. PMID:10747130

Detection of Foodborne Pathogenic Bacteria using Bacteriophage Tail Spike Proteins

NASA Astrophysics Data System (ADS)

Poshtiban, Somayyeh

Foodborne infections are worldwide health problem with tremendous social and financial impacts. Efforts are focused on developing accurate and reliable technologies for detection of food contaminations in early stages preferably on-site. This thesis focuses on interfacing engineering and biology by combining phage receptor binding proteins (RBPs) with engineered platforms including microresonator-based biosensors, magnetic particles and polymerase chain reaction (PCR) to develop bacterial detection sensors. We used phage RBPs as target specific bioreceptors to develop an enhanced microresonator array for bacterial detection. These resonator beams are optimized to feature a high natural frequency while offer large surface area for capture of bacteria. Theoretical analysis indicates a high mass sensitivity with a threshold for the detection of a single bacterial cell. We used phage RBPs as target specific bioreceptors, and successfully demonstrated the application of these phage RBB-immobilized arrays for specific detection of C. jejuni cells. We also developed a RBP-derivatized magnetic pre-enrichment method as an upstream sample preparation method to improve sensitivity and specificity of PCR for detection of bacterial cells in various food samples. The combination of RBP-based magnetic separation and real-time PCR allowed the detection of small number of bacteria in artificially contaminated food samples without any need for time consuming pre-enrichment step through culturing. We also looked into integration of the RBP-based magnetic separation with PCR onto a single microfluidic lab-on-a-chip to reduce the overall turnaround time.

Analysis of LDLR mutations in familial hypercholesterolemia patients in Greece by use of the NanoChip microelectronic array technology.

PubMed

Laios, Eleftheria; Drogari, Euridiki

2006-12-01

Three mutations in the low density lipoprotein receptor (LDLR) gene account for 49% of familial hypercholesterolemia (FH) cases in Greece. We used the microelectronic array technology of the NanoChip Molecular Biology Workstation to develop a multiplex method to analyze these single-nucleotide polymorphisms (SNPs). Primer pairs amplified the region encompassing each SNP. The biotinylated PCR amplicon was electronically addressed to streptavidin-coated microarray sites. Allele-specific fluorescently labeled oligonucleotide reporters were designed and used for detection of wild-type and SNP sequences. Genotypes were compared to PCR-restriction fragment length polymorphism (PCR-RFLP). We developed three monoplex assays (1 SNP/site) and an optimized multiplex assay (3SNPs/site). We performed 92 Greece II, 100 Genoa, and 98 Afrikaner-2 NanoChip monoplex assays (addressed to duplicate sites and analyzed separately). Of the 580 monoplex genotypings (290 samples), 579 agreed with RFLP. Duplicate sites of one sample were not in agreement with each other. Of the 580 multiplex genotypings, 576 agreed with the monoplex results. Duplicate sites of three samples were not in agreement with each other, indicating requirement for repetition upon which discrepancies were resolved. The multiplex assay detects common LDLR mutations in Greek FH patients and can be extended to accommodate additional mutations.

A group arrival retrial G - queue with multi optional stages of service, orbital search and server breakdown

NASA Astrophysics Data System (ADS)

Radha, J.; Indhira, K.; Chandrasekaran, V. M.

2017-11-01

A group arrival feedback retrial queue with k optional stages of service and orbital search policy is studied. Any arriving group of customer finds the server free, one from the group enters into the first stage of service and the rest of the group join into the orbit. After completion of the i th stage of service, the customer under service may have the option to choose (i+1)th stage of service with θi probability, with pI probability may join into orbit as feedback customer or may leave the system with {q}i=≤ft\\{\\begin{array}{l}1-{p}i-{θ }i,i=1,2,\\cdots k-1\\ 1-{p}i,i=k\\end{array}\\right\\} probability. Busy server may get to breakdown due to the arrival of negative customers and the service channel will fail for a short interval of time. At the completion of service or repair, the server searches for the customer in the orbit (if any) with probability α or remains idle with probability 1-α. By using the supplementary variable method, steady state probability generating function for system size, some system performance measures are discussed.

A human fecal contamination score for ranking recreational sites using the HF183/BacR287 quantitative real-time PCR method.

PubMed

Cao, Yiping; Sivaganesan, Mano; Kelty, Catherine A; Wang, Dan; Boehm, Alexandria B; Griffith, John F; Weisberg, Stephen B; Shanks, Orin C

2018-01-01

Human fecal pollution of recreational waters remains a public health concern worldwide. As a result, there is a growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for water quality research and management. However, there are currently no standardized approaches for field implementation and interpretation of qPCR data. In this study, a standardized HF183/BacR287 qPCR method was combined with a water sampling strategy and a novel Bayesian weighted average approach to establish a human fecal contamination score (HFS) that can be used to prioritize sampling sites for remediation based on measured human waste levels. The HFS was then used to investigate 975 study design scenarios utilizing different combinations of sites with varying sampling intensities (daily to once per week) and number of qPCR replicates per sample (2-14 replicates). Findings demonstrate that site prioritization with HFS is feasible and that both sampling intensity and number of qPCR replicates influence reliability of HFS estimates. The novel data analysis strategy presented here provides a prescribed approach for the implementation and interpretation of human-associated HF183/BacR287 qPCR data with the goal of site prioritization based on human fecal pollution levels. In addition, information is provided for future users to customize study designs for optimal HFS performance. Published by Elsevier Ltd.

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

PubMed Central

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

2014-01-01

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

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

PubMed

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

2014-01-01

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

Effect of human rhinovirus infection on airway epithelium tight junction protein disassembly and transepithelial permeability.

PubMed

Looi, Kevin; Troy, Niamh M; Garratt, Luke W; Iosifidis, Thomas; Bosco, Anthony; Buckley, Alysia G; Ling, Kak-Ming; Martinovich, Kelly M; Kicic-Starcevich, Elizabeth; Shaw, Nicole C; Sutanto, Erika N; Zosky, Graeme R; Rigby, Paul J; Larcombe, Alexander N; Knight, Darryl A; Kicic, Anthony; Stick, Stephen M

2016-10-11

No studies have assessed the effects of human rhinovirus (HRV) infection on epithelial tight junctions (TJs) and resultant barrier function. To correlate viral infection with TJ disassembly, epithelial barrier integrity, and function. Human airway epithelial cells were infected with HRV minor serotype 1B (HRV-1B) at various 50% tissue culture infectivity doses (TCID 50 ) over 72 hours. HRV replication was assessed by quantitative-polymerase chain reaction (qPCR) while cell viability and apoptosis were assessed by proliferation and apoptotic assays, respectively. Protein expression of claudin-1, occludin, and zonula occludens protein-1 (ZO-1) was assessed using In-Cell™ Western assays. Transepithelial permeability assays were performed to assess effects on barrier functionality. RT 2 Profiler focused qPCR arrays and pathway analysis evaluating associations between human TJ and antiviral response were performed to identify potential interactions and pathways between genes of interests. HRV-1B infection affected viability that was both time and TCID 50 dependent. Significant increases in apoptosis and viral replication post-infection correlated with viral titer. Viral infection significantly decreased claudin-1 protein expression at the lower TCID 50 , while a significant decrease in all three TJ protein expressions occurred at higher TCID 50 . Decrease in protein expression was concomitant with significant increases in epithelial permeability of fluorescein isothiocynate labeled-dextran 4 and 20 kDa. Analysis of focused qPCR arrays demonstrated a significant decrease in ZO-1 gene expression. Furthermore, network analysis between human TJ and antiviral response genes revealed possible interactions and regulation of TJ genes via interleukin (IL)-15 in response to HRV-1B infection. HRV-1B infection directly alters human airway epithelial TJ expression leading to increased epithelial permeability potentially via an antiviral response of IL-15.

PCR amplification and genetic analysis in a microwell cell culturing chip.

PubMed

Lindström, Sara; Hammond, Maria; Brismar, Hjalmar; Andersson-Svahn, Helene; Ahmadian, Afshin

2009-12-21

We have previously described a microwell chip designed for high throughput, long-term single-cell culturing and clonal analysis in individual wells providing a controlled way of studying high numbers of individual adherent or non-adherent cells. Here we present a method for the genetic analysis of cells cultured on-chip by PCR and minisequencing, demonstrated using two human adherent cell lines: one wild type and one with a single-base mutation in the p53 gene. Five wild type or mutated cells were seeded per well (in a defined set of wells, each holding 500 nL of culture medium) in a 672-microwell chip. The cell chip was incubated overnight, or cultured for up to five days, depending on the desired colony size, after which the cells were lysed and subjected to PCR directly in the wells. PCR products were detected, in the wells, using a biotinylated primer and a fluorescently labelled primer, allowing the products to be captured on streptavidin-coated magnetic beads and detected by a fluorescence microscope. In addition, to enable genetic analysis by minisequencing, the double-stranded PCR products were denatured and the immobilized strands were kept in the wells by applying a magnetic field from the bottom of the wells while the wells were washed, a minisequencing reaction mixture was added, and after incubation in appropriate conditions the expected genotypes were detected in the investigated microwells, simultaneously, by an array scanner. We anticipate that the technique could be used in mutation frequency screening, providing the ability to correlate cells' proliferative heterogeneity to their genetic heterogeneity, in hundreds of samples simultaneously. The presented method of single-cell culture and DNA amplification thus offers a potentially powerful alternative to single-cell PCR, with advantageous robustness and sensitivity.

Low SWaP multispectral sensors using dichroic filter arrays

NASA Astrophysics Data System (ADS)

Dougherty, John; Varghese, Ron

2015-06-01

The benefits of multispectral imaging are well established in a variety of applications including remote sensing, authentication, satellite and aerial surveillance, machine vision, biomedical, and other scientific and industrial uses. However, many of the potential solutions require more compact, robust, and cost-effective cameras to realize these benefits. The next generation of multispectral sensors and cameras needs to deliver improvements in size, weight, power, portability, and spectral band customization to support widespread deployment for a variety of purpose-built aerial, unmanned, and scientific applications. A novel implementation uses micro-patterning of dichroic filters1 into Bayer and custom mosaics, enabling true real-time multispectral imaging with simultaneous multi-band image acquisition. Consistent with color image processing, individual spectral channels are de-mosaiced with each channel providing an image of the field of view. This approach can be implemented across a variety of wavelength ranges and on a variety of detector types including linear, area, silicon, and InGaAs. This dichroic filter array approach can also reduce payloads and increase range for unmanned systems, with the capability to support both handheld and autonomous systems. Recent examples and results of 4 band RGB + NIR dichroic filter arrays in multispectral cameras are discussed. Benefits and tradeoffs of multispectral sensors using dichroic filter arrays are compared with alternative approaches - including their passivity, spectral range, customization options, and scalable production.

Nuclear Matrix Proteins in Disparity of Prostate Cancer

DTIC Science & Technology

2013-09-01

nuclear coactivator-3 (NCOA3). 5 Methods Patients and Prostate Cancer Specimens Fresh, flash -frozen specimens were obtained from age- (50 to...for reliable data interpretation. Gene Array Analysis Total RNA isolated from LCM-procured normal epithelium and tumor cells from flash -frozen...PCR Briefly, RNA was extracted from matched LCM procured normal epithelium and tumor cells of age-, tumor grade-matched flash -frozen sections (n=24

Non-lethal sampling for the detection of Myxobolus cerebralis in asymptomatic rainbow trout

USGS Publications Warehouse

Schill, Bane; Waldrop, Thomas; Densmore, Christine; Blazer, Vicki

1999-01-01

We have described in previous reports (Schill et al., 1998) the development of a polymerase chain reaction (PCR) amplification of 18S ribosomal RNA for the detection of Myxozoan parasites. Oligonucleotide primers were developed by multiple alignment of Myxozoan sequence information and analysis by a custom-written computer program (PRIM). Candidate pairs of primer sequences were then analyzed for specificity by BLAST (Basic Local Alignment Search Tool). From these, a set of promising primers (MYXFWD and MYXREV) was chosen for further testing. These were chosen because they should direct detection of a number of Myxozoan species (Table 1). PCR using MXYFWD and MYXREV proved to be robust and relatively free of artifact products. Further, we were able to routinely detect Myxobolus cerebralis in fish tissues (Figure 1).

SMN1 and SMN2 copy numbers in cell lines derived from patients with spinal muscular atrophy as measured by array digital PCR.

PubMed

Stabley, Deborah L; Harris, Ashlee W; Holbrook, Jennifer; Chubbs, Nicholas J; Lozo, Kevin W; Crawford, Thomas O; Swoboda, Kathryn J; Funanage, Vicky L; Wang, Wenlan; Mackenzie, William; Scavina, Mena; Sol-Church, Katia; Butchbach, Matthew E R

2015-07-01

Proximal spinal muscular atrophy (SMA) is an early-onset motor neuron disease characterized by loss of α-motor neurons and associated muscle atrophy. SMA is caused by deletion or other disabling mutation of survival motor neuron 1 (SMN1). In the human genome, a large duplication of the SMN-containing region gives rise to a second copy of this gene (SMN2) that is distinguishable by a single nucleotide change in exon 7. Within the SMA population, there is substantial variation in SMN2 copy number; in general, those individuals with SMA who have a high SMN2 copy number have a milder disease. Because SMN2 functions as a disease modifier, its accurate copy number determination may have clinical relevance. In this study, we describe the development of an assay to assess SMN1 and SMN2 copy numbers in DNA samples using an array-based digital PCR (dPCR) system. This dPCR assay can accurately and reliably measure the number of SMN1 and SMN2 copies in DNA samples. In a cohort of SMA patient-derived cell lines, the assay confirmed a strong inverse correlation between SMN2 copy number and disease severity. Array dPCR is a practical technique to determine, accurately and reliably, SMN1 and SMN2 copy numbers from SMA samples.

Evaluation of a Real-Time Reverse Transcription-PCR Assay for Detection of Enterovirus D68 in Clinical Samples from an Outbreak in New York State in 2014.

PubMed

Zhuge, Jian; Vail, Eric; Bush, Jeffrey L; Singelakis, Lauren; Huang, Weihua; Nolan, Sheila M; Haas, Janet P; Engel, Helen; Della Posta, Millicent; Yoon, Esther C; Fallon, John T; Wang, Guiqing

2015-06-01

An outbreak of severe respiratory illness associated with enterovirus D68 (EV-D68) infection was reported in mid-August 2014 in the United States. In this study, we evaluated the diagnostic utility of an EV-D68-specific real-time reverse transcription-PCR (rRT-PCR) that was recently developed by the Centers for Disease Control and Prevention in clinical samples. Nasopharyngeal (NP) swab specimens from patients in a recent outbreak of respiratory illness in the lower Hudson Valley, New York State, were collected and examined for the presence of human rhinovirus or enterovirus using the FilmArray Respiratory Panel (RP) assay. Samples positive by RP were assessed using EV-D68 rRT-PCR, and the data were compared to results from sequencing analysis of partial VP1 and 5' untranslated region (5'-UTR) sequences of the EV genome. A total of 285 RP-positive NP specimens (260 from the 2014 outbreak and 25 from 2013) were analyzed by rRT-PCR; EV-D68 was detected in 74 of 285 (26.0%) specimens examined. Data for comparisons between rRT-PCR and sequencing analysis were obtained from 194 NP specimens. EV-D68 detection was confirmed by sequencing analysis in 71 of 74 positive and in 1 of 120 randomly selected negative specimens by rRT-PCR. The EV-D68 rRT-PCR showed diagnostic sensitivity and specificity of 98.6% and 97.5%, respectively. Our data suggest that the EV-D68 rRT-PCR is a reliable assay for detection of EV-D68 in clinical samples and has a potential to be used as a tool for rapid diagnosis and outbreak investigation of EV-D68-associated infections in clinical and public health laboratories. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Evaluation of a Real-Time Reverse Transcription-PCR Assay for Detection of Enterovirus D68 in Clinical Samples from an Outbreak in New York State in 2014

PubMed Central

Zhuge, Jian; Vail, Eric; Bush, Jeffrey L.; Singelakis, Lauren; Huang, Weihua; Nolan, Sheila M.; Haas, Janet P.; Engel, Helen; Della Posta, Millicent; Yoon, Esther C.; Fallon, John T.

2015-01-01

An outbreak of severe respiratory illness associated with enterovirus D68 (EV-D68) infection was reported in mid-August 2014 in the United States. In this study, we evaluated the diagnostic utility of an EV-D68-specific real-time reverse transcription-PCR (rRT-PCR) that was recently developed by the Centers for Disease Control and Prevention in clinical samples. Nasopharyngeal (NP) swab specimens from patients in a recent outbreak of respiratory illness in the lower Hudson Valley, New York State, were collected and examined for the presence of human rhinovirus or enterovirus using the FilmArray Respiratory Panel (RP) assay. Samples positive by RP were assessed using EV-D68 rRT-PCR, and the data were compared to results from sequencing analysis of partial VP1 and 5′ untranslated region (5′-UTR) sequences of the EV genome. A total of 285 RP-positive NP specimens (260 from the 2014 outbreak and 25 from 2013) were analyzed by rRT-PCR; EV-D68 was detected in 74 of 285 (26.0%) specimens examined. Data for comparisons between rRT-PCR and sequencing analysis were obtained from 194 NP specimens. EV-D68 detection was confirmed by sequencing analysis in 71 of 74 positive and in 1 of 120 randomly selected negative specimens by rRT-PCR. The EV-D68 rRT-PCR showed diagnostic sensitivity and specificity of 98.6% and 97.5%, respectively. Our data suggest that the EV-D68 rRT-PCR is a reliable assay for detection of EV-D68 in clinical samples and has a potential to be used as a tool for rapid diagnosis and outbreak investigation of EV-D68-associated infections in clinical and public health laboratories. PMID:25854481

Aerosol sampling system for collection of Capstone depleted uranium particles in a high-energy environment.

PubMed

Holmes, Thomas D; Guilmette, Raymond A; Cheng, Yung Sung; Parkhurst, Mary Ann; Hoover, Mark D

2009-03-01

The Capstone Depleted Uranium (DU) Aerosol Study was undertaken to obtain aerosol samples resulting from a large-caliber DU penetrator striking an Abrams or Bradley test vehicle. The sampling strategy was designed to (1) optimize the performance of the samplers and maintain their integrity in the extreme environment created during perforation of an armored vehicle by a DU penetrator, (2) collect aerosols as a function of time post perforation, and (3) obtain size-classified samples for analysis of chemical composition, particle morphology, and solubility in lung fluid. This paper describes the experimental setup and sampling methodologies used to achieve these objectives. Custom-designed arrays of sampling heads were secured to the inside of the target in locations approximating the breathing zones of the crew locations in the test vehicles. Each array was designed to support nine filter cassettes and nine cascade impactors mounted with quick-disconnect fittings. Shielding and sampler placement strategies were used to minimize sampler loss caused by the penetrator impact and the resulting fragments of eroded penetrator and perforated armor. A cyclone train was used to collect larger quantities of DU aerosol for measurement of chemical composition and solubility. A moving filter sample was used to obtain semicontinuous samples for DU concentration determination. Control for the air samplers was provided by five remotely located valve control and pressure monitoring units located inside and around the test vehicle. These units were connected to a computer interface chassis and controlled using a customized LabVIEW engineering computer control program. The aerosol sampling arrays and control systems for the Capstone study provided the needed aerosol samples for physicochemical analysis, and the resultant data were used for risk assessment of exposure to DU aerosol.

Aerosol Sampling System for Collection of Capstone Depleted Uranium Particles in a High-Energy Environment

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

Holmes, Thomas D.; Guilmette, Raymond A.; Cheng, Yung-Sung

2009-03-01

The Capstone Depleted Uranium Aerosol Study was undertaken to obtain aerosol samples resulting from a kinetic-energy cartridge with a large-caliber depleted uranium (DU) penetrator striking an Abrams or Bradley test vehicle. The sampling strategy was designed to (1) optimize the performance of the samplers and maintain their integrity in the extreme environment created during perforation of an armored vehicle by a DU penetrator, (2) collect aerosols as a function of time post-impact, and (3) obtain size-classified samples for analysis of chemical composition, particle morphology, and solubility in lung fluid. This paper describes the experimental setup and sampling methodologies used tomore » achieve these objectives. Custom-designed arrays of sampling heads were secured to the inside of the target in locations approximating the breathing zones of the vehicle commander, loader, gunner, and driver. Each array was designed to support nine filter cassettes and nine cascade impactors mounted with quick-disconnect fittings. Shielding and sampler placement strategies were used to minimize sampler loss caused by the penetrator impact and the resulting fragments of eroded penetrator and perforated armor. A cyclone train was used to collect larger quantities of DU aerosol for chemical composition and solubility. A moving filter sample was used to obtain semicontinuous samples for depleted uranium concentration determination. Control for the air samplers was provided by five remotely located valve control and pressure monitoring units located inside and around the test vehicle. These units were connected to a computer interface chassis and controlled using a customized LabVIEW engineering computer control program. The aerosol sampling arrays and control systems for the Capstone study provided the needed aerosol samples for physicochemical analysis, and the resultant data were used for risk assessment of exposure to DU aerosol.« less

PTCH1 expression at diagnosis predicts imatinib failure in chronic myeloid leukaemia patients in chronic phase.

PubMed

Alonso-Dominguez, Juan M; Grinfeld, Jacob; Alikian, Mary; Marin, David; Reid, Alistair; Daghistani, Mustafa; Hedgley, Corinne; O'Brien, Stephen; Clark, Richard E; Apperley, Jane; Foroni, Letizia; Gerrard, Gareth

2015-01-01

The tyrosine kinase inhibitor (TKI) imatinib has revolutionized the management of chronic myeloid leukaemia (CML). However, around 25% of patients fail to sustain an adequate response. We sought to identify gene-expression biomarkers that could be used to predict imatinib response. The expression of 29 genes, previously implicated in CML pathogenesis, were measured by TaqMan Low Density Array in 73 CML patient samples. Patients were divided into low and high expression for each gene and imatinib failure (IF), probability of achieving CCyR, progression free survival and CML related OS were compared by Kaplan-Meier and log-rank. Results were validated in a second cohort of 56 patients, with a further technical validation using custom gene-expression assays in a conventional RT-qPCR in a sub-cohort of 37 patients. Patients with low PTCH1 expression showed a worse clinical response for all variables in all cohorts. PTCH1 was the most significant predictor in the multivariate analysis compared with Sokal, age and EUTOS. PTCH1 expression assay showed the adequate sensitivity, specificity and predictive values to predict for IF. Given the different treatments available for CML, measuring PTCH1 expression at diagnosis may help establish who will benefit best from imatinib and who is better selected for second generation TKI. © 2014 Wiley Periodicals, Inc.

Transcriptome discovery in non-model wild fish species for the development of quantitative transcript abundance assays.

PubMed

Hahn, Cassidy M; Iwanowicz, Luke R; Cornman, Robert S; Mazik, Patricia M; Blazer, Vicki S

2016-12-01

Environmental studies increasingly identify the presence of both contaminants of emerging concern (CECs) and legacy contaminants in aquatic environments; however, the biological effects of these compounds on resident fishes remain largely unknown. High throughput methodologies were employed to establish partial transcriptomes for three wild-caught, non-model fish species; smallmouth bass (Micropterus dolomieu), white sucker (Catostomus commersonii) and brown bullhead (Ameiurus nebulosus). Sequences from these transcriptome databases were utilized in the development of a custom nCounter CodeSet that allowed for direct multiplexed measurement of 50 transcript abundance endpoints in liver tissue. Sequence information was also utilized in the development of quantitative real-time PCR (qPCR) primers. Cross-species hybridization allowed the smallmouth bass nCounter CodeSet to be used for quantitative transcript abundance analysis of an additional non-model species, largemouth bass (Micropterus salmoides). We validated the nCounter analysis data system with qPCR for a subset of genes and confirmed concordant results. Changes in transcript abundance biomarkers between sexes and seasons were evaluated to provide baseline data on transcript modulation for each species of interest. Published by Elsevier Inc.

Delivery of video-on-demand services using local storages within passive optical networks.

PubMed

Abeywickrama, Sandu; Wong, Elaine

2013-01-28

At present, distributed storage systems have been widely studied to alleviate Internet traffic build-up caused by high-bandwidth, on-demand applications. Distributed storage arrays located locally within the passive optical network were previously proposed to deliver Video-on-Demand services. As an added feature, a popularity-aware caching algorithm was also proposed to dynamically maintain the most popular videos in the storage arrays of such local storages. In this paper, we present a new dynamic bandwidth allocation algorithm to improve Video-on-Demand services over passive optical networks using local storages. The algorithm exploits the use of standard control packets to reduce the time taken for the initial request communication between the customer and the central office, and to maintain the set of popular movies in the local storage. We conduct packet level simulations to perform a comparative analysis of the Quality-of-Service attributes between two passive optical networks, namely the conventional passive optical network and one that is equipped with a local storage. Results from our analysis highlight that strategic placement of a local storage inside the network enables the services to be delivered with improved Quality-of-Service to the customer. We further formulate power consumption models of both architectures to examine the trade-off between enhanced Quality-of-Service performance versus the increased power requirement from implementing a local storage within the network.

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

PubMed Central

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

2013-01-01

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

Inhibition of inflammatory cytokine-induced response in human islet cells by withaferin A.

PubMed

Peng, H; Olsen, G; Tamura, Y; Noguchi, H; Matsumoto, S; Levy, M F; Naziruddin, B

2010-01-01

After islet cell transplantation, a substantial mass of islets are lost owing to nonspecific inflammatory reactions. Cytokine exposure before or after transplantation can upregulate expression of proinflammatory genes via the nuclear factor-kappaB signaling pathway, eventually resulting in islet loss. To test the effects of a naturally occurring nuclear factor-kappaB inhibitor, withaferin A, on regulation of inflammatory genes in human islets. Human pancreatic islets were isolated using a modified Ricordi protocol. Purified islets were cultured for 2 days. The effect of withaferin A treatment on islet cell viability was examined using the fluorescein diacetate-propidium iodide dye exclusion test, and on function using a static glucose stimulation assay. Islet cells were treated with a cytokine mixture (50 U/mL of interleukin-1beta, 1000 U/mL of tumor necrosis factor-alpha, and 1000 U/mL of interferon-gamma) for 48 hours with or without withaferin A, 1 microg/mL. Treated islets were used for real-time polymerase chain reaction (PCR) array analysis for expression of inflammatory genes, and expression of other selected genes was analyzed using real-time PCR with single primers. Glucose stimulation and viability assays demonstrated that withaferin A was not toxic to islet cells. Of 84 inflammation-related genes examined using real-time PCR array analysis, 9 were significantly upregulated by cytokine treatment compared with the control group. However, addition of withaferin A to the culture significantly inhibited expression of all genes. Withaferin A significantly inhibits the inflammatory response of islet cells with cytokine exposure. Copyright 2010 Elsevier Inc. All rights reserved.

Retrospective Evaluation of Infants Aged 1-60 Days With Residual CSF Tested Using the FilmArray® Meningitis/Encephalitis (ME) Panel.

PubMed

Blaschke, Anne J; Holmberg, Kristen M; Daly, Judy A; Leber, Amy L; Dien Bard, Jennifer; Korgenski, Ernest K; Bourzac, Kevin M; Kanack, Kristen J

2018-04-18

In pediatric practice it is common for infants under 2 months of age to undergo evaluation for sepsis when they are ill, often including lumbar puncture (LP) to assess for central nervous system (CNS) infection. The FilmArray® Meningitis/Encephalitis (ME) Panel is a newly approved test for rapid identification of CNS pathogens. Our objective was to study the epidemiology of CNS infection in young infants and the potential impact of rapid multiplex PCR on their care.A performance evaluation of the FilmArray ME Panel was conducted from 2/2014-9/2014 at 11 sites. FilmArray ME Panel results were compared to reference standards but not shared with providers. In our study, medical records for infants (aged 1-60 days) enrolled at 3 sites were reviewed for clinical, laboratory and outcome data.145 infants were reviewed. Median age was 25 days. Most were hospitalized [134/145 (92%)], received antibiotics [123/145 (85%)] and almost half [71/145 (49%)] received acyclovir. One infant had a bacterial pathogen, likely false-positive, identified by the FilmArray ME Panel. Thirty-six infants (25%) had a viral pathogen detected, including 21 enteroviruses. All infants with enteroviral meningitis detected by the FilmArray ME Panel and conventional PCR were hospitalized, but 20% were discharged in less than 24 hours when conventional PCR results became available.The FilmArray ME Panel may play a role in the evaluation of young infants for CNS infection. Results may be used to guide management, possibly resulting in decreased length of stay and antimicrobial exposure for infants with low-risk viral infection detected. Copyright © 2018 Blaschke et al.

Isolation and measurement of the features of arrays of cell aggregates formed by dielectrophoresis using the user-specified Multi Regions Masking (MRM) technique

NASA Astrophysics Data System (ADS)

Yusvana, Rama; Headon, Denis; Markx, Gerard H.

2009-08-01

The use of dielectrophoresis for the construction of artificial skin tissue with skin cells in follicle-like 3D cell aggregates in well-defined patterns is demonstrated. To analyse the patterns produced and to study their development after their formation a Virtual Instrument (VI) system was developed using the LabVIEW IMAQ Vision Development Module. A series of programming functions (algorithms) was used to isolate the features on the image (in our case; the patterned aggregates) and separate them from all other unwanted regions on the image. The image was subsequently converted into a binary version, covering only the desired microarray regions which could then be analysed by computer for automatic object measurements. The analysis utilized the simple and easy-to-use User-Specified Multi-Regions Masking (MRM) technique, which allows one to concentrate the analysis on the desired regions specified in the mask. This simplified the algorithms for the analysis of images of cell arrays having similar geometrical properties. By having a collection of scripts containing masks of different patterns, it was possible to quickly and efficiently develop sets of custom virtual instruments for the offline or online analysis of images of cell arrays in the database.

A Custom Robotic System for Inspecting HEPA Filters in the Payload Changeout Room at the NASA Kennedy Space Center

NASA Technical Reports Server (NTRS)

Spencer, James E., Jr.; Looney, Joe

1994-01-01

In this paper, the prime objective is to describe a custom 4-dof (degree-of-freedom) robotic arm capable of autonomously or telerobotically performing systematic HEPA filter inspection and certification in the Shuttle Launch Pad Payload Changeout Rooms (PCR's) on pads A and B at the Kennedy Space Center, Florida. This HEPA filter inspection robot (HFIR) has been designed to be easily deployable and is equipped with the necessary sensory devices, control hardware, software and man-machine interfaces needed to implement HEPA filter inspection reliably and efficiently without damaging the filters or colliding with existing PCR structures or filters. The main purpose of the HFIR is to implement an automated positioning system to move special inspection sensors in pre-defined or manual patterns for the purpose of verifying filter integrity and efficiency. This will ultimately relieve NASA Payload Operations from significant problems associated with time, cost and personnel safety, impacts realized during non-automated PCR HFIR filter certification.

ICE: A Scalable, Low-Cost FPGA-Based Telescope Signal Processing and Networking System

NASA Astrophysics Data System (ADS)

Bandura, K.; Bender, A. N.; Cliche, J. F.; de Haan, T.; Dobbs, M. A.; Gilbert, A. J.; Griffin, S.; Hsyu, G.; Ittah, D.; Parra, J. Mena; Montgomery, J.; Pinsonneault-Marotte, T.; Siegel, S.; Smecher, G.; Tang, Q. Y.; Vanderlinde, K.; Whitehorn, N.

2016-03-01

We present an overview of the ‘ICE’ hardware and software framework that implements large arrays of interconnected field-programmable gate array (FPGA)-based data acquisition, signal processing and networking nodes economically. The system was conceived for application to radio, millimeter and sub-millimeter telescope readout systems that have requirements beyond typical off-the-shelf processing systems, such as careful control of interference signals produced by the digital electronics, and clocking of all elements in the system from a single precise observatory-derived oscillator. A new generation of telescopes operating at these frequency bands and designed with a vastly increased emphasis on digital signal processing to support their detector multiplexing technology or high-bandwidth correlators — data rates exceeding a terabyte per second — are becoming common. The ICE system is built around a custom FPGA motherboard that makes use of an Xilinx Kintex-7 FPGA and ARM-based co-processor. The system is specialized for specific applications through software, firmware and custom mezzanine daughter boards that interface to the FPGA through the industry-standard FPGA mezzanine card (FMC) specifications. For high density applications, the motherboards are packaged in 16-slot crates with ICE backplanes that implement a low-cost passive full-mesh network between the motherboards in a crate, allow high bandwidth interconnection between crates and enable data offload to a computer cluster. A Python-based control software library automatically detects and operates the hardware in the array. Examples of specific telescope applications of the ICE framework are presented, namely the frequency-multiplexed bolometer readout systems used for the South Pole Telescope (SPT) and Simons Array and the digitizer, F-engine, and networking engine for the Canadian Hydrogen Intensity Mapping Experiment (CHIME) and Hydrogen Intensity and Real-time Analysis eXperiment (HIRAX) radio interferometers.

Fine-mapping identifies multiple prostate cancer risk loci at 5p15, one of which associates with TERT expression

PubMed Central

Kote-Jarai, Zsofia; Saunders, Edward J.; Leongamornlert, Daniel A.; Tymrakiewicz, Malgorzata; Dadaev, Tokhir; Jugurnauth-Little, Sarah; Ross-Adams, Helen; Al Olama, Ali Amin; Benlloch, Sara; Halim, Silvia; Russel, Roslin; Dunning, Alison M.; Luccarini, Craig; Dennis, Joe; Neal, David E.; Hamdy, Freddie C.; Donovan, Jenny L.; Muir, Ken; Giles, Graham G.; Severi, Gianluca; Wiklund, Fredrik; Gronberg, Henrik; Haiman, Christopher A.; Schumacher, Fredrick; Henderson, Brian E.; Le Marchand, Loic; Lindstrom, Sara; Kraft, Peter; Hunter, David J.; Gapstur, Susan; Chanock, Stephen; Berndt, Sonja I.; Albanes, Demetrius; Andriole, Gerald; Schleutker, Johanna; Weischer, Maren; Canzian, Federico; Riboli, Elio; Key, Tim J.; Travis, Ruth C.; Campa, Daniele; Ingles, Sue A.; John, Esther M.; Hayes, Richard B.; Pharoah, Paul; Khaw, Kay-Tee; Stanford, Janet L.; Ostrander, Elaine A.; Signorello, Lisa B.; Thibodeau, Stephen N.; Schaid, Dan; Maier, Christiane; Vogel, Walther; Kibel, Adam S.; Cybulski, Cezary; Lubinski, Jan; Cannon-Albright, Lisa; Brenner, Hermann; Park, Jong Y.; Kaneva, Radka; Batra, Jyotsna; Spurdle, Amanda; Clements, Judith A.; Teixeira, Manuel R.; Govindasami, Koveela; Guy, Michelle; Wilkinson, Rosemary A.; Sawyer, Emma J.; Morgan, Angela; Dicks, Ed; Baynes, Caroline; Conroy, Don; Bojesen, Stig E.; Kaaks, Rudolf; Vincent, Daniel; Bacot, François; Tessier, Daniel C.; Easton, Douglas F.; Eeles, Rosalind A.

2013-01-01

Associations between single nucleotide polymorphisms (SNPs) at 5p15 and multiple cancer types have been reported. We have previously shown evidence for a strong association between prostate cancer (PrCa) risk and rs2242652 at 5p15, intronic in the telomerase reverse transcriptase (TERT) gene that encodes TERT. To comprehensively evaluate the association between genetic variation across this region and PrCa, we performed a fine-mapping analysis by genotyping 134 SNPs using a custom Illumina iSelect array or Sequenom MassArray iPlex, followed by imputation of 1094 SNPs in 22 301 PrCa cases and 22 320 controls in The PRACTICAL consortium. Multiple stepwise logistic regression analysis identified four signals in the promoter or intronic regions of TERT that independently associated with PrCa risk. Gene expression analysis of normal prostate tissue showed evidence that SNPs within one of these regions also associated with TERT expression, providing a potential mechanism for predisposition to disease. PMID:23535824

Analysis of X chromosome genomic DNA sequence copy number variation associated with premature ovarian failure (POF)

PubMed Central

Quilter, C.R.; Karcanias, A.C.; Bagga, M.R.; Duncan, S.; Murray, A.; Conway, G.S.; Sargent, C.A.; Affara, N.A.

2013-01-01

BACKGROUND Premature ovarian failure (POF) is a heterogeneous disease defined as amenorrhoea for >6 months before age 40, with an FSH serum level >40 mIU/ml (menopausal levels). While there is a strong genetic association with POF, familial studies have also indicated that idiopathic POF may also be genetically linked. Conventional cytogenetic analyses have identified regions of the X chromosome that are strongly associated with ovarian function, as well as several POF candidate genes. Cryptic chromosome abnormalities that have been missed might be detected by array comparative genomic hybridization. METHODS In this study, samples from 42 idiopathic POF patients were subjected to a complete end-to-end X/Y chromosome tiling path array to achieve a detailed copy number variation (CNV) analysis of X chromosome involvement in POF. The arrays also contained a 1 Mb autosomal tiling path as a reference control. Quantitative PCR for selected genes contained within the CNVs was used to confirm the majority of the changes detected. The expression pattern of some of these genes in human tissue RNA was examined by reverse transcription (RT)–PCR. RESULTS A number of CNVs were identified on both Xp and Xq, with several being shared among the POF cases. Some CNVs fall within known polymorphic CNV regions, and others span previously identified POF candidate regions and genes. CONCLUSIONS The new data reported in this study reveal further discrete X chromosome intervals not previously associated with the disease and therefore implicate new clusters of candidate genes. Further studies will be required to elucidate their involvement in POF. PMID:20570974

Gene expression profiling of single cells on large-scale oligonucleotide arrays

PubMed Central

Hartmann, Claudia H.; Klein, Christoph A.

2006-01-01

Over the last decade, important insights into the regulation of cellular responses to various stimuli were gained by global gene expression analyses of cell populations. More recently, specific cell functions and underlying regulatory networks of rare cells isolated from their natural environment moved to the center of attention. However, low cell numbers still hinder gene expression profiling of rare ex vivo material in biomedical research. Therefore, we developed a robust method for gene expression profiling of single cells on high-density oligonucleotide arrays with excellent coverage of low abundance transcripts. The protocol was extensively tested with freshly isolated single cells of very low mRNA content including single epithelial, mature and immature dendritic cells and hematopoietic stem cells. Quantitative PCR confirmed that the PCR-based global amplification method did not change the relative ratios of transcript abundance and unsupervised hierarchical cluster analysis revealed that the histogenetic origin of an individual cell is correctly reflected by the gene expression profile. Moreover, the gene expression data from dendritic cells demonstrate that cellular differentiation and pathway activation can be monitored in individual cells. PMID:17071717

DNA methylation pattern of apoptosis-related genes in ameloblastoma.

PubMed

Costa, Sfs; Pereira, N B; Pereira, Kma; Campos, K; de Castro, W H; Diniz, M G; Gomes, C C; Gomez, R S

2017-09-01

DNA methylation is an important mechanism of gene control expression, and it has been poorly addressed in odontogenic tumours. On this basis, we aimed to assess the methylation pattern of 22 apoptosis-related genes in solid ameloblastomas. Ameloblastoma fresh samples (n = 10) and dental follicles (n = 8) were included in the study. The percentage fraction of methylated and unmethylated DNA promoter of 22 apoptosis-related genes was determined using enzymatic restriction digestion and quantitative real-time PCR (qPCR) array. The relative expressions of the genes that showed the most discrepant methylation profile between tumours and controls were analysed by reverse-transcription quantitative PCR (RT-qPCR). Lower methylation percentages of TNFRSF25 (47.2%) and BCL2L11 (33.2%) were observed in ameloblastomas compared with dental follicles (79.3% and 59.5%, respectively). The RT-qPCR analysis showed increased expression of BCL2L11 in ameloblastomas compared with dental follicles, in agreement with the methylation analysis results, while there was no difference between the expression levels of TNFRSF25 between both groups. On the basis of our results, the transcription of the apoptosis-related gene BCL2L11 is possibly regulated by promoter DNA methylation in ameloblastoma. The biological significance of this finding in ameloblastoma pathobiology remains to be clarified. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth disease virus and look-alike disease viruses

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

Hindson, B J; Reid, S M; Baker, B R

2007-07-26

A high-throughput multiplexed assay was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspect cases of foot-and-mouthmore » disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRT-PCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.« less

Defense Industrial Base Assessment: U.S. Integrated Circuit Design and Fabrication Capability

DTIC Science & Technology

2009-05-01

in the U.S for the period 2003-2006, with projections to 2011.6 The resulting draft OTE survey was field tested for accuracy and usability with a...custom application specific integrated circuits (ASICs) to field programmable gate arrays (FPGAs). Companies of all sizes can manufacture these IC...able to design one-time Electronically Programmable Gate Arrays (EPGAs) while nine are able to design Field Programmable Gate Arrays (FPGAs). Eight

Combined array CGH plus SNP genome analyses in a single assay for optimized clinical testing

PubMed Central

Wiszniewska, Joanna; Bi, Weimin; Shaw, Chad; Stankiewicz, Pawel; Kang, Sung-Hae L; Pursley, Amber N; Lalani, Seema; Hixson, Patricia; Gambin, Tomasz; Tsai, Chun-hui; Bock, Hans-Georg; Descartes, Maria; Probst, Frank J; Scaglia, Fernando; Beaudet, Arthur L; Lupski, James R; Eng, Christine; Wai Cheung, Sau; Bacino, Carlos; Patel, Ankita

2014-01-01

In clinical diagnostics, both array comparative genomic hybridization (array CGH) and single nucleotide polymorphism (SNP) genotyping have proven to be powerful genomic technologies utilized for the evaluation of developmental delay, multiple congenital anomalies, and neuropsychiatric disorders. Differences in the ability to resolve genomic changes between these arrays may constitute an implementation challenge for clinicians: which platform (SNP vs array CGH) might best detect the underlying genetic cause for the disease in the patient? While only SNP arrays enable the detection of copy number neutral regions of absence of heterozygosity (AOH), they have limited ability to detect single-exon copy number variants (CNVs) due to the distribution of SNPs across the genome. To provide comprehensive clinical testing for both CNVs and copy-neutral AOH, we enhanced our custom-designed high-resolution oligonucleotide array that has exon-targeted coverage of 1860 genes with 60 000 SNP probes, referred to as Chromosomal Microarray Analysis – Comprehensive (CMA-COMP). Of the 3240 cases evaluated by this array, clinically significant CNVs were detected in 445 cases including 21 cases with exonic events. In addition, 162 cases (5.0%) showed at least one AOH region >10 Mb. We demonstrate that even though this array has a lower density of SNP probes than other commercially available SNP arrays, it reliably detected AOH events >10 Mb as well as exonic CNVs beyond the detection limitations of SNP genotyping. Thus, combining SNP probes and exon-targeted array CGH into one platform provides clinically useful genetic screening in an efficient manner. PMID:23695279

Friction Stir Weld Inspection Through Conductivity Imaging Using Shaped Field MWM(Registered Trademark) - Arrays

NASA Technical Reports Server (NTRS)

Goldfine, Neil; Grundy, David; Zilberstein, Vladimir; Kinchen, David G.; McCool, Alex (Technical Monitor)

2002-01-01

Friction Stir Welds (FSW) of Al 2195-T8 and Al 2219-T8, provided by Lockheed Martin Michoud Operations, were inspected for lack-of-penetration (LOP) defects using a custom designed MWM-Array, a multi-element eddy-current sensor. MWM (registered trademark) electrical conductivity mapping demonstrated high sensitivity to LOP as small as 0.75 mm (0.03 in.), as confirmed by metallographic data that characterized the extent of LOP defects. High sensitivity and high spatial resolution was achieved via a 37-element custom designed MWM-Array allowing LOP detection using the normalized longitudinal component of the MWM measured conductivity. This permitted both LOP detection and correlation of MWM conductivity features with the LOP defect size, as changes in conductivity were apparently associated with metallurgical features within the near-surface layer of the LOP defect zone. MWM conductivity mapping reveals information similar to macro-etching as the MWM-Array is sensitive to small changes in conductivity due to changes in microstructure associated with material thermal processing, in this case welding. The electrical conductivity measured on the root side of FSWs varies across the weld due to microstructural differences introduced by the FSW process, as well as those caused by planar flaws. Weld metal, i.e., dynamically recrystallized zone (DXZ), thermomechanically affected zone (TMZ), heat-affected zone (HAZ), and parent metal (PM) are all evident in the conductivity maps. While prior efforts had met with limited success for NDE (Nondestructive Evaluation) of dissimilar alloy, Al2219 to Al2195 FSW, the new custom designed multi-element MWM-Array achieved detection of all LOP defects even in dissimilar metal welds.

High-speed RNA microextraction technology using magnetic oligo-dT beads and lateral magnetophoresis.

PubMed

Lee, Hwanyong; Jung, Jinhee; Han, Song-I; Han, Ki-Ho

2010-10-21

This paper presents a high-speed RNA microextractor for the direct isolation of RNA from peripheral blood lysate using magnetic oligo-dT beads. The extraction is achieved through lateral magnetophoresis, generated by a ferromagnetic wire array inlaid on a glass substrate. This RNA microextractor separated more than 80% of magnetic beads with a flow rate up to 20 ml h(-1), and the overall extraction procedure was completed within 1 min. The absorbance ratio of RNA to protein (A(260)/A(280)) was >1.7, indicating that the extraction technology yielded nearly pure RNA. The feasibility of this technique was evaluated further for its applicability to reverse transcription polymerase chain reaction (RT-PCR) procedures by performing cDNA synthesis and PCR. The analysis verified that the RNA microextractor is a practical method for easy, rapid, and high-precision RT-PCR using minimal reagent volumes without requiring highly trained personnel. In addition, it can be readily incorporated into genetic analysis procedures for realizing automated on-chip genetic platforms in a micro format.

Loss of Chromosome 18 in Neuroendocrine Tumors of the Small Intestine: The Enigma Remains.

PubMed

Nieser, Maike; Henopp, Tobias; Brix, Joachim; Stoß, Laura; Sitek, Barbara; Naboulsi, Wael; Anlauf, Martin; Schlitter, Anna M; Klöppel, Günter; Gress, Thomas; Moll, Roland; Bartsch, Detlef K; Heverhagen, Anna E; Knoefel, Wolfram T; Kaemmerer, Daniel; Haybaeck, Johannes; Fend, Falko; Sperveslage, Jan; Sipos, Bence

2017-01-01

Neuroendocrine tumors of the small intestine (SI-NETs) exhibit an increasing incidence and high mortality rate. Until now, no fundamental molecular event has been linked to the tumorigenesis and progression of these tumors. Only the loss of chromosome 18 (Chr18) has been shown in up to two thirds of SI-NETs, whereby the significance of this alteration is still not understood. We therefore performed the first comprehensive study to identify Chr18-related events at the genetic, epigenetic and gene/protein expression levels. We did expression analysis of all seven putative Chr18-related tumor suppressors by quantitative real-time PCR (qRT-PCR), Western blot and immunohistochemistry. Next-generation exome sequencing and SNP array analysis were performed with five SI-NETs with (partial) loss of Chr18. Finally, we analyzed all microRNAs (miRNAs) located on Chr18 by qRT-PCR, comparing Chr18+/- and Chr18+/+ SI-NETs. Only DCC (deleted in colorectal cancer) revealed loss of/greatly reduced expression in 6/21 cases (29%). No relevant loss of SMAD2, SMAD4, elongin A3 and CABLES was detected. PMAIP1 and maspin were absent at the protein level. Next-generation sequencing did not reveal relevant recurrent somatic mutations on Chr18 either in an exploratory cohort of five SI-NETs, or in a validation cohort (n = 30). SNP array analysis showed no additional losses. The quantitative analysis of all 27 Chr18-related miRNAs revealed no difference in expression between Chr18+/- and Chr18+/+ SI-NETs. DCC seems to be the only Chr18-related tumor suppressor affected by the monoallelic loss of Chr18 resulting in a loss of DCC protein expression in one third of SI-NETs. No additional genetic or epigenetic alterations were present on Chr18. © 2016 S. Karger AG, Basel.

Investigation into the effect of high concentrations of volatile fatty acids in anaerobic digestion on methanogenic communities

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

Franke-Whittle, Ingrid H., E-mail: ingrid.whittle@uibk.ac.at; Walter, Andreas; Ebner, Christian

Highlights: • Different methanogenic communities in mesophilic and thermophilic reactors. • High VFA levels do not cause major changes in archaeal communities. • Real-time PCR indicated greater diversity than ANAEROCHIP microarray. - Abstract: A study was conducted to determine whether differences in the levels of volatile fatty acids (VFAs) in anaerobic digester plants could result in variations in the indigenous methanogenic communities. Two digesters (one operated under mesophilic conditions, the other under thermophilic conditions) were monitored, and sampled at points where VFA levels were high, as well as when VFA levels were low. Physical and chemical parameters were measured, andmore » the methanogenic diversity was screened using the phylogenetic microarray ANAEROCHIP. In addition, real-time PCR was used to quantify the presence of the different methanogenic genera in the sludge samples. Array results indicated that the archaeal communities in the different reactors were stable, and that changes in the VFA levels of the anaerobic digesters did not greatly alter the dominating methanogenic organisms. In contrast, the two digesters were found to harbour different dominating methanogenic communities, which appeared to remain stable over time. Real-time PCR results were inline with those of microarray analysis indicating only minimal changes in methanogen numbers during periods of high VFAs, however, revealed a greater diversity in methanogens than found with the array.« less

[Development of a universal primers PCR-coupled liquid bead array to detect biothreat bacteria].

PubMed

Wen, Hai-yan; Wang, Jing; Liu, Heng-chuan; Sun, Xiao-hong; Yang, Yu; Hu, Kong-xin; Shan, Lin-jun

2009-10-01

To develop a fast, high-throughput screening method with suspension array technique for simultaneous detection of biothreat bacteria. 16 S rDNA universal primers for Bacillus anthracis, Francisella tularensis, Yersinia pestis, Brucella spp.and Burkholderia pseudomallei were selected to amplify corresponding regions and the genus-specific or species-specific probes were designed. After amplification of chromosomal DNA by 16 S rDNA primers 341A and 519B, the PCR products were detected by suspension array technique. The sensitivity, specificity, reproducibility and detection power were also analyzed. After PCR amplification by 16 S rDNA primers and specific probe hybridization, the target microorganisms could be identified at genus level, cross reaction was recognized in the same genus. The detection sensitivity of the assay was 1.5 pg/microl (Burkholderia pseudomallei), 20 pg/microl (Brucella spp.), 7 pg/microl (Bacillus anthracis), 0.1 pg/microl (Francisella tularensis), and 1.1 pg/microl (Yersinia pestis), respectively. The coefficient of variation for 15 test of different probes was ranged from 5.18% to 17.88%, it showed good reproducibility. The assay could correctly identify Bacillus anthracis and Yersinia pestis strains in simulated white powder samples. The suspension array technique could be served as an opening screening method for biothreat bacteria rapid detection.

Using a whole-body 31P birdcage transmit coil and 16-element receive array for human cardiac metabolic imaging at 7T

PubMed Central

Dragonu, Iulius; Almujayyaz, Salam; Batzakis, Alex; Young, Liam A. J.; Purvis, Lucian A. B.; Clarke, William T.; Wichmann, Tobias; Lanz, Titus; Neubauer, Stefan; Robson, Matthew D.; Klomp, Dennis W. J.; Rodgers, Christopher T.

2017-01-01

Purpose Cardiac phosphorus magnetic resonance spectroscopy (31P-MRS) provides unique insight into the mechanisms of heart failure. Yet, clinical applications have been hindered by the restricted sensitivity of the surface radiofrequency-coils normally used. These permit the analysis of spectra only from the interventricular septum, or large volumes of myocardium, which may not be meaningful in focal disease. Löring et al. recently presented a prototype whole-body (52 cm diameter) transmit/receive birdcage coil for 31P at 7T. We now present a new, easily-removable, whole-body 31P transmit radiofrequency-coil built into a patient-bed extension combined with a 16-element receive array for cardiac 31P-MRS. Materials and methods A fully-removable (55 cm diameter) birdcage transmit coil was combined with a 16-element receive array on a Magnetom 7T scanner (Siemens, Germany). Electro-magnetic field simulations and phantom tests of the setup were performed. In vivo maps of B1+, metabolite signals, and saturation-band efficiency were acquired across the torsos of eight volunteers. Results The combined (volume-transmit, local receive array) setup increased signal-to-noise ratio 2.6-fold 10 cm below the array (depth of the interventricular septum) compared to using the birdcage coil in transceiver mode. The simulated coefficient of variation for B1+ of the whole-body coil across the heart was 46.7% (surface coil 129.0%); and the in vivo measured value was 38.4%. Metabolite images of 2,3-diphosphoglycerate clearly resolved the ventricular blood pools, and muscle tissue was visible in phosphocreatine (PCr) maps. Amplitude-modulated saturation bands achieved 71±4% suppression of phosphocreatine PCr in chest-wall muscles. Subjects reported they were comfortable. Conclusion This easy-to-assemble, volume-transmit, local receive array coil combination significantly improves the homogeneity and field-of-view for metabolic imaging of the human heart at 7T. PMID:29073228

Application specific serial arithmetic arrays

NASA Technical Reports Server (NTRS)

Winters, K.; Mathews, D.; Thompson, T.

1990-01-01

High performance systolic arrays of serial-parallel multiplier elements may be rapidly constructed for specific applications by applying hardware description language techniques to a library of full-custom CMOS building blocks. Single clock pre-charged circuits have been implemented for these arrays at clock rates in excess of 100 Mhz using economical 2-micron (minimum feature size) CMOS processes, which may be quickly configured for a variety of applications. A number of application-specific arrays are presented, including a 2-D convolver for image processing, an integer polynomial solver, and a finite-field polynomial solver.

Design and operation of a portable scanner for high performance microchip capillary array electrophoresis.

PubMed

Scherer, James R; Liu, Peng; Mathies, Richard A

2010-11-01

We have developed a compact, laser-induced fluorescence detection scanner, the multichannel capillary array electrophoresis portable scanner (McCAEPs) as a platform for electrophoretic detection and control of high-throughput, integrated microfluidic devices for genetic and other analyses. The instrument contains a confocal optical system with a rotary objective for detecting four different fluorescence signals, a pneumatic system consisting of two pressure/vacuum pumps and 28 individual addressable solenoid valves for control of on-chip microvalves and micropumps, four Polymerase Chain Reaction (PCR) temperature control systems, and four high voltage power supplies for electrophoresis. The detection limit of the instrument is ~20 pM for on-chip capillary electrophoresis of fluorescein dyes. To demonstrate the system performance for forensic short tandem repeat (STR) analysis, two experiments were conducted: (i) electrophoretic separation and detection of STR samples on a 96-lane microfabricated capillary array electrophoresis microchip. Fully resolved PowerPlex(®) 16 STR profiles amplified from 1 ng of 9947A female standard DNA were successfully obtained; (ii) nine-plex STR amplification, sample injection, separation, and fluorescence detection of 100-copy 9948 male standard DNA in a single integrated PCR- capillary electrophoresis microchip. These results demonstrate that the McCAEPs can be used as a versatile control and detection instrument that operates integrated microfluidic devices for high-performance forensic human identification.

Design and operation of a portable scanner for high performance microchip capillary array electrophoresis

NASA Astrophysics Data System (ADS)

Scherer, James R.; Liu, Peng; Mathies, Richard A.

2010-11-01

We have developed a compact, laser-induced fluorescence detection scanner, the multichannel capillary array electrophoresis portable scanner (McCAEPs) as a platform for electrophoretic detection and control of high-throughput, integrated microfluidic devices for genetic and other analyses. The instrument contains a confocal optical system with a rotary objective for detecting four different fluorescence signals, a pneumatic system consisting of two pressure/vacuum pumps and 28 individual addressable solenoid valves for control of on-chip microvalves and micropumps, four Polymerase Chain Reaction (PCR) temperature control systems, and four high voltage power supplies for electrophoresis. The detection limit of the instrument is ˜20 pM for on-chip capillary electrophoresis of fluorescein dyes. To demonstrate the system performance for forensic short tandem repeat (STR) analysis, two experiments were conducted: (i) electrophoretic separation and detection of STR samples on a 96-lane microfabricated capillary array electrophoresis microchip. Fully resolved PowerPlex® 16 STR profiles amplified from 1 ng of 9947A female standard DNA were successfully obtained; (ii) nine-plex STR amplification, sample injection, separation, and fluorescence detection of 100-copy 9948 male standard DNA in a single integrated PCR- capillary electrophoresis microchip. These results demonstrate that the McCAEPs can be used as a versatile control and detection instrument that operates integrated microfluidic devices for high-performance forensic human identification.

Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth and look-alike disease viruses

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

Hindson, B J; Baker, B R; Bentley Tammero, L F

2007-09-18

A high-throughput multiplexed assay (Multiplex Version 1.0) was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspectmore » cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRTPCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.« less

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.

