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.

Customizable PCR-microplate array for differential identification of multiple pathogens

PubMed Central

Woubit, Abdela; Yehualaeshet, Teshome; Roberts, Sherrelle; Graham, Martha; Kim, Moonil; Samuel, Temesgen

2014-01-01

Customizable PCR-microplate arrays were developed for the rapid identification of Francisella tularensis subsp. tularensis, Salmonella Typhi, Shigella dysenteriae, Yersinia pestis, Vibrio cholerae Escherichia coli O157:H7, Salmonella Typhimurium, Salmonella Saintpaul, Francisella tularensis subsp. novicida, Vibrio parahaemolyticus, and Yersinia pseudotuberculosis. Previously, we identified highly specific primers targeting each of the pathogens above. Here, we report the development of customizable PCR-microplate arrays for simultaneous identification of the pathogens using the primers. A mixed aliquot of genomic DNA from 38 different strains was used to validate three PCR-microplate array formats. Identical PCR conditions were used to run all the samples on the three formats. Results show specific amplifications on all the three custom plates. In a preliminary test to evaluate the sensitivity of these assays in laboratory-inoculated samples, detection limits as low as 9 cfu/g/ml S. Typhimurium were obtained from beef hot dog, and 78 cfu/ml from milk. Such microplate arrays could serve as valuable tools for initial identification or secondary confirmation of these pathogens. PMID:24215700

Overexpression of miR-9 in mast cells is associated with invasive behavior and spontaneous metastasis

PubMed Central

2014-01-01

Background While microRNA (miRNA) expression is known to be altered in a variety of human malignancies contributing to cancer development and progression, the potential role of miRNA dysregulation in malignant mast cell disease has not been previously explored. The purpose of this study was to investigate the potential contribution of miRNA dysregulation to the biology of canine mast cell tumors (MCTs), a well-established spontaneous model of malignant mast cell disease. Methods We evaluated the miRNA expression profiles from biologically low-grade and biologically high-grade primary canine MCTs using real-time PCR-based TaqMan Low Density miRNA Arrays and performed real-time PCR to evaluate miR-9 expression in primary canine MCTs, malignant mast cell lines, and normal bone marrow-derived mast cells (BMMCs). Mouse mast cell lines and BMMCs were transduced with empty or pre-miR-9 expressing lentiviral constructs and cell proliferation, caspase 3/7 activity, and invasion were assessed. Transcriptional profiling of cells overexpressing miR-9 was performed using Affymetrix GeneChip Mouse Gene 2.0 ST arrays and real-time PCR was performed to validate changes in mRNA expression. Results Our data demonstrate that unique miRNA expression profiles correlate with the biological behavior of primary canine MCTs and that miR-9 expression is increased in biologically high grade canine MCTs and malignant cell lines compared to biologically low grade tumors and normal canine BMMCs. In transformed mouse malignant mast cell lines expressing either wild-type (C57) or activating (P815) KIT mutations and mouse BMMCs, miR-9 overexpression significantly enhanced invasion but had no effect on cell proliferation or apoptosis. Transcriptional profiling of normal mouse BMMCs and P815 cells possessing enforced miR-9 expression demonstrated dysregulation of several genes, including upregulation of CMA1, a protease involved in activation of matrix metalloproteases and extracellular matrix remodeling. Conclusions Our findings demonstrate that unique miRNA expression profiles correlate with the biological behavior of canine MCTs. Furthermore, dysregulation of miR-9 is associated with MCT metastasis potentially through the induction of an invasive phenotype, identifying a potentially novel pathway for therapeutic intervention. PMID:24517413

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.

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.

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

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

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.

Customizable PCR-microplate array for differential identification of multiple pathogens.

PubMed

Woubit, Abdela; Yehualaeshet, Teshome; Roberts, Sherrelle; Graham, Martha; Kim, Moonil; Samuel, Temesgen

2013-11-01

Customizable PCR-microplate arrays were developed for the rapid identification of Salmonella Typhimurium, Salmonella Saintpaul, Salmonella Typhi, Shigella dysenteriae, Escherichia coli O157:H7, Francisella tularensis subsp. tularensis, Francisella tularensis subsp. novicida, Vibrio cholerae, Vibrio parahaemolyticus, Yersinia pestis, and Yersinia pseudotuberculosis. Previously, we identified highly specific primers targeting each of these pathogens. Here, we report the development of customizable PCR-microplate arrays for simultaneous identification of the pathogens using the primers identified. A mixed aliquot of genomic DNA from 38 strains was used to validate three PCR-microplate array formats. Identical PCR conditions were used to run all the samples on the three formats. Specific amplifications were obtained on all three custom plates. In preliminary tests performed to evaluate the sensitivity of these assays in samples inoculated in the laboratory with Salmonella Typhimurium, amplifications were obtained from 1 g of beef hot dog inoculated at as low as 9 CFU/ml or from milk inoculated at as low as 78 CFU/ml. Such microplate arrays could be valuable tools for initial identification or secondary confirmation of contamination by these pathogens.

Real-time PCR array as a universal platform for the detection of genetically modified crops and its application in identifying unapproved genetically modified crops in Japan.

PubMed

Mano, Junichi; Shigemitsu, Natsuki; Futo, Satoshi; Akiyama, Hiroshi; Teshima, Reiko; Hino, Akihiro; Furui, Satoshi; Kitta, Kazumi

2009-01-14

We developed a novel type of real-time polymerase chain reaction (PCR) array with TaqMan chemistry as a platform for the comprehensive and semiquantitative detection of genetically modified (GM) crops. Thirty primer-probe sets for the specific detection of GM lines, recombinant DNA (r-DNA) segments, endogenous reference genes, and donor organisms were synthesized, and a 96-well PCR plate was prepared with a different primer-probe in each well as the real-time PCR array. The specificity and sensitivity of the array were evaluated. A comparative analysis with the data and publicly available information on GM crops approved in Japan allowed us to assume the possibility of unapproved GM crop contamination. Furthermore, we designed a Microsoft Excel spreadsheet application, Unapproved GMO Checker version 2.01, which helps process all the data of real-time PCR arrays for the easy assumption of unapproved GM crop contamination. The spreadsheet is available free of charge at http://cse.naro.affrc.go.jp/jmano/index.html .

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

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.

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

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.

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

Tissue Gene Expression Analysis Using Arrayed Normalized cDNA Libraries

PubMed Central

Eickhoff, Holger; Schuchhardt, Johannes; Ivanov, Igor; Meier-Ewert, Sebastian; O'Brien, John; Malik, Arif; Tandon, Neeraj; Wolski, Eryk-Witold; Rohlfs, Elke; Nyarsik, Lajos; Reinhardt, Richard; Nietfeld, Wilfried; Lehrach, Hans

2000-01-01

We have used oligonucleotide-fingerprinting data on 60,000 cDNA clones from two different mouse embryonic stages to establish a normalized cDNA clone set. The normalized set of 5,376 clones represents different clusters and therefore, in almost all cases, different genes. The inserts of the cDNA clones were amplified by PCR and spotted on glass slides. The resulting arrays were hybridized with mRNA probes prepared from six different adult mouse tissues. Expression profiles were analyzed by hierarchical clustering techniques. We have chosen radioactive detection because it combines robustness with sensitivity and allows the comparison of multiple normalized experiments. Sensitive detection combined with highly effective clustering algorithms allowed the identification of tissue-specific expression profiles and the detection of genes specifically expressed in the tissues investigated. The obtained results are publicly available (http://www.rzpd.de) and can be used by other researchers as a digital expression reference. [The sequence data described in this paper have been submitted to the EMBL data library under accession nos. AL360374–AL36537.] PMID:10958641

Nano-cone optical fiber array sensors for MiRNA profiling

NASA Astrophysics Data System (ADS)

Wang, Yunshan; Senapati, Satyajyoti; Stoddart, Paul; Howard, Scott; Chang, Hsueh-Chia

2013-09-01

Up/down regulation of microRNA panels has been correlated to cardiovascular diseases and cancer. Frequent miRNA profiling at home can hence allow early cancer diagnosis and home-use chronic disease monitoring, thus reducing both mortality rate and healthcare cost. However, lifetime of miRNAs is less than 1 hour without preservation and their concentrations range from pM to mM. Despite rapid progress in the last decade, modern nucleic acid analysis methods still do not allow personalized miRNA profiling---Real-time PCR and DNA micro-array both require elaborate miRNA preservation steps and expensive equipment and nano pore sensors cannot selectively quantify a large panel with a large dynamic range. We report a novel and low-cost optical fiber sensing platform, which has the potential to profile a panel of miRNA with simple LED light sources and detectors. The individual tips of an optical imaging fiber bundle (mm in diameter with 7000 fiber cores) were etched into cones with 10 nm radius of curvature and coated with Au. FRET (Forster Resonant Energy Transfer) hairpin oligo probes, with the loop complementary to a specific miRNA that can release the hairpin, were functionalized onto the conic tips. Exciting light in the optical fiber waveguide is optimally coupled to surface plasmonics on the gold surface, which then converges to the conic tips with two orders of magnitude enhancement in intensity. Unlike nanoparticle plasmonics, tip plasmonics can be excited over a large band width and hence the plasmonic enhanced fluorescence signal of the FRET reporter is also focused towards the tip--- and is further enhanced with the periodic resonant grid of the fiber array which gives rise to pronounced standing wave interference patterns. Multiplexing is realized by functionalizing different probes onto one fiber bundle using a photoactivation process.

Differential Expression of CHL1 Gene during Development of Major Human Cancers

PubMed Central

Senchenko, Vera N.; Krasnov, George S.; Dmitriev, Alexey A.; Kudryavtseva, Anna V.; Anedchenko, Ekaterina A.; Braga, Eleonora A.; Pronina, Irina V.; Kondratieva, Tatiana T.; Ivanov, Sergey V.; Zabarovsky, Eugene R.; Lerman, Michael I.

2011-01-01

Background CHL1 gene (also known as CALL) on 3p26.3 encodes a one-pass trans-membrane cell adhesion molecule (CAM). Previously CAMs of this type, including L1, were shown to be involved in cancer growth and metastasis. Methodology/Principal Findings We used Clontech Cancer Profiling Arrays (19 different types of cancers, 395 samples) to analyze expression of the CHL1 gene. The results were further validated by RT-qPCR for breast, renal and lung cancer. Cancer Profiling Arrays revealed differential expression of the gene: down-regulation/silencing in a majority of primary tumors and up-regulation associated with invasive/metastatic growth. Frequent down-regulation (>40% of cases) was detected in 11 types of cancer (breast, kidney, rectum, colon, thyroid, stomach, skin, small intestine, bladder, vulva and pancreatic cancer) and frequent up-regulation (>40% of cases) – in 5 types (lung, ovary, uterus, liver and trachea) of cancer. Using real-time quantitative PCR (RT-qPCR) we found that CHL1 expression was decreased in 61% of breast, 60% of lung, 87% of clear cell and 89% papillary renal cancer specimens (P<0.03 for all the cases). There was a higher frequency of CHL1 mRNA decrease in lung squamous cell carcinoma compared to adenocarcinoma (81% vs. 38%, P = 0.02) without association with tumor progression. Conclusions/Significance Our results suggested that CHL1 is involved in the development of different human cancers. Initially, during the primary tumor growth CHL1 could act as a putative tumor suppressor and is silenced to facilitate in situ tumor growth for 11 cancer types. We also suggested that re-expression of the gene on the edge of tumor mass might promote local invasive growth and enable further metastatic spread in ovary, colon and breast cancer. Our data also supported the role of CHL1 as a potentially novel specific biomarker in the early pathogenesis of two major histological types of renal cancer. PMID:21408220

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

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.

Luteolin and apigenin activate the Oct-4/Sox2 signal via NFATc1 in human periodontal ligament cells.

PubMed

Liu, Lu; Peng, Zhengjun; Huang, Haoquan; Xu, Zhezhen; Wei, Xi

2016-10-01

Identifying small molecules to activate the Oct-4/Sox2-derived pluripotency network represents a hopeful and safe method to pluripotency without genetic manipulation. Luteolin and apigenin, two major bioactive flavonoids, enhance reprogramming efficiency and increase expression of Oct-4/Sox2/c-Myc, albeit the detailed mechanism regulating pluripotency in dental-derived cells remains unknown. In the present study, to elucidate the effect of luteolin/apigenin on pluripotency of periodontal ligament cells (PDLCs) through interaction with downstream signals, we examined cell cycle, proliferation, apoptosis, expression of Oct-4/Sox2/c-Myc, and multilineage differentiation of PDLCs with luteolin/apigenin treatment. Moreover, we profiled the differentially expressed pluripotency genes by PCR arrays. Our results demonstrated that luteolin/apigenin restrained cell proliferation, increased apoptosis, and arrested PDLCs in G2/M and S phase. Luteolin and apigenin activated expression of Oct-4, Sox2, and c-Myc in a time- and dose-dependent pattern, and repressed lineage-specific differentiation. PCR arrays profiled multiple signals in PDLCs with luteolin/apigenin treatment, among which NFATc1 was the major upregulated gene. Notably, blocking of the NFATc1 signal with INCA-6 significantly decreased mRNA and protein expression of Oct-4, Sox2, and c-Myc in PDLCs with luteolin/apigenin treatment, indicating that NFATc1 may act as an upstream modulator of Oct-4/Sox2 signal. Taken together, this study showed that luteolin and apigenin effectively maintain pluripotency of PDLCs through activation of Oct-4/Sox2 signal via NFATc1. © 2016 International Federation for Cell Biology.

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.

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.

Gene expression patterns in rainbow trout, Oncorhynchus mykiss, exposed to a suite of model toxicants

PubMed Central

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

2008-01-01

The increased availability and use of DNA microarrays has allowed the characterization of gene expression patterns associated with exposure to different toxicants. An important question is whether toxicant induced changes in gene expression in fish are sufficiently diverse to allow for identification of specific modes of action and/or specific contaminants. In theory, each class of toxicant may generate a gene expression profile unique to its mode of toxic action. In this study, isogenic (cloned) rainbow trout Oncorhynchus mykiss were exposed to sublethal levels of a series of model toxicants with varying modes of action, including ethynylestradiol (xeno-estrogen), 2,2,4,4′-tetrabromodiphenyl ether (BDE-47, thyroid active), diquat (oxidant stressor), chromium VI, and benzo[a]pyrene (BaP) for a period of 1–3 weeks. An additional experiment measured trenbolone (anabolic steroid; model androgen) induced gene expression changes in sexually mature female trout. Following exposure, fish were euthanized, livers removed and RNA 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 cDNA’s. The slides were scanned to measure abundance of a given transcript in each sample relative to controls. Data were analyzed via Genespring (Silicon Genetics) to identify a list of up- and downregulated genes, as well as to determine gene clustering patterns that can be used as “expression signatures”. The results indicate each toxicant exposure caused between 64 and 222 genes to be significantly altered in expression. Most genes exhibiting altered expression responded to only one of the toxicants and relatively few were co-expressed in multiple treatments. For example, BaP and Diquat, both of which exert toxicity via oxidative stress, upregulated 28 of the same genes, of over 100 genes altered by either treatment. Other genes associated with steroidogenesis, p450 and estrogen responsive genes appear to be useful for selectively identifying toxicant mode of action in fish, suggesting a link between gene expression profile and mode of toxicity. Our array results showed good agreement with quantitative real time polymerase chain reaction (qRT PCR), which demonstrates that the arrays are an accurate measure of gene expression. The specificity of the gene expression profile in response to a model toxicant, the link between genes with altered expression and mode of toxic action, and the consistency between array and qRT PCR results all suggest that cDNA microarrays have the potential to screen environmental contaminants for biomarkers and mode of toxic action. PMID:16488489

Gene expression patterns in rainbow trout, Oncorhynchus mykiss, exposed to a suite of model toxicants.

PubMed

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

2006-05-25

The increased availability and use of DNA microarrays has allowed the characterization of gene expression patterns associated with exposure to different toxicants. An important question is whether toxicant induced changes in gene expression in fish are sufficiently diverse to allow for identification of specific modes of action and/or specific contaminants. In theory, each class of toxicant may generate a gene expression profile unique to its mode of toxic action. In this study, isogenic (cloned) rainbow trout Oncorhynchus mykiss were exposed to sublethal levels of a series of model toxicants with varying modes of action, including ethynylestradiol (xeno-estrogen), 2,2,4,4'-tetrabromodiphenyl ether (BDE-47, thyroid active), diquat (oxidant stressor), chromium VI, and benzo[a]pyrene (BaP) for a period of 1-3 weeks. An additional experiment measured trenbolone (anabolic steroid; model androgen) induced gene expression changes in sexually mature female trout. Following exposure, fish were euthanized, livers removed and RNA 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 cDNA's. The slides were scanned to measure abundance of a given transcript in each sample relative to controls. Data were analyzed via Genespring (Silicon Genetics) to identify a list of up- and downregulated genes, as well as to determine gene clustering patterns that can be used as "expression signatures". The results indicate each toxicant exposure caused between 64 and 222 genes to be significantly altered in expression. Most genes exhibiting altered expression responded to only one of the toxicants and relatively few were co-expressed in multiple treatments. For example, BaP and Diquat, both of which exert toxicity via oxidative stress, upregulated 28 of the same genes, of over 100 genes altered by either treatment. Other genes associated with steroidogenesis, p450 and estrogen responsive genes appear to be useful for selectively identifying toxicant mode of action in fish, suggesting a link between gene expression profile and mode of toxicity. Our array results showed good agreement with quantitative real time polymerase chain reaction (qRT PCR), which demonstrates that the arrays are an accurate measure of gene expression. The specificity of the gene expression profile in response to a model toxicant, the link between genes with altered expression and mode of toxic action, and the consistency between array and qRT PCR results all suggest that cDNA microarrays have the potential to screen environmental contaminants for biomarkers and mode of toxic action.

Comparison of array comparative genomic hybridization and quantitative real-time PCR-based aneuploidy screening of blastocyst biopsies.

PubMed

Capalbo, Antonio; Treff, Nathan R; Cimadomo, Danilo; Tao, Xin; Upham, Kathleen; Ubaldi, Filippo Maria; Rienzi, Laura; Scott, Richard T

2015-07-01

Comprehensive chromosome screening (CCS) methods are being extensively used to select chromosomally normal embryos in human assisted reproduction. Some concerns related to the stage of analysis and which aneuploidy screening method to use still remain. In this study, the reliability of blastocyst-stage aneuploidy screening and the diagnostic performance of the two mostly used CCS methods (quantitative real-time PCR (qPCR) and array comparative genome hybridization (aCGH)) has been assessed. aCGH aneuploid blastocysts were rebiopsied, blinded, and evaluated by qPCR. Discordant cases were subsequently rebiopsied, blinded, and evaluated by single-nucleotide polymorphism (SNP) array-based CCS. Although 81.7% of embryos showed the same diagnosis when comparing aCGH and qPCR-based CCS, 18.3% (22/120) of embryos gave a discordant result for at least one chromosome. SNP array reanalysis showed that a discordance was reported in ten blastocysts for aCGH, mostly due to false positives, and in four cases for qPCR. The discordant aneuploidy call rate per chromosome was significantly higher for aCGH (5.7%) compared with qPCR (0.6%; P<0.01). To corroborate these findings, 39 embryos were simultaneously biopsied for aCGH and qPCR during blastocyst-stage aneuploidy screening cycles. 35 matched including all 21 euploid embryos. Blinded SNP analysis on rebiopsies of the four embryos matched qPCR. These findings demonstrate the high reliability of diagnosis performed at the blastocyst stage with the use of different CCS methods. However, the application of aCGH can be expected to result in a higher aneuploidy rate than other contemporary methods of CCS.

Underwater Application of Quantitative PCR on an Ocean Mooring

PubMed Central

Preston, Christina M.; Harris, Adeline; Ryan, John P.; Roman, Brent; Marin, Roman; Jensen, Scott; Everlove, Cheri; Birch, James; Dzenitis, John M.; Pargett, Douglas; Adachi, Masao; Turk, Kendra; Zehr, Jonathon P.; Scholin, Christopher A.

2011-01-01

The Environmental Sample Processor (ESP) is a device that allows for the underwater, autonomous application of DNA and protein probe array technologies as a means to remotely identify and quantify, in situ, marine microorganisms and substances they produce. Here, we added functionality to the ESP through the development and incorporation of a module capable of solid-phase nucleic acid extraction and quantitative PCR (qPCR). Samples collected by the instrument were homogenized in a chaotropic buffer compatible with direct detection of ribosomal RNA (rRNA) and nucleic acid purification. From a single sample, both an rRNA community profile and select gene abundances were ascertained. To illustrate this functionality, we focused on bacterioplankton commonly found along the central coast of California and that are known to vary in accordance with different oceanic conditions. DNA probe arrays targeting rRNA revealed the presence of 16S rRNA indicative of marine crenarchaea, SAR11 and marine cyanobacteria; in parallel, qPCR was used to detect 16S rRNA genes from the former two groups and the large subunit RuBisCo gene (rbcL) from Synecchococcus. The PCR-enabled ESP was deployed on a coastal mooring in Monterey Bay for 28 days during the spring-summer upwelling season. The distributions of the targeted bacterioplankon groups were as expected, with the exception of an increase in abundance of marine crenarchaea in anomalous nitrate-rich, low-salinity waters. The unexpected co-occurrence demonstrated the utility of the ESP in detecting novel events relative to previously described distributions of particular bacterioplankton groups. The ESP can easily be configured to detect and enumerate genes and gene products from a wide range of organisms. This study demonstrated for the first time that gene abundances could be assessed autonomously, underwater in near real-time and referenced against prevailing chemical, physical and bulk biological conditions. PMID:21829630

Single cell transcriptomic analysis of prostate cancer cells.

PubMed

Welty, Christopher J; Coleman, Ilsa; Coleman, Roger; Lakely, Bryce; Xia, Jing; Chen, Shu; Gulati, Roman; Larson, Sandy R; Lange, Paul H; Montgomery, Bruce; Nelson, Peter S; Vessella, Robert L; Morrissey, Colm

2013-02-16

The ability to interrogate circulating tumor cells (CTC) and disseminated tumor cells (DTC) is restricted by the small number detected and isolated (typically <10). To determine if a commercially available technology could provide a transcriptomic profile of a single prostate cancer (PCa) cell, we clonally selected and cultured a single passage of cell cycle synchronized C4-2B PCa cells. Ten sets of single, 5-, or 10-cells were isolated using a micromanipulator under direct visualization with an inverted microscope. Additionally, two groups of 10 individual DTC, each isolated from bone marrow of 2 patients with metastatic PCa were obtained. RNA was amplified using the WT-Ovation™ One-Direct Amplification System. The amplified material was hybridized on a 44K Whole Human Gene Expression Microarray. A high stringency threshold, a mean Alexa Fluor® 3 signal intensity above 300, was used for gene detection. Relative expression levels were validated for select genes using real-time PCR (RT-qPCR). Using this approach, 22,410, 20,423, and 17,009 probes were positive on the arrays from 10-cell pools, 5-cell pools, and single-cells, respectively. The sensitivity and specificity of gene detection on the single-cell analyses were 0.739 and 0.972 respectively when compared to 10-cell pools, and 0.814 and 0.979 respectively when compared to 5-cell pools, demonstrating a low false positive rate. Among 10,000 randomly selected pairs of genes, the Pearson correlation coefficient was 0.875 between the single-cell and 5-cell pools and 0.783 between the single-cell and 10-cell pools. As expected, abundant transcripts in the 5- and 10-cell samples were detected by RT-qPCR in the single-cell isolates, while lower abundance messages were not. Using the same stringency, 16,039 probes were positive on the patient single-cell arrays. Cluster analysis showed that all 10 DTC grouped together within each patient. A transcriptomic profile can be reliably obtained from a single cell using commercially available technology. As expected, fewer amplified genes are detected from a single-cell sample than from pooled-cell samples, however this method can be used to reliably obtain a transcriptomic profile from DTC isolated from the bone marrow of patients with PCa.

Genomic profiling of human penile carcinoma predicts worse prognosis and survival.

PubMed

Busso-Lopes, Ariane F; Marchi, Fábio A; Kuasne, Hellen; Scapulatempo-Neto, Cristovam; Trindade-Filho, José Carlos S; de Jesus, Carlos Márcio N; Lopes, Ademar; Guimarães, Gustavo C; Rogatto, Silvia R

2015-02-01

The molecular mechanisms underlying penile carcinoma are still poorly understood, and the detection of genetic markers would be of great benefit for these patients. In this study, we assessed the genomic profile aiming at identifying potential prognostic biomarkers in penile carcinoma. Globally, 46 penile carcinoma samples were considered to evaluate DNA copy-number alterations via array comparative genomic hybridization (aCGH) combined with human papillomavirus (HPV) genotyping. Specific genes were investigated by using qPCR, FISH, and RT-qPCR. Genomic alterations mapped at 3p and 8p were related to worse prognostic features, including advanced T and clinical stage, recurrence and death from the disease. Losses of 3p21.1-p14.3 and gains of 3q25.31-q29 were associated with reduced cancer-specific and disease-free survival. Genomic alterations detected for chromosome 3 (LAMP3, PPARG, TNFSF10 genes) and 8 (DLC1) were evaluated by qPCR. DLC1 and PPARG losses were associated with poor prognosis characteristics. Losses of DLC1 were an independent risk factor for recurrence on multivariate analysis. The gene-expression analysis showed downexpression of DLC1 and PPARG and overexpression of LAMP3 and TNFSF10 genes. Chromosome Y losses and MYC gene (8q24) gains were confirmed by FISH. HPV infection was detected in 34.8% of the samples, and 19 differential genomic regions were obtained related to viral status. At first time, we described recurrent copy-number alterations and its potential prognostic value in penile carcinomas. We also showed a specific genomic profile according to HPV infection, supporting the hypothesis that penile tumors present distinct etiologies according to virus status. ©2014 American Association for Cancer Research.

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.

Increased expression of resistin in ectopic endometrial tissue of women with endometriosis.

PubMed

Oh, Yoon Kyung; Ha, Young Ran; Yi, Kyong Wook; Park, Hyun Tae; Shin, Jung-Ho; Kim, Tak; Hur, Jun-Young

2017-11-01

Inflammation is a key process in the establishment and progression of endometriosis. Resistin, an adipocytokine, has biological properties linked to immunologic functions, but its role in endometriosis is unclear. Resistin gene expression was examined in eutopic and ectopic endometrial tissues from women with (n=25) or without (n=25) endometriosis. Resistin mRNA and protein levels were determined in endometrial tissue using quantitative real-time reverse transcription PCR and Western blotting, following adipokine profiling arrays. Resistin protein was detected in human endometrial tissues using an adipokine array test. Resistin mRNA and protein levels were significantly higher in ectopic endometrial tissue of patients with endometriosis than in normal eutopic endometrial tissue. Our results indicate that resistin is differentially expressed in endometrial tissues from women with endometriosis and imply a role for resistin in endometriosis-associated pelvic inflammation. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Profound Effect of Profiling Platform and Normalization Strategy on Detection of Differentially Expressed MicroRNAs – A Comparative Study

PubMed Central

Meyer, Swanhild U.; Kaiser, Sebastian; Wagner, Carola; Thirion, Christian; Pfaffl, Michael W.

2012-01-01

Background Adequate normalization minimizes the effects of systematic technical variations and is a prerequisite for getting meaningful biological changes. However, there is inconsistency about miRNA normalization performances and recommendations. Thus, we investigated the impact of seven different normalization methods (reference gene index, global geometric mean, quantile, invariant selection, loess, loessM, and generalized procrustes analysis) on intra- and inter-platform performance of two distinct and commonly used miRNA profiling platforms. Methodology/Principal Findings We included data from miRNA profiling analyses derived from a hybridization-based platform (Agilent Technologies) and an RT-qPCR platform (Applied Biosystems). Furthermore, we validated a subset of miRNAs by individual RT-qPCR assays. Our analyses incorporated data from the effect of differentiation and tumor necrosis factor alpha treatment on primary human skeletal muscle cells and a murine skeletal muscle cell line. Distinct normalization methods differed in their impact on (i) standard deviations, (ii) the area under the receiver operating characteristic (ROC) curve, (iii) the similarity of differential expression. Loess, loessM, and quantile analysis were most effective in minimizing standard deviations on the Agilent and TLDA platform. Moreover, loess, loessM, invariant selection and generalized procrustes analysis increased the area under the ROC curve, a measure for the statistical performance of a test. The Jaccard index revealed that inter-platform concordance of differential expression tended to be increased by loess, loessM, quantile, and GPA normalization of AGL and TLDA data as well as RGI normalization of TLDA data. Conclusions/Significance We recommend the application of loess, or loessM, and GPA normalization for miRNA Agilent arrays and qPCR cards as these normalization approaches showed to (i) effectively reduce standard deviations, (ii) increase sensitivity and accuracy of differential miRNA expression detection as well as (iii) increase inter-platform concordance. Results showed the successful adoption of loessM and generalized procrustes analysis to one-color miRNA profiling experiments. PMID:22723911

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.

Changes in the transcriptional profile in response to overexpression of the osteopontin-c splice isoform in ovarian (OvCar-3) and prostate (PC-3) cancer cell lines

PubMed Central

2014-01-01

Background Especially in human tumor cells, the osteopontin (OPN) primary transcript is subject to alternative splicing, generating three isoforms termed OPNa, OPNb and OPNc. We previously demonstrated that the OPNc splice variant activates several aspects of the progression of ovarian and prostate cancers. The goal of the present study was to develop cell line models to determine the impact of OPNc overexpression on main cancer signaling pathways and thus obtain insights into the mechanisms of OPNc pro-tumorigenic roles. Methods Human ovarian and prostate cancer cell lines, OvCar-3 and PC-3 cells, respectively, were stably transfected to overexpress OPNc. Transcriptomic profiling was performed on these cells and compared to controls, to identify OPNc overexpression-dependent changes in gene expression levels and pathways by qRT-PCR analyses. Results Among 84 genes tested by using a multiplex real-time PCR Cancer Pathway Array approach, 34 and 16, respectively, were differentially expressed between OvCar-3 and PC-3 OPNc-overexpressing cells in relation to control clones. Differentially expressed genes are included in all main hallmarks of cancer, and several interacting proteins have been identified using an interactome network analysis. Based on marked up-regulation of Vegfa transcript in response to OPNc overexpression, we partially validated the array data by demonstrating that conditioned medium (CM) secreted from OvCar-3 and PC-3 OPNc-overexpressing cells significantly induced endothelial cell adhesion, proliferation and migration, compared to CM secreted from control cells. Conclusions Overall, the present study elucidated transcriptional changes of OvCar-3 and PC-3 cancer cell lines in response to OPNc overexpression, which provides an assessment for predicting the molecular mechanisms by which this splice variant promotes tumor progression features. PMID:24928374

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.

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.

Measurement of indicator genes using global complementary DNA (cDNA) amplification, by polyadenylic acid reverse transcriptase polymerase chain reaction (poly A RT-PCR): A feasibility study using paired samples from tissue and ductal juice in patients undergoing pancreatoduodenectomy.

PubMed

Sanyal, Sudip; Siriwardena, Ajith K; Byers, Richard

2018-06-01

The aim of this study is to compare gene expression profiles in RNA isolated from pancreatic ductal juice with the RNA expression profiles of the same genes from matched intra-operative tissue samples from pancreatic tumours. Intra-operative sampling of pancreatic juice and collection of matched tissue samples was undertaken in patients undergoing pancreatoduodenectomy for clinically suspected pancreatic cancer and a precursor lesion, main-duct intraductal papillary mucinous neoplasm. RNA was isolated and Poly A PCR was used to globally amplify the RNA. Real-time polymerase chain reaction (RT-PCR) was used to measure expression levels of 17 genes selected from microarray studies. Spearman's rank correlation test was used to examine the relationship of gene expression between pancreatic juice and tissue. The study was approved by Regional Ethics Committee. Mesothelin (MSLN) showed significant correlation (p < 0.008) in expression levels between paired pancreatic juice and tissue samples in pancreas cancer. In intraductal papillary mucinous neoplasms (IPMN), Matrix Metalloproteinase 7 (MMP7), showed significant correlation (p < 0.01) in the expression levels between paired pancreatic juice and tissue samples. This study confirms that RNA analysis of paired pancreatic juice and tissue samples and establishment of cDNA using poly A PCR is technically feasible. Application of the technique to non-invasively obtained pancreatic juice during endoscopic assessment of tumours and the use of gene arrays of cancer indicator genes are the next steps in development of this technique. Copyright © 2018 IAP and EPC. Published by Elsevier B.V. All rights reserved.

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

PubMed

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

2017-12-01

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

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

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.

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

PubMed

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

2009-07-01

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

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.

Identification of Mouse Serum miRNA Endogenous References by Global Gene Expression Profiles

PubMed Central

Mi, Qing-Sheng; Weiland, Matthew; Qi, Rui-Qun; Gao, Xing-Hua; Poisson, Laila M.; Zhou, Li

2012-01-01

MicroRNAs (miRNAs) are recently discovered small non-coding RNAs and can serve as serum biomarkers for disease diagnosis and prognoses. Lack of reliable serum miRNA endogenous references for normalization in miRNA gene expression makes single miRNA assays inaccurate. Using TaqMan® real-time PCR miRNA arrays with a global gene expression normalization strategy, we have analyzed serum miRNA expression profiles of 20 female mice of NOD/ShiLtJ (n = 8), NOR/LtJ (n = 6), and C57BL/6J (n = 6) at different ages and disease conditions. We identified five miRNAs, miR-146a, miR-16, miR-195, miR-30e and miR-744, to be stably expressed in all strains, which could serve as mouse serum miRNA endogenous references for single assay experiments. PMID:22348064

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.

Gene expression profile of steroid-induced necrosis of femoral head of rats.

PubMed

Tong, Peijian; Wu, Chengliang; Jin, Hongting; Mao, Qiang; Yu, Nanze; Holz, Jonathan D; Shan, Letian; Liu, Hui; Xiao, Luwei

2011-10-01

The key to treating steroid-induced necrosis of femoral heads (SINFH) is early diagnosis. Dramatic improvements in diagnosis could be made if the pathogenesis of SINFH was more fully understood; however, the underlying mechanism of this disease is currently unknown. To explore the potential mechanism of SINFH, we performed gene array analysis on a rat model of the disease and compare the expression profile with that of normal rats. A quantitative RT-PCR and immunohistochemistry (IHC) assays were used to confirm the microarray results. Compared to the control group, 190 genes in the experimental group were differentially expressed, with 52 up-regulated and 138 down-regulated. Of these genes, 102 are known (deposited in GenBank), while 88 of them are unknown. The known genes can be divided into several families according to their biological functions, such as oxidative stress, apoptosis, signal transduction, angiogenesis, extracellular matrix, lipid metabolism, and transcription related genes. The results of quantitative RT-PCR and IHC were consistent with gene chip results. Our findings indicate that many genes involved in diverse signaling pathways were differentially expressed between SINFH rats and normal rats. Furthermore, our findings suggest that the development of SINFH is a complicated and dynamic process affected by multiple factors and signaling pathways and regulated by various genes.

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

Use of a custom RT-PCR array to analyze toxicity pathways at different life stages in Brown Norway Rat Brain following acute Toluene exposure.

EPA Science Inventory

To investigate the contribution of different life stages on response to toxicants, we utilized a custom designed RT-PCR array to examine the effects of acute exposure by oral gavage of the volatile organic solvent toluene (0.00, 0.65 or 1.0 glkg) in the brains of ma1e Brown Norwa...

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.

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.

Inflammation and Epstein-Barr Virus Infection Are Common Features of Myasthenia Gravis Thymus: Possible Roles in Pathogenesis

PubMed Central

Cavalcante, Paola; Maggi, Lorenzo; Colleoni, Lara; Caldara, Rosa; Motta, Teresio; Giardina, Carmelo; Antozzi, Carlo; Berrih-Aknin, Sonia; Bernasconi, Pia; Mantegazza, Renato

2011-01-01

The thymus plays a major role in myasthenia gravis (MG). Our recent finding of a persistent Epstein-Barr (EBV) virus infection in some MG thymuses, combined with data showing that the thymus is in a proinflammatory state in most patients, supports a viral contribution to the pathogenesis of MG. Aim of this study was to gain further evidence for intrathymic chronic inflammation and EBV infection in MG patients. Transcriptional profiling by low density array and real-time PCR showed overexpression of genes involved in inflammatory and immune response in MG thymuses. Real-time PCR for EBV genome, latent (EBER1, EBNA1, LMP1) and lytic (BZLF1) transcripts, and immunohistochemistry for LMP1 and BZLF1 proteins confirmed an active intrathymic EBV infection, further supporting the hypothesis that EBV might contribute to onset or perpetuation of the autoimmune response in MG. Altogether, our results support a role of inflammation and EBV infection as pathogenic features of MG thymus. PMID:21961056

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

PubMed

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

2017-01-01

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

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.

Expression signature as a biomarker for prenatal diagnosis of trisomy 21.

PubMed

Volk, Marija; Maver, Aleš; Lovrečić, Luca; Juvan, Peter; Peterlin, Borut

2013-01-01

A universal biomarker panel with the potential to predict high-risk pregnancies or adverse pregnancy outcome does not exist. Transcriptome analysis is a powerful tool to capture differentially expressed genes (DEG), which can be used as biomarker-diagnostic-predictive tool for various conditions in prenatal setting. In search of biomarker set for predicting high-risk pregnancies, we performed global expression profiling to find DEG in Ts21. Subsequently, we performed targeted validation and diagnostic performance evaluation on a larger group of case and control samples. Initially, transcriptomic profiles of 10 cultivated amniocyte samples with Ts21 and 9 with normal euploid constitution were determined using expression microarrays. Datasets from Ts21 transcriptomic studies from GEO repository were incorporated. DEG were discovered using linear regression modelling and validated using RT-PCR quantification on an independent sample of 16 cases with Ts21 and 32 controls. The classification performance of Ts21 status based on expression profiling was performed using supervised machine learning algorithm and evaluated using a leave-one-out cross validation approach. Global gene expression profiling has revealed significant expression changes between normal and Ts21 samples, which in combination with data from previously performed Ts21 transcriptomic studies, were used to generate a multi-gene biomarker for Ts21, comprising of 9 gene expression profiles. In addition to biomarker's high performance in discriminating samples from global expression profiling, we were also able to show its discriminatory performance on a larger sample set 2, validated using RT-PCR experiment (AUC=0.97), while its performance on data from previously published studies reached discriminatory AUC values of 1.00. Our results show that transcriptomic changes might potentially be used to discriminate trisomy of chromosome 21 in the prenatal setting. As expressional alterations reflect both, causal and reactive cellular mechanisms, transcriptomic changes may thus have future potential in the diagnosis of a wide array of heterogeneous diseases that result from genetic disturbances.

Prospective technical validation and assessment of intra-tumour heterogeneity of a low density array hypoxia gene profile in head and neck squamous cell carcinoma.

PubMed

Betts, Guy N J; Eustace, Amanda; Patiar, Shalini; Valentine, Helen R; Irlam, Joely; Ramachandran, Anassuya; Merve, Ashirwad; Homer, Jarrod J; Möller-Levet, Carla; Buffa, Francesca M; Hall, Gillian; Miller, Crispin J; Harris, Adrian L; West, Catharine M L

2013-01-01

Tumour hypoxia is associated with a poor prognosis in head and neck squamous cell carcinoma (HNSCC), however there is no accepted method for assessing hypoxia clinically. We aimed to conduct a technical validation of a hypoxia gene expression signature using the TaqMan Low Density Array (TLDA) platform to investigate if this approach reliably identified hypoxic tumours. Tumour samples (n=201) from 80 HNSCC patients were collected prospectively from two centres. Fifty-three patients received pimonidazole prior to surgery. TaqMan Low Density Array-Hypoxia Scores (TLDA-HS) were obtained by quantitative real-time PCR (qPCR) using a 25-gene signature and customised TLDA cards. Assay performance was assessed as coefficient of variation (CoV). The assay was sensitive with linear reaction efficiencies across a 4 log(10) range of inputted cDNA (0.001-10 ng/μl). Intra- (CoV=6.9%) and inter- (CoV=2.0%) assay reproducibility were excellent. Intra-tumour heterogeneity was lower for TLDA-HS (23.2%) than for pimonidazole (67.2%) or single gene measurements of CA9 (62.2%), VEGFA (45.0%) or HIG2 (39.4%). TLDA-HS in HNSCC cell lines increased with decreasing pO(2). TLDA-HS correlated with Affymetrix U133 Plus 2.0 microarray HS (p<0.01) and positive pimonidazole scores (p=0.005). Gene expression measurements of hypoxia using a 25-gene signature and TLDA cards are sensitive, reproducible and associated with lower intra-tumour heterogeneity than assaying individual genes or pimonidazole binding. The approach is suitable for further assessment of prognostic and predictive capability in clinical trial material. Copyright © 2012 Elsevier Ltd. All rights reserved.

Method for replicating an array of nucleic acid probes

DOEpatents

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

1998-08-18

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

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

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.

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.

Fidelity and enhanced sensitivity of differential transcription profiles following linear amplification of nanogram amounts of endothelial mRNA

NASA Technical Reports Server (NTRS)

Polacek, Denise C.; Passerini, Anthony G.; Shi, Congzhu; Francesco, Nadeene M.; Manduchi, Elisabetta; Grant, Gregory R.; Powell, Steven; Bischof, Helen; Winkler, Hans; Stoeckert, Christian J Jr;

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

MicroRNA profiling identifies miR-7-5p and miR-26b-5p as differentially expressed in hypertensive patients with left ventricular hypertrophy

PubMed Central

Kaneto, C.M.; Nascimento, J.S.; Moreira, M.C.R.; Ludovico, N.D.; Santana, A.P.; Silva, R.A.A.; Silva-Jardim, I.; Santos, J.L.; Sousa, S.M.B.; Lima, P.S.P.

2017-01-01

Recent evidence suggests that cell-derived circulating miRNAs may serve as biomarkers of cardiovascular diseases. However, a few studies have investigated the potential of circulating miRNAs as biomarkers for left ventricular hypertrophy (LVH). In this study, we aimed to characterize the miRNA profiles that could distinguish hypertensive patients with LHV, hypertensive patients without LVH and control subjects, and identify potential miRNAs as biomarkers of LVH. LVH was defined by left ventricular mass indexed to body surface area >125 g/m2 in men and >110 g/m2 in women and patients were classified as hypertensive when presenting a systolic blood pressure of 140 mmHg or more, or a diastolic blood pressure of 90 mmHg or more. We employed miRNA PCR array to screen serum miRNAs profiles of patients with LVH, essential hypertension and healthy subjects. We identified 75 differentially expressed miRNAs, including 49 upregulated miRNAs and 26 downregulated miRNAs between LVH and control patients. We chose 2 miRNAs with significant differences for further testing in 59 patients. RT-PCR analysis of serum samples confirmed that miR-7-5p and miR-26b-5p were upregulated in the serum of LVH hypertensive patients compared with healthy subjects. Our findings suggest that these miRNAs may play a role in the pathogenesis of hypertensive LVH and may represent novel biomarkers for this disease. PMID:29069223

Unique gene expression profiles of donor-matched human retinal and choroidal vascular endothelial cells.

PubMed

Smith, Justine R; Choi, Dongseok; Chipps, Timothy J; Pan, Yuzhen; Zamora, David O; Davies, Michael H; Babra, Bobby; Powers, Michael R; Planck, Stephen R; Rosenbaum, James T

2007-06-01

Consistent with clinical observations that posterior uveitis frequently involves the retinal vasculature and recent recognition of vascular heterogeneity, the hypothesis for this study was that retinal vascular endothelium was a cell population of unique molecular phenotype. Donor-matched cultures of primary retinal and choroidal endothelial cells from six human cadavers were incubated with either Toxoplasma gondii tachyzoites (10:1, parasites per cell) or Escherichia coli lipopolysaccharide (100 ng/mL); control cultures were simultaneously incubated with medium. Gene expression profiling of endothelial cells was performed using oligonucleotide arrays containing probes designed to detect 8746 human transcripts. After normalization, differential gene expression was assessed by the significance analysis of microarrays, with the false-discovery rate set at 5%. For selected genes, differences in the level of expression between retinal and choroidal cells were evaluated by real-time RT-PCR. Graphic descriptive analysis demonstrated a strong correlation between gene expression of unstimulated retinal and choroidal endothelial cells, but also highlighted distinctly different patterns of expression that were greater than differences noted between donors or between unstimulated and stimulated cells. Overall, 779 (8.9%) of 8746 transcripts were differentially represented. Of note, the 330 transcripts that were present at higher levels in retinal cells included a larger percentage of transcripts encoding molecules involved in the immune response. Differential gene expression was confirmed for 12 transcripts by RT-PCR. Retinal and choroidal vascular endothelial cells display distinctive gene expression profiles. The findings suggest the possibility of treating posterior uveitis by targeting specific interactions between the retinal endothelial cell and an infiltrating leukocyte.

Analysis of microRNAs expressions in chondrosarcoma.

PubMed

Yoshitaka, Teruhito; Kawai, Akira; Miyaki, Shigeru; Numoto, Kunihiko; Kikuta, Kazutaka; Ozaki, Toshifumi; Lotz, Martin; Asahara, Hiroshi

2013-12-01

MicroRNAs (miRNAs) are small non-coding RNAs capable of inhibiting gene expression post-transcriptionally and expression profiling can provide therapeutic targets and tools for cancer diagnosis. Chondrosarcoma is a mesenchymal tumor with unknown cause and differentiation status. Here, we profiled miRNA expression of chondrosarcoma, namely clinical samples from human conventional chondrosarcoma tissue, established chondrosarcoma cell lines, and primary non-tumorous adult articular chondrocytes, by miRNA array and quantitative real-time PCR. A wide variety of miRNAs were differently downregulated in chondrosarcoma compared to non-tumorous articular chondrocytes; 27 miRNAs: miR-10b, 23b, 24-1*, 27b, 100, 134, 136, 136*, 138, 181d, 186, 193b, 221*, 222, 335, 337-5p, 376a, 376a*, 376b, 376c, 377, 454, 495, 497, 505, 574-3p, and 660, were significantly downregulated in chondrosarcoma and only 2: miR-96 and 183, were upregulated. We further validated the expression levels of miRNAs by quantitative real-time PCR for miR-181a, let-7a, 100, 222, 136, 376a, and 335 in extended number of chondrosarcoma clinical samples. Among them, all except miR-181a were found to be significantly downregulated in chondrosarcoma derived samples. The findings provide potential diagnostic value and new molecular understanding of chondrosarcoma. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

High-throughput STR analysis for DNA database using direct PCR.

PubMed

Sim, Jeong Eun; Park, Su Jeong; Lee, Han Chul; Kim, Se-Yong; Kim, Jong Yeol; Lee, Seung Hwan

2013-07-01

Since the Korean criminal DNA database was launched in 2010, we have focused on establishing an automated DNA database profiling system that analyzes short tandem repeat loci in a high-throughput and cost-effective manner. We established a DNA database profiling system without DNA purification using a direct PCR buffer system. The quality of direct PCR procedures was compared with that of conventional PCR system under their respective optimized conditions. The results revealed not only perfect concordance but also an excellent PCR success rate, good electropherogram quality, and an optimal intra/inter-loci peak height ratio. In particular, the proportion of DNA extraction required due to direct PCR failure could be minimized to <3%. In conclusion, the newly developed direct PCR system can be adopted for automated DNA database profiling systems to replace or supplement conventional PCR system in a time- and cost-saving manner. © 2013 American Academy of Forensic Sciences Published 2013. This article is a U.S. Government work and is in the public domain in the U.S.A.

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.

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

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.

Messenger RNA profiling of human platelets by microarray hybridization.

PubMed

Bugert, Peter; Dugrillon, Alex; Günaydin, Ayse; Eichler, Hermann; Klüter, Harald

2003-10-01

Platelets are generally believed to be inactive in terms of de novo protein synthesis. On the other hand, the presence of ribosomes and mRNA molecules is well established. Many studies have used reverse transcriptase (RT) -PCR for detection of gene transcripts in platelets. As RT-PCR is a very sensitive method, any leukocyte contamination of platelet preparations can lead to false results. We performed three filtration procedures to minimize leukocyte contamination of pooled buffy-coat platelet concentrates prior to RNA isolation. Furthermore, by applying a genomic PCR approach with 50 amplification cycles we demonstrated that nucleated cells were not detectable. Microarray hybridization was used to analyze 9,850 individual human genes in RNA from purified platelets. In total we identified 1,526 (15.5%) positive genes. The data were confirmed in six individual experiments each performed on a PC pooled from four individual blood donations. Genes specific for nucleated blood cells such as CD4, CD83 and others were negative and verified the purity of PC. Overrepresentation of positive genes was found in the functional categories of glycoproteins/integrins (22.6% vs. 15.5%, p=0.029) and receptors (20.7% vs. 15.5%, p<0.001). Gene transcripts encoding RANTES, GRO-alpha, MIP-1alpha, MIP-1beta, and others were found at high levels of signal intensity and confirmed literature data. This work provides a mRNA profile of human platelets and a complete list of results can be downloaded from the website of our institute www.ma.uni-heidelberg.de/inst/iti/plt_array.xls. The knowledge about gene transcripts may have an impact on the characterization of novel proteins and their functions in platelets.

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

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.

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.

MicroRNA Expression Profiling to Identify and Validate Reference Genes for the Relative Quantification of microRNA in Rectal Cancer.

PubMed

Eriksen, Anne Haahr Mellergaard; Andersen, Rikke Fredslund; Pallisgaard, Niels; Sørensen, Flemming Brandt; Jakobsen, Anders; Hansen, Torben Frøstrup

2016-01-01

MicroRNAs (miRNAs) play important roles in regulating biological processes at the post-transcriptional level. Deregulation of miRNAs has been observed in cancer, and miRNAs are being investigated as potential biomarkers regarding diagnosis, prognosis and prediction in cancer management. Real-time quantitative polymerase chain reaction (RT-qPCR) is commonly used, when measuring miRNA expression. Appropriate normalisation of RT-qPCR data is important to ensure reliable results. The aim of the present study was to identify stably expressed miRNAs applicable as normaliser candidates in future studies of miRNA expression in rectal cancer. We performed high-throughput miRNA profiling (OpenArray®) on ten pairs of laser micro-dissected rectal cancer tissue and adjacent stroma. A global mean expression normalisation strategy was applied to identify the most stably expressed miRNAs for subsequent validation. In the first validation experiment, a panel of miRNAs were analysed on 25 pairs of micro dissected rectal cancer tissue and adjacent stroma. Subsequently, the same miRNAs were analysed in 28 pairs of rectal cancer tissue and normal rectal mucosa. From the miRNA profiling experiment, miR-645, miR-193a-5p, miR-27a and let-7g were identified as stably expressed, both in malignant and stromal tissue. In addition, NormFinder confirmed high expression stability for the four miRNAs. In the RT-qPCR based validation experiments, no significant difference between tumour and stroma/normal rectal mucosa was detected for the mean of the normaliser candidates miR-27a, miR-193a-5p and let-7g (first validation P = 0.801, second validation P = 0.321). MiR-645 was excluded from the data analysis, because it was undetected in 35 of 50 samples (first validation) and in 24 of 56 samples (second validation), respectively. Significant difference in expression level of RNU6B was observed between tumour and adjacent stromal (first validation), and between tumour and normal rectal mucosa (second validation). We recommend the mean expression of miR-27a, miR-193a-5p and let-7g as normalisation factor, when performing miRNA expression analyses by RT-qPCR on rectal cancer tissue.

Cellular Levels of Oxidative Stress Affect the Response of Cervical Cancer Cells to Chemotherapeutic Agents

PubMed Central

Williams, Vonetta M.; Kokoza, Anatolii; Bashkirova, Svetlana; Duerksen-Hughes, Penelope

2014-01-01

Treatment of advanced and relapsed cervical cancer is frequently ineffective, due in large part to chemoresistance. To examine the pathways responsible, we employed the cervical carcinoma-derived SiHa and CaSki cells as cellular models of resistance and sensitivity, respectively, to treatment with chemotherapeutic agents, doxorubicin, and cisplatin. We compared the proteomic profiles of SiHa and CaSki cells and identified pathways with the potential to contribute to the differential response. We then extended these findings by comparing the expression level of genes involved in reactive oxygen species (ROS) metabolism through the use of a RT-PCR array. The analyses demonstrated that the resistant SiHa cells expressed higher levels of antioxidant enzymes. Decreasing or increasing oxidative stress led to protection or sensitization, respectively, in both cell lines, supporting the idea that cellular levels of oxidative stress affect responsiveness to treatment. Interestingly, doxorubicin and cisplatin induced different profiles of ROS, and these differences appear to contribute to the sensitivity to treatment displayed by cervical cancer cells. Overall, our findings demonstrate that cervical cancer cells display variable profiles with respect to their redox-generating and -adaptive systems, and that these different profiles have the potential to contribute to their responses to treatments with chemotherapy. PMID:25478571

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

Prevalence and genetic characterization of Vibrio vulnificus in raw seafood and seawater in Malaysia.

PubMed

Paydar, Mohammadjavad; Thong, Kwai Lin

2013-10-01

Vibrio vulnificus is a highly invasive human pathogen that exists naturally in estuarine environment and coastal waters. In this study, we used different PCR assays to detect V. vulnificus in 260 seafood and 80 seawater samples. V. vulnificus was present in about 34 (13%) of the 260 seafood samples and 18 (23%) of the 80 seawater samples. Repetitive extragenic palindromic PCR (REP-PCR) and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) were applied to subtype the V. vulnificus isolates. Twenty-five REP profiles and 45 ERIC profiles were observed, and the isolates were categorized into 9 and 10 distinct clusters at the similarity of 80%, by REP-PCR and ERIC-PCR, respectively. ERIC-PCR is more discriminative than REP-PCR in subtyping V. vulnificus, demonstrating high genetic diversity among the isolates.

Profiling and bioinformatics analyses reveal differential circular RNA expression in radioresistant esophageal cancer cells.

PubMed

Su, Huafang; Lin, Fuqiang; Deng, Xia; Shen, Lanxiao; Fang, Ya; Fei, Zhenghua; Zhao, Lihao; Zhang, Xuebang; Pan, Huanle; Xie, Deyao; Jin, Xiance; Xie, Congying

2016-07-28

Acquired radioresistance during radiotherapy is considered as the most important reason for local tumor recurrence or treatment failure. Circular RNAs (circRNAs) have recently been identified as microRNA sponges and involve in various biological processes. The purpose of this study is to investigate the role of circRNAs in the radioresistance of esophageal cancer. Total RNA was isolated from human parental cell line KYSE-150 and self-established radioresistant esophageal cancer cell line KYSE-150R, and hybridized to Arraystar Human circRNA Array. Quantitative real-time PCR was used to confirm the circRNA expression profiles obtained from the microarray data. Bioinformatic tools including gene ontology (GO) analysis, KEGG pathway analysis and network analysis were done for further assessment. Among the detected candidate 3752 circRNA genes, significant upregulation of 57 circRNAs and downregulation of 17 circRNAs in human radioresistant esophageal cancer cell line KYSE-150R were observed compared with the parental cell line KYSE-150 (fold change ≥2.0 and P < 0.05). There were 9 out of these candidate circRNAs were validated by real-time PCR. GO analysis revealed that numerous target genes, including most microRNAs were involved in the biological processes. There were more than 400 target genes enrichment on Wnt signaling pathway. CircRNA_001059 and circRNA_000167 were the two largest nodes in circRNA/microRNA co-expression network. Our study revealed a comprehensive expression and functional profile of differentially expressed circRNAs in radioresistant esophageal cancer cells, indicating possible involvement of these dysregulated circRNAs in the development of radiation resistance.

Effects of strenuous exercise on Th1/Th2 gene expression from human peripheral blood mononuclear cells of marathon participants.

PubMed

Xiang, Lianbin; Rehm, Kristina E; Marshall, Gailen D

2014-08-01

Physical stressors, such as strenuous exercise, can have numerous effects on the human body including the immune system. The aim of this study was to evaluate the gene expression profile of Th1/Th2 cytokines and related transcription factor genes in order to investigate possible immune imbalances before and after a marathon. Blood samples were collected from 16 normal volunteers 24-48 h before and one week after completing a marathon race. Gene expression of Th1 and Th2 related cytokines from human peripheral blood mononuclear cells (PBMC) was analyzed using Human Th1-Th2-Th3 RT(2) Profiler PCR Array and qRT-PCR that measured the transcript levels of 84 genes related to T cell activation. We found that PBMC express a characteristic Th2-like gene profile one week post-marathon compared to pre-marathon. The majority of genes up-regulated one week post-marathon such as IL-4, GATA3, and CCR4 were Th2 associated. For Th1-related genes, CXCR3 and IRF1 were up-regulated one week post-marathon. There was a trend of down-regulation of two Th1 related genes, T-bet and STAT1. Th3-related gene expression patterns did not change in the study. The ratios of both IFN-γ/IL-4 and T-bet/GATA3 gene expressions were significantly lower one week after marathon. These findings suggest that a Th1/Th2 immune imbalance persisted at least 1 week after completion of a marathon which offers a mechanistic rationale for the increased risk of upper respiratory tract infections often reported after strenuous exercise. Copyright © 2014 Elsevier Ltd. All rights reserved.

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 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.

Molecular profiling reveals frequent gain of MYCN and anaplasia-specific loss of 4q and 14q in Wilms tumor.

PubMed

Williams, Richard D; Al-Saadi, Reem; Natrajan, Rachael; Mackay, Alan; Chagtai, Tasnim; Little, Suzanne; Hing, Sandra N; Fenwick, Kerry; Ashworth, Alan; Grundy, Paul; Anderson, James R; Dome, Jeffrey S; Perlman, Elizabeth J; Jones, Chris; Pritchard-Jones, Kathy

2011-12-01

Anaplasia in Wilms tumor, a distinctive histology characterized by abnormal mitoses, is associated with poor patient outcome. While anaplastic tumors frequently harbour TP53 mutations, little is otherwise known about their molecular biology. We have used array comparative genomic hybridization (aCGH) and cDNA microarray expression profiling to compare anaplastic and favorable histology Wilms tumors to determine their common and differentiating features. In addition to changes on 17p, consistent with TP53 deletion, recurrent anaplasia-specific genomic loss and under-expression were noted in several other regions, most strikingly 4q and 14q. Further aberrations, including gain of 1q and loss of 16q were common to both histologies. Focal gain of MYCN, initially detected by high resolution aCGH profiling in 6/61 anaplastic samples, was confirmed in a significant proportion of both tumor types by a genomic quantitative PCR survey of over 400 tumors. Overall, these results are consistent with a model where anaplasia, rather than forming an entirely distinct molecular entity, arises from the general continuum of Wilms tumor by the acquisition of additional genomic changes at multiple loci. Copyright © 2011 Wiley Periodicals, Inc.

Coherent and incoherent combination of Gaussian beams employing lens array distributed on the spherical chamber.

PubMed

Huang, Zhihua; Wei, Xiaofeng; Li, Mingzhong; Wang, Jianjun; Lin, Honghuan; Xu, Dangpeng; Deng, Ying; Zhang, Rui

2012-04-01

Coherent and incoherent combination of Gaussian beams employing a lens array distributed on the spherical chamber is theoretically analyzed. The output field of each source in the array is coupled through an individual optical system whose local optical axis coincides with the radial direction of the chamber. The resulting intensity profile near the origin is derived. The intensity profile and power in the bucket on the target for rectangular and hexagonal arrangement are numerically calculated. The influences of the center-to-center separation and the ring number of the focusing lens array are given. The synthetic intensity profile of incoherent combination changes little for a lens array scale much smaller than the chamber size. In contrast, the synthetic intensity profile of coherent combination shows an interference pattern with a sharp central peak and sidelobes.

Ephrin-B2 is differentially expressed in the intestinal epithelium in Crohn’s disease and contributes to accelerated epithelial wound healing in vitro

PubMed Central

Hafner, Christian; Meyer, Stefanie; Langmann, Thomas; Schmitz, Gerd; Bataille, Frauke; Hagen, Ilja; Becker, Bernd; Roesch, Alexander; Rogler, Gerhard; Landthaler, Michael; Vogt, Thomas

2005-01-01

AIM: Eph receptor tyrosine kinases and their membrane bound receptor-like ligands, the ephrins, represent a bi-directional cell-cell contact signaling system that directs epithelial movements in development. The meaning of this system in the adult human gut is unknown. We investigated the Eph/ephrin mRNA expression in the intestinal epithelium of healthy controls and patients with inflammatory bowel disease (IBD). METHODS: mRNA expression profiles of all Eph/ephrin family members in normal small intestine and colon were established by real-time RT-PCR. In addition, differential expression in IBD was investigated by cDNA array technology, and validated by both real-time RT-PCR and immunohistochemistry. Potential effects of enhanced EphB/ephrin-B signaling were analyzed in an in vitro IEC-6 cell scratch wound model. RESULTS: Human adult intestinal mucosa exhibits a complex pattern of Eph receptors and ephrins. Beside the known prominent co-expression of EphA2 and ephrinA1, we found abundantly co-expressed EphB2 and ephrin-B1/2. Interestingly, cDNA array data, validated by real-time PCR and immunohistochemistry, showed upregulation of ephrin-B2 in both perilesional and lesional intestinal epithelial cells of IBD patients, suggesting a role in epithelial homeostasis. Stimulation of ephrin-B signaling in ephrin-B1/2 expressing rat IEC-6-cells with recombinant EphB1-Fc resulted in a significant dose-dependent acceleration of wound closure. Furthermore, fluorescence microscopy showed that EphB1-Fc induced coordinated migration of wound edge cells is associated with enhanced formation of lamellipodial protrusions into the wound, increased actin stress fiber assembly and production of laminin at the wound edge. CONCLUSION: EphB/ephrin-B signaling might represent a novel protective mechanism that promotes intestinal epithelial wound healing, with potential impact on epithelial restitution in IBD. PMID:15996027

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

PubMed

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

2018-03-20

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

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.

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

REPETITIVE SEQUENCE BASED-PCR PROFILING OF ESCHERICHIA COLI O157 STRAINS FROM BEEF IN SOUTHERN THAILAND.

PubMed

Sukhumungoon, Pharanai; Tantadapan, Rujira; Rattanachuay, Pattamarat

2016-01-01

Beef and its products are potential vehicles of Escherichia coli O157, the most important serotype implicated in many large outbreaks of diarrheal infection in humans worldwide. There is a need for rapid detection of contaminated food in order to implement appropriate and effective control measures. In this study, repetitive sequence (rep)-PCR, using three different primers, BOXA1R, ERIC2 and (GTG)5, singly and in combinations, were employed to compare the genetic relatedness among E. coli O157 group with other diarrheagenic E. coli strains as controls. Although a combination of BOXA1R + ERIC2 + (GTG)5 primers generated a rep-PCR profile containing the highest number of amplicon bands among the DEC strains tested, dendrogram (at 80% similarity) exhibited the lowest DEC classification of 5 clusters, whereas that from BOXA1R or BOXA1R+ (GTG)5 rep-PCR profiling produced 8 clusters. Nevertheless, focusing E. coli O157 strains were grouped into 4 clusters irrespective of the rep-PCR profiles analyzed, and all 14 but two, PSU60 and PSU132, E. coli O157 strains isolated from beef in southern Thailand during 2012 to 2014 fell into a single cluster. Thus, rep-PCR profiling generated with BOXA1R or BOXA1R + (GTG)5 is sufficient for distinguishing among DEC strains, including E. coli O157 in southern Thailand.

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

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.

Typing DNA profiles from previously enhanced fingerprints using direct PCR.

PubMed

Templeton, Jennifer E L; Taylor, Duncan; Handt, Oliva; Linacre, Adrian

2017-07-01

Fingermarks are a source of human identification both through the ridge patterns and DNA profiling. Typing nuclear STR DNA markers from previously enhanced fingermarks provides an alternative method of utilising the limited fingermark deposit that can be left behind during a criminal act. Dusting with fingerprint powders is a standard method used in classical fingermark enhancement and can affect DNA data. The ability to generate informative DNA profiles from powdered fingerprints using direct PCR swabs was investigated. Direct PCR was used as the opportunity to generate usable DNA profiles after performing any of the standard DNA extraction processes is minimal. Omitting the extraction step will, for many samples, be the key to success if there is limited sample DNA. DNA profiles were generated by direct PCR from 160 fingermarks after treatment with one of the following dactyloscopic fingerprint powders: white hadonite; silver aluminium; HiFi Volcano silk black; or black magnetic fingerprint powder. This was achieved by a combination of an optimised double-swabbing technique and swab media, omission of the extraction step to minimise loss of critical low-template DNA, and additional AmpliTaq Gold ® DNA polymerase to boost the PCR. Ninety eight out of 160 samples (61%) were considered 'up-loadable' to the Australian National Criminal Investigation DNA Database (NCIDD). The method described required a minimum of working steps, equipment and reagents, and was completed within 4h. Direct PCR allows the generation of DNA profiles from enhanced prints without the need to increase PCR cycle numbers beyond manufacturer's recommendations. Particular emphasis was placed on preventing contamination by applying strict protocols and avoiding the use of previously used fingerprint brushes. Based on this extensive survey, the data provided indicate minimal effects of any of these four powders on the chance of obtaining DNA profiles from enhanced fingermarks. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

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.

MicroRNA profiling in intraocular medulloepitheliomas.

PubMed

Edward, Deepak P; Alkatan, Hind; Rafiq, Qundeel; Eberhart, Charles; Al Mesfer, Saleh; Ghazi, Nicola; Al Safieh, Leen; Kondkar, Altaf A; Abu Amero, Khaled K

2015-01-01

To study the differential expression of microRNA (miRNA) profiles between intraocular medulloepithelioma (ME) and normal control tissue (CT). Total RNA was extracted from formalin fixed paraffin embedded (FFPE) intraocular ME (n=7) and from age matched ciliary body controls (n=8). The clinical history and phenotype was recorded. MiRNA profiles were determined using the Affymetrix GeneChip miRNA Arrays analyzed using expression console 1.3 software. Validation of significantly dysregulated miRNA was confirmed by quantitative real-time PCR. The web-based DNA Intelligent Analysis (DIANA)-miRPath v2.0 was used to perform enrichment analysis of differentially expressed (DE) miRNA gene targets in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. The pathologic evaluation revealed one benign (benign non-teratoid, n=1) and six malignant tumors (malignant teratoid, n=2; malignant non-teratoid, n = 4). A total of 88 miRNAs were upregulated and 43 miRNAs were downregulated significantly (P<0.05) in the tumor specimens. Many of these significantly dysregulated miRNAs were known to play various roles in carcinogenesis and tumor behavior. RT-PCR validated three significantly upregulated miRNAs and three significantly downregulated miRNAs namely miR-217, miR-216a, miR-216b, miR-146a, miR-509-3p and miR-211. Many DE miRNAs that were significant in ME tumors showed dysregulation in retinoblastoma, glioblastoma, and precursor, normal and reactive human cartilage. Enriched pathway analysis suggested a significant association of upregulated miRNAs with 15 pathways involved in prion disease and several types of cancer. The pathways involving significantly downregulated miRNAs included the toll-like receptor (TLR) (p<4.36E-16) and Nuclear Factor kappa B (NF-κB) signaling pathways (p<9.00E-06). We report significantly dysregulated miRNAs in intraocular ME tumors, which exhibited abnormal profiles in other cancers as well such as retinoblastoma and glioblastoma. Pathway analysis of all dysregulated miRNAs shared commonalities with other cancer pathways.

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

PubMed

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

2007-12-01

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

Characterization of MicroRNA Expression Profiles and Identification of Potential Biomarkers in Leprosy.

PubMed

Jorge, Karina T O S; Souza, Renan P; Assis, Marieta T A; Araújo, Marcelo G; Locati, Massimo; Jesus, Amélia M R; Dias Baptista, Ida M F; Lima, Cristiano X; Teixeira, Antônio L; Teixeira, Mauro M; Soriani, Frederico M

2017-05-01

Leprosy is an important cause of disability in the developing world. Early diagnosis is essential to allow for cure and to interrupt transmission of this infection. MicroRNAs (miRNAs) are important factors for host-pathogen interaction and they have been identified as biomarkers for various infectious diseases. The expression profile of 377 microRNAs were analyzed by TaqMan low-density array (TLDA) in skin lesions of tuberculoid and lepromatous leprosy patients as well as skin specimens from healthy controls. In a second step, 16 microRNAs were selected for validation experiments with reverse transcription-quantitative PCR (qRT-PCR) in skin samples from new individuals. Principal-component analysis followed by logistic regression model and receiver operating characteristic (ROC) curve analyses were performed to evaluate the diagnostic potential of selected miRNAs. Four patterns of differential expression were identified in the TLDA experiment, suggesting a diagnostic potential of miRNAs in leprosy. After validation experiments, a combination of four miRNAs (miR-101, miR-196b, miR-27b, and miR-29c) was revealed as able to discriminate between healthy control and leprosy patients with 80% sensitivity and 91% specificity. This set of miRNAs was also able to discriminate between lepromatous and tuberculoid patients with a sensitivity of 83% and 80% specificity. In this work, it was possible to identify a set of miRNAs with good diagnostic potential for leprosy. Copyright © 2017 American Society for Microbiology.

Development of a fast PCR protocol enabling rapid generation of AmpFℓSTR® Identifiler® profiles for genotyping of human DNA

PubMed Central

2012-01-01

Background Traditional PCR methods for forensic STR genotyping require approximately 2.5 to 4 hours to complete, contributing a significant portion of the time required to process forensic DNA samples. The purpose of this study was to develop and validate a fast PCR protocol that enabled amplification of the 16 loci targeted by the AmpFℓSTR® Identifiler® primer set, allowing decreased cycling times. Methods Fast PCR conditions were achieved by substituting the traditional Taq polymerase for SpeedSTAR™ HS DNA polymerase which is designed for fast PCR, by upgrading to a thermal cycler with faster temperature ramping rates and by modifying cycling parameters (less time at each temperature) and adopting a two-step PCR approach. Results The total time required for the optimized protocol is 26 min. A total of 147 forensically relevant DNA samples were amplified using the fast PCR protocol for Identifiler. Heterozygote peak height ratios were not affected by fast PCR conditions, and full profiles were generated for single-source DNA amounts between 0.125 ng and 2.0 ng. Individual loci in profiles produced with the fast PCR protocol exhibited average n-4 stutter percentages ranging from 2.5 ± 0.9% (THO1) to 9.9 ± 2.7% (D2S1338). No increase in non-adenylation or other amplification artefacts was observed. Minor contributor alleles in two-person DNA mixtures were reliably discerned. Low level cross-reactivity (monomorphic peaks) was observed with some domestic animal DNA. Conclusions The fast PCR protocol presented offers a feasible alternative to current amplification methods and could aid in reducing the overall time in STR profile production or could be incorporated into a fast STR genotyping procedure for time-sensitive situations. PMID:22394458

Development of a fast PCR protocol enabling rapid generation of AmpFℓSTR® Identifiler® profiles for genotyping of human DNA.

PubMed

Foster, Amanda; Laurin, Nancy

2012-03-06

Traditional PCR methods for forensic STR genotyping require approximately 2.5 to 4 hours to complete, contributing a significant portion of the time required to process forensic DNA samples. The purpose of this study was to develop and validate a fast PCR protocol that enabled amplification of the 16 loci targeted by the AmpFℓSTR® Identifiler® primer set, allowing decreased cycling times. Fast PCR conditions were achieved by substituting the traditional Taq polymerase for SpeedSTAR™ HS DNA polymerase which is designed for fast PCR, by upgrading to a thermal cycler with faster temperature ramping rates and by modifying cycling parameters (less time at each temperature) and adopting a two-step PCR approach. The total time required for the optimized protocol is 26 min. A total of 147 forensically relevant DNA samples were amplified using the fast PCR protocol for Identifiler. Heterozygote peak height ratios were not affected by fast PCR conditions, and full profiles were generated for single-source DNA amounts between 0.125 ng and 2.0 ng. Individual loci in profiles produced with the fast PCR protocol exhibited average n-4 stutter percentages ranging from 2.5 ± 0.9% (THO1) to 9.9 ± 2.7% (D2S1338). No increase in non-adenylation or other amplification artefacts was observed. Minor contributor alleles in two-person DNA mixtures were reliably discerned. Low level cross-reactivity (monomorphic peaks) was observed with some domestic animal DNA. The fast PCR protocol presented offers a feasible alternative to current amplification methods and could aid in reducing the overall time in STR profile production or could be incorporated into a fast STR genotyping procedure for time-sensitive situations.

Effective formation method for an aspherical microlens array based on an aperiodic moving mask during exposure.

PubMed

Shi, Lifang; Du, Chunlei; Dong, Xiaochun; Deng, Qiling; Luo, Xiangang

2007-12-01

An aperiodic mask design method for fabricating a microlens array with an aspherical profile is proposed. The nonlinear relationship between exposure doses and lens profile is considered, and the select criteria of quantization interval and fabrication range of the method are given. The mask function of a quadrangle microlens array with a hyperboloid profile used in the infrared was constructed by using this method. The microlens array can be effectively fabricated during a one time exposure process using the mask. Reactive ion etching was carried out to transfer the structure into the substrate of germanium. The measurement results indicate that the roughness is less than 10 nm (pv), and the profile error is less than 40 nm (rms).

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

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

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.

Biomarker discovery for colon cancer using a 761 gene RT-PCR assay.

PubMed

Clark-Langone, Kim M; Wu, Jenny Y; Sangli, Chithra; Chen, Angela; Snable, James L; Nguyen, Anhthu; Hackett, James R; Baker, Joffre; Yothers, Greg; Kim, Chungyeul; Cronin, Maureen T

2007-08-15

Reverse transcription PCR (RT-PCR) is widely recognized to be the gold standard method for quantifying gene expression. Studies using RT-PCR technology as a discovery tool have historically been limited to relatively small gene sets compared to other gene expression platforms such as microarrays. We have recently shown that TaqMan RT-PCR can be scaled up to profile expression for 192 genes in fixed paraffin-embedded (FPE) clinical study tumor specimens. This technology has also been used to develop and commercialize a widely used clinical test for breast cancer prognosis and prediction, the Onco typeDX assay. A similar need exists in colon cancer for a test that provides information on the likelihood of disease recurrence in colon cancer (prognosis) and the likelihood of tumor response to standard chemotherapy regimens (prediction). We have now scaled our RT-PCR assay to efficiently screen 761 biomarkers across hundreds of patient samples and applied this process to biomarker discovery in colon cancer. This screening strategy remains attractive due to the inherent advantages of maintaining platform consistency from discovery through clinical application. RNA was extracted from formalin fixed paraffin embedded (FPE) tissue, as old as 28 years, from 354 patients enrolled in NSABP C-01 and C-02 colon cancer studies. Multiplexed reverse transcription reactions were performed using a gene specific primer pool containing 761 unique primers. PCR was performed as independent TaqMan reactions for each candidate gene. Hierarchal clustering demonstrates that genes expected to co-express form obvious, distinct and in certain cases very tightly correlated clusters, validating the reliability of this technical approach to biomarker discovery. We have developed a high throughput, quantitatively precise multi-analyte gene expression platform for biomarker discovery that approaches low density DNA arrays in numbers of genes analyzed while maintaining the high specificity, sensitivity and reproducibility that are characteristics of RT-PCR. Biomarkers discovered using this approach can be transferred to a clinical reference laboratory setting without having to re-validate the assay on a second technology platform.

Electronic transport characterization of silicon wafers by spatially resolved steady-state photocarrier radiometric imaging

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

Wang, Qian; University of the Chinese Academy of Sciences, Beijing 100039; Li, Bincheng, E-mail: bcli@ioe.ac.cn

2015-09-28

Spatially resolved steady-state photocarrier radiometric (PCR) imaging technique is developed to characterize the electronic transport properties of silicon wafers. Based on a nonlinear PCR theory, simulations are performed to investigate the effects of electronic transport parameters (the carrier lifetime, the carrier diffusion coefficient, and the front surface recombination velocity) on the steady-state PCR intensity profiles. The electronic transport parameters of an n-type silicon wafer are simultaneously determined by fitting the measured steady-state PCR intensity profiles to the three-dimensional nonlinear PCR model. The determined transport parameters are in good agreement with the results obtained by the conventional modulated PCR technique withmore » multiple pump beam radii.« less

Real-Time PCR (qPCR) Primer Design Using Free Online Software

ERIC Educational Resources Information Center

Thornton, Brenda; Basu, Chhandak

2011-01-01

Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

Compression dynamics of quasi-spherical wire arrays with different linear mass profiles

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

Mitrofanov, K. N., E-mail: mitrofan@triniti.ru; Aleksandrov, V. V.; Gritsuk, A. N.

Results of experimental studies of the implosion of quasi-spherical wire (or metalized fiber) arrays are presented. The goal of the experiments was to achieve synchronous three-dimensional compression of the plasma produced in different regions of a quasi-spherical array into its geometrical center. To search for optimal synchronization conditions, quasi-spherical arrays with different initial profiles of the linear mass were used. The following dependences of the linear mass on the poloidal angle were used: m{sub l}(θ) ∝ sin{sup –1}θ and m{sub l}(θ) ∝ sin{sup –2}θ. The compression dynamics of such arrays was compared with that of quasi-spherical arrays without linear massmore » profiling, m{sub l}(θ) = const. To verify the experimental data, the spatiotemporal dynamics of plasma compression in quasi-spherical arrays was studied using various diagnostics. The experiments on three-dimensional implosion of quasi-spherical arrays made it possible to study how the frozen-in magnetic field of the discharge current penetrates into the array. By measuring the magnetic field in the plasma of a quasi-spherical array, information is obtained on the processes of plasma production and formation of plasma flows from the wire/fiber regions with and without an additionally deposited mass. It is found that penetration of the magnetic flux depends on the initial linear mass profile m{sub l}(θ) of the quasi-spherical array. From space-resolved spectral measurements and frame imaging of plasma X-ray emission, information is obtained on the dimensions and shape of the X-ray source formed during the implosion of a quasi-spherical array. The intensity of this source is estimated and compared with that of the Z-pinch formed during the implosion of a cylindrical array.« less

Gene expression profiling in patients with polymyalgia rheumatica before and after symptom-abolishing glucocorticoid treatment.

PubMed

Kreiner, Frederik Flindt; Borup, Rehannah; Nielsen, Finn Cilius; Schjerling, Peter; Galbo, Henrik

2017-08-07

The pathophysiology, including the impact of gene expression, of polymyalgia rheumatica (PMR) remains elusive. We profiled the gene expression in muscle tissue in PMR patients before and after glucocorticoid treatment. Gene expression was measured using Affymetrix Human Genome U133 Plus 2.0 arrays in muscle biopsies from 8 glucocorticoid-naive patients with PMR and 10 controls before and after prednisolone-treatment for 14 days. For 14 genes, quantitative real-time PCR (qRT-PCR, n = 9 in both groups) was used to validate the microarray findings and to further investigate the expression of genes of particular interest. Prednisolone normalized erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) in PMR patients. A total of 165 putatively clinically relevant, differentially expressed genes were identified (cut-off: fold difference > ±1.2, difference of mean > 30, and p < 0.05); of these, 78 genes differed between patients and controls before treatment, 131 genes responded to treatment in a given direction only in patients, and 44 fulfilled both these criteria. In 43 of the 44 genes, treatment counteracted the initial difference. Functional clustering identified themes of biological function, including regulation of protein biosynthesis, and regulation of transcription and of extracellular matrix processes. Overall, qRT-PCR confirmed the microarray findings: Microarray-detected group differences were confirmed for 9 genes in 17 of 18 comparisons (same magnitude and direction of change); lack of group differences in microarray testing was confirmed for 5 genes in 8 of 10 comparisons. Before treatment, using qRT-PCR, expression of interleukin 6 (IL-6) was found to be 4-fold higher in patients (p < 0.05). This study identifies genes in muscle, the expression of which may impact the pathophysiology of PMR. Moreover, the study adds further evidence of the importance of IL-6 in the disease. Follow-up studies are needed to establish the exact pathophysiological relevance of the identified genes. The study was retrospectively listed on the ISRCTN registry with study ID ISRCTN69503018 and date of registration the 26th of July 2017.

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

Gene Transcription Profile of the Detached Retina (An AOS Thesis)

PubMed Central

Zacks, David N.

2009-01-01

Purpose: Separation of the neurosensory retina from the retinal pigment epithelium (RPE) yields many morphologic and functional consequences, including death of the photoreceptor cells, Müller cell hypertrophy, and inner retinal rewiring. Many of these changes are due to the separation-induced activation of specific genes. In this work, we define the gene transcription profile within the retina as a function of time after detachment. We also define the early activation of kinases that might be responsible for the detachment-induced changes in gene transcription. Methods: Separation of the retina from the RPE was induced in Brown-Norway rats by the injection of 1% hyaluronic acid into the subretinal space. Retinas were harvested at 1, 7, and 28 days after separation. Gene transcription profiles for each time point were determined using the Affymetrix Rat 230A gene microarray chip. Transcription levels in detached retinas were compared to those of nondetached retinas with the BRB-ArrayTools Version 3.6.0 using a random variance analysis of variance (ANOVA) model. Confirmation of the significant transcriptional changes for a subset of the genes was performed using microfluidic quantitative real-time polymerase chain reaction (qRT-PCR) assays. Kinase activation was explored using Western blot analysis to look for early phosphorylation of any of the 3 main families of mitogen-activated protein kinases (MAPK): the p38 family, the Janus kinase family, and the p42/p44 family. Results: Retinas separated from the RPE showed extensive alterations in their gene transcription profile. Many of these changes were initiated as early as 1 day after separation, with significant increases by 7 days. ANOVA analysis defined 144 genes that had significantly altered transcription levels as a function of time after separation when setting a false discovery rate at ≤0.1. Confirmatory RT-PCR was performed on 51 of these 144 genes. Differential transcription detected on the microarray chip was confirmed by qRT-PCR for all 51 genes. Western blot analysis showed that the p42/p44 family of MAPK was phosphorylated within 2 hours of retinal-RPE separation. This phosphorylation was detachment-induced and could be inhibited by specific inhibitors of MAPK phosphorylation. Conclusions: Separation of the retina from the RPE induces significant alteration in the gene transcription profile within the retina. These profiles are not static, but change as a function of time after detachment. These gene transcription changes are preceded by the activation of the p42/p44 family of MAPK. This altered transcription may serve as the basis for many of the morphologic, biochemical, and functional changes seen within the detached retina. PMID:20126507

Indel analysis by droplet digital PCR: a sensitive method for DNA mixture detection and chimerism analysis.

PubMed

Santurtún, Ana; Riancho, José A; Arozamena, Jana; López-Duarte, Mónica; Zarrabeitia, María T

2017-01-01

Several methods have been developed to determinate genetic profiles from a mixed samples and chimerism analysis in transplanted patients. The aim of this study was to explore the effectiveness of using the droplet digital PCR (ddPCR) for mixed chimerism detection (a mixture of genetic profiles resulting after allogeneic hematopoietic stem cell transplantation (HSCT)). We analyzed 25 DNA samples from patients who had undergone HSCT and compared the performance of ddPCR and two established methods for chimerism detection, based upon the Indel and STRs analysis, respectively. Additionally, eight artificial mixture DNA samples were created to evaluate the sensibility of ddPCR. Our results show that the chimerism percentages estimated by the analysis of a single Indel using ddPCR were very similar to those calculated by the amplification of 15 STRs (r 2  = 0.970) and with the results obtained by the amplification of 38 Indels (r 2  = 0.975). Moreover, the amplification of a single Indel by ddPCR was sensitive enough to detect a minor DNA contributor comprising down to 0.5 % of the sample. We conclude that ddPCR can be a powerful tool for the determination of a genetic profile of forensic mixtures and clinical chimerism analysis when traditional techniques are not sensitive enough.