Detection of Pathogenic Vibrio spp. in Shellfish by Using Multiplex PCR and DNA Microarrays

PubMed Central

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

2004-01-01

This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50°C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 102 to 103 CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers. PMID:15574946

Comparison of two suspension arrays for simultaneous detection of five biothreat bacterial in powder samples.

PubMed

Yang, Yu; Wang, Jing; Wen, Haiyan; Liu, Hengchuan

2012-01-01

We have developed novel Bio-Plex assays for simultaneous detection of Bacillus anthracis, Yersinia pestis, Brucella spp., Francisella tularensis, and Burkholderia pseudomallei. Universal primers were used to amplify highly conserved region located within the 16S rRNA amplicon, followed by hybridized to pathogen-specific probes for identification of these five organisms. The other assay is based on multiplex PCR to simultaneously amplify five species-specific pathogen identification-targeted regions unique to individual pathogen. Both of the two arrays are validated to be flexible and sensitive for simultaneous detection of bioterrorism bacteria. However, universal primer PCR-based array could not identify Bacillus anthracis, Yersinia pestis, and Brucella spp. at the species level because of the high conservation of 16S rDNA of the same genus. The two suspension arrays can be utilized to detect Bacillus anthracis sterne spore and Yersinia pestis EV76 from mimic "write powder" samples, they also proved that the suspension array system will be valuable tools for diagnosis of bacterial biothreat agents in environmental samples.

Repression of anti-proliferative factor Tob1 in osteoarthritic cartilage

PubMed Central

Gebauer, Mathias; Saas, Joachim; Haag, Jochen; Dietz, Uwe; Takigawa, Masaharu; Bartnik, Eckart; Aigner, Thomas

2005-01-01

Osteoarthritis is the most common degenerative disorder of the modern world. However, many basic cellular features and molecular processes of the disease are poorly understood. In the present study we used oligonucleotide-based microarray analysis of genes of known or assumed relevance to the cellular phenotype to screen for relevant differences in gene expression between normal and osteoarthritic chondrocytes. Custom made oligonucleotide DNA arrays were used to screen for differentially expressed genes in normal (n = 9) and osteoarthritic (n = 10) cartilage samples. Real-time polymerase chain reaction (PCR) with gene-specific primers was used for quantification. Primary human adult articular chondrocytes and chondrosarcoma cell line HCS-2/8 were used to study changes in gene expression levels after stimulation with interleukin-1β and bone morphogenetic protein, as well as the dependence on cell differentiation. In situ hybridization with a gene-specific probe was applied to detect mRNA expression levels in fetal growth plate cartilage. Overall, more than 200 significantly regulated genes were detected between normal and osteoarthritic cartilage (P < 0.01). One of the significantly repressed genes, Tob1, encodes a protein belonging to a family involved in silencing cells in terms of proliferation and functional activity. The repression of Tob1 was confirmed by quantitative PCR and correlated to markers of chondrocyte activity and proliferation in vivo. Tob1 expression was also detected at a decreased level in isolated chondrocytes and in the chondrosarcoma cell line HCS-2/8. Again, in these cells it was negatively correlated with proliferative activity and positively with cellular differentiation. Altogether, the downregulation of the expression of Tob1 in osteoarthritic chondrocytes might be an important aspect of the cellular processes taking place during osteoarthritic cartilage degeneration. Activation, the reinitiation of proliferative activity and the loss of a stable phenotype are three major changes in osteoarthritic chondrocytes that are highly significantly correlated with the repression of Tob1 expression. PMID:15743474

Replica amplification of nucleic acid arrays

DOEpatents

Church, George M.; Mitra, Robi D.

2010-08-31

Disclosed are improved methods of making and using immobilized arrays of nucleic acids, particularly methods for producing replicas of such arrays. Included are methods for producing high density arrays of nucleic acids and replicas of such arrays, as well as methods for preserving the resolution of arrays through rounds of replication. Also included are methods which take advantage of the availability of replicas of arrays for increased sensitivity in detection of sequences on arrays. Improved methods of sequencing nucleic acids immobilized on arrays utilizing single copies of arrays and methods taking further advantage of the availability of replicas of arrays are disclosed. The improvements lead to higher fidelity and longer read lengths of sequences immobilized on arrays. Methods are also disclosed which improve the efficiency of multiplex PCR using arrays of immobilized nucleic acids.

Patient-Specific Circulating Tumor DNA Detection during Neoadjuvant Chemotherapy in Triple-Negative Breast Cancer.

PubMed

Riva, Francesca; Bidard, Francois-Clement; Houy, Alexandre; Saliou, Adrien; Madic, Jordan; Rampanou, Aurore; Hego, Caroline; Milder, Maud; Cottu, Paul; Sablin, Marie-Paule; Vincent-Salomon, Anne; Lantz, Olivier; Stern, Marc-Henri; Proudhon, Charlotte; Pierga, Jean-Yves

2017-03-01

In nonmetastatic triple-negative breast cancer (TNBC) patients, we investigated whether circulating tumor DNA (ctDNA) detection can reflect the tumor response to neoadjuvant chemotherapy (NCT) and detect minimal residual disease after surgery. Ten milliliters of plasma were collected at 4 time points: before NCT; after 1 cycle; before surgery; after surgery. Customized droplet digital PCR (ddPCR) assays were used to track tumor protein p53 ( TP53 ) mutations previously characterized in tumor tissue by massively parallel sequencing (MPS). Forty-six patients with nonmetastatic TNBC were enrolled. TP53 mutations were identified in 40 of them. Customized ddPCR probes were validated for 38 patients, with excellent correlation with MPS ( r = 0.99), specificity (≥2 droplets/assay), and sensitivity (at least 0.1%). At baseline, ctDNA was detected in 27/36 patients (75%). Its detection was associated with mitotic index ( P = 0.003), tumor grade ( P = 0.003), and stage ( P = 0.03). During treatment, we observed a drop of ctDNA levels in all patients but 1. No patient had detectable ctDNA after surgery. The patient with rising ctDNA levels experienced tumor progression during NCT. Pathological complete response (16/38 patients) was not correlated with ctDNA detection at any time point. ctDNA positivity after 1 cycle of NCT was correlated with shorter disease-free ( P < 0.001) and overall ( P = 0.006) survival. Customized ctDNA detection by ddPCR achieved a 75% detection rate at baseline. During NCT, ctDNA levels decreased quickly and minimal residual disease was not detected after surgery. However, a slow decrease of ctDNA level during NCT was strongly associated with shorter survival. © 2016 American Association for Clinical Chemistry.

A microfluidic approach to parallelized transcriptional profiling of single cells.

PubMed

Sun, Hao; Olsen, Timothy; Zhu, Jing; Tao, Jianguo; Ponnaiya, Brian; Amundson, Sally A; Brenner, David J; Lin, Qiao

2015-12-01

The ability to correlate single-cell genetic information with cellular phenotypes is of great importance to biology and medicine, as it holds the potential to gain insight into disease pathways that is unavailable from ensemble measurements. We present a microfluidic approach to parallelized, rapid, quantitative analysis of messenger RNA from single cells via RT-qPCR. The approach leverages an array of single-cell RT-qPCR analysis units formed by a set of parallel microchannels concurrently controlled by elastomeric pneumatic valves, thereby enabling parallelized handling and processing of single cells in a drastically simplified operation procedure using a relatively small number of microvalves. All steps for single-cell RT-qPCR, including cell isolation and immobilization, cell lysis, mRNA purification, reverse transcription and qPCR, are integrated on a single chip, eliminating the need for off-chip manual cell and reagent transfer and qPCR amplification as commonly used in existing approaches. Additionally, the approach incorporates optically transparent microfluidic components to allow monitoring of single-cell trapping without the need for molecular labeling that can potentially alter the targeted gene expression and utilizes a polycarbonate film as a barrier against evaporation to minimize the loss of reagents at elevated temperatures during the analysis. We demonstrate the utility of the approach by the transcriptional profiling for the induction of the cyclin-dependent kinase inhibitor 1a and the glyceraldehyde 3-phosphate dehydrogenase in single cells from the MCF-7 breast cancer cell line. Furthermore, the methyl methanesulfonate is employed to allow measurement of the expression of the genes in individual cells responding to a genotoxic stress.

Preconcentration of DNA using magnetic ionic liquids that are compatible with real-time PCR for rapid nucleic acid quantification.

PubMed

Emaus, Miranda N; Clark, Kevin D; Hinners, Paige; Anderson, Jared L

2018-04-28

Nucleic acid extraction and purification represents a major bottleneck in DNA analysis. Traditional methods for DNA purification often require reagents that may inhibit quantitative polymerase chain reaction (qPCR) if not sufficiently removed from the sample. Approaches that employ magnetic beads may exhibit lower extraction efficiencies due to sedimentation and aggregation. In this study, four hydrophobic magnetic ionic liquids (MILs) were investigated as DNA extraction solvents with the goal of improving DNA enrichment factors and compatibility with downstream bioanalytical techniques. By designing custom qPCR buffers, we directly incorporated DNA-enriched MILs including trihexyl(tetradecyl)phosphonium tris(hexafluoroacetylaceto)nickelate(II) ([P 6,6,6,14 + ][Ni(hfacac) 3 - ]), [P 6,6,6,14 + ] tris(hexafluoroacetylaceto)colbaltate(II) ([Co(hfacac) 3 - ]), [P 6,6,6,14 + ] tris(hexafluoroacetylaceto)manganate(II) ([Mn(hfacac) 3 - ]), or [P 6,6,6,14 + ] tetrakis(hexafluoroacetylaceto)dysprosate(III) ([Dy(hfacac) 4 - ]) into reaction systems, thereby circumventing the need for time-consuming DNA recovery steps. Incorporating MILs into the reaction buffer did not significantly impact the amplification efficiency of the reaction (91.1%). High enrichment factors were achieved using the [P 6,6,6,14 + ][Ni(hfacac) 3 - ] MIL for the extraction of single-stranded and double-stranded DNA with extraction times as short as 2 min. When compared to a commercial magnetic bead-based platform, the [P 6,6,6,14 + ][Ni(hfacac) 3 - ] MIL was capable of producing higher enrichment factors for single-stranded DNA and similar enrichment factors for double-stranded DNA. The MIL-based method was applied for the extraction and direct qPCR amplification of mutation prone-KRAS oncogene fragment in plasma samples. Graphical abstract Magnetic ionic liquid solvents are shown to preconcentrate sufficient KRAS DNA template from an aqueous solution in as short as 2 min without using chaotropic salts or toxic organic solvents. By using custom-designed qPCR buffers, DNA can be directly amplified and quantified from four MILs examined in this study.

Developing custom fire behavior fuel models from ecologically complex fuel structures for upper Atlantic Coastal Plain forests

Treesearch

Bernard R. Parresol; Joe H. Scott; Anne Andreu; Susan Prichard; Laurie Kurth

2012-01-01

Currently geospatial fire behavior analyses are performed with an array of fire behavior modeling systems such as FARSITE, FlamMap, and the Large Fire Simulation System. These systems currently require standard or customized surface fire behavior fuel models as inputs that are often assigned through remote sensing information. The ability to handle hundreds or...

Serial multiplier arrays for parallel computation

NASA Technical Reports Server (NTRS)

Winters, Kel

1990-01-01

Arrays of systolic serial-parallel multiplier elements are proposed as an alternative to conventional SIMD mesh serial adder arrays for applications that are multiplication intensive and require few stored operands. The design and operation of a number of multiplier and array configurations featuring locality of connection, modularity, and regularity of structure are discussed. A design methodology combining top-down and bottom-up techniques is described to facilitate development of custom high-performance CMOS multiplier element arrays as well as rapid synthesis of simulation models and semicustom prototype CMOS components. Finally, a differential version of NORA dynamic circuits requiring a single-phase uncomplemented clock signal introduced for this application.

Nucleic acid amplification using modular branched primers

DOEpatents

Ulanovsky, Levy; Raja, Mugasimangalam C.

2001-01-01

Methods and compositions expand the options for making primers for use in amplifying nucleic acid segments. The invention eliminates the step of custom synthesis of primers for Polymerase Chain Reactions (PCR). Instead of being custom-synthesized, a primer is replaced by a combination of several oligonucleotide modules selected from a pre-synthesized library. A modular combination of just a few oligonucleotides essentially mimics the performance of a conventional, custom-made primer by matching the sequence of the priming site in the template. Each oligonucleotide module has a segment that matches one of the stretches within the priming site.

How Well Does Physician Selection of Microbiologic Tests Identify Clostridium difficile and other Pathogens in Paediatric Diarrhoea? Insights Using Multiplex PCR-Based Detection

PubMed Central

Stockmann, Chris; Rogatcheva, Margarita; Harrel, Brian; Vaughn, Mike; Crisp, Rob; Poritz, Mark; Thatcher, Stephanie; Korgenski, Ernest K; Barney, Trenda; Daly, Judy; Pavia, Andrew T

2014-01-01

The objective of this study was to compare the aetiologic yield of standard of care microbiologic testing ordered by physicians with that of a multiplex PCR platform. Stool specimens obtained from children and young adults with gastrointestinal illness were evaluated by standard laboratory methods and a developmental version of the FilmArray Gastrointestinal Diagnostic System (FilmArray GI Panel), a rapid multiplex PCR platform that detects 23 bacterial, viral, and protozoal agents. Results were classified according to the microbiologic tests requested by the treating physician. A median of 3 (range 1-10) microbiologic tests were performed by the clinical laboratory during 378 unique diarrhoeal episodes. A potential aetiologic agent was identified in 46% of stool specimens by standard laboratory methods and in 65% of specimens tested using the FilmArray GI Panel (P<0.001). For those patients who only had Clostridium difficile testing requested, an alternative pathogen was identified in 29% of cases with the FilmArray GI Panel. Notably, 11 (12%) cases of norovirus were identified among children who only had testing for C. difficile ordered. Among those who had C. difficile testing ordered in combination with other tests, an additional pathogen was identified in 57% of stool specimens with the FilmArray GI Panel. For patients who had no C. difficile testing performed, the FilmArray GI Panel identified a pathogen in 63% of cases, including C. difficile in 8%. Physician-specified laboratory testing may miss important diarrhoeal pathogens. Additionally, standard laboratory testing is likely to underestimate co-infections with multiple infectious diarrhoeagenic agents. PMID:25599941

Transcriptome sequencing and microarray development for the woodrat (Neotoma spp.): custom genetic tools for exploring herbivore ecology.

PubMed

Malenke, J R; Milash, B; Miller, A W; Dearing, M D

2013-07-01

Massively parallel sequencing has enabled the creation of novel, in-depth genetic tools for nonmodel, ecologically important organisms. We present the de novo transcriptome sequencing, analysis and microarray development for a vertebrate herbivore, the woodrat (Neotoma spp.). This genus is of ecological and evolutionary interest, especially with respect to ingestion and hepatic metabolism of potentially toxic plant secondary compounds. We generated a liver transcriptome of the desert woodrat (Neotoma lepida) using the Roche 454 platform. The assembled contigs were well annotated using rodent references (99.7% annotation), and biotransformation function was reflected in the gene ontology. The transcriptome was used to develop a custom microarray (eArray, Agilent). We tested the microarray with three experiments: one across species with similar habitat (thus, dietary) niches, one across species with different habitat niches and one across populations within a species. The resulting one-colour arrays had high technical and biological quality. Probes designed from the woodrat transcriptome performed significantly better than functionally similar probes from the Norway rat (Rattus norvegicus). There were a multitude of expression differences across the woodrat treatments, many of which related to biotransformation processes and activities. The pattern and function of the differences indicate shared ecological pressures, and not merely phylogenetic distance, play an important role in shaping gene expression profiles of woodrat species and populations. The quality and functionality of the woodrat transcriptome and custom microarray suggest these tools will be valuable for expanding the scope of herbivore biology, as well as the exploration of conceptual topics in ecology. © 2013 John Wiley & Sons Ltd.

Methods for producing partially digested restriction DNA fragments and for producing a partially modified PCR product

DOEpatents

Wong, Kwong-Kwok

2000-01-01

The present invention is an improved method of making a partially modified PCR product from a DNA fragment with a polymerase chain reaction (PCR). In a standard PCR process, the DNA fragment is combined with starting deoxynucleoside triphosphates, a primer, a buffer and a DNA polymerase in a PCR mixture. The PCR mixture is then reacted in the PCR producing copies of the DNA fragment. The improvement of the present invention is adding an amount of a modifier at any step prior to completion of the PCR process thereby randomly and partially modifying the copies of the DNA fragment as a partially modified PCR product. The partially modified PCR product may then be digested with an enzyme that cuts the partially modified PCR product at unmodified sites thereby producing an array of DNA restriction fragments.

Quantifying EGFR alterations in the lung cancer genome with nanofluidic digital PCR arrays.

PubMed

Wang, Jun; Ramakrishnan, Ramesh; Tang, Zhe; Fan, Weiwen; Kluge, Amy; Dowlati, Afshin; Jones, Robert C; Ma, Patrick C

2010-04-01

The EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] gene is known to harbor genomic alterations in advanced lung cancer involving gene amplification and kinase mutations that predict the clinical response to EGFR-targeted inhibitors. Methods for detecting such molecular changes in lung cancer tumors are desirable. We used a nanofluidic digital PCR array platform and 16 cell lines and 20 samples of genomic DNA from resected tumors (stages I-III) to quantify the relative numbers of copies of the EGFR gene and to detect mutated EGFR alleles in lung cancer. We assessed the relative number of EGFR gene copies by calculating the ratio of the number of EGFR molecules (measured with a 6-carboxyfluorescein-labeled Scorpion assay) to the number of molecules of the single-copy gene RPP30 (ribonuclease P/MRP 30kDa subunit) (measured with a 6-carboxy-X-rhodamine-labeled TaqMan assay) in each panel. To assay for the EGFR L858R (exon 21) mutation and exon 19 in-frame deletions, we used the ARMS and Scorpion technologies in a DxS/Qiagen EGFR29 Mutation Test Kit for the digital PCR array. The digital array detected and quantified rare gefitinib/erlotinib-sensitizing EGFR mutations (0.02%-9.26% abundance) that were present in formalin-fixed, paraffin-embedded samples of early-stage resectable lung tumors without an associated increase in gene copy number. Our results also demonstrated the presence of intratumor molecular heterogeneity for the clinically relevant EGFR mutated alleles in these early-stage lung tumors. The digital PCR array platform allows characterization and quantification of oncogenes, such as EGFR, at the single-molecule level. Use of this nanofluidics platform may provide deeper insight into the specific roles of clinically relevant kinase mutations during different stages of lung tumor progression and may be useful in predicting the clinical response to EGFR-targeted inhibitors.

Altered serum microRNAs as biomarkers for the early diagnosis of pulmonary tuberculosis infection

PubMed Central

2012-01-01

Background Pulmonary tuberculosis (TB) is a highly lethal infectious disease and early diagnosis of TB is critical for the control of disease progression. The objective of this study was to profile a panel of serum microRNAs (miRNAs) as potential biomarkers for the early diagnosis of pulmonary TB infection. Methods Using TaqMan Low-Density Array (TLDA) analysis followed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) validation, expression levels of miRNAs in serum samples from 30 patients with active tuberculosis and 60 patients with Bordetella pertussis (BP), varicella-zoster virus (VZV) and enterovirus (EV) were analyzed. Results The Low-Density Array data showed that 97 miRNAs were differentially expressed in pulmonary TB patient sera compared with healthy controls (90 up-regulated and 7 down-regulated). Following qRT-PCR confirmation and receiver operational curve (ROC) analysis, three miRNAs (miR-361-5p, miR-889 and miR-576-3p) were shown to distinguish TB infected patients from healthy controls and other microbial infections with moderate sensitivity and specificity (area under curve (AUC) value range, 0.711-0.848). Multiple logistic regression analysis of a combination of these three miRNAs showed an enhanced ability to discriminate between these two groups with an AUC value of 0.863. Conclusions Our study suggests that altered levels of serum miRNAs have great potential to serve as non-invasive biomarkers for early detection of pulmonary TB infection. PMID:23272999

Development and validation of a high density SNP genotyping array for Atlantic salmon (Salmo salar).

PubMed

Houston, Ross D; Taggart, John B; Cézard, Timothé; Bekaert, Michaël; Lowe, Natalie R; Downing, Alison; Talbot, Richard; Bishop, Stephen C; Archibald, Alan L; Bron, James E; Penman, David J; Davassi, Alessandro; Brew, Fiona; Tinch, Alan E; Gharbi, Karim; Hamilton, Alastair

2014-02-06

Dense single nucleotide polymorphism (SNP) genotyping arrays provide extensive information on polymorphic variation across the genome of species of interest. Such information can be used in studies of the genetic architecture of quantitative traits and to improve the accuracy of selection in breeding programs. In Atlantic salmon (Salmo salar), these goals are currently hampered by the lack of a high-density SNP genotyping platform. Therefore, the aim of the study was to develop and test a dense Atlantic salmon SNP array. SNP discovery was performed using extensive deep sequencing of Reduced Representation (RR-Seq), Restriction site-Associated DNA (RAD-Seq) and mRNA (RNA-Seq) libraries derived from farmed and wild Atlantic salmon samples (n = 283) resulting in the discovery of > 400 K putative SNPs. An Affymetrix Axiom® myDesign Custom Array was created and tested on samples of animals of wild and farmed origin (n = 96) revealing a total of 132,033 polymorphic SNPs with high call rate, good cluster separation on the array and stable Mendelian inheritance in our sample. At least 38% of these SNPs are from transcribed genomic regions and therefore more likely to include functional variants. Linkage analysis utilising the lack of male recombination in salmonids allowed the mapping of 40,214 SNPs distributed across all 29 pairs of chromosomes, highlighting the extensive genome-wide coverage of the SNPs. An identity-by-state clustering analysis revealed that the array can clearly distinguish between fish of different origins, within and between farmed and wild populations. Finally, Y-chromosome-specific probes included on the array provide an accurate molecular genetic test for sex. This manuscript describes the first high-density SNP genotyping array for Atlantic salmon. This array will be publicly available and is likely to be used as a platform for high-resolution genetics research into traits of evolutionary and economic importance in salmonids and in aquaculture breeding programs via genomic selection.

Development and validation of a high density SNP genotyping array for Atlantic salmon (Salmo salar)

PubMed Central

2014-01-01

Background Dense single nucleotide polymorphism (SNP) genotyping arrays provide extensive information on polymorphic variation across the genome of species of interest. Such information can be used in studies of the genetic architecture of quantitative traits and to improve the accuracy of selection in breeding programs. In Atlantic salmon (Salmo salar), these goals are currently hampered by the lack of a high-density SNP genotyping platform. Therefore, the aim of the study was to develop and test a dense Atlantic salmon SNP array. Results SNP discovery was performed using extensive deep sequencing of Reduced Representation (RR-Seq), Restriction site-Associated DNA (RAD-Seq) and mRNA (RNA-Seq) libraries derived from farmed and wild Atlantic salmon samples (n = 283) resulting in the discovery of > 400 K putative SNPs. An Affymetrix Axiom® myDesign Custom Array was created and tested on samples of animals of wild and farmed origin (n = 96) revealing a total of 132,033 polymorphic SNPs with high call rate, good cluster separation on the array and stable Mendelian inheritance in our sample. At least 38% of these SNPs are from transcribed genomic regions and therefore more likely to include functional variants. Linkage analysis utilising the lack of male recombination in salmonids allowed the mapping of 40,214 SNPs distributed across all 29 pairs of chromosomes, highlighting the extensive genome-wide coverage of the SNPs. An identity-by-state clustering analysis revealed that the array can clearly distinguish between fish of different origins, within and between farmed and wild populations. Finally, Y-chromosome-specific probes included on the array provide an accurate molecular genetic test for sex. Conclusions This manuscript describes the first high-density SNP genotyping array for Atlantic salmon. This array will be publicly available and is likely to be used as a platform for high-resolution genetics research into traits of evolutionary and economic importance in salmonids and in aquaculture breeding programs via genomic selection. PMID:24524230

Design of ultrathin dual-resonant reflective polarization converter with customized bandwidths

NASA Astrophysics Data System (ADS)

Kundu, Debidas; Mohan, Akhilesh; Chakrabarty, Ajay

2017-10-01

In this paper, an ultrathin dual-resonant reflective polarization converter is proposed to obtain customized bandwidths using precise space-filling technique to its top geometry. The unit cell of the dual-resonant prototype consists of conductive square ring with two diagonally arranged slits, supported by metal-backed thin dielectric layer. It offers two narrow bands with fractional bandwidths of 3.98 and 6.65% and polarization conversion ratio (PCR) of 97.16 and 98.87% at 4.52 and 6.97 GHz, respectively. The resonances are brought in proximity to each other by changing the length of surface current paths of the two resonances. By virtue of this mechanism, two polarization converters with two different types of bandwidths are obtained. One polarization converter produces a full-width at half-maxima PCR bandwidth of 34%, whereas another polarization converter produces a 90% PCR bandwidth of 19%. All the proposed polarization converters are insensitive to wide variations of incident angle for both TE- and TM-polarized incident waves. Measured results show good agreement with the numerically simulated results.

Development and Calibration of a Field-Deployable Microphone Phased Array for Propulsion and Airframe Noise Flyover Measurements

NASA Technical Reports Server (NTRS)

Humphreys, William M., Jr.; Lockard, David P.; Khorrami, Mehdi R.; Culliton, William G.; McSwain, Robert G.; Ravetta, Patricio A.; Johns, Zachary

2016-01-01

A new aeroacoustic measurement capability has been developed consisting of a large channelcount, field-deployable microphone phased array suitable for airframe noise flyover measurements for a range of aircraft types and scales. The array incorporates up to 185 hardened, weather-resistant sensors suitable for outdoor use. A custom 4-mA current loop receiver circuit with temperature compensation was developed to power the sensors over extended cable lengths with minimal degradation of the signal to noise ratio and frequency response. Extensive laboratory calibrations and environmental testing of the sensors were conducted to verify the design's performance specifications. A compact data system combining sensor power, signal conditioning, and digitization was assembled for use with the array. Complementing the data system is a robust analysis system capable of near real-time presentation of beamformed and deconvolved contour plots and integrated spectra obtained from array data acquired during flyover passes. Additional instrumentation systems needed to process the array data were also assembled. These include a commercial weather station and a video monitoring / recording system. A detailed mock-up of the instrumentation suite (phased array, weather station, and data processor) was performed in the NASA Langley Acoustic Development Laboratory to vet the system performance. The first deployment of the system occurred at Finnegan Airfield at Fort A.P. Hill where the array was utilized to measure the vehicle noise from a number of sUAS (small Unmanned Aerial System) aircraft. A unique in-situ calibration method for the array microphones using a hovering aerial sound source was attempted for the first time during the deployment.

FPGA Vision Data Architecture

NASA Technical Reports Server (NTRS)

Morfopoulos, Arin C.; Pham, Thang D.

2013-01-01

JPL has produced a series of FPGA (field programmable gate array) vision algorithms that were written with custom interfaces to get data in and out of each vision module. Each module has unique requirements on the data interface, and further vision modules are continually being developed, each with their own custom interfaces. Each memory module had also been designed for direct access to memory or to another memory module.

High-throughput mouse genotyping using robotics automation.

PubMed

Linask, Kaari L; Lo, Cecilia W

2005-02-01

The use of mouse models is rapidly expanding in biomedical research. This has dictated the need for the rapid genotyping of mutant mouse colonies for more efficient utilization of animal holding space. We have established a high-throughput protocol for mouse genotyping using two robotics workstations: a liquid-handling robot to assemble PCR and a microfluidics electrophoresis robot for PCR product analysis. This dual-robotics setup incurs lower start-up costs than a fully automated system while still minimizing human intervention. Essential to this automation scheme is the construction of a database containing customized scripts for programming the robotics workstations. Using these scripts and the robotics systems, multiple combinations of genotyping reactions can be assembled simultaneously, allowing even complex genotyping data to be generated rapidly with consistency and accuracy. A detailed protocol, database, scripts, and additional background information are available at http://dir.nhlbi.nih.gov/labs/ldb-chd/autogene/.

A sub-millimeter resolution PET detector module using a multi-pixel photon counter array

NASA Astrophysics Data System (ADS)

Song, Tae Yong; Wu, Heyu; Komarov, Sergey; Siegel, Stefan B.; Tai, Yuan-Chuan

2010-05-01

A PET block detector module using an array of sub-millimeter lutetium oxyorthosilicate (LSO) crystals read out by an array of surface-mount, semiconductor photosensors has been developed. The detector consists of a LSO array, a custom acrylic light guide, a 3 × 3 multi-pixel photon counter (MPPC) array (S10362-11-050P, Hamamatsu Photonics, Japan) and a readout board with a charge division resistor network. The LSO array consists of 100 crystals, each measuring 0.8 × 0.8 × 3 mm3 and arranged in 0.86 mm pitches. A Monte Carlo simulation was used to aid the design and fabrication of a custom light guide to control distribution of scintillation light over the surface of the MPPC array. The output signals of the nine MPPC are multiplexed by a charge division resistor network to generate four position-encoded analog outputs. Flood image, energy resolution and timing resolution measurements were performed using standard NIM electronics. The linearity of the detector response was investigated using gamma-ray sources of different energies. The 10 × 10 array of 0.8 mm LSO crystals was clearly resolved in the flood image. The average energy resolution and standard deviation were 20.0% full-width at half-maximum (FWHM) and ±5.0%, respectively, at 511 keV. The timing resolution of a single MPPC coupled to a LSO crystal was found to be 857 ps FWHM, and the value for the central region of detector module was 1182 ps FWHM when ±10% energy window was applied. The nonlinear response of a single MPPC when used to read out a single LSO was observed among the corner crystals of the proposed detector module. However, the central region of the detector module exhibits significantly less nonlinearity (6.5% for 511 keV). These results demonstrate that (1) a charge-sharing resistor network can effectively multiplex MPPC signals and reduce the number of output signals without significantly degrading the performance of a PET detector and (2) a custom light guide to permit light sharing among multiple MPPC and to diffuse and direct scintillation light can reduce the nonlinearity of the detector response within the limited dynamic range of a typical MPPC. As a result, the proposed PET detector module has the potential to be refined for use in high-resolution PET insert applications.

A sub-millimeter resolution PET detector module using a multi-pixel photon counter array.

PubMed

Song, Tae Yong; Wu, Heyu; Komarov, Sergey; Siegel, Stefan B; Tai, Yuan-Chuan

2010-05-07

A PET block detector module using an array of sub-millimeter lutetium oxyorthosilicate (LSO) crystals read out by an array of surface-mount, semiconductor photosensors has been developed. The detector consists of a LSO array, a custom acrylic light guide, a 3 x 3 multi-pixel photon counter (MPPC) array (S10362-11-050P, Hamamatsu Photonics, Japan) and a readout board with a charge division resistor network. The LSO array consists of 100 crystals, each measuring 0.8 x 0.8 x 3 mm(3) and arranged in 0.86 mm pitches. A Monte Carlo simulation was used to aid the design and fabrication of a custom light guide to control distribution of scintillation light over the surface of the MPPC array. The output signals of the nine MPPC are multiplexed by a charge division resistor network to generate four position-encoded analog outputs. Flood image, energy resolution and timing resolution measurements were performed using standard NIM electronics. The linearity of the detector response was investigated using gamma-ray sources of different energies. The 10 x 10 array of 0.8 mm LSO crystals was clearly resolved in the flood image. The average energy resolution and standard deviation were 20.0% full-width at half-maximum (FWHM) and +/-5.0%, respectively, at 511 keV. The timing resolution of a single MPPC coupled to a LSO crystal was found to be 857 ps FWHM, and the value for the central region of detector module was 1182 ps FWHM when +/-10% energy window was applied. The nonlinear response of a single MPPC when used to read out a single LSO was observed among the corner crystals of the proposed detector module. However, the central region of the detector module exhibits significantly less nonlinearity (6.5% for 511 keV). These results demonstrate that (1) a charge-sharing resistor network can effectively multiplex MPPC signals and reduce the number of output signals without significantly degrading the performance of a PET detector and (2) a custom light guide to permit light sharing among multiple MPPC and to diffuse and direct scintillation light can reduce the nonlinearity of the detector response within the limited dynamic range of a typical MPPC. As a result, the proposed PET detector module has the potential to be refined for use in high-resolution PET insert applications.

A sub-millimeter resolution PET detector module using a multi-pixel photon counter array

PubMed Central

Song, Tae Yong; Wu, Heyu; Komarov, Sergey; Siegel, Stefan B; Tai, Yuan-Chuan

2010-01-01

A PET block detector module using an array of sub-millimeter lutetium oxyorthosilicate (LSO) crystals read out by an array of surface-mount, semiconductor photosensors has been developed. The detector consists of a LSO array, a custom acrylic light guide, a 3 × 3 multi-pixel photon counter (MPPC) array (S10362-11-050P, Hamamatsu Photonics, Japan) and a readout board with a charge division resistor network. The LSO array consists of 100 crystals, each measuring 0.8 × 0.8 × 3 mm3 and arranged in 0.86 mm pitches. A Monte Carlo simulation was used to aid the design and fabrication of a custom light guide to control distribution of scintillation light over the surface of the MPPC array. The output signals of the nine MPPC are multiplexed by a charge division resistor network to generate four position-encoded analog outputs. Flood image, energy resolution and timing resolution measurements were performed using standard NIM electronics. The linearity of the detector response was investigated using gamma-ray sources of different energies. The 10 × 10 array of 0.8 mm LSO crystals was clearly resolved in the flood image. The average energy resolution and standard deviation were 20.0% full-width at half-maximum (FWHM) and ±5.0%, respectively, at 511 keV. The timing resolution of a single MPPC coupled to a LSO crystal was found to be 857 ps FWHM, and the value for the central region of detector module was 1182 ps FWHM when ±10% energy window was applied. The nonlinear response of a single MPPC when used to read out a single LSO was observed among the corner crystals of the proposed detector module. However, the central region of the detector module exhibits significantly less nonlinearity (6.5% for 511 keV). These results demonstrate that (1) a charge-sharing resistor network can effectively multiplex MPPC signals and reduce the number of output signals without significantly degrading the performance of a PET detector and (2) a custom light guide to permit light sharing among multiple MPPC and to diffuse and direct scintillation light can reduce the nonlinearity of the detector response within the limited dynamic range of a typical MPPC. As a result, the proposed PET detector module has the potential to be refined for use in high-resolution PET insert applications. PMID:20393236

A 1-MHz 2-D CMUT array for HIFU thermal ablation

NASA Astrophysics Data System (ADS)

Yoon, Hyo-Seon; Vaithilingam, Srikant; Park, Kwan Kyu; Nikoozadeh, Amin; Firouzi, Kamyar; Choe, Jung Woo; Watkins, Ronald D.; Oguz, Huseyin Kagan; Kupnik, Mario; Pauly, Kim Butts; Khuri-Yakub, Pierre

2017-03-01

We developed a fully-populated 2-D capacitive micromachined ultrasonic transducer (CMUT) array for high intensity focused ultrasound (HIFU) treatment. The 2-D CMUT array, which consists of 20 × 20 square CMUT elements with an element-to-element pitch of 1 mm, was designed and fabricated using the thick-buried-oxide (BOX) fabrication process. It was then assembled on a custom interface board that can provide various array configurations depending on the desired applications. In this study, the interface board groups the CMUT array elements into eight channels, based on the phase delay from the element to the targeted focal point at a 20-mm distance from the array surface, which corresponds to an F-number of 1. An 8-channel phase generating system supplies continuous waves with eight different phases to the eight channels of the CMUT array through bias-tees and amplifiers. This array aperture, grouped into eight channels, gives a focusing gain of 6.09 according to field simulation using Field II. Assuming a peak-to-peak pressure of 1 MPa at the surface of the array, our custom temperature simulator predicts successful tissue ablation at the focus. During the measurements, each channel was tuned with a series inductor for an operational frequency of 1 MHz. With a CMUT DC bias of 100 V and a 1-MHz AC input voltage of 55 V, we achieved peak-to-peak output pressures of 173.9 kPa and 568.7 kPa at the array surface and at the focus, respectively. The focusing gain calculated from this measurement is 3.27, which is lower than the simulated gain of 6.09 because of the mutual radiation impedance among the CMUT cells. Further optimization of the operating condition of this array and design improvements for reducing the effect of mutual radiation impedance are currently on-going.

C-MOS bulk metal design handbook. [LSI standard cell (circuits)

NASA Technical Reports Server (NTRS)

Edge, T. M.

1977-01-01

The LSI standard cell array technique was used in the fabrication of more than 20 CMOS custom arrays. This technique consists of a series of computer programs and design automation techniques referred to as the Computer Aided Design And Test (CADAT) system that automatically translate a partitioned logic diagram into a set of instructions for driving an automatic plotter which generates precision mask artwork for complex LSI arrays of CMOS standard cells. The standard cell concept for producing LSI arrays begins with the design, layout, and validation of a group of custom circuits called standard cells. Once validated, these cells are given identification or pattern numbers and are permanently stored. To use one of these cells in a logic design, the user calls for the desired cell by pattern number. The Place, Route in Two Dimension (PR2D) computer program is then used to automatically generate the metalization and/or tunnels to interconnect the standard cells into the required function. Data sheets that describe the function, artwork, and performance of each of the standard cells, the general procedure for implementation of logic in CMOS standard cells, and additional detailed design information are presented.

DNA microarray-based PCR ribotyping of Clostridium difficile.

PubMed

Schneeberg, Alexander; Ehricht, Ralf; Slickers, Peter; Baier, Vico; Neubauer, Heinrich; Zimmermann, Stefan; Rabold, Denise; Lübke-Becker, Antina; Seyboldt, Christian

2015-02-01

This study presents a DNA microarray-based assay for fast and simple PCR ribotyping of Clostridium difficile strains. Hybridization probes were designed to query the modularly structured intergenic spacer region (ISR), which is also the template for conventional and PCR ribotyping with subsequent capillary gel electrophoresis (seq-PCR) ribotyping. The probes were derived from sequences available in GenBank as well as from theoretical ISR module combinations. A database of reference hybridization patterns was set up from a collection of 142 well-characterized C. difficile isolates representing 48 seq-PCR ribotypes. The reference hybridization patterns calculated by the arithmetic mean were compared using a similarity matrix analysis. The 48 investigated seq-PCR ribotypes revealed 27 array profiles that were clearly distinguishable. The most frequent human-pathogenic ribotypes 001, 014/020, 027, and 078/126 were discriminated by the microarray. C. difficile strains related to 078/126 (033, 045/FLI01, 078, 126, 126/FLI01, 413, 413/FLI01, 598, 620, 652, and 660) and 014/020 (014, 020, and 449) showed similar hybridization patterns, confirming their genetic relatedness, which was previously reported. A panel of 50 C. difficile field isolates was tested by seq-PCR ribotyping and the DNA microarray-based assay in parallel. Taking into account that the current version of the microarray does not discriminate some closely related seq-PCR ribotypes, all isolates were typed correctly. Moreover, seq-PCR ribotypes without reference profiles available in the database (ribotype 009 and 5 new types) were correctly recognized as new ribotypes, confirming the performance and expansion potential of the microarray. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Impact of Early Detection of Respiratory Viruses by Multiplex PCR Assay on Clinical Outcomes in Adult Patients

PubMed Central

Schuetz, Audrey N.; Jenkins, Stephen G.; Calfee, David P.; Walsh, Thomas J.; Wells, Martin T.; Hollenberg, James P.; Glesby, Marshall J.

2016-01-01

Rapid and definitive diagnosis of viral respiratory infections is imperative in patient triage and management. We compared the outcomes for adult patients with positive tests for respiratory viruses at a tertiary care center across two consecutive influenza seasons (winters of 2010-2011 and 2012). Infections were diagnosed by conventional methods in the first season and by multiplex PCR (FilmArray) in the second season. FilmArray decreased the time to diagnosis of influenza compared to conventional methods (median turnaround times of 1.7 h versus 7.7 h, respectively; P = 0.015); FilmArray also decreased the time to diagnosis of non-influenza viruses (1.5 h versus 13.5 h, respectively; P < 0.0001). Multivariate logistic regression found that a diagnosis of influenza by FilmArray was associated with significantly lower odds ratios (ORs) for admission (P = 0.046), length of stay (P = 0.040), duration of antimicrobial use (P = 0.032), and number of chest radiographs (P = 0.005), when controlling for potential confounders. We conclude that the rapid turnaround time, multiplex nature of the test (allowing simultaneous detection of an array of viruses), and superior sensitivity of FilmArray may improve the evaluation and management of patients suspected of having respiratory virus infections. PMID:27225406

Electrophoresis for genotyping: microtiter array diagonal gel electrophoresis on horizontal polyacrylamide gels, hydrolink, or agarose.

PubMed

Day, I N; Humphries, S E

1994-11-01

Electrophoresis of DNA has been performed traditionally in either an agarose or acrylamide gel matrix. Considerable effort has been directed to improved quality agaroses capable of high resolution, but for small fragments, such as those from polymerase chain reaction (PCR) and post-PCR digests, acrylamide still offers the highest resolution. Although agarose gels can easily be prepared in an open-faced format to gain the conveniences of horizontal electrophoresis, acrylamide does not polymerize in the presence of air and the usual configurations for gel preparation lead to electrophoresis in the vertical dimension. We describe here a very simple device and method to prepare and manipulate horizontal polyacrylamide gels (H-PAGE). In addition, the open-faced horizontal arrangement enables loading of arrays of wells. Since many procedures are undertaken in standard 96-well microtiter plates, we have also designed a device which preserves the exact configuration of the 8 x 12 array and enables electrophoresis in tracks following a 71.6 degrees diagonal between wells (MADGE, microtiter array diagonal gel electrophoresis), using either acrylamide or agarose. This eliminates almost all of the staff time taken in setup, loading, and recordkeeping and offers high resolution for genotyping pattern recognition. The nature and size of the gels allow direct stacking of gels in one tank, so that a tank used typically to analyze 30-60 samples can readily be used to analyze 1000-2000 samples. The gels would also enable robotic loading. Electrophoresis allows analysis of size and charge, parameters inaccessible to liquid-phase methods: thus, genotyping size patterns, variable length repeats, and haplotypes is possible, as well as adaptability to typing of point variations using protocols which create a difference detectable by electrophoresis.

Carious Dentine Provides a Habitat for a Complex Array of Novel Prevotella-Like Bacteria

PubMed Central

Nadkarni, Mangala A.; Caldon, C. Elizabeth; Chhour, Kim-Ly; Fisher, Ilana P.; Martin, F. Elizabeth; Jacques, Nicholas A.; Hunter, Neil

2004-01-01

Previous analysis of the microbiology of advanced caries by culture and real-time PCR emphasized the high incidence and abundance of gram-negative anaerobic species, particularly Prevotella-like bacteria. The diversity of Prevotella-like bacteria was further explored by analyzing pooled bacterial DNA from lesions of carious dentine. This was achieved by amplification of a region of the 16S ribosomal DNA with a Prevotella genus-specific forward primer and a universal bacterial reverse primer, followed by cloning and sequencing. Cultured Prevotella species commonly associated with oral tissues constituted only 12% of the Prevotella clones isolated from advanced carious lesions. The remaining 88% consisted of a diverse range of phylotypes. These included five clusters of previously recognized but uncultured oral Prevotella spp. and a major cluster containing Prevotella-like bacteria most closely related to uncharacterized rumen bacteria. Cluster-specific primers were designed, and the numbers of bacteria within clusters were quantified by real-time PCR, confirming the abundance of these organisms. The data indicated that advanced dental caries provides a unique environment for a complex array of novel and uncultured Prevotella and Prevotella-like bacteria which, in some cases, may dominate the diverse polymicrobial community associated with the disease. PMID:15528720

Investigation into the effect of high concentrations of volatile fatty acids in anaerobic digestion on methanogenic communities

PubMed Central

Franke-Whittle, Ingrid H.; Walter, Andreas; Ebner, Christian; Insam, Heribert

2014-01-01

A study was conducted to determine whether differences in the levels of volatile fatty acids (VFAs) in anaerobic digester plants could result in variations in the indigenous methanogenic communities. Two digesters (one operated under mesophilic conditions, the other under thermophilic conditions) were monitored, and sampled at points where VFA levels were high, as well as when VFA levels were low. Physical and chemical parameters were measured, and the methanogenic diversity was screened using the phylogenetic microarray ANAEROCHIP. In addition, real-time PCR was used to quantify the presence of the different methanogenic genera in the sludge samples. Array results indicated that the archaeal communities in the different reactors were stable, and that changes in the VFA levels of the anaerobic digesters did not greatly alter the dominating methanogenic organisms. In contrast, the two digesters were found to harbour different dominating methanogenic communities, which appeared to remain stable over time. Real-time PCR results were inline with those of microarray analysis indicating only minimal changes in methanogen numbers during periods of high VFAs, however, revealed a greater diversity in methanogens than found with the array. PMID:25164858

Performance and characterization of new micromachined high-frequency linear arrays.

PubMed

Lukacs, Marc; Yin, Jianhua; Pang, Guofeng; Garcia, Richard C; Cherin, Emmanuel; Williams, Ross; Mehi, Jim; Foster, F Stuart

2006-10-01

A new approach for fabricating high frequency (> 20 MHz) linear array transducers, based on laser micromachining, has been developed. A 30 MHz, 64-element, 74-microm pitch, linear array design is presented. The performance of the device is demonstrated by comparing electrical and acoustic measurements with analytical, equivalent circuit, and finite-element analysis (FEA) simulations. All FEA results for array performance have been generated using one global set of material parameters. Each fabricated array has been integrated onto a flex circuit for ease of handling, and the flex has been integrated onto a custom printed circuit board test card for ease of testing. For a fully assembled array, with an acoustic lens, the center frequency was 28.7 MHz with a one-way -3 dB and -6 dB bandwidth of 59% and 83%, respectively, and a -20 dB pulse width of -99 ns. The per-element peak acoustic power, for a +/- 30 V single cycle pulse, measured at the 10 mm focal length of the lens was 590 kPa with a -6 dB directivity span of about 30 degrees. The worst-case total cross talk of the combined array and flex assembly is for nearest neighboring elements and was measured to have an average level -40 dB across the -6 dB bandwidth of the device. Any significant deviation from simulation can be explained through limitations in apparatus calibration and in device packaging.

The Long Noncoding RNA Landscape of the Mouse Eye.

PubMed

Chen, Weiwei; Yang, Shuai; Zhou, Zhonglou; Zhao, Xiaoting; Zhong, Jiayun; Reinach, Peter S; Yan, Dongsheng

2017-12-01

Long noncoding RNAs (lncRNAs) are important regulators of diverse biological functions. However, an extensive in-depth analysis of their expression profile and function in mammalian eyes is still lacking. Here we describe comprehensive landscapes of stage-dependent and tissue-specific lncRNA expression in the mouse eye. Affymetrix transcriptome array profiled lncRNA signatures from six different ocular tissue subsets (i.e., cornea, lens, retina, RPE, choroid, and sclera) in newborn and 8-week-old mice. Quantitative RT-PCR analysis validated array findings. Cis analyses and Gene Ontology (GO) annotation of protein-coding genes adjacent to signature lncRNA loci clarified potential lncRNA roles in maintaining tissue identity and regulating eye maturation during the aforementioned phase. In newborn and 8-week-old mice, we identified 47,332 protein-coding and noncoding gene transcripts. LncRNAs comprise 19,313 of these transcripts annotated in public data banks. During this maturation phase of these six different tissue subsets, more than 1000 lncRNAs expression levels underwent ≥2-fold changes. qRT-PCR analysis confirmed part of the gene microarray analysis results. K-means clustering identified 910 lncRNAs in the P0 groups and 686 lncRNAs in the postnatal 8-week-old groups, suggesting distinct tissue-specific lncRNA clusters. GO analysis of protein-coding genes proximal to lncRNA signatures resolved close correlations with their tissue-specific functional maturation between P0 and 8 weeks of age in the 6 tissue subsets. Characterizating maturational changes in lncRNA expression patterns as well as tissue-specific lncRNA signatures in six ocular tissues suggest important contributions made by lncRNA to the control of developmental processes in the mouse eye.

PCR/LDR/universal array platforms for the diagnosis of infectious disease.

PubMed

Pingle, Maneesh; Rundell, Mark; Das, Sanchita; Golightly, Linnie M; Barany, Francis

2010-01-01

Infectious diseases account for between 14 and 17 million deaths worldwide each year. Accurate and rapid diagnosis of bacterial, fungal, viral, and parasitic infections is therefore essential to reduce the morbidity and mortality associated with these diseases. Classical microbiological and serological methods have long served as the gold standard for diagnosis but are increasingly being replaced by molecular diagnostic methods that demonstrate increased sensitivity and specificity and provide an identification of the etiologic agent in a shorter period of time. PCR/LDR coupled with universal array detection provides a highly sensitive and specific platform for the detection and identification of bacterial and viral infections.

PCR/LDR/Universal Array Platforms for the Diagnosis of Infectious Disease

PubMed Central

Pingle, Maneesh; Rundell, Mark; Das, Sanchita; Golightly, Linnie M.; Barany, Francis

2015-01-01

Infectious diseases account for between 14 and 17 million deaths worldwide each year. Accurate and rapid diagnosis of bacterial, fungal, viral, and parasitic infections is therefore essential to reduce the morbidity and mortality associated with these diseases. Classical microbiological and serological methods have long served as the gold standard for diagnosis but are increasingly being replaced by molecular diagnostic methods that demonstrate increased sensitivity and specificity and provide an identification of the etiologic agent in a shorter period of time. PCR/LDR coupled with universal array detection provides a highly sensitive and specific platform for the detection and identification of bacterial and viral infections. PMID:20217576

A customized metal oxide semiconductor-based gas sensor array for onion quality evaluation: system development and characterization.

PubMed

Konduru, Tharun; Rains, Glen C; Li, Changying

2015-01-12

A gas sensor array, consisting of seven Metal Oxide Semiconductor (MOS) sensors that are sensitive to a wide range of organic volatile compounds was developed to detect rotten onions during storage. These MOS sensors were enclosed in a specially designed Teflon chamber equipped with a gas delivery system to pump volatiles from the onion samples into the chamber. The electronic circuit mainly comprised a microcontroller, non-volatile memory chip, and trickle-charge real time clock chip, serial communication chip, and parallel LCD panel. User preferences are communicated with the on-board microcontroller through a graphical user interface developed using LabVIEW. The developed gas sensor array was characterized and the discrimination potential was tested by exposing it to three different concentrations of acetone (ketone), acetonitrile (nitrile), ethyl acetate (ester), and ethanol (alcohol). The gas sensor array could differentiate the four chemicals of same concentrations and different concentrations within the chemical with significant difference. Experiment results also showed that the system was able to discriminate two concentrations (196 and 1964 ppm) of methlypropyl sulfide and two concentrations (145 and 1452 ppm) of 2-nonanone, two key volatile compounds emitted by rotten onions. As a proof of concept, the gas sensor array was able to achieve 89% correct classification of sour skin infected onions. The customized low-cost gas sensor array could be a useful tool to detect onion postharvest diseases in storage.

A Customized Metal Oxide Semiconductor-Based Gas Sensor Array for Onion Quality Evaluation: System Development and Characterization

PubMed Central

Konduru, Tharun; Rains, Glen C.; Li, Changying

2015-01-01

A gas sensor array, consisting of seven Metal Oxide Semiconductor (MOS) sensors that are sensitive to a wide range of organic volatile compounds was developed to detect rotten onions during storage. These MOS sensors were enclosed in a specially designed Teflon chamber equipped with a gas delivery system to pump volatiles from the onion samples into the chamber. The electronic circuit mainly comprised a microcontroller, non-volatile memory chip, and trickle-charge real time clock chip, serial communication chip, and parallel LCD panel. User preferences are communicated with the on-board microcontroller through a graphical user interface developed using LabVIEW. The developed gas sensor array was characterized and the discrimination potential was tested by exposing it to three different concentrations of acetone (ketone), acetonitrile (nitrile), ethyl acetate (ester), and ethanol (alcohol). The gas sensor array could differentiate the four chemicals of same concentrations and different concentrations within the chemical with significant difference. Experiment results also showed that the system was able to discriminate two concentrations (196 and 1964 ppm) of methlypropyl sulfide and two concentrations (145 and 1452 ppm) of 2-nonanone, two key volatile compounds emitted by rotten onions. As a proof of concept, the gas sensor array was able to achieve 89% correct classification of sour skin infected onions. The customized low-cost gas sensor array could be a useful tool to detect onion postharvest diseases in storage. PMID:25587975

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

PubMed Central

Sun, Lan; Irudayaraj, Joseph

2009-01-01

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

RNA-based determination of ESR1 and HER2 expression and response to neoadjuvant chemotherapy.

PubMed

Denkert, C; Loibl, S; Kronenwett, R; Budczies, J; von Törne, C; Nekljudova, V; Darb-Esfahani, S; Solbach, C; Sinn, B V; Petry, C; Müller, B M; Hilfrich, J; Altmann, G; Staebler, A; Roth, C; Ataseven, B; Kirchner, T; Dietel, M; Untch, M; von Minckwitz, G

2013-03-01

Hormone and human epidermal growth factor receptor 2 (HER2) receptors are the most important breast cancer biomarkers, and additional objective and quantitative test methods such as messenger RNA (mRNA)-based quantitative analysis are urgently needed. In this study, we investigated the clinical validity of RT-PCR-based evaluation of estrogen receptor (ESR1) and HER2 mRNA expression. A total of 1050 core biopsies from two retrospective (GeparTrio, GeparQuattro) and one prospective (PREDICT) neoadjuvant studies were evaluated by quantitative RT-PCR for ESR1 and HER2. ESR1 mRNA was significantly predictive for reduced response to neoadjuvant chemotherapy in univariate and multivariate analysis in all three cohorts. The complete pathologically documented response (pathological complete response, pCR) rate for ESR1+/HER2- tumors was 7.3%, 8.0% and 8.6%; for ESR1-/HER2- tumors it was 34.4%, 33.7% and 37.3% in GeparTrio, GeparQuattro and PREDICT, respectively (P < 0.001 in each cohort). In the Kaplan-Meier analysis in GeparTrio patients with ESR1+/HER2- tumors had the best prognosis, compared with ESR1-/HER2- and ESR1-/HER2+ tumors [disease-free survival (DFS): P < 0.0005, overall survival (OS): P < 0.0005]. Our results suggest that mRNA levels of ESR1 and HER2 predict response to neoadjuvant chemotherapy and are significantly associated with long-term outcome. As an additional option to standard immunohistochemistry and gene-array-based analysis, quantitative RT-PCR analysis might be useful for determination of the receptor status in breast cancer.

Digital detection of multiple minority mutants in stool DNA for noninvasive colorectal cancer diagnosis.

PubMed

Deng, Lili; Qi, Zongtai; Zou, Binjie; Wu, Haiping; Huang, Huan; Kajiyama, Tomoharu; Kambara, Hideki; Zhou, Guohua

2012-07-03

Somatic mutations in stool DNA are quite specific to colorectal cancer (CRC), but a method being able to detect the extraordinarily low amounts of mutants is challengeable in sensitivity. We proposed a hydrogel bead-array to digitally count CRC-specific mutants in stool at a low cost. At first, multiplex amplification of targets containing multiple mutation loci of interest is carried out by a target enriched multiplex PCR (Tem-PCR), yielding the templates qualified for emulsion PCR (emPCR). Then, after immobilizing the beads from emPCR on a glass surface, the incorporation of Cy3-dUTP into the mutant-specific probes, which are specifically hybridized with the amplified beads from emPCR, is used to color the beads coated with mutants. As all amplified beads are hybridized with the Cy5-labeled universal probe, a mutation rate is readily obtained by digitally counting the beads with different colors (yellow and red). A high specificity of the method is achieved by removing the mismatched probes in a bead-array with electrophoresis. The approach has been used to simultaneously detect 8 mutation loci within the APC, TP53, and KRAS genes in stools from eight CRC patients, and 50% of CRC patients were positively diagnosed; therefore, our method can be a potential tool for the noninvasive diagnosis of CRC.

High-performance genetic analysis on microfabricated capillary array electrophoresis plastic chips fabricated by injection molding.

PubMed

Dang, Fuquan; Tabata, Osamu; Kurokawa, Masaya; Ewis, Ashraf A; Zhang, Lihua; Yamaoka, Yoshihisa; Shinohara, Shouji; Shinohara, Yasuo; Ishikawa, Mitsuru; Baba, Yoshinobu

2005-04-01

We have developed a novel technique for mass production of microfabricated capillary array electrophoresis (mu-CAE) plastic chips for high-speed, high-throughput genetic analysis. The mu-CAE chips, containing 10 individual separation channels of 50-microm width, 50-microm depth, and a 100-microm lane-to-lane spacing at the detection region and a sacrificial channel network, were fabricated on a poly(methyl methacrylate) substrate by injection molding and then bonded manually using a pressure-sensitive sealing tape within several seconds at room temperature. The conditions for injection molding and bonding were carefully characterized to yield mu-CAE chips with well-defined channel and injection structures. A CCD camera equipped with an image intensifier was used to monitor simultaneously the separation in a 10-channel array with laser-induced fluorescence detection. High-performance electrophoretic separations of phiX174 HaeIII DNA restriction fragments and PCR products related to the human beta-globin gene and SP-B gene (the surfactant protein B) have been demonstrated on mu-CAE plastic chips using a methylcellulose sieving matrix in individual channels. The current work demonstrated greatly simplified the fabrication process as well as a detection scheme for mu-CAE chips and will bring the low-cost mass production and application of mu-CAE plastic chips for genetic analysis.