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

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

Functional Genomics Using the Saccharomyces cerevisiae Yeast Deletion Collections.

PubMed

Nislow, Corey; Wong, Lai Hong; Lee, Amy Huei-Yi; Giaever, Guri

2016-09-01

Constructed by a consortium of 16 laboratories, the Saccharomyces genome-wide deletion collections have, for the past decade, provided a powerful, rapid, and inexpensive approach for functional profiling of the yeast genome. Loss-of-function deletion mutants were systematically created using a polymerase chain reaction (PCR)-based gene deletion strategy to generate a start-to-stop codon replacement of each open reading frame by homologous recombination. Each strain carries two molecular barcodes that serve as unique strain identifiers, enabling their growth to be analyzed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays or through the use of next-generation sequencing technologies. Functional profiling of the deletion collections, using either strain-by-strain or parallel assays, provides an unbiased approach to systematically survey the yeast genome. The Saccharomyces yeast deletion collections have proved immensely powerful in contributing to the understanding of gene function, including functional relationships between genes and genetic pathways in response to diverse genetic and environmental perturbations. © 2016 Cold Spring Harbor Laboratory Press.

Simultaneous isolation of high-quality DNA, RNA, miRNA and proteins from tissues for genomic applications

PubMed Central

Peña-Llopis, Samuel; Brugarolas, James

2014-01-01

Genomic technologies have revolutionized our understanding of complex Mendelian diseases and cancer. Solid tumors present several challenges for genomic analyses, such as tumor heterogeneity and tumor contamination with surrounding stroma and infiltrating lymphocytes. We developed a protocol to (i) select tissues of high cellular purity on the basis of histological analyses of immediately flanking sections and (ii) simultaneously extract genomic DNA (gDNA), messenger RNA (mRNA), noncoding RNA (ncRNA; enriched in microRNA (miRNA)) and protein from the same tissues. After tissue selection, about 12–16 extractions of DNA/RNA/protein can be obtained per day. Compared with other similar approaches, this fast and reliable methodology allowed us to identify mutations in tumors with remarkable sensitivity and to perform integrative analyses of whole-genome and exome data sets, DNA copy numbers (by single-nucleotide polymorphism (SNP) arrays), gene expression data (by transcriptome profiling and quantitative PCR (qPCR)) and protein levels (by western blotting and immunohistochemical analysis) from the same samples. Although we focused on renal cell carcinoma, this protocol may be adapted with minor changes to any human or animal tissue to obtain high-quality and high-yield nucleic acids and proteins. PMID:24136348

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

Spiral biasing adaptor for use in Si drift detectors and Si drift detector arrays

DOEpatents

Li, Zheng; Chen, Wei

2016-07-05

A drift detector array, preferably a silicon drift detector (SDD) array, that uses a low current biasing adaptor is disclosed. The biasing adaptor is customizable for any desired geometry of the drift detector single cell with minimum drift time of carriers. The biasing adaptor has spiral shaped ion-implants that generate the desired voltage profile. The biasing adaptor can be processed on the same wafer as the drift detector array and only one biasing adaptor chip/side is needed for one drift detector array to generate the voltage profiles on the front side and back side of the detector array.

Improved intra-array and interarray normalization of peptide microarray phosphorylation for phosphorylome and kinome profiling by rational selection of relevant spots

PubMed Central

Scholma, Jetse; Fuhler, Gwenny M.; Joore, Jos; Hulsman, Marc; Schivo, Stefano; List, Alan F.; Reinders, Marcel J. T.; Peppelenbosch, Maikel P.; Post, Janine N.

2016-01-01

Massive parallel analysis using array technology has become the mainstay for analysis of genomes and transcriptomes. Analogously, the predominance of phosphorylation as a regulator of cellular metabolism has fostered the development of peptide arrays of kinase consensus substrates that allow the charting of cellular phosphorylation events (often called kinome profiling). However, whereas the bioinformatical framework for expression array analysis is well-developed, no advanced analysis tools are yet available for kinome profiling. Especially intra-array and interarray normalization of peptide array phosphorylation remain problematic, due to the absence of “housekeeping” kinases and the obvious fallacy of the assumption that different experimental conditions should exhibit equal amounts of kinase activity. Here we describe the development of analysis tools that reliably quantify phosphorylation of peptide arrays and that allow normalization of the signals obtained. We provide a method for intraslide gradient correction and spot quality control. We describe a novel interarray normalization procedure, named repetitive signal enhancement, RSE, which provides a mathematical approach to limit the false negative results occuring with the use of other normalization procedures. Using in silico and biological experiments we show that employing such protocols yields superior insight into cellular physiology as compared to classical analysis tools for kinome profiling. PMID:27225531

Genome wide profiling in oral squamous cell carcinoma identifies a four genetic marker signature of prognostic significance

PubMed Central

Vincent-Chong, Vui King; Salahshourifar, Iman; Woo, Kar Mun; Anwar, Arif; Razali, Rozaimi; Gudimella, Ranganath; Rahman, Zainal Ariff Abdul; Ismail, Siti Mazlipah; Kallarakkal, Thomas George; Ramanathan, Anand; Wan Mustafa, Wan Mahadzir; Abraham, Mannil Thomas; Tay, Keng Kiong; Zain, Rosnah Binti

2017-01-01

Background Cancers of the oral cavity are primarily oral squamous cell carcinomas (OSCCs). Many of the OSCCs present at late stages with an exceptionally poor prognosis. A probable limitation in management of patients with OSCC lies in the insufficient knowledge pertaining to the linkage between copy number alterations in OSCC and oral tumourigenesis thereby resulting in an inability to deliver targeted therapy. Objectives The current study aimed to identify copy number alterations (CNAs) in OSCC using array comparative genomic hybridization (array CGH) and to correlate the CNAs with clinico-pathologic parameters and clinical outcomes. Materials and methods Using array CGH, genome-wide profiling was performed on 75 OSCCs. Selected genes that were harboured in the frequently amplified and deleted regions were validated using quantitative polymerase chain reaction (qPCR). Thereafter, pathway and network functional analysis were carried out using Ingenuity Pathway Analysis (IPA) software. Results Multiple chromosomal regions including 3q, 5p, 7p, 8q, 9p, 10p, 11q were frequently amplified, while 3p and 8p chromosomal regions were frequently deleted. These findings were in confirmation with our previous study using ultra-dense array CGH. In addition, amplification of 8q, 11q, 7p and 9p and deletion of 8p chromosomal regions showed a significant correlation with clinico-pathologic parameters such as the size of the tumour, metastatic lymph nodes and pathological staging. Co-amplification of 7p, 8q, 9p and 11q regions that harbored amplified genes namely CCND1, EGFR, TPM2 and LRP12 respectively, when combined, continues to be an independent prognostic factor in OSCC. Conclusion Amplification of 3q, 5p, 7p, 8q, 9p, 10p, 11q and deletion of 3p and 8p chromosomal regions were recurrent among OSCC patients. Co-alteration of 7p, 8q, 9p and 11q was found to be associated with clinico-pathologic parameters and poor survival. These regions contain genes that play critical roles in tumourigenesis pathways. PMID:28384287

Intra-Platform Repeatability and Inter-Platform Comparability of MicroRNA Microarray Technology

PubMed Central

Sato, Fumiaki; Tsuchiya, Soken; Terasawa, Kazuya; Tsujimoto, Gozoh

2009-01-01

Over the last decade, DNA microarray technology has provided a great contribution to the life sciences. The MicroArray Quality Control (MAQC) project demonstrated the way to analyze the expression microarray. Recently, microarray technology has been utilized to analyze a comprehensive microRNA expression profiling. Currently, several platforms of microRNA microarray chips are commercially available. Thus, we compared repeatability and comparability of five different microRNA microarray platforms (Agilent, Ambion, Exiqon, Invitrogen and Toray) using 309 microRNAs probes, and the Taqman microRNA system using 142 microRNA probes. This study demonstrated that microRNA microarray has high intra-platform repeatability and comparability to quantitative RT-PCR of microRNA. Among the five platforms, Agilent and Toray array showed relatively better performances than the others. However, the current lineup of commercially available microRNA microarray systems fails to show good inter-platform concordance, probably because of lack of an adequate normalization method and severe divergence in stringency of detection call criteria between different platforms. This study provided the basic information about the performance and the problems specific to the current microRNA microarray systems. PMID:19436744

Loss of heterozygosity assay for molecular detection of cancer using energy-transfer primers and capillary array electrophoresis.

PubMed

Medintz, I L; Lee, C C; Wong, W W; Pirkola, K; Sidransky, D; Mathies, R A

2000-08-01

Microsatellite DNA loci are useful markers for the detection of loss of heterozygosity (LOH) and microsatellite instability (MI) associated with primary cancers. To carry out large-scale studies of LOH and MI in cancer progression, high-throughput instrumentation and assays with high accuracy and sensitivity need to be validated. DNA was extracted from 26 renal tumor and paired lymphocyte samples and amplified with two-color energy-transfer (ET) fluorescent primers specific for loci associated with cancer-induced chromosomal changes. PCR amplicons were separated on the MegaBACE-1000 96 capillary array electrophoresis (CAE) instrument and analyzed with MegaBACE Genetic Profiler v.1.0 software. Ninety-six separations were achieved in parallel in 75 minutes. Loss of heterozygosity was easily detected in tumor samples as was the gain/loss of microsatellite core repeats. Allelic ratios were determined with a precision of +/- 10% or better. Prior analysis of these samples with slab gel electrophoresis and radioisotope labeling had not detected these changes with as much sensitivity or precision. This study establishes the validity of this assay and the MegaBACE instrument for large-scale, high-throughput studies of the molecular genetic changes associated with cancer.

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.

Alteration of gene expression profiling including GPR174 and GNG2 is associated with vasovagal syncope.

PubMed

Huang, Yu-Juan; Zhou, Zai-wei; Xu, Miao; Ma, Qing-wen; Yan, Jing-bin; Wang, Jian-yi; Zhang, Quo-qin; Huang, Min; Bao, Liming

2015-03-01

Vasovagal syncope (VVS) causes accidental harm for susceptible patients. However, pathophysiology of this disorder remains largely unknown. In an effort to understanding of molecular mechanism for VVS, genome-wide gene expression profiling analyses were performed on VVS patients at syncope state. A total of 66 Type 1 VVS child patients and the same number healthy controls were enrolled in this study. Peripheral blood RNAs were isolated from all subjects, of which 10 RNA samples were randomly selected from each groups for gene expression profile analysis using Gene ST 1.0 arrays (Affymetrix). The results revealed that 103 genes were differently expressed between the patients and controls. Significantly, two G-proteins related genes, GPR174 and GNG2 that have not been related to VVS were among the differently expressed genes. The microarray results were confirmed by qRT-PCR in all the tested individuals. Ingenuity pathway analysis and gene ontology annotation study showed that the differently expressed genes are associated with stress response and apoptosis, suggesting that the alteration of some gene expression including G-proteins related genes is associated with VVS. This study provides new insight into the molecular mechanism of VVS and would be helpful to further identify new molecular biomarkers for the disease.

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.

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.

Genome-Wide Mapping of Cystitis Due to Streptococcus agalactiae and Escherichia coli in Mice Identifies a Unique Bladder Transcriptome That Signifies Pathogen-Specific Antimicrobial Defense against Urinary Tract Infection

PubMed Central

Tan, Chee K.; Carey, Alison J.; Cui, Xiangqin; Webb, Richard I.; Ipe, Deepak; Crowley, Michael; Cripps, Allan W.; Benjamin, William H.; Ulett, Kimberly B.; Schembri, Mark A.

2012-01-01

The most common causes of urinary tract infections (UTIs) are Gram-negative pathogens such as Escherichia coli; however, Gram-positive organisms, including Streptococcus agalactiae, or group B streptococcus (GBS), also cause UTI. In GBS infection, UTI progresses to cystitis once the bacteria colonize the bladder, but the host responses triggered in the bladder immediately following infection are largely unknown. Here, we used genome-wide expression profiling to map the bladder transcriptome of GBS UTI in mice infected transurethrally with uropathogenic GBS that was cultured from a 35-year-old women with cystitis. RNA from bladders was applied to Affymetrix Gene-1.0ST microarrays; quantitative reverse transcriptase PCR (qRT-PCR) was used to analyze selected gene responses identified in array data sets. A surprisingly small significant-gene list of 172 genes was identified at 24 h; this compared to 2,507 genes identified in a side-by-side comparison with uropathogenic E. coli (UPEC). No genes exhibited significantly altered expression at 2 h in GBS-infected mice according to arrays despite high bladder bacterial loads at this early time point. The absence of a marked early host response to GBS juxtaposed with broad-based bladder responses activated by UPEC at 2 h. Bioinformatics analyses, including integrative system-level network mapping, revealed multiple activated biological pathways in the GBS bladder transcriptome that regulate leukocyte activation, inflammation, apoptosis, and cytokine-chemokine biosynthesis. These findings define a novel, minimalistic type of bladder host response triggered by GBS UTI, which comprises collective antimicrobial pathways that differ dramatically from those activated by UPEC. Overall, this study emphasizes the unique nature of bladder immune activation mechanisms triggered by distinct uropathogens. PMID:22733575

Prediction of pharmacokinetic and toxicological parameters of a 4-phenylcoumarin isolated from geopropolis: In silico and in vitro approaches.

PubMed

da Cunha, Marcos Guilherme; Franco, Gilson César Nobre; Franchin, Marcelo; Beutler, John A; de Alencar, Severino Matias; Ikegaki, Masaharu; Rosalen, Pedro Luiz

2016-11-30

In silico and in vitro methodologies have been used as important tools in the drug discovery process, including from natural sources. The aim of this study was to predict pharmacokinetic and toxicity (ADME/Tox) properties of a coumarin isolated from geopropolis using in silico and in vitro approaches. Cinnamoyloxy-mammeisin (CNM) isolated from Brazilian M. scutellaris geopropolis was evaluated for its pharmacokinetic parameters by in silico models (ACD/Percepta™ and MetaDrug™ software). Genotoxicity was assessed by in vitro DNA damage signaling PCR array. CNM did not pass all parameters of Lipinski's rule of five, with a predicted low oral bioavailability and high plasma protein binding, but with good predicted blood brain barrier penetration. CNM was predicted to show low affinity to cytochrome P450 family members. Furthermore, the predicted Ames test indicated potential mutagenicity of CNM. Also, the probability of toxicity for organs and tissues was classified as moderate and high for liver and kidney, and moderate and low for skin and eye irritation, respectively. The PCR array analysis showed that CNM significantly upregulated about 7% of all DNA damage-related genes. By exploring the biological function of these genes, it was found that the predicted CNM genotoxicity is likely to be mediated by apoptosis. The predicted ADME/Tox profile suggests that external use of CNM may be preferable to systemic exposure, while its genotoxicity was characterized by the upregulation of apoptosis-related genes after treatment. The combined use of in silico and in vitro approaches to evaluate these parameters generated useful hypotheses to guide further preclinical studies. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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.

Technical aspects and recommendations for single-cell qPCR.

PubMed

Ståhlberg, Anders; Kubista, Mikael

2018-02-01

Single cells are basic physiological and biological units that can function individually as well as in groups in tissues and organs. It is central to identify, characterize and profile single cells at molecular level to be able to distinguish different kinds, to understand their functions and determine how they interact with each other. During the last decade several technologies for single-cell profiling have been developed and used in various applications, revealing many novel findings. Quantitative PCR (qPCR) is one of the most developed methods for single-cell profiling that can be used to interrogate several analytes, including DNA, RNA and protein. Single-cell qPCR has the potential to become routine methodology but the technique is still challenging, as it involves several experimental steps and few molecules are handled. Here, we discuss technical aspects and provide recommendation for single-cell qPCR analysis. The workflow includes experimental design, sample preparation, single-cell collection, direct lysis, reverse transcription, preamplification, qPCR and data analysis. Detailed reporting and sharing of experimental details and data will promote further development and make validation studies possible. Efforts aiming to standardize single-cell qPCR open up means to move single-cell analysis from specialized research settings to standard research laboratories. Copyright © 2017 Elsevier Ltd. All rights reserved.

A New Method for Rapid Screening of End-Point PCR Products: Application to Single Genome Amplified HIV and SIV Envelope Amplicons

PubMed Central

Houzet, Laurent; Deleage, Claire; Satie, Anne-Pascale; Merlande, Laetitia; Mahe, Dominique; Dejucq-Rainsford, Nathalie

2015-01-01

PCR is the most widely applied technique for large scale screening of bacterial clones, mouse genotypes, virus genomes etc. A drawback of large PCR screening is that amplicon analysis is usually performed using gel electrophoresis, a step that is very labor intensive, tedious and chemical waste generating. Single genome amplification (SGA) is used to characterize the diversity and evolutionary dynamics of virus populations within infected hosts. SGA is based on the isolation of single template molecule using limiting dilution followed by nested PCR amplification and requires the analysis of hundreds of reactions per sample, making large scale SGA studies very challenging. Here we present a novel approach entitled Long Amplicon Melt Profiling (LAMP) based on the analysis of the melting profile of the PCR reactions using SYBR Green and/or EvaGreen fluorescent dyes. The LAMP method represents an attractive alternative to gel electrophoresis and enables the quick discrimination of positive reactions. We validate LAMP for SIV and HIV env-SGA, in 96- and 384-well plate formats. Because the melt profiling allows the screening of several thousands of PCR reactions in a cost-effective, rapid and robust way, we believe it will greatly facilitate any large scale PCR screening. PMID:26053379

Structural diversity of Burkholderia pseudomallei lipopolysaccharides affects innate immune signaling

PubMed Central

Norris, Michael H.; Schweizer, Herbert P.

2017-01-01

Burkholderia pseudomallei (Bp) causes the disease melioidosis. The main cause of mortality in this disease is septic shock triggered by the host responding to lipopolysaccharide (LPS) components of the Gram-negative outer membrane. Bp LPS is thought to be a weak inducer of the host immune system. LPS from several strains of Bp were purified and their ability to induce the inflammatory mediators TNF-α and iNOS in murine macrophages at low concentrations was investigated. Innate and adaptive immunity qPCR arrays were used to profile expression patterns of 84 gene targets in response to the different LPS types. Additional qPCR validation confirmed large differences in macrophage response. LPS from a high-virulence serotype B strain 576a and a virulent rough central nervous system tropic strain MSHR435 greatly induced the innate immune response indicating that the immunopathogenesis of these strains is different than in infections with strains similar to the prototype strain 1026b. The accumulation of autophagic vesicles was also increased in macrophages challenged with highly immunogenic Bp LPS. Gene induction and concomitant cytokine secretion profiles of human PBMCs in response to the various LPS were also investigated. MALDI-TOF/TOF was used to probe the lipid A portions of the LPS, indicating substantial structural differences that likely play a role in host response to LPS. These findings add to the evolving knowledge of host-response to bacterial LPS, which can be used to better understand septic shock in melioidosis patients and in the rational design of vaccines. PMID:28453531

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

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

PubMed

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

2004-09-01

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

Generation of miniaturized planar ecombinant antibody arrays using a microcantilever-based printer

NASA Astrophysics Data System (ADS)

Petersson, Linn; Berthet Duroure, Nathalie; Auger, Angèle; Dexlin-Mellby, Linda; Borrebaeck, Carl AK; Ait Ikhlef, Ali; Wingren, Christer

2014-07-01

Miniaturized (Ø 10 μm), multiplexed (>5-plex), and high-density (>100 000 spots cm-2) antibody arrays will play a key role in generating protein expression profiles in health and disease. However, producing such antibody arrays is challenging, and it is the type and range of available spotters which set the stage. This pilot study explored the use of a novel microspotting tool, BioplumeTM—consisting of an array of micromachined silicon cantilevers with integrated microfluidic channels—to produce miniaturized, multiplexed, and high-density planar recombinant antibody arrays for protein expression profiling which targets crude, directly labelled serum. The results demonstrated that 16-plex recombinant antibody arrays could be produced—based on miniaturized spot features (78.5 um2, Ø 10 μm) at a 7-125-times increased spot density (250 000 spots cm-2), interfaced with a fluorescent-based read-out. This prototype platform was found to display adequate reproducibility (spot-to-spot) and an assay sensitivity in the pM range. The feasibility of the array platform for serum protein profiling was outlined.

The von Hippel-Lindau (VHL) tumor-suppressor gene is down-regulated by selenium deficiency in Caco-2 cells and rat colon mucosa.

PubMed

Uthus, Eric; Begaye, Adrienne; Ross, Sharon; Zeng, Huawei

2011-08-01

To test the hypothesis that selenium affects DNA methylation and hence gene regulation, we employed a methylation array (Panomics) in the human colonic epithelial Caco-2 cell model. The array profiles DNA methylation from promoter regions of 82 human genes. After conditioning cells to repeatedly reduced concentrations of fetal bovine serum, a serum-free culture was established. Se-methylselenocysteine (SeMSC) was added at 0 (deficient Se) or 250 (control Se) nM to cells maintained in DMEM. After 7 days, cells were collected and stored at -80 °C until analysis; experiments were replicated three times. Glutathione peroxidase activity was significantly decreased in cells grown in low SeMSC. Cells grown in 250 nM SeMSC had maximal GPx activity. Genomic DNA from cells grown in the low-SeMSC media and media containing 250 nM SeMSC was incubated with methylation-binding protein followed by isolation of methylated DNA. The methylated DNA was labeled with biotin and hybridized to the methylation array. Thus, genes with promoter methylation will produce a higher chemiluminescence signal than those genes with no promoter methylation. Of the genes profiled, the von Hippel-Lindau (VHL) gene was most different as indicated by quantification following chemiluminescence detection demonstrating that the promoter region of VHL was hypermethylated in cells from the low-SeMSC media. To determine whether promoter methylation affected transcription, we isolated RNA from replicate samples and performed real-time RT PCR. VHL (mRNA) was down-regulated (fold change significantly <1) in cells grown in low SeMSC compared to cells grown in 250 nM SeMSC (control; fold change = 1). We also show that (mRNA) Vhl expression is significantly reduced in mucosa from rats fed a diet deficient in Se. Our results suggest that low Se status affects DNA promoter region methylation and that this can result in down-regulation of the tumor suppressor gene VHL.

Projection-based estimation and nonuniformity correction of sensitivity profiles in phased-array surface coils.

PubMed

Yun, Sungdae; Kyriakos, Walid E; Chung, Jun-Young; Han, Yeji; Yoo, Seung-Schik; Park, Hyunwook

2007-03-01

To develop a novel approach for calculating the accurate sensitivity profiles of phased-array coils, resulting in correction of nonuniform intensity in parallel MRI. The proposed intensity-correction method estimates the accurate sensitivity profile of each channel of the phased-array coil. The sensitivity profile is estimated by fitting a nonlinear curve to every projection view through the imaged object. The nonlinear curve-fitting efficiently obtains the low-frequency sensitivity profile by eliminating the high-frequency image contents. Filtered back-projection (FBP) is then used to compute the estimates of the sensitivity profile of each channel. The method was applied to both phantom and brain images acquired from the phased-array coil. Intensity-corrected images from the proposed method had more uniform intensity than those obtained by the commonly used sum-of-squares (SOS) approach. With the use of the proposed correction method, the intensity variation was reduced to 6.1% from 13.1% of the SOS. When the proposed approach was applied to the computation of the sensitivity maps during sensitivity encoding (SENSE) reconstruction, it outperformed the SOS approach in terms of the reconstructed image uniformity. The proposed method is more effective at correcting the intensity nonuniformity of phased-array surface-coil images than the conventional SOS method. In addition, the method was shown to be resilient to noise and was successfully applied for image reconstruction in parallel imaging.

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

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

PubMed

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

2016-10-22

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

Development of internal amplification controls for DNA profiling with the AmpFℓSTR(®) SGM Plus(®) kit.

PubMed

Zahra, Nathalie; Hadi, Sibte; Smith, Judith A; Iyengar, Arati; Goodwin, William

2011-06-01

DNA extracted from forensic samples can be degraded and also contain co-extracted contaminants that inhibit PCR. The effects of DNA degradation and PCR inhibition are often indistinguishable when examining a DNA profile. Two internal amplification controls (IACs) were developed to improve quality control of PCR using the AmpFℓSTR® SGM Plus® kit. The co-amplification of these controls with DNA samples was used to monitor amplification efficiency and detect PCR inhibitors. IAC fragments of 90 and 410 bp (IAC₉₀ and IAC₄₁₀) were generated from the plasmid pBR322 using tailed primers and then amplified with ROX-labelled primers. Co-amplification of IAC₉₀ and IAC₄₁₀ was performed with varying amounts of template DNA, degraded DNA and DNA contaminated with humic acid, heme and indigo dye. Both IAC₉₀ and IAC₄₁₀ were successfully amplified with human DNA without significantly affecting the quality of the DNA profile, even with DNA amounts lower than 0.5 ng. In the presence of inhibitors, the IAC₉₀ signal was still present after all human DNA loci fail to amplify; in contrast, the IAC₄₁₀ signal was reduced or absent at low levels of inhibition. Amplification of the two IACs provided an internal PCR control and allowed partial profiles caused by inhibition to be distinguished from degraded DNA profiles. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development of PCR internal controls for DNA profiling with the AmpFℓSTR® SGM Plus® amplification kit.

PubMed

Nathalie, Zahra; Hadi, Sibte; Goodwin, William

2012-09-01

Forensic DNA profiling uses a series of commercial kits that co-amplify several loci in one reaction; the products of the PCR are fluorescently labelled and analysed using CE. Before CE, an aliquot of the PCR is mixed with formamide and an internal lane size standard. Using the SGM Plus amplification kit, we have developed two internal non-amplified controls of 80 bp and 380 bp that are labelled with ROX fluorescent dye and added to the PCR. Combined with two internal amplification controls of 90 bp and 410 bp, they provide additional controls for the PCR, electrokinetic injection, and CE and also function as an internal size standard. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

lpxC and yafS are the most suitable internal controls to normalize real time RT-qPCR expression in the phytopathogenic bacteria Dickeya dadantii.

PubMed

Hommais, Florence; Zghidi-Abouzid, Ouafa; Oger-Desfeux, Christine; Pineau-Chapelle, Emilie; Van Gijsegem, Frederique; Nasser, William; Reverchon, Sylvie

2011-01-01

Quantitative RT-PCR is the method of choice for studying, with both sensitivity and accuracy, the expression of genes. A reliable normalization of the data, using several reference genes, is critical for an accurate quantification of gene expression. Here, we propose a set of reference genes, of the phytopathogenic bacteria Dickeya dadantii and Pectobacterium atrosepticum, which are stable in a wide range of growth conditions. We extracted, from a D. dadantii micro-array transcript profile dataset comprising thirty-two different growth conditions, an initial set of 49 expressed genes with very low variation in gene expression. Out of these, we retained 10 genes representing different functional categories, different levels of expression (low, medium, and high) and with no systematic variation in expression correlating with growth conditions. We measured the expression of these reference gene candidates using quantitative RT-PCR in 50 different experimental conditions, mimicking the environment encountered by the bacteria in their host and directly during the infection process in planta. The two most stable genes (ABF-0017965 (lpxC) and ABF-0020529 (yafS) were successfully used for normalization of RT-qPCR data. Finally, we demonstrated that the ortholog of lpxC and yafS in Pectobacterium atrosepticum also showed stable expression in diverse growth conditions. We have identified at least two genes, lpxC (ABF-0017965) and yafS (ABF-0020509), whose expressions are stable in a wide range of growth conditions and during infection. Thus, these genes are considered suitable for use as reference genes for the normalization of real-time RT-qPCR data of the two main pectinolytic phytopathogenic bacteria D. dadantii and P. atrosepticum and, probably, of other Enterobacteriaceae. Moreover, we defined general criteria to select good reference genes in bacteria.

MicroRNAs Involved in Asthma After Mesenchymal Stem Cells Treatment

PubMed Central

Tang, Guan-Nan; Li, Cheng-Lin; Yao, Yin; Xu, Zhi-Bin; Deng, Meng-Xia; Wang, Shu-Yue; Sun, Yue-Qi; Shi, Jian-Bo

2016-01-01

Administration of human bone marrow-derived mesenchymal stem cells (BM-MSCs) significantly alleviates allergic airway inflammation. There are no studies that refer to the role of microRNAs (miRNAs) after the BM-MSCs treatment in airway allergic inflammation. We induced a mouse model of asthma and performed the transplantation of BM-MSCs. We analyzed aberrant miRNAs and key immune regulators using both miRNA and messenger RNA (mRNA) polymerase chain reaction (PCR) arrays. We identified that 296 miRNAs were differently expressed after the induction of asthma and/or the treatment of BM-MSCs, in which 14 miRNAs presented the reverse variation tendency between asthma induction and BM-MSCs transplantation. Mmu-miR-21a-3p, mmu-miR-449c-5p, and mmu-miR-496a-3p were further confirmed to be differently expressed with additional samples and quantitative real-time PCR. With an mRNA PCR array, we identified 19 genes to be involved in the allergy induction and the administration of BM-MSCs. Further target genes analysis revealed that mmu-miR-21a-3p was significantly correlated with the immune regulator activin A receptor, Type IIA (Acvr2a). Mmu-miR-21a-3p had opposite expression with Acvr2a after asthma and BM-MSCs treatment. Acvr2a had binding sites for miR-21a for both mice and human, suggesting that miR-21/Acvr2a axis is conserved between human and mice. Dual-luciferase reporter assay showed that mmu-miR-21a-3p negatively regulated the transcript of Acvr2a. In addition, has-miR-21a inhibitor significantly increased the expression of Acvr2a mRNA in BEAS-2B cells under lipopolysaccharide stimulation. Our results suggest that there were different miRNA and mRNA profiles after asthma induction and BM-MSCs treatment, and the miR-21/Acvr2a axis is an important mechanism for the induction of asthmatic inflammation. PMID:27106170

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.

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 microfabricated low-profile wideband antenna array for terahertz communications.

PubMed

Luk, K M; Zhou, S F; Li, Y J; Wu, F; Ng, K B; Chan, C H; Pang, S W

2017-04-28

While terahertz communications are considered to be the future solutions for the increasing demands on bandwidth, terahertz equivalents of radio frequency front-end components have not been realized. It remains challenging to achieve wideband, low profile antenna arrays with highly directive beams of radiation. Here, based on the complementary antenna approach, a wideband 2 × 2 cavity-backed slot antenna array with a corrugated surface is proposed. The approach is based on a unidirectional antenna with a cardiac radiation pattern and stable frequency characteristics that is achieved by integrating a series-resonant electric dipole with a parallel-resonant magnetic dipole. In this design, the slots work as magnetic dipoles while the corrugated surface radiates as an array of electric dipoles. The proposed antenna is realized at 1 THz operating frequency by stacking multiple metallized layers using the microfabrication technology. S-parameter measurements of this terahertz low-profile metallic antenna array demonstrate high efficiency at terahertz frequencies. Fractional bandwidth and gain are measured to be 26% and 14 dBi which are consistent with the simulated results. The proposed antenna can be used as the building block for larger antenna arrays with more directive beams, paving the way to develop high gain low-profile antennas for future communication needs.

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

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

PubMed

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

2017-01-01

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

Evaluations of Different Hypervariable Regions of Archaeal 16S rRNA Genes in Profiling of Methanogens by Archaea-Specific PCR and Denaturing Gradient Gel Electrophoresis▿

PubMed Central

Yu, Zhongtang; García-González, Rubén; Schanbacher, Floyd L.; Morrison, Mark

2008-01-01

Different hypervariable (V) regions of the archaeal 16S rRNA gene (rrs) were compared systematically to establish a preferred V region(s) for use in Archaea-specific PCR-denaturing gradient gel electrophoresis (DGGE). The PCR products of the V3 region produced the most informative DGGE profiles and permitted identification of common methanogens from rumen samples from sheep. This study also showed that different methanogens might be detected when different V regions are targeted by PCR-DGGE. Dietary fat appeared to transiently stimulate Methanosphaera stadtmanae but inhibit Methanobrevibacter sp. strain AbM4 in rumen samples. PMID:18083874

Gene expression profiling defined pathways correlated with fibroblast cell proliferation induced by Opisthorchis viverrini excretory/secretory product.

PubMed

Thuwajit, Chanitra; Thuwajit, Peti; Uchida, Kazuhiko; Daorueang, Daoyot; Kaewkes, Sasithorn; Wongkham, Sopit; Miwa, Masanao

2006-06-14

To investigate the mechanism of fibroblast cell proliferation stimulated by the Opisthorchis viverrini excretory/secretory (ES) product. NIH-3T3, mouse fibroblast cells were treated with O. viverrini ES product by non-contact co-cultured with the adult parasites. Total RNA from NIH-3T3 treated and untreated with O. viverrini was extracted, reverse transcribed and hybridized with the mouse 15K complementary DNA (cDNA) array. The result was analyzed by ArrayVision version 5 and GeneSpring version 5 softwares. After normalization, the ratios of gene expression of parasite treated to untreated NIH-3T3 cells of 2-and more-fold upregulated was defined as the differentially expressed genes. The expression levels of the signal transduction genes were validated by semi-quantitative SYBR-based real-time RT-PCR. Among a total of 15,000 genes/ESTs, 239 genes with established cell proliferation-related function were 2 fold-and more-up-regulated by O. viverrini ES product compared to those in cells without exposure to the parasitic product. These genes were classified into groups including energy and metabolism, signal transduction, protein synthesis and translation, matrix and structural protein, transcription control, cell cycle and DNA replication. Moreover, the expressions of serine-threonine kinase receptor, receptor tyrosine kinase and collagen production-related genes were up-regulated by O. viverrini ES product. The expression level of signal transduction genes; pkC, pdgfr alpha, jak 1, eps 8, tgf beta 1i4, strap and h ras measured by real-time RT-PCR confirmed their expression levels to those obtained from cDNA array. However, only the up-regulated expression of pkC, eps 8 and tgfbeta 1i4 which are the downstream signaling molecules of either epidermal growth factor (EGF) or transforming growth factor-beta (TGF-beta) showed statistical significance (P < 0.05). O. viverrini ES product stimulates the significant changes of gene expression in several functional categories and these mainly include transcripts related to cell proliferation. The TGF-beta and EGF signal transduction pathways are indicated as the possible pathways of O. viverrini-driven cell proliferation.

N-(3-Benzoylphenyl)-1H-Indole-2-Carboxamide decreases triglyceride levels by downregulation of Apoc3 gene expression in acute hyperlipidemic rat model.

PubMed

Hamadneh, Lama; Al-Essa, Luay; Hikmat, Suhair; Al-Qirim, Tariq; Abu Sheikha, Ghassan; Al-Hiari, Yusuf; Azmy, Nisrin; Shattat, Ghassan

2017-07-01

Hyperlipidemia is a known cause of coronary vascular diseases, which is a major cause of death in many parts of the world. Targeting several pathways that lead to increase in lipid profiles is of great potential to control diseases. 1H-indole-2-carboxamide derivatives were tested for their hypolipidemic activity at the molecular level in comparison with bezafibrate. The gene expression profiles of lipoprotein signaling and cholesterol metabolism and fatty acid metabolism PCR arrays were determined in rats with acute hyperlipidemia induced by Triton WR1339. Lipid profiles of serum from treated rats showed significant hypolipidemic effect by the compounds. Several genes of potential interest were reported to be overexpressed by Triton WR1339 including Apoc3, Apob, Hmgcs2, Apoa1, Apoe, Apof, acsl1, and Decr1. Most of the overexpressed genes were downregulated by N-(3-Benzoylphenyl)-1H-Indole-2-Carboxamide with significant decreases in Apoc3, Apob, Acaa2, Acsl1, and Slc247a5 gene expression levels. N-(4-Benzoylphenyl)-1H-Indole-2-Carboxamide and bezafibrate did not significantly affect the gene expression levels which were increased with acute hyperlipidemia induced by Triton WR1339. In conclusion, gene expression profiling identified the possible mechanism in which Triton WR1339 induces its acute hyperlipidemic effect which was reversed by the use of N-(3-Benzoylphenyl)-1H-Indole-2-Carboxamide.

Manipulation of a two-photon state in a χ(2)-modulated nonlinear waveguide array

NASA Astrophysics Data System (ADS)

Yang, Y.; Xu, P.; Lu, L. L.; Zhu, S. N.

2014-10-01

We propose to engineer the quantum state in a high-dimensional Hilbert space by taking advantage of a χ(2)-modulated nonlinear waveguide array. By varying the pump condition and the waveguide array length, the momentum correlation between the signal and idler photons can be manipulated, exhibiting bunching, antibunching, and the evolution between these two states, which are characterized by the Schmidt number. We find the Schmidt number is dependent on a structure parameter, namely the ratio of the array length and the number of channels pumped. By designing the linear profile waveguide array, the degree of spatial entanglement shows a periodic relationship with the slope of linear profile, during which a high degree of position-bunching state is suggested. The two-photon self-focusing effect is disclosed when the χ(2) modulation in the waveguide array contains a parabolic profile, which can be designed for efficient coupling between a waveguide array and fibers. These results shed light on a feasible way to achieve desirable quantum state on a single waveguide chip by a compact engineering of χ(2) and also suggest a degree of freedom for quantum walk and other related applications.

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.

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

Construction, database integration, and application of an Oenothera EST library.

PubMed

Mrácek, Jaroslav; Greiner, Stephan; Cho, Won Kyong; Rauwolf, Uwe; Braun, Martha; Umate, Pavan; Altstätter, Johannes; Stoppel, Rhea; Mlcochová, Lada; Silber, Martina V; Volz, Stefanie M; White, Sarah; Selmeier, Renate; Rudd, Stephen; Herrmann, Reinhold G; Meurer, Jörg

2006-09-01

Coevolution of cellular genetic compartments is a fundamental aspect in eukaryotic genome evolution that becomes apparent in serious developmental disturbances after interspecific organelle exchanges. The genus Oenothera represents a unique, at present the only available, resource to study the role of the compartmentalized plant genome in diversification of populations and speciation processes. An integrated approach involving cDNA cloning, EST sequencing, and bioinformatic data mining was chosen using Oenothera elata with the genetic constitution nuclear genome AA with plastome type I. The Gene Ontology system grouped 1621 unique gene products into 17 different functional categories. Application of arrays generated from a selected fraction of ESTs revealed significantly differing expression profiles among closely related Oenothera species possessing the potential to generate fertile and incompatible plastid/nuclear hybrids (hybrid bleaching). Furthermore, the EST library provides a valuable source of PCR-based polymorphic molecular markers that are instrumental for genotyping and molecular mapping approaches.

Strand-specific transcriptome profiling with directly labeled RNA on genomic tiling microarrays

PubMed Central

2011-01-01

Background With lower manufacturing cost, high spot density, and flexible probe design, genomic tiling microarrays are ideal for comprehensive transcriptome studies. Typically, transcriptome profiling using microarrays involves reverse transcription, which converts RNA to cDNA. The cDNA is then labeled and hybridized to the probes on the arrays, thus the RNA signals are detected indirectly. Reverse transcription is known to generate artifactual cDNA, in particular the synthesis of second-strand cDNA, leading to false discovery of antisense RNA. To address this issue, we have developed an effective method using RNA that is directly labeled, thus by-passing the cDNA generation. This paper describes this method and its application to the mapping of transcriptome profiles. Results RNA extracted from laboratory cultures of Porphyromonas gingivalis was fluorescently labeled with an alkylation reagent and hybridized directly to probes on genomic tiling microarrays specifically designed for this periodontal pathogen. The generated transcriptome profile was strand-specific and produced signals close to background level in most antisense regions of the genome. In contrast, high levels of signal were detected in the antisense regions when the hybridization was done with cDNA. Five antisense areas were tested with independent strand-specific RT-PCR and none to negligible amplification was detected, indicating that the strong antisense cDNA signals were experimental artifacts. Conclusions An efficient method was developed for mapping transcriptome profiles specific to both coding strands of a bacterial genome. This method chemically labels and uses extracted RNA directly in microarray hybridization. The generated transcriptome profile was free of cDNA artifactual signals. In addition, this method requires fewer processing steps and is potentially more sensitive in detecting small amount of RNA compared to conventional end-labeling methods due to the incorporation of more fluorescent molecules per RNA fragment. PMID:21235785

Distinct AGO1 and AGO2 associated miRNA profiles in human cells and blood plasma

PubMed Central

Turchinovich, Andrey; Burwinkel, Barbara

2012-01-01

Studies of miRNA association with Argonaute (AGO) proteins in mammalian cells have indicated lack of bias toward particular AGO. However, to our knowledge, the use of quantitative methods for studying miRNA association with different AGOs has not been reported so far. In this work we compared the total miRNA content in AGO1 and AGO2 immunoprecipitates obtained from MCF7 adenocarcinoma cells using TaqMan Low Density miRNA Arrays and successfully verified selected miRNAs with qPCR. For most of the miRNA species AGO1 and AGO2 profiles were well correlated, however, some miRNAs demonstrated consistent biases toward one of the Argonautes. Furthermore, miRNAs which were predominantly AGO2-associated derived mostly from sense strands of the corresponding pre-miRNAs while the majority of AGO1 biased miRNAs originated from antisense strands of the pre-miRNAs. Additionally, we show that circulating miRNA in human blood plasma can be immunoprecipitated with both AGO1 and AGO2 antibody. However, unlike in cell lysates, AGO1 and AGO2 associated miRNA profiles in plasma did not correlate, indicating that many cell types contribute to circulating miRNA (given that expression of AGO proteins is tissue specific). Furthermore, AGO-specific miRNA profiles in blood cells differed significantly from miRNAs profiles in plasma indicating that most circulating miRNAs are likely to derive from non-blood cells. Since circulating miRNAs hold great promise as biomarkers for numerous cancers and other diseases, we hypothesize that AGO-specific miRNA profiles might add an additional dimension to circulating miRNA-based diagnostics. PMID:22858679

Molecular typing of Salmonella enterica serovar typhi isolates from various countries in Asia by a multiplex PCR assay on variable-number tandem repeats.

PubMed

Liu, Yichun; Lee, May-Ann; Ooi, Eng-Eong; Mavis, Yeo; Tan, Ai-Ling; Quek, Hung-Hiang

2003-09-01

A multiplex PCR method incorporating primers flanking three variable-number tandem repeat (VNTR) loci (arbitrarily labeled TR1, TR2, and TR3) in the CT18 strain of Salmonella enterica serovar Typhi has been developed for molecular typing of S. enterica serovar Typhi clinical isolates from several Asian countries, including Singapore, Indonesia, India, Bangladesh, Malaysia, and Nepal. We have demonstrated that the multiplex PCR could be performed on crude cell lysates and that the VNTR banding profiles produced could be easily analyzed by visual inspection after conventional agarose gel electrophoresis. The assay was highly discriminative in identifying 49 distinct VNTR profiles among 59 individual isolates. A high level of VNTR profile heterogeneity was observed in isolates from within the same country and among countries. These VNTR profiles remained stable after the strains were passaged extensively under routine laboratory culture conditions. In contrast to the S. enterica serovar Typhi isolates, an absence of TR3 amplicons and a lack of length polymorphisms in TR1 and TR2 amplicons were observed for other S. enterica serovars, such as Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Paratyphi A, B, and C. DNA sequencing of the amplified VNTR regions substantiated these results, suggesting the high stability of the multiplex PCR assay. The multiplex-PCR-based VNTR profiling developed in this study provides a simple, rapid, reproducible, and high-resolution molecular tool for the epidemiological analysis of S. enterica serovar Typhi strains.

[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.

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

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.

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.

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.

Expression Profile of DNA Damage Signaling Genes in Proton Exposed Mouse Brain

NASA Astrophysics Data System (ADS)

Ramesh, Govindarajan; Wu, Honglu

Exposure of living systems to radiation results in a wide assortment of lesions, the most signif-icant of is damage to genomic DNA which induce several cellular functions such as cell cycle arrest, repair, apoptosis etc. The radiation induced DNA damage investigation is one of the im-portant area in biology, but still the information available regarding the effects of proton is very limited. In this report, we investigated the differential gene expression pattern of DNA damage signaling genes particularly, damaged DNA binding, repair, cell cycle arrest, checkpoints and apoptosis using quantitative real-time RT-PCR array in proton exposed mouse brain tissues. The expression profiles showed significant changes in DNA damage related genes in 2Gy proton exposed mouse brain tissues as compared with control brain tissues. Furthermore, we also show that significantly increased levels of apoptotic related genes, caspase-3 and 8 activities in these cells, suggesting that in addition to differential expression of DNA damage genes, the alteration of apoptosis related genes may also contribute to the radiation induced DNA damage followed by programmed cell death. In summary, our findings suggest that proton exposed brain tissue undergo severe DNA damage which in turn destabilize the chromatin stability.

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.

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

RNA-Seq Differentiates Tumour and Host mRNA Expression Changes Induced by Treatment of Human Tumour Xenografts with the VEGFR Tyrosine Kinase Inhibitor Cediranib

PubMed Central

Bradford, James R.; Farren, Matthew; Powell, Steve J.; Runswick, Sarah; Weston, Susie L.; Brown, Helen; Delpuech, Oona; Wappett, Mark; Smith, Neil R.; Carr, T. Hedley; Dry, Jonathan R.; Gibson, Neil J.; Barry, Simon T.

2013-01-01

Pre-clinical models of tumour biology often rely on propagating human tumour cells in a mouse. In order to gain insight into the alignment of these models to human disease segments or investigate the effects of different therapeutics, approaches such as PCR or array based expression profiling are often employed despite suffering from biased transcript coverage, and a requirement for specialist experimental protocols to separate tumour and host signals. Here, we describe a computational strategy to profile transcript expression in both the tumour and host compartments of pre-clinical xenograft models from the same RNA sample using RNA-Seq. Key to this strategy is a species-specific mapping approach that removes the need for manipulation of the RNA population, customised sequencing protocols, or prior knowledge of the species component ratio. The method demonstrates comparable performance to species-specific RT-qPCR and a standard microarray platform, and allowed us to quantify gene expression changes in both the tumour and host tissue following treatment with cediranib, a potent vascular endothelial growth factor receptor tyrosine kinase inhibitor, including the reduction of multiple murine transcripts associated with endothelium or vessels, and an increase in genes associated with the inflammatory response in response to cediranib. In the human compartment, we observed a robust induction of hypoxia genes and a reduction in cell cycle associated transcripts. In conclusion, the study establishes that RNA-Seq can be applied to pre-clinical models to gain deeper understanding of model characteristics and compound mechanism of action, and to identify both tumour and host biomarkers. PMID:23840389

Global microRNA profiling of peripheral blood mononuclear cells in patients with Behçet's disease.

PubMed

Erre, Gian Luca; Piga, Matteo; Carru, Ciriaco; Angius, Andrea; Carcangiu, Laura; Piras, Marco; Sotgia, Salvatore; Zinellu, Angelo; Mathieu, Alessandro; Passiu, Giuseppe; Pescatori, Mario

2015-01-01

To explore the post-transcriptional regulation of the peripheral blood mononuclear cells (PBMCs) transcriptome by microRNAs in Behçet's disease (BD). Using TaqMan Low Density Array-based microRNAs expression profiling, the expression of 750 mature human microRNAs in PBMCs from 5 BD patients and 3 healthy controls (HC) was compared. The expression of deregulated microRNAs was then validated by quantitative real time-polymerase chain reaction (qRT-PCR), in 42 BD patients and 8 HC. In the initial screening, 13 microRNAs appeared deregulated in BD vs HC. Among them, the differential expression of miR-720 and miR-139-3p was confirmed by qRT-PCR, (p<0.05 and FDR<5%). Areas under the receiver operating characteristic curve for miR-139-3p, miR-720 and miR-139-3p+miR-720 in the validation cohort were 0.84, 0.87 and 0.92 respectively, indicating good discrimination between BD patients and HC. Post-hoc analysis showed that 9 out of 13 microRNAs from the discovery phase were significantly upregulated in active vs. quiescent BD, suggesting inflammation as a key regulator of microRNAs machinery in BD. In silico analysis revealed that several BD candidate susceptibility genes are predicted target of significantly deregulated microRNAs in active BD. A significant enrichment in microRNAs targeting elements of the Toll-like receptor (TLR) and T-cell receptor signalling pathways was also assumed. miR199-3p and miR720 deserve further confirmation as biomarkers of BD in larger studies. PBMCs from active BD displayed a unique signature of microRNAs which may be implicated in regulation of innate immunity activation and T-cell function.

Characterization of Colistin-Resistant Escherichia coli Isolated from Diseased Pigs in France

PubMed Central

Delannoy, Sabine; Le Devendec, Laetitia; Jouy, Eric; Fach, Patrick; Drider, Djamel; Kempf, Isabelle

2017-01-01

We studied a collection of 79 colistin-resistant Escherichia coli isolates isolated from diseased pigs in France between 2009 and 2013. We determined a number of phenotypic and genetic characters using broth microdilution to characterize their antimicrobial susceptibility. We performed pulse field gel electrophoresis (PFGE) to assess their genetic diversity and assign them to phylogroups. High-throughput real-time PCR micro-array was used to screen for a selection of genetic markers of virulence, and PCR and sequencing of the main recognized resistance genes allowed us to investigate the mechanisms of colistin resistance. Results showed that isolates belonged to several phylogroups and most had a unique PFGE profile. More than 50% of the isolates were also resistant to sulfonamides, trimethoprim, tetracycline, ampicillin or chloramphenicol. The mcr-1 gene was detected in 70 out of 79 isolates and was transferred by conjugation in 33 of them, sometimes together with resistance to sulfonamides, trimethoprim, tetracycline, ampicillin, chloramphenicol, cefotaxime, or gentamicin. Mutations in the amino-acid sequences of proteins MgrB, PhoP, PhoQ, PmrB, but not PmrA, were detected in isolates with or without the mcr-1 gene. More than one-third of the isolates harbored the F18, F4, astA, hlyA, estI, estII, elt, stx2e, iha, orfA, orfB, paa, terE, ecs1763, or ureD virulence markers. In conclusion, although most isolates had a unique PFGE profile, a few particular combinations of phylogenetic groups, virulence genes and mutations in the sequenced genes involved in colistin resistance were identified on a number of occasions, suggesting the persistence of certain isolates over several years. PMID:29209292

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

PubMed Central

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

2011-01-01

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

Differential Gene Expression in Gonadotropin-Releasing Hormone Neurons of Male and Metestrous Female Mice.

PubMed

Vastagh, Csaba; Rodolosse, Annie; Solymosi, Norbert; Farkas, Imre; Auer, Herbert; Sárvári, Miklós; Liposits, Zsolt

2015-01-01

Gonadotropin-releasing hormone (GnRH) neurons play a pivotal role in the regulation of the hypothalamic-pituitary gonadal axis in a sex-specific manner. We hypothesized that the differences seen in reproductive functions of males and females are associated with a sexually dimorphic gene expression profile of GnRH neurons. We compared the transcriptome of GnRH neurons obtained from intact metestrous female and male GnRH-green fluorescent protein transgenic mice. About 1,500 individual GnRH neurons from each sex were sampled with laser capture microdissection followed by whole-transcriptome amplification for gene expression profiling. Under stringent selection criteria (fold change >1.6, adjusted p value 0.01), Affymetrix Mouse Genome 430 PM array analysis identified 543 differentially expressed genes. Sexual dimorphism was most apparent in gene clusters associated with synaptic communication, signal transduction, cell adhesion, vesicular transport and cell metabolism. To validate microarray results, 57 genes were selected, and 91% of their differential expression was confirmed by real-time PCR. Similarly, 88% of microarray results were confirmed with PCR from independent samples obtained by patch pipette harvesting and pooling of 30 GnRH neurons from each sex. We found significant differences in the expression of genes involved in vesicle priming and docking (Syt1, Cplx1), GABAergic (Gabra3, Gabrb3, Gabrg2) and glutamatergic (Gria1, Grin1, Slc17a6) neurotransmission, peptide signaling (Sstr3, Npr2, Cxcr4) and the regulation of intracellular ion homeostasis (Cacna1, Cacnb1, Cacng5, Kcnq2, Kcnc1). The striking sexual dimorphism of the GnRH neuron transcriptome we report here contributes to a better understanding of the differences in cellular mechanisms of GnRH neurons in the two sexes. © 2015 S. Karger AG, Basel.

Unravelling site-specific breast cancer metastasis: a microRNA expression profiling study

PubMed Central

Schrijver, Willemijne A.M.E.; van Diest, Paul J.; Moelans, Cathy B

2017-01-01

Distant metastasis is still the main cause of death from breast cancer. MicroRNAs (miRs) are important regulators of many physiological and pathological processes, including metastasis. Molecular breast cancer subtypes are known to show a site-specific pattern of metastases formation. In this study, we set out to determine the underlying molecular mechanisms of site-specific breast cancer metastasis by microRNA expression profiling. To identify a miR signature for metastatic breast carcinoma that could predict metastatic localization, we compared global miR expression in 23 primary breast cancer specimens with their corresponding multiple distant metastases to ovary (n=9), skin (n=12), lung (n=10), brain (n=4) and gastrointestinal tract (n=10) by miRCURY microRNA expression arrays. For validation, we performed quantitative real-time (qRT) PCR on the discovery cohort and on an independent validation cohort of 29 primary breast cancer specimens and their matched metastases. miR expression was highly patient specific and miR signatures in the primary tumor were largely retained in the metastases, with the exception of several differentially expressed, location specific miRs. Validation with qPCR demonstrated that hsa-miR-106b-5p was predictive for the development of lung metastases. In time, the second metastasis often showed a miR upregulation compared to the first metastasis. This study discovered a metastatic site-specific miR and found miR expression to be highly patient specific. This may lead to novel biomarkers predicting site of distant metastases, and to adjuvant, personalized targeted therapy strategies that could prevent such metastases from becoming clinically manifest. PMID:27902972

Unravelling site-specific breast cancer metastasis: a microRNA expression profiling study.

PubMed

Schrijver, Willemijne A M E; van Diest, Paul J; Moelans, Cathy B

2017-01-10

Distant metastasis is still the main cause of death from breast cancer. MicroRNAs (miRs) are important regulators of many physiological and pathological processes, including metastasis. Molecular breast cancer subtypes are known to show a site-specific pattern of metastases formation. In this study, we set out to determine the underlying molecular mechanisms of site-specific breast cancer metastasis by microRNA expression profiling.To identify a miR signature for metastatic breast carcinoma that could predict metastatic localization, we compared global miR expression in 23 primary breast cancer specimens with their corresponding multiple distant metastases to ovary (n=9), skin (n=12), lung (n=10), brain (n=4) and gastrointestinal tract (n=10) by miRCURY microRNA expression arrays. For validation, we performed quantitative real-time (qRT) PCR on the discovery cohort and on an independent validation cohort of 29 primary breast cancer specimens and their matched metastases.miR expression was highly patient specific and miR signatures in the primary tumor were largely retained in the metastases, with the exception of several differentially expressed, location specific miRs. Validation with qPCR demonstrated that hsa-miR-106b-5p was predictive for the development of lung metastases. In time, the second metastasis often showed a miR upregulation compared to the first metastasis.This study discovered a metastatic site-specific miR and found miR expression to be highly patient specific. This may lead to novel biomarkers predicting site of distant metastases, and to adjuvant, personalized targeted therapy strategies that could prevent such metastases from becoming clinically manifest.

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

PubMed Central

2010-01-01

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

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

PubMed

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

2010-04-22

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

Immunomediator expression profiling in two beluga whale (delphinapterus leucas) clinical cases

USDA-ARS?s Scientific Manuscript database

Cytokines and other immunomediators can be biomarkers of inflammation. Quantitative real-time PCR (qPCR) has been used to examine cytokine gene expression in beluga whale (Delphinapterus leucas) peripheral blood mononuclear cells (PBMC). Thus, qPCR-based immunomediator assays could supplement clinic...

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.

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

Perturbation of polyamine catabolism affects grape ripening of Vitis vinifera cv. Trincadeira.

PubMed

Agudelo-Romero, Patricia; Ali, Kashif; Choi, Young H; Sousa, Lisete; Verpoorte, Rob; Tiburcio, Antonio F; Fortes, Ana M

2014-01-01

Grapes are economically the most important fruit worldwide. However, the complexity of biological events that lead to ripening of nonclimacteric fruits is not fully understood, particularly the role of polyamines' catabolism. The transcriptional and metabolic profilings complemented with biochemical data were studied during ripening of Trincadeira grapes submitted to guazatine treatment, a potent inhibitor of polyamine oxidase activity. The mRNA expression profiles of one time point (EL 38) corresponding to harvest stage was compared between mock and guazatine treatments using Affymetrix GrapeGen(®) genome array. A total of 2113 probesets (1880 unigenes) were differentially expressed between these samples. Quantitative RT-PCR validated microarrays results being carried out for EL 35 (véraison berries), EL 36 (ripe berries) and EL 38 (harvest stage berries). Metabolic profiling using HPLC and (1)H NMR spectroscopy showed increase of putrescine, proline, threonine and 1-O-ethyl-β-glucoside in guazatine treated samples. Genes involved in amino acid, carbohydrate and water transport were down-regulated in guazatine treated samples suggesting that the strong dehydrated phenotype obtained in guazatine treated samples may be due to impaired transport mechanisms. Genes involved in terpenes' metabolism were differentially expressed between guazatine and mock treated samples. Altogether, results support an important role of polyamine catabolism in grape ripening namely in cell expansion and aroma development. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Performance parameters of a liquid filled ionization chamber array

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

Poppe, B.; Stelljes, T. S.; Looe, H. K.

2013-08-15

Purpose: In this work, the properties of the two-dimensional liquid filled ionization chamber array Octavius 1000SRS (PTW-Freiburg, Germany) for use in clinical photon-beam dosimetry are investigated.Methods: Measurements were carried out at an Elekta Synergy and Siemens Primus accelerator. For measurements of stability, linearity, and saturation effects of the 1000SRS array a Semiflex 31013 ionization chamber (PTW-Freiburg, Germany) was used as a reference. The effective point of measurement was determined by TPR measurements of the array in comparison with a Roos chamber (type 31004, PTW-Freiburg, Germany). The response of the array with varying field size and depth of measurement was evaluatedmore » using a Semiflex 31010 ionization chamber as a reference. Output factor measurements were carried out with a Semiflex 31010 ionization chamber, a diode (type 60012, PTW-Freiburg, Germany), and the detector array under investigation. The dose response function for a single detector of the array was determined by measuring 1 cm wide slit-beam dose profiles and comparing them against diode-measured profiles. Theoretical aspects of the low pass properties and of the sampling frequency of the detector array were evaluated. Dose profiles measured with the array and the diode detector were compared, and an intensity modulated radiation therapy (IMRT) field was verified using the Gamma-Index method and the visualization of line dose profiles.Results: The array showed a short and long term stability better than 0.1% and 0.2%, respectively. Fluctuations in linearity were found to be within ±0.2% for the vendor specified dose range. Saturation effects were found to be similar to those reported in other studies for liquid-filled ionization chambers. The detector's relative response varied with field size and depth of measurement, showing a small energy dependence accounting for maximum signal deviations of ±2.6% from the reference condition for the setup used. The σ-values of the Gaussian dose response function for a single detector of the array were found to be (0.72 ± 0.25) mm at 6 MV and (0.74 ± 0.25) mm at 15 MV and the corresponding low pass cutoff frequencies are 0.22 and 0.21 mm{sup −1}, respectively. For the inner 5 × 5 cm{sup 2} region and the outer 11 × 11 cm{sup 2} region of the array the Nyquist theorem is fulfilled for maximum sampling frequencies of 0.2 and 0.1 mm{sup −1}, respectively. An IMRT field verification with a Gamma-Index analysis yielded a passing rate of 95.2% for a 3 mm/3% criterion with a TPS calculation as reference.Conclusions: This study shows the applicability of the Octavius 1000SRS in modern dosimetry. Output factor and dose profile measurements illustrated the applicability of the array in small field and stereotactic dosimetry. The high spatial resolution ensures adequate measurements of dose profiles in regular and intensity modulated photon-beam fields.« less

Differentially expressed genes in nonsmall cell lung cancer: expression profiling of cancer-related genes in squamous cell lung cancer.

PubMed

Kettunen, Eeva; Anttila, Sisko; Seppänen, Jouni K; Karjalainen, Antti; Edgren, Henrik; Lindström, Irmeli; Salovaara, Reijo; Nissén, Anna-Maria; Salo, Jarmo; Mattson, Karin; Hollmén, Jaakko; Knuutila, Sakari; Wikman, Harriet

2004-03-01

The expression patterns of cancer-related genes in 13 cases of squamous cell lung cancer (SCC) were characterized and compared with those in normal lung tissue and 13 adenocarcinomas (AC), the other major type of nonsmall cell lung cancer (NSCLC). cDNA array was used to screen the gene expression levels and the array results were verified using a real-time reverse-transcriptase-polymerase chain reaction (RT-PCR). Thirty-nine percent of the 25 most upregulated and the 25 most downregulated genes were common to SCC and AC. Of these genes, DSP, HMGA1 (alias HMGIY), TIMP1, MIF, CCNB1, TN, MMP11, and MMP12 were upregulated and COPEB (alias CPBP), TYROBP, BENE, BMPR2, SOCS3, TIMP3, CAV1, and CAV2 were downregulated. The expression levels of several genes from distinct protein families (cytokeratins and hemidesmosomal proteins) were markedly increased in SCC compared with AC and normal lung. In addition, several genes, overexpressed in SCC, such as HMGA1, CDK4, IGFBP3, MMP9, MMP11, MMP12, and MMP14, fell into distinct chromosomal loci, which we have detected as gained regions on the basis of comparative genomic hybridization data. Our study revealed new candidate genes involved in NSCLC.

Physiological Adjustments and Circulating MicroRNA Reprogramming Are Involved in Early Acclimatization to High Altitude in Chinese Han Males

PubMed Central

Liu, Bao; Huang, He; Wang, Shou-Xian; Wu, Gang; Xu, Gang; Sun, Bing-Da; Zhang, Er-Long; Gao, Yu-Qi

2016-01-01

Background: Altitude acclimatization is a physiological process that restores oxygen delivery to the tissues and promotes oxygen use under high altitude hypoxia. High altitude sickness occurs in individuals without acclimatization. Unraveling the molecular underpinnings of altitude acclimatization could help understand the beneficial body responses to high altitude hypoxia as well as the altered biological events in un-acclimatized individuals. This study assessed physiological adjustments and circulating microRNA (cmiRNA) profiles in individuals exposed to high altitude, aiming to explore altitude acclimatization in humans. Methods: Ninety volunteers were enrolled in this study. Among them, 22 individuals provided samples for microRNA arrays; 68 additional individuals constituted the validation set. Un-acclimatized individuals were identified by the Lake Louise Scoring System. Thirty-three phenotypes were recorded pre- and post-exposure to high altitude, including stress hormones, lipid profiles, hematological indices, myocardial enzyme spectrum, and liver and kidney function related enzymes. CmiRNA expression profiles were assessed using miRCURYTM LNA Array (v.18.0) screening, with data validated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Then, associations of plasma microRNA expression with physiological adjustments were evaluated. The biological relevance of the main differentially expressed cmiRNAs was explored by bioinformatics prediction. Results: Nineteen of the 33 phenotypes were significantly altered during early altitude acclimatization, including hematological indices, lipid profiles, and stress hormones; meanwhile, 86 cmiRNAs (79 up-regulated and 7 down-regulated) showed differential expression with statistical significance. Among them, 32 and 25 microRNAs were strongly correlated with low-density lipoprotein-cholesterol and total cholesterol elevations, respectively. In addition, 22 microRNAs were closely correlated with cortisol increase. In un-acclimatized individuals, 55 cmiRNAs were up-regulated and 36 down-regulated, compared with acclimatized individuals. The HIF signaling pathway was suppressed in un-acclimatized individuals. Conclusion: Physiological adjustments, including the hematological system, stress hormones, and lipid molecules contributed to early altitude acclimatization, and showed strong correlations with cmiRNA reprogramming. Moreover, acclimatized and un-acclimatized individuals showed different cmiRNA profile. Suppression of the HIF-1 signaling pathway by microRNA regulation may play a key role in the pathogenesis of un-acclimatization with high altitude hypoxia. PMID:27994555

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

Experimental demonstration of a multi-wavelength distributed feedback semiconductor laser array with an equivalent chirped grating profile based on the equivalent chirp technology.

PubMed

Li, Wangzhe; Zhang, Xia; Yao, Jianping

2013-08-26

We report, to the best of our knowledge, the first realization of a multi-wavelength distributed feedback (DFB) semiconductor laser array with an equivalent chirped grating profile based on equivalent chirp technology. All the lasers in the laser array have an identical grating period with an equivalent chirped grating structure, which are realized by nonuniform sampling of the gratings. Different wavelengths are achieved by changing the sampling functions. A multi-wavelength DFB semiconductor laser array is fabricated and the lasing performance is evaluated. The results show that the equivalent chirp technology is an effective solution for monolithic integration of a multi-wavelength laser array with potential for large volume fabrication.

The Design and Characterization of Wideband Spline-profiled Feedhorns for Advanced Actpol

NASA Technical Reports Server (NTRS)

Simon, Sara M.; Austermann, Jason; Beall, James A.; Choi, Steve K.; Coughlin, Kevin P.; Duff, Shannon M.; Gallardo, Patricio A.; Henderson, Shawn W.; Hills, Felicity B.; Ho, Shuay-Pwu Patty;

Molecular characterization of Salmonella enterica serotype Enteritidis isolates from food and human samples by serotyping, antimicrobial resistance, plasmid profiling, (GTG)5-PCR and ERIC-PCR.

PubMed

Fardsanei, F; Nikkhahi, F; Bakhshi, B; Salehi, T Z; Tamai, I A; Soltan Dallal, M M

2016-11-01

In recent years, Salmonella enterica serovar Enteritidis has been a primary cause of human salmonellosis in many countries. The major objective of this study was to investigate genetic diversity among Salmonella Enteritidis strains from different origins (food and human) by Enterobacterial Repetitive Intergenic Consensus (ERIC) -PCR, as well as to assess their plasmid profiling and antimicrobial resistance. A total of 30 Salmonella Enteritidis isolates, 15 from food samples (chicken, lamb, beef and duck meats) and 15 from clinical samples were collected in Tehran. Identification of isolates as Salmonella was confirmed by using conventional standard biochemical and serological tests. Multiplex-PCR was used for serotyping of isolates to identify Salmonella Enteritidis. Antimicrobial susceptibility testing to 16 agents founds drug resistance patterns among Salmonella Enteritidis isolates. No resistance was observed to cephalexin, ceftriaxone, ceftazidime and cefotaxime, ciprofloxacin, imipenem or meropenem, chloramphenicol and gentamicin. The highest resistance (96.7%) was observed to nitrofurantoin. Seven plasmid profiles (P1-P7) were detected, and a 68-kb plasmid was found in all isolates. Two different primers; ERIC and (GTG)5 were used for genotyping, which each produced four profiles. The majority of clinical and food isolates fell into two separate common types (CTs) with a similar percentage of 95% by ERIC-PCR. Using primer (GTG)5, 29 isolates incorporated in three CTs with 70% of isolates showing a single banding pattern. Limited genetic diversity among human and food isolates of Salmonella Enteritidis may indicate that contaminated foods were possibly the source of human salmonellosis. These results confirmed that ERIC-PCR genotyping has limited discriminatory power for Salmonella Enteritidis of different origin.

Comparison of modeled and experimental PV array temperature profiles for accurate interpretation of module performance and degradation

NASA Astrophysics Data System (ADS)

Elwood, Teri; Simmons-Potter, Kelly

2017-08-01

Quantification of the effect of temperature on photovoltaic (PV) module efficiency is vital to the correct interpretation of PV module performance under varied environmental conditions. However, previous work has demonstrated that PV module arrays in the field are subject to significant location-based temperature variations associated with, for example, local heating/cooling and array edge effects. Such thermal non-uniformity can potentially lead to under-prediction or over-prediction of PV array performance due to an incorrect interpretation of individual module temperature de-rating. In the current work, a simulated method for modeling the thermal profile of an extended PV array has been investigated through extensive computational modeling utilizing ANSYS, a high-performance computational fluid dynamics (CFD) software tool. Using the local wind speed as an input, simulations were run to determine the velocity at particular points along modular strings corresponding to the locations of temperature sensors along strings in the field. The point velocities were utilized along with laminar flow theories in order to calculate Nusselt's number for each point. These calculations produced a heat flux profile which, when combined with local thermal and solar radiation profiles, were used as inputs in an ANSYS Thermal Transient model that generated a solar string operating temperature profile. A comparison of the data collected during field testing, and the data fabricated by ANSYS simulations, will be discussed in order to authenticate the accuracy of the model.

Outbreak of hepatitis E virus infection in Darfur, Sudan: effectiveness of real-time reverse transcription-PCR analysis of dried blood spots.

PubMed

Mérens, Audrey; Guérin, Philippe Jean; Guthmann, Jean-Paul; Nicand, Elisabeth

2009-06-01

Biological samples collected in refugee camps during an outbreak of hepatitis E were used to compare the accuracy of hepatitis E virus RNA amplification by real-time reverse transcription-PCR (RT-PCR) for sera and dried blood spots (concordance of 90.6%). Biological profiles (RT-PCR and serology) of asymptomatic individuals were also analyzed.

Field testing of a convergent array of acoustic Doppler profilers for high-resolution velocimetry in energetic tidal currents

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

Harding, Samuel F.; Sellar, Brian; Richmond, Marshall C.

An array of single-beam acoustic Doppler profilers has been developed for the high resolution measurement of three-dimensional tidal flow velocities and subsequently tested in an energetic tidal site. This configuration has been developed to increase spatial resolution of velocity measurements in comparison to conventional acoustic Doppler profilers (ADPs) which characteristically use divergent acoustic beams emanating from a single instrument. This is achieved using geometrically convergent acoustic beams creating a sample volume at the focal point of 0.03 m3. Away from the focal point, the array is also able to simultaneously reconstruct three-dimensional velocity components in a profile throughout the watermore » column, and is referred to herein as a convergent-beam acoustic Doppler profiler (C-ADP). Mid-depth profiling is achieved through integration of the sensor platform with the operational commercial-scale Alstom 1MW DeepGen-IV Tidal Turbine deployed at the European Marine Energy Center, Orkney Isles, UK. This proof-of-concept paper outlines the C-ADP system configuration and comparison to measurements provided by co-installed reference instrumentation.« less

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

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.

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.

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.

Barcode DNA length polymorphisms vs fatty acid profiling for adulteration detection in olive oil.

PubMed

Uncu, Ali Tevfik; Uncu, Ayse Ozgur; Frary, Anne; Doganlar, Sami

2017-04-15

The aim of this study was to compare the performance of a DNA-barcode assay with fatty acid profile analysis to authenticate the botanical origin of olive oil. To achieve this aim, we performed a PCR-capillary electrophoresis (PCR-CE) approach on olive oil: seed oil blends using the plastid trnL (UAA) intron barcode. In parallel to genomic analysis, we subjected the samples to gas chromatography analysis of fatty acid composition. While the PCR-CE assay proved equally efficient as gas chromatography analysis in detecting adulteration with soybean, palm, rapeseed, sunflower, sesame, cottonseed and peanut oils, it was superior to the widely utilized analytical chemistry approach in revealing the adulterant species and detecting small quantities of corn and safflower oils in olive oil. Moreover, the DNA-based test correctly identified all tested olive oil: hazelnut oil blends whereas it was not feasible to detect hazelnut oil adulteration through fatty acid profile analysis. Thus, the present research has shown the feasibility of a PCR-CE barcode assay to detect adulteration in olive oil. Copyright © 2016 Elsevier Ltd. All rights reserved.

Wideband Array for C, X, and Ku-Band Applications with 5.3:1 Bandwidth

NASA Technical Reports Server (NTRS)

Novak, Markus H.; Volakis, John L.; Miranda, Felix A.

2015-01-01

Planar arrays that exploit strong intentional coupling between elements have allowed for very wide bandwidths in low-profile configurations. However, such designs also require complex impedance matching networks that must also be very compact. For many space applications, typically occurring at C-, X-, Ku-, and most recently at Ka-band, such designs require specialized and expensive fabrication techniques. To address this issue, a novel ultra-wideband array is presented, using a simplified feed network to reduce fabrication cost. The array operates from 3.5-18.5 GHz with VSWR less than 2.4 at broadside, and is of very low profile, having a total height of lambda/10 at the lowest frequency of operation. Validation is provided using a 64-element prototype array, fabricated using common Printed Circuit Board (PCB) technology. The low size, weight, and cost of this array make it attractive for space-borne applications.

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.

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

Profiling differential gene expression of corals along a transect of waters adjacent to the Bermuda municipal dump.

PubMed

Morgan, Michael B; Edge, Sara E; Snell, Terry W

2005-01-01

A coral cDNA array containing 32 genes was used to examine the gene expression profiles of coral populations located at four sites that varied with distance from a semi-submerged municipal dump in Castle Harbour, Bermuda (previously identified as a point source of anthropogenic stressors). Genes on the array represent transcripts induced under controlled laboratory conditions to a variety of stressors both natural (temperature, sediment, salinity, darkness) and xenobiotic (heavy metals, pesticides, PAH) in origin. The gene expression profiles produced revealed information about the types of stressors. Consistent with other studies undertaken in Castle Harbour, the coral cDNA array detected responses to heavy metals, sedimentation, as well as oxidative stress.

Circulating microRNAs as biomarkers of disease and typification of the atherothrombotic status in antiphospholipid syndrome

PubMed Central

Pérez-Sánchez, Carlos; Arias-de la Rosa, Iván; Aguirre, María Ángeles; Luque-Tévar, María; Ruiz-Limón, Patricia; Barbarroja, Nuria; Jiménez-Gómez, Yolanda; Ábalos-Aguilera, María Carmen; Collantes-Estévez, Eduardo; Segui, Pedro; Velasco, Francisco; Herranz, María Teresa; Lozano-Herrero, Jesús; Hernandez-Vidal, María Julia; Martínez, Constantino; González-Conejero, Rocío; Radin, Massimo; Sciascia, Savino; Cecchi, Irene; Cuadrado, María José; López-Pedrera, Chary

2018-01-01

We aimed to identify the plasma miRNA profile of antiphospholipid syndrome (APS) patients and to investigate the potential role of specific circulating miRNAs as non-invasive disease biomarkers. Ninety APS patients and 42 healthy donors were recruited. Profiling of miRNAs by PCR-array in plasma of APS patients identified a set of miRNAs differentially expressed and collectively involved in clinical features. Logistic regression and ROC analysis identified a signature of 10 miRNA ratios as biomarkers of disease. In addition, miRNA signature was related to fetal loss, atherosclerosis, and type of thrombosis, and correlated with parameters linked to inflammation, thrombosis, and autoimmunity. Hard clustering analysis differentiated 3 clusters representing different thrombotic risk profile groups. Significant differences between groups for several miRNA ratios were found. Moreover, miRNA signature remained stable over time, demonstrated by their analysis three months after the first sample collection. Parallel analysis in two additional cohorts of patients, including thrombosis without autoimmune disease, and systemic lupus erythematosus without antiphospholipid antibodies, each displayed specific miRNA profiles that were distinct from those of APS patients. In vitro, antiphospholipid antibodies of IgG isotype promoted deregulation in selected miRNAs and their potential atherothrombotic protein targets in monocytes and endothelial cells. Taken together, differentially expressed circulating miRNAs in APS patients, modulated at least partially by antiphospholipid antibodies of IgG isotype, might have the potential to serve as novel biomarkers of disease features and to typify patients’ atherothrombotic status, thus constituting a useful tool in the management of the disease. PMID:29545345

IL-17A Mediates a Selective Gene Expression Profile in Asthmatic Human Airway Smooth Muscle Cells

PubMed Central

Dragon, Stéphane; Hirst, Stuart J.; Lee, Tak H.

2014-01-01

Airway smooth muscle (ASM) cells are thought to contribute to the pathogenesis of allergic asthma by orchestrating and perpetuating airway inflammation and remodeling responses. In this study, we evaluated the IL-17RA signal transduction and gene expression profile in ASM cells from subjects with mild asthma and healthy individuals. Human primary ASM cells were treated with IL-17A and probed by the Affymetrix GeneChip array, and gene targets were validated by real-time quantitative RT-PCR. Genomic analysis underlined the proinflammatory nature of IL-17A, as multiple NF-κB regulatory factors and chemokines were induced in ASM cells. Transcriptional regulators consisting of primary response genes were overrepresented and displayed dynamic expression profiles. IL-17A poorly enhanced IL-1β or IL-22 gene responses in ASM cells from both subjects with mild asthma and healthy donors. Interestingly, protein modifications to the NF-κB regulatory network were not observed after IL-17A stimulation, although oscillations in IκBε expression were detected. ASM cells from subjects with mild asthma up-regulated more genes with greater overall variability in response to IL-17A than from healthy donors. Finally, in response to IL-17A, ASM cells displayed rapid activation of the extracellular signal–regulated kinase/ribosomal S6 kinase signaling pathway and increased nuclear levels of phosphorylated extracellular signal–regulated kinase. Taken together, our results suggest that IL-17A mediated modest gene expression response, which, in cooperation with the NF-κB signaling network, may regulate the gene expression profile in ASM cells. PMID:24393021

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.

Isolating cells from female/male blood mixtures using florescence in situ hybridization combined with low volume PCR and its application in forensic science.

PubMed

Feng, Lei; Li, Cai-Xia; Han, Jun-Ping; Xu, Cheng; Hu, Lan

2015-11-01

To obtain single-source short tandem repeat (STR) profiles in trace female/male blood mixture samples, we combined florescence in situ hybridization (FISH), laser microdissection, and low volume PCR (LV-PCR) to isolate male/female cells and improve sensitivity. The results showed that isolation of as few as 10 leukocytes was sufficient to yield full STR profiles in fresh female or male blood samples for 32 independent tests with a low additional alleles rate (3.91%) and drop-out alleles rate (5.01%). Moreover, this procedure was tested in two fresh blood mixture series at three ratios (1:5, 1:10, and 1:20), two mock female/male blood mixture casework samples, and one practical casework sample. Male and female STR profiles were successfully detected in all of these samples, showing that this procedure could be used in forensic casework in the future.

Identification of Three Kinds of Citri Reticulatae Pericarpium Based on Deoxyribonucleic Acid Barcoding and High-performance Liquid Chromatography-diode Array Detection-electrospray Ionization/Mass Spectrometry/Mass Spectrometry Combined with Chemometric Analysis

PubMed Central

Yu, Xiaoxue; Zhang, Yafeng; Wang, Dongmei; Jiang, Lin; Xu, Xinjun

2018-01-01

Background: Citri Reticulatae Pericarpium is the dried mature pericarp of Citrus reticulata Blanco which can be divided into “Chenpi” and “Guangchenpi.” “Guangchenpi” is the genuine Chinese medicinal material in Xinhui, Guangdong province; based on the greatest quality and least amount, it is most expensive among others. Hesperidin is used as the marker to identify Citri Reticulatae Pericarpium described in the Chinese Pharmacopoeia 2010. However, both “Chenpi” and “Guangchenpi” contain hesperidin so that it is impossible to differentiate them by measuring hesperidin. Objective: Our study aims to develop an efficient and accurate method to separate and identify “Guangchenpi” from other Citri Reticulatae Pericarpium. Materials and Methods: The genomic deoxyribonucleic acid (DNA) of all the materials was extracted and then the internal transcribed spacer 2 was amplified, sequenced, aligned, and analyzed. The secondary structures were created in terms of the database and website established by Jörg Schultz et al. High-performance liquid chromatography-diode array detection-electrospray Ionization/mass spectrometry (HPLC-DAD-ESI-MS)/MS coupled with chemometric analysis was applied to compare the differences in chemical profiles of the three kinds of Citri Reticulatae Pericarpium. Results: A total of 22 samples were classified into three groups. The results of DNA barcoding were in accordance with principal component analysis and hierarchical cluster analysis. Eight compounds were deduced from HPLC-DAD-ESI-MS/MS. Conclusions: This method is a reliable and effective tool to differentiate the three Citri Reticulatae Pericarpium. SUMMARY The internal transcribed spacer 2 regions and the secondary structure among three kinds of Citri Reticulatae Pericarpium varied considerablyAll the 22 samples were analyzed by high-performance liquid chromatography (HPLC) to obtain the chemical profilesPrincipal component analysis and hierarchical cluster analysis were used in the chemometric analysisdeoxyribonucleic acid barcoding and HPLC-diode array detection-electrospray ionization/mass spectrometry/MS coupled with chemometric analysis provided an accurate and strong proof to identify these three herbs. Abbreviations used: CTAB: Hexadecyltrimethylammonium bromide, DNA: Deoxyribonucleic acid, ITS2: Internal transcribed spacer 2, PCR: Polymerase chain reaction. PMID:29576703

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.

Integration Profile and Safety of an Adenovirus Hybrid-Vector Utilizing Hyperactive Sleeping Beauty Transposase for Somatic Integration

PubMed Central

Zhang, Wenli; Muck-Hausl, Martin; Wang, Jichang; Sun, Chuanbo; Gebbing, Maren; Miskey, Csaba; Ivics, Zoltan; Izsvak, Zsuzsanna; Ehrhardt, Anja

2013-01-01

We recently developed adenovirus/transposase hybrid-vectors utilizing the previously described hyperactive Sleeping Beauty (SB) transposase HSB5 for somatic integration and we could show stabilized transgene expression in mice and a canine model for hemophilia B. However, the safety profile of these hybrid-vectors with respect to vector dose and genotoxicity remains to be investigated. Herein, we evaluated this hybrid-vector system in C57Bl/6 mice with escalating vector dose settings. We found that in all mice which received the hyperactive SB transposase, transgene expression levels were stabilized in a dose-dependent manner and that the highest vector dose was accompanied by fatalities in mice. To analyze potential genotoxic side-effects due to somatic integration into host chromosomes, we performed a genome-wide integration site analysis using linker-mediated PCR (LM-PCR) and linear amplification-mediated PCR (LAM-PCR). Analysis of genomic DNA samples obtained from HSB5 treated female and male mice revealed a total of 1327 unique transposition events. Overall the chromosomal distribution pattern was close-to-random and we observed a random integration profile with respect to integration into gene and non-gene areas. Notably, when using the LM-PCR protocol, 27 extra-chromosomal integration events were identified, most likely caused by transposon excision and subsequent transposition into the delivered adenoviral vector genome. In total, this study provides a careful evaluation of the safety profile of adenovirus/Sleeping Beauty transposase hybrid-vectors. The obtained information will be useful when designing future preclinical studies utilizing hybrid-vectors in small and large animal models. PMID:24124483

Differential expression of miRNAs in the seminal plasma and serum of testicular cancer patients.