Customized Oligonucleotide Array-Based Comparative Genomic Hybridization as a Clinical Assay for Genomic Profiling of Chronic Lymphocytic Leukemia

PubMed Central

Sargent, Rachel; Jones, Dan; Abruzzo, Lynne V.; Yao, Hui; Bonderover, Jaime; Cisneros, Marissa; Wierda, William G.; Keating, Michael J.; Luthra, Rajyalakshmi

2009-01-01

Chromosome gains and losses used for risk stratification in chronic lymphocytic leukemia (CLL) are commonly assessed by multiprobe fluorescence in situ hybridization (FISH) studies. We designed and validated a customized array-comparative genomic hybridization (aCGH) platform as a clinical assay for CLL genomic profiling. A 60-mer, 44,000-probe oligonucleotide array with a 50-kb average spatial resolution was augmented with high-density probe tiling at loci that are frequently aberrant in CLL. Aberrations identified by aCGH were compared with those identified by a FISH panel, including locus-specific probes to ATM (11q22.3), the centromeric region of chromosome 12 (12p11.1–q11), D13S319 (13q14.3), LAMP1 (13q34), and TP53 (17p13.1). In 100 CLL samples, aCGH/FISH concordance was seen for 89% of FISH-called aberrations at the ATM (n = 18), D13S319 (n = 42), LAMP (n = 12), and TP53 (n = 22) loci and for chromosome 12 (n = 14). Eighty-four percentage of FISH/aCGH discordant calls were in samples either at or below the limit of aCGH sensitivity (10% to 25% FISH aberration-containing cells). Therefore, aCGH profiling is a feasible routine clinical test with comparable results to multiprobe FISH studies; however, it may be less sensitive than FISH in cases with low-level aberrations. Further, a customized array design can provide comprehensive genomic profiling with additional accuracy in both identifying and defining the extent of small aberrations at target loci. PMID:19074592

KSC-00pp1732

NASA Image and Video Library

2000-11-14

KENNEDY SPACE CENTER, FLA. -- On Launch Pad 39B, the payload transport canister, with the P6 integrated truss segment inside, is lifted toward the payload changeout room (PCR). The PCR is the enclosed, environmentally controlled portion of the Rotating Service Structure that supports payload delivery at the pad and subsequent vertical installation in the orbiter payload bay. Attached to the canister are the red umbilical lines that maintain the controlled environment inside. The P6, payload on mission STS-97, comprises Solar Array Wing-3 and the Integrated Electronic Assembly, to be installed on the International Space Station. The Station’s electrical power system will use eight photovoltaic solar arrays, each 112 feet long by 39 feet wide, to convert sunlight to electricity. The solar arrays are mounted on a “blanket” that can be folded like an accordion for delivery. Once in orbit, astronauts will deploy the blankets to their full size. Gimbals will be used to rotate the arrays so that they will face the Sun to provide maximum power to the Space Station. Launch of STS-97 is scheduled for Nov. 30 at 10:06 p.m. EST

KSC00pp1732

NASA Image and Video Library

2000-11-14

KENNEDY SPACE CENTER, FLA. -- On Launch Pad 39B, the payload transport canister, with the P6 integrated truss segment inside, is lifted toward the payload changeout room (PCR). The PCR is the enclosed, environmentally controlled portion of the Rotating Service Structure that supports payload delivery at the pad and subsequent vertical installation in the orbiter payload bay. Attached to the canister are the red umbilical lines that maintain the controlled environment inside. The P6, payload on mission STS-97, comprises Solar Array Wing-3 and the Integrated Electronic Assembly, to be installed on the International Space Station. The Station’s electrical power system will use eight photovoltaic solar arrays, each 112 feet long by 39 feet wide, to convert sunlight to electricity. The solar arrays are mounted on a “blanket” that can be folded like an accordion for delivery. Once in orbit, astronauts will deploy the blankets to their full size. Gimbals will be used to rotate the arrays so that they will face the Sun to provide maximum power to the Space Station. Launch of STS-97 is scheduled for Nov. 30 at 10:06 p.m. EST

Targeting Quiescent Cancer Cells to Eliminate Tumor Recurrence After Therapy

DTIC Science & Technology

2016-12-01

promoter regions cloned can recapitulate its entire promoter activities. RBL2 promoter constructs are not commercially available. We attempted to...customer order the RBL2 promoter commercially, but the vendors failed to amplify or clone the RBL2 promoter. Therefore we had to clone the promoter...exponential megapriming PCR (EMP), we cloned the product into PGL4 vector. For this approach as shown in Figure 2, in the first EMP PCR a forward primer F1

Direct-to-PCR tissue preservation for DNA profiling.

PubMed

Sorensen, Amy; Berry, Clare; Bruce, David; Gahan, Michelle Elizabeth; Hughes-Stamm, Sheree; McNevin, Dennis

2016-05-01

Disaster victim identification (DVI) often occurs in remote locations with extremes of temperatures and humidities. Access to mortuary facilities and refrigeration are not always available. An effective and robust DNA sampling and preservation procedure would increase the probability of successful DNA profiling and allow faster repatriation of bodies and body parts. If the act of tissue preservation also released DNA into solution, ready for polymerase chain reaction (PCR), the DVI process could be further streamlined. In this study, we explored the possibility of obtaining DNA profiles without DNA extraction, by adding aliquots of preservative solutions surrounding fresh human muscle and decomposing human muscle and skin tissue samples directly to PCR. The preservatives consisted of two custom preparations and two proprietary solutions. The custom preparations were a salt-saturated solution of dimethyl sulfoxide (DMSO) with ethylenediaminetetraacetic (EDTA) and TENT buffer (Tris, EDTA, NaCl, Tween 20). The proprietary preservatives were DNAgard (Biomatrica(®)) and Tissue Stabilising Kit (DNA Genotek). We obtained full PowerPlex(®) 21 (Promega) and GlobalFiler(®) (Life Technologies) DNA profiles from fresh and decomposed tissue preserved at 35 °C for up to 28 days for all four preservatives. The preservative aliquots removed from the fresh muscle tissue samples had been stored at -80 °C for 4 years, indicating that long-term archival does not diminish the probability of successful DNA typing. Rather, storage at -80 °C seems to reduce PCR inhibition.

DASH-2: Flexible, Low-Cost, and High-Throughput SNP Genotyping by Dynamic Allele-Specific Hybridization on Membrane Arrays

PubMed Central

Jobs, Magnus; Howell, W. Mathias; Strömqvist, Linda; Mayr, Torsten; Brookes, Anthony J.

2003-01-01

Genotyping technologies need to be continually improved in terms of their flexibility, cost-efficiency, and throughput, to push forward genome variation analysis. To this end, we have leveraged the inherent simplicity of dynamic allele-specific hybridization (DASH) and coupled it to recent innovations of centrifugal arrays and iFRET. We have thereby created a new genotyping platform we term DASH-2, which we demonstrate and evaluate in this report. The system is highly flexible in many ways (any plate format, PCR multiplexing, serial and parallel array processing, spectral-multiplexing of hybridization probes), thus supporting a wide range of application scales and objectives. Precision is demonstrated to be in the range 99.8–100%, and assay costs are 0.05 USD or less per genotype assignment. DASH-2 thus provides a powerful new alternative for genotyping practice, which can be used without the need for expensive robotics support. PMID:12727908

[Analysis of genetics mechanism for the phenotypic diversity in a patient carrying a rare ring chromosome 9].

PubMed

Qin, Shengfang; Wang, Xueyan; Li, Yunxing; Wei, Ping; Chen, Chun; Zeng, Lan

2016-02-01

To explore the genetics mechanism for the phenotypic variability in a patient carrying a rare ring chromosome 9. The karyotype of the patient was analyzed with cytogenetics method. Presence of sex chromosome was confirmed with fluorescence in situ hybridization. The SRY gene was subjected to PCR amplification and direct sequencing. Potential deletion and duplication were detected with array-based comparative genomic hybridization (array-CGH). The karyotype of the patient has comprised 6 types of cell lines containing a ring chromosome 9. The SRY gene sequence was normal. By array-CGH, the patient has carried a hemizygous deletion at 9p24.3-p23 (174 201-9 721 761) encompassing 30 genes from Online Mendelian Inheritance in Man. The phenotypic variability of the 9p deletion syndrome in conjunct with ring chromosome 9 may be attributable to multiple factors including loss of chromosomal material, insufficient dosage of genes, instability of ring chromosome, and pattern of inheritance.

Two Siblings with Alternate Unbalanced Recombinants Derived from a Large Cryptic Maternal Pericentric Inversion of Chromosome 20

PubMed Central

DeScipio, Cheryl; Morrissette, Jennifer J.D.; Conlin, Laura K.; Clark, Dinah; Kaur, Maninder; Coplan, James; Riethman, Harold; Spinner, Nancy B.; Krantz, Ian D.

2009-01-01

Two brothers, with dissimilar clinical features, were each found to have different abnormalities of chromosome 20 by subtelomere fluorescence in situ hybridization (FISH). The proband had deletion of 20p subtelomere and duplication of 20q subtelomere, while his brother was found to have a duplication of 20p subtelomere and deletion of 20q subtelomere. Parental cytogenetic studies were initially thought to be normal, both by G-banding and by subtelomere FISH analysis. Since chromosome 20 is a metacentric chromosome and an inversion was suspected, we used anchored FISH to assist in identifying a possible inversion. This approach employed concomitant hybridization of a FISH probe to the short (p) arm of chromosome 20 with the 20q subtelomere probe. We identified a cytogenetically non-visible, mosaic pericentric inversion of one of the maternal chromosome 20 homologues, providing a mechanistic explanation for the chromosomal abnormalities present in these brothers. Array comparative genomic hybridization (CGH) with both a custom-made BAC and cosmid-based subtelomere specific array (TEL array) and a commercially-available SNP-based array confirmed and further characterized these rearrangements, identifying this as the largest pericentric inversion of chromosome 20 described to date. TEL array data indicate that the 20p breakpoint is defined by BAC RP11-978M13, ~900 kb from the pter; SNP array data reveal this breakpoint to occur within BAC RP11-978M13. The 20q breakpoint is defined by BAC RP11-93B14, ~1.7 Mb from the qter, by TEL array; SNP array data refine this breakpoint to within a gap between BACs on the TEL array (i.e. between RP11-93B14 and proximal BAC RP11-765G16). PMID:20101690

Two siblings with alternate unbalanced recombinants derived from a large cryptic maternal pericentric inversion of chromosome 20.

PubMed

Descipio, Cheryl; Morrissette, Jennifer D; Conlin, Laura K; Clark, Dinah; Kaur, Maninder; Coplan, James; Riethman, Harold; Spinner, Nancy B; Krantz, Ian D

2010-02-01

Two brothers, with dissimilar clinical features, were each found to have different abnormalities of chromosome 20 by subtelomere fluorescence in situ hybridization (FISH). The proband had deletion of 20p subtelomere and duplication of 20q subtelomere, while his brother was found to have a duplication of 20p subtelomere and deletion of 20q subtelomere. Parental cytogenetic studies were initially thought to be normal, both by G-banding and by subtelomere FISH analysis. Since chromosome 20 is a metacentric chromosome and an inversion was suspected, we used anchored FISH to assist in identifying a possible inversion. This approach employed concomitant hybridization of a FISH probe to the short (p) arm of chromosome 20 with the 20q subtelomere probe. We identified a cytogenetically non-visible, mosaic pericentric inversion of one of the maternal chromosome 20 homologs, providing a mechanistic explanation for the chromosomal abnormalities present in these brothers. Array comparative genomic hybridization (CGH) with both a custom-made BAC and cosmid-based subtelomere specific array (TEL array) and a commercially available SNP-based array confirmed and further characterized these rearrangements, identifying this as the largest pericentric inversion of chromosome 20 described to date. TEL array data indicate that the 20p breakpoint is defined by BAC RP11-978M13, approximately 900 kb from the pter; SNP array data reveal this breakpoint to occur within BAC RP11-978M13. The 20q breakpoint is defined by BAC RP11-93B14, approximately 1.7 Mb from the qter, by TEL array; SNP array data refine this breakpoint to within a gap between BACs on the TEL array (i.e., between RP11-93B14 and proximal BAC RP11-765G16). Copyright 2010 Wiley-Liss, Inc.

NuGO contributions to GenePattern

PubMed Central

Reiff, C.; Mayer, C.; Müller, M.

2008-01-01

NuGO, the European Nutrigenomics Organization, utilizes 31 powerful computers for, e.g., data storage and analysis. These so-called black boxes (NBXses) are located at the sites of different partners. NuGO decided to use GenePattern as the preferred genomic analysis tool on each NBX. To handle the custom made Affymetrix NuGO arrays, new NuGO modules are added to GenePattern. These NuGO modules execute the latest Bioconductor version ensuring up-to-date annotations and access to the latest scientific developments. The following GenePattern modules are provided by NuGO: NuGOArrayQualityAnalysis for comprehensive quality control, NuGOExpressionFileCreator for import and normalization of data, LimmaAnalysis for identification of differentially expressed genes, TopGoAnalysis for calculation of GO enrichment, and GetResultForGo for retrieval of information on genes associated with specific GO terms. All together, these NuGO modules allow comprehensive, up-to-date, and user friendly analysis of Affymetrix data. A special feature of the NuGO modules is that for analysis they allow the use of either the standard Affymetrix or the MBNI custom CDF-files, which remap probes based on current knowledge. In both cases a .chip-file is created to enable GSEA analysis. The NuGO GenePattern installations are distributed as binary Ubuntu (.deb) packages via the NuGO repository. PMID:19034553

NuGO contributions to GenePattern.

PubMed

De Groot, P J; Reiff, C; Mayer, C; Müller, M

2008-12-01

NuGO, the European Nutrigenomics Organization, utilizes 31 powerful computers for, e.g., data storage and analysis. These so-called black boxes (NBXses) are located at the sites of different partners. NuGO decided to use GenePattern as the preferred genomic analysis tool on each NBX. To handle the custom made Affymetrix NuGO arrays, new NuGO modules are added to GenePattern. These NuGO modules execute the latest Bioconductor version ensuring up-to-date annotations and access to the latest scientific developments. The following GenePattern modules are provided by NuGO: NuGOArrayQualityAnalysis for comprehensive quality control, NuGOExpressionFileCreator for import and normalization of data, LimmaAnalysis for identification of differentially expressed genes, TopGoAnalysis for calculation of GO enrichment, and GetResultForGo for retrieval of information on genes associated with specific GO terms. All together, these NuGO modules allow comprehensive, up-to-date, and user friendly analysis of Affymetrix data. A special feature of the NuGO modules is that for analysis they allow the use of either the standard Affymetrix or the MBNI custom CDF-files, which remap probes based on current knowledge. In both cases a .chip-file is created to enable GSEA analysis. The NuGO GenePattern installations are distributed as binary Ubuntu (.deb) packages via the NuGO repository.

Profiling of the yak skeletal muscle tissue gene expression and comparison with the domestic cattle by genome array.

PubMed

Wang, H B; Zan, L S; Zhang, Y Y

2014-01-01

Of all the mammals of the world, the yak lives at the highest altitude area of more than 3000 m. Comparison between yak and cattle of the low-altitude areas will be informative in studying animal adaptation to higher altitudes. To investigate the molecular mechanism involved in meat quality differences between the two Chinese special varieties Qinghai yak and Qinchuan cattle, 12 chemical-physical characteristics of the longissimus dorsi muscle related to meat quality were compared at the age of 36 months, and the gene expression profiles were constructed by utilizing the bovine genome array. Significant analysis of microarrays was used to identify the differentially expressed genes. Gene ontology and pathway analysis were performed by a free Web-based Molecular Annotation System 2.0. The results reveal ~11 000 probes representing about 10 000 genes that were detected in both the Qinghai yak and Qinchuan cattle. A total of 1922 genes were shown to be differentially expressed, 633 probes were upregulated and 1259 probes were downregulated in the muscle tissue of Qinghai yak that were mainly involved in ubiquitin-mediated proteolysis, muscle growth regulation, glucose metabolism, immune response and so on. Quantitative real-time PCR (qRT-PCR) was performed to validate some differentially expressed genes identified by microarray. Further analysis implied that animals living at a high altitude may supply energy by more active protein catabolism and glycolysis compared with those living in the plain areas. Our results establish the groundwork for further studies on yaks' meat quality and will be beneficial in improving the yaks' breeding by molecular biotechnology.

Quality control, analysis and secure sharing of Luminex® immunoassay data using the open source LabKey Server platform

PubMed Central

2013-01-01

Background Immunoassays that employ multiplexed bead arrays produce high information content per sample. Such assays are now frequently used to evaluate humoral responses in clinical trials. Integrated software is needed for the analysis, quality control, and secure sharing of the high volume of data produced by such multiplexed assays. Software that facilitates data exchange and provides flexibility to perform customized analyses (including multiple curve fits and visualizations of assay performance over time) could increase scientists’ capacity to use these immunoassays to evaluate human clinical trials. Results The HIV Vaccine Trials Network and the Statistical Center for HIV/AIDS Research and Prevention collaborated with LabKey Software to enhance the open source LabKey Server platform to facilitate workflows for multiplexed bead assays. This system now supports the management, analysis, quality control, and secure sharing of data from multiplexed immunoassays that leverage Luminex xMAP® technology. These assays may be custom or kit-based. Newly added features enable labs to: (i) import run data from spreadsheets output by Bio-Plex Manager™ software; (ii) customize data processing, curve fits, and algorithms through scripts written in common languages, such as R; (iii) select script-defined calculation options through a graphical user interface; (iv) collect custom metadata for each titration, analyte, run and batch of runs; (v) calculate dose–response curves for titrations; (vi) interpolate unknown concentrations from curves for titrated standards; (vii) flag run data for exclusion from analysis; (viii) track quality control metrics across runs using Levey-Jennings plots; and (ix) automatically flag outliers based on expected values. Existing system features allow researchers to analyze, integrate, visualize, export and securely share their data, as well as to construct custom user interfaces and workflows. Conclusions Unlike other tools tailored for Luminex immunoassays, LabKey Server allows labs to customize their Luminex analyses using scripting while still presenting users with a single, graphical interface for processing and analyzing data. The LabKey Server system also stands out among Luminex tools for enabling smooth, secure transfer of data, quality control information, and analyses between collaborators. LabKey Server and its Luminex features are freely available as open source software at http://www.labkey.com under the Apache 2.0 license. PMID:23631706

Quality control, analysis and secure sharing of Luminex® immunoassay data using the open source LabKey Server platform.

PubMed

Eckels, Josh; Nathe, Cory; Nelson, Elizabeth K; Shoemaker, Sara G; Nostrand, Elizabeth Van; Yates, Nicole L; Ashley, Vicki C; Harris, Linda J; Bollenbeck, Mark; Fong, Youyi; Tomaras, Georgia D; Piehler, Britt

2013-04-30

Immunoassays that employ multiplexed bead arrays produce high information content per sample. Such assays are now frequently used to evaluate humoral responses in clinical trials. Integrated software is needed for the analysis, quality control, and secure sharing of the high volume of data produced by such multiplexed assays. Software that facilitates data exchange and provides flexibility to perform customized analyses (including multiple curve fits and visualizations of assay performance over time) could increase scientists' capacity to use these immunoassays to evaluate human clinical trials. The HIV Vaccine Trials Network and the Statistical Center for HIV/AIDS Research and Prevention collaborated with LabKey Software to enhance the open source LabKey Server platform to facilitate workflows for multiplexed bead assays. This system now supports the management, analysis, quality control, and secure sharing of data from multiplexed immunoassays that leverage Luminex xMAP® technology. These assays may be custom or kit-based. Newly added features enable labs to: (i) import run data from spreadsheets output by Bio-Plex Manager™ software; (ii) customize data processing, curve fits, and algorithms through scripts written in common languages, such as R; (iii) select script-defined calculation options through a graphical user interface; (iv) collect custom metadata for each titration, analyte, run and batch of runs; (v) calculate dose-response curves for titrations; (vi) interpolate unknown concentrations from curves for titrated standards; (vii) flag run data for exclusion from analysis; (viii) track quality control metrics across runs using Levey-Jennings plots; and (ix) automatically flag outliers based on expected values. Existing system features allow researchers to analyze, integrate, visualize, export and securely share their data, as well as to construct custom user interfaces and workflows. Unlike other tools tailored for Luminex immunoassays, LabKey Server allows labs to customize their Luminex analyses using scripting while still presenting users with a single, graphical interface for processing and analyzing data. The LabKey Server system also stands out among Luminex tools for enabling smooth, secure transfer of data, quality control information, and analyses between collaborators. LabKey Server and its Luminex features are freely available as open source software at http://www.labkey.com under the Apache 2.0 license.

Multicapillary Gas Chromatography-Temperature Modulated Metal Oxide Semiconductor Sensors Array Detector for Monitoring of Volatile Organic Compounds in Closed Atmosphere Using Gaussian Apodization Factor Analysis.

PubMed

Alinoori, Amir Hossein; Masoum, Saeed

2018-05-22

A unique metal oxide semiconductor sensor (MOS) array detector with eight sensors was designed and fabricated in a PTFE chamber as an interface for coupling with multicapillary gas chromatography. This design consists of eight transfer lines with equal length between the multicapillary columns (MCC) and sensors. The deactivated capillary columns were passed through each transfer line and homemade flow splitter to distribute the same gas flow on each sensor. Using the eight ports flow splitter design helps us to equal the length of carrier gas path and flow for each sensor, minimizing the dead volume of the sensor's chamber and increasing chromatographic resolution. In addition to coupling of MCC to MOS array detector and other considerations in hardware design, modulation of MOS temperature was used to increase sensitivity and selectivity, and data analysis was enhanced with adapted Gaussian apodization factor analysis (GAFA) as a multivariate curve resolution algorithm. Continues air sampling and injecting system (CASI) design provides a fast and easily applied method for continues injection of air sample with no additional sample preparation. The analysis cycle time required for each run is less than 300 s. The high sample load and sharp injection with the fast separation by MCC decrease the peak widths and improve detection limits. This homemade customized instrument is an alternative to other time-consuming and expensive technologies for continuous monitoring of outgassing in air samples.

Space Environment Testing of Photovoltaic Array Systems at NASA's Marshall Space Flight Center

NASA Technical Reports Server (NTRS)

Phillips, Brandon S.; Schneider, Todd A.; Vaughn, Jason A.; Wright, Kenneth H., Jr.

2015-01-01

To successfully operate a photovoltaic (PV) array system in space requires planning and testing to account for the effects of the space environment. It is critical to understand space environment interactions not only on the PV components, but also the array substrate materials, wiring harnesses, connectors, and protection circuitry (e.g. blocking diodes). Key elements of the space environment which must be accounted for in a PV system design include: Solar Photon Radiation, Charged Particle Radiation, Plasma, and Thermal Cycling. While solar photon radiation is central to generating power in PV systems, the complete spectrum includes short wavelength ultraviolet components, which photo-ionize materials, as well as long wavelength infrared which heat materials. High energy electron radiation has been demonstrated to significantly reduce the output power of III-V type PV cells; and proton radiation damages material surfaces - often impacting coverglasses and antireflective coatings. Plasma environments influence electrostatic charging of PV array materials, and must be understood to ensure that long duration arcs do not form and potentially destroy PV cells. Thermal cycling impacts all components on a PV array by inducing stresses due to thermal expansion and contraction. Given such demanding environments, and the complexity of structures and materials that form a PV array system, mission success can only be ensured through realistic testing in the laboratory. NASA's Marshall Space Flight Center has developed a broad space environment test capability to allow PV array designers and manufacturers to verify their system's integrity and avoid costly on-orbit failures. The Marshall Space Flight Center test capabilities are available to government, commercial, and university customers. Test solutions are tailored to meet the customer's needs, and can include performance assessments, such as flash testing in the case of PV cells.

KSC-2009-1096

NASA Image and Video Library

2009-01-11

CAPE CANAVERAL, Fla. -- With red umbilical lines attached, the payload containing space shuttle Discovery's S6 truss and solar arrays is lifted up to the Payload Changeout Room, or PCR, on Launch Pad 39A at NASA's Kennedy Space Center in Florida. The payload will be transferred inside the PCR where it will wait until Discovery rolls out to the pad. Then the payload will be installed in the shuttle's payload bay. Launch of Discovery on the STS-119 mission is scheduled for Feb. 12. During Discovery's 14-day mission, the crew will install the S6 truss segment and its solar arrays to the starboard side of the station, completing the station's backbone, or truss. Photo credit: NASA/Jim Grossmann

KSC-2009-1097

NASA Image and Video Library

2009-01-11

CAPE CANAVERAL, Fla. -- With red umbilical lines attached, the payload containing space shuttle Discovery's S6 truss and solar arrays is lifted up to the Payload Changeout Room, or PCR, on Launch Pad 39A at NASA's Kennedy Space Center in Florida. The payload will be transferred inside the PCR where it will wait until Discovery rolls out to the pad. Then the payload will be installed in the shuttle's payload bay. Launch of Discovery on the STS-119 mission is scheduled for Feb. 12. During Discovery's 14-day mission, the crew will install the S6 truss segment and its solar arrays to the starboard side of the station, completing the station's backbone, or truss Photo credit: NASA/Jim Grossmann

KSC-2009-1098

NASA Image and Video Library

2009-01-11

CAPE CANAVERAL, Fla. -- With red umbilical lines attached, the payload containing space shuttle Discovery's S6 truss and solar arrays is lifted up to the Payload Changeout Room, or PCR, on Launch Pad 39A at NASA's Kennedy Space Center in Florida. The payload will be transferred inside the PCR where it will wait until Discovery rolls out to the pad. Then the payload will be installed in the shuttle's payload bay. Launch of Discovery on the STS-119 mission is scheduled for Feb. 12. During Discovery's 14-day mission, the crew will install the S6 truss segment and its solar arrays to the starboard side of the station, completing the station's backbone, or truss Photo credit: NASA/Jim Grossmann

A novel scalable manufacturing process for the production of hydrogel-forming microneedle arrays.

PubMed

Lutton, Rebecca E M; Larrañeta, Eneko; Kearney, Mary-Carmel; Boyd, Peter; Woolfson, A David; Donnelly, Ryan F

2015-10-15

A novel manufacturing process for fabricating microneedle arrays (MN) has been designed and evaluated. The prototype is able to successfully produce 14×14 MN arrays and is easily capable of scale-up, enabling the transition from laboratory to industry and subsequent commercialisation. The method requires the custom design of metal MN master templates to produce silicone MN moulds using an injection moulding process. The MN arrays produced using this novel method was compared with centrifugation, the traditional method of producing aqueous hydrogel-forming MN arrays. The results proved that there was negligible difference between either methods, with each producing MN arrays with comparable quality. Both types of MN arrays can be successfully inserted in a skin simulant. In both cases the insertion depth was approximately 60% of the needle length and the height reduction after insertion was in both cases approximately 3%. Copyright © 2015 Elsevier B.V. All rights reserved.

A novel scalable manufacturing process for the production of hydrogel-forming microneedle arrays

PubMed Central

Lutton, Rebecca E.M.; Larrañeta, Eneko; Kearney, Mary-Carmel; Boyd, Peter; Woolfson, A.David; Donnelly, Ryan F.

2015-01-01

A novel manufacturing process for fabricating microneedle arrays (MN) has been designed and evaluated. The prototype is able to successfully produce 14 × 14 MN arrays and is easily capable of scale-up, enabling the transition from laboratory to industry and subsequent commercialisation. The method requires the custom design of metal MN master templates to produce silicone MN moulds using an injection moulding process. The MN arrays produced using this novel method was compared with centrifugation, the traditional method of producing aqueous hydrogel-forming MN arrays. The results proved that there was negligible difference between either methods, with each producing MN arrays with comparable quality. Both types of MN arrays can be successfully inserted in a skin simulant. In both cases the insertion depth was approximately 60% of the needle length and the height reduction after insertion was in both cases approximately 3%. PMID:26302858

Mechanical Designs and Developement of Advanced ACT: A Transfomative Upgrade to the ACTPol Receiver on the Atacama Cosmology Telescope.

NASA Astrophysics Data System (ADS)

Ward, Jonathan; Advanced ACT Collaboration, NASA Space Technology Research Fellowship

2017-06-01

The Atacama Cosmology Telescope is a six-meter diameter telescope located at 17,000 feet (5,200 meters) on Cerro Toco in the Andes Mountains of northern Chile. The next generation Advanced ACT (AdvACT) experiment is currently underway and will consist of three multichroic TES bolometer arrays operating together, totaling 5800 detectors on the sky. Each array will be sensitive to two frequency bands: a high frequency (HF) array at 150 and 230 GHz, two middle frequency (MF) arrays at 90 and 150 GHz, and a low frequency (LF) array at 28 and 41 GHz. The AdACT detector arrays will feature a revamped design when compared to ACTPol, including a transition to 150mm wafers equipped with multichroic pixels, allowing for a more densely packed focal plane. Each set of detectors consists of a feedhorn array of stacked silicon wafers which form a corrugated profile leading to each pixel. This is then followed by a four-piece detector stack assembly of silicon wafers which includes a waveguide interface plate, detector wafer, backshort cavity plate, and backshort cap. Each array is housed in a custom designed structure manufactured out of gold-plated, high purity copper. In addition to the detector array assembly, the array package also encloses the majority of our readout electronics. We present the full mechanical design of the AdvACT HF and MF detector array packages along with a detailed look at the detector array assemblies. We also highlight the use of continuously rotating warm half-wave plates (HWPs) at the front of the AdvACT receiver. We review the design of the rotation system and also early pipeline data analysis results. This experiment will also make use of extensive hardware and software previously developed for ACT, which will be modified to incorporate the new AdvACT instruments. Therefore, we discuss the integration of all AdvACT instruments with pre-existing ACTPol infrastructure.

High-Reliability Pump Module for Non-Planar Ring Oscillator Laser

NASA Technical Reports Server (NTRS)

Liu, Duncan T.; Qiu, Yueming; Wilson, Daniel W.; Dubovitsky, Serge; Forouhar, Siamak

2007-01-01

We propose and have demonstrated a prototype high-reliability pump module for pumping a Non-Planar Ring Oscillator (NPRO) laser suitable for space missions. The pump module consists of multiple fiber-coupled single-mode laser diodes and a fiber array micro-lens array based fiber combiner. The reported Single-Mode laser diode combiner laser pump module (LPM) provides a higher normalized brightness at the combined beam than multimode laser diode based LPMs. A higher brightness from the pump source is essential for efficient NPRO laser pumping and leads to higher reliability because higher efficiency requires a lower operating power for the laser diodes, which in turn increases the reliability and lifetime of the laser diodes. Single-mode laser diodes with Fiber Bragg Grating (FBG) stabilized wavelength permit the pump module to be operated without a thermal electric cooler (TEC) and this further improves the overall reliability of the pump module. The single-mode laser diode LPM is scalable in terms of the number of pump diodes and is capable of combining hundreds of fiber-coupled laser diodes. In the proof-of-concept demonstration, an e-beam written diffractive micro lens array, a custom fiber array, commercial 808nm single mode laser diodes, and a custom NPRO laser head are used. The reliability of the proposed LPM is discussed.

GLOBECOM '84 - Global Telecommunications Conference, Atlanta, GA, November 26-29, 1984, Conference Record. Volume 1

NASA Astrophysics Data System (ADS)

The subjects discussed are related to LSI/VLSI based subscriber transmission and customer access for the Integrated Services Digital Network (ISDN), special applications of fiber optics, ISDN and competitive telecommunication services, technical preparations for the Geostationary-Satellite Orbit Conference, high-capacity statistical switching fabrics, networking and distributed systems software, adaptive arrays and cancelers, synchronization and tracking, speech processing, advances in communication terminals, full-color videotex, and a performance analysis of protocols. Advances in data communications are considered along with transmission network plans and progress, direct broadcast satellite systems, packet radio system aspects, radio-new and developing technologies and applications, the management of software quality, and Open Systems Interconnection (OSI) aspects of telematic services. Attention is given to personal computers and OSI, the role of software reliability measurement in information systems, and an active array antenna for the next-generation direct broadcast satellite.

A Double-Blinded Randomized Study Investigating a Possible Anti-Inflammatory Effect of Saxagliptin versus Placebo as Add-On Therapy in Patients with Both Type 2 Diabetes And Stable Coronary Artery Disease.

PubMed

Njerve, Ida Unhammer; Åkra, Sissel; Weiss, Thomas W; Solheim, Svein; Øvstebø, Reidun; Aass, Hans Christian D; Byrkjeland, Rune; Arnesen, Harald; Seljeflot, Ingebjørg

2017-01-01

Promising results regarding potential anti-inflammatory and antiatherosclerotic effects of gliptins have been reported. Our aim was to investigate whether saxagliptin treatment modifies expression of inflammatory markers, primarily in peripheral blood mononuclear cells (PBMCs) and in circulating leukocytes in patients with stable coronary artery disease (CAD) and T2DM. Patients ( n = 12) were randomized to saxagliptin 5 mg daily or placebo for 3 months. Samples were taken at baseline and end of study in fasting state prior to intake of medications. PBMCs were isolated and cryopreserved at -150°C until ex vivo exposed to 1 ng/mL of lipopolysaccharide (LPS) for 4 hours. Gene expression was performed with custom-designed TaqMan® Arrays and relative quantification by real-time PCR (RT-qPCR). HbA1c was reduced in the saxagliptin-treated group compared to that in the change with placebo ( p = 0.042). In unstimulated PBMCs and in circulating leukocytes, we observed a significant increase in IL-10 expression in the saxagliptin group ( p = 0.043, both), significantly different from that in the placebo ( p = 0.009 and p = 0.032, resp.). No between group differences in changes were observed in any of the selected proinflammatory markers. In our small cohort of patients with combined T2DM and CAD, a possible anti-inflammatory effect of saxagliptin, observed in the present study by upregulation of IL-10 in leukocytes, needs to be confirmed in larger studies.

Betaine supplement enhances skeletal muscle differentiation in murine myoblasts via IGF-1 signaling activation.

PubMed

Senesi, Pamela; Luzi, Livio; Montesano, Anna; Mazzocchi, Nausicaa; Terruzzi, Ileana

2013-07-19

Betaine (BET) is a component of many foods, including spinach and wheat. It is an essential osmolyte and a source of methyl groups. Recent studies have hypothesized that BET might play a role in athletic performance. However, BET effects on skeletal muscle differentiation and hypertrophy are still poorly understood. We examined BET action on neo myotubes maturation and on differentiation process, using C2C12 murine myoblastic cells. We used RT2-PCR array, Western blot and immunofluorescence analysis to study the BET effects on morphological features of C2C12 and on signaling pathways involved in muscle differentiation and hypertrophy. We performed a dose-response study, establishing that 10 mM BET was the dose able to stimulate morphological changes and hypertrophic process in neo myotubes. RT2-PCR array methodology was used to identify the expression profile of genes encoding proteins involved in IGF-1 pathway. A dose of 10 mM BET was found to promote IGF-1 receptor (IGF-1 R) expression. Western blot and immunofluorescence analysis, performed in neo myotubes, pointed out that 10 mM BET improved IGF-1 signaling, synthesis of Myosin Heavy Chain (MyHC) and neo myotubes length. Our findings provide the first evidence that BET could promote muscle fibers differentiation and increase myotubes size by IGF-1 pathway activation, suggesting that BET might represent a possible new drug/integrator strategy, not only in sport performance but also in clinical conditions characterized by muscle function impairment.

Exploring target-specific primer extension in combination with a bead-based suspension array for multiplexed detection and typing using Streptococcus suis as a model pathogen

PubMed Central

van der Wal, Fimme J.; Achterberg, René P.; van Solt-Smits, Conny; Bergervoet, Jan H. W.; de Weerdt, Marjanne; Wisselink, Henk J.

2017-01-01

We investigated the feasibility of an assay based on target-specific primer extension, combined with a suspension array, for the multiplexed detection and typing of a veterinary pathogen in animal samples, using Streptococcus suis as a model pathogen. A procedure was established for simultaneous detection of 6 S. suis targets in pig tonsil samples (i.e., 4 genes associated with serotype 1, 2, 7, or 9, the generic S. suis glutamate dehydrogenase gene [gdh], and the gene encoding the extracellular protein factor [epf]). The procedure was set up as a combination of protocols: DNA isolation from porcine tonsils, a multiplex PCR, a multiplex target-specific primer extension, and finally a suspension array as the readout. The resulting assay was compared with a panel of conventional PCR assays. The proposed multiplex assay can correctly identify the serotype of isolates and is capable of simultaneous detection of multiple targets in porcine tonsillar samples. The assay is not as sensitive as the current conventional PCR assays, but with the correct sampling strategy, the assay can be useful for screening pig herds to establish which S. suis serotypes are circulating in a pig population. PMID:28980519

Multiplex PCR-based DNA array for simultaneous detection of three human herpesviruses, EVB, CMV and KSHV.

PubMed

Fujimuro, Masahiro; Nakaso, Kazuhiro; Nakashima, Kenji; Sadanari, Hidetaka; Hisanori, Inoue; Teishikata, Yasuhiro; Hayward, S Diane; Yokosawa, Hideyoshi

2006-04-01

Human lymphotropic herpesviruses, Epstein-Barr virus (EBV), cytomegalovirus (CMV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are responsible for a wide variety of human diseases. Due to an increase in diseased states associated with immunosuppression, more instances of co-morbid infections with these herpesviruses have resulted in viral reactivations that have caused numerous fatalities. Therefore, the development of rapid and accurate method to detect these viruses in immunocompromised patients is vital for immediate treatment with antiviral prophylactic drugs. In this study, we developed a new multiplex PCR method coupled to DNA array hybridization, which can simultaneously detect all three human herpesviruses in one single cell sample. Multiplex PCR primers were designed to amplify specific regions of the EBV (EBER1), CMV (IE) and KSHV (LANA) viral genomes. Pre-clinical application of this method revealed that this approach is capable of detecting as few as 1 copy of the viral genomes for KSHV and CMV and 100 copies of the genome for EBV. Furthermore, this highly sensitive test showed no cross-reactivity among the three viruses and is capable of detecting both KSHV and EBV viral genomes simultaneously in the lymphoblastoid cells that have been double infected with both viruses. Thus, this array-based approach serves as a rapid and reliable diagnostic tool for clinical applications.

Genetics Home Reference: cri-du-chat syndrome

MedlinePlus

... Pinkel D. High-resolution mapping of genotype-phenotype relationships in cri du chat syndrome using array comparative ... for Links Data Files & API Site Map Subscribe Customer Support USA.gov Copyright Privacy Accessibility FOIA Viewers & ...

Best face forward.

PubMed

Rayport, Jeffrey F; Jaworski, Bernard J

2004-12-01

Most companies serve customers through a broad array of interfaces, from retail sales clerks to Web sites to voice-response telephone systems. But while the typical company has an impressive interface collection, it doesn't have an interface system. That is, the whole set does not add up to the sum of its parts in its ability to provide service and build customer relationships. Too many people and too many machines operating with insufficient coordination (and often at cross-purposes) mean rising complexity, costs, and customer dissatisfaction. In a world where companies compete not on what they sell but on how they sell it, turning that liability into an asset is what separates winners from losers. In this adaptation of their forthcoming book by the same title, Jeffrey Rayport and Bernard Jaworski explain how companies must reengineer their customer interface systems for optimal efficiency and effectiveness. Part of that transformation, they observe, will involve a steady encroachment by machine interfaces into areas that have long been the sacred province of humans. Managers now have opportunities unprecedented in the history of business to use machines, not just people, to credibly manage their interactions with customers. Because people and machines each have their strengths and weaknesses, company executives must identify what people do best, what machines do best, and how to deploy them separately and together. Front-office reengineering subjects every current and potential service interface to an analysis of opportunities for substitution (using machines instead of people), complementarity (using a mix of machines and people), and displacement (using networks to shift physical locations of people and machines), with the twin objectives of compressing costs and driving top-line growth through increased customer value.

Utah optrode array customization using stereotactic brain atlases and 3-D CAD modeling for optogenetic neocortical interrogation in small rodents and nonhuman primates.

PubMed

Boutte, Ronald W; Merlin, Sam; Yona, Guy; Griffiths, Brandon; Angelucci, Alessandra; Kahn, Itamar; Shoham, Shy; Blair, Steve

2017-10-01

As the optogenetic field expands, the need for precise targeting of neocortical circuits only grows more crucial. This work demonstrates a technique for using Solidworks ® computer-aided design (CAD) and readily available stereotactic brain atlases to create a three-dimensional (3-D) model of the dorsal region of area visual cortex 4 (V4D) of the macaque monkey ( Macaca fascicularis ) visual cortex. The 3-D CAD model of the brain was used to customize an [Formula: see text] Utah optrode array (UOA) after it was determined that a high-density ([Formula: see text]) UOA caused extensive damage to marmoset ( Callithrix jacchus ) primary visual cortex as assessed by electrophysiological recording of spiking activity through a 1.5-mm-diameter through glass via. The [Formula: see text] UOA was customized for optrode length ([Formula: see text]), optrode width ([Formula: see text]), optrode pitch ([Formula: see text]), backplane thickness ([Formula: see text]), and overall form factor ([Formula: see text]). Two [Formula: see text] UOAs were inserted into layer VI of macaque V4D cortices with minimal damage as assessed in fixed tissue cytochrome oxidase staining in nonrecoverable surgeries. Additionally, two [Formula: see text] arrays were implanted in mice ( Mus musculus ) motor cortices, providing early evidence for long-term tolerability (over 6 months), and for the ability to integrate the UOA with a Holobundle light delivery system toward patterned optogenetic stimulation of cortical networks.

NAFLD and Atherosclerosis Are Prevented by a Natural Dietary Supplement Containing Curcumin, Silymarin, Guggul, Chlorogenic Acid and Inulin in Mice Fed a High-Fat Diet.

PubMed

Amato, Antonella; Caldara, Gaetano-Felice; Nuzzo, Domenico; Baldassano, Sara; Picone, Pasquale; Rizzo, Manfredi; Mulè, Flavia; Di Carlo, Marta

2017-05-13

Non-alcoholic fatty liver disease (NAFLD) confers an increased risk of cardiovascular diseases. NAFDL is associated with atherogenic dyslipidemia, inflammation and renin-angiotensin system (RAS) imbalance, which in turn lead to atherosclerotic lesions. In the present study, the impact of a natural dietary supplement (NDS) containing Curcuma longa , silymarin, guggul, chlorogenic acid and inulin on NAFLD and atherosclerosis was evaluated, and the mechanism of action was examined. C57BL/6 mice were fed an HFD for 16 weeks; half of the mice were simultaneously treated with a daily oral administration (os) of the NDS. NAFLD and atherogenic lesions in aorta and carotid artery (histological analysis), hepatic expression of genes involved in the NAFLD (PCR array), hepatic angiotensinogen (AGT) and AT₁R mRNA expression (real-time PCR) and plasma angiotensin (ANG)-II levels (ELISA) were evaluated. In the NDS group, steatosis, aortic lesions or carotid artery thickening was not observed. PCR array showed upregulation of some genes involved in lipid metabolism and anti-inflammatory activity (Cpt2, Ifng) and downregulation of some genes involved in pro-inflammatory response and in free fatty acid up-take (Fabp5, Socs3). Hepatic AGT, AT₁R mRNA and ANG II plasma levels were significantly lower with respect to the untreated-group. Furthermore, NDS inhibited the dyslipidemia observed in the untreated animals. Altogether, these results suggest that NDS prevents NAFLD and atherogenesis by modulating the expression of different genes involved in NAFLD and avoiding RAS imbalance.

NAFLD and Atherosclerosis Are Prevented by a Natural Dietary Supplement Containing Curcumin, Silymarin, Guggul, Chlorogenic Acid and Inulin in Mice Fed a High-Fat Diet

PubMed Central

Amato, Antonella; Caldara, Gaetano-Felice; Nuzzo, Domenico; Baldassano, Sara; Picone, Pasquale; Rizzo, Manfredi; Mulè, Flavia; Di Carlo, Marta

2017-01-01

Non-alcoholic fatty liver disease (NAFLD) confers an increased risk of cardiovascular diseases. NAFDL is associated with atherogenic dyslipidemia, inflammation and renin-angiotensin system (RAS) imbalance, which in turn lead to atherosclerotic lesions. In the present study, the impact of a natural dietary supplement (NDS) containing Curcuma longa, silymarin, guggul, chlorogenic acid and inulin on NAFLD and atherosclerosis was evaluated, and the mechanism of action was examined. C57BL/6 mice were fed an HFD for 16 weeks; half of the mice were simultaneously treated with a daily oral administration (os) of the NDS. NAFLD and atherogenic lesions in aorta and carotid artery (histological analysis), hepatic expression of genes involved in the NAFLD (PCR array), hepatic angiotensinogen (AGT) and AT1R mRNA expression (real-time PCR) and plasma angiotensin (ANG)-II levels (ELISA) were evaluated. In the NDS group, steatosis, aortic lesions or carotid artery thickening was not observed. PCR array showed upregulation of some genes involved in lipid metabolism and anti-inflammatory activity (Cpt2, Ifng) and downregulation of some genes involved in pro-inflammatory response and in free fatty acid up-take (Fabp5, Socs3). Hepatic AGT, AT1R mRNA and ANG II plasma levels were significantly lower with respect to the untreated-group. Furthermore, NDS inhibited the dyslipidemia observed in the untreated animals. Altogether, these results suggest that NDS prevents NAFLD and atherogenesis by modulating the expression of different genes involved in NAFLD and avoiding RAS imbalance. PMID:28505074

Analysis of gene expression profiles in tympanic membrane following perforation using PCR Array in rats--preliminary investigation.

PubMed

Hassmann-Poznańska, Elżbieta; Taranta, Andrzej; Bialuk, Izabela; Poznańska, Maria; Zajączkiewicz, Hanna; Winnicka, Maria Małgorzata

2013-10-01

The goal of this work was to identify genes, known to be involved in the skin wound healing, that express differentially in the healthy and injured tympanic membrane (TM), and designate the molecules potentially beneficial for treatment of TM perforation. The molecular mechanisms controlling the course of TM regeneration are far from being elucidated. Twenty rats had their tympanic membranes perforated, while four served as a control. Animals were sacrificed on either days 1, 2, 3, 5 and 10 post injury, and TMs were immediately dissected and frozen in liquid nitrogen. Total TM RNA was isolated and reversely transcribed. qPCR was performed using Rat Wound Healing RT(2) Profiler PCR Array (QIAGEN) containing primers for 84 genes. Statistically significant changes in the expression of 42 genes were found in various stages of TM healing. The increased expression of genes taking part in the inflammatory reaction (interleukin 6, granulocyte and macrophage chemotactic proteins) was observed from day 2. The expression of several genes of extracellular matrix components and their remodeling enzymes was also changed. Among growth factor genes: Vegfa, Igf1 and Hbegf showed increased expression at the beginning of the healing process, while Hgf expression was highest on day 3. Several changes in the expression of genes involved in remodeling of extracellular matrix point to important role of connective tissue in TM healing. The molecules accelerating this process, like HbEGF and HGF, seem to be good candidates for further evaluation of their possible use in clinical treatment. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

microRNA-7 down-regulation mediates excessive collagen expression in localized scleroderma.

PubMed

Etoh, Mitsuhiko; Jinnin, Masatoshi; Makino, Katsunari; Yamane, Keitaro; Nakayama, Wakana; Aoi, Jun; Honda, Noritoshi; Kajihara, Ikko; Makino, Takamitsu; Fukushima, Satoshi; Ihn, Hironobu

2013-01-01

Localized scleroderma (LSc), a connective tissue disorder restricted to the skin and subcutaneous tissue, is characterized by skin fibrosis due to an excessive deposition of types I collagen. The mechanism of such fibrosis is still unknown, but epigenetics may play some roles in the excessive collagen expression. In the present study, we investigated the mechanism of fibrosis seen in LSc, focusing on microRNA (miRNA). miRNA expression was determined by PCR array, real-time PCR, and in situ hybridization. The function of miRNA was evaluated using specific inhibitor. Immunoblotting was performed to detect α2(I) collagen protein. PCR array analysis using tissue miRNA demonstrated miR-7 level was significantly decreased in LSc skin as well as keloid tissue compared to normal skin in vivo. In situ hybridization also showed miR-7 expression in dermal fibroblasts was decreased in LSc dermis. The transfection of specific inhibitor for miR-7 into cultured normal dermal fibroblasts resulted in the up-regulation of α2(I) collagen protein in vitro. Also, the serum levels of miR-7 were significantly decreased in LSc patients compared with healthy controls, but serum miR-29a levels not. Systemic or local down-regulation of miR-7 may contribute to the pathogenesis of LSc via the overexpression of α2(I) collagen, and serum miR-7 may be useful as a disease marker. Investigation of the regulatory mechanisms of LSc by miRNA may lead to new treatments by the transfection into the lesional skin of this disease.

DNA-based techniques for authentication of processed food and food supplements.

PubMed

Lo, Yat-Tung; Shaw, Pang-Chui

2018-02-01

Authentication of food or food supplements with medicinal values is important to avoid adverse toxic effects, provide consumer rights, as well as for certification purpose. Compared to morphological and spectrometric techniques, molecular authentication is found to be accurate, sensitive and reliable. However, DNA degradation and inclusion of inhibitors may lead to failure in PCR amplification. This paper reviews on the existing DNA extraction and PCR protocols, and the use of small size DNA markers with sufficient discriminative power for molecular authentication. Various emerging new molecular techniques such as isothermal amplification for on-site diagnosis, next-generation sequencing for high-throughput species identification, high resolution melting analysis for quick species differentiation, DNA array techniques for rapid detection and quantitative determination in food products are also discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

High-throughput multiplex HLA-typing by ligase detection reaction (LDR) and universal array (UA) approach.

PubMed

Consolandi, Clarissa

2009-01-01

One major goal of genetic research is to understand the role of genetic variation in living systems. In humans, by far the most common type of such variation involves differences in single DNA nucleotides, and is thus termed single nucleotide polymorphism (SNP). The need for improvement in throughput and reliability of traditional techniques makes it necessary to develop new technologies. Thus the past few years have witnessed an extraordinary surge of interest in DNA microarray technology. This new technology offers the first great hope for providing a systematic way to explore the genome. It permits a very rapid analysis of thousands genes for the purpose of gene discovery, sequencing, mapping, expression, and polymorphism detection. We generated a series of analytical tools to address the manufacturing, detection and data analysis components of a microarray experiment. In particular, we set up a universal array approach in combination with a PCR-LDR (polymerase chain reaction-ligation detection reaction) strategy for allele identification in the HLA gene.

Focal-plane detector system for the KATRIN experiment

NASA Astrophysics Data System (ADS)

Amsbaugh, J. F.; Barrett, J.; Beglarian, A.; Bergmann, T.; Bichsel, H.; Bodine, L. I.; Bonn, J.; Boyd, N. M.; Burritt, T. H.; Chaoui, Z.; Chilingaryan, S.; Corona, T. J.; Doe, P. J.; Dunmore, J. A.; Enomoto, S.; Formaggio, J. A.; Fränkle, F. M.; Furse, D.; Gemmeke, H.; Glück, F.; Harms, F.; Harper, G. C.; Hartmann, J.; Howe, M. A.; Kaboth, A.; Kelsey, J.; Knauer, M.; Kopmann, A.; Leber, M. L.; Martin, E. L.; Middleman, K. J.; Myers, A. W.; Oblath, N. S.; Parno, D. S.; Peterson, D. A.; Petzold, L.; Phillips, D. G.; Renschler, P.; Robertson, R. G. H.; Schwarz, J.; Steidl, M.; Tcherniakhovski, D.; Thümmler, T.; Van Wechel, T. D.; VanDevender, B. A.; Vöcking, S.; Wall, B. L.; Wierman, K. L.; Wilkerson, J. F.; Wüstling, S.

2015-04-01

The focal-plane detector system for the KArlsruhe TRItium Neutrino (KATRIN) experiment consists of a multi-pixel silicon p-i-n-diode array, custom readout electronics, two superconducting solenoid magnets, an ultra high-vacuum system, a high-vacuum system, calibration and monitoring devices, a scintillating veto, and a custom data-acquisition system. It is designed to detect the low-energy electrons selected by the KATRIN main spectrometer. We describe the system and summarize its performance after its final installation.

Surface invasive cleavage assay on a maskless light-directed diamond DNA microarray for genome-wide human SNP mapping.

PubMed

Nie, Bei; Yang, Min; Fu, Weiling; Liang, Zhiqing

2015-07-07

The surface invasive cleavage assay, because of its innate accuracy and ability for self-signal amplification, provides a potential route for the mapping of hundreds of thousands of human SNP sites. However, its performance on a high density DNA array has not yet been established, due to the unusual "hairpin" probe design on the microarray and the lack of chemical stability of commercially available substrates. Here we present an applicable method to implement a nanocrystalline diamond thin film as an alternative substrate for fabricating an addressable DNA array using maskless light-directed photochemistry, producing the most chemically stable and biocompatible system for genetic analysis and enzymatic reactions. The surface invasive cleavage reaction, followed by degenerated primer ligation and post-rolling circle amplification is consecutively performed on the addressable diamond DNA array, accurately mapping SNP sites from PCR-amplified human genomic target DNA. Furthermore, a specially-designed DNA array containing dual probes in the same pixel is fabricated by following a reverse light-directed DNA synthesis protocol. This essentially enables us to decipher thousands of SNP alleles in a single-pot reaction by the simple addition of enzyme, target and reaction buffers.

Custom microarray construction and analysis for determining potential biomarkers of subchronic androgen exposure in the Eastern Mosquitofish (Gambusia holbrooki)

USGS Publications Warehouse

Brockmeier, Erica K.; Yu, Fahong; Amador, David Moraga; Bargar, Timothy A.; Denslow, Nancy D.

2013-01-01

Coupling microarray data with phenotypic changes driven by androgen exposure in mosquitofish is key for developing this organism into a bioindicator for EDCs. Future studies using this array will enhance knowledge of the biology and toxicological response of this species. This work provides a foundation of molecular knowledge and tools that can be used to delve further into understanding the biology of G. holbrooki and how this organism can be used as a bioindicator organism for endocrine disrupting pollutants in the environment.

A miniaturized and integrated gel post platform for multiparameter PCR detection of herpes simplex viruses from raw genital swabs.

PubMed

Manage, Dammika P; Lauzon, Jana; Atrazhev, Alexey; Morrissey, Yuen C; Edwards, Ann L; Stickel, Alexander J; Crabtree, H John; Pabbaraju, Kanti; Zahariadis, George; Yanow, Stephanie K; Pilarski, Linda M

2012-05-07

Herpes simplex virus (HSV) is one of the most prevalent viruses, with acute and recurrent infections in humans. The current gold standard for the diagnosis of HSV is viral culture which takes 2-14 days and has low sensitivity. In contrast, DNA amplification by polymerase chain reaction (PCR) can be performed within 1-2 h. We here describe a multiparameter PCR assay to simultaneously detect HSV-1 and HSV-2 DNA templates, together with integrated positive and negative controls, with product detection by melting curve analysis (MCA), in an array of semi-solid polyacrylamide gel posts. Each gel post is 0.67 μL in volume, and polymerized with all the components required for PCR. Both PCR and MCA can currently be performed in one hour and 20 min. Unprocessed genital swabs collected in universal transport medium were directly added to the reagents before or after polymerization, diffusing from atop the gel posts. The gel post platform detects HSV templates in as little as 2.5 nL of raw sample. In this study, 45 genital swab specimens were tested blindly as a preliminary validation of this platform. The concordance of PCR on gel posts with conventional PCR was 91%. The primer sequestration method introduced here (wherein different primers are placed in different sets of posts) enables the simultaneous detection of multiple pathogens for the same sample, together with positive and negative controls, on a single chip. This platform accepts unprocessed samples and is readily adaptable to detection of multiple different pathogens or biomarkers for point-of-care diagnostics.

Analysis of Multiplexed Nanosensor Arrays Based on Near-Infrared Fluorescent Single-Walled Carbon Nanotubes.

PubMed

Dong, Juyao; Salem, Daniel P; Sun, Jessica H; Strano, Michael S

2018-04-24

The high-throughput, label-free detection of biomolecules remains an important challenge in analytical chemistry with the potential of nanosensors to significantly increase the ability to multiplex such assays. In this work, we develop an optical sensor array, printable from a single-walled carbon nanotube/chitosan ink and functionalized to enable a divalent ion-based proximity quenching mechanism for transducing binding between a capture protein or an antibody with the target analyte. Arrays of 5 × 6, 200 μm near-infrared (nIR) spots at a density of ≈300 spots/cm 2 are conjugated with immunoglobulin-binding proteins (proteins A, G, and L) for the detection of human IgG, mouse IgM, rat IgG2a, and human IgD. Binding kinetics are measured in a parallel, multiplexed fashion from each sensor spot using a custom laser scanning imaging configuration with an nIR photomultiplier tube detector. These arrays are used to examine cross-reactivity, competitive and nonspecific binding of analyte mixtures. We find that protein G and protein L functionalized sensors report selective responses to mouse IgM on the latter, as anticipated. Optically addressable platforms such as the one examined in this work have potential to significantly advance the real-time, multiplexed biomolecular detection of complex mixtures.