PubMed

Pelloni, Marianna; Coltrinari, Giulia; Paoli, Donatella; Pallotti, Francesco; Lombardo, Francesco; Lenzi, Andrea; Gandini, Loredana

2017-09-01

Various microRNAs from the miR-371-3 and miR-302a-d clusters have recently been proposed as markers for testicular germ cell tumours. Upregulation of these miRNAs has been found in both the tissue and serum of testicular cancer patients, but they have never been studied in human seminal plasma. The aim of this study was, therefore, to assess the differences in the expression of miR-371-3 and miR-302a-d between the seminal plasma and serum of testicular cancer patients, and to identify new potential testicular cancer markers in seminal plasma. We investigated the serum and seminal plasma of 28 pre-orchiectomy patients subsequently diagnosed with testicular cancer, the seminal plasma of another 20 patients 30 days post-orchiectomy and a control group consisting of 28 cancer-free subjects attending our centre for an andrological check-up. Serum microRNA expression was analysed using RT-qPCR. TaqMan Array Card 3.0 platform was used for microRNA profiling in the seminal plasma of cancer patients. Results for both miR-371-3 and the miR-302 cluster in the serum of testicular cancer patients were in line with literature reports, while miR-371and miR-372 expression in seminal plasma showed the opposite trend to serum. On array analysis, 37 miRNAs were differentially expressed in the seminal plasma of cancer patients, and the upregulated miR-142 and the downregulated miR-34b were validated using RT-qPCR. Our study investigated the expression of miRNAs in the seminal plasma of patients with testicular cancer for the first time. Unlike in serum, miR-371-3 cannot be considered as markers in seminal plasma, whereas miR-142 levels in seminal plasma may be a potential marker for testicular cancer.

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

PubMed Central

2013-01-01

Background 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. Methods 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. Results 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. In addition, we investigated BET role on myoblasts proliferation and differentiation. During proliferation, BET did not modify C2C12 proliferative rate, but promoted myogenic induction, enhancing MyoD protein content and cellular elongation. During differentiation, BET caused an increase of muscle-specific markers and IGF-1 R protein levels. Conclusions 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. PMID:23870626

Functional convergence of Akt protein with VEGFR-1 in human endothelial progenitor cells exposed to sera from patient with type 2 diabetes mellitus.

PubMed

Hassanpour, Mehdi; Rezabakhsh, Aysa; Rahbarghazi, Reza; Nourazarian, Alireza; Nouri, Mohammad; Avci, Çığır Biray; Ghaderi, Shahrooz; Alidadyani, Neda; Bagca, Bakiye Goker; Bagheri, Hesam Saghaei

2017-11-01

Diabetes mellitus type 2 predisposes patients to various microvascular complications. In the current experiment, the potent role of diabetes mellitus was investigated on the content of VEGFR-1, -2, Tie-1 and -2, and Akt in human endothelial progenitor cells. The gene expression profile of mTOR and Hedgehog signaling pathways were measured by PCR array. The possible crosstalk between RTKs, mTOR and Hedgehog signaling was also studied by bioinformatic analysis. Endothelial progenitor cells were incubated with serum from normal and diabetic for 7days. Compared to non-treated cells, diabetic serum-induced cell apoptosis (~2-fold) and prohibited cell migration toward bFGF (p<0.001). ELISA analysis showed that diabetes exposed cells had increased abundance of Tie-1, -2 and VEGFR-2 and reduced amount of VEGFR-1 (p<0.0001) in diabetic cells. Western blotting showed a marked reduction in the protein level of Akt after cells exposure to serum from diabetic subjects (p<0.0001). PCR array revealed a significant stimulation of both mTOR and Hedgehog signaling pathways in diabetic cells (p<0.05). According to data from bioinformatic datasets, we showed VEGFR-1, -2 and Tie-2, but not Tie-1, are master regulators of angiogenesis. There is a crosstalk between RTKs and mTOR signaling by involving P62, GABARAPL1, and HTT genes. It seems that physical interaction and co-expression of Akt decreased the level of VEGFR-1 in diabetic cells. Regarding data from the present experiment, diabetic serum contributed to uncontrolled induction of both mTOR and Hedgehog signaling in endothelial progenitor cells. Diabetes mellitus induces mTOR pathway by involving receptor tyrosine kinases while Hedgehog stimulation is independent of these receptors. Copyright © 2017 Elsevier Inc. All rights reserved.

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

Phased Array Theory and Technology

DTIC Science & Technology

1981-07-01

Generalized Array Coordinates 2. Linear, Planar and Circular Art -ays 3. Periodic fwo Dimensional ^rras 4. Grating Lobe Lattices 5. 1’llenienl...formal and low profile antennas, antennas for limited sector coverage, and wide- band array feeds. To aid designers, there is an attempt to give ...ol Vol. 2, Elliott gives convenient formulas lor the directivity of Imear dipole arrays, and derives an especially simple form tor arrays

Evaluation of LSSP-PCR for identification of Leptospira spp. in urine samples of cattle with clinical suspicion of leptospirosis.

PubMed

Bomfim, Maria Rosa Quaresma; Koury, Matilde Cota

2006-12-20

We evaluated the use of low-stringency single specific primer PCR (LSSP-PCR) for genetically typing Leptospira directly from urine samples of cattle with clinical suspicion of leptospirosis. Urine samples obtained from 40 cattle with clinical suspicion of leptospirosis were amplified by specific PCR using the following primers: Internal 1/Internal 2 and G1/G2. The internal primers were designed from the gene sequence of the outer membrane lipoprotein Lip32 from Leptospira kirschneri, strain RM52. The PCR products were amplified with these two pairs of primers, which had approximately 497 and 285bp, respectively, and were subsequently used as a template for LSSP-PCR analysis. The genetic signatures from the leptospires which were present in the urine samples allowed us to make a preliminary identification of the leptospires by comparing the LSSP-PCR profiles obtained directly from urine samples with those from reference leptospires. The LSSP-PCR profiles obtained with the Internal 1 primer or with the G1 primer allowed the grouping of the leptospires into serogroups. LSSP-PCR was found to be a useful and sensitive approach capable of identifying leptospires directly from biological samples without the need for prior bacterial isolation. In conclusion, the LSSP-PCR technique may still be helpful in discriminating serogroups of Leptospira from different animal reservoirs, since the early identification of carrier animals and information on the shedding state are crucial to prevent the spread of leptospiral infection to other animals and humans.

The effects of DRIE operational parameters on vertically aligned micropillar arrays

NASA Astrophysics Data System (ADS)

Miller, Kane; Li, Mingxiao; Walsh, Kevin M.; Fu, Xiao-An

2013-03-01

Vertically aligned silicon micropillar arrays have been created by deep reactive ion etching (DRIE) and used for a number of microfabricated devices including microfluidic devices, micropreconcentrators and photovoltaic cells. This paper delineates an experimental design performed on the Bosch process of DRIE of micropillar arrays. The arrays are fabricated with direct-write optical lithography without photomask, and the effects of DRIE process parameters, including etch cycle time, passivation cycle time, platen power and coil power on profile angle, scallop depth and scallop peak-to-peak distance are studied by statistical design of experiments. Scanning electron microscope images are used for measuring the resultant profile angles and characterizing the scalloping effect on the pillar sidewalls. The experimental results indicate the effects of the determining factors, etch cycle time, passivation cycle time and platen power, on the micropillar profile angles and scallop depths. An optimized DRIE process recipe for creating nearly 90° and smooth surface (invisible scalloping) has been obtained as a result of the statistical design of experiments.

Attitude maneuvers of a solar-powered electric orbital transfer vehicle

NASA Astrophysics Data System (ADS)

Jenkin, Alan B.

1992-08-01

Attitude maneuver requirements of a solar-powered electric orbital transfer vehicle have been studied in detail. This involved evaluation of the yaw, pitch, and roll profiles and associated angular accelerations needed to simultaneously steer the vehicle thrust vector and maintain the solar array pointed toward the sun. Maintaining the solar array pointed exactly at the sun leads to snap roll maneuvers which have very high (theoretically unbounded) accelerations, thereby imposing large torque requirements. The problem is exacerbated by the large solar arrays which are needed to generate the high levels of power needed by electric propulsion devices. A method of eliminating the snap roll maneuvers is presented. The method involves the determination of relaxed roll profiles which approximate a forced transition between alternate exact roll profiles and incur only small errors in solar array pointing. The method makes it feasible to perform the required maneuvers using currently available attitude control technology such as reaction wheels, hot gas jets, or gimballed main engines.

Selection and Validation of Reference Genes for qRT-PCR Expression Analysis of Candidate Genes Involved in Olfactory Communication in the Butterfly Bicyclus anynana

PubMed Central

Arun, Alok; Baumlé, Véronique; Amelot, Gaël; Nieberding, Caroline M.

2015-01-01

Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera) have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae), two developmental stages (pupal and adult) and two sexes (male and female), all of which were subjected to two food treatments (food stress and control feeding ad libitum). The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm. Both methods identified RpS8 as the best suitable reference gene for expression data normalization. We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana. Finally, we tested the effect of choosing reference genes with different stability on the normalization of the transcript abundance of a candidate gene involved in olfactory communication in B. anynana, the Fatty Acyl Reductase 2, and we confirmed that using an unstable reference gene can drastically alter the expression profile of the target candidate genes. PMID:25793735

Selection and validation of reference genes for qRT-PCR expression analysis of candidate genes involved in olfactory communication in the butterfly Bicyclus anynana.

PubMed

Arun, Alok; Baumlé, Véronique; Amelot, Gaël; Nieberding, Caroline M

2015-01-01

Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera) have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae), two developmental stages (pupal and adult) and two sexes (male and female), all of which were subjected to two food treatments (food stress and control feeding ad libitum). The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm. Both methods identified RpS8 as the best suitable reference gene for expression data normalization. We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana. Finally, we tested the effect of choosing reference genes with different stability on the normalization of the transcript abundance of a candidate gene involved in olfactory communication in B. anynana, the Fatty Acyl Reductase 2, and we confirmed that using an unstable reference gene can drastically alter the expression profile of the target candidate genes.

Tapered microelectrode array system for dielectrophoretically filtration: fabrication, characterization, and simulation study

NASA Astrophysics Data System (ADS)

Buyong, Muhamad Ramdzan; Larki, Farhad; Takamura, Yuzuru; Majlis, Burhanuddin Yeop

2017-10-01

This paper presents the fabrication, characterization, and simulation of microelectrode arrays system with tapered profile having an aluminum surface for dielectrophoresis (DEP)-based manipulation of particles. The proposed structure demonstrates more effective electric field gradient compared with its counterpart with untapered profile. Therefore, according to the asymmetric distribution of the electric field in the active region of microelectrode, it produces more effective particle manipulation. The tapered aluminum microelectrode array (TAMA) fabrication process uses a state-of-the-art technique in the formation of the resist's taper profile. The performance of TAMA with various sidewall profile angles (5 deg to 90 deg) was analyzed through finite-element method numerical simulations to offer a better understanding of the origin of the sidewall profile effect. The ability of capturing and manipulating of the device was examined through modification of the Clausius-Mossotti factor and cross-over frequency (f). The fabricated system has been particularly implemented for filtration of particles with a desired diameter from a mixture of particles with three different diameters in an aqueous medium. The microelectrode system with tapered side wall profile offers a more efficient platform for particle manipulation and sensing applications compared with the conventional microelectrode systems.

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.

High-resolution melt PCR analysis for genotyping of Ureaplasma parvum isolates directly from clinical samples.

PubMed

Payne, Matthew S; Tabone, Tania; Kemp, Matthew W; Keelan, Jeffrey A; Spiller, O Brad; Newnham, John P

2014-02-01

Ureaplasma sp. infection in neonates and adults underlies a variety of disease pathologies. Of the two human Ureaplasma spp., Ureaplasma parvum is clinically the most common. We have developed a high-resolution melt (HRM) PCR assay for the differentiation of the four serovars of U. parvum in a single step. Currently U. parvum strains are separated into four serovars by sequencing the promoter and coding region of the multiple-banded antigen (MBA) gene. We designed primers to conserved sequences within this region for PCR amplification and HRM analysis to generate reproducible and distinct melt profiles that distinguish clonal representatives of serovars 1, 3, 6, and 14. Furthermore, our HRM PCR assay could classify DNA extracted from 74 known (MBA-sequenced) test strains with 100% accuracy. Importantly, HRM PCR was also able to identify U. parvum serovars directly from 16 clinical swabs. HRM PCR performed with DNA consisting of mixtures of combined known serovars yielded profiles that were easily distinguished from those for single-serovar controls. These profiles mirrored clinical samples that contained mixed serovars. Unfortunately, melt curve analysis software is not yet robust enough to identify the composition of mixed serovar samples, only that more than one serovar is present. HRM PCR provides a single-step, rapid, cost-effective means to differentiate the four serovars of U. parvum that did not amplify any of the known 10 serovars of Ureaplasma urealyticum tested in parallel. Choice of reaction reagents was found to be crucial to allow sufficient sensitivity to differentiate U. parvum serovars directly from clinical swabs rather than requiring cell enrichment using microbial culture techniques.

Expression of stress/defense-related genes in barley grown under space environment

NASA Astrophysics Data System (ADS)

Sugimoto, Manabu; Shagimardanova, Elena; Gusev, Oleg; Bingham, Gail; Levinskikh, Margarita; Sychev, Vladimir

Plants are exposed to the extreme environment in space, especially space radiation is suspected to induce oxidative stress by generating high-energy free radicals and microgravity would enhance the effect of space radiation, however, current understandings of plant growth and responses on this synergistic effect of radiation and microgravity is limited to a few experiments. In this study, expression of stress/defense-related genes in barley grown under space environment was analyzed by RT-PCR and DNA microarray experiments to understand plant responses and adaptation to space environment and to develop the space stress-tolerant plants. The seeds of barley, Hordeum vulgare L. cv. Haruna nijo, kept in the international space station (ISS) over 4 months, were germinated after 3 days of irrigation in LADA plant growth chamber onboard Russian segment of ISS and the final germination ratio was over 90 %. The height of plants was about 50 to 60 cm and flag leaf has been opened after 26 days of irrigation under 24 hr lighting, showing the similar growth to ground-grown barley. Expression levels of stress/defense-related genes in space-grown barley were compared to those in ground-grown barley by semi-quantitative RT-PCR. In 17 stress/defense-related genes that are up-regulated by oxidative stress or other abiotic stress, only catalase, pathogenesis-related protein 13, chalcone synthase, and phenylalanine ammonia-lyase genes were increased in space-grown barley. DNA microarrya analysis with the GeneChip Barley Genome Array showed the similar expression profiles of the stress/defense-related genes to those by RT-PCR experiment, suggesting that the barley germinated and grown in LADA onboard ISS is not damaged by space environment, especially oxidative stress induced by space radiation and microgravity.

The genomic transcriptional response of female fathead minnows (Pimephales promelas) to an acute exposure to the androgen, 17β-trenbolone

USGS Publications Warehouse

Dorts, Jennifer; Richter, Catherine A.; Wright-Osment, Maureen K.; Ellersieck, Mark R.; Carter, Barbara J.; Tillitt, Donald E.

2009-01-01

We investigated the genomic transcriptional response of female fathead minnows (Pimephales promelas) to an acute (4 days) exposure to 0.1 or 1.0 ??g/L of 17??-trenbolone (TB), the active metabolite of an anabolic androgenic steroid used as a growth promoter in cattle and a contaminant of concern in aquatic systems. Our objectives were to investigate the gene expression profile induced by TB, define biomarkers of exposure to TB, and increase our understanding of the mechanisms of adverse effects of TB on fish reproduction. In female gonad tissue, microarray analysis using a 22 K oligonucleotide microarray (EcoArray Inc., Gainesville, FL) showed 99 significantly upregulated genes and 741 significantly downregulated genes in response to 1 ??g TB/L. In particular, hydroxysteroid (17??) dehydrogenase 12a (hsd17b12a), zona pellucida glycoprotein 2.2 (zp2.2), and protein inhibitor of activated STAT, 2 (pias2) were all downregulated in gonad. Q-PCR measurements in a larger sample set were consistent with the microarray results in the direction and magnitude of these changes in gene expression. However, several novel potential biomarkers were verified by Q-PCR in the same samples, but could not be validated in independent samples. In liver, Q-PCR measurements showed a significant decrease in vitellogenin 1 (vtg1) mRNA expression. In brain, cytochrome P450, family 19, subfamily A, polypeptide 1b (cyp19a1b, previously known as aromatase B) transcript levels were significantly reduced following TB exposure. Our study provides a candidate gene involved in mediating the action of TB, hsd17b12a, and two potential biomarkers sensitive to acute TB exposure, hepatic vtg1 and brain cyp19a1b.

Prevalence and antimicrobial susceptibility of Salmonella isolated from a variety of raw meat sausages in Gaborone (Botswana) retail stores.

PubMed

Samaxa, Ronald Gaelekolwe; Matsheka, Maitshwarelo Ignatius; Mpoloka, Sununguko Wata; Gashe, Berhanu Abegaz

2012-04-01

The objective of the study was to provide baseline data on the prevalence and antimicrobial susceptibility of Salmonella in different types of raw meat sausages directly accessible to the consumers in Gaborone, Botswana. A total of 300 raw sausages comprising 79 beef, 78 pork, 72 chicken, and 71 mutton samples were concurrently analyzed for the presence of Salmonella using a conventional culture method and a validated PCR method. The PCR assay results were in full concordance with those of the conventional culture method for the detection of Salmonella. Sixty-five (21.7%) of 300 samples were positive for Salmonella by both the conventional culture method and PCR assay. Even though more chicken samples contained Salmonella than did any other sausage type, the difference in the presence of Salmonella among the four sausages types was not significant. Eleven serotypes were identified, and Salmonella enterica subsp. salamae II was most prevalent in all the sausage types. Beef sausages generally had higher mesophilic bacterial counts than did the other three sausage types. However, higher microbial counts were not reflective of the presence of salmonellae. Susceptibility of the Salmonella enterica serotypes to 20 antimicrobial agents was determined, and Salmonella Muenchen was resistant to the widest array of agents and was mostly isolated from chicken sausages. Regardless of the meat of origin, all 65 Salmonella isolates were resistant to at least four antimicrobial agents: amikacin, gentamicin, cefuroxime, and tombramycin. This resistance profile group was the most common in all four sausage types, comprising 90% of all Salmonella isolates from beef, 71% from pork, 63% from mutton, and 35% from chicken. These results suggest that raw sausages pose a risk of transmitting multidrug-resistant Salmonella isolates to consumers.

Indirubin-3′-(2,3 dihydroxypropyl)-oximether (E804) is a potent modulator of LPS-stimulated macrophage functions

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

Babcock, Abigail S.; Anderson, Amy L.; Graduate Program in Environmental Toxicology, Clemson University, Clemson, SC 29634

2013-01-01

Indirubin is a deep-red bis-indole isomer of indigo blue, both of which are biologically active ingredients in Danggui Longhui Wan, an ancient Chinese herbal tea mixture used to treat neoplasia and chronic inflammation and to enhance detoxification of xenobiotics. Multiple indirubin derivatives have been synthesized and shown to inhibit cyclin-dependent kinases (CDKs) and glycogen-synthase kinase (GSK-3β) with varying degrees of potency. Several indirubins are also aryl hydrocarbon receptor (AhR) agonists, with AhR-associated activities covering a wide range of potencies, depending on molecular structure. This study examined the effects of indirubin-3′-(2,3 dihydroxypropyl)-oximether (E804), a novel indirubin with potent STAT3 inhibitory properties,more » on basal and LPS-inducible activities in murine RAW264.7 macrophages. Using a focused commercial qRT-PCR array platform (SuperArray®), the effects of E804 on expression of a suite of genes associated with stress and toxicity were determined. Most genes up-regulated by LPS treatment were suppressed by E804; including LPS-induced expression of pro-inflammatory cytokines and receptors, apoptosis control genes, and oxidative stress response genes. Using qRT-PCR as a follow up to the commercial arrays, E804 treatment suppressed LPS-induced COX-2, iNOS, IL-6 and IL-10 gene expression, though the effects on iNOS and COX-2 protein expression were less dramatic. E804 also inhibited LPS-induced secretion of IL-6 and IL-10. Functional endpoints, including iNOS and lysozyme enzymatic activity, phagocytosis of fluorescent latex beads, and intracellular killing of bacteria, were also examined, and in each experimental condition E804 suppressed activities. Collectively, these results indicate that E804 is a potent modulator of pro-inflammatory profiles in LPS-treated macrophages. -- Highlights: ► RAW 264.7 macrophages were treated with 1 μM Indirubin E804, 1 μg/ml LPS, or both. ► E804 suppresses LPS-induced expression of iNOS, IL-6, COX-2, and IL-10. ► E804 suppresses LPS-induced iNOS and lysozyme activity, and IL-6 and IL-10 secretion. ► E804 suppressed phagocytosis and intracellular killing by macrophages.« less

A Practical Platform for Blood Biomarker Study by Using Global Gene Expression Profiling of Peripheral Whole Blood

PubMed Central

Schmid, Patrick; Yao, Hui; Galdzicki, Michal; Berger, Bonnie; Wu, Erxi; Kohane, Isaac S.

2009-01-01

Background Although microarray technology has become the most common method for studying global gene expression, a plethora of technical factors across the experiment contribute to the variable of genome gene expression profiling using peripheral whole blood. A practical platform needs to be established in order to obtain reliable and reproducible data to meet clinical requirements for biomarker study. Methods and Findings We applied peripheral whole blood samples with globin reduction and performed genome-wide transcriptome analysis using Illumina BeadChips. Real-time PCR was subsequently used to evaluate the quality of array data and elucidate the mode in which hemoglobin interferes in gene expression profiling. We demonstrated that, when applied in the context of standard microarray processing procedures, globin reduction results in a consistent and significant increase in the quality of beadarray data. When compared to their pre-globin reduction counterparts, post-globin reduction samples show improved detection statistics, lowered variance and increased sensitivity. More importantly, gender gene separation is remarkably clearer in post-globin reduction samples than in pre-globin reduction samples. Our study suggests that the poor data obtained from pre-globin reduction samples is the result of the high concentration of hemoglobin derived from red blood cells either interfering with target mRNA binding or giving the pseudo binding background signal. Conclusion We therefore recommend the combination of performing globin mRNA reduction in peripheral whole blood samples and hybridizing on Illumina BeadChips as the practical approach for biomarker study. PMID:19381341

Gene-expression profiles of epithelial cells treated with EMD in vitro: analysis using complementary DNA arrays.

PubMed

Kapferer, I; Schmidt, S; Gstir, R; Durstberger, G; Huber, L A; Vietor, I

2011-02-01

During surgical periodontal treatment, EMD is topically applied in order to facilitate regeneration of the periodontal ligament, acellular cementum and alveolar bone. Suppresion of epithelial down-growth is essential for successful periodontal regeneration; however, the underlying mechanisms of how EMD influences epithelial wound healing are poorly understood. In the present study, the effects of EMD on gene-expression profiling in an epithelial cell line (HSC-2) model were investigated. Gene-expression modifications, determined using a comparative genome-wide expression-profiling strategy, were independently validated by quantitative real-time RT-PCR. Additionally, cell cycle, cell growth and in vitro wound-healing assays were conducted. A set of 43 EMD-regulated genes was defined, which may be responsible for the reduced epithelial down-growth upon EMD application. Gene ontology analysis revealed genes that could be attributed to pathways of locomotion, developmental processes and associated processes such as regulation of cell size and cell growth. Additionally, eight regulated genes have previously been reported to take part in the process of epithelial-to-mesenchymal transition. Several independent experimental assays revealed significant inhibition of cell migration, growth and cell cycle by EMD. The set of EMD-regulated genes identified in this study offers the opportunity to clarify mechanisms underlying the effects of EMD on epithelial cells. Reduced epithelial repopulation of the dental root upon periodontal surgery may be the consequence of reduced migration and cell growth, as well as epithelial-to-mesenchymal transition. © 2010 John Wiley & Sons A/S.

Next-Generation Sequencing Combined with Specific PCR Assays To Determine the Bacterial 16S rRNA Gene Profiles of Middle Ear Fluid Collected from Children with Acute Otitis Media

PubMed Central

Kramna, Lenka; Oikarinen, Sami; Sipilä, Markku; Rautiainen, Markus; Aittoniemi, Janne; Laranne, Jussi; Hyöty, Heikki; Cinek, Ondrej

2017-01-01

ABSTRACT The aim of the study was to analyze the bacteriome of acute otitis media with a novel modification of next-generation sequencing techniques. Outpatient children with acute otitis media were enrolled in the study, and middle ear fluids were collected during 90 episodes from 79 subjects aged 5 to 42 months (median age, 19 months). The bacteriome profiles of middle ear fluid samples were determined by a nested-PCR amplification of the 16S rRNA gene (V4 region), followed by mass sequencing. The profiling results were compared to the results of specific PCR assays targeting selected prevalent pathogens. Bacteriome profiling using nested amplification of low-volume samples was aided by a bioinformatic subtraction of signal contaminants from the recombinant polymerase, achieving a sensitivity slightly lower than that of specific PCR detection. Streptococcus pneumoniae was detected in 28 (31%) samples, Haemophilus influenzae in 24 (27%), Moraxella catarrhalis in 18 (20%), Staphylococcus spp. in 21 (23%), Turicella otitidis in 5 (5.6%), Alloiococcus otitidis in 3 (3.3%), and other bacteria in 14 (16%) using bacteriome profiling. S. pneumoniae was the dominant pathogen in 14 (16%) samples, H. influenzae in 15 (17%), M. catarrhalis in 5 (5.6%), T. otitidis in 2, and Staphylococcus auricularis in 2. Weaker signals of Prevotella melaninogenica, Veillonella dispar, and Veillonella montpellierensis were noted in several samples. Fourteen samples (16%) were not explainable by bacterial pathogens; novel causative agents were not detected. In conclusion, unbiased bacteriome profiling helped in depicting the true mutual quantitative ratios of ear bacteria, but at present, its complicated protocol impedes its routine clinical use. IMPORTANCE Although S. pneumoniae, H. influenzae, and M. catarrhalis have been long established as the most important pathogens in acute otitis media using culture and specific PCR assays, the knowledge of their mutual quantitative relations and possible roles of other bacteria is incomplete. The advent of unbiased bacteriome 16S rRNA gene profiling has allowed the detection of nearly all bacteria present in the sample, and it helps in depicting their mutual quantitative ratios. Due to the difficulties in performing mass sequencing in low-volume samples, only a few bacteriome-profiling studies of otitis media have been published, all limited to cases of chronic otitis media. Here, we present a study on samples obtained from young children with acute otitis media, successfully using a strategy of nested PCR coupled with mass sequencing, and demonstrate that the method can confer quantitative information hardly obtainable by other methods. PMID:28357413

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

Large-Scale Mapping and Validation of Escherichia coli Transcriptional Regulation from a Compendium of Expression Profiles

PubMed Central

Thaden, Joshua T; Mogno, Ilaria; Wierzbowski, Jamey; Cottarel, Guillaume; Kasif, Simon; Collins, James J; Gardner, Timothy S

2007-01-01

Machine learning approaches offer the potential to systematically identify transcriptional regulatory interactions from a compendium of microarray expression profiles. However, experimental validation of the performance of these methods at the genome scale has remained elusive. Here we assess the global performance of four existing classes of inference algorithms using 445 Escherichia coli Affymetrix arrays and 3,216 known E. coli regulatory interactions from RegulonDB. We also developed and applied the context likelihood of relatedness (CLR) algorithm, a novel extension of the relevance networks class of algorithms. CLR demonstrates an average precision gain of 36% relative to the next-best performing algorithm. At a 60% true positive rate, CLR identifies 1,079 regulatory interactions, of which 338 were in the previously known network and 741 were novel predictions. We tested the predicted interactions for three transcription factors with chromatin immunoprecipitation, confirming 21 novel interactions and verifying our RegulonDB-based performance estimates. CLR also identified a regulatory link providing central metabolic control of iron transport, which we confirmed with real-time quantitative PCR. The compendium of expression data compiled in this study, coupled with RegulonDB, provides a valuable model system for further improvement of network inference algorithms using experimental data. PMID:17214507

Genome-Wide Requirements for Resistance to Functionally Distinct DNA-Damaging Agents

PubMed Central

Proctor, Michael; Flaherty, Patrick; Jordan, Michael I; Arkin, Adam P; Davis, Ronald W; Nislow, Corey; Giaever, Guri

2005-01-01

The mechanistic and therapeutic differences in the cellular response to DNA-damaging compounds are not completely understood, despite intense study. To expand our knowledge of DNA damage, we assayed the effects of 12 closely related DNA-damaging agents on the complete pool of ~4,700 barcoded homozygous deletion strains of Saccharomyces cerevisiae. In our protocol, deletion strains are pooled together and grown competitively in the presence of compound. Relative strain sensitivity is determined by hybridization of PCR-amplified barcodes to an oligonucleotide array carrying the barcode complements. These screens identified genes in well-characterized DNA-damage-response pathways as well as genes whose role in the DNA-damage response had not been previously established. High-throughput individual growth analysis was used to independently confirm microarray results. Each compound produced a unique genome-wide profile. Analysis of these data allowed us to determine the relative importance of DNA-repair modules for resistance to each of the 12 profiled compounds. Clustering the data for 12 distinct compounds uncovered both known and novel functional interactions that comprise the DNA-damage response and allowed us to define the genetic determinants required for repair of interstrand cross-links. Further genetic analysis allowed determination of epistasis for one of these functional groups. PMID:16121259

The placental immune milieu is characterized by a Th2- and anti-inflammatory transcription profile, regardless of maternal allergy, and associates with neonatal immunity.

PubMed

Abelius, Martina S; Janefjord, Camilla; Ernerudh, Jan; Berg, Göran; Matthiesen, Leif; Duchén, Karel; Nilsson, Lennart J; Jenmalm, Maria C

2015-05-01

How maternal allergy affects the systemic and local immunological environment during pregnancy and the immune development of the offspring is unclear. Expression of 40 genes was quantified by PCR arrays in placenta, peripheral blood mononuclear cells (PBMC), and cord blood mononuclear cells (CBMC) from 7 allergic and 12 non-allergic women and their offspring. Placental gene expression was dominated by a Th2-/anti-inflammatory profile, irrespectively of maternal allergy, as compared to gene expression in PBMC. p35 expression in placenta correlated with fetal Tbx21 (ρ = -0.88, P < 0.001) and IL-5 expression in PBMC with fetal galectin1 (ρ = 0.91, P < 0.001). Increased expression of Th2-associated CCL22 in CBMC preceded allergy development. Gene expression locally and systemically during pregnancy was partly associated with the offspring's gene expression, possibly indicating that the immunological milieu is important for fetal immune development. Maternal allergy was not associated with an enhanced Th2 immunity in placenta or PBMC, while a marked prenatal Th2 skewing, shown as increased CCL22 mRNA expression, might contribute to postnatal allergy development. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Gene expression profiles reveal that DCN, DIO1, and DIO2 are underexpressed in benign and malignant thyroid tumors.

PubMed

Arnaldi, L A T; Borra, R C; Maciel, R M B; Cerutti, J M

2005-03-01

To investigate the molecular events involved in the pathogenesis and/or progression of thyroid tumors, we compared the gene expression profiles of three thyroid carcinoma cell lines, which represent major tumor subtypes of thyroid cancer and normal thyroid tissue. Using cDNA array methodology, we investigated the expression of 1807 open reading frame expressed sequence tags (ORESTES), selected from head and neck tumor libraries generated through the Brazilian Human Cancer Project-LICR/FAPESP. We found that 505 transcripts were differentially expressed in the thyroid carcinoma cell lines. Using a more stringent criterion, transcripts underexpressed or overexpressed more than fivefold in 1 of 3 or 3 of 3 carcinoma cell lines, a list of 55 ESTs were detected. Five candidate genes were further validated by quantitative polymerase chain reaction (qPCR) in an independent set of 52 thyroid tumors and 22 matched normal thyroid tissues. DCN was found underexpressed in a high percentage of the follicular thyroid adenomas, follicular thyroid carcinomas, and follicular variant of papillary thyroid carcinomas. DIO1 and DIO2 were underexpressed in nearly all papillary thyroid carcinomas. These genes not only could help to better define a tumor signature for thyroid tumors, but may, in part, also become useful as potential targets for thyroid tumor treatment.

Kidney Transplant Rejection and Tissue Injury by Gene Profiling of Biopsies and Peripheral Blood Lymphocytes

PubMed Central

Flechner, Stuart M.; Kurian, Sunil M.; Head, Steven R.; Sharp, Starlette M.; Whisenant, Thomas C.; Zhang, Jie; Chismar, Jeffrey D.; Horvath, Steve; Mondala, Tony; Gilmartin, Timothy; Cook, Daniel J.; Kay, Steven A.; Walker, John R.; Salomon, Daniel R.

2007-01-01

A major challenge for kidney transplantation is balancing the need for immunosuppression to prevent rejection, while minimizing drug-induced toxicities. We used DNA microarrays (HG-U95Av2 GeneChips, Affymetrix) to determine gene expression profiles for kidney biopsies and peripheral blood lymphocytes (PBLs) in transplant patients including normal donor kidneys, well-functioning transplants without rejection, kidneys undergoing acute rejection, and transplants with renal dysfunction without rejection. We developed a data analysis schema based on expression signal determination, class comparison and prediction, hierarchical clustering, statistical power analysis and real-time quantitative PCR validation. We identified distinct gene expression signatures for both biopsies and PBLs that correlated significantly with each of the different classes of transplant patients. This is the most complete report to date using commercial arrays to identify unique expression signatures in transplant biopsies distinguishing acute rejection, acute dysfunction without rejection and well-functioning transplants with no rejection history. We demonstrate for the first time the successful application of high density DNA chip analysis of PBL as a diagnostic tool for transplantation. The significance of these results, if validated in a multicenter prospective trial, would be the establishment of a metric based on gene expression signatures for monitoring the immune status and immunosuppression of transplanted patients. PMID:15307835

Apoptosis Induction and Gene Expression Profile Alterations of Cutaneous T-Cell Lymphoma Cells following Their Exposure to Bortezomib and Methotrexate

PubMed Central

Kontsioti, Frieda; Konsta, Eugene; Vikentiou, Miriam; Spathis, Aris; Papageorgiou, Sotiris; Vasilatou, Diamantina; Gkontopoulos, Konstantinos; Mpazani, Efthimia; Karakitsos, Petros; Rigopoulos, Dimitrios; Dimitriadis, George

2017-01-01

Mycosis fungoides (MF) and its leukemic variant Sézary syndrome (SS) comprise the majority of CTCL, a heterogenous group of non-Hodgkins lymphomas involving the skin. The CTCL’s resistance to chemotherapy and the lack of full understanding of their pathogenesis request further investigation. With the view of a more targeted therapy, we evaluated in vitro the effectiveness of bortezomib and methotrexate, as well as their combination in CTCL cell lines, regarding apoptosis induction. Our data are of clinical value and indicate that the bortezomib/methotrexate combinational therapy has an inferior impact on the apoptosis of CTCL compared to monotherapy, with bortezomib presenting as the most efficient treatment option for SS and methotrexate for MF. Using PCR arrays technology, we also investigated the alterations in the expression profile of genes related to DNA repair pathways in CTCL cell lines after treatment with bortezomib or methotrexate. We found that both agents, but mostly bortezomib, significantly deregulate a large number of genes in SS and MF cell lines, suggesting another pathway through which these agents could induce apoptosis in CTCL. Finally, we show that SS and MF respond differently to treatment, verifying their distinct nature and further emphasizing the need for discrete treatment approaches. PMID:28107479

Multi-channel infrared thermometer

DOEpatents

Ulrickson, Michael A.

1986-01-01

A device for measuring the two-dimensional temperature profile of a surface comprises imaging optics for generating an image of the light radiating from the surface; an infrared detector array having a plurality of detectors; and a light pipe array positioned between the imaging optics and the detector array for sampling, transmitting, and distributing the image over the detector surfaces. The light pipe array includes one light pipe for each detector in the detector array.

Regulation of inflammatory factors by double-stranded RNA receptors in breast cancer cells.

PubMed

Venkatesh, Amritha; Nandigam, Harika; Muccioli, Maria; Singh, Manindra; Loftus, Tiffany; Lewis, Deana; Pate, Michelle; Benencia, Fabian

Malignant cells are not the only components of a tumor mass since other cells (e.g., fibroblasts, infiltrating leukocytes and endothelial cells) are also part of it. In combination with the extracellular matrix, all these cells constitute the tumor microenvironment. In the last decade the role of the tumor microenvironment in cancer progression has gained increased attention and prompted efforts directed to abrogate its deleterious effects on anti-cancer therapies. The immune system can detect and attack tumor cells, and tumor-infiltrating lymphocytes (particularly CD8 T cells) have been associated with improved survival or better response to therapies in colorectal, melanoma, breast, prostate and ovarian cancer patients among others. Contrariwise, tumor-associated myeloid cells (myeloid-derived suppressor cells [MDSCs], dendritic cells [DCs], macrophages) or lymphoid cells such as regulatory T cells can stimulate tumor growth via inhibition of immune responses against the tumor or by participating in tumor neoangiogenesis. Herewith we analyzed the chemokine profile of mouse breast tumors regarding their capacity to generate factors capable of attracting and sequestering DCs to their midst. Chemoattractants from tumors were investigated by molecular biology and immunological techniques and tumor infiltrating DCs were investigated for matched chemokine receptors. In addition, we investigated the inflammatory response of breast cancer cells, a major component of the tumor microenvironment, to double-stranded RNA stimulation. By using molecular biology techniques such as qualitative and quantitative PCR, PCR arrays, and immunological techniques (ELISA, cytokine immunoarrays) we examined the effects of dsRNA treatment on the cytokine secretion profiles of mouse and human breast cancer cells and non-transformed cells. We were able to determine that tumors generate chemokines that are able to interact with receptors present on the surface of tumor infiltrating DCs. We observed that PRR signaling is able to modify the production of chemokines by breast tumor cells and normal breast cells, thereby constituting a possible player in shaping the profile of the leukocyte population in the TME. Copyright © 2017 Elsevier GmbH. All rights reserved.

Contribution of Avian Salmonella enterica Isolates to Human Salmonellosis Cases in Constantine (Algeria)

PubMed Central

Elgroud, Rachid; Granier, Sophie A.; Marault, Muriel; Kerouanton, Annaëlle; Lezzar, Abdesslem; Bouzitouna-Bentchouala, Chafia; Brisabois, Anne; Millemann, Yves

2015-01-01

An epidemiological investigation was carried out on one hundred Salmonella isolates from broiler farms, slaughterhouses, and human patients in the Constantine region of Algeria, in order to explore the contribution of avian strains to human salmonellosis cases in this region over the same period of time. The isolates were characterized by phenotypic as well as genotypic methods. A large variety of antimicrobial resistance profiles was found among human isolates, while only seven profiles were found among avian isolates. Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR), Insertion Sequence 200-PCR (IS200-PCR), and Pulsed Field Gel Electrophoresis (PFGE) resulted in the allocation of the isolates to 16, 20, and 34 different profiles, respectively. The 3 genotyping methods led to complementary results by underlining the clonality of some serovars with the diffusion and persistence of a single clone in the Constantine area as well as stressing the polymorphism present in isolates belonging to other serovars, indicating the diversity of potential reservoirs of nontyphoidal Salmonella. Altogether, our results seem to indicate that nontyphoidal avian Salmonella may play an important role in human salmonellosis in the Constantine region. PMID:26543858

DNA Microarray Profiling of a Diverse Collection of Nosocomial Methicillin-Resistant Staphylococcus aureus Isolates Assigns the Majority to the Correct Sequence Type and Staphylococcal Cassette Chromosome mec (SCCmec) Type and Results in the Subsequent Identification and Characterization of Novel SCCmec-SCCM1 Composite Islands

PubMed Central

Brennan, Orla M.; Deasy, Emily C.; Rossney, Angela S.; Kinnevey, Peter M.; Ehricht, Ralf; Monecke, Stefan; Coleman, David C.

2012-01-01

One hundred seventy-five isolates representative of methicillin-resistant Staphylococcus aureus (MRSA) clones that predominated in Irish hospitals between 1971 and 2004 and that previously underwent multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCCmec) typing were characterized by spa typing (175 isolates) and DNA microarray profiling (107 isolates). The isolates belonged to 26 sequence type (ST)-SCCmec types and subtypes and 35 spa types. The array assigned all isolates to the correct MLST clonal complex (CC), and 94% (100/107) were assigned an ST, with 98% (98/100) correlating with MLST. The array assigned all isolates to the correct SCCmec type, but subtyping of only some SCCmec elements was possible. Additional SCCmec/SCC genes or DNA sequence variation not detected by SCCmec typing was detected by array profiling, including the SCC-fusidic acid resistance determinant Q6GD50/fusC. Novel SCCmec/SCC composite islands (CIs) were detected among CC8 isolates and comprised SCCmec IIA-IIE, IVE, IVF, or IVg and a ccrAB4-SCC element with 99% DNA sequence identity to SCCM1 from ST8/t024-MRSA, SCCmec VIII, and SCC-CI in Staphylococcus epidermidis. The array showed that the majority of isolates harbored one or more superantigen (94%; 100/107) and immune evasion cluster (91%; 97/107) genes. Apart from fusidic acid and trimethoprim resistance, the correlation between isolate antimicrobial resistance phenotype and the presence of specific resistance genes was ≥97%. Array profiling allowed high-throughput, accurate assignment of MRSA to CCs/STs and SCCmec types and provided further evidence of the diversity of SCCmec/SCC. In most cases, array profiling can accurately predict the resistance phenotype of an isolate. PMID:22869569

One-year monthly quantitative survey of noroviruses, enteroviruses, and adenoviruses in wastewater collected from six plants in Japan.