Killer whale caller localization using a hydrophone array in an oceanarium pool

NASA Astrophysics Data System (ADS)

Bowles, Ann E.; Greenlaw, Charles F.; McGehee, Duncan E.; van Holliday, D.

2004-05-01

A system to localize calling killer whales was designed around a ten-hydrophone array in a pool at SeaWorld San Diego. The array consisted of nine ITC 8212 and one ITC 6050H hydrophones mounted in recessed 30×30 cm2 niches. Eight of the hydrophones were connected to a Compaq Armada E500 laptop computer through a National Instruments DAQ 6024E PCMCIA A/D data acquisition card and a BNC-2120 signal conditioner. The system was calibrated with a 139-dB, 4.5-kHz pinger. Acoustic data were collected during four 48-72 h recording sessions, simultaneously with video recorded from a four-camera array. Calling whales were localized by one of two methods, (1) at the hydrophone reporting the highest sound exposure level and (2) using custom-designed 3-D localization software based on time-of-arrival (ORCA). Complex reverberations in the niches and pool made locations based on time of arrival difficult to collect. Based on preliminary analysis of data from four sessions (400+ calls/session), the hydrophone reporting the highest level reliably attributed callers 51%-100% of the time. This represents a substantial improvement over attribution rates of 5%-15% obtained with single hydrophone recordings. [Funding provided by Hubbs-SeaWorld Research Institute and the Hubbs Society.

Neurovascular and Neuroimmune Aspects in the Pathophysiology of Rosacea

PubMed Central

Schwab, Verena D.; Sulk, Mathias; Seeliger, Stephan; Nowak, Pawel; Aubert, Jerome; Mess, Christian; Rivier, Michel; Carlavan, Isabelle; Rossio, Patricia; Metze, Dieter; Buddenkotte, Jörg; Cevikbas, Ferda; Voegel, Johannes J.; Steinhoff, Martin

2013-01-01

Rosacea is a common skin disease with a high impact on quality of life. Characterized by erythema, edema, burning pain, immune infiltration, and facial skin fibrosis, rosacea has all the characteristics of neurogenic inflammation, a condition induced by sensory nerves via antidromically released neuromediators. To investigate the hypothesis of a central role of neural interactions in the pathophysiology, we analyzed molecular and morphological characteristics in the different subtypes of rosacea by immunohistochemistry, double immunofluorescence, morphometry, real-time PCR, and gene array analysis, and compared the findings with those for lupus erythematosus or healthy skin. Our results showed significantly dilated blood and lymphatic vessels. Signs of angiogenesis were only evident in phymatous rosacea. The number of mast cells and fibroblasts was increased in rosacea, already in subtypes in which fibrosis is not clinically apparent, indicating early activation. Sensory nerves were closely associated with blood vessels and mast cells, and were increased in erythematous rosacea. Gene array studies and qRT-PCR confirmed upregulation of genes involved in vasoregulation and neurogenic inflammation. Thus, dysregulation of mediators and receptors implicated in neurovascular and neuroimmune communication may be crucial at early stages of rosacea. Drugs that function on neurovascular and/or neuroimmune communication may be beneficial for the treatment of rosacea. PMID:22076328

A novel deletion in the thyrotropin Beta-subunit gene identified by array comparative genomic hybridization analysis causes central congenital hypothyroidism in a boy originating from Turkey.

PubMed

Hermanns, Pia; Couch, Robert; Leonard, Norma; Klotz, Cherise; Pohlenz, Joachim

2014-01-01

Isolated central congenital hypothyroidism (ICCH) is rare but important. Most ICCH patients are diagnosed later, which results in severe growth failure and intellectual disability. We describe a boy with ICCH due to a large homozygous TSHβ gene deletion. A 51-day-old male Turkish infant, whose parents were first cousins, was admitted for evaluation of prolonged jaundice. His clinical appearance was compatible with hypothyroidism. Venous thyrotropin (TSH) was undetectably low, with a subsequent low free T4 and a low free T3, suggestive of central hypothyroidism. Using different PCR protocols, we could not amplify both coding exons of the boy's TSHβ gene, which suggested a deletion. An array comparative genomic hybridization (aCGH) using specific probes around the TSHβ gene locus showed him to be homozygous for a 6-kb deletion spanning all exons and parts of the 5' untranslated region of the gene. Infants who are clinically suspected of having hypothyroidism should be evaluated thoroughly, even if their TSH-based screening result is normal. In cases with ICCH and undetectably low TSH serum concentrations, a TSHβ gene deletion should be considered; aCGH should be performed when gene deletions are suspected. In such cases, PCR-based sequencing techniques give negative results.

Improving efficiency of a small forensic DNA laboratory: validation of robotic assays and evaluation of microcapillary array device.

PubMed

Crouse, Cecelia A; Yeung, Stephanie; Greenspoon, Susan; McGuckian, Amy; Sikorsky, Julie; Ban, Jeff; Mathies, Richard

2005-08-01

To present validation studies performed for the implementation of existing and new technologies to increase the efficiency in the forensic DNA Section of the Palm Beach County Sheriff's Office (PBSO) Crime Laboratory. Using federally funded grants, internal support, and an external Process Mapping Team, the PBSO collaborated with forensic vendors, universities, and other forensic laboratories to enhance DNA testing procedures, including validation of the DNA IQ magnetic bead extraction system, robotic DNA extraction using the BioMek2000, the ABI7000 Sequence Detection System, and is currently evaluating a micro Capillary Array Electrophoresis device. The PBSO successfully validated and implemented both manual and automated Promega DNA IQ magnetic bead extractions system, which have increased DNA profile results from samples with low DNA template concentrations. The Beckman BioMek2000 DNA robotic workstation has been validated for blood, tissue, bone, hair, epithelial cells (touch evidence), and mixed stains such as semen. There has been a dramatic increase in the number of samples tested per case since implementation of the robotic extraction protocols. The validation of the ABI7000 real-time quantitative polymerase chain reaction (qPCR) technology and the single multiplex short tandem repeat (STR) PowerPlex16 BIO amplification system has provided both a time and a financial benefit. In addition, the qPCR system allows more accurate DNA concentration data and the PowerPlex 16 BIO multiplex generates DNA profiles data in half the time when compared to PowerPlex1.1 and PowerPlex2.1 STR systems. The PBSO's future efficiency requirements are being addressed through collaboration with the University of California at Berkeley and the Virginia Division of Forensic Science to validate microcapillary array electrophoresis instrumentation. Initial data demonstrated the electrophoresis of 96 samples in less than twenty minutes. The PBSO demonstrated, through the validation of more efficient extraction and quantification technology, an increase in the number of evidence samples tested using robotic/DNA IQ magnetic bead DNA extraction, a decrease in the number of negative samples amplified due to qPCR and implementation of a single multiplex amplification system. In addition, initial studies show the microcapillary array electrophoresis device (microCAE) evaluation results provide greater sensitivity and faster STR analysis output than current platforms.

Detection of Leptospira interrogans DNA and antigen in fixed equine eyes affected with end-stage equine recurrent uveitis.

PubMed

Pearce, Jacqueline W; Galle, Laurence E; Kleiboeker, Steve B; Turk, James R; Schommer, Susan K; Dubielizig, Richard R; Mitchell, William J; Moore, Cecil P; Giuliano, Elizabeth A

2007-11-01

Equine recurrent uveitis (ERU) is the most frequent cause of blindness in horses worldwide. Leptospira has been implicated as an etiologic agent in some cases of ERU and has been detected in fresh ocular tissues of affected horses. The objective of this study was to determine the presence of Leptospira antigen and DNA in fixed equine ocular tissues affected with end-stage ERU. Sections of eyes from 30 horses were obtained. Controls included 1) 10 normal equine eyes and 2) 10 equine eyes with a nonrecurrent form of uveitis. The experimental group consisted of 10 eyes diagnosed with ERU based on clinical signs and histologic lesions. Sections were subjected to immunohistochemical staining with an array of rabbit anti-Leptospira polyclonal antibodies. DNA extractions were performed by using a commercial kit designed for fixed tissue. Real-time PCR analysis was completed on extracted DNA. The target sequence for PCR was designed from alignments of available Leptospira 16S rDNA partial sequences obtained from GenBank. Two of 10 test samples were positive for Leptospira antigen by immunohistochemical assay. Zero of 20 controls were positive for Leptospira antigen. All test samples and controls were negative for Leptospira DNA by real-time PCR analysis. Leptospira was detected at a lower frequency than that previously reported for fresh ERU-affected aqueous humor and vitreous samples. Leptospira is not frequently detectable in fixed ocular tissues of horses affected with ERU when using traditional immunohistochemical and real-time PCR techniques.

Determining the analytical specificity of PCR-based assays for the diagnosis of IA: What is Aspergillus?

PubMed

Morton, C Oliver; White, P Lewis; Barnes, Rosemary A; Klingspor, Lena; Cuenca-Estrella, Manuel; Lagrou, Katrien; Bretagne, Stéphane; Melchers, Willem; Mengoli, Carlo; Caliendo, Angela M; Cogliati, Massimo; Debets-Ossenkopp, Yvette; Gorton, Rebecca; Hagen, Ferry; Halliday, Catriona; Hamal, Petr; Harvey-Wood, Kathleen; Jaton, Katia; Johnson, Gemma; Kidd, Sarah; Lengerova, Martina; Lass-Florl, Cornelia; Linton, Chris; Millon, Laurence; Morrissey, C Orla; Paholcsek, Melinda; Talento, Alida Fe; Ruhnke, Markus; Willinger, Birgit; Donnelly, J Peter; Loeffler, Juergen

2017-06-01

A wide array of PCR tests has been developed to aid the diagnosis of invasive aspergillosis (IA), providing technical diversity but limiting standardisation and acceptance. Methodological recommendations for testing blood samples using PCR exist, based on achieving optimal assay sensitivity to help exclude IA. Conversely, when testing more invasive samples (BAL, biopsy, CSF) emphasis is placed on confirming disease, so analytical specificity is paramount. This multicenter study examined the analytical specificity of PCR methods for detecting IA by blind testing a panel of DNA extracted from a various fungal species to explore the range of Aspergillus species that could be detected, but also potential cross reactivity with other fungal species. Positivity rates were calculated and regression analysis was performed to determine any associations between technical specifications and performance. The accuracy of Aspergillus genus specific assays was 71.8%, significantly greater (P < .0001) than assays specific for individual Aspergillus species (47.2%). For genus specific assays the most often missed species were A. lentulus (25.0%), A. versicolor (24.1%), A. terreus (16.1%), A. flavus (15.2%), A. niger (13.4%), and A. fumigatus (6.2%). There was a significant positive association between accuracy and using an Aspergillus genus PCR assay targeting the rRNA genes (P = .0011). Conversely, there was a significant association between rRNA PCR targets and false positivity (P = .0032). To conclude current Aspergillus PCR assays are better suited for detecting A. fumigatus, with inferior detection of most other Aspergillus species. The use of an Aspergillus genus specific PCR assay targeting the rRNA genes is preferential. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Development of MATLAB software to control data acquisition from a multichannel systems multi-electrode array.

PubMed

Messier, Erik

2016-08-01

A Multichannel Systems (MCS) microelectrode array data acquisition (DAQ) unit is used to collect multichannel electrograms (EGM) from a Langendorff perfused rabbit heart system to study sudden cardiac death (SCD). MCS provides software through which data being processed by the DAQ unit can be displayed and saved, but this software's combined utility with MATLAB is not very effective. MCSs software stores recorded EGM data in a MathCad (MCD) format, which is then converted to a text file format. These text files are very large, and it is therefore very time consuming to import the EGM data into MATLAB for real-time analysis. Therefore, customized MATLAB software was developed to control the acquisition of data from the MCS DAQ unit, and provide specific laboratory accommodations for this study of SCD. The developed DAQ unit control software will be able to accurately: provide real time display of EGM signals; record and save EGM signals in MATLAB in a desired format; and produce real time analysis of the EGM signals; all through an intuitive GUI.

Automated PCR setup for forensic casework samples using the Normalization Wizard and PCR Setup robotic methods.

PubMed

Greenspoon, S A; Sykes, K L V; Ban, J D; Pollard, A; Baisden, M; Farr, M; Graham, N; Collins, B L; Green, M M; Christenson, C C

2006-12-20

Human genome, pharmaceutical and research laboratories have long enjoyed the application of robotics to performing repetitive laboratory tasks. However, the utilization of robotics in forensic laboratories for processing casework samples is relatively new and poses particular challenges. Since the quantity and quality (a mixture versus a single source sample, the level of degradation, the presence of PCR inhibitors) of the DNA contained within a casework sample is unknown, particular attention must be paid to procedural susceptibility to contamination, as well as DNA yield, especially as it pertains to samples with little biological material. The Virginia Department of Forensic Science (VDFS) has successfully automated forensic casework DNA extraction utilizing the DNA IQ(trade mark) System in conjunction with the Biomek 2000 Automation Workstation. Human DNA quantitation is also performed in a near complete automated fashion utilizing the AluQuant Human DNA Quantitation System and the Biomek 2000 Automation Workstation. Recently, the PCR setup for casework samples has been automated, employing the Biomek 2000 Automation Workstation and Normalization Wizard, Genetic Identity version, which utilizes the quantitation data, imported into the software, to create a customized automated method for DNA dilution, unique to that plate of DNA samples. The PCR Setup software method, used in conjunction with the Normalization Wizard method and written for the Biomek 2000, functions to mix the diluted DNA samples, transfer the PCR master mix, and transfer the diluted DNA samples to PCR amplification tubes. Once the process is complete, the DNA extracts, still on the deck of the robot in PCR amplification strip tubes, are transferred to pre-labeled 1.5 mL tubes for long-term storage using an automated method. The automation of these steps in the process of forensic DNA casework analysis has been accomplished by performing extensive optimization, validation and testing of the software methods.

Copy number analysis of NIPBL in a cohort of 510 patients reveals rare copy number variants and a mosaic deletion.

PubMed

Cheng, Yu-Wei; Tan, Christopher A; Minor, Agata; Arndt, Kelly; Wysinger, Latrice; Grange, Dorothy K; Kozel, Beth A; Robin, Nathaniel H; Waggoner, Darrel; Fitzpatrick, Carrie; Das, Soma; Del Gaudio, Daniela

2014-03-01

Cornelia de Lange syndrome (CdLS) is a genetically heterogeneous disorder characterized by growth retardation, intellectual disability, upper limb abnormalities, hirsutism, and characteristic facial features. In this study we explored the occurrence of intragenic NIPBL copy number variations (CNVs) in a cohort of 510 NIPBL sequence-negative patients with suspected CdLS. Copy number analysis was performed by custom exon-targeted oligonucleotide array-comparative genomic hybridization and/or MLPA. Whole-genome SNP array was used to further characterize rearrangements extending beyond the NIPBL gene. We identified NIPBL CNVs in 13 patients (2.5%) including one intragenic duplication and a deletion in mosaic state. Breakpoint sequences in two patients provided further evidence of a microhomology-mediated replicative mechanism as a potential predominant contributor to CNVs in NIPBL. Patients for whom clinical information was available share classical CdLS features including craniofacial and limb defects. Our experience in studying the frequency of NIBPL CNVs in the largest series of patients to date widens the mutational spectrum of NIPBL and emphasizes the clinical utility of performing NIPBL deletion/duplication analysis in patients with CdLS.

Performance of PCR-based and Bioluminescent assays for mycoplasma detection.

PubMed

Falagan-Lotsch, Priscila; Lopes, Talíria Silva; Ferreira, Nívea; Balthazar, Nathália; Monteiro, Antônio M; Borojevic, Radovan; Granjeiro, José Mauro

2015-11-01

Contaminated eukaryotic cell cultures are frequently responsible for unreliable results. Regulatory entities request that cell cultures must be mycoplasma-free. Mycoplasma contamination remains a significant problem for cell cultures and may have an impact on biological analysis since they affect many cell parameters. The gold standard microbiological assay for mycoplasma detection involves laborious and time-consuming protocols. PCR-based and Bioluminescent assays have been considered for routine cell culture screening in research laboratories since they are fast, easy and sensitive. Thus, the aim of this work is to compare the performance of two popular commercial assays, PCR-based and Bioluminescent assays, by assessing the level of mycoplasma contamination in cell cultures from Rio de Janeiro Cell Bank (RJCB) and also from customers' laboratories. The results obtained by both performed assays were confirmed by scanning electron microscopy. In addition, we evaluated the limit of detection of the PCR kit under our laboratory conditions and the storage effects on mycoplasma detection in frozen cell culture supernatants. The performance of both assays for mycoplasma detection was not significantly different and they showed very good agreement. The Bioluminescent assay for mycoplasma detection was slightly more dependable than PCR-based due to the lack of inconclusive results produced by the first technique, especially considering the ability to detect mycoplasma contamination in frozen cell culture supernatants. However, cell lines should be precultured for four days or more without antibiotics to obtain safe results. On the other hand, a false negative result was obtained by using this biochemical approach. The implementation of fast and reliable mycoplasma testing methods is an important technical and regulatory issue and PCR-based and Bioluminescent assays may be good candidates. However, validation studies are needed. Copyright © 2015 Elsevier B.V. All rights reserved.

Combined RT-qPCR of mRNA and microRNA Targets within One Fluidigm Integrated Fluidic Circuit.

PubMed

Baldwin, Don A; Horan, Annamarie D; Hesketh, Patrick J; Mehta, Samir

2016-07-01

The ability to profile expression levels of a large number of mRNAs and microRNAs (miRNAs) within the same sample, using a single assay method, would facilitate investigations of miRNA effects on mRNA abundance and streamline biomarker screening across multiple RNA classes. A protocol is described for reverse transcription of long RNA and miRNA targets, followed by preassay amplification of the pooled cDNAs and quantitative PCR (qPCR) detection for a mixed panel of candidate RNA biomarkers. The method provides flexibility for designing custom target panels, is robust over a range of input RNA amounts, and demonstrated a high assay success rate.

miRNA expression profiling in formalin-fixed paraffin-embedded endometriosis and ovarian cancer samples

PubMed Central

Braicu, Ovidiu-Leonard; Budisan, Liviuta; Buiga, Rares; Jurj, Ancuta; Achimas-Cadariu, Patriciu; Pop, Laura Ancuta; Braicu, Cornelia; Irimie, Alexandru; Berindan-Neagoe, Ioana

2017-01-01

Endometriosis is an inflammatory pathology associated with a negative effect on life quality. Recently, this pathology was connected to ovarian cancer, in particular with endometrioid ovarian cancer. microRNAs (miRNAs) are a class of RNA transcripts ~19–22 nucleotides in length, the altered miRNA pattern being connected to pathological status. miRNAs are highly stable transcripts, and these can be assessed from formalin-fixed paraffin-embedded (FFPE) samples leading to the identification of miRNAs that could be developed as diagnostic and prognostic biomarkers, in particular those involved in malignant transformation. The aim of our study was to evaluate miRNA expression pattern in FFPE samples from endometriosis and ovarian cancer patients using PCR-array technology and also to compare the differential expression pattern in ovarian cancer versus endometriosis. For the PCR-array study, we have used nine macrodissected FFPE samples from endometriosis tissue, eight samples of ovarian cancers and five normal ovarian tissues. Quantitative real-time PCR (qRT-PCR) was used for data validation in a new patient cohort of 17 normal samples, 33 endometriosis samples and 28 ovarian cancer macrodissected FFPE samples. Considering 1.5-fold expression difference as a cut-off level and a P-value <0.05, we have identified four miRNAs being overexpressed in endometrial tissue, while in ovarian cancer 15 were differentially expressed (nine overexpressed and six downregulated). The expression level was confirmed by qRT-PCR for miR-93, miR-141, miR-155, miR-429, miR-200c, miR-205 and miR-492. Using the interpretative program Ingenuity Pathway Analysis revealed several deregulated pathways due to abnormal miRNA expression in endometriosis and ovarian cancer, which in turn is responsible for pathogenesis; this differential expression of miRNAs can be exploited as a therapeutic target. A higher number of altered miRNAs were detected in endometriosis versus ovarian cancer tissue, most of them being linked with epithelial-to-mesenchymal transition. PMID:28894379

Clinical Significance of Human Herpesvirus 6 Positivity on the FilmArray Meningitis/Encephalitis Multiplex PCR Panel.

PubMed

Green, Daniel A; Pereira, Marcus; Miko, Benjamin; Radmard, Sara; Whittier, Susan; Thakur, Kiran

2018-04-09

A review of 15 patients who tested positive for HHV-6 on the FilmArray Meningitis/Encephalitis (M/E) panel revealed that the majority were unlikely to have HHV-6 encephalitis. Interpreting HHV-6 positive results on the M/E panel requires careful clinical judgment, as this finding may be clinically insignificant for many patients.

STS-97 P6 truss payload canister is lifted into payload changeout room

NASA Technical Reports Server (NTRS)

2000-01-01

On Launch Pad 39B, the payload transport canister, with the P6 integrated truss segment inside, is lifted toward the payload changeout room (PCR). The PCR is the enclosed, environmentally controlled portion of the Rotating Service Structure that supports payload delivery at the pad and subsequent vertical installation in the orbiter payload bay. Attached to the canister are the red umbilical lines that maintain the controlled environment inside. The P6, payload on mission STS-97, comprises Solar Array Wing-3 and the Integrated Electronic Assembly, to be installed on the International Space Station. The Station'''s electrical power system will use eight photovoltaic solar arrays, each 112 feet long by 39 feet wide, to convert sunlight to electricity. The solar arrays are mounted on a '''blanket''' that can be folded like an accordion for delivery. Once in orbit, astronauts will deploy the blankets to their full size. Gimbals will be used to rotate the arrays so that they will face the Sun to provide maximum power to the Space Station. Launch of STS-97 is scheduled for Nov. 30 at 10:06 p.m. EST.

KSC00pp1730

NASA Image and Video Library

2000-11-14

KENNEDY SPACE CENTER, FLA. -- On Launch Pad 39B, the payload transport canister waits at the base of the Rotating Service Structure (RSS) with the P6 integrated truss segment inside. The canister will be lifted up to the payload changeout room (PCR) where the P6 will be removed for transfer to Space Shuttle Endeavour’s payload bay. The PCR is the enclosed, environmentally controlled portion of the RSS that supports payload delivery at the pad and subsequent vertical installation in the orbiter payload bay. The P6, payload on mission STS-97, comprises Solar Array Wing-3 and the Integrated Electronic Assembly, to be installed on the International Space Station. The Station’s electrical power system will use eight photovoltaic solar arrays, each 112 feet long by 39 feet wide, to convert sunlight to electricity. The solar arrays are mounted on a “blanket” that can be folded like an accordion for delivery. Once in orbit, astronauts will deploy the blankets to their full size. Gimbals will be used to rotate the arrays so that they will face the Sun to provide maximum power to the Space Station. Launch of STS-97 is scheduled for Nov. 30 at 10:06 p.m. EST

KSC-00pp1730

NASA Image and Video Library

2000-11-14

KENNEDY SPACE CENTER, FLA. -- On Launch Pad 39B, the payload transport canister waits at the base of the Rotating Service Structure (RSS) with the P6 integrated truss segment inside. The canister will be lifted up to the payload changeout room (PCR) where the P6 will be removed for transfer to Space Shuttle Endeavour’s payload bay. The PCR is the enclosed, environmentally controlled portion of the RSS that supports payload delivery at the pad and subsequent vertical installation in the orbiter payload bay. The P6, payload on mission STS-97, comprises Solar Array Wing-3 and the Integrated Electronic Assembly, to be installed on the International Space Station. The Station’s electrical power system will use eight photovoltaic solar arrays, each 112 feet long by 39 feet wide, to convert sunlight to electricity. The solar arrays are mounted on a “blanket” that can be folded like an accordion for delivery. Once in orbit, astronauts will deploy the blankets to their full size. Gimbals will be used to rotate the arrays so that they will face the Sun to provide maximum power to the Space Station. Launch of STS-97 is scheduled for Nov. 30 at 10:06 p.m. EST

ChIP-chip.

PubMed

Kim, Tae Hoon; Dekker, Job

2018-05-01

ChIP-chip can be used to analyze protein-DNA interactions in a region-wide and genome-wide manner. DNA microarrays contain PCR products or oligonucleotide probes that are designed to represent genomic sequences. Identification of genomic sites that interact with a specific protein is based on competitive hybridization of the ChIP-enriched DNA and the input DNA to DNA microarrays. The ChIP-chip protocol can be divided into two main sections: Amplification of ChIP DNA and hybridization of ChIP DNA to arrays. A large amount of DNA is required to hybridize to DNA arrays, and hybridization to a set of multiple commercial arrays that represent the entire human genome requires two rounds of PCR amplifications. The relative hybridization intensity of ChIP DNA and that of the input DNA is used to determine whether the probe sequence is a potential site of protein-DNA interaction. Resolution of actual genomic sites bound by the protein is dependent on the size of the chromatin and on the genomic distance between the probes on the array. As with expression profiling using gene chips, ChIP-chip experiments require multiple replicates for reliable statistical measure of protein-DNA interactions. © 2018 Cold Spring Harbor Laboratory Press.

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

PubMed

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

2013-07-01

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

An unreliable group arrival queue with k stages of service, retrial under variant vacation policy

NASA Astrophysics Data System (ADS)

Radha, J.; Indhira, K.; Chandrasekaran, V. M.

2017-11-01

In this research work we considered repairable retrial queue with group arrival and the server utilize the variant vacations. A server gives service in k stages. Any arriving group of units finds the server free, one from the group entering the first stage of service and the rest are joining into the orbit. After completion of the i th stage of service, the customer may have the option to choose (i+1)th stage of service with probability θi , with probability pi may join into orbit as feedback customer or may leave the system with probability {q}i=≤ft\\{\\begin{array}{l}1-{p}i-{θ }i,i=1,2,\\cdots k-1\\ 1-{p}i,i=k\\end{array}\\right\\}. If the orbit is empty at the service completion of each stage service, the server takes modified vacation until at least one customer appears in the orbit on the server returns from a vacation. Busy server may get to breakdown and the service channel will fail for a short interval of time. By using the supplementary variable method, steady state probability generating function for system size, some system performance measures are discussed.

Focal plane subsystem design and performance for atmospheric chemistry from geostationary orbit tropospheric emissions monitoring of pollution

NASA Astrophysics Data System (ADS)

Gilmore, A. S.; Philbrick, R. H.; Funderburg, J.

2017-09-01

Remote sensing of pollutants are enabled from a satellite in a geostationary orbit containing an imaging spectrometer encompassing the wavelength ranges of 290 - 490 nm and 540 - 740 nm. As the first of NASA's Earth Venture Instrument Program, the Tropospheric Emissions: Monitoring of Pollution (TEMPO) program will utilize this instrument to measure hourly air quality over a large portion of North America. The focal plane subsystem (FPS) contains two custom designed and critically aligned full frame transfer charge coupled devices (active area: 1028 x 2048, 18 μm) within a focal plane array package designed for radiation tolerance and space charging rejection. In addition, the FPS contains custom distributed focal plane electronics that provide all necessary clocks and biases to the sensors, receives all analog data from the sensors and performs 14 bit analog to digital conversion for upstream processing. Finally, the FPS encompasses custom low noise cables connecting the focal plane array and associated electronics. This paper discusses the design and performance of this novel focal plane subsystem with particular emphasis on the optical performance achieved including alignment, quantum efficiency, and modulation transfer function.

Differential gene expression profiles of peripheral blood mononuclear cells in childhood asthma.

PubMed

Kong, Qian; Li, Wen-Jing; Huang, Hua-Rong; Zhong, Ying-Qiang; Fang, Jian-Pei

2015-05-01

Asthma is a common childhood disease with strong genetic components. This study compared whole-genome expression differences between asthmatic young children and healthy controls to identify gene signatures of childhood asthma. Total RNA extracted from peripheral blood mononuclear cells (PBMC) was subjected to microarray analysis. QRT-PCR was performed to verify the microarray results. Classification and functional characterization of differential genes were illustrated by hierarchical clustering and gene ontology analysis. Multiple logistic regression (MLR) analysis, receiver operating characteristic (ROC) curve analysis, and discriminate power were used to scan asthma-specific diagnostic markers. For fold-change>2 and p < 0.05, there were 758 named differential genes. The results of QRT-PCR confirmed successfully the array data. Hierarchical clustering divided 29 highly possible genes into seven categories and the genes in the same cluster were likely to possess similar expression patterns or functions. Gene ontology analysis presented that differential genes primarily enriched in immune response, response to stress or stimulus, and regulation of apoptosis in biological process. MLR and ROC curve analysis revealed that the combination of ADAM33, Smad7, and LIGHT possessed excellent discriminating power. The combination of ADAM33, Smad7, and LIGHT would be a reliable and useful childhood asthma model for prediction and diagnosis.

Focal-plane detector system for the KATRIN experiment

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

Amsbaugh, J. F.; Barrett, J.; Beglarian, A.

Here, the local plane detector system for the KArlsiuhe TRItium Neutrino (KATRIN) experiment consists of a multi-pixel silicon p-i-n-diode array, custom readout electronics, two superconducting solenoid magnets, an ultra high vacuum system, a high vacuum system, calibration and monitoring devices, a scintillating veto, and a custom data-acquisition system, It is designed to detect the low-energy electrons selected by the KATRIN main spectrometer. We describe the system and summarize its performance after its final installation.

Focal-plane detector system for the KATRIN experiment

DOE PAGES

Amsbaugh, J. F.; Barrett, J.; Beglarian, A.; ...

2015-01-09

Here, the local plane detector system for the KArlsiuhe TRItium Neutrino (KATRIN) experiment consists of a multi-pixel silicon p-i-n-diode array, custom readout electronics, two superconducting solenoid magnets, an ultra high vacuum system, a high vacuum system, calibration and monitoring devices, a scintillating veto, and a custom data-acquisition system, It is designed to detect the low-energy electrons selected by the KATRIN main spectrometer. We describe the system and summarize its performance after its final installation.

Convolutional Neural Network on Embedded Linux(trademark) System-on-Chip: A Methodology and Performance Benchmark

DTIC Science & Technology

2016-05-01

A9 CPU and 15 W for the i7 CPU. A method of accelerating this computation is by using a customized hardware unit called a field- programmable gate...implementation of custom logic to accelerate com- putational workloads. This FPGA fabric, in addition to the standard programmable logic, contains 220...chip; field- programmable gate array Daniel Gebhardt U U U U 18 (619) 553-2786 INITIAL DISTRIBUTION 84300 Library (2) 85300 Archive/Stock (1

Convolutional Neural Network on Embedded Linux System-on-Chip: A Methodology and Performance Benchmark

DTIC Science & Technology

2016-05-01

A9 CPU and 15 W for the i7 CPU. A method of accelerating this computation is by using a customized hardware unit called a field- programmable gate...implementation of custom logic to accelerate com- putational workloads. This FPGA fabric, in addition to the standard programmable logic, contains 220...chip; field- programmable gate array Daniel Gebhardt U U U U 18 (619) 553-2786 INITIAL DISTRIBUTION 84300 Library (2) 85300 Archive/Stock (1

Arraying proteins by cell-free synthesis.

PubMed

He, Mingyue; Wang, Ming-Wei

2007-10-01

Recent advances in life science have led to great motivation for the development of protein arrays to study functions of genome-encoded proteins. While traditional cell-based methods have been commonly used for generating protein arrays, they are usually a time-consuming process with a number of technical challenges. Cell-free protein synthesis offers an attractive system for making protein arrays, not only does it rapidly converts the genetic information into functional proteins without the need for DNA cloning, but also presents a flexible environment amenable to production of folded proteins or proteins with defined modifications. Recent advancements have made it possible to rapidly generate protein arrays from PCR DNA templates through parallel on-chip protein synthesis. This article reviews current cell-free protein array technologies and their proteomic applications.

KRAS detection in colonic tumors by DNA extraction from FTA paper: the molecular touch-prep.

PubMed

Petras, Melissa L; Lefferts, Joel A; Ward, Brian P; Suriawinata, Arief A; Tsongalis, Gregory J

2011-12-01

DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissue is usually more degraded and contains more polymerase chain reaction (PCR) inhibitors than DNA isolated from nonfixed tissue. In addition, the tumor size and cellular heterogeneity found in tissue sections can often impact testing for molecular biomarkers. As a potential remedy to this situation, we evaluated the use of Whatman FTA paper cards for collection of colorectal tumor samples before tissue fixation and for isolation of DNA for use in a real-time PCR-based KRAS mutation assay. Eleven colon tumor samples were collected by making a cut into the fresh tumor and applying the Whatman FTA paper to the cut surface. Matched FFPE tissue blocks from these tumors were also collected for comparison. KRAS mutation analysis was carried out using the Applied Biosystems 7500 Fast Real-time PCR System using 7 independent custom TaqMan PCR assays. Of the 11 colon tumors sampled, 6 were positive for KRAS mutations in both the Whatman FTA paper preparations and corresponding FFPE samples. Whatman FTA paper cards for collection of colorectal tumor samples before tissue fixation and for isolation of DNA have many advantages including ease of use, intrinsic antimicrobial properties, long storage potential (stability of DNA over time), and a faster turnaround time for results. Extracted DNA should be suitable for most molecular diagnostic assays that use PCR techniques. This novel means of DNA preservation from surgical specimens would benefit from additional study and validation as a dependable and practical technique to preserve specimens for molecular testing.

Plenoptic Ophthalmoscopy: A Novel Imaging Technique.

PubMed

Adam, Murtaza K; Aenchbacher, Weston; Kurzweg, Timothy; Hsu, Jason

2016-11-01

This prospective retinal imaging case series was designed to establish feasibility of plenoptic ophthalmoscopy (PO), a novel mydriatic fundus imaging technique. A custom variable intensity LED array light source adapter was created for the Lytro Gen1 light-field camera (Lytro, Mountain View, CA). Initial PO testing was performed on a model eye and rabbit fundi. PO image acquisition was then performed on dilated human subjects with a variety of retinal pathology and images were subjected to computational enhancement. The Lytro Gen1 light-field camera with custom LED array captured fundus images of eyes with diabetic retinopathy, age-related macular degeneration, retinal detachment, and other diagnoses. Post-acquisition computational processing allowed for refocusing and perspective shifting of retinal PO images, resulting in improved image quality. The application of PO to image the ocular fundus is feasible. Additional studies are needed to determine its potential clinical utility. [Ophthalmic Surg Lasers Imaging Retina. 2016;47:1038-1043.]. Copyright 2016, SLACK Incorporated.

Identification of 23 new prostate cancer susceptibility loci using the iCOGS custom genotyping array

PubMed Central

Eeles, Rosalind A; Olama, Ali Amin Al; Benlloch, Sara; Saunders, Edward J; Leongamornlert, Daniel A; Tymrakiewicz, Malgorzata; Ghoussaini, Maya; Luccarini, Craig; Dennis, Joe; Jugurnauth-Little, Sarah; Dadaev, Tokhir; Neal, David E; Hamdy, Freddie C; Donovan, Jenny L; Muir, Ken; Giles, Graham G; Severi, Gianluca; Wiklund, Fredrik; Gronberg, Henrik; Haiman, Christopher A; Schumacher, Fredrick; Henderson, Brian; Le Marchand, Loic; Lindstrom, Sara; Kraft, Peter; Hunter, David J; Gapstur, Susan; Chanock, Stephen J; Berndt, Sonja I; Albanes, Demetrius; Andriole, Gerald; Schleutker, Johanna; Weischer, Maren; Canzian, Federico; Riboli, Elio; Key, Tim J; Travis, Ruth; Campa, Daniele; Ingles, Sue A; John, Esther M; Hayes, Richard B; Pharoah, Paul DP; Pashayan, Nora; Khaw, Kay-Tee; Stanford, Janet; Ostrander, Elaine A; Signorello, Lisa B; Thibodeau, Stephen N; Schaid, Dan; Maier, Christiane; Vogel, Walther; Kibel, Adam S; Cybulski, Cezary; Lubinski, Jan; Cannon-Albright; Brenner, Hermann; Park, Jong Y; Kaneva, Radka; Batra, Jyotsna; Spurdle, Amanda B; Clements, Judith A; Teixeira, Manuel R; Dicks, Ed; Lee, Andrew; Dunning, Alison; Baynes, Caroline; Conroy, Don; Maranian, Melanie J; Ahmed, Shahana; Govindasami, Koveela; Guy, Michelle; Wilkinson, Rosemary A; Sawyer, Emma J; Morgan, Angela; Dearnaley, David P; Horwich, Alan; Huddart, Robert A; Khoo, Vincent S; Parker, Christopher C; Van As, Nicholas J; Woodhouse, J; Thompson, Alan; Dudderidge, Tim; Ogden, Chris; Cooper, Colin; Lophatananon, Artitaya; Cox, Angela; Southey, Melissa; Hopper, John L; English, Dallas R; Aly, Markus; Adolfsson, Jan; Xu, Jiangfeng; Zheng, Siqun; Yeager, Meredith; Kaaks, Rudolf; Diver, W Ryan; Gaudet, Mia M; Stern, Mariana; Corral, Roman; Joshi, Amit D; Shahabi, Ahva; Wahlfors, Tiina; Tammela, Teuvo J; Auvinen, Anssi; Virtamo, Jarmo; Klarskov, Peter; Nordestgaard, Børge G; Røder, Andreas; Nielsen, Sune F; Bojesen, Stig E; Siddiq, Afshan; FitzGerald, Liesel; Kolb, Suzanne; Kwon, Erika; Karyadi, Danielle; Blot, William J; Zheng, Wei; Cai, Qiuyin; McDonnell, Shannon K; Rinckleb, Antje; Drake, Bettina; Colditz, Graham; Wokolorczyk, Dominika; Stephenson, Robert A; Teerlink, Craig; Muller, Heiko; Rothenbacher, Dietrich; Sellers, Thomas A; Lin, Hui-Yi; Slavov, Chavdar; Mitev, Vanio; Lose, Felicity; Srinivasan, Srilakshmi; Maia, Sofia; Paulo, Paula; Lange, Ethan; Cooney, Kathleen A; Antoniou, Antonis; Vincent, Daniel; Bacot, François; Tessier; Kote-Jarai, Zsofia; Easton, Douglas F

2013-01-01

Prostate cancer is the most frequently diagnosed cancer in males in developed countries. To identify common prostate cancer susceptibility alleles, we genotyped 211,155 SNPs on a custom Illumina array (iCOGS) in blood DNA from 25,074 prostate cancer cases and 24,272 controls from the international PRACTICAL Consortium. Twenty-three new prostate cancer susceptibility loci were identified at genome-wide significance (P < 5 × 10−8). More than 70 prostate cancer susceptibility loci, explaining ~30% of the familial risk for this disease, have now been identified. On the basis of combined risks conferred by the new and previously known risk loci, the top 1% of the risk distribution has a 4.7-fold higher risk than the average of the population being profiled. These results will facilitate population risk stratification for clinical studies. PMID:23535732

An acceleration framework for synthetic aperture radar algorithms

NASA Astrophysics Data System (ADS)

Kim, Youngsoo; Gloster, Clay S.; Alexander, Winser E.

2017-04-01

Algorithms for radar signal processing, such as Synthetic Aperture Radar (SAR) are computationally intensive and require considerable execution time on a general purpose processor. Reconfigurable logic can be used to off-load the primary computational kernel onto a custom computing machine in order to reduce execution time by an order of magnitude as compared to kernel execution on a general purpose processor. Specifically, Field Programmable Gate Arrays (FPGAs) can be used to accelerate these kernels using hardware-based custom logic implementations. In this paper, we demonstrate a framework for algorithm acceleration. We used SAR as a case study to illustrate the potential for algorithm acceleration offered by FPGAs. Initially, we profiled the SAR algorithm and implemented a homomorphic filter using a hardware implementation of the natural logarithm. Experimental results show a linear speedup by adding reasonably small processing elements in Field Programmable Gate Array (FPGA) as opposed to using a software implementation running on a typical general purpose processor.

Plasmodium Genus Assay Transition to the Joint Biological Agent Identification and Diagnostic System (JBAIDS)

DTIC Science & Technology

2012-07-12

readily transferable to diverse real-time PCR instrumentation. These assays are used regularly for vector surveillance and are the primary...instrumentation ( FilmArray ) is under assessment. Assay oligonucleotide sequences and formulations are available for use in future joint projects...Plasmodium real-time PCR detection capability has been challenging. During 2006, the Division of Entomology, WRAIR designed and developed a

Transcriptional expression of type-I interferon response genes and stability of housekeeping genes in the human endometrium and endometriosis.

PubMed

Vestergaard, Anna L; Knudsen, Ulla B; Munk, Torben; Rosbach, Hanne; Martensen, Pia M

2011-04-01

Endometriosis is a painful chronic female disease defined by the presence of endometrial tissue implants in ectopic (Ec) locations. The pathogenesis is much debated, and type-I interferons (IFNs) could be involved. The expression of genes of the type-I IFN response were profiled by a specific PCR array of RNA obtained from Ec and eutopic (Eu) endometrium collected from nine endometriosis patients and nine healthy control women. Transcriptional expression levels of selected IFN-regulated and housekeeping genes (HKGs) were investigated by real-time quantitative reverse transcriptase PCR (qRT-PCR). Stably expressed HKGs for valid normalization of transcriptional studies of endometrium and endometriosis have not yet been published. Here, seven HKGs were evaluated for stability using the GeNorm and NormFinder software. A normalization factor based on HMBS, TBP and YWHAZ expression was suitable for normalization of qRT-PCR studies of Eu versus Ec endometrium. In the endometrial cell lines HEC1A, HEC1B, Ishikawa and RL95-2, HMBS and HPRT1 were the most stably expressed. The IFN-specific PCR array indicated significantly different expression of the genes BST2, COL16A1, HOXB2 and ISG20 between the endometrial tissue types. However, by correctly normalized qRT-PCR, levels of BST2, COL16A1 and the highly type-I IFN-stimulated genes ISG12A and 6-16 displayed insignificant variations. Conversely, HOXB2 and ISG20 transcriptions were significantly reduced in endometriosis lesions compared with endometrium from endometriosis patients and healthy controls. In conclusion, appropriate HKGs for normalization of qRT-PCR studies of endometrium and endometriosis have been identified here. Abolished expression of ISG20 and HOX genes could be important in endometriosis.

Characteristics of a semi-custom library development system

NASA Technical Reports Server (NTRS)

Yancey, M.; Cannon, R.

1990-01-01

Standard cell and gate array macro libraries are in common use with workstation computer aided design (CAD) tools for application specific integrated circuit (ASIC) semi-custom application and have resulted in significant improvements in the overall design efficiencies as contrasted with custom design methodologies. Similar design methodology enhancements in providing for the efficient development of the library cells is an important factor in responding to the need for continuous technology improvement. The characteristics of a library development system that provides design flexibility and productivity enhancements for the library development engineer as he provides libraries in the state-of-the-art process technologies are presented. An overview of Gould's library development system ('Accolade') is also presented.

Dose Response and Prediction Characteristics of a Methylation Sensitive Digital PCR Assay for Cigarette Consumption in Adults.

PubMed

Philibert, Robert; Dogan, Meesha; Noel, Amanda; Miller, Shelly; Krukow, Brianna; Papworth, Emma; Cowley, Joseph; Long, Jeffrey D; Beach, Steven R H; Black, Donald W

2018-01-01

The tobacco use disorders are the largest preventable cause of morbidity and mortality in the world. A substantial barrier to the development of better intervention and screening measures is the lack of clinically employable biomarkers to detect the existence and extent of tobacco consumption. In prior work, we and others have shown that array based assessment of DNA methylation status at cg05575921 is a sensitive and quantitative method for assessing cigarette consumption. Unfortunately, in general, arrays are not practical clinical tools. Herein, we detail the prediction performance metrics and dose dependency of a clinically implementable droplet digital PCR (ddPCR) assay for cigarette consumption in adults. First, we demonstrate that measurements of cg05575921 as determined by Illumina array and ddPCR are highly correlated ( R 2 = 0.98, n = 92). Second, using clinical data and biomaterial from 177 subjects ranging from 18 to 78 years of age, we show that the Receiver Operating Characteristic (ROC) area under the curve (AUC) for classifying smoking status using methylation status at cg05575921 is 0.99. Finally, we conduct modeling analyses of cigarette consumption over discrete time periods to show that methylation status is best correlated with mean cigarette consumption over the past year ( R 2 = 0.5) and that demethylation at cg05575921 is dose dependent with a demethylation (delta beta) of 1% being equivalent to 1.2 cigarettes per day. But we do not find a relationship between Fagerstrom score and DNA methylation. We conclude that ddPCR assessment of cg05575921 methylation is an accurate method for assessing the presence and extent of cigarette consumption in adult subjects. We suggest that skillful clinical implementation of this approach alone or in combination with other assessment methods could lead to substantial reduction of cigarette consumption related morbidity and mortality.

Dose Response and Prediction Characteristics of a Methylation Sensitive Digital PCR Assay for Cigarette Consumption in Adults

PubMed Central

Philibert, Robert; Dogan, Meesha; Noel, Amanda; Miller, Shelly; Krukow, Brianna; Papworth, Emma; Cowley, Joseph; Long, Jeffrey D.; Beach, Steven R. H.; Black, Donald W.

2018-01-01

The tobacco use disorders are the largest preventable cause of morbidity and mortality in the world. A substantial barrier to the development of better intervention and screening measures is the lack of clinically employable biomarkers to detect the existence and extent of tobacco consumption. In prior work, we and others have shown that array based assessment of DNA methylation status at cg05575921 is a sensitive and quantitative method for assessing cigarette consumption. Unfortunately, in general, arrays are not practical clinical tools. Herein, we detail the prediction performance metrics and dose dependency of a clinically implementable droplet digital PCR (ddPCR) assay for cigarette consumption in adults. First, we demonstrate that measurements of cg05575921 as determined by Illumina array and ddPCR are highly correlated (R2 = 0.98, n = 92). Second, using clinical data and biomaterial from 177 subjects ranging from 18 to 78 years of age, we show that the Receiver Operating Characteristic (ROC) area under the curve (AUC) for classifying smoking status using methylation status at cg05575921 is 0.99. Finally, we conduct modeling analyses of cigarette consumption over discrete time periods to show that methylation status is best correlated with mean cigarette consumption over the past year (R2 = 0.5) and that demethylation at cg05575921 is dose dependent with a demethylation (delta beta) of 1% being equivalent to 1.2 cigarettes per day. But we do not find a relationship between Fagerstrom score and DNA methylation. We conclude that ddPCR assessment of cg05575921 methylation is an accurate method for assessing the presence and extent of cigarette consumption in adult subjects. We suggest that skillful clinical implementation of this approach alone or in combination with other assessment methods could lead to substantial reduction of cigarette consumption related morbidity and mortality. PMID:29740475

Analysis of Solar Census Remote Solar Access Value Calculation Methodology

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

Nangle, J.; Dean, J.; Van Geet, O.

2015-03-01

The costs of photovoltaic (PV) system hardware (PV panels, inverters, racking, etc.) have fallen dramatically over the past few years. Nonhardware (soft) costs, however, have failed to keep pace with the decrease in hardware costs, and soft costs have become a major driver of U.S. PV system prices. Upfront or 'sunken' customer acquisition costs make up a portion of an installation's soft costs and can be addressed through software solutions that aim to streamline sales and system design aspects of customer acquisition. One of the key soft costs associated with sales and system design is collecting information on solar accessmore » for a particular site. Solar access, reported in solar access values (SAVs), is a measurement of the available clear sky over a site and is used to characterize the impacts of local shading objects. Historically, onsite shading studies have been required to characterize the SAV of the proposed array and determine the potential energy production of a photovoltaic system.« less

Copy number variations in Saudi family with intellectual disability and epilepsy.

PubMed

Naseer, Muhammad I; Chaudhary, Adeel G; Rasool, Mahmood; Kalamegam, Gauthaman; Ashgan, Fai T; Assidi, Mourad; Ahmed, Farid; Ansari, Shakeel A; Zaidi, Syed Kashif; Jan, Mohammed M; Al-Qahtani, Mohammad H

2016-10-17

Epilepsy is genetically complex but common brain disorder of the world affecting millions of people with almost of all age groups. Novel Copy number variations (CNVs) are considered as important reason for the numerous neurodevelopmental disorders along with intellectual disability and epilepsy. DNA array based studies contribute to explain a more severe clinical presentation of the disease but interoperation of many detected CNVs are still challenging. In order to study novel CNVs with epilepsy related genes in Saudi family with six affected and two normal individuals with several forms of epileptic seizures, intellectual disability (ID), and minor dysmorphism, we performed the high density whole genome Agilent sure print G3 Hmn CGH 2x 400 K array-CGH chips analysis. Our results showed de novo deletions, duplications and deletion plus duplication on differential chromosomal regions in the affected individuals that were not shown in the normal fathe and normal kids by using Agilent CytoGenomics 3.0.6.6 softwear. Copy number gain were observed in the chromosome 1, 16 and 22 with LCE3C, HPR, GSTT2, GSTTP2, DDT and DDTL genes respectively whereas the deletions observed in the chromosomal regions 8p23-p21 (4303127-4337759) and the potential gene in this region is CSMD1 (OMIM: 612279). Moreover, the array CGH results deletions and duplication were also validated by using primer design of deleted regions utilizing the flanked SNPs using simple PCR and also by using quantitative real time PCR. We found some of the de novo deletions and duplication in our study in Saudi family with intellectual disability and epilepsy. Our results suggest that array-CGH should be used as a first line of genetic test for epilepsy except there is a strong indication for a monogenic syndrome. The advanced high through put array-CGH technique used in this study aim to collect the data base and to identify new mechanisms describing epileptic disorder, may help to improve the clinical management of individual cases in decreasing the burden of epilepsy in Saudi Arabia.

Comparison of global brain gene expression profiles between inbred long-sleep and inbred short-sleep mice by high-density gene array hybridization.

PubMed

Xu, Y; Ehringer, M; Yang, F; Sikela, J M

2001-06-01

Inbred long-sleep (ILS) and short-sleep (ISS) mice show significant central nervous system-mediated differences in sleep time for sedative dose of ethanol and are frequently used as a rodent model for ethanol sensitivity. In this study, we have used complementary DNA (cDNA) array hybridization methodology to identify genes that are differentially expressed between the brains of ILS and ISS mice. To carry out this analysis, we used both the gene discovery array (GDA) and the Mouse GEM 1 Microarray. GDA consists of 18,378 nonredundant mouse cDNA clones on a single nylon filter. Complex probes were prepared from total brain mRNA of ILS or ISS mice by using reverse transcription and 33P labeling. The labeled probes were hybridized in parallel to the gene array filters. Data from GDA experiments were analyzed with SQL-Plus and Oracle 8. The GEM microarray includes 8,730 sequence-verified clones on a glass chip. Two fluorescently labeled probes were used to hybridize a microarray simultaneously. Data from GEM experiments were analyzed by using the GEMTools software package (Incyte). Differentially expressed genes identified from each method were confirmed by relative quantitative reverse transcription-polymerase chain reaction (RT-PCR). A total of 41 genes or expressed sequence tags (ESTs) display significant expression level differences between brains of ILS and ISS mice after GDA, GEM1 hybridization, and quantitative RT-PCR confirmation. Among them, 18 clones were expressed higher in ILS mice, and 23 clones were expressed higher in ISS mice. The individual gene or EST's function and mapping information have been analyzed. This study identified 41 genes that are differentially expressed between brains of ILS and ISS mice. Some of them may have biological relevance in mediation of phenotypic variation between ILS and ISS mice for ethanol sensitivity. This study also demonstrates that parallel gene expression comparison with high-density cDNA arrays is a rapid and efficient way to discover potential genes and pathways involved in alcoholism and alcohol-related physiologic processes.

Serum Autoantibodies in Chronic Prostate Inflammation in Prostate Cancer Patients.

PubMed

Schlick, Bettina; Massoner, Petra; Lueking, Angelika; Charoentong, Pornpimol; Blattner, Mirjam; Schaefer, Georg; Marquart, Klaus; Theek, Carmen; Amersdorfer, Peter; Zielinski, Dirk; Kirchner, Matthias; Trajanoski, Zlatko; Rubin, Mark A; Müllner, Stefan; Schulz-Knappe, Peter; Klocker, Helmut

2016-01-01

Chronic inflammation is frequently observed on histological analysis of malignant and non-malignant prostate specimens. It is a suspected supporting factor for prostate diseases and their progression and a main cause of false positive PSA tests in cancer screening. We hypothesized that inflammation induces autoantibodies, which may be useful biomarkers. We aimed to identify and validate prostate inflammation associated serum autoantibodies in prostate cancer patients and evaluate the expression of corresponding autoantigens. Radical prostatectomy specimens of prostate cancer patients (N = 70) were classified into high and low inflammation groups according to the amount of tissue infiltrating lymphocytes. The corresponding pre-surgery blood serum samples were scrutinized for autoantibodies using a low-density protein array. Selected autoantigens were identified in prostate tissue and their expression pattern analyzed by immunohistochemistry and qPCR. The identified autoantibody profile was cross-checked in an independent sample set (N = 63) using the Luminex-bead protein array technology. Protein array screening identified 165 autoantibodies differentially abundant in the serum of high compared to low inflammation patients. The expression pattern of three corresponding antigens were established in benign and cancer tissue by immunohistochemistry and qPCR: SPAST (Spastin), STX18 (Syntaxin 18) and SPOP (speckle-type POZ protein). Of these, SPAST was significantly increased in prostate tissue with high inflammation. All three autoantigens were differentially expressed in primary and/or castration resistant prostate tumors when analyzed in an inflammation-independent tissue microarray. Cross-validation of the inflammation autoantibody profile on an independent sample set using a Luminex-bead protein array, retrieved 51 of the significantly discriminating autoantibodies. Three autoantibodies were significantly upregulated in both screens, MUT, RAB11B and CSRP2 (p>0.05), two, SPOP and ZNF671, close to statistical significance (p = 0.051 and 0.076). We provide evidence of an inflammation-specific autoantibody profile and confirm the expression of corresponding autoantigens in prostate tissue. This supports evaluation of autoantibodies as non-invasive markers for prostate inflammation.

Serum Autoantibodies in Chronic Prostate Inflammation in Prostate Cancer Patients

PubMed Central

Schlick, Bettina; Massoner, Petra; Lueking, Angelika; Charoentong, Pornpimol; Blattner, Mirjam; Schaefer, Georg; Marquart, Klaus; Theek, Carmen; Amersdorfer, Peter; Zielinski, Dirk; Kirchner, Matthias; Trajanoski, Zlatko; Rubin, Mark A.; Müllner, Stefan; Schulz-Knappe, Peter; Klocker, Helmut

2016-01-01

Background Chronic inflammation is frequently observed on histological analysis of malignant and non-malignant prostate specimens. It is a suspected supporting factor for prostate diseases and their progression and a main cause of false positive PSA tests in cancer screening. We hypothesized that inflammation induces autoantibodies, which may be useful biomarkers. We aimed to identify and validate prostate inflammation associated serum autoantibodies in prostate cancer patients and evaluate the expression of corresponding autoantigens. Methods Radical prostatectomy specimens of prostate cancer patients (N = 70) were classified into high and low inflammation groups according to the amount of tissue infiltrating lymphocytes. The corresponding pre-surgery blood serum samples were scrutinized for autoantibodies using a low-density protein array. Selected autoantigens were identified in prostate tissue and their expression pattern analyzed by immunohistochemistry and qPCR. The identified autoantibody profile was cross-checked in an independent sample set (N = 63) using the Luminex-bead protein array technology. Results Protein array screening identified 165 autoantibodies differentially abundant in the serum of high compared to low inflammation patients. The expression pattern of three corresponding antigens were established in benign and cancer tissue by immunohistochemistry and qPCR: SPAST (Spastin), STX18 (Syntaxin 18) and SPOP (speckle-type POZ protein). Of these, SPAST was significantly increased in prostate tissue with high inflammation. All three autoantigens were differentially expressed in primary and/or castration resistant prostate tumors when analyzed in an inflammation-independent tissue microarray. Cross-validation of the inflammation autoantibody profile on an independent sample set using a Luminex-bead protein array, retrieved 51 of the significantly discriminating autoantibodies. Three autoantibodies were significantly upregulated in both screens, MUT, RAB11B and CSRP2 (p>0.05), two, SPOP and ZNF671, close to statistical significance (p = 0.051 and 0.076). Conclusions We provide evidence of an inflammation-specific autoantibody profile and confirm the expression of corresponding autoantigens in prostate tissue. This supports evaluation of autoantibodies as non-invasive markers for prostate inflammation. PMID:26863016

Generation of Custom DSP Transform IP Cores: Case Study Walsh-Hadamard Transform

DTIC Science & Technology

2002-09-01

mathematics and hardware design What I know: Finite state machine Pipelining Systolic array … What I know: Linear algebra Digital signal processing...state machine Pipelining Systolic array … What I know: Linear algebra Digital signal processing Adaptive filter theory … A math guy A hardware engineer...Synthesis Technology Libary Bit-width (8) HF factor (1,2,3,6) VF factor (1,2,4, ... 32) Xilinx FPGA Place&Route Xilinx FPGA Place&Route Performance

High Speed Imaging using Nanoprobe Arrays

DTIC Science & Technology

2010-06-23

Gotsmann and U. Dürig, Appl. Phys. Lett. 87, 194102 2005. 9 W. P. King, S. Saxena, B. A. Nelson, R. Pitchimani, and B. L. Weeks, Nano Lett. 6, 2145...microcantilevers with selective coatings has been applied as an artificial nose to recognize and characterize alcohol vapors either in a static mode...doped resistive heater. Fig. 4(c) shows a custom printed circuit board (PCB) to mount the array chip and a flexible ribbon cable for the electrical

SU-F-P-49: Comparison of Mapcheck 2 Commission for Photon and Electron Beams

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

Lu, J; Yang, C; Morris, B

2016-06-15

Purpose: We will investigate the performance variation of the MapCheck2 detector array with different array calibration and dose calibration pairs from different radiation therapy machine. Methods: A MapCheck2 detector array was calibrated on 3 Elekta accelerators with different energy of photon (6 MV, 10 MV, 15 MV and 18 MV) and electron (6 MeV, 9 MeV, 12 MeV, 15 MeV, 18 MeV and 20 MeV) beams. Dose calibration was conducted by referring a water phantom measurement following TG-51 protocol and commission data for each accelerator. A 10 cm × 10 cm beam was measured. This measured map was morphed bymore » applying different calibration pairs. Then the difference was quantified by comparing the doses and similarity using gamma analysis of criteria (0.5 %, 0 mm). Profile variation was evaluated on a same dataset with different calibration pairs. The passing rate of an IMRT QA planar dose was calculated by using 3 mm and 3% criteria and compared with respect to each calibration pairs. Results: In this study, a dose variation up to 0.67% for matched photons and 1.0% for electron beams is observed. Differences of flatness and symmetry can be as high as 1% and 0.7% respectively. Gamma analysis shows a passing rate ranging from 34% to 85% for the standard 10 × 10 cm field. Conclusion: Our work demonstrated that a customized array calibration and dose calibration for each machine is preferred to fulfill a high standard patient QA task.« less

Dose-response relationships in gene expression profiles in rainbow trout, Oncorhyncus mykiss, exposed to ethynylestradiol.

PubMed

Hook, Sharon E; Skillman, Ann D; Small, Jack A; Schultz, Irvin R

2006-07-01

Determining how gene expression profiles change with toxicant dose will improve the utility of arrays in identifying biomarkers and modes of toxic action. Isogenic rainbow trout, Oncorhyncus mykiss,were exposed to 10, 50 or 100 ng/L ethynylestradiol (a xeno-estrogen) for 7 days. Following exposure hepatic RNA was extracted. Fluorescently labeled cDNA were generated and hybridized against a commercially available Atlantic Salmon/Trout array (GRASP project, University of Victoria) spotted with 16,000 cDNAs. Transcript expression in treated vs control fish was analyzed via Genespring (Silicon Genetics) to identify genes with altered expression, as well as to determine gene clustering patterns that can be used as "expression signatures". Array results were confirmed via qRT PCR. Our analysis indicates that gene expression profiles varied somewhat with dose. Established biomarkers of exposure to estrogenic chemicals, such as vitellogenin, vitelline envelope proteins, and the estrogen receptor alpha, were induced at every dose. Other genes were dose specific, suggesting that different doses induce distinct physiological responses. These findings demonstrate that cDNA microarrays could be used to identify both toxicant class and relative dose.

A Hermetic Wireless Subretinal Neurostimulator for Vision Prostheses

PubMed Central

Shire, Douglas B.; Chen, Jinghua; Doyle, Patrick; Gingerich, Marcus D.; Cogan, Stuart F.; Drohan, William A.; Behan, Sonny; Theogarajan, Luke; Wyatt, John L.; Rizzo, Joseph F.

2016-01-01

A miniaturized, hermetically encased, wirelessly operated retinal prosthesis has been developed for preclinical studies in the Yucatan minipig, and includes several design improvements over our previously reported device. The prosthesis attaches conformally to the outside of the eye and electrically drives a microfabricated thin-film polyimide array of sputtered iridium oxide film electrodes. This array is implanted into the subretinal space using a customized ab externo surgical technique. The implanted device includes a hermetic titanium case containing a 15-channel stimulator chip and discrete circuit components. Feedthroughs in the case connect the stimulator chip to secondary power and data receiving coils on the eye and to the electrode array under the retina. Long-term in vitro pulse testing of the electrodes projected a lifetime consistent with typical devices in industry. The final assembly was tested in vitro to verify wireless operation of the system in physiological saline using a custom RF transmitter and primary coils. Stimulation pulse strength, duration, and frequency were programmed wirelessly from a Peripheral Component Interconnect eXtensions for Instrumentation (PXI) computer. Operation of the retinal implant has been verified in two pigs for up to five and a half months by detecting stimulus artifacts generated by the implanted device. PMID:21859595

Web-based NGS data analysis using miRMaster: a large-scale meta-analysis of human miRNAs.

PubMed

Fehlmann, Tobias; Backes, Christina; Kahraman, Mustafa; Haas, Jan; Ludwig, Nicole; Posch, Andreas E; Würstle, Maximilian L; Hübenthal, Matthias; Franke, Andre; Meder, Benjamin; Meese, Eckart; Keller, Andreas

2017-09-06

The analysis of small RNA NGS data together with the discovery of new small RNAs is among the foremost challenges in life science. For the analysis of raw high-throughput sequencing data we implemented the fast, accurate and comprehensive web-based tool miRMaster. Our toolbox provides a wide range of modules for quantification of miRNAs and other non-coding RNAs, discovering new miRNAs, isomiRs, mutations, exogenous RNAs and motifs. Use-cases comprising hundreds of samples are processed in less than 5 h with an accuracy of 99.4%. An integrative analysis of small RNAs from 1836 data sets (20 billion reads) indicated that context-specific miRNAs (e.g. miRNAs present only in one or few different tissues / cell types) still remain to be discovered while broadly expressed miRNAs appear to be largely known. In total, our analysis of known and novel miRNAs indicated nearly 22 000 candidates of precursors with one or two mature forms. Based on these, we designed a custom microarray comprising 11 872 potential mature miRNAs to assess the quality of our prediction. MiRMaster is a convenient-to-use tool for the comprehensive and fast analysis of miRNA NGS data. In addition, our predicted miRNA candidates provided as custom array will allow researchers to perform in depth validation of candidates interesting to them. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Prenatal Diagnosis of DNA Copy Number Variations by Genomic Single-Nucleotide Polymorphism Array in Fetuses with Congenital Heart Defects.

PubMed

Tang, Shaohua; Lv, Jiaojiao; Chen, Xiangnan; Bai, Lili; Li, Huanzheng; Chen, Chong; Wang, Ping; Xu, Xueqin; Lu, Jianxin

2016-01-01

To evaluate the usefulness of single-nucleotide polymorphism (SNP) array for prenatal genetic diagnosis of congenital heart defect (CHD), we used this approach to detect clinically significant copy number variants (CNVs) in fetuses with CHDs. A HumanCytoSNP-12 array was used to detect genomic samples obtained from 39 fetuses that exhibited cardiovascular abnormalities on ultrasound and had a normal karyotype. The relationship between CNVs and CHDs was identified by using genotype-phenotype comparisons and searching of chromosomal databases. All clinically significant CNVs were confirmed by real-time PCR. CNVs were detected in 38/39 (97.4%) fetuses: variants of unknown significance were detected in 2/39 (5.1%), and clinically significant CNVs were identified in 7/39 (17.9%). In 3 of the 7 fetuses with clinically significant CNVs, 3 rare and previously undescribed CNVs were detected, and these CNVs encompassed the CHD candidate genes FLNA (Xq28 dup), BCOR (Xp11.4 dup), and RBL2 (16q12.2 del). Compared with conventional cytogenetic genomics, SNP array analysis provides significantly improved detection of submicroscopic genomic aberrations in pregnancies with CHDs. Based on these results, we propose that genomic SNP array is an effective method which could be used in the prenatal diagnostic test to assist genetic counseling for pregnancies with CHDs. © 2015 S. Karger AG, Basel.

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

PubMed Central

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

2008-01-01

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

Optical Fiber Array Assemblies for Space Flight on the Lunar Reconnaissance Orbiter

NASA Technical Reports Server (NTRS)

Ott, Jelanie; Matuszeski, Adam

2011-01-01

Custom fiber optic bundle array assemblies developed by the Photonics Group at NASA Goddard Space Flight Center were an enabling technology for both the Lunar Orbiter Laser Altimeter (LOLA) and the Laser Ranging (LR) Investigation on the Lunar Reconnaissance Orbiter (LRO) currently in operation. The unique assembly array designs provided considerable decrease in size and weight and met stringent system level requirements. This is the first time optical fiber array bundle assemblies were used in a high performance space flight application. This innovation was achieved using customized Diamond Switzerland AVIM optical connectors. For LOLA, a five fiber array was developed for the receiver telescope to maintain precise alignment for each of the 200/220 micron optical fibers collecting 1,064 nm wavelength light being reflected back from the moon. The array splits to five separate detectors replacing the need for multiple telescopes. An image illustration of the LOLA instrument can be found at the top of the figure. For the laser ranging, a seven-optical-fiber array of 400/440 micron fibers was developed to transmit light from behind the LR receiver telescope located on the end of the high gain antenna system (HGAS). The bundle was routed across two moving gimbals, down the HGAS boom arm, over a deployable mandrel and across the spacecraft to a detector on the LOLA instrument. The routing of the optical fiber bundle and its end locations is identified in the figure. The Laser Ranging array and bundle is currently accepting light at a wavelength of 532 nm sent to the moon from laser stations at Greenbelt MD and other stations around the world to gather precision ranging information from the Earth to the LRO spacecraft. The LR bundle assembly is capable of withstanding temperatures down to -55 C at the connectors, and 20,000 mechanical gimbal cycles at temperatures as cold as -20 C along the length of the seven-fiber bundle (that is packaged into the gimbals). The total bundle assembly is 10 meters long with two interconnections requiring precise clocking of the seven-fiber array pattern.

A novel functional class 2 integron in clinical Proteus mirabilis isolates.

PubMed

Wei, Quhao; Hu, Qingfeng; Li, Shanshan; Lu, Huoyang; Chen, Guoqiang; Shen, Beiqiong; Zhang, Ping; Zhou, Yonglie

2014-04-01

To describe a novel functional class 2 integron that was found in clinical Proteus mirabilis isolates. Class 1 and 2 integrons were screened by PCR in 153 clinical Proteus isolates. The variable regions of class 1 and 2 integrons were determined by restriction analysis and sequencing. The mutations of internal stop codons in class 2 integrons and their common promoters were also determined by sequencing. Enterobacterial repetitive intergenic consensus (ERIC)-PCR was used to analyse the phylogenetic relations of class 2 integron-positive P. mirabilis isolates. Class 1 integrons were detected in 96 (63%) of 153 Proteus isolates: eight different gene cassette arrays were detected, including dfrA32-ereA1-aadA2, which was detected for the first time in P. mirabilis. Class 2 integrons were detected in 101 (66%) of 153 Proteus isolates: four different gene cassette arrays were detected, including dfrA1-catB2-sat2-aadA1, which was detected for the first time in a class 2 integron. A novel functional class 2 integron was detected in 38 P. mirabilis isolates with a common promoter (-35 TTTAAT|16 bp|-10 TAAAGT). The variable region of this functional class 2 integron contained dfrA14 and three novel open reading frames with unknown functions. Very similar ERIC-PCR fingerprinting patterns were detected in these 38 P. mirabilis isolates and were different from other class 2 integron-positive isolates. A novel functional class 2 integron was found for the first time in P. mirabilis. These functional class 2 integron-harbouring P. mirabilis isolates were likely to be clonally spread in our hospital.

Genome-Wide Survey of Cold Stress Regulated Alternative Splicing in Arabidopsis thaliana with Tiling Microarray

PubMed Central

Leviatan, Noam; Alkan, Noam; Leshkowitz, Dena; Fluhr, Robert

2013-01-01

Alternative splicing plays a major role in expanding the potential informational content of eukaryotic genomes. It is an important post-transcriptional regulatory mechanism that can increase protein diversity and affect mRNA stability. Alternative splicing is often regulated in a tissue-specific and stress-responsive manner. Cold stress, which adversely affects plant growth and development, regulates the transcription and splicing of plant splicing factors. This can affect the pre-mRNA processing of many genes. To identify cold regulated alternative splicing we applied Affymetrix Arabidopsis tiling arrays to survey the transcriptome under cold treatment conditions. A novel algorithm was used for detection of statistically relevant changes in intron expression within a transcript between control and cold growth conditions. A reverse transcription polymerase chain reaction (RT-PCR) analysis of a number of randomly selected genes confirmed the changes in splicing patterns under cold stress predicted by tiling array. Our analysis revealed new types of cold responsive genes. While their expression level remains relatively unchanged under cold stress their splicing pattern shows detectable changes in the relative abundance of isoforms. The majority of cold regulated alternative splicing introduced a premature termination codon (PTC) into the transcripts creating potential targets for degradation by the nonsense mediated mRNA decay (NMD) process. A number of these genes were analyzed in NMD-defective mutants by RT-PCR and shown to evade NMD. This may result in new and truncated proteins with altered functions or dominant negative effects. The results indicate that cold affects both quantitative and qualitative aspects of gene expression. PMID:23776682

Genome-wide and digital polymerase chain reaction epigenetic assessments of alcohol consumption.

PubMed

Philibert, Robert; Dogan, Meesha; Noel, Amanda; Miller, Shelly; Krukow, Brianna; Papworth, Emma; Cowley, Joseph; Knudsen, April; Beach, Steven R H; Black, Donald

2018-04-28

The lack of readily employable biomarkers of alcohol consumption is a problem for clinicians and researchers. In 2014, we published a preliminary DNA methylation signature of heavy alcohol consumption that remits as a function of abstinence. Herein, we present new genome-wide methylation findings from a cohort of additional subjects and a meta-analysis of the data. Using DNA from 47 consecutive heavy drinkers admitted for alcohol detoxification in the context of alcohol treatment and 47 abstinent controls, we replicate the 2014 results and show that 21,221 CpG residues are differentially methylated in active heavy drinkers. Meta-analysis of all data from the 448,058 probes common to the two methylation platforms shows a similarly profound signature with confirmation of findings from other groups. Principal components analyses show that genome-wide methylation changes in response to alcohol consumption load on two major factors with one component accounting at least 50% of the total variance in both smokers and nonsmoking alcoholics. Using data from the arrays, we derive a panel of five methylation probes that classifies use status with a receiver operator characteristic area under the curve (AUC) of 0.97. Finally, using droplet digital polymerase chain reaction (PCR), we convert these array-based findings to two marker assays with an AUC of 0.95 and a four marker set AUC of 0.98. We conclude that DNA methylation assessments are capable of quantifying alcohol use status and suggest that readily employable digital PCR approaches for substance consumption may find widespread use in alcohol-related research and patient care. © 2018 Wiley Periodicals, Inc.

High throughput ion-channel pharmacology: planar-array-based voltage clamp.

PubMed

Kiss, Laszlo; Bennett, Paul B; Uebele, Victor N; Koblan, Kenneth S; Kane, Stefanie A; Neagle, Brad; Schroeder, Kirk

2003-02-01

Technological advances often drive major breakthroughs in biology. Examples include PCR, automated DNA sequencing, confocal/single photon microscopy, AFM, and voltage/patch-clamp methods. The patch-clamp method, first described nearly 30 years ago, was a major technical achievement that permitted voltage-clamp analysis (membrane potential control) of ion channels in most cells and revealed a role for channels in unimagined areas. Because of the high information content, voltage clamp is the best way to study ion-channel function; however, throughput is too low for drug screening. Here we describe a novel breakthrough planar-array-based HT patch-clamp technology developed by Essen Instruments capable of voltage-clamping thousands of cells per day. This technology provides greater than two orders of magnitude increase in throughput compared with the traditional voltage-clamp techniques. We have applied this method to study the hERG K(+) channel and to determine the pharmacological profile of QT prolonging drugs.

Inflammatory peeling skin syndrome caused by homozygous genomic deletion in the PSORS1 region encompassing the CDSN gene.

PubMed

Ishida-Yamamoto, Akemi; Furio, Laetitia; Igawa, Satomi; Honma, Masaru; Tron, Elodie; Malan, Valerie; Murakami, Masamoto; Hovnanian, Alain

2014-01-01

Peeling skin syndrome (PSS) type B is a rare recessive genodermatosis characterized by lifelong widespread, reddish peeling of the skin with pruritus. The disease is caused by small-scale mutations in the Corneodesmosin gene (CDSN) leading to premature termination codons. We report for the first time a Japanese case resulting from complete deletion of CDSN. Corneodesmosin was undetectable in the epidermis, and CDSN was unamplifiable by PCR. QMPSF analysis demonstrated deletion of CDSN exons inherited from each parent. Deletion mapping using microsatellite haplotyping, CGH array and PCR analysis established that the genomic deletion spanned 49-72 kb between HCG22 and TCF19, removing CDSN as well as five other genes within the psoriasis susceptibility region 1 (PSORS1) on 6p21.33. This observation widens the spectrum of molecular defects underlying PSS type B and shows that loss of these five genes from the PSORS1 region does not result in an additional cutaneous phenotype. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A Double-Blinded Randomized Study Investigating a Possible Anti-Inflammatory Effect of Saxagliptin versus Placebo as Add-On Therapy in Patients with Both Type 2 Diabetes And Stable Coronary Artery Disease

PubMed Central

Åkra, Sissel; Weiss, Thomas W.; Øvstebø, Reidun; Aass, Hans Christian D.; Byrkjeland, Rune; Arnesen, Harald

2017-01-01

Background Promising results regarding potential anti-inflammatory and antiatherosclerotic effects of gliptins have been reported. Our aim was to investigate whether saxagliptin treatment modifies expression of inflammatory markers, primarily in peripheral blood mononuclear cells (PBMCs) and in circulating leukocytes in patients with stable coronary artery disease (CAD) and T2DM. Methods Patients (n = 12) were randomized to saxagliptin 5 mg daily or placebo for 3 months. Samples were taken at baseline and end of study in fasting state prior to intake of medications. PBMCs were isolated and cryopreserved at −150°C until ex vivo exposed to 1 ng/mL of lipopolysaccharide (LPS) for 4 hours. Gene expression was performed with custom-designed TaqMan® Arrays and relative quantification by real-time PCR (RT-qPCR). Results HbA1c was reduced in the saxagliptin-treated group compared to that in the change with placebo (p = 0.042). In unstimulated PBMCs and in circulating leukocytes, we observed a significant increase in IL-10 expression in the saxagliptin group (p = 0.043, both), significantly different from that in the placebo (p = 0.009 and p = 0.032, resp.). No between group differences in changes were observed in any of the selected proinflammatory markers. Conclusion In our small cohort of patients with combined T2DM and CAD, a possible anti-inflammatory effect of saxagliptin, observed in the present study by upregulation of IL-10 in leukocytes, needs to be confirmed in larger studies. PMID:28596642

The effects of whole ovarian perfusion and cryopreservation on endothelial cell-related gene expression in the ovarian medulla and pedicle.

PubMed

Onions, V J; Webb, R; Pincott-Allen, C; Picton, H M; Campbell, B K

2013-04-01

Fertility preservation by whole ovarian cryopreservation requires successful cryopreservation of both the ovary and its vascular supply. Previous work has indicated detrimental effects of both perfusion and cryopreservation on the ovarian vasculature. This study assessed the effects of blood perfusion, alone or in combination with cryopreservation, on functional effects in the follicle population and ovarian function in vivo following short-term autotransplantation of the tissue after vascular reanastomosis and measured acute changes in endothelial cell-related gene expression within the ovarian medulla and pedicle. Following autotransplantation for 7 days, primordial, transitional and primary follicle densities were significantly reduced (P < 0.05) and stromal Ki67 and caspase-3 expression significantly increased (P < 0.05) in cryopreserved but not fresh or perfused whole ovaries. There was evidence of clot formation and fluorescent microsphere (FMS) extravasation in the medulla of all cryopreserved ovaries, indicating vascular damage. Utilizing a customized RT-PCR array or conventional RT-PCR, we found that perfusion alone resulted in down-regulation in the expression of caspase 6 and thrombospondin 1 (THBS1) genes in the medulla. Following additional cryopreservation, endothelial nitric oxide synthase (eNOS), endothelin 1, endothelin receptor A and Bcl-2 expression were significantly (P < 0.05) down-regulated. In the pedicle, both perfusion and cryopreservation caused a (P < 0.05) down-regulation of eNOS and THBS1, and an up-regulation in Bax expression. Perfusion also caused a down-regulation of TNF and up-regulation of endothelin-2 expression (P < 0.05). In conclusion, this study has identified a number of endothelial cell-related genes expressed in the medulla which are acutely affected by both cryopreservation and perfusion, supporting the hypothesis that both interventions have deleterious effects on endothelial cell function.

Taking advantage of modern turbines

NASA Astrophysics Data System (ADS)

Thresher, Robert

2018-06-01

Wind facilities have generally deployed turbines of the same power and height in regular uniform arrays. Now, the modern generation of turbines, with customer-selectable tower heights and larger rotors, can significantly increase wind energy's economic potential using less land to generate cheaper electricity.

Molecular identification of common Salmonella serovars using multiplex DNA sensor-based suspension array.

PubMed

Aydin, Muhsin; Carter-Conger, Jacqueline; Gao, Ning; Gilmore, David F; Ricke, Steven C; Ahn, Soohyoun

2018-04-01

Salmonella is one of major foodborne pathogens and the leading cause of foodborne illness-related hospitalizations and deaths. It is critical to develop a sensitive and rapid detection assay that can identify Salmonella to ensure food safety. In this study, a DNA sensor-based suspension array system of high multiplexing ability was developed to identify eight Salmonella serovars commonly associated with foodborne outbreaks to the serotype level. Each DNA sensor was prepared by activating pre-encoded microspheres with oligonucleotide probes that are targeting virulence genes and serovar-specific regions. The mixture of 12 different types of DNA sensors were loaded into a 96-well microplate and used as a 12-plex DNA sensor array platform. DNA isolated from Salmonella was amplified by multiplex polymerase chain reaction (mPCR), and the presence of Salmonella was determined by reading fluorescent signals from hybridization between probes on DNA sensors and fluorescently labeled target DNA using the Bio-Plex® system. The developed multiplex array was able to detect synthetic DNA at the concentration as low as 100 fM and various Salmonella serovars as low as 100 CFU/mL within 1 h post-PCR. Sensitivity of this assay was further improved to 1 CFU/mL with 6-h enrichment. The array system also correctly and specifically identified serotype of tested Salmonella strains without any cross-reactivity with other common foodborne pathogens. Our results indicate the developed DNA sensor suspension array can be a rapid and reliable high-throughput method for simultaneous detection and molecular identification of common Salmonella serotypes.

Array-based comparative genomic hybridization-guided identification of reference genes for normalization of real-time quantitative polymerase chain reaction assay data for lymphomas, histiocytic sarcomas, and osteosarcomas of dogs.

PubMed

Tsai, Pei-Chien; Breen, Matthew

2012-09-01

To identify suitable reference genes for normalization of real-time quantitative PCR (RT-qPCR) assay data for common tumors of dogs. Malignant lymph node (n = 8), appendicular osteosarcoma (9), and histiocytic sarcoma (12) samples and control samples of various nonneoplastic canine tissues. Array-based comparative genomic hybridization (aCGH) data were used to guide selection of 9 candidate reference genes. Expression stability of candidate reference genes and 4 commonly used reference genes was determined for tumor samples with RT-qPCR assays and 3 software programs. LOC611555 was the candidate reference gene with the highest expression stability among the 3 tumor types. Of the commonly used reference genes, expression stability of HPRT was high in histiocytic sarcoma samples, and expression stability of Ubi and RPL32 was high in osteosarcoma samples. Some of the candidate reference genes had higher expression stability than did the commonly used reference genes. Data for constitutively expressed genes with high expression stability are required for normalization of RT-qPCR assay results. Without such data, accurate quantification of gene expression in tumor tissue samples is difficult. Results of the present study indicated LOC611555 may be a useful RT-qPCR assay reference gene for multiple tissue types. Some commonly used reference genes may be suitable for normalization of gene expression data for tumors of dogs, such as lymphomas, osteosarcomas, or histiocytic sarcomas.

Detection of Neisseria meningitidis in cerebrospinal fluid using a multiplex PCR and the Luminex detection technology.

PubMed

Møller, Jens Kjølseth

2012-01-01

Rapid clinical and laboratory diagnoses are the foundation for a successful management of serious infections with Neisseria meningitidis. A species-specific multiplex polymerase chain reaction (PCR) coupled with fluidic microarrays using microbeads (the Luminex xMAP™ Technology) can detect pathogens most frequently found in the cerebrospinal fluid of patients. The Luminex suspension array system uniquely combines flow cytometry, microspheres, laser technology, digital signal processing, and traditional chemistry. In this method, the reaction is carried out in one vessel, in which distinctly color-coded bead sets, each conjugated with a different specific nucleic acid reactant, are hybridized with the PCR products, and a reporter molecule is used to quantify the interaction. The flow-based Luminex array reader identifies each reaction (bead set) after excitation by a red classification laser. Reporter signals from each reaction are simultaneously quantified by fluorescence generated by a green reporter laser. This nonculture, multiplex assay may prove to be an important tool for optimal laboratory diagnosis, not only of meningococcal meningitis, but also of meningitis caused by other bacterial or viral pathogens.

Multistage WDM access architecture employing cascaded AWGs

NASA Astrophysics Data System (ADS)

El-Nahal, F. I.; Mears, R. J.

2009-03-01

Here we propose passive/active arrayed waveguide gratings (AWGs) with enhanced performance for system applications mainly in novel access architectures employing cascaded AWG technology. Two technologies were considered to achieve space wavelength switching in these networks. Firstly, a passive AWG with semiconductor optical amplifiers array, and secondly, an active AWG. Active AWG is an AWG with an array of phase modulators on its arrayed-waveguides section, where a programmable linear phase-profile or a phase hologram is applied across the arrayed-waveguide section. This results in a wavelength shift at the output section of the AWG. These architectures can address up to 6912 customers employing only 24 wavelengths, coarsely separated by 1.6 nm. Simulation results obtained here demonstrate that cascaded AWGs access architectures have a great potential in future local area networks. Furthermore, they indicate for the first time that active AWGs architectures are more efficient in routing signals to the destination optical network units than passive AWG architectures.

Development of a 35-MHz piezo-composite ultrasound array for medical imaging.

PubMed

Cannata, Jonathan M; Williams, Jay A; Zhou, Qifa; Ritter, Timothy A; Shung, K Kirk

2006-01-01

This paper discusses the development of a 64-element 35-MHz composite ultrasonic array. This array was designed primarily for ocular imaging applications, and features 2-2 composite elements mechanically diced out of a fine-grain high-density Navy Type VI ceramic. Array elements were spaced at a 50-micron pitch, interconnected via a custom flexible circuit and matched to the 50-ohm system electronics via a 75-ohm transmission line coaxial cable. Elevation focusing was achieved using a cylindrically shaped epoxy lens. One functional 64-element array was fabricated and tested. Bandwidths averaging 55%, 23-dB insertion loss, and crosstalk less than -24 dB were measured. An image of a tungsten wire target phantom was acquired using a synthetic aperture reconstruction algorithm. The results from this imaging test demonstrate resolution exceeding 50 microm axially and 100 microm laterally.

Clinical prototype of a plastic water-equivalent scintillating fiber dosimeter array for QA applications.

PubMed

Lacroix, Fréderic; Archambault, Louis; Gingras, Luc; Guillot, Mathieu; Beddar, A Sam; Beaulieu, Luc

2008-08-01

A clinical prototype of a scintillating fiber dosimeter array for quality assurance applications is presented. The array consists of a linear array of 29 plastic scintillation detectors embedded in a water-equivalent plastic sheet coupled to optical fibers used to guide optical photons to a charge coupled device (CCD) camera. The CCD is packaged in a light-tight, radiation-shielded housing designed for convenient transport. A custom designed connector is used to ensure reproducible mechanical positioning of the optical fibers relative to the CCD. Profile and depth dose characterization measurements are presented and show that the prototype provides excellent dose measurement reproducibility (+/-0.8%) in-field and good accuracy (+/-1.6% maximum deviation) relative to the dose measured with an IC10 ionization chamber.

Real-time, multiplexed electrochemical DNA detection using an active complementary metal-oxide-semiconductor biosensor array with integrated sensor electronics.

PubMed

Levine, Peter M; Gong, Ping; Levicky, Rastislav; Shepard, Kenneth L

2009-03-15

Optical biosensing based on fluorescence detection has arguably become the standard technique for quantifying extents of hybridization between surface-immobilized probes and fluorophore-labeled analyte targets in DNA microarrays. However, electrochemical detection techniques are emerging which could eliminate the need for physically bulky optical instrumentation, enabling the design of portable devices for point-of-care applications. Unlike fluorescence detection, which can function well using a passive substrate (one without integrated electronics), multiplexed electrochemical detection requires an electronically active substrate to analyze each array site and benefits from the addition of integrated electronic instrumentation to further reduce platform size and eliminate the electromagnetic interference that can result from bringing non-amplified signals off chip. We report on an active electrochemical biosensor array, constructed with a standard complementary metal-oxide-semiconductor (CMOS) technology, to perform quantitative DNA hybridization detection on chip using targets conjugated with ferrocene redox labels. A 4 x 4 array of gold working electrodes and integrated potentiostat electronics, consisting of control amplifiers and current-input analog-to-digital converters, on a custom-designed 5 mm x 3 mm CMOS chip drive redox reactions using cyclic voltammetry, sense DNA binding, and transmit digital data off chip for analysis. We demonstrate multiplexed and specific detection of DNA targets as well as real-time monitoring of hybridization, a task that is difficult, if not impossible, with traditional fluorescence-based microarrays.

KSC-00pp1734

NASA Image and Video Library

2000-11-14

KENNEDY SPACE CENTER, FLA. -- On Launch Pad 39B, the payload transport canister, with the P6 integrated truss segment inside, moves higher toward the payload changeout room (PCR). The PCR is the enclosed, environmentally controlled portion of the Rotating Service Structure (RSS) (at left) that supports payload delivery at the pad. At right is Space Shuttle Endeavour with its orange external tank and solid rocket boosters showing behind it. When the RSS is closed around Endeavour, the P6 truss will be able to be moved into the orbiter’s payload bay. The P6, payload on mission STS-97, comprises Solar Array Wing-3 and the Integrated Electronic Assembly, to be installed on the International Space Station. The Station’s electrical power system will use eight photovoltaic solar arrays, each 112 feet long by 39 feet wide, to convert sunlight to electricity. The solar arrays are mounted on a “blanket” that can be folded like an accordion for delivery. Once in orbit, astronauts will deploy the blankets to their full size. Gimbals will be used to rotate the arrays so that they will face the Sun to provide maximum power to the Space Station. Launch of STS-97 is scheduled for Nov. 30 at 10:06 p.m. EST

KSC00pp1734

NASA Image and Video Library

2000-11-14

KENNEDY SPACE CENTER, FLA. -- On Launch Pad 39B, the payload transport canister, with the P6 integrated truss segment inside, moves higher toward the payload changeout room (PCR). The PCR is the enclosed, environmentally controlled portion of the Rotating Service Structure (RSS) (at left) that supports payload delivery at the pad. At right is Space Shuttle Endeavour with its orange external tank and solid rocket boosters showing behind it. When the RSS is closed around Endeavour, the P6 truss will be able to be moved into the orbiter’s payload bay. The P6, payload on mission STS-97, comprises Solar Array Wing-3 and the Integrated Electronic Assembly, to be installed on the International Space Station. The Station’s electrical power system will use eight photovoltaic solar arrays, each 112 feet long by 39 feet wide, to convert sunlight to electricity. The solar arrays are mounted on a “blanket” that can be folded like an accordion for delivery. Once in orbit, astronauts will deploy the blankets to their full size. Gimbals will be used to rotate the arrays so that they will face the Sun to provide maximum power to the Space Station. Launch of STS-97 is scheduled for Nov. 30 at 10:06 p.m. EST

KSC00pp1735

NASA Image and Video Library

2000-11-14

KENNEDY SPACE CENTER, FLA. -- On Launch Pad 39B, the payload transport canister, with the P6 integrated truss segment inside, is lifted higher toward the payload changeout room (PCR) above it. The PCR is the enclosed, environmentally controlled portion of the Rotating Service Structure (RSS) (at left) that supports payload delivery at the pad. At right is Space Shuttle Endeavour with its orange external tank and one solid rocket booster showing behind it. When the RSS is closed around Endeavour, the P6 truss will be moved into the orbiter’s payload bay. The P6, payload on mission STS-97, comprises Solar Array Wing-3 and the Integrated Electronic Assembly, to be installed on the International Space Station. The Station’s electrical power system will use eight photovoltaic solar arrays, each 112 feet long by 39 feet wide, to convert sunlight to electricity. The solar arrays are mounted on a “blanket” that can be folded like an accordion for delivery. Once in orbit, astronauts will deploy the blankets to their full size. Gimbals will be used to rotate the arrays so that they will face the Sun to provide maximum power to the Space Station. Launch of STS-97 is scheduled for Nov. 30 at 10:06 p.m. EST

KSC-00pp1733

NASA Image and Video Library

2000-11-14

KENNEDY SPACE CENTER, FLA. -- On Launch Pad 39B, the payload transport canister, with the P6 integrated truss segment inside, is lifted toward the payload changeout room (PCR). The PCR is the enclosed, environmentally controlled portion of the Rotating Service Structure (RSS) (on the left) that supports payload delivery at the pad. At right is Space Shuttle Endeavour with its orange external tank and one solid rocket booster showing behind it. When the RSS is closed around Endeavour, the P6 truss will be able to be moved into the orbiter’s payload bay. The P6, payload on mission STS-97, comprises Solar Array Wing-3 and the Integrated Electronic Assembly, to be installed on the International Space Station. The Station’s electrical power system will use eight photovoltaic solar arrays, each 112 feet long by 39 feet wide, to convert sunlight to electricity. The solar arrays are mounted on a “blanket” that can be folded like an accordion for delivery. Once in orbit, astronauts will deploy the blankets to their full size. Gimbals will be used to rotate the arrays so that they will face the Sun to provide maximum power to the Space Station. Launch of STS-97 is scheduled for Nov. 30 at 10:06 p.m. EST

KSC00pp1733

NASA Image and Video Library

2000-11-14

KENNEDY SPACE CENTER, FLA. -- On Launch Pad 39B, the payload transport canister, with the P6 integrated truss segment inside, is lifted toward the payload changeout room (PCR). The PCR is the enclosed, environmentally controlled portion of the Rotating Service Structure (RSS) (on the left) that supports payload delivery at the pad. At right is Space Shuttle Endeavour with its orange external tank and one solid rocket booster showing behind it. When the RSS is closed around Endeavour, the P6 truss will be able to be moved into the orbiter’s payload bay. The P6, payload on mission STS-97, comprises Solar Array Wing-3 and the Integrated Electronic Assembly, to be installed on the International Space Station. The Station’s electrical power system will use eight photovoltaic solar arrays, each 112 feet long by 39 feet wide, to convert sunlight to electricity. The solar arrays are mounted on a “blanket” that can be folded like an accordion for delivery. Once in orbit, astronauts will deploy the blankets to their full size. Gimbals will be used to rotate the arrays so that they will face the Sun to provide maximum power to the Space Station. Launch of STS-97 is scheduled for Nov. 30 at 10:06 p.m. EST

KSC-00pp1735

NASA Image and Video Library

2000-11-14

KENNEDY SPACE CENTER, FLA. -- On Launch Pad 39B, the payload transport canister, with the P6 integrated truss segment inside, is lifted higher toward the payload changeout room (PCR) above it. The PCR is the enclosed, environmentally controlled portion of the Rotating Service Structure (RSS) (at left) that supports payload delivery at the pad. At right is Space Shuttle Endeavour with its orange external tank and one solid rocket booster showing behind it. When the RSS is closed around Endeavour, the P6 truss will be moved into the orbiter’s payload bay. The P6, payload on mission STS-97, comprises Solar Array Wing-3 and the Integrated Electronic Assembly, to be installed on the International Space Station. The Station’s electrical power system will use eight photovoltaic solar arrays, each 112 feet long by 39 feet wide, to convert sunlight to electricity. The solar arrays are mounted on a “blanket” that can be folded like an accordion for delivery. Once in orbit, astronauts will deploy the blankets to their full size. Gimbals will be used to rotate the arrays so that they will face the Sun to provide maximum power to the Space Station. Launch of STS-97 is scheduled for Nov. 30 at 10:06 p.m. EST

Gene ARMADA: an integrated multi-analysis platform for microarray data implemented in MATLAB.

PubMed

Chatziioannou, Aristotelis; Moulos, Panagiotis; Kolisis, Fragiskos N

2009-10-27

The microarray data analysis realm is ever growing through the development of various tools, open source and commercial. However there is absence of predefined rational algorithmic analysis workflows or batch standardized processing to incorporate all steps, from raw data import up to the derivation of significantly differentially expressed gene lists. This absence obfuscates the analytical procedure and obstructs the massive comparative processing of genomic microarray datasets. Moreover, the solutions provided, heavily depend on the programming skills of the user, whereas in the case of GUI embedded solutions, they do not provide direct support of various raw image analysis formats or a versatile and simultaneously flexible combination of signal processing methods. We describe here Gene ARMADA (Automated Robust MicroArray Data Analysis), a MATLAB implemented platform with a Graphical User Interface. This suite integrates all steps of microarray data analysis including automated data import, noise correction and filtering, normalization, statistical selection of differentially expressed genes, clustering, classification and annotation. In its current version, Gene ARMADA fully supports 2 coloured cDNA and Affymetrix oligonucleotide arrays, plus custom arrays for which experimental details are given in tabular form (Excel spreadsheet, comma separated values, tab-delimited text formats). It also supports the analysis of already processed results through its versatile import editor. Besides being fully automated, Gene ARMADA incorporates numerous functionalities of the Statistics and Bioinformatics Toolboxes of MATLAB. In addition, it provides numerous visualization and exploration tools plus customizable export data formats for seamless integration by other analysis tools or MATLAB, for further processing. Gene ARMADA requires MATLAB 7.4 (R2007a) or higher and is also distributed as a stand-alone application with MATLAB Component Runtime. Gene ARMADA provides a highly adaptable, integrative, yet flexible tool which can be used for automated quality control, analysis, annotation and visualization of microarray data, constituting a starting point for further data interpretation and integration with numerous other tools.

Induction of apoptosis by pistachio (Pistacia vera L.) hull extract and its molecular mechanisms of action in human hepatoma cell line HepG2.

PubMed

Fathalizadeh, J; Bagheri, V; Khorramdelazad, H; Kazemi Arababadi, M; Jafarzadeh, A; Mirzaei, M R; Shamsizadeh, A; Hajizadeh, M R

2015-11-30

Several important Pistacia species such as P. vera have been traditionally used for treating a wide range of diseases (for instance, liver-related disorders). There is a relative lack of research into pharmacological aspects of pistachio hull. Hence, this study was aimed at investigating whether pistachio rosy hull (PRH) extract exerts apoptotic impacts on HepG2 liver cancer cell line. In order to evaluate cell viability and apoptosis in response to treatment with the extract, MTT assay and Annexin-V-fluorescein/propidium iodide (PI) double staining were performed, respectively. Moreover, molecular mechanism of apoptosis induced by the extract was determined using human apoptosis PCR array. Our findings showed that PRH extract treatment reduced cell viability (IC50 ~ 0.3 mg/ml) in a dose-dependent manner. Flow cytometric analysis revealed that the extract significantly induced apoptosis in HepG2 cells. In addition, quantitative PCR array results demonstrated the regulation of a considerable number of apoptosis-related genes belonging to the TNF, BCL2, IAP, TRAF, and caspase families. We observed altered expression of both pro-apoptotic and anti-apoptotic genes associated with the extrinsic and intrinsic apoptosis signaling pathways. These results suggest that the aqueous extract of PRH possesses apoptotic activity through cytotoxic and apoptosis-inducing effects on HepG2 cells.

Evolutionary multidimensional access architecture featuring cost-reduced components

NASA Astrophysics Data System (ADS)

Farjady, Farsheed; Parker, Michael C.; Walker, Stuart D.

1998-12-01

We describe a three-stage wavelength-routed optical access network, utilizing coarse passband-flattened arrayed- waveguide grating routers. An N-dimensional addressing strategy enables 6912 customers to be bi-directionally addressed with multi-Gb/s data using only 24 wavelengths spaced by 1.6 nm. Coarse wavelength separation allows use of increased tolerance WDM components at the exchange and customer premises. The architecture is designed to map onto standard access network topologies, allowing elegant upgradability from legacy PON infrastructures at low cost. Passband-flattening of the routers is achieved through phase apodization.

Quantifying the effects of disorder on switching of perpendicular spin ice arrays

NASA Astrophysics Data System (ADS)

Kempinger, Susan; Fraleigh, Robert; Lammert, Paul; Crespi, Vincent; Samarth, Nitin; Zhang, Sheng; Schiffer, Peter

There is much contemporary interest in probing custom designed, frustrated systems such as artificial spin ice. To that end, we study arrays of lithographically patterned, single-domain Pt/Co multilayer islands. Due to the perpendicular anisotropy of these materials, we are able to use diffraction-limited magneto-optical Kerr effect microscopy to access the magnetic state in situ with an applied field. As we tune the interaction strength by adjusting the lattice spacing, we observe the switching field distribution broadening with increasing dipolar interactions. Using a simple mathematical analysis we extract the intrinsic disorder (the disorder that would be present without interactions) from these switching field distributions. We also characterize the intrinsic disorder by systematically removing neighbor effects from the switching field distribution. Understanding this disorder contribution as well as the interaction strength allows us to more accurately characterize the moment correlation. This project was funded by the US Department of Energy, Office of Basic Energy Sciences, Materials Sciences and Engineering Division under Grant No. DE- SC0010778

Infrared emission of a freestanding plasmonic membrane

NASA Astrophysics Data System (ADS)

Monshat, Hosein; Liu, Longju; McClelland, John; Biswas, Rana; Lu, Meng

2018-01-01

This paper reports a free-standing plasmonic membrane as a thermal emitter in the near- and mid-infrared regions. The plasmonic membrane consists of an ultrathin gold film perforated with a two-dimensional array of holes. The device was fabricated using an imprint and transfer process and fixed on a low-emissivity metal grid. The thermal radiation characteristics of the plasmonic membrane can be engineered by controlling the array period and the thickness of the gold membrane. Plasmonic membranes with two different periods were designed using electromagnetic simulation and then characterized for their transmission and infrared radiation properties. The free-standing membranes exhibit extraordinary optical transmissions with the resonant transmission coefficient as high as 76.8%. After integration with a customized heater, the membranes demonstrate narrowband thermal emission in the wavelength range of 2.5 μm to 5.5 μm. The emission signatures, including peak emission wavelength and bandwidth, are associated with the membrane geometry. The ultrathin membrane infrared emitter can be adopted in applications, such as chemical analysis and thermal imaging.

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

PubMed

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

2002-05-01

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

Diagnostic Performance of a Multiplex PCR assay for meningitis in an HIV-infected population in Uganda

PubMed Central

Rhein, Joshua; Bahr, Nathan C; Hemmert, Andrew C; Cloud, Joann L; Bellamkonda, Satya; Oswald, Cody; Lo, Eric; Nabeta, Henry; Kiggundu, Reuben; Akampurira, Andrew; Musubire, Abdu; Williams, Darlisha; Meya, David B; Boulware, David R

2015-01-01

Meningitis remains a worldwide problem, and rapid diagnosis is essential to optimize survival. We evaluated the utility of a multiplex PCR test in differentiating possible etiologies of meningitis. Cerebrospinal fluid (CSF) from 69 HIV-infected Ugandan adults with meningitis was collected at diagnosis (n=51) and among persons with cryptococcal meningitis during therapeutic lumbar punctures (n=68). Cryopreserved CSF specimens were analyzed with BioFire FilmArray® Meningitis/Encephalitis panel, which targets 17 pathogens. The panel detected Cryptococcus in the CSF of patients diagnosed with a first-episode of cryptococcal meningitis by fungal culture with 100% sensitivity and specificity, and differentiated between fungal relapse and paradoxical immune reconstitution inflammatory syndrome in recurrent episodes. A negative FilmArray result was predictive of CSF sterility on follow-up lumbar punctures for cryptococcal meningitis. EBV was frequently detected in this immunosuppressed population (n=45). Other pathogens detected included: CMV (n=2), VZV (n=2), HHV-6 (n=1), and Streptococcus pneumoniae (n=1). The FilmArray Meningitis/Encephalitis panel offers a promising platform for rapid meningitis diagnosis. PMID:26711635

Arrays of probes for positional sequencing by hybridization

DOEpatents

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

2008-01-15

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

Taking advantage of modern turbines

DOE PAGES

Thresher, Robert

2018-05-14

Here, wind facilities have generally deployed turbines of the same power and height in regular uniform arrays. Now, the modern generation of turbines, with customer-selectable tower heights and larger rotors, can significantly increase wind energy's economic potential using less land to generate cheaper electricity.

Taking advantage of modern turbines

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

Thresher, Robert

Here, wind facilities have generally deployed turbines of the same power and height in regular uniform arrays. Now, the modern generation of turbines, with customer-selectable tower heights and larger rotors, can significantly increase wind energy's economic potential using less land to generate cheaper electricity.

A Transcription Activator-Like Effector (TALE) Toolbox for Genome Engineering

PubMed Central

Sanjana, Neville E.; Cong, Le; Zhou, Yang; Cunniff, Margaret M.; Feng, Guoping; Zhang, Feng

2013-01-01

Transcription activator-like effectors (TALEs) are a class of naturally occurring DNA binding proteins found in the plant pathogen Xanthomonas sp. The DNA binding domain of each TALE consists of tandem 34-amino acid repeat modules that can be rearranged according to a simple cipher to target new DNA sequences. Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing. Here we describe a toolbox for rapid construction of custom TALE transcription factors (TALE-TFs) and nucleases (TALENs) using a hierarchical ligation procedure. This toolbox facilitates affordable and rapid construction of custom TALE-TFs and TALENs within one week and can be easily scaled up to construct TALEs for multiple targets in parallel. We also provide details for testing the activity in mammalian cells of custom TALE-TFs and TALENs using, respectively, qRT-PCR and Surveyor nuclease. The TALE toolbox described here will enable a broad range of biological applications. PMID:22222791

Unraveling unusual X-chromosome patterns during fragile-X syndrome genetic testing.

PubMed

Esposito, Gabriella; Tremolaterra, Maria Roberta; Savarese, Maria; Spiniello, Michele; Patrizio, Maria Pia; Lombardo, Barbara; Pastore, Lucio; Salvatore, Francesco; Carsana, Antonella

2018-01-01

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability (ID). Together with fragile X-associated tremor and ataxia (FXTAS) and fragile X-associated premature ovarian failure (POF)/primary ovarian insufficiency (POI), FXS depends on dysfunctional expression of the FMR1 gene on Xq27.3. In most cases, FXS is caused by a >200 CGG repeats in FMR1 5'-untranslated region (UTR) and by promoter hypermethylation that results in gene silencing. Males and females with unmethylated premutated alleles (repeats between 55 and 200) are at risk for FXTAS and POF/POI. FXS molecular testing relied on PCR and methylation-specific Southern blot analysis of the FMR1 5'UTR. Atypical Southern blot patterns were studied by X-chromosome microsatellite analysis, copy number dosage at DMD locus, amelogenin gender-marker analysis and array-comparative genomic hybridization (array-CGH). Six men affected by ID and three women affected by ID and POF/POI underwent FXS molecular testing. They had normal FMR1 CGG repeats, but atypical X chromosome patterns. Further investigations revealed that the six males had Klinefelter syndrome (XXY), one female was a Turner mosaic (X0/XX) and two women had novel rearrangements involving X chromosome. Diagnostic investigation of atypical patterns at FMR1 locus can address patients and/or their relatives to further verify the condition by performing karyotyping and/or array-CGH. Copyright © 2017. Published by Elsevier B.V.

Identification of DNA copy number aberrations by array comparative genomic hybridization in patients with ruptured intracranial aneurysms.

PubMed

Choi, Jin Soo; Kim, Seong-Rim; Jeon, Yang-Whan; Lee, Kweon-Haeng; Rha, Hyoung Kyun

2009-02-01

We aimed to use array comparative genomic hybridization (CGH) to identify chromosomal loci that contribute to the pathogenesis of ruptured intracranial aneurysms (IAs) in a Korean population and to confirm the results using real-time polymerase chain reaction (PCR). Twenty-three patients with ruptured IAs were enrolled in this study. Array CGH revealed copy number aberrations in 19 chromosomal regions. Chromosomal gains were identified at a high frequency in regions 1p12, 4q24, 5p15.31, 5p15.33, 6p12.2, 6q22.33, 7p21.1, 9q22.1, 10q24.32, 10q26.3, 12q13.13, 17p12, 18q12.3, 18q23, 19p13.3, 20q13.33, 21q11.2, and 21q22.3, whereas chromosomal losses were identified at 15q11.2 and 22q11.21. Real-time PCR confirmed the results of the array CGH studies of the COL6A2, GRIN3B, MUC17, and PRODH genes. This is the first study to identify candidate regions by array CGH in patients with IAs. The identification of genes that may predispose an individual to the development of IAs may lead to a better understanding of the mechanism of IA formation. Multicenter studies comparing cohorts of patients of different ethnicities are needed to better understand the mechanism of IA formation.

Carbon Nanotube Nanoelectrode Array as an Electronic Chip for Ultrasensitive Label-free DNA Detection

NASA Technical Reports Server (NTRS)

Li, Jun; Koehne, Jessica; Chen, Hua; Cassell, Alan; Ng, Hou Tee; Fan, Wendy; Ye, Qi; Han, Jie; Meyyappan, M.

2003-01-01

A reliable nanoelectrode array based on vertically aligned multi-walled carbon nanotubes (MWNTs) embedded in SiO2 is used for ultrasensitive DNA detection. Characteristic nanoelectrode behavior is observed using low-density MWNT arrays for measuring both bulk and surface immobilized redox species such as K4Fe(CN)6 and ferrocene derivatives. The open-end of MWNTs are found to present similar properties as graphite edge-plane electrodes with wide potential window, flexible chemical functionalities, and good biocompatibility. BRCA1 related oligonucleotide probes with 18 bp are selectively functionalized at the open ends of the nanotube array and specifically hybridized with oligonucleotide targets incorporated with a polyG tag. The guanine groups are employed as the signal moieties in the electrochemical measurements. R(bpy)(sup 2+, sub 3) mediator is used to further amplify the guanine oxidation signal. The hybridization of sub-attomoles of DNA targets is detected electrochemically by combining the MWNT nanoelectrode array with the R(bpy)(sup 2+, sub 3) amplification mechanism. This technique was employed for direct electrochemical detection of label-free PCR amplicon from a healthy donor through specific hybridization with the BRCA1 probe. The detection limit is estimated to be less than 1000 DNA molecules since abundant guanine bases in the PCR amplicon provides a large signal. This system provides a general platform for rapid molecular diagnostics in applications requiring ultrahigh sensitivity, high-degree of miniaturization, and simple sample preparation, and low-cost operation.

Clinical application of 2.7M Cytogenetics array for CNV detection in subjects with idiopathic autism and/or intellectual disability.

PubMed

Qiao, Y; Tyson, C; Hrynchak, M; Lopez-Rangel, E; Hildebrand, J; Martell, S; Fawcett, C; Kasmara, L; Calli, K; Harvard, C; Liu, X; Holden, J J A; Lewis, S M E; Rajcan-Separovic, E

2013-02-01

Higher resolution whole-genome arrays facilitate the identification of smaller copy number variations (CNVs) and their integral genes contributing to autism and/or intellectual disability (ASD/ID). Our study describes the use of one of the highest resolution arrays, the Affymetrix(®) Cytogenetics 2.7M array, coupled with quantitative multiplex polymerase chain reaction (PCR) of short fluorescent fragments (QMPSF) for detection and validation of small CNVs. We studied 82 subjects with ASD and ID in total (30 in the validation and 52 in the application cohort) and detected putatively pathogenic CNVs in 6/52 cases from the application cohort. This included a 130-kb maternal duplication spanning exons 64-79 of the DMD gene which was found in a 3-year-old boy manifesting autism and mild neuromotor delays. Other pathogenic CNVs involved 4p14, 12q24.31, 14q32.31, 15q13.2-13.3, and 17p13.3. We established the optimal experimental conditions which, when applied to select small CNVs for QMPSF confirmation, reduced the false positive rate from 60% to 25%. Our work suggests that selection of small CNVs based on the function of integral genes, followed by review of array experimental parameters resulting in highest confirmation rate using multiplex PCR, may enhance the usefulness of higher resolution platforms for ASD and ID gene discovery. © 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

Photoacoustic Imaging with a Commercial Ultrasound System and a Custom Probe

PubMed Central

Wang, Xueding; Fowlkes, J. Brian; Cannata, Jonathan M.; Hu, Changhong; Carson, Paul L.

2010-01-01

Building photoacoustic imaging (PAI) systems by using stand-alone ultrasound (US) units makes it convenient to take advantage of the state-of-the-art ultrasonic technologies. However, the sometimes limited receiving sensitivity and the comparatively narrow bandwidth of commercial US probes may not be sufficient to acquire high quality photoacoustic images. In this work, a high-speed PAI system has been developed using a commercial US unit and a custom built 128-element piezoelectric-polymer array (PPA) probe using a P(VDF-TrFE) film and flexible circuit to define the elements. Since the US unit supports simultaneous signal acquisition from 64 parallel receive channels, PAI data for synthetic image formation from a 64 or 128 element array aperture can be acquired after a single or dual laser firing, respectively. Therefore, 2D B-scan imaging can be achieved with a maximum frame rate up to 10 Hz, limited only by the laser repetition rate. The uniquely properties of P(VDF-TrFE) facilitated a wide -6 dB receiving bandwidth of over 120 % for the array. A specially designed 128-channel preamplifier board made the connection between the array and the system cable which not only enabled element electrical impedance matching but also further elevated the signal-to-noise ratio (SNR) to further enhance the detection of weak photoacoustic signals. Through the experiments on phantoms and rabbit ears, the good performance of this PAI system was demonstrated. PMID:21276653

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

PubMed

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

2013-07-01

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

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

PubMed Central

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

2013-01-01

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

High-voltage integrated active quenching circuit for single photon count rate up to 80 Mcounts/s.

PubMed

Acconcia, Giulia; Rech, Ivan; Gulinatti, Angelo; Ghioni, Massimo

2016-08-08

Single photon avalanche diodes (SPADs) have been subject to a fast improvement in recent years. In particular, custom technologies specifically developed to fabricate SPAD devices give the designer the freedom to pursue the best detector performance required by applications. A significant breakthrough in this field is represented by the recent introduction of a red enhanced SPAD (RE-SPAD) technology, capable of attaining a good photon detection efficiency in the near infrared range (e.g. 40% at a wavelength of 800 nm) while maintaining a remarkable timing resolution of about 100ps full width at half maximum. Being planar, the RE-SPAD custom technology opened the way to the development of SPAD arrays particularly suited for demanding applications in the field of life sciences. However, to achieve such excellent performance custom SPAD detectors must be operated with an external active quenching circuit (AQC) designed on purpose. Next steps toward the development of compact and practical multichannel systems will require a new generation of monolithically integrated AQC arrays. In this paper we present a new, fully integrated AQC fabricated in a high-voltage 0.18 µm CMOS technology able to provide quenching pulses up to 50 Volts with fast leading and trailing edges. Although specifically designed for optimal operation of RE-SPAD devices, the new AQC is quite versatile: it can be used with any SPAD detector, regardless its fabrication technology, reaching remarkable count rates up to 80 Mcounts/s and generating a photon detection pulse with a timing jitter as low as 119 ps full width at half maximum. The compact design of our circuit has been specifically laid out to make this IC a suitable building block for monolithically integrated AQC arrays.