PubMed

Katayama, Hiroyuki; Haramoto, Eiji; Oguma, Kumiko; Yamashita, Hiromasa; Tajima, Atsushi; Nakajima, Hideichiro; Ohgaki, Shinichiro

2008-03-01

Sewerage systems are important nodes to monitor human enteric pathogens transmitted via water. A quantitative virus survey was performed once a month for a year to understand the seasonal profiles of noroviruses genotype 1 and genotype 2, enteroviruses, and adenoviruses in sewerage systems. A total of 72 samples of influent, secondary-treated wastewater before chlorination and effluent were collected from six wastewater treatment plants in Japan. Viruses were successfully recovered from 100ml of influent and 1000ml of the secondary-treated wastewater and effluent using the acid rinse method. Viruses were determined by the RT-PCR or PCR method to obtain the most probable number for each sample. All the samples were also assayed for fecal coliforms (FCs) by a double-layer method. The seasonal profiles of noroviruses genotype 1 and genotype 2 in influent were very similar, i.e. they were abundant in winter (from November to March) at a geometric mean value of 190 and 200 RT-PCR units/ml, respectively, and less frequent in summer (from June to September), at 4.9 and 9.1 RT-PCR units/ml, respectively. The concentrations of enteroviruses and adenoviruses were mostly constant all the year round, 17 RT-PCR units/ml and 320 PCR units/ml in influent, and 0.044 RT-PCR units/ml and 7.0 PCR units/ml in effluent, respectively.

Extended PCR conditions to reduce drop-out frequencies in low template STR typing including unequal mixtures.

PubMed

Weiler, Natalie E C; Matai, Anuska S; Sijen, Titia

2012-01-01

Forensic laboratories employ various approaches to obtain short tandem repeat (STR) profiles from minimal traces (<100 pg DNA input). Most approaches aim to sensitize DNA profiling by increasing the amplification level by a higher cycle number or enlarging the amount of PCR products analyzed during capillary electrophoresis. These methods have limitations when unequal mixtures are genotyped, since the major component will be over-amplified or over-loaded. This study explores an alternative strategy for improved detection of the minor components in low template (LT) DNA typing that may be better suited for the detection of the minor component in mixtures. The strategy increases the PCR amplification efficiency by extending the primer annealing time several folds. When the AmpFℓSTR(®) Identifiler(®) amplification parameters are changed to an annealing time of 20 min during all 28 cycles, the drop-out frequency is reduced for both pristine DNA and single or multiple donor mock case work samples. In addition, increased peak heights and slightly more drop-ins are observed while the heterozygous peak balance remains similar as with the conventional Identifiler protocol. By this extended protocol, full DNA profiles were obtained from only 12 sperm heads (which corresponds to 36 pg of DNA) that were collected by laser micro dissection. Notwithstanding the improved detection, allele drop-outs do persist, albeit in lower frequencies. Thus a LT interpretation strategy such as deducing consensus profiles from multiple independent amplifications is appropriate. The use of extended PCR conditions represents a general approach to improve detection of unequal mixtures as shown using four commercially available kits (AmpFℓSTR(®) Identifiler, SEfiler Plus, NGM and Yfiler). The extended PCR protocol seems to amplify more of the molecules in LT samples during PCR, which results in a lower drop-out frequency. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Circular BANP, an upregulated circular RNA that modulates cell proliferation in colorectal cancer.

PubMed

Zhu, Mingchen; Xu, Yijun; Chen, Yun; Yan, Feng

2017-04-01

Circular RNAs (circRNAs) are recently identified as widespread and diverse endogenous noncoding RNAs that may harbor vital functions in human and animals. However, the role of circRNAs in the process of tumorigenesis and development of colorectal cancer (CRC) remains vague. Here we characterized the circRNA expression profile from three paired CRC cancerous and adjacent normal tissues by human circRNA array, and identified 136 significantly overexpressed circRNAs and 243 downregulated circRNAs in CRC cancerous tissues (>2-fold changes). We further validated one circRNA generated from Exon 5-11 of BANP gene, termed circ-BANP. In addition, RT-PCR result showed that circ-BANP was overexpressed in 35 CRC cancerous tissues. Knockdown of circ-BANP with siRNA significantly attenuate the proliferation of CRC cells. In summary, our findings demonstrated that dysregulated circ-BANP appears to have an important role in CRC cells and could serve as a prognostic and therapeutic marker for CRC. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

EPConDB: a web resource for gene expression related to pancreatic development, beta-cell function and diabetes.

PubMed

Mazzarelli, Joan M; Brestelli, John; Gorski, Regina K; Liu, Junmin; Manduchi, Elisabetta; Pinney, Deborah F; Schug, Jonathan; White, Peter; Kaestner, Klaus H; Stoeckert, Christian J

2007-01-01

EPConDB (http://www.cbil.upenn.edu/EPConDB) is a public web site that supports research in diabetes, pancreatic development and beta-cell function by providing information about genes expressed in cells of the pancreas. EPConDB displays expression profiles for individual genes and information about transcripts, promoter elements and transcription factor binding sites. Gene expression results are obtained from studies examining tissue expression, pancreatic development and growth, differentiation of insulin-producing cells, islet or beta-cell injury, and genetic models of impaired beta-cell function. The expression datasets are derived using different microarray platforms, including the BCBC PancChips and Affymetrix gene expression arrays. Other datasets include semi-quantitative RT-PCR and MPSS expression studies. For selected microarray studies, lists of differentially expressed genes, derived from PaGE analysis, are displayed on the site. EPConDB provides database queries and tools to examine the relationship between a gene, its transcriptional regulation, protein function and expression in pancreatic tissues.

Identification of discriminant proteins through antibody profiling, methods and apparatus for identifying an individual

DOEpatents

Apel, William A.; Thompson, Vicki S; Lacey, Jeffrey A.; Gentillon, Cynthia A.

2016-08-09

A method for determining a plurality of proteins for discriminating and positively identifying an individual based from a biological sample. The method may include profiling a biological sample from a plurality of individuals against a protein array including a plurality of proteins. The protein array may include proteins attached to a support in a preselected pattern such that locations of the proteins are known. The biological sample may be contacted with the protein array such that a portion of antibodies in the biological sample reacts with and binds to the proteins forming immune complexes. A statistical analysis method, such as discriminant analysis, may be performed to determine discriminating proteins for distinguishing individuals. Proteins of interest may be used to form a protein array. Such a protein array may be used, for example, to compare a forensic sample from an unknown source with a sample from a known source.

Identification of discriminant proteins through antibody profiling, methods and apparatus for identifying an individual

DOEpatents

Thompson, Vicki S; Lacey, Jeffrey A; Gentillon, Cynthia A; Apel, William A

2015-03-03

A method for determining a plurality of proteins for discriminating and positively identifying an individual based from a biological sample. The method may include profiling a biological sample from a plurality of individuals against a protein array including a plurality of proteins. The protein array may include proteins attached to a support in a preselected pattern such that locations of the proteins are known. The biological sample may be contacted with the protein array such that a portion of antibodies in the biological sample reacts with and binds to the proteins forming immune complexes. A statistical analysis method, such as discriminant analysis, may be performed to determine discriminating proteins for distinguishing individuals. Proteins of interest may be used to form a protein array. Such a protein array may be used, for example, to compare a forensic sample from an unknown source with a sample from a known source.

Application of denaturing gradient gel electrophoresis (DGGE) to the analysis of microbial communities of subgingival plaque.

PubMed

Fujimoto, C; Maeda, H; Kokeguchi, S; Takashiba, S; Nishimura, F; Arai, H; Fukui, K; Murayama, Y

2003-08-01

Denaturing gradient gel electrophoresis (DGGE) was applied to the microbiologic examination of subgingival plaque. The PCR primers were designed from conserved nucleotide sequences on 16S ribosomal RNA gene (16SrDNA) with GC rich clamp at the 5'-end. Polymerase chain reaction (PCR) was performed using the primers and genomic DNAs of typical periodontal bacteria. The generated 16SrDNA fragments were separated by denaturing gel. Although the sizes of the amplified DNA fragments were almost the same among the species, 16SrDNAs of the periodontal bacteria were distinguished according to their specific sequences. The microflora of clinical plaque samples were profiled by the PCR-DGGE method, and the dominant 16SrDNA bands were cloned and sequenced. Simultaneously, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia were detected by an ordinary PCR method. In the deep periodontal pockets, the bacterial community structures were complicated and P. gingivalis was the most dominant species, whereas the DGGE profiles were simple and Streptococcus or Neisseria species were dominant in the shallow pockets. The species-specific PCR method revealed the presence of A. actinomycetemcomitans, P. gingivalis and P. intermedia in the clinical samples. However, corresponding bands were not always observed in the DGGE profiles, indicating a lower sensitivity of the DGGE method. Although the DGGE method may have a lower sensitivity than the ordinary PCR methods, it could visualize the bacterial qualitative compositions and reveal the major species of the plaque. The DGGE analysis and following sequencing may have the potential to be a promising bacterial examination procedure in periodontal diseases.

Validation of MALDI-TOF MS for rapid classification and identification of lactic acid bacteria, with a focus on isolates from traditional fermented foods in Northern Vietnam.

PubMed

Doan, N T L; Van Hoorde, K; Cnockaert, M; De Brandt, E; Aerts, M; Le Thanh, B; Vandamme, P

2012-10-01

To evaluate the potential use of MALDI-TOF MS for fast and reliable classification and identification of lactic acid bacteria (LAB) from traditional fermented foods. A total of 119 strains of LAB from fermented meat (nem chua) were analysed with both (GTG)(5)-PCR fingerprinting and MALDI-TOF MS. Cluster analysis of the profiles revealed five species represented by a single isolate both in (GTG)(5)-PCR and in MALDI-TOF MS; five species grouped alike for (GTG)(5)-PCR and for MALDI-TOF MS; however, differences in minimal similarity between the delineated (GTG)(5)-PCR and MALDI-TOF MS clusters could be observed; three species showed more heterogeneity in their MALDI-TOF MS profiles compared to their (GTG)(5)-PCR profiles; two species, each represented by a single MALDI-TOF cluster, were subdivided in the corresponding (GTG)(5)-PCR dendrogram. As proof of the identification potential of MALDI-TOF MS, LAB diversity from one fermented mustard sample was analysed using MALDI-TOF MS. PheS gene sequencing was used for validation. MALDI-TOF MS is a powerful, fast, reliable and cost-effective technique for the identification of LAB associated with the production of fermented foods. Food LAB can be identified using MALDI-TOF MS, and its application could possibly be extended to other food matrices and/or other food-derived micro-organisms. © 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

Rapid identification of dairy lactic acid bacteria by M13-generated, RAPD-PCR fingerprint databases.

PubMed

Rossetti, Lia; Giraffa, Giorgio

2005-11-01

About a thousand lactic acid bacteria (LAB) isolated from dairy products, especially cheeses, were identified and typed by species-specific PCR and RAPD-PCR, respectively. RAPD-PCR profiles, which were obtained by using the M13 sequence as a primer, allowed us to implement a large database of different fingerprints, which were analysed by BioNumerics software. Cluster analysis of the combined RAPD-PCR fingerprinting profiles enabled us to implement a library, which is a collection of library units, which in turn is a selection of representative database entries. A library unit, in this case, can be considered to be a definable taxon. The strains belonged to 11 main RAPD-PCR fingerprinting library units identified as Lactobacillus casei/paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus brevis, Enterococcus faecium, Enterococcus faecalis, Streptococcus thermophilus and Lactococcus lactis. The possibility to routinely identify newly typed, bacterial isolates by consulting the library of the software was valued. The proposed method could be suggested to refine previous strain identifications, eliminate redundancy and dispose of a technologically useful LAB strain collection. The same approach could also be applied to identify LAB strains isolated from other food ecosystems.

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 Profiling Technique to Accelerate Stem Cell Therapies for Eye Diseases

MedlinePlus

... like RPE. They also use a technique called quantitative RT-PCR to measure the expression of genes ... higher in iPS cells than mature RPE. But quantitative RT-PCR only permits the simultaneous measurement of ...

The Development and Use of Internal Amplification Controls (IACs) with DNA Profiling Kits for Forensic DNA Analysis.

PubMed

Zahra, Nathalie; Goodwin, William

2016-01-01

Biological samples recovered for forensic investigations are often degraded and/or have low amounts of DNA; in addition, in some instances the samples may be contaminated with chemicals that can act as PCR inhibitors. As a consequence this can make interpretation of the results challenging with the possibility of having partial profiles and false negative results. Because of the impact of DNA analysis on forensic investigations, it is important to monitor the process of DNA profiling, in particular the amplification reaction. In this chapter we describe a method for the in-house generation and use of internal amplification controls (IACs) with DNA profiling kits to monitor the success of the PCR proces. In the example we show the use of the SGM Plus® kit. These controls can also be used to aid the interpretation of the DNA profile.

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

Clustered, regularly interspaced short palindromic repeat (CRISPR) diversity and virulence factor distribution in avian Escherichia coli.

PubMed

Fu, Qiang; Su, Zhixin; Cheng, Yuqiang; Wang, Zhaofei; Li, Shiyu; Wang, Heng'an; Sun, Jianhe; Yan, Yaxian

In order to investigate the diverse characteristics of clustered, regularly interspaced short palindromic repeat (CRISPR) arrays and the distribution of virulence factor genes in avian Escherichia coli, 80 E. coli isolates obtained from chickens with avian pathogenic E. coli (APEC) or avian fecal commensal E. coli (AFEC) were identified. Using the multiplex polymerase chain reaction (PCR), five genes were subjected to phylogenetic typing and examined for CRISPR arrays to study genetic relatedness among the strains. The strains were further analyzed for CRISPR loci and virulence factor genes to determine a possible association between their CRISPR elements and their potential virulence. The strains were divided into five phylogenetic groups: A, B1, B2, D and E. It was confirmed that two types of CRISPR arrays, CRISPR1 and CRISPR2, which contain up to 246 distinct spacers, were amplified in most of the strains. Further classification of the isolates was achieved by sorting them into nine CRISPR clusters based on their spacer profiles, which indicates a candidate typing method for E. coli. Several significant differences in invasion-associated gene distribution were found between the APEC isolates and the AFEC isolates. Our results identified the distribution of 11 virulence genes and CRISPR diversity in 80 strains. It was demonstrated that, with the exception of iucD and aslA, there was no sharp demarcation in the gene distribution between the pathogenic (APEC) and commensal (AFEC) strains, while the total number of indicated CRISPR spacers may have a positive correlation with the potential pathogenicity of the E. coli isolates. Copyright © 2016. Published by Elsevier Masson SAS.

Identification of genetic aberrations on chromosome 22 outside the NF2 locus in schwannomatosis and neurofibromatosis type 2.

PubMed

Buckley, Patrick G; Mantripragada, Kiran K; Díaz de Ståhl, Teresita; Piotrowski, Arkadiusz; Hansson, Caisa M; Kiss, Hajnalka; Vetrie, David; Ernberg, Ingemar T; Nordenskjöld, Magnus; Bolund, Lars; Sainio, Markku; Rouleau, Guy A; Niimura, Michihito; Wallace, Andrew J; Evans, D Gareth R; Grigelionis, Gintautas; Menzel, Uwe; Dumanski, Jan P

2005-12-01

Schwannomatosis is characterized by multiple peripheral and cranial nerve schwannomas that occur in the absence of bilateral 8th cranial nerve schwannomas. The latter is the main diagnostic criterion of neurofibromatosis type 2 (NF2), which is a related but distinct disorder. The genetic factors underlying the differences between schwannomatosis and NF2 are poorly understood, although available evidence implicates chromosome 22 as the primary location of the gene(s) of interest. To investigate this, we comprehensively profiled the DNA copy number in samples from sporadic and familial schwannomatosis, NF2, and a large cohort of normal controls. Using a tiling-path chromosome 22 genomic array, we identified two candidate regions of copy number variation, which were further characterized by a PCR-based array with higher resolution. The latter approach allows the detection of minute alterations in total genomic DNA, with as little as 1.5 kb per measurement point of nonredundant sequence on the array. In DNA derived from peripheral blood from a schwannomatosis patient and a sporadic schwannoma sample, we detected rearrangements of the immunoglobulin lambda (IGL) locus, which is unlikely to be due to a B-cell specific somatic recombination of IGL. Analysis of normal controls indicated that these IGL rearrangements were restricted to schwannomatosis/schwannoma samples. In the second candidate region spanning GSTT1 and CABIN1 genes, we observed a frequent copy number polymorphism at the GSTT1 locus. We further describe missense mutations in the CABIN1 gene that are specific to samples from schwannomatosis and NF2 and make this gene a plausible candidate for contributing to the pathogenesis of these disorders. Copyright 2005 Wiley-Liss, Inc.

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.

Using a coherent hydrophone array for observing sperm whale range, classification, and shallow-water dive profiles.

PubMed

Tran, Duong D; Huang, Wei; Bohn, Alexander C; Wang, Delin; Gong, Zheng; Makris, Nicholas C; Ratilal, Purnima

2014-06-01

Sperm whales in the New England continental shelf and slope were passively localized, in both range and bearing, and classified using a single low-frequency (<2500 Hz), densely sampled, towed horizontal coherent hydrophone array system. Whale bearings were estimated using time-domain beamforming that provided high coherent array gain in sperm whale click signal-to-noise ratio. Whale ranges from the receiver array center were estimated using the moving array triangulation technique from a sequence of whale bearing measurements. Multiple concurrently vocalizing sperm whales, in the far-field of the horizontal receiver array, were distinguished and classified based on their horizontal spatial locations and the inter-pulse intervals of their vocalized click signals. The dive profile was estimated for a sperm whale in the shallow waters of the Gulf of Maine with 160 m water-column depth located close to the array's near-field where depth estimation was feasible by employing time difference of arrival of the direct and multiply reflected click signals received on the horizontal array. By accounting for transmission loss modeled using an ocean waveguide-acoustic propagation model, the sperm whale detection range was found to exceed 60 km in low to moderate sea state conditions after coherent array processing.

Reduction of Powerplex(®) Y23 reaction volume for genotyping buccal cell samples on FTA(TM) cards.

PubMed

Raziel, Aliza; Dell'Ariccia-Carmon, Aviva; Zamir, Ashira

2015-01-01

PowerPlex(®) Y23 is a novel kit for Y-STR typing that includes new highly discriminating loci. The Israel DNA Database laboratory has recently adopted it for routine Y-STR analysis. This study examined PCR amplification from 1.2-mm FTA punch in reduced volumes of 5 and 10 μL. Direct amplification and washing of the FTA punches were examined in different PCR cycle numbers. One short robotically performed wash was found to improve the quality and the percent of profiles obtained. The optimal PCR cycle number was determined for 5 and 10 μL reaction volumes. The percent of obtained profiles, color balance, and reproducibility were examined. High-quality profiles were achieved in 90% and 88% of the samples amplified in 5 and 10 μL, respectively, in the first attempt. Volume reduction to 5 μL has a vast economic impact especially for DNA database laboratories. © 2014 American Academy of Forensic Sciences.

Molecular typing of Vibrio parahaemolyticus isolated from seafood harvested along the south-west coast of India.

PubMed

Bhowmick, P P; Khushiramani, R; Raghunath, P; Karunasagar, I; Karunasagar, I

2008-02-01

Evaluation of protein profiling for typing Vibrio parahaemolyticus using 71 strains isolated from different seafood and comparison with other molecular typing techniques such as random amplified polymorphic DNA analysis (RAPD) and enterobacterial repetitive intergenic consensus sequence (ERIC)-PCR. Three molecular typing methods were used for the typing of 71 V. parahaemolyticus isolates from seafood. RAPD had a discriminatory index (DI) of 0.95, while ERIC-PCR showed a DI of 0.94. Though protein profiling had less discriminatory power, use of this method can be helpful in identifying new proteins which might have a role in establishment in the host or virulence of the organism. The use of protein profiling in combination with other established typing methods such as RAPD and ERIC-PCR generates useful information in the case of V. parahaemolyticus associated with seafood. The study demonstrates the usefulness of nucleic acid and protein-based studies in understanding the relationship between various isolates from seafood.

Enhanced low-template DNA analysis conditions and investigation of allele dropout patterns.

PubMed

Hedell, Ronny; Dufva, Charlotte; Ansell, Ricky; Mostad, Petter; Hedman, Johannes

2015-01-01

Forensic DNA analysis applying PCR enables profiling of minute biological samples. Enhanced analysis conditions can be applied to further push the limit of detection, coming with the risk of visualising artefacts and allele imbalances. We have evaluated the consecutive increase of PCR cycles from 30 to 35 to investigate the limitations of low-template (LT) DNA analysis, applying the short tandem repeat (STR) analysis kit PowerPlex ESX 16. Mock crime scene DNA extracts of four different quantities (from around 8-84 pg) were tested. All PCR products were analysed using 5, 10 and 20 capillary electrophoresis (CE) injection seconds. Bayesian models describing allele dropout patterns, allele peak heights and heterozygote balance were developed to assess the overall improvements in EPG quality with altered PCR/CE settings. The models were also used to evaluate the impact of amplicon length, STR marker and fluorescent label on the risk for allele dropout. The allele dropout probability decreased for each PCR cycle increment from 30 to 33 PCR cycles. Irrespective of DNA amount, the dropout probability was not affected by further increasing the number of PCR cycles. For the 42 and 84 pg samples, mainly complete DNA profiles were generated applying 32 PCR cycles. For the 8 and 17 pg samples, the allele dropouts decreased from 100% using 30 cycles to about 75% and 20%, respectively. The results for 33, 34 and 35 PCR cycles indicated that heterozygote balance and stutter ratio were mainly affected by DNA amount, and not directly by PCR cycle number and CE injection settings. We found 32 and 33 PCR cycles with 10 CE injection seconds to be optimal, as 34 and 35 PCR cycles did not improve allele detection and also included CE saturation problems. We find allele dropout probability differences between several STR markers. Markers labelled with the fluorescent dyes CXR-ET (red in electropherogram) and TMR-ET (shown as black) generally have higher dropout risks compared with those labelled with JOE (green) and fluorescein (blue). Overall, the marker D10S1248 has the lowest allele dropout probability and D8S1179 the highest. The marker effect is mainly pronounced for 30-32 PCR cycles. Such effects would not be expected if the amplification efficiencies were identical for all markers. Understanding allele dropout risks and the variability in peak heights and balances is important for correct interpretation of forensic DNA profiles. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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.

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

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.

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

Circulating microRNAs as biomarkers of disease and typification of the atherothrombotic status in antiphospholipid syndrome.

PubMed

Pérez-Sánchez, Carlos; Arias-de la Rosa, Iván; Aguirre, María Ángeles; Luque-Tévar, María; Ruiz-Limón, Patricia; Barbarroja, Nuria; Jiménez-Gómez, Yolanda; Ábalos-Aguilera, María Carmen; Collantes-Estévez, Eduardo; Segui, Pedro; Velasco, Francisco; Herranz, María Teresa; Lozano-Herrero, Jesús; Hernandez-Vidal, María Julia; Martínez, Constantino; González-Conejero, Rocío; Radin, Massimo; Sciascia, Savino; Cecchi, Irene; Cuadrado, María José; López-Pedrera, Chary

2018-05-01

We aimed to identify the plasma miRNA profile of antiphospholipid syndrome (APS) patients and to investigate the potential role of specific circulating miRNAs as non-invasive disease biomarkers. Ninety APS patients and 42 healthy donors were recruited. Profiling of miRNAs by PCR-array in plasma of APS patients identified a set of miRNAs differentially expressed and collectively involved in clinical features. Logistic regression and ROC analysis identified a signature of 10 miRNA ratios as biomarkers of disease. In addition, miRNA signature was related to fetal loss, atherosclerosis, and type of thrombosis, and correlated with parameters linked to inflammation, thrombosis, and autoimmunity. Hard clustering analysis differentiated 3 clusters representing different thrombotic risk profile groups. Significant differences between groups for several miRNA ratios were found. Moreover, miRNA signature remained stable over time, demonstrated by their analysis three months after the first sample collection. Parallel analysis in two additional cohorts of patients, including thrombosis without autoimmune disease, and systemic lupus erythematosus without antiphospholipid antibodies, each displayed specific miRNA profiles that were distinct from those of APS patients. In vitro , antiphospholipid antibodies of IgG isotype promoted deregulation in selected miRNAs and their potential atherothrombotic protein targets in monocytes and endothelial cells. Taken together, differentially expressed circulating miRNAs in APS patients, modulated at least partially by antiphospholipid antibodies of IgG isotype, might have the potential to serve as novel biomarkers of disease features and to typify patients' atherothrombotic status, thus constituting a useful tool in the management of the disease. Copyright © 2018 Ferrata Storti Foundation.

Clinical application of RT-nested PCR integrated with RFLP in Hantavirus detection and genotyping: a prospective study in Shandong Province, PR China.

PubMed

Liu, Yun-Xi; Zhao, Zhong-Tang; Cao, Wu-Chun; Xu, Xiao-Qun; Suo, Ji-Jiang; Xing, Yu-Bin; Jia, Ning; Du, Ming-Mei; Liu, Bo-Wei; Yao, Yuan

2013-01-01

The aim of the present study was to evaluate the clinical usefulness of applying RT-nested PCR along with RFLP as a method for diagnosis and genotypic differentiation of Hantavirus in the acute-stage sera of HFRS patients as compared to the ELISA technique. A prospective study of patients with suspected HFRS patients was carried out. Sera were collected for serological evaluation by ELISA and RT-nested PCR testing. Primers were selected from the published sequence of the S segment of HTNV strain 76-118 and SEOV strain SR-11, which made it possible to obtain an amplicon of 403 bp by RT-nested PCR. The genotypic differentiations of the RT-nested PCR amplicons were carried out by RFLP. Sequence analyses of the amplicons were used to confirm the accuracy of the results obtained by RFLP. Of the 48 acute-stage sera from suspected HFRS patients, 35 were ELISA-positive while 41 were positive by RT-nested PCR. With Hind III and Hinf I, RFLP profiles of the RT-nested PCR amplicons of the 41 positive sera exhibited two patterns. 33 had RFLP profiles similar to the reference strain R22, and thus belonged to the SEOV type. The other 8 samples which were collected during October-December had RFLP profiles similar to the reference strain 76-118, and thus belonged to the HTNV type. Sequence phylogenetic analysis of RT-nested PCR amplicons revealed sdp1, sdp2 YXL-2008, and sdp3 as close relatives of HTNV strain 76-118, while sdp22 and sdp37 as close relatives of SEOV strain Z37 and strain R22 located in two separate clusters in the phylogenetic tree. These results were identical to those acquired by RFLP. RT-nested PCR integrated with RFLP was a rapid, simple, accurate method for detecting and differentiating the genotypes of Hantavirus in the acute-stage sera of suspected HFRS patients. In Shandong province, the main genotypes of Hantavirus belonged to the SEOV types, while the HTNV types were observed during the autumn-winter season.

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

Energetic Profile of the Basketball Exercise Simulation Test in Junior Elite Players.

PubMed

Latzel, Richard; Hoos, Olaf; Stier, Sebastian; Kaufmann, Sebastian; Fresz, Volker; Reim, Dominik; Beneke, Ralph

2017-11-28

To analyze the energetic profile of the basketball exercise simulation test (BEST). 10 male elite junior basketball players (age: 15.5±0.6yrs, height: 180±9cm, body mass: 66.1±11.2kg) performed a modified BEST (20 circuits consisting of jumping, sprinting, jogging, shuffling, and short breaks) simulating professional basketball game play. Circuit time, sprint time, sprint decrement, oxygen uptake (VO2), heart rate (HR), and blood lactate concentration (BLC) were obtained. Metabolic energy and metabolic power above rest (W tot , P tot ) as well as energy share in terms of aerobic (W aer ), glycolytic (W blc ), and high energy phosphates (W PCr ) were calculated from VO2 during exercise, net lactate production, and the fast component of post-exercise VO2 kinetics, respectively. W aer , W blc , and W PCr reflect 89±2%, 5±1%, and 6±1% of total energy needed, respectively. Assuming an aerobic replenishment of PCr energy stores during short breaks, the adjusted energy share yielded W aer : 66±4%, W blc : 5±1%, and W PCr : 29±1%. W aer and W PCr were negatively correlated (-0.72, -0.59) with sprint time, which was not the case for W blc . Consistent with general findings on energy system interaction during repeated high intensity exercise bouts, the intermittent profile of the BEST relies primarily on aerobic energy combined with repetitive supplementation by anaerobic utilization of high energy phosphates.

Development and validation of a gene profile predicting benefit of postmastectomy radiotherapy in patients with high-risk breast cancer: a study of gene expression in the DBCG82bc cohort.

PubMed

Tramm, Trine; Mohammed, Hayat; Myhre, Simen; Kyndi, Marianne; Alsner, Jan; Børresen-Dale, Anne-Lise; Sørlie, Therese; Frigessi, Arnoldo; Overgaard, Jens

2014-10-15

To identify genes predicting benefit of radiotherapy in patients with high-risk breast cancer treated with systemic therapy and randomized to receive or not receive postmastectomy radiotherapy (PMRT). The study was based on the Danish Breast Cancer Cooperative Group (DBCG82bc) cohort. Gene-expression analysis was performed in a training set of frozen tumor tissue from 191 patients. Genes were identified through the Lasso method with the endpoint being locoregional recurrence (LRR). A weighted gene-expression index (DBCG-RT profile) was calculated and transferred to quantitative real-time PCR (qRT-PCR) in corresponding formalin-fixed, paraffin-embedded (FFPE) samples, before validation in FFPE from 112 additional patients. Seven genes were identified, and the derived DBCG-RT profile divided the 191 patients into "high LRR risk" and "low LRR risk" groups. PMRT significantly reduced risk of LRR in "high LRR risk" patients, whereas "low LRR risk" patients showed no additional reduction in LRR rate. Technical transfer of the DBCG-RT profile to FFPE/qRT-PCR was successful, and the predictive impact was successfully validated in another 112 patients. A DBCG-RT gene profile was identified and validated, identifying patients with very low risk of LRR and no benefit from PMRT. The profile may provide a method to individualize treatment with PMRT. ©2014 American Association for Cancer Research.

Modeling, Simulation, and Control of a Solar Electric Propulsion Vehicle in Near-Earth Vicinity Including Solar Array Degradation

NASA Technical Reports Server (NTRS)

Witzberger, Kevin (Inventor); Hojnicki, Jeffery (Inventor); Manzella, David (Inventor)

2016-01-01

Modeling and control software that integrates the complexities of solar array models, a space environment, and an electric propulsion system into a rigid body vehicle simulation and control model is provided. A rigid body vehicle simulation of a solar electric propulsion (SEP) vehicle may be created using at least one solar array model, at least one model of a space environment, and at least one model of a SEP propulsion system. Power availability and thrust profiles may be determined based on the rigid body vehicle simulation as the SEP vehicle transitions from a low Earth orbit (LEO) to a higher orbit or trajectory. The power availability and thrust profiles may be displayed such that a user can use the displayed power availability and thrust profiles to determine design parameters for an SEP vehicle mission.

Simultaneous Profiling of DNA Mutation and Methylation by Melting Analysis Using Magnetoresistive Biosensor Array.

PubMed

Rizzi, Giovanni; Lee, Jung-Rok; Dahl, Christina; Guldberg, Per; Dufva, Martin; Wang, Shan X; Hansen, Mikkel F

2017-09-26

Epigenetic modifications, in particular DNA methylation, are gaining increasing interest as complementary information to DNA mutations for cancer diagnostics and prognostics. We introduce a method to simultaneously profile DNA mutation and methylation events for an array of sites with single site specificity. Genomic (mutation) or bisulphite-treated (methylation) DNA is amplified using nondiscriminatory primers, and the amplicons are then hybridized to a giant magnetoresistive (GMR) biosensor array followed by melting curve measurements. The GMR biosensor platform offers scalable multiplexed detection of DNA hybridization, which is insensitive to temperature variation. The melting curve approach further enhances the assay specificity and tolerance to variations in probe length. We demonstrate the utility of this method by simultaneously profiling five mutation and four methylation sites in human melanoma cell lines. The method correctly identified all mutation and methylation events and further provided quantitative assessment of methylation density validated by bisulphite pyrosequencing.

Fabrication of parabolic cylindrical microlens array by shaped femtosecond laser

NASA Astrophysics Data System (ADS)

Luo, Zhi; Yin, Kai; Dong, Xinran; Duan, Ji'an

2018-04-01

A simple and efficient technique for fabricating parabolic cylindrical microlens arrays (CMLAs) on the surface of fused silica by shaped femtosecond (fs) laser direct-writing is demonstrated. By means of spatially shaping of a Gaussian fs laser beam to a Bessel distribution, an inversed cylindrical shape laser intensity profile is formed in a specific cross-sectional plane among the shaped optical field. Applying it to experiments, large area close-packed parabolic CMLAs with line-width of 37.5 μm and array size of about 5 × 5 mm are produced. The cross-sectional outline of obtained lenslets has a satisfied parabolic profile and the numerical aperture (NA) of lenslets is more than 0.35. Furthermore, the focusing performance of the fabricated CMLA is also tested in this work and it has been demonstrated that the focusing power of the CMLA with a parabolic profile is better than that with a semi-circular one.

Massively parallel sequencing of 17 commonly used forensic autosomal STRs and amelogenin with small amplicons.

PubMed

Kim, Eun Hye; Lee, Hwan Young; Yang, In Seok; Jung, Sang-Eun; Yang, Woo Ick; Shin, Kyoung-Jin

2016-05-01

The next-generation sequencing (NGS) method has been utilized to analyze short tandem repeat (STR) markers, which are routinely used for human identification purposes in the forensic field. Some researchers have demonstrated the successful application of the NGS system to STR typing, suggesting that NGS technology may be an alternative or additional method to overcome limitations of capillary electrophoresis (CE)-based STR profiling. However, there has been no available multiplex PCR system that is optimized for NGS analysis of forensic STR markers. Thus, we constructed a multiplex PCR system for the NGS analysis of 18 markers (13CODIS STRs, D2S1338, D19S433, Penta D, Penta E and amelogenin) by designing amplicons in the size range of 77-210 base pairs. Then, PCR products were generated from two single-sources, mixed samples and artificially degraded DNA samples using a multiplex PCR system, and were prepared for sequencing on the MiSeq system through construction of a subsequent barcoded library. By performing NGS and analyzing the data, we confirmed that the resultant STR genotypes were consistent with those of CE-based typing. Moreover, sequence variations were detected in targeted STR regions. Through the use of small-sized amplicons, the developed multiplex PCR system enables researchers to obtain successful STR profiles even from artificially degraded DNA as well as STR loci which are analyzed with large-sized amplicons in the CE-based commercial kits. In addition, successful profiles can be obtained from mixtures up to a 1:19 ratio. Consequently, the developed multiplex PCR system, which produces small size amplicons, can be successfully applied to STR NGS analysis of forensic casework samples such as mixtures and degraded DNA samples. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Longitudinal Analysis of Vaginal Microbiome Dynamics in Women with Recurrent Bacterial Vaginosis: Recognition of the Conversion Process

PubMed Central

Lambert, Janet A.; John, Susan; Sobel, Jack D.; Akins, Robert A.

2013-01-01

Bacterial vaginosis (BV) affects ∼30% of women of reproductive age, has a high rate of recurrence, and is associated with miscarriage, preterm birth, and increased risk of acquiring other sexually transmitted infections, including HIV-1. Little is known of the daily changes in the vaginal bacterial composition as it progresses from treatment to recurrence, or whether any of these might be useful in its prediction or an understanding of its causes. We used phylogenetic branch-inclusive quantitative PCR (PB-qPCR) and Lactobacillus blocked/unblocked qPCR (Lb-qPCR) to characterize longitudinal changes in the vaginal microbiota in sequential vaginal self-swabs from five women with recurrent BV, from diagnosis through remission to recurrence. Both patients with acute BV samples dominated by G. vaginalis recurred during the study with similar profiles, whereas the three patients with acute BV samples dominated by other anaerobes did not recur or recurred to an intermediate Nugent score. L. iners dominated remission phases, with intermittent days of abnormal microbial profiles typically associated with menses. The exception was a newly discovered phenomenon, a sustained period of abnormal profiles, termed conversion, which preceded symptomatic acute BV. Species known to have antagonistic activity towards Lactobacillus were detected in pre-conversion samples, possibly contributing to the decline in Lactobacillus. Lb-qPCR scores define two categories of response in the initial post-treatment visit samples; scores <5 may correspond with poor response to treatment or rapid recurrence, whereas scores >8 may predict delayed or no recurrence. Amsel criteria or Nugent scores did not have this potential predictive capability. Larger studies are warranted to evaluate the prognostic potential of detecting conversion and poor Lb-qPCR scores at the post-treatment visit of recurrent BV patients. PMID:24376552

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.

Improved Selection of Internal Transcribed Spacer-Specific Primers Enables Quantitative, Ultra-High-Throughput Profiling of Fungal Communities

PubMed Central

Bokulich, Nicholas A.

2013-01-01

Ultra-high-throughput sequencing (HTS) of fungal communities has been restricted by short read lengths and primer amplification bias, slowing the adoption of newer sequencing technologies to fungal community profiling. To address these issues, we evaluated the performance of several common internal transcribed spacer (ITS) primers and designed a novel primer set and work flow for simultaneous quantification and species-level interrogation of fungal consortia. Primer comparison and validation were predicted in silico and by sequencing a “mock community” of mixed yeast species to explore the challenges of amplicon length and amplification bias for reconstructing defined yeast community structures. The amplicon size and distribution of this primer set are smaller than for all preexisting ITS primer sets, maximizing sequencing coverage of hypervariable ITS domains by very-short-amplicon, high-throughput sequencing platforms. This feature also enables the optional integration of quantitative PCR (qPCR) directly into the HTS preparatory work flow by substituting qPCR with these primers for standard PCR, yielding quantification of individual community members. The complete work flow described here, utilizing any of the qualified primer sets evaluated, can rapidly profile mixed fungal communities and capably reconstructed well-characterized beer and wine fermentation fungal communities. PMID:23377949

Massively parallel digital high resolution melt for rapid and absolutely quantitative sequence profiling

NASA Astrophysics Data System (ADS)

Velez, Daniel Ortiz; Mack, Hannah; Jupe, Julietta; Hawker, Sinead; Kulkarni, Ninad; Hedayatnia, Behnam; Zhang, Yang; Lawrence, Shelley; Fraley, Stephanie I.

2017-02-01

In clinical diagnostics and pathogen detection, profiling of complex samples for low-level genotypes represents a significant challenge. Advances in speed, sensitivity, and extent of multiplexing of molecular pathogen detection assays are needed to improve patient care. We report the development of an integrated platform enabling the identification of bacterial pathogen DNA sequences in complex samples in less than four hours. The system incorporates a microfluidic chip and instrumentation to accomplish universal PCR amplification, High Resolution Melting (HRM), and machine learning within 20,000 picoliter scale reactions, simultaneously. Clinically relevant concentrations of bacterial DNA molecules are separated by digitization across 20,000 reactions and amplified with universal primers targeting the bacterial 16S gene. Amplification is followed by HRM sequence fingerprinting in all reactions, simultaneously. The resulting bacteria-specific melt curves are identified by Support Vector Machine learning, and individual pathogen loads are quantified. The platform reduces reaction volumes by 99.995% and achieves a greater than 200-fold increase in dynamic range of detection compared to traditional PCR HRM approaches. Type I and II error rates are reduced by 99% and 100% respectively, compared to intercalating dye-based digital PCR (dPCR) methods. This technology could impact a number of quantitative profiling applications, especially infectious disease diagnostics.

Optimization of analytical parameters for inferring relationships among Escherichia coli isolates from repetitive-element PCR by maximizing correspondence with multilocus sequence typing data.

PubMed

Goldberg, Tony L; Gillespie, Thomas R; Singer, Randall S

2006-09-01

Repetitive-element PCR (rep-PCR) is a method for genotyping bacteria based on the selective amplification of repetitive genetic elements dispersed throughout bacterial chromosomes. The method has great potential for large-scale epidemiological studies because of its speed and simplicity; however, objective guidelines for inferring relationships among bacterial isolates from rep-PCR data are lacking. We used multilocus sequence typing (MLST) as a "gold standard" to optimize the analytical parameters for inferring relationships among Escherichia coli isolates from rep-PCR data. We chose 12 isolates from a large database to represent a wide range of pairwise genetic distances, based on the initial evaluation of their rep-PCR fingerprints. We conducted MLST with these same isolates and systematically varied the analytical parameters to maximize the correspondence between the relationships inferred from rep-PCR and those inferred from MLST. Methods that compared the shapes of densitometric profiles ("curve-based" methods) yielded consistently higher correspondence values between data types than did methods that calculated indices of similarity based on shared and different bands (maximum correspondences of 84.5% and 80.3%, respectively). Curve-based methods were also markedly more robust in accommodating variations in user-specified analytical parameter values than were "band-sharing coefficient" methods, and they enhanced the reproducibility of rep-PCR. Phylogenetic analyses of rep-PCR data yielded trees with high topological correspondence to trees based on MLST and high statistical support for major clades. These results indicate that rep-PCR yields accurate information for inferring relationships among E. coli isolates and that accuracy can be enhanced with the use of analytical methods that consider the shapes of densitometric profiles.

Design, Construction, and Initial Test of High Spatial Resolution Thermometry Arrays for Detection of Surface Temperature Profiles on SRF Cavities in Super Fluid Helium

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

Ari Palczewski, Rongli Geng, Grigory Eremeev

2011-07-01

We designed and built two high resolution (0.6-0.55mm special resolution [1.1-1.2mm separation]) thermometry arrays prototypes out of the Allen Bradley 90-120 ohm 1/8 watt resistor to measure surface temperature profiles on SRF cavities. One array was designed to be physically flexible and conform to any location on a SRF cavity; the other was modeled after the common G-10/stycast 2850 thermometer and designed to fit on the equator of an ILC (Tesla 1.3GHz) SRF cavity. We will discuss the advantages and disadvantages of each array and their construction. In addition we will present a case study of the arrays performance onmore » a real SRF cavity TB9NR001. TB9NR001 presented a unique opportunity to test the performance of each array as it contained a dual (4mm separation) cat eye defect which conventional methods such as OST (Oscillating Superleak second-sound Transducers) and full coverage thermometry mapping were unable to distinguish between. We will discuss the new arrays ability to distinguish between the two defects and their preheating performance.« less

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.

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

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.

Comparison of Thermal Detector Arrays for Off-Axis THz Holography and Real-Time THz Imaging

PubMed Central

Hack, Erwin; Valzania, Lorenzo; Gäumann, Gregory; Shalaby, Mostafa; Hauri, Christoph P.; Zolliker, Peter

2016-01-01

In terahertz (THz) materials science, imaging by scanning prevails when low power THz sources are used. However, the application of array detectors operating with high power THz sources is increasingly reported. We compare the imaging properties of four different array detectors that are able to record THz radiation directly. Two micro-bolometer arrays are designed for infrared imaging in the 8–14 μm wavelength range, but are based on different absorber materials (i) vanadium oxide; (ii) amorphous silicon; (iii) a micro-bolometer array optimized for recording THz radiation based on silicon nitride; and (iv) a pyroelectric array detector for THz beam profile measurements. THz wavelengths of 96.5 μm, 118.8 μm, and 393.6 μm from a powerful far infrared laser were used to assess the technical performance in terms of signal to noise ratio, detector response and detectivity. The usefulness of the detectors for beam profiling and digital holography is assessed. Finally, the potential and limitation for real-time digital holography are discussed. PMID:26861341

Comparison of Thermal Detector Arrays for Off-Axis THz Holography and Real-Time THz Imaging.

PubMed

Hack, Erwin; Valzania, Lorenzo; Gäumann, Gregory; Shalaby, Mostafa; Hauri, Christoph P; Zolliker, Peter

2016-02-06

In terahertz (THz) materials science, imaging by scanning prevails when low power THz sources are used. However, the application of array detectors operating with high power THz sources is increasingly reported. We compare the imaging properties of four different array detectors that are able to record THz radiation directly. Two micro-bolometer arrays are designed for infrared imaging in the 8-14 μm wavelength range, but are based on different absorber materials (i) vanadium oxide; (ii) amorphous silicon; (iii) a micro-bolometer array optimized for recording THz radiation based on silicon nitride; and (iv) a pyroelectric array detector for THz beam profile measurements. THz wavelengths of 96.5 μm, 118.8 μm, and 393.6 μm from a powerful far infrared laser were used to assess the technical performance in terms of signal to noise ratio, detector response and detectivity. The usefulness of the detectors for beam profiling and digital holography is assessed. Finally, the potential and limitation for real-time digital holography are discussed.

The transcriptome of corona radiata cells from individual MІІ oocytes that after ICSI developed to embryos selected for transfer: PCOS women compared to healthy women.

PubMed

Wissing, Marie Louise; Sonne, Si Brask; Westergaard, David; Nguyen, Kho do; Belling, Kirstine; Høst, Thomas; Mikkelsen, Anne Lis

2014-11-29

Corona radiata cells (CRCs) refer to the fraction of cumulus cells just adjacent to the oocyte. The CRCs are closely connected to the oocyte throughout maturation and their gene expression profiles might reflect oocyte quality. Polycystic ovary syndrome (PCOS) is a common cause of infertility. It is controversial whether PCOS associate with diminished oocyte quality. The purpose of this study was to compare individual human CRC samples between PCOS patients and controls. All patients were stimulated by the long gonadotropin-releasing hormone (GnRH) agonist protocol. The CRC samples originated from individual oocytes developing into embryos selected for transfer. CRCs were isolated in a two-step denudation procedure, separating outer cumulus cells from the inner CRCs. Extracted RNA was amplified and transcriptome profiling was performed with Human Agilent® arrays. The transcriptomes of CRCs showed no individual genes with significant differential expression between PCOS and controls, but gene set enrichment analysis identified several cell cycle- and DNA replication pathways overexpressed in PCOS CRCs (FDR < 0.05). Five of the genes contributing to the up-regulated cell cycle pathways in the PCOS CRCs were selected for qRT-PCR validation in ten PCOS and ten control CRC samples. qRT-PCR confirmed significant up-regulation in PCOS CRCs of cell cycle progression genes HIST1H4C (FC = 2.7), UBE2C (FC = 2.6) and cell cycle related transcription factor E2F4 (FC = 2.5). The overexpression of cell cycle-related genes and cell cycle pathways in PCOS CRCs could indicate a disturbed or delayed final maturation and differentiation of the CRCs in response to the human chorionic gonadotropin (hCG) surge. However, this had no effect on the in vitro development of the corresponding embryos. Future studies are needed to clarify whether the up-regulated cell cycle pathways in PCOS CRCs have any clinical implications.

The Peripheral Whole Blood Transcriptome of Acute Pyelonephritis in Human Pregnancy

PubMed Central

Madan, Ichchha; Than, Nandor Gabor; Romero, Roberto; Chaemsaithong, Piya; Miranda, Jezid; Tarca, Adi L.; Bhatti, Gaurav; Draghici, Sorin; Yeo, Lami; Mazor, Moshe; Hassan, Sonia S.; Chaiworapongsa, Tinnakorn

2018-01-01

Objective Human pregnancy is characterized by activation of the innate immune response and suppression of adaptive immunity. The former is thought to provide protection against infection to the mother, and the latter, tolerance against paternal antigens expressed in fetal cells. Acute pyelonephritis is associated with an increased risk of acute respiratory distress syndrome and sepsis in pregnant (vs. nonpregnant) women. The objective of this study was to describe the gene expression profile (transcriptome) of maternal whole blood in acute pyelonephritis. Method A case-control study was conducted to include pregnant women with acute pyelonephritis (n=15) and women with a normal pregnancy (n=34). Affymetrix HG-U133 Plus 2.0 arrays (Affymetrix, Santa Clara, CA, USA) were used for gene expression profiling. A linear model was used to test the association between the presence of pyelonephritis and gene expression levels while controlling for white blood cell count and gestational age. A fold change of 1.5 was considered significant at a false discovery rate of 0.1. A subset of differentially expressed genes (n=56) was tested with real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) (cases, n=19; controls, n=59). Gene ontology and pathway analysis were applied. Results A total of 983 genes were differentially expressed in acute pyelonephritis: 457 were up-regulated and 526 were down-regulated. Significant enrichment of 300 biological processes and 63 molecular functions was found in pyelonephritis. Significantly impacted pathways in pyelonephritis included a) cytokine-cytokine receptor interaction; b) T-cell receptor signaling; c) Jak-STAT signaling; and d) complement and coagulation cascades. Of 56 genes tested by qRT-PCR, 48 (85.7%) had confirmation of differential expression. Conclusion This is the first study of the transcriptomic signature of whole blood in pregnant women with acute pyelonephritis. Acute infection during pregnancy is associated with the increased expression of genes involved in innate immunity and the decreased expression of genes involved in lymphocyte function. PMID:24293448

Compact, low profile antennas for MSAT and mini-M and Std-M land mobile satellite communications

NASA Technical Reports Server (NTRS)

Strickland, P. C.

1995-01-01

CAL Corporation has developed a new class of low profile radiating elements for use in planar phased array antennas. These new elements have been used in the design of a low cost, compact, low profile antenna unit for MSAT and INMARSAT Mini-M land mobile satellite communications. The antenna unit which measures roughly 32 cm in diameter by 5 cm deep incorporates a compact LNA and diplexer unit as well as a complete, low cost, beam steering system. CAL has also developed a low profile antenna unit for INMARSAT-M land mobile satellite communications. A number of these units, which utilize a microstrip patch array design, were put into service in 1994.

CRISPR-cas loci profiling of Cronobacter sakazakii pathovars.

PubMed

Ogrodzki, Pauline; Forsythe, Stephen James

2016-12-01

Cronobacter sakazakii sequence types 1, 4, 8 and 12 are associated with outbreaks of neonatal meningitis and necrotizing enterocolitis infections. However clonality results in strains which are indistinguishable using conventional methods. This study investigated the use of clustered regularly interspaced short palindromic repeats (CRISPR)-cas loci profiling for epidemiological investigations. Seventy whole genomes of C. sakazakii strains from four clonal complexes which were widely distributed temporally, geographically and origin of source were profiled. All strains encoded the same type I-E subtype CRISPR-cas system with a total of 12 different CRISPR spacer arrays. This study demonstrated the greater discriminatory power of CRISPR spacer array profiling compared with multilocus sequence typing, which will be of use in source attribution during Cronobacter outbreak investigations.

915-MHz Wind Profiler for Cloud Forecasting at Brookhaven National Laboratory

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

Jensen, M.; Bartholomew, M. J.; Giangrande, S.

When considering the amount of shortwave radiation incident on a photovoltaic solar array and, therefore, the amount and stability of the energy output from the system, clouds represent the greatest source of short-term (i.e., scale of minutes to hours) variability through scattering and reflection of incoming solar radiation. Providing estimates of this short-term variability is important for determining and regulating the output from large solar arrays as they connect with the larger power infrastructure. In support of the installation of a 37-MW solar array on the grounds of Brookhaven National Laboratory (BNL), a study of the impacts of clouds onmore » the output of the solar array has been undertaken. The study emphasis is on predicting the change in surface solar radiation resulting from the observed/forecast cloud field on a 5-minute time scale. At these time scales, advection of cloud elements over the solar array is of particular importance. As part of the BNL Aerosol Life Cycle Intensive Operational Period (IOP), a 915-MHz Radar Wind Profiler (RWP) was deployed to determine the profile of low-level horizontal winds and the depth of the planetary boundary layer. The initial deployment mission of the 915-MHz RWP for cloud forecasting has been expanded the deployment to provide horizontal wind measurements for estimating and constraining cloud advection speeds. A secondary focus is on the observation of dynamics and microphysics of precipitation during cold season/winter storms on Long Island. In total, the profiler was deployed at BNL for 1 year from May 2011 through May 2012.« less

915-Mhz Wind Profiler for Cloud Forecasting at Brookhaven National Laboratory

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

Jensen, M.; Bartholomew, M. J.; Giangrande, S.

When considering the amount of shortwave radiation incident on a photovoltaic solar array and, therefore, the amount and stability of the energy output from the system, clouds represent the greatest source of short-term (i.e., scale of minutes to hours) variability through scattering and reflection of incoming solar radiation. Providing estimates of this short-term variability is important for determining and regulating the output from large solar arrays as they connect with the larger power infrastructure. In support of the installation of a 37-MW solar array on the grounds of Brookhaven National Laboratory (BNL), a study of the impacts of clouds onmore » the output of the solar array has been undertaken. The study emphasis is on predicting the change in surface solar radiation resulting from the observed/forecast cloud field on a 5-minute time scale. At these time scales, advection of cloud elements over the solar array is of particular importance. As part of the BNL Aerosol Life Cycle Intensive Operational Period (IOP), a 915-MHz Radar Wind Profiler (RWP) was deployed to determine the profile of low-level horizontal winds and the depth of the planetary boundary layer. The initial deployment mission of the 915-MHz RWP for cloud forecasting has been expanded the deployment to provide horizontal wind measurements for estimating and constraining cloud advection speeds. A secondary focus is on the observation of dynamics and microphysics of precipitation during cold season/winter storms on Long Island. In total, the profiler was deployed at BNL for 1 year from May 2011 through May 2012.« less

An OSSE Study for Deep Argo Array using the GFDL Ensemble Coupled Data Assimilation System

NASA Astrophysics Data System (ADS)

Chang, You-Soon; Zhang, Shaoqing; Rosati, Anthony; Vecchi, Gabriel A.; Yang, Xiaosong

2018-03-01

An observing system simulation experiment (OSSE) using an ensemble coupled data assimilation system was designed to investigate the impact of deep ocean Argo profile assimilation in a biased numerical climate system. Based on the modern Argo observational array and an artificial extension to full depth, "observations" drawn from one coupled general circulation model (CM2.0) were assimilated into another model (CM2.1). Our results showed that coupled data assimilation with simultaneous atmospheric and oceanic constraints plays a significant role in preventing deep ocean drift. However, the extension of the Argo array to full depth did not significantly improve the quality of the oceanic climate estimation within the bias magnitude in the twin experiment. Even in the "identical" twin experiment for the deep Argo array from the same model (CM2.1) with the assimilation model, no significant changes were shown in the deep ocean, such as in the Atlantic meridional overturning circulation and the Antarctic bottom water cell. The small ensemble spread and corresponding weak constraints by the deep Argo profiles with medium spatial and temporal resolution may explain why the deep Argo profiles did not improve the deep ocean features in the assimilation system. Additional studies using different assimilation methods with improved spatial and temporal resolution of the deep Argo array are necessary in order to more thoroughly understand the impact of the deep Argo array on the assimilation system.

Combination of Multiplex PCR and PCR-Denaturing Gradient Gel Electrophoresis for Monitoring Common Sourdough-Associated Lactobacillus Species

PubMed Central

Settanni, Luca; Valmorri, Sara; van Sinderen, Douwe; Suzzi, Giovanna; Paparella, Antonello; Corsetti, Aldo

2006-01-01

A combination of denaturing gradient gel electrophoresis (DGGE) and a previously described multiplex PCR approach was employed to detect sourdough lactobacilli. Primers specific for certain groups of Lactobacillus spp. were used to amplify fragments, which were analyzed by DGGE. DGGE profiles obtained from Lactobacillus type strains acted as standards to analyze lactobacilli from four regional Abruzzo (central Italy) sourdoughs. PMID:16672538

Evaluation of a PCR melting profile method for intraspecies differentiation of Trichophyton rubrum and Trichophyton interdigitale.

PubMed

Leibner-Ciszak, Justyna; Dobrowolska, Anita; Krawczyk, Beata; Kaszuba, Aleksandra; Staczek, Paweł

2010-02-01

In order to identify the source of infections caused by dermatophytes, as well as the pathogen transmission pathway, there is a need to determine methods that allow detailed genetic differentiation of the strains within the dermatophyte genera. In this work, a PCR melting profile (PCR-MP) technique based on the ligation of adaptors and the difference in melting temperatures of DNA restriction fragments was used for the first time for intraspecies genotyping of dermatophytes. Clinical isolates and reference strains of dermatophytes isolated from skin, scalp, toenails and fingernails were used for this study. PCR-MP and random amplification of polymorphic DNA (RAPD) were used to type 11 isolates of Trichophyton rubrum, 40 isolates of Trichophyton interdigitale and 14 isolates of Microsporum canis. The results distinguished five types (containing one subtype) characteristic for T. rubrum and seven types characteristic for T. interdigitale using the PCR-MP technique. Analysis conducted using RAPD revealed five types for T. rubrum and four types for T. interdigitale isolates. No differentiation was observed for the M. canis isolates with either method. These results demonstrate that PCR-MP is a reliable method for the differentiation of T. rubrum and T. interdigitale strains and yields a discriminatory power that is at least equal to that of RAPD.

Comparison of pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus PCR and biochemical tests to characterize Lactococcus garvieae.

PubMed

Ture, M; Altinok, I; Capkin, E

2015-01-01

Biochemical test, pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) were used to compare 42 strains of Lactococcus garvieae isolated from different regions of Turkey, Italy, France and Spain. Twenty biotypes of L. garvieae were formed based on 54 biochemical tests. ERIC-PCR of genomic DNA from different L. garvieae strains resulted in amplification of multiple fragments of DNA in sizes ranging between 200 and 5000 bp with various band intensities. After cutting DNA with ApaI restriction enzyme and running on the PFGE, 11–22 resolvable bands ranging from 2 to 194 kb were observed. Turkish isolates were grouped into two clusters, and only A58 (Italy) strain was connected with Turkish isolates. Similarities between Turkish, Spanish, Italian and French isolates were <50% except 216-6 Rize strain. In Turkey, first lactococcosis occurred in Mugla, and then, it has been spread all over the country. Based on ERIC-PCR, Spanish and Italian strains of L. garvieae were related to Mugla strains. Therefore, after comparing PFGE profiles, ERIC-PCR profiles and phenotypic characteristics of 42 strains of L. garvieae, there were no relationships found between these three typing methods. PFGE method was more discriminative than the other methods. © 2014 John Wiley & Sons Ltd.

Microstrip Yagi array antenna for mobile satellite vehicle application

NASA Technical Reports Server (NTRS)

Huang, John; Densmore, Arthur C.

1991-01-01

A novel antenna structure formed by combining the Yagi-Uda array concept and the microstrip radiator technique is discussed. This antenna, called the microstrip Yagi array, has been developed for the mobile satellite (MSAT) system as a low-profile, low-cost, and mechanically steered medium-gain land-vehicle antenna. With the antenna's active patches (driven elements) and parasitic patches (reflector and director elements) located on the same horizontal plane, the main beam of the array can be tilted, by the effect of mutual coupling, in the elevation direction providing optimal coverage for users in the continental United States. Because the parasitic patches are not connected to any of the lossy RF power distributing circuit the antenna is an efficient radiating system. With the complete monopulse beamforming and power distributing circuits etched on a single thin stripline board underneath the microstrip Yagi array, the overall L-band antenna system has achieved a very low profile for vehicle's rooftop mounting, as well as a low manufacturing cost. Experimental results demonstrate the performance of this antenna.

An experimental investigation of S-duct flow control using arrays of low-profile vortex generators

NASA Technical Reports Server (NTRS)

Reichert, Bruce A.; Wendt, Bruce J.

1993-01-01

An experimental investigation was undertaken to measure the effect of various configurations of low-profile vortex generator arrays on the flow in a diffusing S-duct. Three parameters that characterize the vortex generator array were systematically varied to determine their effect: (1) the vortex generator height; (2) the streamwise location of the vortex generator array; and (3) the vortex generator spacing. Detailed measurements of total pressure at the duct exit, surface static pressure, and surface flow visualization were gathered for each vortex generator configuration. These results are reported here along with total pressure recovery and distortion coefficients determined from the experimental data. Each array of vortex generators tested improved total pressure recovery. The configuration employing the largest vortex generators was the most effective in reducing total pressure recovery. No configuration of vortex generators completely eliminated the flow separation that naturally occurs in the S-duct, however the extent of the separated flow region was reduced.

A Cabled, High Bandwidth Instrument Platform for Continuous Scanning of the Upper Ocean Water Column

NASA Astrophysics Data System (ADS)

McRae, E.; Delaney, J. R.; Kelly, D.; Daly, K. L.; Luther, D. S.; Harkins, G.; Harrington, M.; McGuire, C.; Tilley, J.; Dosher, J.; Waite, P.; Cram, G.; Kawka, O. E.

2016-02-01

The Cabled Array portion of the National Science Foundation funded Ocean Observatories Initiative is a large scale, high bandwidth and high power subsea science network designed by the University of Washington Applied Physics Laboratory. Part of that system is a set of winched profilers which continuously scan the upper 200m of the ocean at their deployment sites. The custom built profilers leverage the Cabled Array's technology for interfacing collections of science instruments and add the ability to run predefined missions and to switch missions or mission parameters on the fly via command from shore. The profilers were designed to operate continuously for up to two years after deployment after which certain wearing components must be replaced. The data from the profiler's science and engineering sensors are streamed to shore via the seafloor network in real time. Data channel capacity from the profilers exceeds 40 Mbps. For profiler safety, mission execution is controlled within the platform. Inputs such as 3D gyro, pressure depth and deployed cable calculations are monitored to assure safe operation during any sea state. The profilers never surface but are designed to approach within 5m of the surface if conditions allow. Substantial engineering effort was focused on reliable cable handling under all ocean conditions. The profilers are currently operated from subsea moorings which also contain sets of fixed science and engineering sensors. The profilers and their associated mooring instrument assemblies are designed for rapid replacement using ROVs. We have operated this system for two years, including one annual maintenance turn and information relative to that experience will be included in the paper.[Image Caption] Cabled Array Shallow Profiler shown in its parking position.

Oligonucleotide arrays vs. metaphase-comparative genomic hybridisation and BAC arrays for single-cell analysis: first applications to preimplantation genetic diagnosis for Robertsonian translocation carriers.

PubMed

Ramos, Laia; del Rey, Javier; Daina, Gemma; García-Aragonés, Manel; Armengol, Lluís; Fernandez-Encinas, Alba; Parriego, Mònica; Boada, Montserrat; Martinez-Passarell, Olga; Martorell, Maria Rosa; Casagran, Oriol; Benet, Jordi; Navarro, Joaquima

2014-01-01

Comprehensive chromosome analysis techniques such as metaphase-Comparative Genomic Hybridisation (CGH) and array-CGH are available for single-cell analysis. However, while metaphase-CGH and BAC array-CGH have been widely used for Preimplantation Genetic Diagnosis, oligonucleotide array-CGH has not been used in an extensive way. A comparison between oligonucleotide array-CGH and metaphase-CGH has been performed analysing 15 single fibroblasts from aneuploid cell-lines and 18 single blastomeres from human cleavage-stage embryos. Afterwards, oligonucleotide array-CGH and BAC array-CGH were also compared analysing 16 single blastomeres from human cleavage-stage embryos. All three comprehensive analysis techniques provided broadly similar cytogenetic profiles; however, non-identical profiles appeared when extensive aneuploidies were present in a cell. Both array techniques provided an optimised analysis procedure and a higher resolution than metaphase-CGH. Moreover, oligonucleotide array-CGH was able to define extra segmental imbalances in 14.7% of the blastomeres and it better determined the specific unbalanced chromosome regions due to a higher resolution of the technique (≈ 20 kb). Applicability of oligonucleotide array-CGH for Preimplantation Genetic Diagnosis has been demonstrated in two cases of Robertsonian translocation carriers 45,XY,der(13;14)(q10;q10). Transfer of euploid embryos was performed in both cases and pregnancy was achieved by one of the couples. This is the first time that an oligonucleotide array-CGH approach has been successfully applied to Preimplantation Genetic Diagnosis for balanced chromosome rearrangement carriers.

Oligonucleotide Arrays vs. Metaphase-Comparative Genomic Hybridisation and BAC Arrays for Single-Cell Analysis: First Applications to Preimplantation Genetic Diagnosis for Robertsonian Translocation Carriers

PubMed Central

Ramos, Laia; del Rey, Javier; Daina, Gemma; García-Aragonés, Manel; Armengol, Lluís; Fernandez-Encinas, Alba; Parriego, Mònica; Boada, Montserrat; Martinez-Passarell, Olga; Martorell, Maria Rosa; Casagran, Oriol; Benet, Jordi; Navarro, Joaquima

2014-01-01

Comprehensive chromosome analysis techniques such as metaphase-Comparative Genomic Hybridisation (CGH) and array-CGH are available for single-cell analysis. However, while metaphase-CGH and BAC array-CGH have been widely used for Preimplantation Genetic Diagnosis, oligonucleotide array-CGH has not been used in an extensive way. A comparison between oligonucleotide array-CGH and metaphase-CGH has been performed analysing 15 single fibroblasts from aneuploid cell-lines and 18 single blastomeres from human cleavage-stage embryos. Afterwards, oligonucleotide array-CGH and BAC array-CGH were also compared analysing 16 single blastomeres from human cleavage-stage embryos. All three comprehensive analysis techniques provided broadly similar cytogenetic profiles; however, non-identical profiles appeared when extensive aneuploidies were present in a cell. Both array techniques provided an optimised analysis procedure and a higher resolution than metaphase-CGH. Moreover, oligonucleotide array-CGH was able to define extra segmental imbalances in 14.7% of the blastomeres and it better determined the specific unbalanced chromosome regions due to a higher resolution of the technique (≈20 kb). Applicability of oligonucleotide array-CGH for Preimplantation Genetic Diagnosis has been demonstrated in two cases of Robertsonian translocation carriers 45,XY,der(13;14)(q10;q10). Transfer of euploid embryos was performed in both cases and pregnancy was achieved by one of the couples. This is the first time that an oligonucleotide array-CGH approach has been successfully applied to Preimplantation Genetic Diagnosis for balanced chromosome rearrangement carriers. PMID:25415307

Shear-wave velocity profile and seismic input derived from ambient vibration array measurements: the case study of downtown L'Aquila

NASA Astrophysics Data System (ADS)

Di Giulio, Giuseppe; Gaudiosi, Iolanda; Cara, Fabrizio; Milana, Giuliano; Tallini, Marco

2014-08-01

Downtown L'Aquila suffered severe damage (VIII-IX EMS98 intensity) during the 2009 April 6 Mw 6.3 earthquake. The city is settled on a top flat hill, with a shear-wave velocity profile characterized by a reversal of velocity at a depth of the order of 50-100 m, corresponding to the contact between calcareous breccia and lacustrine deposits. In the southern sector of downtown, a thin unit of superficial red soils causes a further shallow impedance contrast that may have influenced the damage distribution during the 2009 earthquake. In this paper, the main features of ambient seismic vibrations have been studied in the entire city centre by using array measurements. We deployed six 2-D arrays of seismic stations and 1-D array of vertical geophones. The 2-D arrays recorded ambient noise, whereas the 1-D array recorded signals produced by active sources. Surface-wave dispersion curves have been measured by array methods and have been inverted through a neighbourhood algorithm, jointly with the H/V ambient noise spectral ratios related to Rayleigh waves ellipticity. We obtained shear-wave velocity (Vs) profiles representative of the southern and northern sectors of downtown L'Aquila. The theoretical 1-D transfer functions for the estimated Vs profiles have been compared to the available empirical transfer functions computed from aftershock data analysis, revealing a general good agreement. Then, the Vs profiles have been used as input for a deconvolution analysis aimed at deriving the ground motion at bedrock level. The deconvolution has been performed by means of EERA and STRATA codes, two tools commonly employed in the geotechnical engineering community to perform equivalent-linear site response studies. The waveform at the bedrock level has been obtained deconvolving the 2009 main shock recorded at a strong motion station installed in downtown. Finally, this deconvolved waveform has been used as seismic input for evaluating synthetic time-histories in a strong-motion target site located in the middle Aterno river valley. As a target site, we selected the strong-motion station of AQV 5 km away from downtown L'Aquila. For this site, the record of the 2009 L'Aquila main shock is available and its surface stratigraphy is adequately known making possible to propagate the deconvolved bedrock motion back to the surface, and to compare recorded and synthetic waveforms.

Use of PCR-Based Methods for Rapid Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis

PubMed Central

Torriani, Sandra; Zapparoli, Giacomo; Dellaglio, Franco

1999-01-01

Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412T, which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains. PMID:10508059

Use of PCR-based methods for rapid differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis.

PubMed

Torriani, S; Zapparoli, G; Dellaglio, F

1999-10-01

Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412(T), which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains.

Development of a Standardized Approach for Environmental Microbiota Investigations related to Asthma Development in Children

PubMed Central

Fujimura, Kei E.; Rauch, Marcus; Matsui, Elizabeth; Iwai, Shoko; Calatroni, Agustin; Lynn, Henry; Mitchell, Herman; Johnson, Christine C.; Gern, James E.; Togias, Alkis; Boushey, Homer A.; Kennedy, Suzanne; Lynch, Susan V.

2013-01-01

Summary Standardized studies examining environmental microbial exposure in populations at risk for asthma are necessary to improve our understanding of the role this factor plays in disease development. Here we describe studies aimed at developing guidelines for high-resolution culture-independent microbiome profiling, using a phylogenetic microarray (PhyloChip), of house dust samples in a cohort collected as part of the NIH-funded Inner City Asthma Consortium (ICAC). We demonstrate that though extracted DNA concentrations varied across dust samples, the majority produced sufficient 16S rRNA to be profiled by the array. Comparison of array and 454-pyrosequencing performed in parallel on a subset of samples, illustrated that increasingly deeper sequencing efforts validated greater numbers of array-detected taxa. Community composition agreement across samples exhibited a hierarchy in concordance, with the highest level of agreement in replicate array profiles followed by samples collected from adjacent 1×1 m2 sites in the same room, adjacent sites with different sized sampling quadrants (1×1 and 2×2 m2), different sites within homes (living and bedroom) to lowest in living room samples collected from different homes. The guidelines for sample collection and processing in this pilot study extend beyond PhyloChip based studies of house-associated microbiota, and bear relevance for other microbiome profiling approaches such as next-generation sequencing. PMID:22975469

Development of a High-Throughput Resequencing Array for the Detection of Pathogenic Mutations in Osteogenesis Imperfecta

PubMed Central

Wang, Yao; Cui, Yazhou; Zhou, Xiaoyan; Han, Jinxiang

2015-01-01

Objective Osteogenesis imperfecta (OI) is a rare inherited skeletal disease, characterized by bone fragility and low bone density. The mutations in this disorder have been widely reported to be on various exonal hotspots of the candidate genes, including COL1A1, COL1A2, CRTAP, LEPRE1, and FKBP10, thus creating a great demand for precise genetic tests. However, large genome sizes make the process daunting and the analyses, inefficient and expensive. Therefore, we aimed at developing a fast, accurate, efficient, and cheaper sequencing platform for OI diagnosis; and to this end, use of an advanced array-based technique was proposed. Method A CustomSeq Affymetrix Resequencing Array was established for high-throughput sequencing of five genes simultaneously. Genomic DNA extraction from 13 OI patients and 85 normal controls and amplification using long-range PCR (LR-PCR) were followed by DNA fragmentation and chip hybridization, according to standard Affymetrix protocols. Hybridization signals were determined using GeneChip Sequence Analysis Software (GSEQ). To examine the feasibility, the outcome from new resequencing approach was validated by conventional capillary sequencing method. Result Overall call rates using resequencing array was 96–98% and the agreement between microarray and capillary sequencing was 99.99%. 11 out of 13 OI patients with pathogenic mutations were successfully detected by the chip analysis without adjustment, and one mutation could also be identified using manual visual inspection. Conclusion A high-throughput resequencing array was developed that detects the disease-associated mutations in OI, providing a potential tool to facilitate large-scale genetic screening for OI patients. Through this method, a novel mutation was also found. PMID:25742658

Estrogen-Dependent Nrf2 Expression Protects Against Reflux-Induced Esophagitis.

PubMed

Torihata, Yudai; Asanuma, Kiyotaka; Iijima, Katsunori; Mikami, Tetsuhiko; Hamada, Shin; Asano, Naoki; Koike, Tomoyuki; Imatani, Akira; Masamune, Atsushi; Shimosegawa, Tooru

2018-02-01

Gastroesophageal reflux disease is more common in males than in females. The enhanced antioxidative capacity of estrogen in females might account for the gender difference. Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a pivotal role in the host defense mechanism against oxidative stress. This study aimed to clarify the role of Nrf2 in reflux-induced esophageal inflammation, focusing on the gender difference and nitric oxide. Gastroesophageal reflux was surgically induced in male and female rats. Nitrite and ascorbic acid were administered for 1 week to provoke nitric oxide in the esophageal lumen. Male rats with gastroesophageal reflux were supplemented with 17β-estradiol or tert-butylhydroquinone, an Nrf2-inducing reagent. Esophageal squamous cell carcinoma KYSE30 cells were treated with 17β-estradiol. Nrf2 expression was examined by Western blotting and quantitative real-time PCR. Antioxidant gene expression profiles were examined by a PCR array. In the presence of nitric oxide, reflux-induced esophageal damage was less evident, whereas esophageal expression of Nrf2 and its target genes such as Nqo1 was more evident in female or male rats supplemented with 17β-estradiol than in male rats. 17β-Estradiol increased nuclear Nrf2 expression in KYSE30 cells. tert-Butylhydroquinone increased tissue Nqo1 mRNA expression, leading to a reduction in reflux-induced esophageal damage. Estrogen-dependent Nrf2 expression might contribute to protection against the development of gastroesophageal reflux disease in females.

Curcumin may serve an anticancer role in human osteosarcoma cell line U-2 OS by targeting ITPR1.

PubMed

Luo, Zhanpeng; Li, Dawei; Luo, Xiaobo; Li, Litao; Gu, Suxi; Yu, Long; Ma, Yuanzheng

2018-04-01

The present study aimed to determine the mechanisms of action of curcumin in osteosarcoma. Human osteosarcoma U-2 OS cells was purchased from the Cell Bank of the Chinese Academy of Sciences. RNA sequencing analysis was performed for 2 curcumin-treated samples and 2 control samples using Illumina deep sequencing technology. The differentially expressed genes were identified using Cufflink software. Enrichment and protein-protein interaction network analyses were performed separately using cluster Profiler package and Cytoscape software to identify key genes. Then, the mRNA levels of key genes were detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR) in U-2 OS cells. Finally, cell apoptosis, proliferation, migration and invasion arrays were performed. In total, 201 DEGs were identified in the curcumin-treated group. EEF1A1 (degree=88), ATF7IP, HIF1A, SMAD7, CLTC, MCM10, ITPR1, ADAM15, WWP2 and ATP5C1, which were enriched in 'biological process', exhibited higher degrees than other genes in the PPI network. RT-qPCR demonstrated that treatment with curcumin was able to significantly increase the levels of CLTC and ITPR1 mRNA in curcumin-treated cells compared with control. In addition, targeting ITPR1 with curcumin significantly promoted apoptosis and suppressed proliferation, migration and invasion. Targeting ITPR1 via curcumin may serve an anticancer role by mediating apoptosis, proliferation, migration and invasion in U-2 OS cells.

Innate inflammatory gene expression profiling in potential brain-dead donors: detailed investigation of the effect of common corticosteroid therapy.

PubMed

Gholamnezhadjafari, Reza; Tajik, Nader; Falak, Reza; Aflatoonian, Reza; Dehghan, Sanaz; Rezaei, Abbas

2017-07-01

Our study aimed to assess the influence of common methylprednisolone therapy on innate inflammatory factors in potential brain-dead organ donors (BDDs). The study groups consisted of 50 potential BDDs who received 15 mg/kg/d methylprednisolone and 25 live organ donors (LDs) as control group. Innate immunity gene expression profiling was performed by RT-PCR array. Soluble serum cytokines and chemokines, complement components, heat shock protein 70 (HSP70) and high mobility group box-1 (HMGB1) were measured by ELISA. Surface expression of TLR2 and TLR4 were determined using flow cytometry. Gene expression profiling revealed up-regulation of TLRs 1, 2, 4, 5, 6, 7 and 8, MYD88, NF-κB, NF-κB1A, IRAK1, STAT3, JAK2, TNF-α, IL-1β, CD86 and CD14 in the BDD group. Remarkably, the serum levels of C-reactive protein and HSP70 were considerably higher in the BDD group. In addition, serum amounts of IL-1β, IL-6, TNF-α, HMGB1, HSP70, C3a and C5a, but not IL-8, sCD86 or monocyte chemoattractant protein-1, were significantly increased in the BDD group. Significant differences were observed in flow cytometry analysis of TLR2 and TLR4 between the two groups. In summary, common methylprednisolone therapy in BDDs did not adequately reduce systemic inflammation, which could be due to inadequate doses or inefficient impact on other inflammatory-inducing pathways, for example oxidative stress or production of damage-associated molecules.

MicroRNAs expression profile in solid and unicystic ameloblastomas.

PubMed

Setién-Olarra, A; Marichalar-Mendia, X; Bediaga, N G; Aguirre-Echebarria, P; Aguirre-Urizar, J M; Mosqueda-Taylor, A

2017-01-01

Odontogenic tumors (OT) represent a specific pathological category that includes some lesions with unpredictable biological behavior. Although most of these lesions are benign, some, such as the ameloblastoma, exhibit local aggressiveness and high recurrence rates. The most common types of ameloblastoma are the solid/multicystic (SA) and the unicystic ameloblastoma (UA); the latter considered a much less aggressive entity as compared to the SA. The microRNA system regulates the expression of many human genes while its deregulation has been associated with neoplastic development. The aim of the current study was to determine the expression profiles of microRNAs present in the two most common types of ameloblastomas. MicroRNA expression profiles were assessed using TaqMan® Low Density Arrays (TLDAs) in 24 samples (8 SA, 8 UA and 8 control samples). The findings were validated using quantitative RTqPCR in an independent cohort of 19 SA, 8 UA and 19 dentigerous cysts as controls. We identified 40 microRNAs differentially regulated in ameloblastomas, which are related to neoplastic development and differentiation, and with the osteogenic process. Further validation of the top ranked microRNAs revealed significant differences in the expression of 6 of them in relation to UA, 7 in relation to SA and 1 (miR-489) that was related to both types. We identified a new microRNA signature for the ameloblastoma and for its main types, which may be useful to better understand the etiopathogenesis of this neoplasm. In addition, we identified a microRNA (miR-489) that is suggestive of differentiating among solid from unicystic ameloblastoma.

MicroRNAs expression profile in solid and unicystic ameloblastomas

PubMed Central

Setién-Olarra, A.; Bediaga, N. G.; Aguirre-Echebarria, P.; Aguirre-Urizar, J. M.; Mosqueda-Taylor, A.

2017-01-01

Objectives Odontogenic tumors (OT) represent a specific pathological category that includes some lesions with unpredictable biological behavior. Although most of these lesions are benign, some, such as the ameloblastoma, exhibit local aggressiveness and high recurrence rates. The most common types of ameloblastoma are the solid/multicystic (SA) and the unicystic ameloblastoma (UA); the latter considered a much less aggressive entity as compared to the SA. The microRNA system regulates the expression of many human genes while its deregulation has been associated with neoplastic development. The aim of the current study was to determine the expression profiles of microRNAs present in the two most common types of ameloblastomas. Material & methods MicroRNA expression profiles were assessed using TaqMan® Low Density Arrays (TLDAs) in 24 samples (8 SA, 8 UA and 8 control samples). The findings were validated using quantitative RTqPCR in an independent cohort of 19 SA, 8 UA and 19 dentigerous cysts as controls. Results We identified 40 microRNAs differentially regulated in ameloblastomas, which are related to neoplastic development and differentiation, and with the osteogenic process. Further validation of the top ranked microRNAs revealed significant differences in the expression of 6 of them in relation to UA, 7 in relation to SA and 1 (miR-489) that was related to both types. Conclusion We identified a new microRNA signature for the ameloblastoma and for its main types, which may be useful to better understand the etiopathogenesis of this neoplasm. In addition, we identified a microRNA (miR-489) that is suggestive of differentiating among solid from unicystic ameloblastoma. PMID:29053755

Gene expression profile of endoscopically active and inactive ulcerative colitis: preliminary data.

PubMed

Ţieranu, Cristian George; Dobre, Maria; Mănuc, Teodora Ecaterina; Milanesi, Elena; Pleşea, Iancu Emil; Popa, Caterina; Mănuc, Mircea; Ţieranu, Ioana; Preda, Carmen Monica; Diculescu, Mihai Mircea; Ionescu, Elena Mirela; Becheanu, Gabriel

2017-01-01

Multiple cytokines and chemokines related to immune response, apoptosis and inflammation have been identified as molecules implicated in ulcerative colitis (UC) pathogenesis. The aim of this study was to identify the differences at gene expression level of a panel of candidate genes in mucosa from patients with active UC (UCA), patients in remission (UCR), and normal controls. Eleven individuals were enrolled in the study: eight UC patients (four with active lesions, four with mucosal healing) and three controls without inflammatory bowel disease (IBD) seen on endoscopy. All the individuals underwent mucosal biopsy during colonoscopy. Gene expression profile was evaluated by polymerase chain reaction (PCR) array, investigating 84 genes implicated in apoptosis, inflammation, immune response, cellular adhesion, tissue remodeling and mucous secretion. Seventeen and three genes out of 84 were found significantly differentially expressed in UCA and UCR compared to controls, respectively. In particular, REG1A and CHI3L1 genes reported an up-regulation in UCA with a fold difference above 200. In UCR patients, the levels of CASP1, LYZ and ISG15 were different compared to controls. However, since a significant up-regulation of both CASP1 and LYZ was observed also in the UCA group, only ISG15 levels remained associated to the remission state. ISG15, that plays a key role in the innate immune response, seemed to be specifically associated to the UC remission state. These preliminary data represent a starting point for defining the gene profile of UC in different stages in Romanian population. Identification of genes implicated in UC pathogenesis could be useful to select new therapeutic targets.

EFEMP1 as a novel DNA methylation marker for prostate cancer: array-based DNA methylation and expression profiling.

PubMed

Kim, Yong-June; Yoon, Hyung-Yoon; Kim, Seon-Kyu; Kim, Young-Won; Kim, Eun-Jung; Kim, Isaac Yi; Kim, Wun-Jae

2011-07-01

Abnormal DNA methylation is associated with many human cancers. The aim of the present study was to identify novel methylation markers in prostate cancer (PCa) by microarray analysis and to test whether these markers could discriminate normal and PCa cells. Microarray-based DNA methylation and gene expression profiling was carried out using a panel of PCa cell lines and a control normal prostate cell line. The methylation status of candidate genes in prostate cell lines was confirmed by real-time reverse transcriptase-PCR, bisulfite sequencing analysis, and treatment with a demethylation agent. DNA methylation and gene expression analysis in 203 human prostate specimens, including 106 PCa and 97 benign prostate hyperplasia (BPH), were carried out. Further validation using microarray gene expression data from the Gene Expression Omnibus (GEO) was carried out. Epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1) was identified as a lead candidate methylation marker for PCa. The gene expression level of EFEMP1 was significantly higher in tissue samples from patients with BPH than in those with PCa (P < 0.001). The sensitivity and specificity of EFEMP1 methylation status in discriminating between PCa and BPH reached 95.3% (101 of 106) and 86.6% (84 of 97), respectively. From the GEO data set, we confirmed that the expression level of EFEMP1 was significantly different between PCa and BPH. Genome-wide characterization of DNA methylation profiles enabled the identification of EFEMP1 aberrant methylation patterns in PCa. EFEMP1 might be a useful indicator for the detection of PCa.

A large-scale dataset of single and mixed-source short tandem repeat profiles to inform human identification strategies: PROVEDIt.