Design and Use of Microphone Directional Arrays for Aeroacoustic Measurements

NASA Technical Reports Server (NTRS)

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

1998-01-01

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

ASIC Readout Circuit Architecture for Large Geiger Photodiode Arrays

NASA Technical Reports Server (NTRS)

Vasile, Stefan; Lipson, Jerold

2012-01-01

The objective of this work was to develop a new class of readout integrated circuit (ROIC) arrays to be operated with Geiger avalanche photodiode (GPD) arrays, by integrating multiple functions at the pixel level (smart-pixel or active pixel technology) in 250-nm CMOS (complementary metal oxide semiconductor) processes. In order to pack a maximum of functions within a minimum pixel size, the ROIC array is a full, custom application-specific integrated circuit (ASIC) design using a mixed-signal CMOS process with compact primitive layout cells. The ROIC array was processed to allow assembly in bump-bonding technology with photon-counting infrared detector arrays into 3-D imaging cameras (LADAR). The ROIC architecture was designed to work with either common- anode Si GPD arrays or common-cathode InGaAs GPD arrays. The current ROIC pixel design is hardwired prior to processing one of the two GPD array configurations, and it has the provision to allow soft reconfiguration to either array (to be implemented into the next ROIC array generation). The ROIC pixel architecture implements the Geiger avalanche quenching, bias, reset, and time to digital conversion (TDC) functions in full-digital design, and uses time domain over-sampling (vernier) to allow high temporal resolution at low clock rates, increased data yield, and improved utilization of the laser beam.

A wideband software reconfigurable modem

NASA Astrophysics Data System (ADS)

Turner, J. H., Jr.; Vickers, H.

A wideband modem is described which provides signal processing capability for four Lx-band signals employing QPSK, MSK and PPM waveforms and employs a software reconfigurable architecture for maximum system flexibility and graceful degradation. The current processor uses a 2901 and two 8086 microprocessors per channel and performs acquisition, tracking, and data demodulation for JITDS, GPS, IFF and TACAN systems. The next generation processor will be implemented using a VHSIC chip set employing a programmable complex array vector processor module, a GP computer module, customized gate array modules, and a digital array correlator. This integrated processor has application to a wide number of diverse system waveforms, and will bring the benefits of VHSIC technology insertion into avionic antijam communications systems.

Premalignant Genetic and Epigenetic Alterations in Tubal Epithelium from Women with BRCA1 Mutations

DTIC Science & Technology

2010-10-14

Homo sapiens , clone IMAGE:5590287, mRNA Unknown 1.8 .0044 3.0...RT-PCR W T -F T B 1- F T B 1 C an ce r 0 2 4 6 8 10 R el at iv e Q u an ti fi ca ti o n CDKN1C Array W T -F T B 1- F T B 1 C an ce r 0 100 200 300...S p o t In te n si ty CDKN1C RT-PCR W T -F T B 1- F T B 1 C an ce r 0.0 0.2 0.4 0.6 0.8 R el at iv e Q u an ti fi ca ti o n CYP3A5 Array W T

Development and Validation of a High-Density SNP Genotyping Array for African Oil Palm.

PubMed

Kwong, Qi Bin; Teh, Chee Keng; Ong, Ai Ling; Heng, Huey Ying; Lee, Heng Leng; Mohamed, Mohaimi; Low, Joel Zi-Bin; Apparow, Sukganah; Chew, Fook Tim; Mayes, Sean; Kulaveerasingam, Harikrishna; Tammi, Martti; Appleton, David Ross

2016-08-01

High-density single nucleotide polymorphism (SNP) genotyping arrays are powerful tools that can measure the level of genetic polymorphism within a population. To develop a whole-genome SNP array for oil palms, SNP discovery was performed using deep resequencing of eight libraries derived from 132 Elaeis guineensis and Elaeis oleifera palms belonging to 59 origins, resulting in the discovery of >3 million putative SNPs. After SNP filtering, the Illumina OP200K custom array was built with 170 860 successful probes. Phenetic clustering analysis revealed that the array could distinguish between palms of different origins in a way consistent with pedigree records. Genome-wide linkage disequilibrium declined more slowly for the commercial populations (ranging from 120 kb at r(2) = 0.43 to 146 kb at r(2) = 0.50) when compared with the semi-wild populations (19.5 kb at r(2) = 0.22). Genetic fixation mapping comparing the semi-wild and commercial population identified 321 selective sweeps. A genome-wide association study (GWAS) detected a significant peak on chromosome 2 associated with the polygenic component of the shell thickness trait (based on the trait shell-to-fruit; S/F %) in tenera palms. Testing of a genomic selection model on the same trait resulted in good prediction accuracy (r = 0.65) with 42% of the S/F % variation explained. The first high-density SNP genotyping array for oil palm has been developed and shown to be robust for use in genetic studies and with potential for developing early trait prediction to shorten the oil palm breeding cycle. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

MSP-HTPrimer: a high-throughput primer design tool to improve assay design for DNA methylation analysis in epigenetics.

PubMed

Pandey, Ram Vinay; Pulverer, Walter; Kallmeyer, Rainer; Beikircher, Gabriel; Pabinger, Stephan; Kriegner, Albert; Weinhäusel, Andreas

2016-01-01

Bisulfite (BS) conversion-based and methylation-sensitive restriction enzyme (MSRE)-based PCR methods have been the most commonly used techniques for locus-specific DNA methylation analysis. However, both methods have advantages and limitations. Thus, an integrated approach would be extremely useful to quantify the DNA methylation status successfully with great sensitivity and specificity. Designing specific and optimized primers for target regions is the most critical and challenging step in obtaining the adequate DNA methylation results using PCR-based methods. Currently, no integrated, optimized, and high-throughput methylation-specific primer design software methods are available for both BS- and MSRE-based methods. Therefore an integrated, powerful, and easy-to-use methylation-specific primer design pipeline with great accuracy and success rate will be very useful. We have developed a new web-based pipeline, called MSP-HTPrimer, to design primers pairs for MSP, BSP, pyrosequencing, COBRA, and MSRE assays on both genomic strands. First, our pipeline converts all target sequences into bisulfite-treated templates for both forward and reverse strand and designs all possible primer pairs, followed by filtering for single nucleotide polymorphisms (SNPs) and known repeat regions. Next, each primer pairs are annotated with the upstream and downstream RefSeq genes, CpG island, and cut sites (for COBRA and MSRE). Finally, MSP-HTPrimer selects specific primers from both strands based on custom and user-defined hierarchical selection criteria. MSP-HTPrimer produces a primer pair summary output table in TXT and HTML format for display and UCSC custom tracks for resulting primer pairs in GTF format. MSP-HTPrimer is an integrated, web-based, and high-throughput pipeline and has no limitation on the number and size of target sequences and designs MSP, BSP, pyrosequencing, COBRA, and MSRE assays. It is the only pipeline, which automatically designs primers on both genomic strands to increase the success rate. It is a standalone web-based pipeline, which is fully configured within a virtual machine and thus can be readily used without any configuration. We have experimentally validated primer pairs designed by our pipeline and shown a very high success rate of primer pairs: out of 66 BSP primer pairs, 63 were successfully validated without any further optimization step and using the same qPCR conditions. The MSP-HTPrimer pipeline is freely available from http://sourceforge.net/p/msp-htprimer.

Quantitative high-resolution genomic analysis of single cancer cells.

PubMed

Hannemann, Juliane; Meyer-Staeckling, Sönke; Kemming, Dirk; Alpers, Iris; Joosse, Simon A; Pospisil, Heike; Kurtz, Stefan; Görndt, Jennifer; Püschel, Klaus; Riethdorf, Sabine; Pantel, Klaus; Brandt, Burkhard

2011-01-01

During cancer progression, specific genomic aberrations arise that can determine the scope of the disease and can be used as predictive or prognostic markers. The detection of specific gene amplifications or deletions in single blood-borne or disseminated tumour cells that may give rise to the development of metastases is of great clinical interest but technically challenging. In this study, we present a method for quantitative high-resolution genomic analysis of single cells. Cells were isolated under permanent microscopic control followed by high-fidelity whole genome amplification and subsequent analyses by fine tiling array-CGH and qPCR. The assay was applied to single breast cancer cells to analyze the chromosomal region centred by the therapeutical relevant EGFR gene. This method allows precise quantitative analysis of copy number variations in single cell diagnostics.

CMOS gate array characterization procedures

NASA Astrophysics Data System (ADS)

Spratt, James P.

1993-09-01

Present procedures are inadequate for characterizing the radiation hardness of gate array product lines prior to personalization because the selection of circuits to be used, from among all those available in the manufacturer's circuit library, is usually uncontrolled. (Some circuits are fundamentally more radiation resistant than others.) In such cases, differences in hardness can result between different designs of the same logic function. Hardness also varies because many gate arrays feature large custom-designed megacells (e.g., microprocessors and random access memories-MicroP's and RAM's). As a result, different product lines cannot be compared equally. A characterization strategy is needed, along with standardized test vehicle(s), methodology, and conditions, so that users can make informed judgments on which gate arrays are best suited for their needs. The program described developed preferred procedures for the radiation characterization of gate arrays, including a gate array evaluation test vehicle, featuring a canary circuit, designed to define the speed versus hardness envelope of the gate array. A multiplier was chosen for this role, and a baseline multiplier architecture is suggested that could be incorporated into an existing standard evaluation circuit chip.

Design and coverage of high throughput genotyping arrays optimized for individuals of East Asian, African American, and Latino race/ethnicity using imputation and a novel hybrid SNP selection algorithm.

PubMed

Hoffmann, Thomas J; Zhan, Yiping; Kvale, Mark N; Hesselson, Stephanie E; Gollub, Jeremy; Iribarren, Carlos; Lu, Yontao; Mei, Gangwu; Purdy, Matthew M; Quesenberry, Charles; Rowell, Sarah; Shapero, Michael H; Smethurst, David; Somkin, Carol P; Van den Eeden, Stephen K; Walter, Larry; Webster, Teresa; Whitmer, Rachel A; Finn, Andrea; Schaefer, Catherine; Kwok, Pui-Yan; Risch, Neil

2011-12-01

Four custom Axiom genotyping arrays were designed for a genome-wide association (GWA) study of 100,000 participants from the Kaiser Permanente Research Program on Genes, Environment and Health. The array optimized for individuals of European race/ethnicity was previously described. Here we detail the development of three additional microarrays optimized for individuals of East Asian, African American, and Latino race/ethnicity. For these arrays, we decreased redundancy of high-performing SNPs to increase SNP capacity. The East Asian array was designed using greedy pairwise SNP selection. However, removing SNPs from the target set based on imputation coverage is more efficient than pairwise tagging. Therefore, we developed a novel hybrid SNP selection method for the African American and Latino arrays utilizing rounds of greedy pairwise SNP selection, followed by removal from the target set of SNPs covered by imputation. The arrays provide excellent genome-wide coverage and are valuable additions for large-scale GWA studies. Copyright © 2011 Elsevier Inc. All rights reserved.

Gene expression profiling of acute myeloid leukemia samples from adult patients with AML-M1 and -M2 through boutique microarrays, real-time PCR and droplet digital PCR.

PubMed

Handschuh, Luiza; Kaźmierczak, Maciej; Milewski, Marek C; Góralski, Michał; Łuczak, Magdalena; Wojtaszewska, Marzena; Uszczyńska-Ratajczak, Barbara; Lewandowski, Krzysztof; Komarnicki, Mieczysław; Figlerowicz, Marek

2018-03-01

Acute myeloid leukemia (AML) is the most common and severe form of acute leukemia diagnosed in adults. Owing to its heterogeneity, AML is divided into classes associated with different treatment outcomes and specific gene expression profiles. Based on previous studies on AML, in this study, we designed and generated an AML-array containing 900 oligonucleotide probes complementary to human genes implicated in hematopoietic cell differentiation and maturation, proliferation, apoptosis and leukemic transformation. The AML-array was used to hybridize 118 samples from 33 patients with AML of the M1 and M2 subtypes of the French-American‑British (FAB) classification and 15 healthy volunteers (HV). Rigorous analysis of the microarray data revealed that 83 genes were differentially expressed between the patients with AML and the HV, including genes not yet discussed in the context of AML pathogenesis. The most overexpressed genes in AML were STMN1, KITLG, CDK6, MCM5, KRAS, CEBPA, MYC, ANGPT1, SRGN, RPLP0, ENO1 and SET, whereas the most underexpressed genes were IFITM1, LTB, FCN1, BIRC3, LYZ, ADD3, S100A9, FCER1G, PTRPE, CD74 and TMSB4X. The overexpression of the CPA3 gene was specific for AML with mutated NPM1 and FLT3. Although the microarray-based method was insufficient to differentiate between any other AML subgroups, quantitative PCR approaches enabled us to identify 3 genes (ANXA3, S100A9 and WT1) whose expression can be used to discriminate between the 2 studied AML FAB subtypes. The expression levels of the ANXA3 and S100A9 genes were increased, whereas those of WT1 were decreased in the AML-M2 compared to the AML-M1 group. We also examined the association between the STMN1, CAT and ABL1 genes, and the FLT3 and NPM1 mutation status. FLT3+/NPM1- AML was associated with the highest expression of STMN1, and ABL1 was upregulated in FLT3+ AML and CAT in FLT3- AML, irrespectively of the NPM1 mutation status. Moreover, our results indicated that CAT and WT1 gene expression levels correlated with the response to therapy. CAT expression was highest in patients who remained longer under complete remission, whereas WT1 expression increased with treatment resistance. On the whole, this study demonstrates that the AML-array can potentially serve as a first-line screening tool, and may be helpful for the diagnosis of AML, whereas the differentiation between AML subgroups can be more successfully performed with PCR-based analysis of a few marker genes.

An externally head-mounted wireless neural recording device for laboratory animal research and possible human clinical use.

PubMed

Yin, Ming; Li, Hao; Bull, Christopher; Borton, David A; Aceros, Juan; Larson, Lawrence; Nurmikko, Arto V

2013-01-01

In this paper we present a new type of head-mounted wireless neural recording device in a highly compact package, dedicated for untethered laboratory animal research and designed for future mobile human clinical use. The device, which takes its input from an array of intracortical microelectrode arrays (MEA) has ninety-seven broadband parallel neural recording channels and was integrated on to two custom designed printed circuit boards. These house several low power, custom integrated circuits, including a preamplifier ASIC, a controller ASIC, plus two SAR ADCs, a 3-axis accelerometer, a 48MHz clock source, and a Manchester encoder. Another ultralow power RF chip supports an OOK transmitter with the center frequency tunable from 3GHz to 4GHz, mounted on a separate low loss dielectric board together with a 3V LDO, with output fed to a UWB chip antenna. The IC boards were interconnected and packaged in a polyether ether ketone (PEEK) enclosure which is compatible with both animal and human use (e.g. sterilizable). The entire system consumes 17mA from a 1.2Ahr 3.6V Li-SOCl2 1/2AA battery, which operates the device for more than 2 days. The overall system includes a custom RF receiver electronics which are designed to directly interface with any number of commercial (or custom) neural signal processors for multi-channel broadband neural recording. Bench-top measurements and in vivo testing of the device in rhesus macaques are presented to demonstrate the performance of the wireless neural interface.

Database resources of the National Center for Biotechnology

PubMed Central

Wheeler, David L.; Church, Deanna M.; Federhen, Scott; Lash, Alex E.; Madden, Thomas L.; Pontius, Joan U.; Schuler, Gregory D.; Schriml, Lynn M.; Sequeira, Edwin; Tatusova, Tatiana A.; Wagner, Lukas

2003-01-01

In addition to maintaining the GenBank(R) nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides data analysis and retrieval resources for the data in GenBank and other biological data made available through NCBI's Web site. NCBI resources include Entrez, PubMed, PubMed Central (PMC), LocusLink, the NCBITaxonomy Browser, BLAST, BLAST Link (BLink), Electronic PCR (e-PCR), Open Reading Frame (ORF) Finder, References Sequence (RefSeq), UniGene, HomoloGene, ProtEST, Database of Single Nucleotide Polymorphisms (dbSNP), Human/Mouse Homology Map, Cancer Chromosome Aberration Project (CCAP), Entrez Genomes and related tools, the Map Viewer, Model Maker (MM), Evidence Viewer (EV), Clusters of Orthologous Groups (COGs) database, Retroviral Genotyping Tools, SAGEmap, Gene Expression Omnibus (GEO), Online Mendelian Inheritance in Man (OMIM), the Molecular Modeling Database (MMDB), the Conserved Domain Database (CDD), and the Conserved Domain Architecture Retrieval Tool (CDART). Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized data sets. All of the resources can be accessed through the NCBI home page at: http://www.ncbi.nlm.nih.gov. PMID:12519941

A high-speed trapezoid image sensor design for continuous traffic monitoring at signalized intersection approaches.

DOT National Transportation Integrated Search

2014-10-01

The goal of this project is to monitor traffic flow continuously with an innovative camera system composed of a custom : designed image sensor integrated circuit (IC) containing trapezoid pixel array and camera system that is capable of : intelligent...

Simulation and experimental characterization of the point spread function, pixel saturation, and blooming of a mercury cadmium telluride focal plane array.

PubMed

Soehnel, Grant; Tanbakuchi, Anthony

2012-11-20

A custom IR spot scanning experiment was constructed to project subpixel spots on a mercury cadmium telluride focal plane array (FPA). The hardware consists of an FPA in a liquid nitrogen cooled Dewar, high precision motorized stages, a custom aspheric lens, and a 1.55 and 3.39 μm laser source. By controlling the position and intensity of the spot, characterizations of cross talk, saturation, blooming, and (indirectly) the minority carrier lifetime were performed. In addition, a Monte-Carlo-based charge diffusion model was developed to validate experimental data and make predictions. Results show very good agreement between the model and experimental data. Parameters such as wavelength, reverse bias, and operating temperature were found to have little effect on pixel crosstalk in the absorber layer of the detector. Saturation characterizations show that these FPAs, which do not have antiblooming circuitry, exhibit an increase in cross talk due to blooming at ∼39% beyond the flux required for analog saturation.

Developmental Exposure to Estrogen Alters Differentiation and Epigenetic Programming in a Human Fetal Prostate Xenograft Model

PubMed Central

Saffarini, Camelia M.; McDonnell-Clark, Elizabeth V.; Amin, Ali; Huse, Susan M.; Boekelheide, Kim

2015-01-01

Prostate cancer is the most frequent non-cutaneous malignancy in men. There is strong evidence in rodents that neonatal estrogen exposure plays a role in the development of this disease. However, there is little information regarding the effects of estrogen in human fetal prostate tissue. This study explored early life estrogen exposure, with and without a secondary estrogen and testosterone treatment in a human fetal prostate xenograft model. Histopathological lesions, proliferation, and serum hormone levels were evaluated at 7, 30, 90, and 200-day time-points after xenografting. The expression of 40 key genes involved in prostatic glandular and stromal growth, cell-cycle progression, apoptosis, hormone receptors and tumor suppressors was evaluated using a custom PCR array. Epigenome-wide analysis of DNA methylation was performed on whole tissue, and laser capture-microdissection (LCM) isolated epithelial and stromal compartments of 200-day prostate xenografts. Combined initial plus secondary estrogenic exposures had the most severe tissue changes as revealed by the presence of hyperplastic glands at day 200. Gene expression changes corresponded with the cellular events in the KEGG prostate cancer pathway, indicating that initial plus secondary exposure to estrogen altered the PI3K-Akt signaling pathway, ultimately resulting in apoptosis inhibition and an increase in cell cycle progression. DNA methylation revealed that differentially methylated CpG sites significantly predominate in the stromal compartment as a result of estrogen-treatment, thereby providing new targets for future investigation. By using human fetal prostate tissue and eliminating the need for species extrapolation, this study provides novel insights into the gene expression and epigenetic effects related to prostate carcinogenesis following early life estrogen exposure. PMID:25799167

Custom Array Comparative Genomic Hybridization: the Importance of DNA Quality, an Expert Eye, and Variant Validation

PubMed Central

Lantieri, Francesca; Malacarne, Michela; Gimelli, Stefania; Santamaria, Giuseppe; Coviello, Domenico; Ceccherini, Isabella

2017-01-01

The presence of false positive and false negative results in the Array Comparative Genomic Hybridization (aCGH) design is poorly addressed in literature reports. We took advantage of a custom aCGH recently carried out to analyze its design performance, the use of several Agilent aberrations detection algorithms, and the presence of false results. Our study provides a confirmation that the high density design does not generate more noise than standard designs and, might reach a good resolution. We noticed a not negligible presence of false negative and false positive results in the imbalances call performed by the Agilent software. The Aberration Detection Method 2 (ADM-2) algorithm with a threshold of 6 performed quite well, and the array design proved to be reliable, provided that some additional filters are applied, such as considering only intervals with average absolute log2ratio above 0.3. We also propose an additional filter that takes into account the proportion of probes with log2ratio exceeding suggestive values for gain or loss. In addition, the quality of samples was confirmed to be a crucial parameter. Finally, this work raises the importance of evaluating the samples profiles by eye and the necessity of validating the imbalances detected. PMID:28287439

Customization of the acoustic field produced by a piezoelectric array through interelement delays

PubMed Central

Chitnis, Parag V.; Barbone, Paul E.; Cleveland, Robin O.

2008-01-01

A method for producing a prescribed acoustic pressure field from a piezoelectric array was investigated. The array consisted of 170 elements placed on the inner surface of a 15 cm radius spherical cap. Each element was independently driven by using individual pulsers each capable of generating 1.2 kV. Acoustic field customization was achieved by independently controlling the time when each element was excited. The set of time delays necessary to produce a particular acoustic field was determined by using an optimization scheme. The acoustic field at the focal plane was simulated by using the angular spectrum method, and the optimization searched for the time delays that minimized the least squared difference between the magnitudes of the simulated and desired pressure fields. The acoustic field was shaped in two different ways: the −6 dB focal width was increased to different desired widths and the ring-shaped pressure distributions of various prescribed diameters were produced. For both cases, the set of delays resulting from the respective optimization schemes were confirmed to yield the desired pressure distributions by using simulations and measurements. The simulations, however, predicted peak positive pressures roughly half those obtained from the measurements, which was attributed to the exclusion of nonlinearity in the simulations. PMID:18537369

Nuclear Matrix Proteins in Disparity of Prostate Cancer

DTIC Science & Technology

2011-07-01

using G3PDH 3’ primer and PCR primer 1 followed by two rounds of hybridization. In the first hybridization, the RsaI-digested driver cDNA was mixed...determined using β-actin to confirm the reduced relative abundance of the housekeeping gene after SSH. The SSH nested PCR products were cloned using pCR®2.1...variability of DNA concentration. Additionally, negative and positive controls ( housekeeping genes) from the Ambion™ ArrayControls™ Set were included

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

PubMed Central

2013-01-01

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

Mechanical Design and Development of TES Bolometer Detector Arrays for the Advanced ACTPol Experiment

NASA Technical Reports Server (NTRS)

Ward, Jonathan T.; Austermann, Jason; Beall, James A.; Choi, Steve K.; Crowley, Kevin T.; Devlin, Mark J.; Duff, Shannon M.; Gallardo, Patricio M.; Henderson, Shawn W.; Ho, Shuay-Pwu Patty;

Mechanical designs and development of TES bolometer detector arrays for the Advanced ACTPol experiment

NASA Astrophysics Data System (ADS)

Ward, Jonathan T.; Austermann, Jason; Beall, James A.; Choi, Steve K.; Crowley, Kevin T.; Devlin, Mark J.; Duff, Shannon M.; Gallardo, Patricio A.; Henderson, Shawn W.; Ho, Shuay-Pwu Patty; Hilton, Gene; Hubmayr, Johannes; Khavari, Niloufar; Klein, Jeffrey; Koopman, Brian J.; Li, Dale; McMahon, Jeffrey; Mumby, Grace; Nati, Federico; Niemack, Michael D.; Page, Lyman A.; Salatino, Maria; Schillaci, Alessandro; Schmitt, Benjamin L.; Simon, Sara M.; Staggs, Suzanne T.; Thornton, Robert; Ullom, Joel N.; Vavagiakis, Eve M.; Wollack, Edward J.

2016-07-01

The next generation Advanced ACTPol (AdvACT) experiment is currently underway and will consist of four Transition Edge Sensor (TES) bolometer arrays, with three operating together, totaling 5800 detectors on the sky. Building on experience gained with the ACTPol detector arrays, AdvACT will utilize various new technologies, including 150 mm detector wafers equipped with multichroic pixels, allowing for a more densely packed focal plane. Each set of detectors includes a feedhorn array of stacked silicon wafers which form a spline profile leading to each pixel. This is then followed by a waveguide interface plate, detector wafer, back short cavity plate, and backshort cap. Each array is housed in a custom designed structure manufactured from high purity copper and then gold plated. In addition to the detector array assembly, the array package also encloses cryogenic readout electronics. We present the full mechanical design of the AdvACT high frequency (HF) detector array package along with a detailed look at the detector array stack assemblies. This experiment will also make use of extensive hardware and software previously developed for ACT, which will be modified to incorporate the new AdvACT instruments. Therefore, we discuss the integration of all AdvACT arrays with pre-existing ACTPol infrastructure.

Detection of Diarrhea Etiology Among U.S. Military Personnel During Exercise Balikatan 2014, Philippines, Using TaqMan Array Cards.

PubMed

Lertsethtakarn, Paphavee; Nakjarung, Kaewkanya; Silapong, Sasikorn; Neesanant, Pimmnapar; Sakpaisal, Pimmada; Bodhidatta, Ladaporn; Liu, Jie; Houpt, Eric; Velasco, John Mark; Macareo, Louis R; Swierczewski, Brett E; Mason, Carl J

2016-11-01

Military personnel are vulnerable to diarrhea. Diarrheal disease is common when deployed for operations or exercise in developing countries. Although diarrheal disease is transient, cumulative time lost and medical asset can have a significant impact on military operations. Currently, diagnostics of diarrheal etiology typically relies on a mixture of conventional bacteriology, enzyme-linked immunosorbent assay, and polymerase chain reaction (PCR)-based methods including real-time PCR. These methods, however, can be time and labor intensive, although the identification of diarrheal etiology needs to be informative and rapid for treatment and prevention. Real-time PCR has been increasingly used to identify pathogens. Real-time PCR panels of common diarrheal pathogens have been developed, but several diarrheal pathogens are not included in the panel. An expanded and customizable panel to detect diarrhea etiology has been developed employing TaqMan Array Card (TAC) technology. TAC performs 384 real-time PCR reactions simultaneously. As currently configured for diarrheal disease by the University of Virginia, a maximum of 8 samples can be tested simultaneously with approximately 48 target pathogens per sample including bacteria, fungi, helminths, protozoan parasites, and viruses. TAC diarrheal disease panels have been successfully applied to detect pathogens in acute diarrheal stool samples from young children in several international multicenter diarrhea studies. In this study, TAC was applied to stool samples collected under an approved human use protocol from military personnel with acute diarrhea participating in the annual joint military exercise, Balikatan, between the Republic of the Philippines and the United States in 2014. Several established pathogen-specific real-time PCR detection assays were also performed in parallel for comparative purposes. TAC was applied to 7 stool samples. Campylobacter spp. was the most common diarrheal disease pathogen detected. Results from TAC matched 5 out of 6 pathogen specific real-time PCR assays. TAC required a total of 5-6 hours to complete all the procedures from nucleic acid extraction and data analysis, whereas a minimum of 18 hours and 4 hours are required for conventional bacteriology and enzyme-linked immunosorbent assay, respectively, per pathogen. With TAC, pathogen load can be estimated from the amount of nucleic acid present for each pathogen, which can be analyzed further to better determine pathogen attribution and to compare pathogen load between case and control samples. Unfortunately, such correlative analysis was not possible because of the limited sample size available in this study. A larger sample size is needed for further evaluation of TAC on a specific population set, including military personnel. Regardless, TAC was found to be a useful and functional diagnostic platform that is less time-consuming, easy to use with high reproducibility, and costs less per sample compared to the current typically employed methods. The successful application of TAC in acute diarrhea stool samples from a US military population in the Philippines demonstrates its versatility as a potential candidate for a next-generation diagnostics platform. Reprint & Copyright © 2016 Association of Military Surgeons of the U.S.

Optimization of a multi-well array SERS chip

NASA Astrophysics Data System (ADS)

Abell, J. L.; Driskell, J. D.; Dluhy, R. A.; Tripp, R. A.; Zhao, Y.-P.

2009-05-01

SERS-active substrates are fabricated by oblique angle deposition and patterned by a polymer-molding technique to provide a uniform array for high throughput biosensing and multiplexing. Using a conventional SERS-active molecule, 1,2-Bis(4-pyridyl)ethylene (BPE), we show that this device provides a uniform Raman signal enhancement from well to well. The patterning technique employed in this study demonstrates a flexibility allowing for patterning control and customization, and performance optimization of the substrate. Avian influenza is analyzed to demonstrate the ability of this multi-well patterned SERS substrate for biosensing.

Microarray Technology for the Diagnosis of Fetal Chromosomal Aberrations: Which Platform Should We Use?

PubMed Central

Karampetsou, Evangelia; Morrogh, Deborah; Chitty, Lyn

2014-01-01

The advantage of microarray (array) over conventional karyotype for the diagnosis of fetal pathogenic chromosomal anomalies has prompted the use of microarrays in prenatal diagnostics. In this review we compare the performance of different array platforms (BAC, oligonucleotide CGH, SNP) and designs (targeted, whole genome, whole genome, and targeted, custom) and discuss their advantages and disadvantages in relation to prenatal testing. We also discuss the factors to consider when implementing a microarray testing service for the diagnosis of fetal chromosomal aberrations. PMID:26237396

A multiplex reverse transcription PCR and automated electronic microarray assay for detection and differentiation of seven viruses affecting swine.

PubMed

Erickson, A; Fisher, M; Furukawa-Stoffer, T; Ambagala, A; Hodko, D; Pasick, J; King, D P; Nfon, C; Ortega Polo, R; Lung, O

2018-04-01

Microarray technology can be useful for pathogen detection as it allows simultaneous interrogation of the presence or absence of a large number of genetic signatures. However, most microarray assays are labour-intensive and time-consuming to perform. This study describes the development and initial evaluation of a multiplex reverse transcription (RT)-PCR and novel accompanying automated electronic microarray assay for simultaneous detection and differentiation of seven important viruses that affect swine (foot-and-mouth disease virus [FMDV], swine vesicular disease virus [SVDV], vesicular exanthema of swine virus [VESV], African swine fever virus [ASFV], classical swine fever virus [CSFV], porcine respiratory and reproductive syndrome virus [PRRSV] and porcine circovirus type 2 [PCV2]). The novel electronic microarray assay utilizes a single, user-friendly instrument that integrates and automates capture probe printing, hybridization, washing and reporting on a disposable electronic microarray cartridge with 400 features. This assay accurately detected and identified a total of 68 isolates of the seven targeted virus species including 23 samples of FMDV, representing all seven serotypes, and 10 CSFV strains, representing all three genotypes. The assay successfully detected viruses in clinical samples from the field, experimentally infected animals (as early as 1 day post-infection (dpi) for FMDV and SVDV, 4 dpi for ASFV, 5 dpi for CSFV), as well as in biological material that were spiked with target viruses. The limit of detection was 10 copies/μl for ASFV, PCV2 and PRRSV, 100 copies/μl for SVDV, CSFV, VESV and 1,000 copies/μl for FMDV. The electronic microarray component had reduced analytical sensitivity for several of the target viruses when compared with the multiplex RT-PCR. The integration of capture probe printing allows custom onsite array printing as needed, while electrophoretically driven hybridization generates results faster than conventional microarrays that rely on passive hybridization. With further refinement, this novel, rapid, highly automated microarray technology has potential applications in multipathogen surveillance of livestock diseases. © 2017 Her Majesty the Queen in Right of Canada • Transboundary and Emerging Diseases.

Development of advanced micromirror arrays by flip-chip assembly

NASA Astrophysics Data System (ADS)

Michalicek, M. Adrian; Bright, Victor M.

2001-10-01

This paper presents the design, commercial prefabrication, modeling and testing of advanced micromirror arrays fabricated using a novel, simple and inexpensive flip-chip assembly technique. Several polar piston arrays and rectangular cantilever arrays were fabricated using flip-chip assembly by which the upper layers of the array are fabricated on a separate chip and then transferred to a receiving module containing the lower layers. Typical polar piston arrays boast 98.3% active surface area, highly planarized surfaces, low address potentials compatible with CMOS electronics, highly standardized actuation between devices, and complex segmentation of mirror surfaces which allows for custom aberration configurations. Typical cantilever arrays boast large angles of rotation as well as an average surface planarity of only 1.779 nm of RMS roughness across 100 +m mirrors. Continuous torsion devices offer stable operation through as much as six degrees of rotation while binary operation devices offer stable activated positions with as much as 20 degrees of rotation. All arrays have desirable features of costly fabrication services like five structural layers and planarized mirror surfaces, but are prefabricated in the less costly MUMPs process. Models are developed for all devices and used to compare empirical data.

Cell Homogeneity Indispensable for Regenerative Medicine by Cultured Human Corneal Endothelial Cells.

PubMed

Hamuro, Junji; Toda, Munetoyo; Asada, Kazuko; Hiraga, Asako; Schlötzer-Schrehardt, Ursula; Montoya, Monty; Sotozono, Chie; Ueno, Morio; Kinoshita, Shigeru

2016-09-01

To identify the subpopulation (SP) among heterogeneous cultured human corneal endothelial cells (cHCECs) devoid of cell-state transition applicable for cell-based therapy. Subpopulation presence in cHCECs was confirmed via surface CD-marker expression level by flow cytometry. CD markers effective for distinguishing distinct SPs were selected by analyzing those on established cHCECs with a small cell area and high cell density. Contrasting features among three typical cHCEC SPs was confirmed by PCR array for extracellular matrix (ECM). Combined analysis of CD markers was performed to identify the SP (effector cells) applicable for therapy. ZO-1 and Na+/K+ ATPase, CD200, and HLA expression were compared among heterogeneous SPs. Flow cytometry analysis identified the effector cell expressing CD166+CD105-CD44-∼+/-CD26-CD24-, but CD200-, and the presence of other SPs with CD166+ CD105-CD44+++ (CD26 and CD24, either + or -) was confirmed. PCR array revealed three distinct ECM expression profiles. Some SPs expressed ZO-1 and Na+/K+ ATPase at comparable levels with effector cells, while only one SP expressed CD200, but not on effector cells. Human leukocyte antigen expression was most reduced in the effector SP. The proportion of effector cells (E-ratio) inversely paralleled donor age and decreased during prolonged culture passages. The presence of Rho-associated protein kinase (ROCK) inhibitor increased the E-ratio in cHCECs. The average area of effector cells was approximately 200∼220 μm2, and the density of cHCECs exceeded 2500 cells/mm2. A specified cultured effector cell population sharing the surface phenotypes with mature HCECs in corneal tissues may serve as an alternative to donor corneas for the treatment of corneal endothelial dysfunction.

Characterization of canine osteosarcoma by array comparative genomic hybridization and RT-qPCR: signatures of genomic imbalance in canine osteosarcoma parallel the human counterpart.

PubMed

Angstadt, Andrea Y; Motsinger-Reif, Alison; Thomas, Rachael; Kisseberth, William C; Guillermo Couto, C; Duval, Dawn L; Nielsen, Dahlia M; Modiano, Jaime F; Breen, Matthew

2011-11-01

Osteosarcoma (OS) is the most commonly diagnosed malignant bone tumor in humans and dogs, characterized in both species by extremely complex karyotypes exhibiting high frequencies of genomic imbalance. Evaluation of genomic signatures in human OS using array comparative genomic hybridization (aCGH) has assisted in uncovering genetic mechanisms that result in disease phenotype. Previous low-resolution (10-20 Mb) aCGH analysis of canine OS identified a wide range of recurrent DNA copy number aberrations, indicating extensive genomic instability. In this study, we profiled 123 canine OS tumors by 1 Mb-resolution aCGH to generate a dataset for direct comparison with current data for human OS, concluding that several high frequency aberrations in canine and human OS are orthologous. To ensure complete coverage of gene annotation, we identified the human refseq genes that map to these orthologous aberrant dog regions and found several candidate genes warranting evaluation for OS involvement. Specifically, subsequenct FISH and qRT-PCR analysis of RUNX2, TUSC3, and PTEN indicated that expression levels correlated with genomic copy number status, showcasing RUNX2 as an OS associated gene and TUSC3 as a possible tumor suppressor candidate. Together these data demonstrate the ability of genomic comparative oncology to identify genetic abberations which may be important for OS progression. Large scale screening of genomic imbalance in canine OS further validates the use of the dog as a suitable model for human cancers, supporting the idea that dysregulation discovered in canine cancers will provide an avenue for complementary study in human counterparts. Copyright © 2011 Wiley-Liss, Inc.

Dose–response relationships in gene expression profiles in rainbow trout, Oncorhyncus mykiss, exposed to ethynylestradiol

PubMed Central

Hook, Sharon E.; Skillman, Ann D.; Small, Jack A.; Schultz, Irvin R.

2008-01-01

Determining how gene expression profiles change with toxicant dose will improve the utility of arrays in identifying biomarkers and modes of toxic action. Isogenic rainbow trout, Oncorhyncus mykiss, were exposed to 10, 50 or 100 ng/L ethynylestradiol (a xeno-estrogen) for 7 days. Following exposure hepatic RNA was extracted. Fluorescently labeled cDNA were generated and hybridized against a commercially available Atlantic Salmon/Trout array (GRASP project, University of Victoria) spotted with 16,000 cDNAs. Transcript expression in treated vs control fish was analyzed via Genespring (Silicon Genetics) to identify genes with altered expression, as well as to determine gene clustering patterns that can be used as “expression signatures”. Array results were confirmed via qRT PCR. Our analysis indicates that gene expression profiles varied somewhat with dose. Established biomarkers of exposure to estrogenic chemicals, such as vitellogenin, vitelline envelope proteins, and the estrogen receptor alpha, were induced at every dose. Other genes were dose specific, suggesting that diffierent doses induce distinct physiological responses. These findings demonstrate that cDNA microarrays could be used to identify both toxicant class and relative dose. PMID:16725192

Rethinking mobile delivery: using Quick Response codes to access information at the point of need.

PubMed

Lombardo, Nancy T; Morrow, Anne; Le Ber, Jeanne

2012-01-01

This article covers the use of Quick Response (QR) codes to provide instant mobile access to information, digital collections, educational offerings, library website, subject guides, text messages, videos, and library personnel. The array of uses and the value of using QR codes to push customized information to patrons are explained. A case is developed for using QR codes for mobile delivery of customized information to patrons. Applications in use at the Libraries of the University of Utah will be reviewed to provide readers with ideas for use in their library. Copyright © Taylor & Francis Group, LLC

Charge Coupled Devices in Signal Processing Systems. Volume V. Final Report.

DTIC Science & Technology

1979-12-01

the Phase III program. At that time, mutual customer /contractor interest arose in a unique application area, involving manipulation of lists of...using half adders and "or" circuits. 4-35 3 b 2 b3 01 b *3b, *2 b 1 b2b 1 0 1 b, + + + + + + ++ r T 7 7 r* 7FA +-0j FA 147 7 7 1 77 7 7 TL NO.6 NO. 5...which the cell could be step-and- repeated into an array in the CAD system. In practice we found that the attendent custom skewing delay layout

Identification of clinically relevant viridans streptococci by an oligonucleotide array.

PubMed

Chen, Chao Chien; Teng, Lee Jene; Kaiung, Seng; Chang, Tsung Chain

2005-04-01

Viridans streptococci (VS) are common etiologic agents of subacute infective endocarditis and are capable of causing a variety of pyogenic infections. Many species of VS are difficult to differentiate by phenotypic traits. An oligonucleotide array based on 16S-23S rRNA gene intergenic spacer (ITS) sequences was developed to identify 11 clinically relevant VS. These 11 species were Streptococcus anginosus, S. constellatus, S. gordonii, S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguinis, S. salivarius, S. sanguinis, and S. uberis. The method consisted of PCR amplification of the ITS regions by using a pair of universal primers, followed by hybridization of the digoxigenin-labeled PCR products to a panel of species-specific oligonucleotides immobilized on a nylon membrane. After 120 strains of the 11 species of VG and 91 strains of other bacteria were tested, the sensitivity and specificity of the oligonucleotide array were found to be 100% (120 of 120 strains) and 95.6% (87 of 91 strains), respectively. S. pneumoniae cross-hybridized to the probes used for the identification of S. mitis, and simple biochemical tests such as optochin susceptibility or bile solubility should be used to differentiate S. pneumoniae from S. mitis. In conclusion, identification of species of VS by use of the present oligonucleotide array is accurate and could be used as an alternative reliable method for species identification of strains of VS.

Gene expression in developing watermelon fruit

PubMed Central

Wechter, W Patrick; Levi, Amnon; Harris, Karen R; Davis, Angela R; Fei, Zhangjun; Katzir, Nurit; Giovannoni, James J; Salman-Minkov, Ayelet; Hernandez, Alvaro; Thimmapuram, Jyothi; Tadmor, Yaakov; Portnoy, Vitaly; Trebitsh, Tova

2008-01-01

Background Cultivated watermelon form large fruits that are highly variable in size, shape, color, and content, yet have extremely narrow genetic diversity. Whereas a plethora of genes involved in cell wall metabolism, ethylene biosynthesis, fruit softening, and secondary metabolism during fruit development and ripening have been identified in other plant species, little is known of the genes involved in these processes in watermelon. A microarray and quantitative Real-Time PCR-based study was conducted in watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus] in order to elucidate the flow of events associated with fruit development and ripening in this species. RNA from three different maturation stages of watermelon fruits, as well as leaf, were collected from field grown plants during three consecutive years, and analyzed for gene expression using high-density photolithography microarrays and quantitative PCR. Results High-density photolithography arrays, composed of probes of 832 EST-unigenes from a subtracted, fruit development, cDNA library of watermelon were utilized to examine gene expression at three distinct time-points in watermelon fruit development. Analysis was performed with field-grown fruits over three consecutive growing seasons. Microarray analysis identified three hundred and thirty-five unique ESTs that are differentially regulated by at least two-fold in watermelon fruits during the early, ripening, or mature stage when compared to leaf. Of the 335 ESTs identified, 211 share significant homology with known gene products and 96 had no significant matches with any database accession. Of the modulated watermelon ESTs related to annotated genes, a significant number were found to be associated with or involved in the vascular system, carotenoid biosynthesis, transcriptional regulation, pathogen and stress response, and ethylene biosynthesis. Ethylene bioassays, performed with a closely related watermelon genotype with a similar phenotype, i.e. seeded, bright red flesh, dark green rind, etc., determined that ethylene levels were highest during the green fruit stage followed by a decrease during the white and pink fruit stages. Additionally, quantitative Real-Time PCR was used to validate modulation of 127 ESTs that were differentially expressed in developing and ripening fruits based on array analysis. Conclusion This study identified numerous ESTs with putative involvement in the watermelon fruit developmental and ripening process, in particular the involvement of the vascular system and ethylene. The production of ethylene during fruit development in watermelon gives further support to the role of ethylene in fruit development in non-climacteric fruits. PMID:18534026

IRAC test report. Gallium doped silicon band 2: Read noise and dark current

NASA Technical Reports Server (NTRS)

Lamb, Gerald; Shu, Peter; Mather, John; Ewin, Audrey; Bowser, Jeffrey

1987-01-01

A direct readout infrared detector array, a candidate for the Space Infrared Telescope Facility (SIRTF) Infrared Array Camera (IRAC), has been tested. The array has a detector surface of gallium doped silicon, bump bonded to a 58x62 pixel MOSFET multiplexer on a separate chip. Although this chip and system do not meet all the SIRTF requirements, the critically important read noise is within a factor of 3 of the requirement. Significant accomplishments of this study include: (1) development of a low noise correlated double sampling readout system with a readout noise of 127 to 164 electrons (based on the detector integrator capacitance of 0.1 pF); (2) measurement of the readout noise of the detector itself, ranging from 123 to 214 electrons with bias only (best to worst pixel), and 256 to 424 electrons with full clocking in normal operation at 5.4 K where dark current is small. Thirty percent smaller read noises are obtained at a temperature of 15K; (3) measurement of the detector response versus integration time, showing significant nonlinear behavior for large signals, well below the saturation level; and (4) development of a custom computer interface and suitable software for collection, analysis and display of data.

Business Cases for Microgrids: Modeling Interactions of Technology Choice, Reliability, Cost, and Benefit

NASA Astrophysics Data System (ADS)

Hanna, Ryan

Distributed energy resources (DERs), and increasingly microgrids, are becoming an integral part of modern distribution systems. Interest in microgrids--which are insular and autonomous power networks embedded within the bulk grid--stems largely from the vast array of flexibilities and benefits they can offer stakeholders. Managed well, they can improve grid reliability and resiliency, increase end-use energy efficiency by coupling electric and thermal loads, reduce transmission losses by generating power locally, and may reduce system-wide emissions, among many others. Whether these public benefits are realized, however, depends on whether private firms see a "business case", or private value, in investing. To this end, firms need models that evaluate costs, benefits, risks, and assumptions that underlie decisions to invest. The objectives of this dissertation are to assess the business case for microgrids that provide what industry analysts forecast as two primary drivers of market growth--that of providing energy services (similar to an electric utility) as well as reliability service to customers within. Prototypical first adopters are modeled--using an existing model to analyze energy services and a new model that couples that analysis with one of reliability--to explore interactions between technology choice, reliability, costs, and benefits. The new model has a bi-level hierarchy; it uses heuristic optimization to select and size DERs and analytical optimization to schedule them. It further embeds Monte Carlo simulation to evaluate reliability as well as regression models for customer damage functions to monetize reliability. It provides least-cost microgrid configurations for utility customers who seek to reduce interruption and operating costs. Lastly, the model is used to explore the impact of such adoption on system-wide greenhouse gas emissions in California. Results indicate that there are, at present, co-benefits for emissions reductions when customers adopt and operate microgrids for private benefit, though future analysis is needed as the bulk grid continues to transition toward a less carbon intensive system.

Zoonotic Chlamydiaceae Species Associated with Trachoma, Nepal

PubMed Central

Rothschild, James; Ruettger, Anke; Kandel, Ram Prasad; Sachse, Konrad

2013-01-01

Trachoma is the leading cause of preventable blindness. Commercial assays do not discriminate among all Chlamydiaceae species that might be involved in trachoma. We investigated whether a commercial Micro-ArrayTube could discriminate Chlamydiaceae species in DNA extracted directly from conjunctival samples from 101 trachoma patients in Nepal. To evaluate organism viability, we extracted RNA, reverse transcribed it, and subjected it to quantitative real-time PCR. We found that 71 (70.3%) villagers were infected. ArrayTube sensitivity was 91.7% and specificity was 100% compared with that of real-time PCR. Concordance between genotypes detected by microarray and ompA genotyping was 100%. Species distribution included 54 (76%) single infections with Chlamydia trachomatis, C. psittaci, C. suis, or C. pecorum, and 17 (24%) mixed infections that includied C. pneumoniae. Ocular infections were caused by 5 Chlamydiaceae species. Additional studies of trachoma pathogenesis involving Chlamydiaceae species other than C. trachomatis and their zoonotic origins are needed. PMID:24274654

Expression of Reactive Oxygen Species–Related Transcripts in Egyptian Children With Autism

PubMed Central

Meguid, Nagwa A; Ghozlan, Said A S; Mohamed, Magda F; Ibrahim, Marwa K; Dawood, Reham M; Bader El Din, Noha G; Abdelhafez, Tawfeek H; Hemimi, Maha; El Awady, Mostafa K

2017-01-01

The molecular basis of the pathophysiological role of oxidative stress in autism is understudied. Herein, we used polymerase chain reaction (PCR) array to analyze transcriptional pattern of 84 oxidative stress genes in peripheral blood mononuclear cell pools isolated from 32 autistic patients (16 mild/moderate and 16 severe) and 16 healthy subjects (each sample is a pool from 4 autistic patients or 4 controls). The PCR array data were further validated by quantitative real-time PCR in 80 autistic children (55 mild/moderate and 25 severe) and 60 healthy subjects. Our data revealed downregulation in GCLM, SOD2, NCF2, PRNP, and PTGS2 transcripts (1.5, 3.8, 1.2, 1.7, and 2.2, respectively;P < .05 for all) in autistic group compared with controls. In addition, TXN and FTH1 exhibited 1.4- and 1.7-fold downregulation, respectively, in severe autistic patients when compared with mild/moderate group (P = .005 and .0008, respectively). This study helps in a better understanding of the underlying biology and related genetic factors of autism, and most importantly, it presents suggested candidate biomarkers for diagnosis and prognosis purposes as well as targets for therapeutic intervention. PMID:28469396

The analysis of novel microRNA mimic sequences in cancer cells reveals lack of specificity in stem-loop RT-qPCR-based microRNA detection.

PubMed

Winata, Patrick; Williams, Marissa; McGowan, Eileen; Nassif, Najah; van Zandwijk, Nico; Reid, Glen

2017-11-17

MicroRNAs are frequently downregulated in cancer, and restoring expression has tumour suppressive activity in tumour cells. Our recent phase I clinical trial investigated microRNA-based therapy in patients with malignant pleural mesothelioma. Treatment with TargomiRs, microRNA mimics with novel sequence packaged in EGFR antibody-targeted bacterial minicells, revealed clear signs of clinical activity. In order to detect delivery of microRNA mimics to tumour cells in future clinical trials, we tested hydrolysis probe-based assays specific for the sequence of the novel mimics in transfected mesothelioma cell lines using RT-qPCR. The custom assays efficiently and specifically amplified the consensus mimics. However, we found that these assays gave a signal when total RNA from untransfected and control mimic-transfected cells were used as templates. Further investigation revealed that the reverse transcription step using stem-loop primers appeared to introduce substantial non-specific amplification with either total RNA or synthetic RNA templates. This suggests that reverse transcription using stem-loop primers suffers from an intrinsic lack of specificity for the detection of highly similar microRNAs in the same family, especially when analysing total RNA. These results suggest that RT-qPCR is unlikely to be an effective means to detect delivery of microRNA mimic-based drugs to tumour cells in patients.

Estimation and partitioning of (co)heritability of inflammatory bowel disease from GWAS and immunochip data.

PubMed

Chen, Guo-Bo; Lee, Sang Hong; Brion, Marie-Jo A; Montgomery, Grant W; Wray, Naomi R; Radford-Smith, Graham L; Visscher, Peter M

2014-09-01

As custom arrays are cheaper than generic GWAS arrays, larger sample size is achievable for gene discovery. Custom arrays can tag more variants through denser genotyping of SNPs at associated loci, but at the cost of losing genome-wide coverage. Balancing this trade-off is important for maximizing experimental designs. We quantified both the gain in captured SNP-heritability at known candidate regions and the loss due to imperfect genome-wide coverage for inflammatory bowel disease using immunochip (iChip) and imputed GWAS data on 61,251 and 38.550 samples, respectively. For Crohn's disease (CD), the iChip and GWAS data explained 19 and 26% of variation in liability, respectively, and SNPs in the densely genotyped iChip regions explained 13% of the SNP-heritability for both the iChip and GWAS data. For ulcerative colitis (UC), the iChip and GWAS data explained 15 and 19% of variation in liability, respectively, and the dense iChip regions explained 10 and 9% of the SNP-heritability in the iChip and the GWAS data. From bivariate analyses, estimates of the genetic correlation in risk between CD and UC were 0.75 (SE 0.017) and 0.62 (SE 0.042) for the iChip and GWAS data, respectively. We also quantified the SNP-heritability of genomic regions that did or did not contain the previous 163 GWAS hits for CD and UC, and SNP-heritability of the overlapping loci between the densely genotyped iChip regions and the 163 GWAS hits. For both diseases, over different genomic partitioning, the densely genotyped regions on the iChip tagged at least as much variation in liability as in the corresponding regions in the GWAS data, however a certain amount of tagged SNP-heritability in the GWAS data was lost using the iChip due to the low coverage at unselected regions. These results imply that custom arrays with a GWAS backbone will facilitate more gene discovery, both at associated and novel loci. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

SUSD2 is frequently downregulated and functions as a tumor suppressor in RCC and lung cancer.

PubMed

Cheng, Yingying; Wang, Xiaolin; Wang, Pingzhang; Li, Ting; Hu, Fengzhan; Liu, Qiang; Yang, Fan; Wang, Jun; Xu, Tao; Han, Wenling

2016-07-01

Sushi domain containing 2 (SUSD2) is type I membrane protein containing domains inherent to adhesion molecules. There have been few reported studies on SUSD2, and they have mainly focused on breast cancer, colon cancer, and HeLa cells. However, the expression and function of SUSD2 in other cancers remain unclear. In the present study, we conducted an integrated bioinformatics analysis based on the array data from the GEO database and found a significant downregulation of SUSD2 in renal cell carcinoma (RCC) and lung cancer. Western blotting and quantitative RT-PCR (qRT-PCR) confirmed that SUSD2 was frequently decreased in RCC and lung cancer tissues compared with the corresponding levels in normal adjacent tissues. The restoration of SUSD2 expression inhibited the proliferation and clonogenicity of RCC and lung cancer cells, whereas the knockdown of SUSD2 promoted A549 cell growth. Our findings suggested that SUSD2 functions as a tumor suppressor gene (TSG) in RCC and lung cancer.

Incorporating Servqual-QFD with Taguchi Design for optimizing service quality design

NASA Astrophysics Data System (ADS)

Arbi Hadiyat, M.

2018-03-01

Deploying good service design in service companies has been updated issue in improving customer satisfaction, especially based on the level of service quality measured by Parasuraman’s SERVQUAL. Many researchers have been proposing methods in designing the service, and some of them are based on engineering viewpoint, especially by implementing the QFD method or even using robust Taguchi method. The QFD method would found the qualitative solution by generating the “how’s”, while Taguchi method gives more quantitative calculation in optimizing best solution. However, incorporating both QFD and Taguchi has been done in this paper and yields better design process. The purposes of this research is to evaluate the incorporated methods by implemented it to a case study, then analyze the result and see the robustness of those methods to customer perception of service quality. Started by measuring service attributes using SERVQUAL and find the improvement with QFD, the deployment of QFD solution then generated by defining Taguchi factors levels and calculating the Signal-to-noise ratio in its orthogonal array, and optimized Taguchi response then found. A case study was given for designing service in local bank. Afterward, the service design obtained from previous analysis was then evaluated and shows that it was still meet the customer satisfaction. Incorporating QFD and Taguchi has performed well and can be adopted and developed for another research for evaluating the robustness of result.

Generation of dark hollow beam via coherent combination based on adaptive optics.

PubMed

Zheng, Yi; Wang, Xiaohua; Shen, Feng; Li, Xinyang

2010-12-20

A novel method for generating a dark hollow beam (DHB) is proposed and studied both theoretically and experimentally. A coherent combination technique for laser arrays is implemented based on adaptive optics (AO). A beam arraying structure and an active segmented mirror are designed and described. Piston errors are extracted by a zero-order interference detection system with the help of a custom-made photo-detectors array. An algorithm called the extremum approach is adopted to calculate feedback control signals. A dynamic piston error is imported by LiNbO₃ to test the capability of the AO servo. In a closed loop the stable and clear DHB is obtained. The experimental results confirm the feasibility of the concept.

X-ray photon correlation spectroscopy using a fast pixel array detector with a grid mask resolution enhancer.

PubMed

Hoshino, Taiki; Kikuchi, Moriya; Murakami, Daiki; Harada, Yoshiko; Mitamura, Koji; Ito, Kiminori; Tanaka, Yoshihito; Sasaki, Sono; Takata, Masaki; Jinnai, Hiroshi; Takahara, Atsushi

2012-11-01

The performance of a fast pixel array detector with a grid mask resolution enhancer has been demonstrated for X-ray photon correlation spectroscopy (XPCS) measurements to investigate fast dynamics on a microscopic scale. A detecting system, in which each pixel of a single-photon-counting pixel array detector, PILATUS, is covered by grid mask apertures, was constructed for XPCS measurements of silica nanoparticles in polymer melts. The experimental results are confirmed to be consistent by comparison with other independent experiments. By applying this method, XPCS measurements can be carried out by customizing the hole size of the grid mask to suit the experimental conditions, such as beam size, detector size and sample-to-detector distance.

Development of the New DAQ System for the SD Array of TA×4 and TALE

NASA Astrophysics Data System (ADS)

Takahashi, Yuichi; Sahara, Ryosuke; Konishi, Shogo; Goto, Takashi; Ogio, Shoichi

The data acquisition (DAQ) system for the surface detector (SD) arrays of TA×4 and TALE will be presented. Each SD records digital signals with 50 MHz FADCs and sends the data to a central communication center (of the "communication tower") via a wireless network system. The technique employed here is based on the currently-running DAQ system of the Telescope Array, and there are some improvements including i) replacement of a wireless LAN module with a custom protocol to another with TCP/IP, and ii) replacement of the "tower electronics" to a generic Linux board PC of Raspberry Pi Type II B. The details and performance of the new DAQ system are described below.

Optical Links and RF Distribution for Antenna Arrays

NASA Technical Reports Server (NTRS)

Huang, Shouhua; Calhoun, Malcolm; Tjoelker, Robert

2006-01-01

An array of three antennas has recently been developed at the NASA Jet Propulsion Laboratory capable of detecting signals at X and Ka band. The array requires a common frequency reference and high precision phase alignment to correlate received signals. Frequency and timing references are presently provided from a remotely located hydrogen maser and clock through a combination of commercially and custom developed optical links. The selected laser, photodetector, and fiber components have been tested under anticipated thermal and simulated antenna rotation conditions. The resulting stability limitations due to thermal perturbations or induced stress on the optical fiber have been characterized. Distribution of the X band local oscillator includes a loop back and precision phase monitor to enable correlation of signals received from each antenna.

Adaptation of commercial microscopes for advanced imaging applications

NASA Astrophysics Data System (ADS)

Brideau, Craig; Poon, Kelvin; Stys, Peter

2015-03-01

Today's commercially available microscopes offer a wide array of options to accommodate common imaging experiments. Occasionally, an experimental goal will require an unusual light source, filter, or even irregular sample that is not compatible with existing equipment. In these situations the ability to modify an existing microscopy platform with custom accessories can greatly extend its utility and allow for experiments not possible with stock equipment. Light source conditioning/manipulation such as polarization, beam diameter or even custom source filtering can easily be added with bulk components. Custom and after-market detectors can be added to external ports using optical construction hardware and adapters. This paper will present various examples of modifications carried out on commercial microscopes to address both atypical imaging modalities and research needs. Violet and near-ultraviolet source adaptation, custom detection filtering, and laser beam conditioning and control modifications will be demonstrated. The availability of basic `building block' parts will be discussed with respect to user safety, construction strategies, and ease of use.

Arrays of Miniature Microphones for Aeroacoustic Testing

NASA Technical Reports Server (NTRS)

Shams, Qamar A.; Humphreys, William M.; Sealey, Bradley S.; Bartram, Scott M.; Zuckewar, Allan J.; Comeaux, Toby; Adams, James K.

2007-01-01

A phased-array system comprised of custom-made and commercially available microelectromechanical system (MEMS) silicon microphones and custom ancillary hardware has been developed for use in aeroacoustic testing in hard-walled and acoustically treated wind tunnels. Recent advances in the areas of multi-channel signal processing and beam forming have driven the construction of phased arrays containing ever-greater numbers of microphones. Traditional obstacles to this trend have been posed by (1) the high costs of conventional condenser microphones, associated cabling, and support electronics and (2) the difficulty of mounting conventional microphones in the precise locations required for high-density arrays. The present development overcomes these obstacles. One of the hallmarks of the new system is a series of fabricated platforms on which multiple microphones can be mounted. These mounting platforms, consisting of flexible polyimide circuit-board material (see left side of figure), include all the necessary microphone power and signal interconnects. A single bus line connects all microphones to a common power supply, while the signal lines terminate in one or more data buses on the sides of the circuit board. To minimize cross talk between array channels, ground lines are interposed as shields between all the data bus signal lines. The MEMS microphones are electrically connected to the boards via solder pads that are built into the printed wiring. These flexible circuit boards share many characteristics with their traditional rigid counterparts, but can be manufactured much thinner, as small as 0.1 millimeter, and much lighter with boards weighing as much as 75 percent less than traditional rigid ones. For a typical hard-walled wind-tunnel installation, the flexible printed-circuit board is bonded to the tunnel wall and covered with a face sheet that contains precise cutouts for the microphones. Once the face sheet is mounted, a smooth surface is established over the entire array due to the flush mounting of all microphones (see right side of figure). The face sheet is made from a continuous glass-woven-fabric base impregnated with an epoxy resin binder. This material offers a combination of high mechanical strength and low dielectric loss, making it suitable for withstanding the harsh test section environment present in many wind tunnels, while at the same time protecting the underlying polyimide board. Customized signal-conditioning hardware consisting of line drivers and antialiasing filters are coupled with the array. The line drivers are constructed using low-supply-current, high-gain-bandwidth operational amplifiers designed to transmit the microphone signals several dozen feet from the array to external acquisition hardware. The anti-alias filters consist of individual Chebyshev low-pass filters (one for each microphone channel) housed on small printed-circuit boards mounted on one or more motherboards. The mother/daughter board design results in a modular system, which is easy to debug and service and which enables the filter characteristics to be changed by swapping daughter boards with ones containing different filter parameters. The filter outputs are passed to commercially- available acquisition hardware to digitize and store the conditioned microphone signals. Wind-tunnel testing of the new MEMS microphone polyimide mounting system shows that the array performance is comparable to that of traditional arrays, but with significantly less cost of construction.

Accuracy of CNV Detection from GWAS Data.

PubMed

Zhang, Dandan; Qian, Yudong; Akula, Nirmala; Alliey-Rodriguez, Ney; Tang, Jinsong; Gershon, Elliot S; Liu, Chunyu

2011-01-13

Several computer programs are available for detecting copy number variants (CNVs) using genome-wide SNP arrays. We evaluated the performance of four CNV detection software suites--Birdsuite, Partek, HelixTree, and PennCNV-Affy--in the identification of both rare and common CNVs. Each program's performance was assessed in two ways. The first was its recovery rate, i.e., its ability to call 893 CNVs previously identified in eight HapMap samples by paired-end sequencing of whole-genome fosmid clones, and 51,440 CNVs identified by array Comparative Genome Hybridization (aCGH) followed by validation procedures, in 90 HapMap CEU samples. The second evaluation was program performance calling rare and common CNVs in the Bipolar Genome Study (BiGS) data set (1001 bipolar cases and 1033 controls, all of European ancestry) as measured by the Affymetrix SNP 6.0 array. Accuracy in calling rare CNVs was assessed by positive predictive value, based on the proportion of rare CNVs validated by quantitative real-time PCR (qPCR), while accuracy in calling common CNVs was assessed by false positive/false negative rates based on qPCR validation results from a subset of common CNVs. Birdsuite recovered the highest percentages of known HapMap CNVs containing >20 markers in two reference CNV datasets. The recovery rate increased with decreased CNV frequency. In the tested rare CNV data, Birdsuite and Partek had higher positive predictive values than the other software suites. In a test of three common CNVs in the BiGS dataset, Birdsuite's call was 98.8% consistent with qPCR quantification in one CNV region, but the other two regions showed an unacceptable degree of accuracy. We found relatively poor consistency between the two "gold standards," the sequence data of Kidd et al., and aCGH data of Conrad et al. Algorithms for calling CNVs especially common ones need substantial improvement, and a "gold standard" for detection of CNVs remains to be established.

Transparent Fingerprint Sensor System for Large Flat Panel Display.

PubMed

Seo, Wonkuk; Pi, Jae-Eun; Cho, Sung Haeung; Kang, Seung-Youl; Ahn, Seong-Deok; Hwang, Chi-Sun; Jeon, Ho-Sik; Kim, Jong-Uk; Lee, Myunghee

2018-01-19

In this paper, we introduce a transparent fingerprint sensing system using a thin film transistor (TFT) sensor panel, based on a self-capacitive sensing scheme. An armorphousindium gallium zinc oxide (a-IGZO) TFT sensor array and associated custom Read-Out IC (ROIC) are implemented for the system. The sensor panel has a 200 × 200 pixel array and each pixel size is as small as 50 μm × 50 μm. The ROIC uses only eight analog front-end (AFE) amplifier stages along with a successive approximation analog-to-digital converter (SAR ADC). To get the fingerprint image data from the sensor array, the ROIC senses a capacitance, which is formed by a cover glass material between a human finger and an electrode of each pixel of the sensor array. Three methods are reviewed for estimating the self-capacitance. The measurement result demonstrates that the transparent fingerprint sensor system has an ability to differentiate a human finger's ridges and valleys through the fingerprint sensor array.

Transparent Fingerprint Sensor System for Large Flat Panel Display

PubMed Central

Seo, Wonkuk; Pi, Jae-Eun; Cho, Sung Haeung; Kang, Seung-Youl; Ahn, Seong-Deok; Hwang, Chi-Sun; Jeon, Ho-Sik; Kim, Jong-Uk

2018-01-01

In this paper, we introduce a transparent fingerprint sensing system using a thin film transistor (TFT) sensor panel, based on a self-capacitive sensing scheme. An armorphousindium gallium zinc oxide (a-IGZO) TFT sensor array and associated custom Read-Out IC (ROIC) are implemented for the system. The sensor panel has a 200 × 200 pixel array and each pixel size is as small as 50 μm × 50 μm. The ROIC uses only eight analog front-end (AFE) amplifier stages along with a successive approximation analog-to-digital converter (SAR ADC). To get the fingerprint image data from the sensor array, the ROIC senses a capacitance, which is formed by a cover glass material between a human finger and an electrode of each pixel of the sensor array. Three methods are reviewed for estimating the self-capacitance. The measurement result demonstrates that the transparent fingerprint sensor system has an ability to differentiate a human finger’s ridges and valleys through the fingerprint sensor array. PMID:29351218

Chronic, percutaneous connector for electrical recording and stimulation with microelectrode arrays.

PubMed

Shah, Kedar G; Lee, Kye Young; Tolosa, Vanessa; Tooker, Angela; Felix, Sarah; Benett, William; Pannu, Satinderpall

2014-01-01

The translation of advances in neural stimulation and recording research into clinical practice hinges on the ability to perform chronic experiments in awake and behaving animal models. Advances in microelectrode array technology, most notably flexible polymer arrays, have significantly improved reliability of the neural interface. However, electrical connector technology has lagged and is prone to failure from non-biocompatibility, large size, contamination, corrosion, and difficulty of use. We present a novel chronic, percutaneous electrical connector system that is suitable for neural stimulation and recording. This system features biocompatible materials, low connect and disconnect forces, passive alignment, and a protective cap during non-use. We have successfully designed, assembled, and tested in vitro both a 16-channel system and a high density 64-channel system. Custom, polyimide, 16-channel, microelectrode arrays were electrically assembled with the connector system and tested using cyclic voltammetry and electrochemical impedance spectroscopy. This connector system is versatile and can be used with a variety of microelectrode array technologies for chronic studies.

Integrated electronics for time-resolved array of single-photon avalanche diodes

NASA Astrophysics Data System (ADS)

Acconcia, G.; Crotti, M.; Rech, I.; Ghioni, M.

2013-12-01

The Time Correlated Single Photon Counting (TCSPC) technique has reached a prominent position among analytical methods employed in a great variety of fields, from medicine and biology (fluorescence spectroscopy) to telemetry (laser ranging) and communication (quantum cryptography). Nevertheless the development of TCSPC acquisition systems featuring both a high number of parallel channels and very high performance is still an open challenge: to satisfy the tight requirements set by the applications, a fully parallel acquisition system requires not only high efficiency single photon detectors but also a read-out electronics specifically designed to obtain the highest performance in conjunction with these sensors. To this aim three main blocks have been designed: a gigahertz bandwidth front-end stage to directly read the custom technology SPAD array avalanche current, a reconfigurable logic to route the detectors output signals to the acquisition chain and an array of time measurement circuits capable of recording the photon arrival times with picoseconds time resolution and a very high linearity. An innovative architecture based on these three circuits will feature a very high number of detectors to perform a truly parallel spatial or spectral analysis and a smaller number of high performance time-to-amplitude converter offering very high performance and a very high conversion frequency while limiting the area occupation and power dissipation. The routing logic will make the dynamic connection between the two arrays possible in order to guarantee that no information gets lost.

Molecular analysis of microbial diversity in advanced caries.

PubMed

Chhour, Kim-Ly; Nadkarni, Mangala A; Byun, Roy; Martin, F Elizabeth; Jacques, Nicholas A; Hunter, Neil

2005-02-01

Real-time PCR analysis of the total bacterial load in advanced carious lesions has shown that the total load exceeds the number of cultivable bacteria. This suggests that an unresolved complexity exists in bacteria associated with advanced caries. In this report, the profile of the microflora of carious dentine was explored by using DNA extracted from 10 lesions selected on the basis of comparable total microbial load and on the relative abundance of Prevotella spp. Using universal primers for the 16S rRNA gene, PCR amplicons were cloned, and approximately 100 transformants were processed for each lesion. Phylogenetic analysis of 942 edited sequences demonstrated the presence of 75 species or phylotypes in the 10 carious lesions. Up to 31 taxa were represented in each sample. A diverse array of lactobacilli were found to comprise 50% of the species, with prevotellae also abundant, comprising 15% of the species. Other taxa present in a number of lesions or occurring with high abundance included Selenomonas spp., Dialister spp., Fusobacterium nucleatum, Eubacterium spp., members of the Lachnospiraceae family, Olsenella spp., Bifidobacterium spp., Propionibacterium sp., and Pseudoramibacter alactolyticus. The mechanisms by which such diverse patterns of bacteria extend carious lesions, including the aspect of infection of the vital dental pulp, remain unclear.

CIDR

Science.gov Websites

NIH CIDR Program Studies For whole exome sequencing projects, we pretest all samples using a high -density SNP array (>200,000 markers). For custom targeted sequencing, we pretest all samples using a 96 pretest samples using a 96 SNP GoldenGate assay. This extensive pretesting allows us to unambiguously tie

Librarians as Community Partners: An Outreach Handbook

ERIC Educational Resources Information Center

Smallwood, Carol, Ed.

2010-01-01

Including 66 focused snapshots of outreach in action, this resource reflects the creative solutions of librarians searching for new and innovative ways to build programs that meet customer needs while expanding the library's scope into the community. This contributed volume includes: (1) A huge array of program options for partnering with other…

CIDR

Science.gov Websites

variety of arrays appropriate for a wide breadth of study design needs. Genomic coverage of many of the chromosomal anomalies are services offered at NO ADDITIONAL COST to study investigators with GWAS projects be submitted for both the initial GWAS study as well as replication using our custom SNP service

Human minisatellite alleles detectable only after PCR amplification.

PubMed

Armour, J A; Crosier, M; Jeffreys, A J

1992-01-01

We present evidence that a proportion of alleles at two human minisatellite loci is undetected by standard Southern blot hybridization. In each case the missing allele(s) can be identified after PCR amplification and correspond to tandem arrays too short to detect by hybridization. At one locus, there is only one undetected allele (population frequency 0.3), which contains just three repeat units. At the second locus, there are at least five undetected alleles (total population frequency 0.9) containing 60-120 repeats; they are not detected because these tandem repeats give very poor signals when used as a probe in standard Southern blot hybridization, and also cross-hybridize with other sequences in the genome. Under these circumstances only signals from the longest tandemly repeated alleles are detectable above the nonspecific background. The structures of these loci have been compared in human and primate DNA, and at one locus the short human allele containing three repeat units is shown to be an intermediate state in the expansion of a monomeric precursor allele in primates to high copy number in the longer human arrays. We discuss the implications of such loci for studies of human populations, minisatellite isolation by cloning, and the evolution of highly variable tandem arrays.

What is a free customer worth? Armchair calculations of nonpaying customers' value can lead to flawed strategies.

PubMed

Gupta, Sunil; Mela, Carl F

2008-11-01

Free customers who are subsidized by paying customers are essential to a vast array of businesses, such as media companies, employment services, and even IT providers. But because they generate revenue only indirectly, figuring out the true value of those customers--and how much attention to devote them--has always been a challenge. Traditional customer-valuation models don't help; they focus exclusively on paying customers and largely ignore network effects, or how customers help draw other customers to a business. Now a new model, devised by professors Gupta, of Harvard Business School, and Mela, of Fuqua School of Business, takes into account not only direct network effects (where buyers attract more buyers or sellers more sellers) but also indirect network effects (where buyers attract more sellers or vice versa) . The model calculates the precise long-term impact of each additional free customer on a company's profits, factoring in the degree to which he or she brings in other customers--whether free or paying--and the ripple effect of those customers. The model helped an online auction house make several critical decisions. The business made its money on fees charged to sellers but recognized that its free customers--its buyers--were valuable, too. As competition heated up, the company worried that it wasn't wooing enough buyers. Using the model, the business discovered that the network effects of buyers were indeed large and that those customers were worth over $1,000 each--much more than had been assumed. Armed with that information, the firm increased its research on buyers, invested more in targeting them with ads, and improved their experience. The model also helped the company identify the effects of various pricing strategies on sellers, showing that they became less price-sensitive over time. As a result, the company raised the fees it charged them as well.

Development of a c-scan photoacoutsic imaging probe for prostate cancer detection

NASA Astrophysics Data System (ADS)

Valluru, Keerthi S.; Chinni, Bhargava K.; Rao, Navalgund A.; Bhatt, Shweta; Dogra, Vikram S.

2011-03-01

Prostate cancer is the second leading cause of death in American men after lung cancer. The current screening procedures include Digital Rectal Exam (DRE) and Prostate Specific Antigen (PSA) test, along with Transrectal Ultrasound (TRUS). All suffer from low sensitivity and specificity in detecting prostate cancer in early stages. There is a desperate need for a new imaging modality. We are developing a prototype transrectal photoacoustic imaging probe to detect prostate malignancies in vivo that promises high sensitivity and specificity. To generate photoacoustic (PA) signals, the probe utilizes a high energy 1064 nm laser that delivers light pulses onto the prostate at 10Hz with 10ns duration through a fiber optic cable. The designed system will generate focused C-scan planar images using acoustic lens technology. A 5 MHz custom fabricated ultrasound sensor array located in the image plane acquires the focused PA signals, eliminating the need for any synthetic aperture focusing. The lens and sensor array design was optimized towards this objective. For fast acquisition times, a custom built 16 channel simultaneous backend electronics PCB has been developed. It consists of a low-noise variable gain amplifier and a 16 channel ADC. Due to the unavailability of 2d ultrasound arrays, in the current implementation several B-scan (depth-resolved) data is first acquired by scanning a 1d array, which is then processed to reconstruct either 3d volumetric images or several C-scan planar images. Experimental results on excised tissue using a in-vitro prototype of this technology are presented to demonstrate the system capability in terms of resolution and sensitivity.

A high-throughput study on endothelial cell adhesion and growth mediated by adsorbed serum protein via signaling pathway PCR array

PubMed Central

Qu, Yayun; Hong, Ying; Huang, Yan; Zhang, Yiwen; Yang, Dayun; Zhang, Fudan; Xi, Tingfei; Zhang, Deyuan

2018-01-01

Abstract The purpose of this paper is to utilize the signaling pathway polymerase chain reaction (PCR) arrays to investigate the activation of two important biological signaling pathways in endothelial cell adhesion and growth mediated by adsorbed serum protein on the surface of bare and titanium nitride (TiN)-coated nickel titanium (NiTi) alloys. First, the endothelial cells were cultured on the bare and TiN-coated NiTi alloys and chitosan films as control for 4 h and 24 h, respectively. Then, the total RNA of the cells was collected and the PCR arrays were performed. After that, the differentially expressed genes in the transforming growth factor beta (TGF-β) signaling pathway and the regulation of actin cytoskeleton pathway were screened out; and the further bioinformatics analyses were performed. The results showed that both TGF-β signaling pathway and regulation of actin cytoskeleton pathway were activated in the cells after 4 h and 24 h culturing on the surface of bare and TiN-coated NiTi alloys compared to the chitosan group. The activated TGF-β signaling pathway promoted cell adhesion; the activated regulation of actin cytoskeleton pathway promoted cell adhesion, spreading, growth and motility. In addition, the activation of both pathways was much stronger in the cells cultured for 24 h versus 4 h, which indicated that cell adhesion and growth became more favorable with longer time on the surface of two NiTi alloy materials. PMID:29423265

Assembly of ordered contigs of cosmids selected with YACs of human chromosome 13

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

Fischer, S.G.; Cayanis, E.; Boukhgalter, B.

1994-06-01

The authors have developed an efficient method for assembling ordered cosmid contigs aligned to mega-YACs and midi-YACs (average insert sizes of 1.0 and 0.35 Mb, respectively) and used this general method to initiate high-resolution physical mapping of human chromosome 13 (Chr 13). Chr 13-enriched midi-YAC (mYAC) and mega-YAC (MYAC) sublibraries were obtained from corresponding CEPH total human YAC libraries by selecting colonies with inter-Alu PCR probes derived from Chr 13 monochromosomal cell hybrid DNA. These sublibraries were arrayed on filters at high density. In this approach, the MYAC 13 sublibrary is screened by hybridization with cytogenetically assigned Chr 13 DNAmore » probes to select one or a small subset of MYACs. Inter-Alu PCR products from each mYAC are then hybridized to the MYAC and mYAC sublibraries to identify overlapping YACs and to an arrayed Chr 13-specific cosmid library to select corresponding cosmids. The set of selected cosmids, gridded on filters at high density, is hybridized with inter-Alu PCR products from each of the overlapping YACs to identify subsets of cosmids and also with riboprobes from each cosmid of the arrayed set ({open_quotes}cosmid matrix cross-hybridization{close_quotes}). From these data, cosmid contigs are assembled by a specifically designed computer program. Application of this method generates cosmid contigs spanning the length of a MYAC with few gaps. To provide a high-resolution map, ends of cosmids are sequenced at preselected sites to position densely spaced sequence-tagged sites. 33 refs., 7 figs., 1 tab.« less

SAR processing on the MPP

NASA Technical Reports Server (NTRS)

Batcher, K. E.; Eddey, E. E.; Faiss, R. O.; Gilmore, P. A.

1981-01-01

The processing of synthetic aperture radar (SAR) signals using the massively parallel processor (MPP) is discussed. The fast Fourier transform convolution procedures employed in the algorithms are described. The MPP architecture comprises an array unit (ARU) which processes arrays of data; an array control unit which controls the operation of the ARU and performs scalar arithmetic; a program and data management unit which controls the flow of data; and a unique staging memory (SM) which buffers and permutes data. The ARU contains a 128 by 128 array of bit-serial processing elements (PE). Two-by-four surarrays of PE's are packaged in a custom VLSI HCMOS chip. The staging memory is a large multidimensional-access memory which buffers and permutes data flowing with the system. Efficient SAR processing is achieved via ARU communication paths and SM data manipulation. Real time processing capability can be realized via a multiple ARU, multiple SM configuration.

Performance of the Fully Digital FPGA-Based Front-End Electronics for the GALILEO Array

NASA Astrophysics Data System (ADS)

Barrientos, D.; Bellato, M.; Bazzacco, D.; Bortolato, D.; Cocconi, P.; Gadea, A.; González, V.; Gulmini, M.; Isocrate, R.; Mengoni, D.; Pullia, A.; Recchia, F.; Rosso, D.; Sanchis, E.; Toniolo, N.; Ur, C. A.; Valiente-Dobón, J. J.

2015-12-01

In this work we present the architecture and results of a fully digital Front End Electronics (FEE) read out system developed for the GALILEO array. The FEE system, developed in collaboration with the Advanced Gamma Tracking Array (AGATA) collaboration, is composed of three main blocks: preamplifiers, digitizers and preprocessing electronics. The slow control system contains a custom Linux driver, a dynamic library and a server implementing network services. This work presents the first results of the digital FEE system coupled with a GALILEO germanium detector, which has demonstrated the capability to achieve an energy resolution of 1.530/00 at an energy of 1.33 MeV, similar to the one obtained with a conventional analog system. While keeping a good performance in terms of energy resolution, digital electronics will allow to instrument the full GALILEO array with a versatile system with high integration and low power consumption and costs.

Quantitative High-Resolution Genomic Analysis of Single Cancer Cells

PubMed Central

Hannemann, Juliane; Meyer-Staeckling, Sönke; Kemming, Dirk; Alpers, Iris; Joosse, Simon A.; Pospisil, Heike; Kurtz, Stefan; Görndt, Jennifer; Püschel, Klaus; Riethdorf, Sabine; Pantel, Klaus; Brandt, Burkhard

2011-01-01

During cancer progression, specific genomic aberrations arise that can determine the scope of the disease and can be used as predictive or prognostic markers. The detection of specific gene amplifications or deletions in single blood-borne or disseminated tumour cells that may give rise to the development of metastases is of great clinical interest but technically challenging. In this study, we present a method for quantitative high-resolution genomic analysis of single cells. Cells were isolated under permanent microscopic control followed by high-fidelity whole genome amplification and subsequent analyses by fine tiling array-CGH and qPCR. The assay was applied to single breast cancer cells to analyze the chromosomal region centred by the therapeutical relevant EGFR gene. This method allows precise quantitative analysis of copy number variations in single cell diagnostics. PMID:22140428

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

PubMed Central

Eggleston, Paul; Zhao, Yuguang

2001-01-01

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

Selection of internal reference genes for normalization of reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis in the rumen epithelium.

PubMed

Die, Jose V; Baldwin, Ransom L; Rowland, Lisa J; Li, Robert; Oh, Sunghee; Li, Congjun; Connor, Erin E; Ranilla, Maria-Jose

2017-01-01

The rumen is lined on the luminal side by a stratified squamous epithelium that is responsible for not only absorption, but also transport, extensive short-chain fatty acid (SCFA) metabolism and protection. Butyrate has been demonstrated to initiate the differentiation of the tissue following introduction of solid feed to the weaning neonate as well as affecting the metabolism of other nutrients and absorption of nutrients in in vitro experiments. The objective of the present study was to validate expression stability of eight putative reference genes bovine rumen, considering the intrinsic heterogeneity of bovine rumen with regard to different luminal characteristics due to direct infusion of butyrate to double the intra-ruminal content of the rumen liquor. Our focus was on identifying stable reference genes which are suitable to normalize real-time RT-qPCR experiments from rumen samples collected from clinical assays, irrespective of localization within the organ and the across physiological state. The most stably expressed genes included: ACTB, UXT, DBNDD2, RPS9, DDX54 and HMBS. Their high stability values suggest these reference genes will facilitate better evaluation of variation of across an array of conditions including: localization within the rumen, differences among cattle fed an array of rations, as well as response to development in the weaning animal. Moreover, we anticipate these reference genes may be useful for expression studies in other ruminants.

Genetic linkage analysis using pooled DNA and infrared detection of tailed STRP primer patterns

NASA Astrophysics Data System (ADS)

Oetting, William S.; Wildenberg, Scott C.; King, Richard A.

1996-04-01

The mapping of a disease locus to a specific chromosomal region is an important step in the eventual isolation and analysis of a disease causing gene. Conventional mapping methods analyze large multiplex families and/or smaller nuclear families to find linkage between the disease and a chromosome marker that maps to a known chromosomal region. This analysis is time consuming and tedious, typically requiring the determination of 30,000 genotypes or more. For appropriate populations, we have instead utilized pooled DNA samples for gene mapping which greatly reduces the amount of time necessary for an initial chromosomal screen. This technique assumes a common founder for the disease locus of interest and searches for a region of a chromosome shared between affected individuals. Our analysis involves the PCR amplification of short tandem repeat polymorphisms (STRP) to detect these shared regions. In order to reduce the cost of genotyping, we have designed unlabeled tailed PCR primers which, when combined with a labeled universal primer, provides for an alternative to synthesizing custom labeled primers. The STRP pattern is visualized with an infrared fluorescence based automated DNA sequencer and the patterns quantitated by densitometric analysis of the allele pattern. Differences in the distribution of alleles between pools of affected and unaffected individuals, including a reduction in the number of alleles in the affected pool, indicate the sharing of a region of a chromosome. We have found this method effective for markers 10 - 15 cM away from the disease locus for a recessive genetic disease.

Pyrethroid insecticide lambda-cyhalothrin induces hepatic cytochrome P450 enzymes, oxidative stress and apoptosis in rats.

PubMed

Martínez, María-Aránzazu; Ares, Irma; Rodríguez, José-Luis; Martínez, Marta; Roura-Martínez, David; Castellano, Victor; Lopez-Torres, Bernardo; Martínez-Larrañaga, María-Rosa; Anadón, Arturo

2018-08-01

This study aimed to examine in rats the effects of the Type II pyrethroid lambda-cyhalothrin on hepatic microsomal cytochrome P450 (CYP) isoform activities, oxidative stress markers, gene expression of proinflammatory, oxidative stress and apoptosis mediators, and CYP isoform gene expression and metabolism phase I enzyme PCR array analysis. Lambda-cyhalothrin, at oral doses of 1, 2, 4 and 8mg/kg bw for 6days, increased, in a dose-dependent manner, hepatic activities of ethoxyresorufin O-deethylase (CYP1A1), methoxyresorufin O-demethylase (CYP1A2), pentoxyresorufin O-depentylase (CYP2B1/2), testosterone 7α- (CYP2A1), 16β- (CYP2B1), and 6β-hydroxylase (CYP3A1/2), and lauric acid 11- and 12-hydroxylase (CYP4A1/2). Similarly, lambda-cyhalothrin (4 and 8mg/kg bw, for 6days), in a dose-dependent manner, increased significantly hepatic CYP1A1, 1A2, 2A1, 2B1, 2B2, 2E1, 3A1, 3A2 and 4A1 mRNA levels and IL-1β, NFκB, Nrf2, p53, caspase-3 and Bax gene expressions. PCR array analysis showed from 84 genes examined (P<0.05; fold change>1.5), changes in mRNA levels in 18 genes: 13 up-regulated and 5 down-regulated. A greater fold change reversion than 3-fold was observed on the up-regulated ALDH1A1, CYP2B2, CYP2C80 and CYP2D4 genes. Ingenuity Pathway Analysis (IPA) groups the expressed genes into biological mechanisms that are mainly related to drug metabolism. In the top canonical pathways, Oxidative ethanol degradation III together with Fatty Acid α-oxidation may be significant pathways for lambda-cyhalothrin. Our results may provide further understanding of molecular aspects involved in lambda-cyhalothrin-induced liver injury. Copyright © 2018. Published by Elsevier B.V.

Detection of growth hormone doping by gene expression profiling of peripheral blood.

PubMed

Mitchell, Christopher J; Nelson, Anne E; Cowley, Mark J; Kaplan, Warren; Stone, Glenn; Sutton, Selina K; Lau, Amie; Lee, Carol M Y; Ho, Ken K Y

2009-12-01

GH abuse is a significant problem in many sports, and there is currently no robust test that allows detection of doping beyond a short window after administration. Our objective was to evaluate gene expression profiling in peripheral blood leukocytes in-vivo as a test for GH doping in humans. Seven men and thirteen women were administered GH, 2 mg/d sc for 8 wk. Blood was collected at baseline and at 8 wk. RNA was extracted from the white cell fraction. Microarray analysis was undertaken using Agilent 44K G4112F arrays using a two-color design. Quantitative RT-PCR using TaqMan gene expression assays was performed for validation of selected differentially expressed genes. GH induced an approximately 2-fold increase in circulating IGF-I that was maintained throughout the 8 wk of the study. GH induced significant changes in gene expression with 353 in women and 41 in men detected with a false discovery rate of less than 5%. None of the differentially expressed genes were common between men and women. The maximal changes were a doubling for up-regulated or halving for down-regulated genes, similar in magnitude to the variation between individuals. Quantitative RT-PCR for seven target genes showed good concordance between microarray and quantitative PCR data in women but not in men. Gene expression analysis of peripheral blood leukocytes is unlikely to be a viable approach for the detection of GH doping.

Development of a photodiode array biochip using a bipolar semiconductor and its application to detection of human papilloma virus.

PubMed

Baek, Taek Jin; Park, Pan Yun; Han, Kwi Nam; Kwon, Ho Taik; Seong, Gi Hun

2008-03-01

We describe a DNA microarray system using a bipolar integrated circuit photodiode array (PDA) chip as a new platform for DNA analysis. The PDA chip comprises an 8 x 6 array of photodiodes each with a diameter of 600 microm. Each photodiode element acts both as a support for an immobilizing probe DNA and as a two-dimensional photodetector. The usefulness of the PDA microarray platform is demonstrated by the detection of high-risk subtypes of human papilloma virus (HPV). The polymerase chain reaction (PCR)-amplified biotinylated HPV target DNA was hybridized with the immobilized probe DNA on the photodiode surface, and the chip was incubated in an anti-biotin antibody-conjugated gold nanoparticle solution. The silver enhancement by the gold nanoparticles bound to the biotin of the HPV target DNA precipitates silver metal particles at the chip surfaces, which block light irradiated from above. The resulting drop in output voltage depends on the amount of target DNA present in the sample solution, which allows the specific detection and the quantitative analysis of the complementary target DNA. The PDA chip showed high relative signal ratios of HPV probe DNA hybridized with complementary target DNA, indicating an excellent capability in discriminating HPV subtypes. The detection limit for the HPV target DNA analysis improved from 1.2 nM to 30 pM by changing the silver development time from 5 to 10 min. Moreover, the enhanced silver development promoted by the gold nanoparticles could be applied to a broader range of target DNA concentration by controlling the silver development time.

KSC-07pd0363

NASA Image and Video Library

2007-02-12

KENNEDY SPACE CENTER, FLA. -- A worker in the payload changeout room (PCR) on Launch Pad 39A monitors the payload ground-handling mechanism that is used to transfer the payload into the PCR and the shuttle's payload bay. The PCR is the enclosed, environmentally controlled portion of the rotating service structure that supports cargo delivery to the pad and subsequent vertical installation into the orbiter payload bay. The truss is the payload for Space Shuttle Atlantis on mission STS-117 to the International Space Station. The Atlantis crew will install the new truss segment, retract a set of solar arrays and unfold a new set on the starboard side of the station. Launch is targeted for March 15. Photo credit: NASA/Jack Pfaller

A case of 3q29 microdeletion syndrome involving oral cleft inherited from a non-affected mosaic parent: molecular analysis and ethical implications

PubMed Central

Petrin, Aline L.; Daack-Hirsch, Sandra; L’Heureux, Jamie; Murray, Jeffrey C

2010-01-01

Objective The objective of this study was to use array-CGH to detect causal microdeletions in samples of subjects with cleft lip and palate. Subjects We analyzed DNA samples from a male patient and parents that was seen during surgical screening for an Operation Smile medical mission in the Philippines. Method We used Affymetrix Genome Wide Human SNP Array 6.0 followed by sequencing and quantitative PCR using SYBR Green I dye. Results We report the second case of 3q29 microdeletion syndrome including cleft lip with or without cleft palate and the first case of this microdeletion syndrome inherited from a phenotypically normal mosaic parent. Conclusions Our findings confirm the utility of aCGH to detect causal microdeletions; indicate that parental somatic mosaicism should be considered in healthy parents for genetic counseling of the families and discuss important ethical implications of sharing health impact results from research studies with the participant families. PMID:20500065

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

PubMed

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

2016-08-01

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

Testing Microshutter Arrays Using Commercial FPGA Hardware

NASA Technical Reports Server (NTRS)

Rapchun, David

2008-01-01

NASA is developing micro-shutter arrays for the Near Infrared Spectrometer (NIRSpec) instrument on the James Webb Space Telescope (JWST). These micro-shutter arrays allow NIRspec to do Multi Object Spectroscopy, a key part of the mission. Each array consists of 62414 individual 100 x 200 micron shutters. These shutters are magnetically opened and held electrostatically. Individual shutters are then programmatically closed using a simple row/column addressing technique. A common approach to provide these data/clock patterns is to use a Field Programmable Gate Array (FPGA). Such devices require complex VHSIC Hardware Description Language (VHDL) programming and custom electronic hardware. Due to JWST's rapid schedule on the development of the micro-shutters, rapid changes were required to the FPGA code to facilitate new approaches being discovered to optimize the array performance. Such rapid changes simply could not be made using conventional VHDL programming. Subsequently, National Instruments introduced an FPGA product that could be programmed through a Labview interface. Because Labview programming is considerably easier than VHDL programming, this method was adopted and brought success. The software/hardware allowed the rapid change the FPGA code and timely results of new micro-shutter array performance data. As a result, numerous labor hours and money to the project were conserved.

Development of a Microphone Phased Array Capability for the Langley 14- by 22-Foot Subsonic Tunnel

NASA Technical Reports Server (NTRS)

Humphreys, William M.; Brooks, Thomas F.; Bahr, Christopher J.; Spalt, Taylor B.; Bartram, Scott M.; Culliton, William G.; Becker, Lawrence E.

2014-01-01

A new aeroacoustic measurement capability has been developed for use in open-jet testing in the NASA Langley 14- by 22-Foot Subsonic Tunnel (14x22 tunnel). A suite of instruments has been developed to characterize noise source strengths, locations, and directivity for both semi-span and full-span test articles in the facility. The primary instrument of the suite is a fully traversable microphone phased array for identification of noise source locations and strengths on models. The array can be mounted in the ceiling or on either side of the facility test section to accommodate various test article configurations. Complementing the phased array is an ensemble of streamwise traversing microphones that can be placed around the test section at defined locations to conduct noise source directivity studies along both flyover and sideline axes. A customized data acquisition system has been developed for the instrumentation suite that allows for command and control of all aspects of the array and microphone hardware, and is coupled with a comprehensive data reduction system to generate information in near real time. This information includes such items as time histories and spectral data for individual microphones and groups of microphones, contour presentations of noise source locations and strengths, and hemispherical directivity data. The data acquisition system integrates with the 14x22 tunnel data system to allow real time capture of facility parameters during acquisition of microphone data. The design of the phased array system has been vetted via a theoretical performance analysis based on conventional monopole beamforming and DAMAS deconvolution. The performance analysis provides the ability to compute figures of merit for the array as well as characterize factors such as beamwidths, sidelobe levels, and source discrimination for the types of noise sources anticipated in the 14x22 tunnel. The full paper will summarize in detail the design of the instrumentation suite, the construction of the hardware system, and the results of the performance analysis. Although the instrumentation suite is designed to characterize noise for a variety of test articles in the 14x22 tunnel, this paper will concentrate on description of the instruments for two specific test campaigns in the facility, namely a full-span NASA Hybrid Wing Body (HWB) model entry and a semi-span Gulfstream aircraft model entry, tested in the facility in the winter of 2012 and spring of 2013, respectively.

DcR3 binds to ovarian cancer via heparan sulfate proteoglycans and modulates tumor cells response to platinum with corresponding alteration in the expression of BRCA1

PubMed Central

2012-01-01

Background Overcoming platinum resistance is a major obstacle in the treatment of Epithelial Ovarian Cancer (EOC). In our previous work Decoy Receptor 3 (DcR3) was found to be related to platinum resistance. The major objective of this work was to define the cellular interaction of DcR3 with EOC and to explore its effects on platinum responsiveness. Methods We studied cell lines and primary cultures for the expression of and the cells ability to bind DcR3. Cells were cultured with DcR3 and then exposed to platinum. Cell viability was determined by MTT assay. Finally, the cells molecular response to DcR3 was studied using real time RT-PCR based differential expression arrays, standard RT-PCR, and Western blot. Results High DcR3 in the peritoneal cavity of women with EOC is associated with significantly shorter time to first recurrence after platinum based therapy (p = 0.02). None-malignant cells contribute DcR3 in the peritoneal cavity. The cell lines studied do not secrete DcR3; however they all bind exogenous DcR3 to their surface implying that they can be effected by DcR3 from other sources. DcR3s protein binding partners are minimally expressed or negative, however, all cells expressed the DcR3 binding Heparan Sulfate Proteoglycans (HSPGs) Syndecans-2, and CD44v3. DcR3 binding was inhibited by heparin and heparinase. After DcR3 exposure both SKOV-3 and OVCAR-3 became more resistant to platinum with 15% more cells surviving at high doses. On the contrary CaOV3 became more sensitive to platinum with 20–25% more cell death. PCR array analysis showed increase expression of BRCA1 mRNA in SKOV-3 and OVCAR-3 and decreased BRCA1 expression in CaOV-3 after exposure to DcR3. This was confirmed by gene specific real time PCR and Western blot analysis. Conclusions Non-malignant cells contribute to the high levels of DcR3 in ovarian cancer. DcR3 binds readily to EOC cells via HSPGs and alter their responsiveness to platinum chemotherapy. The paradoxical responses seen were related to the expression pattern of HSPGs available on the cells surface to interact with. Although the mechanism behind this is not completely known alterations in DNA repair pathways including the expression of BRCA1 appear to be involved. PMID:22583667

Inter-laboratory evaluation of the EUROFORGEN Global ancestry-informative SNP panel by massively parallel sequencing using the Ion PGM™.

PubMed

Eduardoff, M; Gross, T E; Santos, C; de la Puente, M; Ballard, D; Strobl, C; Børsting, C; Morling, N; Fusco, L; Hussing, C; Egyed, B; Souto, L; Uacyisrael, J; Syndercombe Court, D; Carracedo, Á; Lareu, M V; Schneider, P M; Parson, W; Phillips, C; Parson, W; Phillips, C

2016-07-01

The EUROFORGEN Global ancestry-informative SNP (AIM-SNPs) panel is a forensic multiplex of 128 markers designed to differentiate an individual's ancestry from amongst the five continental population groups of Africa, Europe, East Asia, Native America, and Oceania. A custom multiplex of AmpliSeq™ PCR primers was designed for the Global AIM-SNPs to perform massively parallel sequencing using the Ion PGM™ system. This study assessed individual SNP genotyping precision using the Ion PGM™, the forensic sensitivity of the multiplex using dilution series, degraded DNA plus simple mixtures, and the ancestry differentiation power of the final panel design, which required substitution of three original ancestry-informative SNPs with alternatives. Fourteen populations that had not been previously analyzed were genotyped using the custom multiplex and these studies allowed assessment of genotyping performance by comparison of data across five laboratories. Results indicate a low level of genotyping error can still occur from sequence misalignment caused by homopolymeric tracts close to the target SNP, despite careful scrutiny of candidate SNPs at the design stage. Such sequence misalignment required the exclusion of component SNP rs2080161 from the Global AIM-SNPs panel. However, the overall genotyping precision and sensitivity of this custom multiplex indicates the Ion PGM™ assay for the Global AIM-SNPs is highly suitable for forensic ancestry analysis with massively parallel sequencing. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

PGK1, a glucose metabolism enzyme, may play an important role in rheumatoid arthritis.

PubMed

Zhao, Yan; Yan, Xinfeng; Li, Xia; Zheng, Yabing; Li, Shufeng; Chang, Xiaotian

2016-10-01

Some studies have indicated that glucose metabolism plays an important role in the pathogenesis of rheumatoid arthritis (RA). This study aimed to find the novel genes affecting glucose metabolism in RA. Synovial tissues of collagen-induced arthritis (CIA) were analyzed with Rat Glucose Metabolism RT(2) Profiler™ PCR Array to screen those genes with special expressions in glucose metabolism. Real-time PCR, western blotting, and ELISA were used to confirm the result in synovial tissues and blood of human RA. Culture synovial fibroblast cells (RASF) was treated with siRNA to suppress expressions of the target genes. CCK-8 cell proliferation assay and two-compartment transwell system were performed to examine cell proliferation and cell migration of the treated RASF. Both PCR array and real-time PCR detected the up-regulation of ENO1, HK2, and PGK1 and the down-regulation of PCK1 and PDK4 in synovial tissues of CIA rats. Real-time PCR and western blotting detected the increased expression of ENO1 and PGK1 in RA synovial tissues. ELISA detected a high level of PGK1 in the blood of RA patients. Decreased cell proliferation and cell migration capabilities were significantly detected in RASF following treatment of anti-PGK1 siRNA. IL-1β and IFN-γ rather than TNF-α and IL-1α levels were significantly declined in supernatants of the treated RASF. PGK1, a glycolytic enzyme catalyzing the conversion of 3-phosphoglycerate into 2-phosphoglycerate, has increased expression in synovial tissues and blood of RA, which may be involved in pro-inflammation and synovial hyperplasia of the disease.

A Demonstration of Big Data Technology for Data Intensive Earth Science (Invited)

NASA Astrophysics Data System (ADS)

Kuo, K.; Clune, T.; Ramachandran, R.; Rushing, J.; Fekete, G.; Lin, A.; Doan, K.; Oloso, A. O.; Duffy, D.

2013-12-01

Big Data technologies exhibit great potential to change the way we conduct scientific investigations, especially analysis of voluminous and diverse data sets. Obviously, not all Big Data technologies are applicable to all aspects of scientific data analysis. Our NASA Earth Science Technology Office (ESTO) Advanced Information Systems Technology (AIST) project, Automated Event Service (AES), pioneers the exploration of Big Data technologies for data intensive Earth science. Since Earth science data are largely stored and manipulated in the form of multidimensional arrays, the project first evaluates array performance of several candidate Big Data technologies, including MapReduce (Hadoop), SciDB, and a custom-built Polaris system, which have one important feature in common: shared nothing architecture. The evaluation finds SicDB to be the most promising. In this presentation, we demonstrate SciDB using a couple of use cases, each operating on a distinct data set in the regular latitude-longitude grid. The first use case is the discovery and identification of blizzards using NASA's Modern Era Retrospective-analysis for Research and Application (MERRA) data sets. The other finds diurnal signals in the same 8-year period using SSMI data from three different instruments with different equator crossing times by correlating their retrieved parameters. In addition, the AES project is also developing a collaborative component to enable the sharing of event queries and results. Preliminary capabilities will be presented as well.

An advanced software suite for the processing and analysis of silicon luminescence images

NASA Astrophysics Data System (ADS)

Payne, D. N. R.; Vargas, C.; Hameiri, Z.; Wenham, S. R.; Bagnall, D. M.

2017-06-01

Luminescence imaging is a versatile characterisation technique used for a broad range of research and industrial applications, particularly for the field of photovoltaics where photoluminescence and electroluminescence imaging is routinely carried out for materials analysis and quality control. Luminescence imaging can reveal a wealth of material information, as detailed in extensive literature, yet these techniques are often only used qualitatively instead of being utilised to their full potential. Part of the reason for this is the time and effort required for image processing and analysis in order to convert image data to more meaningful results. In this work, a custom built, Matlab based software suite is presented which aims to dramatically simplify luminescence image processing and analysis. The suite includes four individual programs which can be used in isolation or in conjunction to achieve a broad array of functionality, including but not limited to, point spread function determination and deconvolution, automated sample extraction, image alignment and comparison, minority carrier lifetime calibration and iron impurity concentration mapping.

Development of a nucleic Acid extraction procedure for simultaneous recovery of DNA and RNA from diverse microbes in water.

PubMed

Hill, Vincent R; Narayanan, Jothikumar; Gallen, Rachel R; Ferdinand, Karen L; Cromeans, Theresa; Vinjé, Jan

2015-05-26

Drinking and environmental water samples contain a diverse array of constituents that can interfere with molecular testing techniques, especially when large volumes of water are concentrated to the small volumes needed for effective molecular analysis. In this study, a suite of enteric viruses, bacteria, and protozoan parasites were seeded into concentrated source water and finished drinking water samples, in order to investigate the relative performance of nucleic acid extraction techniques for molecular testing. Real-time PCR and reverse transcription-PCR crossing threshold (CT) values were used as the metrics for evaluating relative performance. Experimental results were used to develop a guanidinium isothiocyanate-based lysis buffer (UNEX buffer) that enabled effective simultaneous extraction and recovery of DNA and RNA from the suite of study microbes. Procedures for bead beating, nucleic acid purification, and PCR facilitation were also developed and integrated in the protocol. The final lysis buffer and sample preparation procedure was found to be effective for a panel of drinking water and source water concentrates when compared to commercial nucleic acid extraction kits. The UNEX buffer-based extraction protocol enabled PCR detection of six study microbes, in 100 L finished water samples from four drinking water treatment facilities, within three CT values (i.e., within 90% difference) of the reagent-grade water control. The results from this study indicate that this newly formulated lysis buffer and sample preparation procedure can be useful for standardized molecular testing of drinking and environmental waters.

Multiplex Polymerase Chain Reaction for Detection of Gastrointestinal Pathogens in Migrant Workers in Qatar.

PubMed

Humphrey, John M; Ranbhise, Sanjay; Ibrahim, Emad; Al-Romaihi, Hamad E; Farag, Elmoubasher; Abu-Raddad, Laith J; Glesby, Marshall J

2016-12-07

The causes of infectious diarrhea among the migrant worker population in Qatar are not well understood. We conducted a prospective observational study to understand the demographic and clinical characteristics and infectious causes of diarrhea among migrant workers in Doha, Qatar. A total of 126 male workers presenting to the Qatar Red Crescent Worker's Health Center outpatient clinic or emergency department were studied over a 5-month period in 2015-2016. Epidemiologic surveys were administered to all subjects and the prevalence of 22 different stool pathogens was determined using multiplex polymerase chain reaction (PCR) (FilmArray ® Gastrointestinal PCR). A target pathogen was identified in 62.7% of subjects. Enteropathogenic Escherichia coli was the most prevalent pathogen and was detected in 24.6% of subjects, followed by Salmonella (22.2%), enteroaggregative E. coli (15.1%), Giardia lamblia (9.5%), and enterotoxigenic E. coli (8.7%). Multiple pathogens were identified in 49.3% of positive stool samples. In a multivariable analysis, the presence of a heart rate ≥ 90 (adjusted odds ratio [OR] = 3.7, 95% confidence interval [CI] = 1.4-10.0) and > 5 fecal leukocytes/high-power field (adjusted OR = 2.8, 95% CI = 1.2-7.0) were significant predictors of detecting an acute inflammatory pathogen by PCR. Use of multiplex PCR enabled the detection of gastrointestinal pathogens in a high proportion of cases, illustrating the utility of this diagnostic tool in epidemiologic studies of infectious diarrhea. © The American Society of Tropical Medicine and Hygiene.

Development of a Nucleic Acid Extraction Procedure for Simultaneous Recovery of DNA and RNA from Diverse Microbes in Water

PubMed Central

Hill, Vincent R.; Narayanan, Jothikumar; Gallen, Rachel R.; Ferdinand, Karen L.; Cromeans, Theresa; Vinjé, Jan

2015-01-01

Drinking and environmental water samples contain a diverse array of constituents that can interfere with molecular testing techniques, especially when large volumes of water are concentrated to the small volumes needed for effective molecular analysis. In this study, a suite of enteric viruses, bacteria, and protozoan parasites were seeded into concentrated source water and finished drinking water samples, in order to investigate the relative performance of nucleic acid extraction techniques for molecular testing. Real-time PCR and reverse transcription-PCR crossing threshold (CT) values were used as the metrics for evaluating relative performance. Experimental results were used to develop a guanidinium isothiocyanate-based lysis buffer (UNEX buffer) that enabled effective simultaneous extraction and recovery of DNA and RNA from the suite of study microbes. Procedures for bead beating, nucleic acid purification, and PCR facilitation were also developed and integrated in the protocol. The final lysis buffer and sample preparation procedure was found to be effective for a panel of drinking water and source water concentrates when compared to commercial nucleic acid extraction kits. The UNEX buffer-based extraction protocol enabled PCR detection of six study microbes, in 100 L finished water samples from four drinking water treatment facilities, within three CT values (i.e., within 90% difference) of the reagent-grade water control. The results from this study indicate that this newly formulated lysis buffer and sample preparation procedure can be useful for standardized molecular testing of drinking and environmental waters. PMID:26016775

Mollusk genes encoding lysine tRNA (UUU) contain introns.

PubMed

Matsuo, M; Abe, Y; Saruta, Y; Okada, N

1995-11-20

New intron-containing genes encoding tRNAs were discovered when genomic DNA isolated from various animal species was amplified by the polymerase chain reaction (PCR) with primers based on sequences of rabbit tRNA(Lys). From sequencing analysis of the products of PCR, we found that introns are present in several genes encoding tRNA(Lys) in mollusks, such as Loligo bleekeri (squid) and Octopus vulgaris (octopus). These introns were specific to genes encoding tRNA(Lys)(CUU) and were not present in genes encoding tRNA(Lys)(CUU). In addition, the sequences of the introns were different from one another. To confirm the results of our initial experiments, we isolated and sequenced genes encoding tRNA(Lys)(CUU) and tRNA(Lys)(UUU). The gene for tRNA(Lys)(UUU) from squid contained an intron, whose sequence was the same as that identified by PCR, and the gene formed a cluster with a corresponding pseudogene. Several DNA regions of 2.1 kb containing this cluster appeared to be tandemly arrayed in the squid genome. By contrast, the gene encoding tRNA(Lys)(CUU) did not contain an intron, as shown also by PCR. The tRNA(Lys)(UUU) that corresponded to the analyzed gene was isolated and characterized. The present study provides the first example of an intron-containing gene encoding a tRNA in mollusks and suggests the universality of introns in such genes in higher eukaryotes.

Salivary extracellular vesicle-associated miRNAs as potential biomarkers in oral squamous cell carcinoma.

PubMed

Gai, Chiara; Camussi, Francesco; Broccoletti, Roberto; Gambino, Alessio; Cabras, Marco; Molinaro, Luca; Carossa, Stefano; Camussi, Giovanni; Arduino, Paolo G

2018-04-18

Several studies in the past have investigated the expression of micro RNAs (miRNAs) in saliva as potential biomarkers. Since miRNAs associated with extracellular vesicles (EVs) are known to be protected from enzymatic degradation, we evaluated whether salivary EVs from patients with oral squamous cell carcinoma (OSCC) were enriched with specific subsets of miRNAs. OSCC patients and controls were matched with regards to age, gender and risk factors. Total RNA was extracted from salivary EVs and the differential expression of miRNAs was evaluated by qRT-PCR array and qRT-PCR. The discrimination power of up-regulated miRNAs as biomarkers in OSCC patients versus controls was evaluated by the Receiver Operating Characteristic (ROC) curves. A preliminary qRT-PCR array was performed on samples from 5 OSCC patients and 5 healthy controls whereby a subset of miRNAs were identified that were differentially expressed. On the basis of these results, a cohort of additional 16 patients and 6 controls were analyzed to further confirm the miRNAs that were up-regulated or selectively expressed in the previous pilot study. The following miRNAs: miR-302b-3p and miR-517b-3p were expressed only in EVs from OSCC patients and miR-512-3p and miR-412-3p were up-regulated in salivary EVs from OSCC patients compared to controls with the ROC curve showing a good discrimination power for OSCC diagnosis. The Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analysis suggested the possible involvement of the miRNAs identified in pathways activated in OSCC. In this work, we suggest that salivary EVs isolated by a simple charge-based precipitation technique can be exploited as a non-invasive source of miRNAs for OSCC diagnosis. Moreover, we have identified a subset of miRNAs selectively enriched in EVs of OSCC patients that could be potential biomarkers.

High temperature, high intensity solar array. [for Venus Radar Mapper mission

NASA Technical Reports Server (NTRS)

Smith, B. S.; Brooks, G. R.; Pinkerton, R.

1985-01-01

The solar array for the Venus Radar Mapper mission will operate in the high temperature, high intensity conditions of a low Venus orbit environment. To fulfill the performance requirements in this environment at minimum cost and mass while maximizing power density and packing factor on the panel surface, several features were introduced into the design. These features included the use of optical surface reflectors (OSR's) to reduce the operating temperature; new adhesives for conductive bonding of OSR's to avoid electrostatic discharges; custom-designed large area cells and novel shunt diode circuit and panel power harness configurations.

Fluorescence-based bioassays for the detection and evaluation of food materials.

PubMed

Nishi, Kentaro; Isobe, Shin-Ichiro; Zhu, Yun; Kiyama, Ryoiti

2015-10-13

We summarize here the recent progress in fluorescence-based bioassays for the detection and evaluation of food materials by focusing on fluorescent dyes used in bioassays and applications of these assays for food safety, quality and efficacy. Fluorescent dyes have been used in various bioassays, such as biosensing, cell assay, energy transfer-based assay, probing, protein/immunological assay and microarray/biochip assay. Among the arrays used in microarray/biochip assay, fluorescence-based microarrays/biochips, such as antibody/protein microarrays, bead/suspension arrays, capillary/sensor arrays, DNA microarrays/polymerase chain reaction (PCR)-based arrays, glycan/lectin arrays, immunoassay/enzyme-linked immunosorbent assay (ELISA)-based arrays, microfluidic chips and tissue arrays, have been developed and used for the assessment of allergy/poisoning/toxicity, contamination and efficacy/mechanism, and quality control/safety. DNA microarray assays have been used widely for food safety and quality as well as searches for active components. DNA microarray-based gene expression profiling may be useful for such purposes due to its advantages in the evaluation of pathway-based intracellular signaling in response to food materials.

Fluorescence-Based Bioassays for the Detection and Evaluation of Food Materials

PubMed Central

Nishi, Kentaro; Isobe, Shin-Ichiro; Zhu, Yun; Kiyama, Ryoiti

2015-01-01

We summarize here the recent progress in fluorescence-based bioassays for the detection and evaluation of food materials by focusing on fluorescent dyes used in bioassays and applications of these assays for food safety, quality and efficacy. Fluorescent dyes have been used in various bioassays, such as biosensing, cell assay, energy transfer-based assay, probing, protein/immunological assay and microarray/biochip assay. Among the arrays used in microarray/biochip assay, fluorescence-based microarrays/biochips, such as antibody/protein microarrays, bead/suspension arrays, capillary/sensor arrays, DNA microarrays/polymerase chain reaction (PCR)-based arrays, glycan/lectin arrays, immunoassay/enzyme-linked immunosorbent assay (ELISA)-based arrays, microfluidic chips and tissue arrays, have been developed and used for the assessment of allergy/poisoning/toxicity, contamination and efficacy/mechanism, and quality control/safety. DNA microarray assays have been used widely for food safety and quality as well as searches for active components. DNA microarray-based gene expression profiling may be useful for such purposes due to its advantages in the evaluation of pathway-based intracellular signaling in response to food materials. PMID:26473869

Development and Comparison of Two Assay Formats for Parallel Detection of Four Biothreat Pathogens by Using Suspension Microarrays

PubMed Central

Janse, Ingmar; Bok, Jasper M.; Hamidjaja, Raditijo A.; Hodemaekers, Hennie M.; van Rotterdam, Bart J.

2012-01-01

Microarrays provide a powerful analytical tool for the simultaneous detection of multiple pathogens. We developed diagnostic suspension microarrays for sensitive and specific detection of the biothreat pathogens Bacillus anthracis, Yersinia pestis, Francisella tularensis and Coxiella burnetii. Two assay chemistries for amplification and labeling were developed, one method using direct hybridization and the other using target-specific primer extension, combined with hybridization to universal arrays. Asymmetric PCR products for both assay chemistries were produced by using a multiplex asymmetric PCR amplifying 16 DNA signatures (16-plex). The performances of both assay chemistries were compared and their advantages and disadvantages are discussed. The developed microarrays detected multiple signature sequences and an internal control which made it possible to confidently identify the targeted pathogens and assess their virulence potential. The microarrays were highly specific and detected various strains of the targeted pathogens. Detection limits for the different pathogen signatures were similar or slightly higher compared to real-time PCR. Probit analysis showed that even a few genomic copies could be detected with 95% confidence. The microarrays detected DNA from different pathogens mixed in different ratios and from spiked or naturally contaminated samples. The assays that were developed have a potential for application in surveillance and diagnostics. PMID:22355407

Development and comparison of two assay formats for parallel detection of four biothreat pathogens by using suspension microarrays.

PubMed

Janse, Ingmar; Bok, Jasper M; Hamidjaja, Raditijo A; Hodemaekers, Hennie M; van Rotterdam, Bart J

2012-01-01

Microarrays provide a powerful analytical tool for the simultaneous detection of multiple pathogens. We developed diagnostic suspension microarrays for sensitive and specific detection of the biothreat pathogens Bacillus anthracis, Yersinia pestis, Francisella tularensis and Coxiella burnetii. Two assay chemistries for amplification and labeling were developed, one method using direct hybridization and the other using target-specific primer extension, combined with hybridization to universal arrays. Asymmetric PCR products for both assay chemistries were produced by using a multiplex asymmetric PCR amplifying 16 DNA signatures (16-plex). The performances of both assay chemistries were compared and their advantages and disadvantages are discussed. The developed microarrays detected multiple signature sequences and an internal control which made it possible to confidently identify the targeted pathogens and assess their virulence potential. The microarrays were highly specific and detected various strains of the targeted pathogens. Detection limits for the different pathogen signatures were similar or slightly higher compared to real-time PCR. Probit analysis showed that even a few genomic copies could be detected with 95% confidence. The microarrays detected DNA from different pathogens mixed in different ratios and from spiked or naturally contaminated samples. The assays that were developed have a potential for application in surveillance and diagnostics.

Analysis of genetically modified organisms by pyrosequencing on a portable photodiode-based bioluminescence sequencer.

PubMed

Song, Qinxin; Wei, Guijiang; Zhou, Guohua

2014-07-01

A portable bioluminescence analyser for detecting the DNA sequence of genetically modified organisms (GMOs) was developed by using a photodiode (PD) array. Pyrosequencing on eight genes (zSSIIb, Bt11 and Bt176 gene of genetically modified maize; Lectin, 35S-CTP4, CP4EPSPS, CaMV35S promoter and NOS terminator of the genetically modified Roundup ready soya) was successfully detected with this instrument. The corresponding limit of detection (LOD) was 0.01% with 35 PCR cycles. The maize and soya available from three different provenances in China were detected. The results indicate that pyrosequencing using the small size of the detector is a simple, inexpensive, and reliable way in a farm/field test of GMO analysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

MicroRNA markers for forensic body fluid identification obtained from microarray screening and quantitative RT-PCR confirmation

PubMed Central

Zubakov, Dmitry; Boersma, Anton W. M.; Choi, Ying; van Kuijk, Patricia F.; Wiemer, Erik A. C.

2010-01-01

MicroRNAs (miRNAs) are non-protein coding molecules with important regulatory functions; many have tissue-specific expression patterns. Their very small size in principle makes them less prone to degradation processes, unlike messenger RNAs (mRNAs), which were previously proposed as molecular tools for forensic body fluid identification. To identify suitable miRNA markers for forensic body fluid identification, we first screened total RNA samples derived from saliva, semen, vaginal secretion, and venous and menstrual blood for the expression of 718 human miRNAs using a microarray platform. All body fluids could be easily distinguished from each other on the basis of complete array-based miRNA expression profiles. Results from quantitative reverse transcription PCR (RT-PCR; TaqMan) assays for microarray candidate markers confirmed strong over-expression in the targeting body fluid of several miRNAs for venous blood and several others for semen. However, no candidate markers from array experiments for other body fluids such as saliva, vaginal secretion, or menstrual blood could be confirmed by RT-PCR. Time-wise degradation of venous blood and semen stains for at least 1 year under lab conditions did not significantly affect the detection sensitivity of the identified miRNA markers. The detection limit of the TaqMan assays tested for selected venous blood and semen miRNA markers required only subpicogram amounts of total RNA per single RT-PCR test, which is considerably less than usually needed for reliable mRNA RT-PCR detection. We therefore propose the application of several stable miRNA markers for the forensic identification of blood stains and several others for semen stain identification, using commercially available TaqMan assays. Additional work remains necessary in search for suitable miRNA markers for other forensically relevant body fluids. Electronic supplementary material The online version of this article (doi:10.1007/s00414-009-0402-3) contains supplementary material, which is available to authorized users. PMID:20145944

Plasma microRNAs serve as biomarkers of therapeutic efficacy and disease progression in hypertension-induced heart failure.

PubMed

Dickinson, Brent A; Semus, Hillary M; Montgomery, Rusty L; Stack, Christianna; Latimer, Paul A; Lewton, Steven M; Lynch, Joshua M; Hullinger, Thomas G; Seto, Anita G; van Rooij, Eva

2013-06-01

Recent studies have shown that microRNAs (miRNAs), besides being potent regulators of gene expression, can additionally serve as circulating biomarkers of disease. The aim of this study is to determine if plasma miRNAs can be used as indicators of disease progression or therapeutic efficacy in hypertension-induced heart disease. In order to define circulating miRNAs that change during hypertension-induced heart failure and that respond to therapeutic treatment, we performed miRNA arrays on plasma RNA from hypertensive rats that show signs of heart failure. Array analysis indicated that approximately one-third of the miRNAs on the array are detectable in plasma. Quantitative real-time polymerase chain reaction (PCR) analysis for a selected panel of miRNAs indicated that circulating levels of miR-16, miR-20b, miR-93, miR-106b, miR-223, and miR-423-5p were significantly increased in response to hypertension-induced heart failure, while this effect was blunted in response to treatment with antimiR-208a as well as an ACE inhibitor. Moreover, treatment with antimiR-208a resulted in a dramatic increase in one miRNA, miR-19b. A time course study indicated that several of these miRNA changes track with disease progression. Circulating levels of miRNAs are responsive to therapeutic interventions and change during the progression of hypertension-induced heart disease.

MobilomeFINDER: web-based tools for in silico and experimental discovery of bacterial genomic islands

PubMed Central

Ou, Hong-Yu; He, Xinyi; Harrison, Ewan M.; Kulasekara, Bridget R.; Thani, Ali Bin; Kadioglu, Aras; Lory, Stephen; Hinton, Jay C. D.; Barer, Michael R.; Rajakumar, Kumar

2007-01-01

MobilomeFINDER (http://mml.sjtu.edu.cn/MobilomeFINDER) is an interactive online tool that facilitates bacterial genomic island or ‘mobile genome’ (mobilome) discovery; it integrates the ArrayOme and tRNAcc software packages. ArrayOme utilizes a microarray-derived comparative genomic hybridization input data set to generate ‘inferred contigs’ produced by merging adjacent genes classified as ‘present’. Collectively these ‘fragments’ represent a hypothetical ‘microarray-visualized genome (MVG)’. ArrayOme permits recognition of discordances between physical genome and MVG sizes, thereby enabling identification of strains rich in microarray-elusive novel genes. Individual tRNAcc tools facilitate automated identification of genomic islands by comparative analysis of the contents and contexts of tRNA sites and other integration hotspots in closely related sequenced genomes. Accessory tools facilitate design of hotspot-flanking primers for in silico and/or wet-science-based interrogation of cognate loci in unsequenced strains and analysis of islands for features suggestive of foreign origins; island-specific and genome-contextual features are tabulated and represented in schematic and graphical forms. To date we have used MobilomeFINDER to analyse several Enterobacteriaceae, Pseudomonas aeruginosa and Streptococcus suis genomes. MobilomeFINDER enables high-throughput island identification and characterization through increased exploitation of emerging sequence data and PCR-based profiling of unsequenced test strains; subsequent targeted yeast recombination-based capture permits full-length sequencing and detailed functional studies of novel genomic islands. PMID:17537813

Biomimetic cell wall model studies to identify new lignin bioengineering targets for improving biomass susceptibility to pretreatment and enzymatic saccharification

USDA-ARS?s Scientific Manuscript database

Increasingly, bioengineering of lignin to contain atypical building blocks from other metabolic pathways is being pursued to custom-design lignin that is easier to remove by chemical pretreatments and less inhibitory toward polysaccharide saccharification. Because plants produce such a diverse array...

Microlens frames for laser diode arrays

DOEpatents

Skidmore, J.A.; Freitas, B.L.

1999-07-13

Monolithic microlens frames enable the fabrication of monolithic laser diode arrays and are manufactured inexpensively with high registration, and with inherent focal length compensation for any lens diameter variation. A monolithic substrate is used to fabricate a low-cost microlens array. The substrate is wet-etched or sawed with a series of v-grooves. The v-grooves can be created by wet-etching, by exploiting the large etch-rate selectivity of different crystal planes. The v-grooves provide a support frame for either cylindrical or custom-shaped microlenses. Because the microlens frames are formed by photolithographic semiconductor batch-processing techniques, they can be formed inexpensively over large areas with precise lateral and vertical registration. The v-groove has an important advantage for preserving the correct focus for lenses of varying diameter. 12 figs.

Microlens frames for laser diode arrays

DOEpatents

Skidmore, Jay A.; Freitas, Barry L.

1999-01-01

Monolithic microlens frames enable the fabrication of monolithic laser diode arrays and are manufactured inexpensively with high registration, and with inherent focal length compensation for any lens diameter variation. A monolithic substrate is used to fabricate a low-cost microlens array. The substrate is wet-etched or sawed with a series of v-grooves. The v-grooves can be created by wet-etching, by exploiting the large etch-rate selectivity of different crystal planes. The v-grooves provide a support frame for either cylindrical or custom-shaped microlenses. Because the microlens frames are formed by photolithographic semiconductor batch-processing techniques, they can be formed inexpensively over large areas with precise lateral and vertical registration. The v-groove has an important advantage for preserving the correct focus for lenses of varying diameter.

Ready to clone: CNV detection and breakpoint fine-mapping in breast and ovarian cancer susceptibility genes by high-resolution array CGH.

PubMed

Hackmann, Karl; Kuhlee, Franziska; Betcheva-Krajcir, Elitza; Kahlert, Anne-Karin; Mackenroth, Luisa; Klink, Barbara; Di Donato, Nataliya; Tzschach, Andreas; Kast, Karin; Wimberger, Pauline; Schrock, Evelin; Rump, Andreas

2016-10-01

Detection of predisposing copy number variants (CNV) in 330 families affected with hereditary breast and ovarian cancer (HBOC). In order to complement mutation detection with Illumina's TruSight Cancer panel, we designed a customized high-resolution 8 × 60k array for CGH (aCGH) that covers all 94 genes from the panel. Copy number variants with immediate clinical relevance were detected in 12 families (3.6%). Besides 3 known CNVs in CHEK2, RAD51C, and BRCA1, we identified 3 novel pathogenic CNVs in BRCA1 (deletion of exons 4-13, deletion of exons 12-18) and ATM (deletion exons 57-63) plus an intragenic duplication of BRCA2 (exons 3-11) and an intronic BRCA1 variant with unknown pathogenicity. The precision of high-resolution aCGH enabled straight forward breakpoint amplification of a BRCA1 deletion which subsequently allowed for fast and economic CNV verification in family members of the index patient. Furthermore, we used our aCGH data to validate an algorithm that was able to detect all identified copy number changes from next-generation sequencing (NGS) data. Copy number detection is a mandatory analysis in HBOC families at least if no predisposing mutations were found by sequencing. Currently, high-resolution array CGH is our first choice of method of analysis due to unmatched detection precision. Although it seems possible to detect CNV from sequencing data, there currently is no satisfying tool to do so in a routine diagnostic setting.

Quantitative trait loci markers derived from whole genome sequence data increases the reliability of genomic prediction.

PubMed

Brøndum, R F; Su, G; Janss, L; Sahana, G; Guldbrandtsen, B; Boichard, D; Lund, M S

2015-06-01

This study investigated the effect on the reliability of genomic prediction when a small number of significant variants from single marker analysis based on whole genome sequence data were added to the regular 54k single nucleotide polymorphism (SNP) array data. The extra markers were selected with the aim of augmenting the custom low-density Illumina BovineLD SNP chip (San Diego, CA) used in the Nordic countries. The single-marker analysis was done breed-wise on all 16 index traits included in the breeding goals for Nordic Holstein, Danish Jersey, and Nordic Red cattle plus the total merit index itself. Depending on the trait's economic weight, 15, 10, or 5 quantitative trait loci (QTL) were selected per trait per breed and 3 to 5 markers were selected to tag each QTL. After removing duplicate markers (same marker selected for more than one trait or breed) and filtering for high pairwise linkage disequilibrium and assaying performance on the array, a total of 1,623 QTL markers were selected for inclusion on the custom chip. Genomic prediction analyses were performed for Nordic and French Holstein and Nordic Red animals using either a genomic BLUP or a Bayesian variable selection model. When using the genomic BLUP model including the QTL markers in the analysis, reliability was increased by up to 4 percentage points for production traits in Nordic Holstein animals, up to 3 percentage points for Nordic Reds, and up to 5 percentage points for French Holstein. Smaller gains of up to 1 percentage point was observed for mastitis, but only a 0.5 percentage point increase was seen for fertility. When using a Bayesian model accuracies were generally higher with only 54k data compared with the genomic BLUP approach, but increases in reliability were relatively smaller when QTL markers were included. Results from this study indicate that the reliability of genomic prediction can be increased by including markers significant in genome-wide association studies on whole genome sequence data alongside the 54k SNP set. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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

PubMed Central

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

2014-01-01

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

Detection, Characterization, and Typing of Shiga Toxin-Producing Escherichia coli.

PubMed

Parsons, Brendon D; Zelyas, Nathan; Berenger, Byron M; Chui, Linda

2016-01-01

Shiga toxin-producing Escherichia coli (STEC) are responsible for gastrointestinal diseases reported in numerous outbreaks around the world. Given the public health importance of STEC, effective detection, characterization and typing is critical to any medical laboratory system. While non-O157 serotypes account for the majority of STEC infections, frontline microbiology laboratories may only screen for STEC using O157-specific agar-based methods. As a result, non-O157 STEC infections are significantly under-reported. This review discusses recent advances on the detection, characterization and typing of STEC with emphasis on work performed at the Alberta Provincial Laboratory for Public Health (ProvLab). Candidates for the detection of all STEC serotypes include chromogenic agars, enzyme immunoassays (EIA) and quantitative real time polymerase chain reaction (qPCR). Culture methods allow further characterization of isolates, whereas qPCR provides the greatest sensitivity and specificity, followed by EIA. The virulence gene profiles using PCR arrays and stx gene subtypes can subsequently be determined. Different non-O157 serotypes exhibit markedly different virulence gene profiles and a greater prevalence of stx1 than stx2 subtypes compared to O157:H7 isolates. Finally, recent innovations in whole genome sequencing (WGS) have allowed it to emerge as a candidate for the characterization and typing of STEC in diagnostic surveillance isolates. Methods of whole genome analysis such as single nucleotide polymorphisms and k-mer analysis are concordant with epidemiological data and standard typing methods, such as pulsed-field gel electrophoresis and multiple-locus variable number tandem repeat analysis while offering additional strain differentiation. Together these findings highlight improved strategies for STEC detection using currently available systems and the development of novel approaches for future surveillance.

Detection, Characterization, and Typing of Shiga Toxin-Producing Escherichia coli

PubMed Central

Parsons, Brendon D.; Zelyas, Nathan; Berenger, Byron M.; Chui, Linda

2016-01-01

Shiga toxin-producing Escherichia coli (STEC) are responsible for gastrointestinal diseases reported in numerous outbreaks around the world. Given the public health importance of STEC, effective detection, characterization and typing is critical to any medical laboratory system. While non-O157 serotypes account for the majority of STEC infections, frontline microbiology laboratories may only screen for STEC using O157-specific agar-based methods. As a result, non-O157 STEC infections are significantly under-reported. This review discusses recent advances on the detection, characterization and typing of STEC with emphasis on work performed at the Alberta Provincial Laboratory for Public Health (ProvLab). Candidates for the detection of all STEC serotypes include chromogenic agars, enzyme immunoassays (EIA) and quantitative real time polymerase chain reaction (qPCR). Culture methods allow further characterization of isolates, whereas qPCR provides the greatest sensitivity and specificity, followed by EIA. The virulence gene profiles using PCR arrays and stx gene subtypes can subsequently be determined. Different non-O157 serotypes exhibit markedly different virulence gene profiles and a greater prevalence of stx1 than stx2 subtypes compared to O157:H7 isolates. Finally, recent innovations in whole genome sequencing (WGS) have allowed it to emerge as a candidate for the characterization and typing of STEC in diagnostic surveillance isolates. Methods of whole genome analysis such as single nucleotide polymorphisms and k-mer analysis are concordant with epidemiological data and standard typing methods, such as pulsed-field gel electrophoresis and multiple-locus variable number tandem repeat analysis while offering additional strain differentiation. Together these findings highlight improved strategies for STEC detection using currently available systems and the development of novel approaches for future surveillance. PMID:27148176

NREL Analysis Identifies Where Commercial Customers Might Benefit from

Science.gov Websites

Battery Energy Storage | NREL | News | NREL NREL Analysis Identifies Where Commercial Customers Might Benefit from Battery Energy Storage News Release: NREL Analysis Identifies Where Commercial reduce operating costs for customers paying demand charges Commercial electricity customers who are

Fiber-optic hydrophone array for acoustic surveillance in the littoral

NASA Astrophysics Data System (ADS)

Hill, David; Nash, Phillip

2005-05-01

We describe a fibre-optic hydrophone array system architecture that can be tailored to meet the underwater acoustic surveillance requirements of the military, counter terrorist and customs authorities in protecting ports and harbours, offshore production facilities or coastal approaches. Physically the fibre-optic hydrophone array is in the form of a lightweight cable, enabling rapid deployment from a small vessel. Based upon an optical architecture of time and wavelength multiplexed interferometric hydrophones, the array is comprised of a series of hydrophone sub-arrays. Using multiple sub-arrays, extended perimeters many tens of kilometres in length can be monitored. Interrogated via a long (~50km) optical fibre data link, the acoustic date is processed using the latest open architecture sonar processing platform, ensuring that acoustic targets below, on and above the surface are detected, tracked and classified. Results obtained from an at sea trial of a 96-channel hydrophone array are given, showing the passive detection and tracking of a diver, small surface craft and big ocean going ships beyond the horizon. Furthermore, we describe how the OptaMarine fibre-optic hydrophone array fits into an integrated multi-layered approach to port and harbour security consisting of active sonar for diver detection and hull imaging, as well as thermal imaging and CCTV for surface monitoring. Finally, we briefly describe a complimentary land perimeter intruder detection system consisting of an array of fibre optic accelerometers.

Design methodology: edgeless 3D ASICs with complex in-pixel processing for pixel detectors

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

Fahim Farah, Fahim Farah; Deptuch, Grzegorz W.; Hoff, James R.

The design methodology for the development of 3D integrated edgeless pixel detectors with in-pixel processing using Electronic Design Automation (EDA) tools is presented. A large area 3 tier 3D detector with one sensor layer and two ASIC layers containing one analog and one digital tier, is built for x-ray photon time of arrival measurement and imaging. A full custom analog pixel is 65μm x 65μm. It is connected to a sensor pixel of the same size on one side, and on the other side it has approximately 40 connections to the digital pixel. A 32 x 32 edgeless array withoutmore » any peripheral functional blocks constitutes a sub-chip. The sub-chip is an indivisible unit, which is further arranged in a 6 x 6 array to create the entire 1.248cm x 1.248cm ASIC. Each chip has 720 bump-bond I/O connections, on the back of the digital tier to the ceramic PCB. All the analog tier power and biasing is conveyed through the digital tier from the PCB. The assembly has no peripheral functional blocks, and hence the active area extends to the edge of the detector. This was achieved by using a few flavors of almost identical analog pixels (minimal variation in layout) to allow for peripheral biasing blocks to be placed within pixels. The 1024 pixels within a digital sub-chip array have a variety of full custom, semi-custom and automated timing driven functional blocks placed together. The methodology uses a modified mixed-mode on-top digital implementation flow to not only harness the tool efficiency for timing and floor-planning but also to maintain designer control over compact parasitically aware layout. The methodology uses the Cadence design platform, however it is not limited to this tool.« less

Design methodology: edgeless 3D ASICs with complex in-pixel processing for pixel detectors

NASA Astrophysics Data System (ADS)

Fahim, Farah; Deptuch, Grzegorz W.; Hoff, James R.; Mohseni, Hooman

2015-08-01

The design methodology for the development of 3D integrated edgeless pixel detectors with in-pixel processing using Electronic Design Automation (EDA) tools is presented. A large area 3 tier 3D detector with one sensor layer and two ASIC layers containing one analog and one digital tier, is built for x-ray photon time of arrival measurement and imaging. A full custom analog pixel is 65μm x 65μm. It is connected to a sensor pixel of the same size on one side, and on the other side it has approximately 40 connections to the digital pixel. A 32 x 32 edgeless array without any peripheral functional blocks constitutes a sub-chip. The sub-chip is an indivisible unit, which is further arranged in a 6 x 6 array to create the entire 1.248cm x 1.248cm ASIC. Each chip has 720 bump-bond I/O connections, on the back of the digital tier to the ceramic PCB. All the analog tier power and biasing is conveyed through the digital tier from the PCB. The assembly has no peripheral functional blocks, and hence the active area extends to the edge of the detector. This was achieved by using a few flavors of almost identical analog pixels (minimal variation in layout) to allow for peripheral biasing blocks to be placed within pixels. The 1024 pixels within a digital sub-chip array have a variety of full custom, semi-custom and automated timing driven functional blocks placed together. The methodology uses a modified mixed-mode on-top digital implementation flow to not only harness the tool efficiency for timing and floor-planning but also to maintain designer control over compact parasitically aware layout. The methodology uses the Cadence design platform, however it is not limited to this tool.

Tandemly repeated sequences in mtDNA control region of whitefish, Coregonus lavaretus.

PubMed

Brzuzan, P

2000-06-01

Length variation of the mitochondrial DNA control region was observed with PCR amplification of a sample of 138 whitefish (Coregonus lavaretus). Nucleotide sequences of representative PCR products showed that the variation was due to the presence of an approximately 100-bp motif tandemly repeated two, three, or five times in the region between the conserved sequence block-3 (CSB-3) and the gene for phenylalanine tRNA. This is the first report on the tandem array composed of long repeat units in mitochondrial DNA of salmonids.

An Array Library for Microsoft SQL Server with Astrophysical Applications

NASA Astrophysics Data System (ADS)

Dobos, L.; Szalay, A. S.; Blakeley, J.; Falck, B.; Budavári, T.; Csabai, I.

2012-09-01

Today's scientific simulations produce output on the 10-100 TB scale. This unprecedented amount of data requires data handling techniques that are beyond what is used for ordinary files. Relational database systems have been successfully used to store and process scientific data, but the new requirements constantly generate new challenges. Moving terabytes of data among servers on a timely basis is a tough problem, even with the newest high-throughput networks. Thus, moving the computations as close to the data as possible and minimizing the client-server overhead are absolutely necessary. At least data subsetting and preprocessing have to be done inside the server process. Out of the box commercial database systems perform very well in scientific applications from the prospective of data storage optimization, data retrieval, and memory management but lack basic functionality like handling scientific data structures or enabling advanced math inside the database server. The most important gap in Microsoft SQL Server is the lack of a native array data type. Fortunately, the technology exists to extend the database server with custom-written code that enables us to address these problems. We present the prototype of a custom-built extension to Microsoft SQL Server that adds array handling functionality to the database system. With our Array Library, fix-sized arrays of all basic numeric data types can be created and manipulated efficiently. Also, the library is designed to be able to be seamlessly integrated with the most common math libraries, such as BLAS, LAPACK, FFTW, etc. With the help of these libraries, complex operations, such as matrix inversions or Fourier transformations, can be done on-the-fly, from SQL code, inside the database server process. We are currently testing the prototype with two different scientific data sets: The Indra cosmological simulation will use it to store particle and density data from N-body simulations, and the Milky Way Laboratory project will use it to store galaxy simulation data.

Transcriptional profiling of host gene expression in chicken embryo lung cells infected with laryngotracheitis virus

PubMed Central

2010-01-01

Background Infection by infectious laryngotracheitis virus (ILTV; gallid herpesvirus 1) causes acute respiratory diseases in chickens often with high mortality. To better understand host-ILTV interactions at the host transcriptional level, a microarray analysis was performed using 4 × 44 K Agilent chicken custom oligo microarrays. Results Microarrays were hybridized using the two color hybridization method with total RNA extracted from ILTV infected chicken embryo lung cells at 0, 1, 3, 5, and 7 days post infection (dpi). Results showed that 789 genes were differentially expressed in response to ILTV infection that include genes involved in the immune system (cytokines, chemokines, MHC, and NF-κB), cell cycle regulation (cyclin B2, CDK1, and CKI3), matrix metalloproteinases (MMPs) and cellular metabolism. Differential expression for 20 out of 789 genes were confirmed by quantitative reverse transcription-PCR (qRT-PCR). A bioinformatics tool (Ingenuity Pathway Analysis) used to analyze biological functions and pathways on the group of 789 differentially expressed genes revealed that 21 possible gene networks with intermolecular connections among 275 functionally identified genes. These 275 genes were classified into a number of functional groups that included cancer, genetic disorder, cellular growth and proliferation, and cell death. Conclusion The results of this study provide comprehensive knowledge on global gene expression, and biological functionalities of differentially expressed genes in chicken embryo lung cells in response to ILTV infections. PMID:20663125

Protein Microarray Analysis in Patients With Asthma*

PubMed Central

Kim, Hyo-Bin; Kim, Chang-Keun; Iijima, Koji; Kobayashi, Takao; Kita, Hirohito

2010-01-01

Background Microarray technology offers a new opportunity to gain insight into global gene and protein expression profiles in asthma. To identify novel factors produced in the asthmatic airway, we analyzed sputum samples by using a membrane-based human cytokine microarray technology in patients with bronchial asthma (BA). Methods Induced sputum was obtained from 28 BA subjects, 20 nonasthmatic atopic control (AC) subjects, and 38 nonasthmatic nonatopic normal control (NC) subjects. The microarray samples of subjects were randomly selected from nine BA subjects, three AC subjects, and six NC subjects. Sputum supernatants were analyzed using a custom human cytokine array (RayBio Custom Human Cytokine Array; RayBiotech; Norcross, GA) designed to analyze 79 specific cytokines simultaneously. The levels of growth-regulated oncogene (GRO)-α, eotaxin-2, and pulmonary and activation-regulated chemokine (PARC)/CCL18 were measured by sandwich enzyme-linked immunosorbent assays (ELISAs), and eosinophil-derived neurotoxin (EDN) was measured by radioimmunoassay. Results By microarray, the signal intensities for GRO-α, eotaxin-2, and PARC were significantly higher in BA subjects than in AC and NC subjects (p = 0.036, p = 0.042, and p = 0.033, respectively). By ELISA, the sputum PARC protein levels were significantly higher in BA subjects than in AC and NC subjects (p < 0.0001). Furthermore, PARC levels correlated significantly with sputum eosinophil percentages (r = 0.570, p < 0.0001) and the levels of EDN(r = 0.633, p < 0.0001), the regulated upon activation, normal T cell expressed and secreted cytokine (r = 0.440, p < 0.001), interleukin-4 (r = 0.415, p < 0.01), and interferon-γ (r = 0.491, p < 0.001). Conclusions By a nonbiased screening approach, a chemokine, PARC, is elevated in sputum specimens from patients with asthma. PARC may play important roles in development of airway eosinophilic inflammation in asthma. PMID:19017877

Enzymatic production of single-molecule FISH and RNA capture probes.

PubMed

Gaspar, Imre; Wippich, Frank; Ephrussi, Anne

2017-10-01

Arrays of singly labeled short oligonucleotides that hybridize to a specific target revolutionized RNA biology, enabling quantitative, single-molecule microscopy analysis and high-efficiency RNA/RNP capture. Here, we describe a simple and efficient method that allows flexible functionalization of inexpensive DNA oligonucleotides by different fluorescent dyes or biotin using terminal deoxynucleotidyl transferase and custom-made functional group conjugated dideoxy-UTP. We show that (i) all steps of the oligonucleotide labeling-including conjugation, enzymatic synthesis, and product purification-can be performed in a standard biology laboratory, (ii) the process yields >90%, often >95% labeled product with minimal carryover of impurities, and (iii) the oligonucleotides can be labeled with different dyes or biotin, allowing single-molecule FISH, RNA affinity purification, and Northern blot analysis to be performed. © 2017 Gaspar et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

ECM1 and TMPRSS4 Are Diagnostic Markers of Malignant Thyroid Neoplasms and Improve the Accuracy of Fine Needle Aspiration Biopsy

PubMed Central

Kebebew, Electron; Peng, Miao; Reiff, Emily; Duh, Quan-Yang; Clark, Orlo H.; McMillan, Alex

2005-01-01

Objective: The objective of this study was to determine whether genes that regulate cellular invasion and metastasis are differentially expressed and could serve as diagnostic markers of malignant thyroid nodules. Summary and Background Data: Patients whose thyroid nodules have indeterminate or suspicious cytologic features on fine needle aspiration (FNA) biopsy require thyroidectomy because of a 20% to 30% risk of thyroid cancer. Cell invasion and metastasis is a hallmark of malignant phenotype; therefore, genes that regulate these processes might be differentially expressed and could serve as diagnostic markers of malignancy. Methods: Differentially expressed genes (2-fold higher or lower) in malignant versus benign thyroid neoplasms were identified by extracellular matrix and adhesion molecule cDNA array analysis and confirmed by real-time quantitative polymerase chain reaction (PCR). The area under the receiver operating characteristic (AUC) curve was calculated to determine diagnostic accuracy of gene expression level cutoffs established by logistic regression analysis. Results: By cDNA array analysis, ADAMTS8, ECM1, MMP8, PLAU, SELP, and TMPRSS4 were upregulated, and by quantitative PCR, ECM1, SELP, and TMPRSS4 mRNA expression was higher in malignant (n = 57) than in benign (n = 38) thyroid neoplasms (P< 0.002). ECM1 and TMPRSS4 mRNA expression levels were independent predictors of a malignant thyroid neoplasm (P < 0.003). The AUC was 0.956 for ECM1 and 0.926 for TMPRSS4. Combining both markers improved their diagnostic use (AUC 0.985; sensitivity, 91.7%; specificity, 89.8%; positive predictive value, 85.7%; negative predictive value, 82.8%). ECM1 and TMPRSS4 expression analysis improved the diagnostic accuracy of FNA biopsy in 35 of 38 indeterminate or suspicious results. The level of ECM1 mRNA expression was higher in TNM stage I differentiated thyroid cancers than in stage II and III tumors (P ≤ 0.031). Conclusions: ECM1 and TMPRSS4 are excellent diagnostic markers of malignant thyroid nodules and may be used to improve the diagnostic accuracy of FNA biopsy. ECM1 is also a marker of the extent of disease in differentiated thyroid cancers. PMID:16135921

Characterization of a chromosome-specific chimpanzee alpha satellite subset: Evolutionary relationship to subsets on human chromosomes

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

Warburton, P.E.; Gosden, J.; Lawson, D.

1996-04-15

Alpha satellite DNA is a tandemly repeated DNA family found at the centromeres of all primate chromosomes examined. The fundamental repeat units of alpha satellite DNA are diverged 169- to 172-bp monomers, often found to be organized in chromosome-specific higher-order repeat units. The chromosomes of human (Homo sapiens (HSA)), chimpanzee (Pan troglodytes (PTR) and Pan paniscus), and gorilla (Gorilla gorilla) share a remarkable similarity and synteny. It is of interest to ask if alpha satellite arrays at centromeres of homologous chromosomes between these species are closely related (evolving in an orthologous manner) or if the evolutionary processes that homogenize andmore » spread these arrays within and between chromosomes result in nonorthologous evolution of arrays. By using PCR primers specific for human chromosome 17-specific alpha satellite DNA, we have amplified, cloned, and characterized a chromosome-specific subset from the PTR chimpanzee genome. Hybridization both on Southern blots and in situ as well as sequence analysis show that this subset is most closely related, as expected, to sequences on HSA 17. However, in situ hybridization reveals that this subset is not found on the homologous chromosome in chimpanzee (PTR 19), but instead on PTR 12, which is homologous to HSA 2p. 40 refs., 3 figs.« less

Laser-ablated poly(methyl methacrylate) microdevices for sub-microliter DNA amplification suitable for micro-total analysis systems

NASA Astrophysics Data System (ADS)

Lounsbury, Jenny A.; Poe, Brian L.; Do, Michael; Landers, James P.

2012-08-01

Biochemical techniques, such as the polymerase chain reaction (PCR), can take up to 3.5 h for completion using a commercial bench-top instrument, creating a bottleneck in sample preparation processes. PCR has been successfully adapted to microfluidic devices, reducing the time needed to as little as 7-10 min. Recently, a trend in the field is to use alternative substrates, such as poly(methyl methacyrlate) (PMMA), for the fabrication of microfluidic devices. PMMA has several advantages over more expensive substrates including rigidity without fragility, disposability, and it is easy to fabricate, using techniques such as hot embossing or CO2 laser ablation. Here, we report the fabrication of PMMA microdevices to explore their effectiveness for PCR amplification. Several types of PMMA microdevices were fabricated using a CO2 laser ablation system, with two or three PMMA layers of different thicknesses. Bonding of the microdevices was significantly improved through the use of a grid system etched into the device, allowing for trapped air to escape, eliminating leakage. Using infrared thermal cycling, the ramping rates were determined to be dependent on the thickness of the PMMA used in fabrication, allowing for customization of cycling conditions. Further reduction of the thermal mass by isolation of the chambers provided a significant increase in the heating and cooling rates (up to 6.19 (±0.32 °C s-1) and -7.99 (±0.06 °C s-1), respectively). Bubble formation, a chronic problem in microfluidic systems in general and problematic during the heating phase of PCR, was minimized through the use of a biocompatible adhesive/manifold combination to seal the reservoirs. Finally, successful PCR amplification was demonstrated with both a fragment from the β-globin gene and 15 tetra-nucleotide repeat regions with a sex-typing marker in a conventional STR kit with the latter facilitated by the dynamic coating of the fluidic architecture with poly(ethylene glycol) (PEG).

Making a chocolate chip: development and evaluation of a 6K SNP array for Theobroma cacao

PubMed Central

Livingstone, Donald; Royaert, Stefan; Stack, Conrad; Mockaitis, Keithanne; May, Greg; Farmer, Andrew; Saski, Christopher; Schnell, Ray; Kuhn, David; Motamayor, Juan Carlos

2015-01-01

Theobroma cacao, the key ingredient in chocolate production, is one of the world's most important tree fruit crops, with ∼4,000,000 metric tons produced across 50 countries. To move towards gene discovery and marker-assisted breeding in cacao, a single-nucleotide polymorphism (SNP) identification project was undertaken using RNAseq data from 16 diverse cacao cultivars. RNA sequences were aligned to the assembled transcriptome of the cultivar Matina 1-6, and 330,000 SNPs within coding regions were identified. From these SNPs, a subset of 6,000 high-quality SNPs were selected for inclusion on an Illumina Infinium SNP array: the Cacao6kSNP array. Using Cacao6KSNP array data from over 1,000 cacao samples, we demonstrate that our custom array produces a saturated genetic map and can be used to distinguish among even closely related genotypes. Our study enhances and expands the genetic resources available to the cacao research community, and provides the genome-scale set of tools that are critical for advancing breeding with molecular markers in an agricultural species with high genetic diversity. PMID:26070980

Photoacoustics and speed-of-sound dual mode imaging with a long depth-of-field by using annular ultrasound array.

PubMed

Ding, Qiuning; Tao, Chao; Liu, Xiaojun

2017-03-20

Speed-of-sound and optical absorption reflect the structure and function of tissues from different aspects. A dual-mode microscopy system based on a concentric annular ultrasound array is proposed to simultaneously acquire the long depth-of-field images of speed-of-sound and optical absorption of inhomogeneous samples. First, speed-of-sound is decoded from the signal delay between each element of the annular array. The measured speed-of-sound could not only be used as an image contrast, but also improve the resolution and accuracy of spatial location of photoacoustic image in inhomogeneous acoustic media. Secondly, benefitting from dynamic focusing of annular array and the measured speed-of-sound, it is achieved an advanced acoustic-resolution photoacoustic microscopy with a precise position and a long depth-of-field. The performance of the dual-mode imaging system has been experimentally examined by using a custom-made annular array. The proposed dual-mode microscopy might have the significances in monitoring the biological physiological and pathological processes.

Estimation of linkage disequilibrium and interspecific gene flow in Ficedula flycatchers by a newly developed 50k single-nucleotide polymorphism array

PubMed Central

Kawakami, Takeshi; Backström, Niclas; Burri, Reto; Husby, Arild; Olason, Pall; Rice, Amber M; Ålund, Murielle; Qvarnström, Anna; Ellegren, Hans

2014-01-01

With the access to draft genome sequence assemblies and whole-genome resequencing data from population samples, molecular ecology studies will be able to take truly genome-wide approaches. This now applies to an avian model system in ecological and evolutionary research: Old World flycatchers of the genus Ficedula, for which we recently obtained a 1.1 Gb collared flycatcher genome assembly and identified 13 million single-nucleotide polymorphism (SNP)s in population resequencing of this species and its sister species, pied flycatcher. Here, we developed a custom 50K Illumina iSelect flycatcher SNP array with markers covering 30 autosomes and the Z chromosome. Using a number of selection criteria for inclusion in the array, both genotyping success rate and polymorphism information content (mean marker heterozygosity = 0.41) were high. We used the array to assess linkage disequilibrium (LD) and hybridization in flycatchers. Linkage disequilibrium declined quickly to the background level at an average distance of 17 kb, but the extent of LD varied markedly within the genome and was more than 10-fold higher in ‘genomic islands’ of differentiation than in the rest of the genome. Genetic ancestry analysis identified 33 F1 hybrids but no later-generation hybrids from sympatric populations of collared flycatchers and pied flycatchers, contradicting earlier reports of backcrosses identified from much fewer number of markers. With an estimated divergence time as recently as <1 Ma, this suggests strong selection against F1 hybrids and unusually rapid evolution of reproductive incompatibility in an avian system. PMID:24784959

Plasmodium copy number variation scan: gene copy numbers evaluation in haploid genomes.

PubMed

Beghain, Johann; Langlois, Anne-Claire; Legrand, Eric; Grange, Laura; Khim, Nimol; Witkowski, Benoit; Duru, Valentine; Ma, Laurence; Bouchier, Christiane; Ménard, Didier; Paul, Richard E; Ariey, Frédéric

2016-04-12

In eukaryotic genomes, deletion or amplification rates have been estimated to be a thousand more frequent than single nucleotide variation. In Plasmodium falciparum, relatively few transcription factors have been identified, and the regulation of transcription is seemingly largely influenced by gene amplification events. Thus copy number variation (CNV) is a major mechanism enabling parasite genomes to adapt to new environmental changes. Currently, the detection of CNVs is based on quantitative PCR (qPCR), which is significantly limited by the relatively small number of genes that can be analysed at any one time. Technological advances that facilitate whole-genome sequencing, such as next generation sequencing (NGS) enable deeper analyses of the genomic variation to be performed. Because the characteristics of Plasmodium CNVs need special consideration in algorithms and strategies for which classical CNV detection programs are not suited a dedicated algorithm to detect CNVs across the entire exome of P. falciparum was developed. This algorithm is based on a custom read depth strategy through NGS data and called PlasmoCNVScan. The analysis of CNV identification on three genes known to have different levels of amplification and which are located either in the nuclear, apicoplast or mitochondrial genomes is presented. The results are correlated with the qPCR experiments, usually used for identification of locus specific amplification/deletion. This tool will facilitate the study of P. falciparum genomic adaptation in response to ecological changes: drug pressure, decreased transmission, reduction of the parasite population size (transition to pre-elimination endemic area).

ASSESSMENT OF DIFFERENTIAL GENE EXPRESSION IN SHEEPSHEAD MINNOWS EXPOSED TO ENVIRONMENTAL ESTROGENS

EPA Science Inventory

Gene arrays and quantitative real-time PCR (Q-PRC) are sensitive methods for assessing exposure of fish and other wildlife to environmental contaminants by measuring changes in gene expression. Several genes normally induced by estradiol in female fish, those for vitellogenins (...

TOXICOGENOMICS AS A TOOL TO ASSESS EXPOSURE OF FISH TO ENVIRONMENTAL POLLUTANTS

EPA Science Inventory

Molecular biological techniques such as gene arrays and quantitative real-time PCR are becoming important tools to study alterations in normal gene expression in fish and other wildlife exposed to such pollutants as endocrine disrupting chemicals (EDCs). An important function fo...

Infralimbic EphB2 Modulates Fear Extinction in Adolescent Rats

PubMed Central

Cruz, Emmanuel; Soler-Cedeño, Omar; Negrón, Geovanny; Criado-Marrero, Marangelie; Chompré, Gladys

2015-01-01

Adolescent rats are prone to impaired fear extinction, suggesting that mechanistic differences in extinction could exist in adolescent and adult rats. Since the infralimbic cortex (IL) is critical for fear extinction, we used PCR array technology to identify gene expression changes in IL induced by fear extinction in adolescent rats. Interestingly, the ephrin type B receptor 2 (EphB2), a tyrosine kinase receptor associated with synaptic development, was downregulated in IL after fear extinction. Consistent with the PCR array results, EphB2 levels of mRNA and protein were reduced in IL after fear extinction compared with fear conditioning, suggesting that EphB2 signaling in IL regulates fear extinction memory in adolescents. Finally, reducing EphB2 synthesis in IL with shRNA accelerated fear extinction learning in adolescent rats, but not in adult rats. These findings identify EphB2 in IL as a key regulator of fear extinction during adolescence, perhaps due to the increase in synaptic remodeling occurring during this developmental phase. PMID:26354908

Investigation of Implantable Multi-Channel Electrode Array in Rat Cerebral Cortex Used for Recording

NASA Astrophysics Data System (ADS)

Taniguchi, Noriyuki; Fukayama, Osamu; Suzuki, Takafumi; Mabuchi, Kunihiko

There have recently been many studies concerning the control of robot movements using neural signals recorded from the brain (usually called the Brain-Machine interface (BMI)). We fabricated implantable multi-electrode arrays to obtain neural signals from the rat cerebral cortex. As any multi-electrode array should have electrode alignment that minimizes invasion, it is necessary to customize the recording site. We designed three types of 22-channel multi-electrode arrays, i.e., 1) wide, 2) three-layered, and 3) separate. The first extensively covers the cerebral cortex. The second has a length of 2 mm, which can cover the area of the primary motor cortex. The third array has a separate structure, which corresponds to the position of the forelimb and hindlimb areas of the primary motor cortex. These arrays were implanted into the cerebral cortex of a rat. We estimated the walking speed from neural signals using our fabricated three-layered array to investigate its feasibility for BMI research. The neural signal of the rat and its walking speed were simultaneously recorded. The results revealed that evaluation using either the anterior electrode group or posterior group provided accurate estimates. However, two electrode groups around the center yielded poor estimates although it was possible to record neural signals.

Fabrication of five-level ultraplanar micromirror arrays by flip-chip assembly

NASA Astrophysics Data System (ADS)

Michalicek, M. Adrian; Bright, Victor M.

2001-10-01

This paper reports a detailed study of the fabrication of various piston, torsion, and cantilever style micromirror arrays using a novel, simple, and inexpensive flip-chip assembly technique. Several rectangular and polar arrays were commercially prefabricated in the MUMPs process and then flip-chip bonded to form advanced micromirror arrays where adverse effects typically associated with surface micromachining were removed. These arrays were bonded by directly fusing the MUMPs gold layers with no complex preprocessing. The modules were assembled using a computer-controlled, custom-built flip-chip bonding machine. Topographically opposed bond pads were designed to correct for slight misalignment errors during bonding and typically result in less than 2 micrometers of lateral alignment error. Although flip-chip micromirror performance is briefly discussed, the means used to create these arrays is the focus of the paper. A detailed study of flip-chip process yield is presented which describes the primary failure mechanisms for flip-chip bonding. Studies of alignment tolerance, bonding force, stress concentration, module planarity, bonding machine calibration techniques, prefabrication errors, and release procedures are presented in relation to specific observations in process yield. Ultimately, the standard thermo-compression flip-chip assembly process remains a viable technique to develop highly complex prototypes of advanced micromirror arrays.

Volumetric Real-Time Imaging Using a CMUT Ring Array

PubMed Central

Choe, Jung Woo; Oralkan, Ömer; Nikoozadeh, Amin; Gencel, Mustafa; Stephens, Douglas N.; O’Donnell, Matthew; Sahn, David J.; Khuri-Yakub, Butrus T.

2012-01-01

A ring array provides a very suitable geometry for forward-looking volumetric intracardiac and intravascular ultrasound imaging. We fabricated an annular 64-element capacitive micromachined ultrasonic transducer (CMUT) array featuring a 10-MHz operating frequency and a 1.27-mm outer radius. A custom software suite was developed to run on a PC-based imaging system for real-time imaging using this device. This paper presents simulated and experimental imaging results for the described CMUT ring array. Three different imaging methods—flash, classic phased array (CPA), and synthetic phased array (SPA)—were used in the study. For SPA imaging, two techniques to improve the image quality—Hadamard coding and aperture weighting—were also applied. The results show that SPA with Hadamard coding and aperture weighting is a good option for ring-array imaging. Compared with CPA, it achieves better image resolution and comparable signal-to-noise ratio at a much faster image acquisition rate. Using this method, a fast frame rate of up to 463 volumes per second is achievable if limited only by the ultrasound time of flight; with the described system we reconstructed three cross-sectional images in real-time at 10 frames per second, which was limited by the computation time in synthetic beamforming. PMID:22718870

Volumetric real-time imaging using a CMUT ring array.

PubMed

Choe, Jung Woo; Oralkan, Ömer; Nikoozadeh, Amin; Gencel, Mustafa; Stephens, Douglas N; O'Donnell, Matthew; Sahn, David J; Khuri-Yakub, Butrus T

2012-06-01

A ring array provides a very suitable geometry for forward-looking volumetric intracardiac and intravascular ultrasound imaging. We fabricated an annular 64-element capacitive micromachined ultrasonic transducer (CMUT) array featuring a 10-MHz operating frequency and a 1.27-mm outer radius. A custom software suite was developed to run on a PC-based imaging system for real-time imaging using this device. This paper presents simulated and experimental imaging results for the described CMUT ring array. Three different imaging methods--flash, classic phased array (CPA), and synthetic phased array (SPA)--were used in the study. For SPA imaging, two techniques to improve the image quality--Hadamard coding and aperture weighting--were also applied. The results show that SPA with Hadamard coding and aperture weighting is a good option for ring-array imaging. Compared with CPA, it achieves better image resolution and comparable signal-to-noise ratio at a much faster image acquisition rate. Using this method, a fast frame rate of up to 463 volumes per second is achievable if limited only by the ultrasound time of flight; with the described system we reconstructed three cross-sectional images in real-time at 10 frames per second, which was limited by the computation time in synthetic beamforming.

Type of Renal Replacement Therapy (Hemodialysis versus Peritoneal Dialysis) Does Not Affect Cytokine Gene Expression or Clinical Parameters of Renal Transplant Candidates

PubMed Central

Kamińska, Dorota; Kościelska-Kasprzak, Katarzyna; Chudoba, Paweł; Mazanowska, Oktawia; Banasik, Mirosław; Żabinska, Marcelina; Boratyńska, Maria; Lepiesza, Agnieszka; Korta, Krzysztof; Gomółkiewicz, Agnieszka; Dzięgiel, Piotr; Klinger, Marian

2015-01-01

Patients with renal failure suffer from immune disturbances, caused by uremic toxins and influenced by dialysis treatment. The aim of the present study was to reveal whether type of dialysis modality (hemodialysis, HD, versus peritoneal dialysis, PD) differentially affects the immunocompetence, particularly the expression of genes involved in the immune response. Material. 87 renal transplant candidates (66 HD, 21 PD) were included in the study. Methods. The peripheral blood RNA samples were obtained with the PAXgene Blood system just before transplantation. The gene expression of CASP3, FAS, TP53, FOXP3, IFNG, IL2, IL6, IL8, IL10, IL17, IL18, LCN2, TGFB1, and TNF was assessed with real-time PCR on custom-designed low density arrays (TaqMan). Gene expression data were analyzed in relation to pretransplant clinical parameters. Results. The mean expression of examined genes showed no significant differences between PD and HD with the exception of FAS, expression of which was 30% higher in PD patients compared to the HD group. There was nonsignificantly higher expression of proinflammatory cytokines in the PD group. The clinical inflammatory parameters (CRP, albumin, cholesterol, and hemoglobin levels) did not differ between the groups. Conclusion. Type of renal replacement therapy exerts no differential effect on cytokine gene expression or inflammatory clinical parameters. PMID:26236736

A Comparative Proteomic Analysis of the Buds and the Young Expanding Leaves of the Tea Plant (Camellia sinensis L.)

PubMed Central

Li, Qin; Li, Juan; Liu, Shuoqian; Huang, Jianan; Lin, Haiyan; Wang, Kunbo; Cheng, Xiaomei; Liu, Zhonghua

2015-01-01

Tea (Camellia sinensis L.) is a perennial woody plant that is widely cultivated to produce a popular non-alcoholic beverage; this beverage has received much attention due to its pleasant flavor and bioactive ingredients, particularly several important secondary metabolites. Due to the significant changes in the metabolite contents of the buds and the young expanding leaves of tea plants, high-performance liquid chromatography (HPLC) analysis and isobaric tags for relative and absolute quantitation (iTRAQ) analysis were performed. A total of 233 differentially expressed proteins were identified. Among these, 116 proteins were up-regulated and 117 proteins were down-regulated in the young expanding leaves compared with the buds. A large array of diverse functions was revealed, including roles in energy and carbohydrate metabolism, secondary metabolite metabolism, nucleic acid and protein metabolism, and photosynthesis- and defense-related processes. These results suggest that polyphenol biosynthesis- and photosynthesis-related proteins regulate the secondary metabolite content of tea plants. The energy and antioxidant metabolism-related proteins may promote tea leaf development. However, reverse transcription quantitative real-time PCR (RT-qPCR) showed that the protein expression levels were not well correlated with the gene expression levels. These findings improve our understanding of the molecular mechanism of the changes in the metabolite content of the buds and the young expanding leaves of tea plants. PMID:26096006

Carbon Nanotube Nanoelectrode Array for Ultrasensitive DNA Detection

NASA Technical Reports Server (NTRS)

Li, Jun; Koehne, Jessica; Chen, Hua; Cassell, Alan; Ng, Hou Tee; Fan, Wendy; Ye, Qi; Han, Jie; Meyyappan, M.

2003-01-01

A reliable nanoelectrode array based on vertically aligned multi-walled carbon nanotubes (MWNTs) embedded in SiO2 is used for ultrasensitive DNA detection. Characteristic nanoelectrode behavior is observed using low-density MWNT arrays for measuring both bulk and surface immobilized redox species such as K4Fe(CN)6. The open-end of MWNTs present similar properties as graphite edge-plane electrodes with wide potential window, flexible chemical functionalities, and good biocompatibility. Oligonucleotide probes are selectively functionalized at the open ends cf the nanotube array and specifically hybridized with oligonucleotide targets. The guanine groups are employed as the signal moieties in the electrochemical measurements. Ru(bpy)3(2+) mediator is used to further amplify the guanine oxidation signal. The hybridization of subattomoles of PCR amplified DNA targets is detected electrochemically by combining the MWNT nanoelectrode array with the Ru(bpy)32' amplification mechanism. This system provides a general platform of molecular diagnostics for applications requiring ultrahigh sensitivity, high-degree of miniaturization, and simple sample preparations.

Childhood and Children: A Compendium of Customs, Superstitions, Theories, Profiles, and Facts.

ERIC Educational Resources Information Center

Bel Geddes, Joan

Children and childhood are almost completely ignored in most history books, encyclopedias, anthologies, and almanacs, which concentrate on the achievement of the adult half of the world's population. This book is intended to fill the gap by focusing on childhood, and presents an array of facts, anecdotes, profiles, and observations about children…

Whole blood miRNA expression analysis reveals miR-3613-3p as a potential biomarker for dedifferentiated liposarcoma.

PubMed

Fricke, A; Cimniak, A F V; Ullrich, P V; Becherer, C; Bickert, C; Pfeifer, D; Heinz, J; Stark, G B; Bannasch, H; Braig, D; Eisenhardt, S U

2018-04-09

Liposarcoma constitute about 13% of all soft tissue sarcoma and are associated with a high risk of metastases. As the preoperative differentiation between benign and malign lipomatous tumors is restricted to magnetic resonance imaging, computed tomography and biopsy, we performed a miRNA array to distinguish dedifferentiated liposarcoma patients from healthy controls and lipoma patients. Blood samples of patients with dedifferentiated liposarcoma, healthy controls and lipoma patients were collected. Whole blood RNA was extracted and samples of patients with dedifferentiated liposarcoma (n= 6) and of healthy donors (n= 4) were analyzed using an Affymetrix GeneChip miRNA Array v. 4.0. qRT-PCR was carried out to confirm the most differentially expressed miRNA; being further analyzed in an independent cohort of healthy controls as well as in lipoma patients. As shown by the microarray, two miRNAs (miR-3613-3p, miR-4668-5p) were shown to be significantly upregulated (fold change: > 2.5; p< 0.05) in patients with dedifferentiated liposarcoma (n= 6) as compared to healthy controls (n= 4). miR-3613-3p was further validated by qRT-PCR to be significantly upregulated in dedifferentiated liposarcoma patients compared to an independent cohort of healthy controls (n= 3) and lipoma patients (n= 5). We identified a specific whole blood miRNA (miR-3613-3p) that may serve to distinguish between dedifferentiated liposarcoma patients and healthy controls, thus potentially serving as a specific biomarker for dedifferentiated liposarcoma.

Performance of the Versatile Array of Neutron Detectors at Low Energy (VANDLE)

DOE PAGES

Peters, W. A.; Ilyushkin, S.; Madurga, M.; ...

2016-08-26

The Versatile Array of Neutron Detectors at Low Energy (VANDLE) is a new, highly efficient plastic-scintillator array constructed for decay and transfer reaction experimental setups that require neutron detection. The versatile and modular design allows for customizable experimental setups including beta-delayed neutron spectroscopy and (d,n) transfer reactions in normal and inverse kinematics. The neutron energy and prompt-photon discrimination is determined through the time of flight technique. Fully digital data acquisition electronics and integrated triggering logic enables some VANDLE modules to achieve an intrinsic efficiency over 70% for 300-keV neutrons, measured through two different methods. A custom Geant4 simulation models aspectsmore » of the detector array and the experimental setups to determine efficiency and detector response. Lastly, a low detection threshold, due to the trigger logic and digitizing data acquisition, allowed us to measure the light-yield response curve from elastically scattered carbon nuclei inside the scintillating plastic from incident neutrons with kinetic energies below 2 MeV.« less

Quantification of larval zebrafish motor function in multiwell plates using open-source MATLAB applications.

PubMed

Zhou, Yangzhong; Cattley, Richard T; Cario, Clinton L; Bai, Qing; Burton, Edward A

2014-07-01

This article describes a method to quantify the movements of larval zebrafish in multiwell plates, using the open-source MATLAB applications LSRtrack and LSRanalyze. The protocol comprises four stages: generation of high-quality, flatly illuminated video recordings with exposure settings that facilitate object recognition; analysis of the resulting recordings using tools provided in LSRtrack to optimize tracking accuracy and motion detection; analysis of tracking data using LSRanalyze or custom MATLAB scripts; and implementation of validation controls. The method is reliable, automated and flexible, requires <1 h of hands-on work for completion once optimized and shows excellent signal:noise characteristics. The resulting data can be analyzed to determine the following: positional preference; displacement, velocity and acceleration; and duration and frequency of movement events and rest periods. This approach is widely applicable to the analysis of spontaneous or stimulus-evoked zebrafish larval neurobehavioral phenotypes resulting from a broad array of genetic and environmental manipulations, in a multiwell plate format suitable for high-throughput applications.