PubMed

Alfonse, Lauren E; Garrett, Amanda D; Lun, Desmond S; Duffy, Ken R; Grgicak, Catherine M

2018-01-01

DNA-based human identity testing is conducted by comparison of PCR-amplified polymorphic Short Tandem Repeat (STR) motifs from a known source with the STR profiles obtained from uncertain sources. Samples such as those found at crime scenes often result in signal that is a composite of incomplete STR profiles from an unknown number of unknown contributors, making interpretation an arduous task. To facilitate advancement in STR interpretation challenges we provide over 25,000 multiplex STR profiles produced from one to five known individuals at target levels ranging from one to 160 copies of DNA. The data, generated under 144 laboratory conditions, are classified by total copy number and contributor proportions. For the 70% of samples that were synthetically compromised, we report the level of DNA damage using quantitative and end-point PCR. In addition, we characterize the complexity of the signal by exploring the number of detected alleles in each profile. Copyright © 2017 Elsevier B.V. All rights reserved.

Purification of crime scene DNA extracts using centrifugal filter devices

PubMed Central

2013-01-01

Background The success of forensic DNA analysis is limited by the size, quality and purity of biological evidence found at crime scenes. Sample impurities can inhibit PCR, resulting in partial or negative DNA profiles. Various DNA purification methods are applied to remove impurities, for example, employing centrifugal filter devices. However, irrespective of method, DNA purification leads to DNA loss. Here we evaluate the filter devices Amicon Ultra 30 K and Microsep 30 K with respect to recovery rate and general performance for various types of PCR-inhibitory crime scene samples. Methods Recovery rates for DNA purification using Amicon Ultra 30 K and Microsep 30 K were gathered using quantitative PCR. Mock crime scene DNA extracts were analyzed using quantitative PCR and short tandem repeat (STR) profiling to test the general performance and inhibitor-removal properties of the two filter devices. Additionally, the outcome of long-term routine casework DNA analysis applying each of the devices was evaluated. Results Applying Microsep 30 K, 14 to 32% of the input DNA was recovered, whereas Amicon Ultra 30 K retained 62 to 70% of the DNA. The improved purity following filter purification counteracted some of this DNA loss, leading to slightly increased electropherogram peak heights for blood on denim (Amicon Ultra 30 K and Microsep 30 K) and saliva on envelope (Amicon Ultra 30 K). Comparing Amicon Ultra 30 K and Microsep 30 K for purification of DNA extracts from mock crime scene samples, the former generated significantly higher peak heights for rape case samples (P-values <0.01) and for hairs (P-values <0.036). In long-term routine use of the two filter devices, DNA extracts purified with Amicon Ultra 30 K were considerably less PCR-inhibitory in Quantifiler Human qPCR analysis compared to Microsep 30 K. Conclusions Amicon Ultra 30 K performed better than Microsep 30 K due to higher DNA recovery and more efficient removal of PCR-inhibitory substances. The different performances of the filter devices are likely caused by the quality of the filters and plastic wares, for example, their DNA binding properties. DNA purification using centrifugal filter devices can be necessary for successful DNA profiling of impure crime scene samples and for consistency between different PCR-based analysis systems, such as quantification and STR analysis. In order to maximize the possibility to obtain complete STR DNA profiles and to create an efficient workflow, the level of DNA purification applied should be correlated to the inhibitor-tolerance of the STR analysis system used. PMID:23618387

Ion Beam Characterization of a NEXT Multi-Thruster Array Plume

NASA Technical Reports Server (NTRS)

Pencil, Eric J.; Foster, John E.; Patterson, Michael J.; Diaz, Esther M.; Van Noord, Jonathan L.; McEwen, Heather K.

2006-01-01

Three operational, engineering model, 7-kW ion thrusters and one instrumented, dormant thruster were installed in a cluster array in a large vacuum facility at NASA Glenn Research Center. A series of engineering demonstration tests were performed to evaluate the system performance impacts of operating various multiple-thruster configurations in an array. A suite of diagnostics was installed to investigate multiple-thruster operation impact on thruster performance and life, thermal interactions, and alternative system modes and architectures. The ion beam characterization included measuring ion current density profiles and ion energy distribution with Faraday probes and retarding potential analyzers, respectively. This report focuses on the ion beam characterization during single thruster operation, multiple thruster operation, various neutralizer configurations, and thruster gimbal articulation. Comparison of beam profiles collected during single and multiple thruster operation demonstrated the utility of superimposing single engine beam profiles to predict multi-thruster beam profiles. High energy ions were detected in the region 45 off the thruster axis, independent of thruster power, number of operating thrusters, and facility background pressure, which indicated that the most probable ion energy was not effected by multiple-thruster operation. There were no significant changes to the beam profiles collected during alternate thruster-neutralizer configurations, therefore supporting the viability of alternative system configuration options. Articulation of one thruster shifted its beam profile, whereas the beam profile of a stationary thruster nearby did not change, indicating there were no beam interactions which was consistent with the behavior of a collisionless beam expansion.

Multi-channel infrared thermometer

DOEpatents

Ulrickson, M.A.

A device for measuring the two-dimensional temperature profile of a surface comprises imaging optics for generating an image of the light radiating from the surface; an infrared detector array having a plurality of detectors; and optical means positioned between the imaging optics and the detector array for sampling, transmitting, and distributing the image over the detector surfaces. The optical means may be a light pipe array having one light pipe for each detector in the detector array.

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.

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

NASA Astrophysics Data System (ADS)

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

2002-06-01

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

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.

Microstrip antenna developments at JPL

NASA Technical Reports Server (NTRS)

Huang, John

1991-01-01

The in-house development of microstrip antennas, initiated in 1981, when a spaceborne lightweight and low-profile planar array was needed for a satellite communication system, is described. The work described covers the prediction of finite-ground-plane effects by the geometric theory of diffraction, higher-order-mode circularly polarized circular patch antennas, circularly polarized microstrip arrays with linearly polarized elements, an impedance-matching teardrop-shaped probe feed, a dual-polarized microstrip array with high isolation and low cross-polarization, a planar microstrip Yagi array, a microstrip reflectarray, a Ka-band MMIC array, and a series-fed linear arrays.

Bulk soil and rhizosphere bacterial community PCR-DGGE profiles and beta-galactosidase activity as indicators of biological quality in soils contaminated by heavy metals and cultivated with Silene vulgaris (Moench) Garcke.

PubMed

Martínez-Iñigo, M J; Pérez-Sanz, A; Ortiz, I; Alonso, J; Alarcón, R; García, P; Lobo, M C

2009-06-01

The biological quality of two heavy metal contaminated soils (soil C: Typic Calcixerept, pH 8.3 and soil H: Typic Haploxeraf, pH 7.3) was investigated after growing the metal-tolerant plant Silene vulgaris (Moench) Garcke for two vegetative periods. The activity of the enzyme beta-galactosidase, which is sensitive to the presence of contaminants in soil, and the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) profiles of 16S rRNA gene fragments of culturable bacteria from bulk soil and rhizosphere were determined. The microbial enzymatic activity was higher in planted soils than in bare soils at the contamination level of 600 mg of total heavy metals kg(-1) soil. After growing S. vulgaris, beta-galactosidase activity was almost recovered in the calcareous soil. In this soil new bands appeared in the PCR-DGGE profiles of the rhizosphere bacterial community as a response to the exposure to heavy metals.

[Effect of genetics, epigenetics and variations in the transcriptional expression of cadherin-E in breast cancer susceptibility].

PubMed

Aristizábal-Pachón, Andrés Felipe; Takahashi, Catarina Satie

2016-12-01

Cadherin-E (CDH1) is an important regulator of epithelial-mesenchymal transition, invasion and metastasis in many carcinomas. However, germinal epimutations and mutations effect in breast cancer susceptibility is not clear. To evaluate rs334558 polymorphism, promoter methylation status and CDH1 expression profile in breast cancer patients. We collected peripheral blood samples from 102 breast cancer patients and 102 healthy subjects. The identification of rs334558 polymorphism was performed using PCR-RFLP, while methylation-specific PCR (MSP) and methylation-sensitive high-resolution melting (MS-HRM) were used to explore CDH1 methylation status; finally, CDH1 transcriptional expression profile was evaluated using RT-qPCR. We found no association between rs334558 polymorphism and breast cancer. Aberrant promoter methylation profile was found in breast cancer patients and it was related with early cancer stages. CDH1 down-regulation was significantly associated with metastasis and promoter methylation. CDH1 alterations were associated with invasion and metastasis in breast cancer. Our results offer further evidence of CDH1 relevance in breast cancer development and progression.

Molecular identification and cluster analysis of homofermentative thermophilic lactobacilli isolated from dairy products.

PubMed

Andrighetto, C; De Dea, P; Lombardi, A; Neviani, E; Rossetti, L; Giraffa, G

1998-10-01

Twenty-five strains of thermophilic lactobacilli isolated from yoghurt and from semi-hard and hard cheeses (in parallel with nine type or reference strains) were identified and grouped according to their genetic relatedness. Strains were identified by sugar fermentation patterns using the "API 50 CHL" galleries, by species-specific DNA probes in dot-blot hybridization experiments, by amplification and restriction analysis of the 16S rRNA gene (ARDRA) and by polymerase chain reaction (PCR) using species-specific oligonucleotide primers. Strains were classified as Lactobacillus delbrueckii subsp. lactis and subsp. bulgaricus, L. helveticus, and L. acidophilus. Strains which were atypical by sugar fermentation patterns were also identified. Most of the strains could not be grouped using carbohydrate fermentation profiles. PCR fingerprinting was used to identify DNA profiles for the 25 lactobacilli. Experimentally obtained PCR profiles enabled discrimination of all strains, which were grouped according to the similarities in their combined patterns. In general, the clustering of the strains corresponded well with species delineation obtained by molecular identification. The dendrogram of genetic relatedness enabled the unambiguous identification of most of the strains which were shown to be atypical by the sugar fermentation profile, except for a discrepancy in one L. delbrueckii subsp. lactis strain and one atypical Lactobacillus sp. strain.

Use of Length Heterogeneity PCR and Fatty Acid Methyl Ester Profiles To Characterize Microbial Communities in Soil†

PubMed Central

Ritchie, Nancy J.; Schutter, Mary E.; Dick, Richard P.; Myrold, David D.

2000-01-01

In length heterogeneity PCR (LH-PCR) a fluorescently labeled primer is used to determine the relative amounts of amplified sequences originating from different microorganisms. Labeled fragments are separated by gel electrophoresis and detected by laser-induced fluorescence with an automated gene sequencer. We used LH-PCR to evaluate the composition of the soil microbial community. Four soils, which differed in terms of soil type and/or crop management practice, were studied. Previous data for microbial biomass, nitrogen and carbon contents, and nitrogen mineralization rates suggested that the microbial characteristics of these soils were different. One site received two different treatments: no-till and conventional till perennial ryegrass. The other sites were no-till continuous grass plots at separate locations with different soil types. Community composition was characterized by assessing the natural length heterogeneity in eubacterial sequences amplified from the 5′ domain of the 16S rRNA gene and by determining fatty acid methyl ester (FAME) profiles. We found that LH-PCR results were reproducible. Both methods distinguished the three sites. The most abundant bacterial community members, based on cloned LH-PCR products, were members of the β subclass of the class Proteobacteria, the Cytophaga-Flexibacter-Bacteriodes group, and the high-G+C-content gram-positive bacterial group. Strong correlations were found between LH-PCR results and FAME results. We found that the LH-PCR method is an efficient, reliable, and highly reproducible method that should be a useful tool in future assessments of microbial community composition. PMID:10742258

An Array-Based Analysis of MicroRNA Expression Comparing Matched Frozen and Formalin-Fixed Paraffin-Embedded Human Tissue Samples

PubMed Central

Zhang, Xiao; Chen, Jiamin; Radcliffe, Tom; LeBrun, Dave P.; Tron, Victor A.; Feilotter, Harriet

2008-01-01

MicroRNAs (miRNAs) are small, noncoding RNAs that suppress gene expression at the posttranscriptional level via an antisense RNA-RNA interaction. miRNAs used for array-based profiling are generally purified from either snap-frozen or fresh samples. Because tissues found in most pathology departments are available only in formalin-fixed and paraffin-embedded (FFPE) states, we sought to evaluate miRNA derived from FFPE samples for microarray analysis. In this study, miRNAs extracted from matched snap-frozen and FFPE samples were profiled using the Agilent miRNA array platform (Agilent, Santa Clara, CA). Each miRNA sample was hybridized to arrays containing probes interrogating 470 human miRNAs. Seven cases were compared in either duplicate or triplicate. Intrachip and interchip analyses demonstrated that the processes of miRNA extraction, labeling, and hybridization from both frozen and FFPE samples are highly reproducible and add little variation to the results; technical replicates showed high correlations with one another (Kendall tau, 0.722 to 0.853; Spearman rank correlation coefficient, 0.891 to 0.954). Our results showed consistent high correlations between matched frozen and FFPE samples (Kendall tau, 0.669 to 0.815; Spearman rank correlation coefficient, 0.847 to 0.948), supporting the use of FFPE-derived miRNAs for array-based, gene expression profiling. PMID:18832457

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

A Tapered Aluminium Microelectrode Array for Improvement of Dielectrophoresis-Based Particle Manipulation

PubMed Central

Buyong, Muhamad Ramdzan; Larki, Farhad; Faiz, Mohd Syafiq; Hamzah, Azrul Azlan; Yunas, Jumrail; Majlis, Burhanuddin Yeop

2015-01-01

In this work, the dielectrophoretic force (FDEP) response of Aluminium Microelectrode Arrays with tapered profile is investigated through experimental measurements and numerical simulations. A standard CMOS processing technique with a step for the formation of a tapered profile resist is implemented in the fabrication of Tapered Aluminium Microelectrode Arrays (TAMA). The FDEP is investigated through analysis of the Clausius-Mossotti factor (CMF) and cross-over frequency (fxo). The performance of TAMA with various side wall angles is compared to that of microelectrodes with a straight cut sidewall profile over a wide range of frequencies through FEM numerical simulations. Additionally, electric field measurement (EFM) is performed through scanning probe microscopy (SPM) in order to obtain the region of force focus in both platforms. Results showed that the tapered profile microelectrodes with angles between 60° and 70° produce the highest electric field gradient on the particles. Also, the region of the strongest electric field in TAMA is located at the bottom and top edge of microelectrode while the strongest electric field in microelectrodes with straight cut profile is found at the top corner of the microelectrode. The latter property of microelectrodes improves the probability of capturing/repelling the particles at the microelectrode’s side wall. PMID:25970255

A tapered aluminium microelectrode array for improvement of dielectrophoresis-based particle manipulation.

PubMed

Buyong, Muhamad Ramdzan; Larki, Farhad; Faiz, Mohd Syafiq; Hamzah, Azrul Azlan; Yunas, Jumrail; Majlis, Burhanuddin Yeop

2015-05-11

In this work, the dielectrophoretic force (F(DEP)) response of Aluminium Microelectrode Arrays with tapered profile is investigated through experimental measurements and numerical simulations. A standard CMOS processing technique with a step for the formation of a tapered profile resist is implemented in the fabrication of Tapered Aluminium Microelectrode Arrays (TAMA). The F(DEP) is investigated through analysis of the Clausius-Mossotti factor (CMF) and cross-over frequency (f(xo)). The performance of TAMA with various side wall angles is compared to that of microelectrodes with a straight cut sidewall profile over a wide range of frequencies through FEM numerical simulations. Additionally, electric field measurement (EFM) is performed through scanning probe microscopy (SPM) in order to obtain the region of force focus in both platforms. Results showed that the tapered profile microelectrodes with angles between 60° and 70° produce the highest electric field gradient on the particles. Also, the region of the strongest electric field in TAMA is located at the bottom and top edge of microelectrode while the strongest electric field in microelectrodes with straight cut profile is found at the top corner of the microelectrode. The latter property of microelectrodes improves the probability of capturing/repelling the particles at the microelectrode's side wall.

Effect of Phenotypic Residual Feed Intake and Dietary Forage Content on the Rumen Microbial Community of Beef Cattle

PubMed Central

Carberry, Ciara A.; Kenny, David A.; Han, Sukkyan; McCabe, Matthew S.

2012-01-01

Feed-efficient animals have lower production costs and reduced environmental impact. Given that rumen microbial fermentation plays a pivotal role in host nutrition, the premise that rumen microbiota may contribute to host feed efficiency is gaining momentum. Since diet is a major factor in determining rumen community structure and fermentation patterns, we investigated the effect of divergence in phenotypic residual feed intake (RFI) on ruminal community structure of beef cattle across two contrasting diets. PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (qPCR) were performed to profile the rumen bacterial population and to quantify the ruminal populations of Entodinium spp., protozoa, Fibrobacter succinogenes, Ruminococcus flavefaciens, Ruminococcus albus, Prevotella brevis, the genus Prevotella, and fungi in 14 low (efficient)- and 14 high (inefficient)-RFI animals offered a low-energy, high-forage diet, followed by a high-energy, low-forage diet. Canonical correspondence and Spearman correlation analyses were used to investigate associations between physiological variables and rumen microbial structure and specific microbial populations, respectively. The effect of RFI on bacterial profiles was influenced by diet, with the association between RFI group and PCR-DGGE profiles stronger for the higher forage diet. qPCR showed that Prevotella abundance was higher (P < 0.0001) in inefficient animals. A higher (P < 0.0001) abundance of Entodinium and Prevotella spp. and a lower (P < 0.0001) abundance of Fibrobacter succinogenes were observed when animals were offered the low-forage diet. Thus, differences in the ruminal microflora may contribute to host feed efficiency, although this effect may also be modulated by the diet offered. PMID:22562991

Effect of phenotypic residual feed intake and dietary forage content on the rumen microbial community of beef cattle.

PubMed

Carberry, Ciara A; Kenny, David A; Han, Sukkyan; McCabe, Matthew S; Waters, Sinead M

2012-07-01

Feed-efficient animals have lower production costs and reduced environmental impact. Given that rumen microbial fermentation plays a pivotal role in host nutrition, the premise that rumen microbiota may contribute to host feed efficiency is gaining momentum. Since diet is a major factor in determining rumen community structure and fermentation patterns, we investigated the effect of divergence in phenotypic residual feed intake (RFI) on ruminal community structure of beef cattle across two contrasting diets. PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (qPCR) were performed to profile the rumen bacterial population and to quantify the ruminal populations of Entodinium spp., protozoa, Fibrobacter succinogenes, Ruminococcus flavefaciens, Ruminococcus albus, Prevotella brevis, the genus Prevotella, and fungi in 14 low (efficient)- and 14 high (inefficient)-RFI animals offered a low-energy, high-forage diet, followed by a high-energy, low-forage diet. Canonical correspondence and Spearman correlation analyses were used to investigate associations between physiological variables and rumen microbial structure and specific microbial populations, respectively. The effect of RFI on bacterial profiles was influenced by diet, with the association between RFI group and PCR-DGGE profiles stronger for the higher forage diet. qPCR showed that Prevotella abundance was higher (P < 0.0001) in inefficient animals. A higher (P < 0.0001) abundance of Entodinium and Prevotella spp. and a lower (P < 0.0001) abundance of Fibrobacter succinogenes were observed when animals were offered the low-forage diet. Thus, differences in the ruminal microflora may contribute to host feed efficiency, although this effect may also be modulated by the diet offered.

CLL Cells Respond to B-Cell Receptor Stimulation with a MicroRNA/mRNA Signature Associated with MYC Activation and Cell Cycle Progression

PubMed Central

Pede, Valerie; Rombout, Ans; Vermeire, Jolien; Naessens, Evelien; Mestdagh, Pieter; Robberecht, Nore; Vanderstraeten, Hanne; Van Roy, Nadine; Vandesompele, Jo; Speleman, Frank; Philippé, Jan; Verhasselt, Bruno

2013-01-01

Chronic lymphocytic leukemia (CLL) is a disease with variable clinical outcome. Several prognostic factors such as the immunoglobulin heavy chain variable genes (IGHV) mutation status are linked to the B-cell receptor (BCR) complex, supporting a role for triggering the BCR in vivo in the pathogenesis. The miRNA profile upon stimulation and correlation with IGHV mutation status is however unknown. To evaluate the transcriptional response of peripheral blood CLL cells upon BCR stimulation in vitro, miRNA and mRNA expression was measured using hybridization arrays and qPCR. We found both IGHV mutated and unmutated CLL cells to respond with increased expression of MYC and other genes associated with BCR activation, and a phenotype of cell cycle progression. Genome-wide expression studies showed hsa-miR-132-3p/hsa-miR-212 miRNA cluster induction associated with a set of downregulated genes, enriched for genes modulated by BCR activation and amplified by Myc. We conclude that BCR triggering of CLL cells induces a transcriptional response of genes associated with BCR activation, enhanced cell cycle entry and progression and suggest that part of the transcriptional profiles linked to IGHV mutation status observed in isolated peripheral blood are not cell intrinsic but rather secondary to in vivo BCR stimulation. PMID:23560086

Epigenetic repression of HOXB cluster in oral cancer cell lines.

PubMed

Xavier, Flávia Caló Aquino; Destro, Maria Fernanda de Souza Setubal; Duarte, Carina Magalhães Esteves; Nunes, Fabio Daumas

2014-08-01

Aberrant DNA methylation is a fundamental transcriptional control mechanism in carcinogenesis. The expression of homeobox genes is usually controlled by an epigenetic mechanism, such as the methylation of CpG islands in the promoter region. The aim of this study was to describe the differential methylation pattern of HOX genes in oral squamous cell carcinoma (OSCC) cell lines and transcript status in a group of hypermethylated and hypomethylated genes. Quantitative analysis of DNA methylation was performed on two OSCC cell lines (SCC4 and SCC9) using a method denominated Human Homeobox Genes EpiTect Methyl qPCR Arrays, which allowed fast, precise methylation detection of 24 HOX specific genes without bisulfite conversion. Methylation greater than 50% was detected in HOXA11, HOXA6, HOXA7, HOXA9, HOXB1, HOXB2, HOXB3, HOXB4, HOXB5, HOXB6, HOXC8 and HOXD10. Both cell lines demonstrated similar hypermethylation status for eight HOX genes. A similar pattern of promoter hypermethylation and hypomethylation was demonstrated for the HOXB cluster and HOXA cluster, respectively. Moreover, the hypermethylation profile of the HOXB cluster, especially HOXB4, was correlated with decreased transcript expression, which was restored following treatment with 5-aza-2'-deoxycytidine. The homeobox methylation profile in OSCC cell lines is consistent with an epigenetic biomarker. Copyright © 2014 Elsevier Ltd. All rights reserved.

Signaling Pathways Involved in Lunar Dust Induced Cytotoxicity

NASA Technical Reports Server (NTRS)

Zhang, Ye; Lam, Chiu-Wing; Scully, Robert R.; Williams, Kyle; Zalesak, Selina; Wu, Honglu; James, John T.

2014-01-01

The Moon's surface is covered by a layer of fine, reactive dust. Lunar dust contain about 1-2% of very fine dust (< 3 micron), that is respirable. The habitable area of any lunar landing vehicle and outpost would inevitably be contaminated with lunar dust that could pose a health risk. The purpose of the study is to evaluate the toxicity of Apollo moon dust in rodents to assess the health risk of dust exposures to humans. One of the particular interests in the study is to evaluate dust-induced changes of the expression of fibrosis-related genes, and to identify specific signaling pathways involved in lunar dust-induced toxicity. F344 rats were exposed for 4 weeks (6h/d; 5d/wk) in nose-only inhalation chambers to concentrations of 0 (control air), 2.1, 6.1, 21, and 61 mg/m(exp 3) of lunar dust. Five rats per group were euthanized 1 day, 1 week, 1 month, and 3 months after the last inhalation exposure. The total RNAs were isolated from the blood or lung tissue after being lavaged, using the Qigen RNeasy kit. The Rat Fibrosis RT2 Profile PCR Array was used to profile the expression of 84 genes relevant to fibrosis. The genes with significant expression changes are identified and the gene expression data were further analyzed using IPA pathway analysis tool to determine the signaling pathways with significant changes.

Alteration of gene expression profiles in skeletal muscle of rats exposed to microgravity during a spaceflight

NASA Technical Reports Server (NTRS)

Taylor, Wayne E.; Bhasin, Shalender; Lalani, Rukhsana; Datta, Anuj; Gonzalez-Cadavid, Nestor F.

2002-01-01

To clarify the mechanism of skeletal muscle wasting during spaceflights, we investigated whether intramuscular gene expression profiles are affected, by using DNA microarray methods. Male rats sent on the 17-day NASA STS-90 Neurolab spaceflight were sacrificed 24 hours after return to earth (MG group). Ground control rats were maintained for 17 days in flight-simulated cages (CS group). Spaceflight induced a 19% and 23% loss of tibialis anterior and gastrocnemius muscle mass, respectively, as compared to ground controls. Muscle RNA was analyzed by the Clontech Atlas DNA expression array in four rats, with two MG/ CS pairs for the tibialis anterior, and one pair for the gastrocnemius. Alterations in gene expression were verified for selected genes by reverse-transcription PCR. In both muscles of MG rats, mRNAs for 12 genes were up-regulated by over 2-fold, and 38 were down-regulated compared to controls. There was inhibition of genes for cell proliferation and growth factor cascades, including cell cycle genes and signal transduction proteins, such as p21 Cip1, retinoblastoma (Rb), cyclins G1/S, -E and -D3, MAP kinase 3, MAD3, and ras related protein RAB2. These data indicate that following exposure to microgravity, there is downregulation of genes involved in regulation of muscle satellite cell replication.

Analysis of ripening-related gene expression in papaya using an Arabidopsis-based microarray

PubMed Central

2012-01-01

Background Papaya (Carica papaya L.) is a commercially important crop that produces climacteric fruits with a soft and sweet pulp that contain a wide range of health promoting phytochemicals. Despite its importance, little is known about transcriptional modifications during papaya fruit ripening and their control. In this study we report the analysis of ripe papaya transcriptome by using a cross-species (XSpecies) microarray technique based on the phylogenetic proximity between papaya and Arabidopsis thaliana. Results Papaya transcriptome analyses resulted in the identification of 414 ripening-related genes with some having their expression validated by qPCR. The transcription profile was compared with that from ripening tomato and grape. There were many similarities between papaya and tomato especially with respect to the expression of genes encoding proteins involved in primary metabolism, regulation of transcription, biotic and abiotic stress and cell wall metabolism. XSpecies microarray data indicated that transcription factors (TFs) of the MADS-box, NAC and AP2/ERF gene families were involved in the control of papaya ripening and revealed that cell wall-related gene expression in papaya had similarities to the expression profiles seen in Arabidopsis during hypocotyl development. Conclusion The cross-species array experiment identified a ripening-related set of genes in papaya allowing the comparison of transcription control between papaya and other fruit bearing taxa during the ripening process. PMID:23256600

Systematic evaluation of RNA quality, microarray data reliability and pathway analysis in fresh, fresh frozen and formalin-fixed paraffin-embedded tissue samples.

PubMed

Wimmer, Isabella; Tröscher, Anna R; Brunner, Florian; Rubino, Stephen J; Bien, Christian G; Weiner, Howard L; Lassmann, Hans; Bauer, Jan

2018-04-20

Formalin-fixed paraffin-embedded (FFPE) tissues are valuable resources commonly used in pathology. However, formalin fixation modifies nucleic acids challenging the isolation of high-quality RNA for genetic profiling. Here, we assessed feasibility and reliability of microarray studies analysing transcriptome data from fresh, fresh-frozen (FF) and FFPE tissues. We show that reproducible microarray data can be generated from only 2 ng FFPE-derived RNA. For RNA quality assessment, fragment size distribution (DV200) and qPCR proved most suitable. During RNA isolation, extending tissue lysis time to 10 hours reduced high-molecular-weight species, while additional incubation at 70 °C markedly increased RNA yields. Since FF- and FFPE-derived microarrays constitute different data entities, we used indirect measures to investigate gene signal variation and relative gene expression. Whole-genome analyses revealed high concordance rates, while reviewing on single-genes basis showed higher data variation in FFPE than FF arrays. Using an experimental model, gene set enrichment analysis (GSEA) of FFPE-derived microarrays and fresh tissue-derived RNA-Seq datasets yielded similarly affected pathways confirming the applicability of FFPE tissue in global gene expression analysis. Our study provides a workflow comprising RNA isolation, quality assessment and microarray profiling using minimal RNA input, thus enabling hypothesis-generating pathway analyses from limited amounts of precious, pathologically significant FFPE tissues.

Characterization of Deletions of the HBA and HBB Loci by Array Comparative Genomic Hybridization

PubMed Central

Sabath, Daniel E.; Bender, Michael A.; Sankaran, Vijay G.; Vamos, Esther; Kentsis, Alex; Yi, Hye-Son; Greisman, Harvey A.

2017-01-01

Thalassemia is among the most common genetic diseases worldwide. α-Thalassemia is usually caused by deletion of one or more of the duplicated HBA genes on chromosome 16. In contrast, most β-thalassemia results from point mutations that decrease or eliminate expression of the HBB gene on chromosome 11. Deletions within the HBB locus result in thalassemia or hereditary persistence of fetal Hb. Although routine diagnostic testing cannot distinguish thalassemia deletions from point mutations, deletional hereditary persistence of fetal Hb is notable for having an elevated HbF level with a normal mean corpuscular volume. A small number of deletions accounts for most α-thalassemias; in contrast, there are no predominant HBB deletions causing β-thalassemia. To facilitate the identification and characterization of deletions of the HBA and HBB globin loci, we performed array-based comparative genomic hybridization using a custom oligonucleotide microarray. We accurately mapped the breakpoints of known and previously uncharacterized HBB deletions defining previously uncharacterized deletion breakpoints by PCR amplification and sequencing. The array also successfully identified the common HBA deletions --SEA and --FIL. In summary, comparative genomic hybridization can be used to characterize deletions of the HBA and HBB loci, allowing high-resolution characterization of novel deletions that are not readily detected by PCR-based methods. PMID:26612711

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.

Expression Comparison of Oil Biosynthesis Genes in Oil Palm Mesocarp Tissue Using Custom Array

PubMed Central

Wong, Yick Ching; Kwong, Qi Bin; Lee, Heng Leng; Ong, Chuang Kee; Mayes, Sean; Chew, Fook Tim; Appleton, David R.; Kulaveerasingam, Harikrishna

2014-01-01

Gene expression changes that occur during mesocarp development are a major research focus in oil palm research due to the economic importance of this tissue and the relatively rapid increase in lipid content to very high levels at fruit ripeness. Here, we report the development of a transcriptome-based 105,000-probe oil palm mesocarp microarray. The expression of genes involved in fatty acid (FA) and triacylglycerol (TAG) assembly, along with the tricarboxylic acid cycle (TCA) and glycolysis pathway at 16 Weeks After Anthesis (WAA) exhibited significantly higher signals compared to those obtained from a cross-species hybridization to the Arabidopsis (p-value < 0.01), and rice (p-value < 0.01) arrays. The oil palm microarray data also showed comparable correlation of expression (r2 = 0.569, p < 0.01) throughout mesocarp development to transcriptome (RNA sequencing) data, and improved correlation over quantitative real-time PCR (qPCR) (r2 = 0.721, p < 0.01) of the same RNA samples. The results confirm the advantage of the custom microarray over commercially available arrays derived from model species. We demonstrate the utility of this custom microarray to gain a better understanding of gene expression patterns in the oil palm mesocarp that may lead to increasing future oil yield. PMID:27600348

Expression Comparison of Oil Biosynthesis Genes in Oil Palm Mesocarp Tissue Using Custom Array.

PubMed

Wong, Yick Ching; Kwong, Qi Bin; Lee, Heng Leng; Ong, Chuang Kee; Mayes, Sean; Chew, Fook Tim; Appleton, David R; Kulaveerasingam, Harikrishna

2014-11-13

Gene expression changes that occur during mesocarp development are a major research focus in oil palm research due to the economic importance of this tissue and the relatively rapid increase in lipid content to very high levels at fruit ripeness. Here, we report the development of a transcriptome-based 105,000-probe oil palm mesocarp microarray. The expression of genes involved in fatty acid (FA) and triacylglycerol (TAG) assembly, along with the tricarboxylic acid cycle (TCA) and glycolysis pathway at 16 Weeks After Anthesis (WAA) exhibited significantly higher signals compared to those obtained from a cross-species hybridization to the Arabidopsis (p-value < 0.01), and rice (p-value < 0.01) arrays. The oil palm microarray data also showed comparable correlation of expression (r² = 0.569, p < 0.01) throughout mesocarp development to transcriptome (RNA sequencing) data, and improved correlation over quantitative real-time PCR (qPCR) (r² = 0.721, p < 0.01) of the same RNA samples. The results confirm the advantage of the custom microarray over commercially available arrays derived from model species. We demonstrate the utility of this custom microarray to gain a better understanding of gene expression patterns in the oil palm mesocarp that may lead to increasing future oil yield.

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

Low Altitude Near-the-Horizon Propagation: A Comparison Between RPO and M-Layer

DTIC Science & Technology

1993-12-01

scaling based on the assumption that a single mode contributes to the complete field strength (Ref. 31, output from M-Layer [Ref. 4, 5] in the over-the...PE. The parabolic equation approximation to the Maxwell wave equations is developed under the optical assumption that the operating frequency is so...profile data are specified (an array) capm zim profile data (modified index of refraction; an array) (a) RPO: from I to n/evs; M-Layer from 0 to nzlayr

The 'PUCE CAFE' Project: the First 15K Coffee Microarray, a New Tool for Discovering Candidate Genes correlated to Agronomic and Quality Traits

PubMed Central

2011-01-01

Background Understanding the genetic elements that contribute to key aspects of coffee biology will have an impact on future agronomical improvements for this economically important tree. During the past years, EST collections were generated in Coffee, opening the possibility to create new tools for functional genomics. Results The "PUCE CAFE" Project, organized by the scientific consortium NESTLE/IRD/CIRAD, has developed an oligo-based microarray using 15,721 unigenes derived from published coffee EST sequences mostly obtained from different stages of fruit development and leaves in Coffea Canephora (Robusta). Hybridizations for two independent experiments served to compare global gene expression profiles in three types of tissue matter (mature beans, leaves and flowers) in C. canephora as well as in the leaves of three different coffee species (C. canephora, C. eugenoides and C. arabica). Microarray construction, statistical analyses and validation by Q-PCR analysis are presented in this study. Conclusion We have generated the first 15 K coffee array during this PUCE CAFE project, granted by Génoplante (the French consortium for plant genomics). This new tool will help study functional genomics in a wide range of experiments on various plant tissues, such as analyzing bean maturation or resistance to pathogens or drought. Furthermore, the use of this array has proven to be valid in different coffee species (diploid or tetraploid), drastically enlarging its impact for high-throughput gene expression in the community of coffee research. PMID:21208403

The 'PUCE CAFE' Project: the first 15K coffee microarray, a new tool for discovering candidate genes correlated to agronomic and quality traits.

PubMed

Privat, Isabelle; Bardil, Amélie; Gomez, Aureliano Bombarely; Severac, Dany; Dantec, Christelle; Fuentes, Ivanna; Mueller, Lukas; Joët, Thierry; Pot, David; Foucrier, Séverine; Dussert, Stéphane; Leroy, Thierry; Journot, Laurent; de Kochko, Alexandre; Campa, Claudine; Combes, Marie-Christine; Lashermes, Philippe; Bertrand, Benoit

2011-01-05

Understanding the genetic elements that contribute to key aspects of coffee biology will have an impact on future agronomical improvements for this economically important tree. During the past years, EST collections were generated in Coffee, opening the possibility to create new tools for functional genomics. The "PUCE CAFE" Project, organized by the scientific consortium NESTLE/IRD/CIRAD, has developed an oligo-based microarray using 15,721 unigenes derived from published coffee EST sequences mostly obtained from different stages of fruit development and leaves in Coffea Canephora (Robusta). Hybridizations for two independent experiments served to compare global gene expression profiles in three types of tissue matter (mature beans, leaves and flowers) in C. canephora as well as in the leaves of three different coffee species (C. canephora, C. eugenoides and C. arabica). Microarray construction, statistical analyses and validation by Q-PCR analysis are presented in this study. We have generated the first 15 K coffee array during this PUCE CAFE project, granted by Génoplante (the French consortium for plant genomics). This new tool will help study functional genomics in a wide range of experiments on various plant tissues, such as analyzing bean maturation or resistance to pathogens or drought. Furthermore, the use of this array has proven to be valid in different coffee species (diploid or tetraploid), drastically enlarging its impact for high-throughput gene expression in the community of coffee research.

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.

Integrated Avalanche Photodiode arrays

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

Harmon, Eric S.

2017-04-18

The present disclosure includes devices for detecting photons, including avalanche photon detectors, arrays of such detectors, and circuits including such arrays. In some aspects, the detectors and arrays include a virtual beveled edge mesa structure surrounded by resistive material damaged by ion implantation and having side wall profiles that taper inwardly towards the top of the mesa structures, or towards the direction from which the ion implantation occurred. Other aspects are directed to masking and multiple implantation and/or annealing steps. Furthermore, methods for fabricating and using such devices, circuits and arrays are disclosed.

Integrated avalanche photodiode arrays

DOEpatents

Harmon, Eric S.

2015-07-07

The present disclosure includes devices for detecting photons, including avalanche photon detectors, arrays of such detectors, and circuits including such arrays. In some aspects, the detectors and arrays include a virtual beveled edge mesa structure surrounded by resistive material damaged by ion implantation and having side wall profiles that taper inwardly towards the top of the mesa structures, or towards the direction from which the ion implantation occurred. Other aspects are directed to masking and multiple implantation and/or annealing steps. Furthermore, methods for fabricating and using such devices, circuits and arrays are disclosed.

Linear array measurements of enhanced dynamic wedge and treatment planning system (TPS) calculation for 15 MV photon beam and comparison with electronic portal imaging device (EPID) measurements.

PubMed

Petrovic, Borislava; Grzadziel, Aleksandra; Rutonjski, Laza; Slosarek, Krzysztof

2010-09-01

Enhanced dynamic wedges (EDW) are known to increase drastically the radiation therapy treatment efficiency. This paper has the aim to compare linear array measurements of EDW with the calculations of treatment planning system (TPS) and the electronic portal imaging device (EPID) for 15 MV photon energy. The range of different field sizes and wedge angles (for 15 MV photon beam) were measured by the linear chamber array CA 24 in Blue water phantom. The measurement conditions were applied to the calculations of the commercial treatment planning system XIO CMS v.4.2.0 using convolution algorithm. EPID measurements were done on EPID-focus distance of 100 cm, and beam parameters being the same as for CA24 measurements. Both depth doses and profiles were measured. EDW linear array measurements of profiles to XIO CMS TPS calculation differ around 0.5%. Profiles in non-wedged direction and open field profiles practically do not differ. Percentage depth doses (PDDs) for all EDW measurements show the difference of not more than 0.2%, while the open field PDD is almost the same as EDW PDD. Wedge factors for 60 deg wedge angle were also examined, and the difference is up to 4%. EPID to linear array differs up to 5%. The implementation of EDW in radiation therapy treatments provides clinicians with an effective tool for the conformal radiotherapy treatment planning. If modelling of EDW beam in TPS is done correctly, a very good agreement between measurements and calculation is obtained, but EPID cannot be used for reference measurements.

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

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

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

Monitoring pressure profiles across an airfoil with a fiber Bragg grating sensor array

NASA Astrophysics Data System (ADS)

Papageorgiou, Anthony W.; Parkinson, Luke A.; Karas, Andrew R.; Hansen, Kristy L.; Arkwright, John W.

2018-02-01

Fluid flow over an airfoil section creates a pressure difference across the upper and lower surfaces, thus generating lift. Successful wing design is a combination of engineering design and experience in the field, with subtleties in design and manufacture having significant impact on the amount of lift produced. Current methods of airfoil optimization and validation typically involve computational fluid dynamics (CFD) and extensive wind tunnel testing with pressure sensors embedded into the airfoil to measure the pressure over the wing. Monitoring pressure along an airfoil in a wind tunnel is typically achieved using surface pressure taps that consist of hollow tubes running from the surface of the airfoil to individual pressure sensors external to the tunnel. These pressure taps are complex to configure and not ideal for in-flight testing. Fiber Bragg grating (FBG) pressure sensing arrays provide a highly viable option for both wind tunnel and inflight pressure measurement. We present a fiber optic sensor array that can detect positive and negative pressure suitable for validating CFD models of airfoil profile sections. The sensing array presented here consists of 6 independent sensing elements, each capable of a pressure resolution of less than 10 Pa over the range of 70 kPa to 120 kPa. The device has been tested with the sensor array attached to a 90mm chord length airfoil section subjected to low velocity flow. Results show that the arrays are capable of accurately detecting variations of the pressure profile along the airfoil as the angle of attack is varied from zero to the point at which stall occurs.

Comparison of X-ray Radiation Process in Single and Nested Wire Array Implosions

NASA Astrophysics Data System (ADS)

Li, Z. H.; Xu, Z. P.; Yang, J. L.; Xu, R. K.; Guo, C.; Grabovsky, E. V.; Oleynic, G. M.; Smirnov, V. P.

2006-01-01

In order to understanding the difference between tungsten single-wire-array and tungsten nested-wire-array Z-pinches, we have measured the x-ray power, the temporal-spatial distributions of x-ray radiation from each of the two loads. The measurements were performed with 0.1mm spatial and 1 ns temporal resolutions at 2.5- and 3.5-MA currents. The experimental conditions, including wire material, number of wires, wire-array length, electrode design, and implosion time, remained unchanged from shot to shot. Analysis of the radiation power profiles suggests that the nested-wire-array radiate slightly less x-ray energy in relatively shorter time interval than the single wire-array, leading to a much greater x-ray power in nested-wire-array implosion. The temporal-spatial distributions of x-ray power show that in both cases, plasmas formed by wire-array ablation radiate not simultaneously along load axis. For nested-wire-array Z-pinch, plasmas near the anode begin to radiate in 2ns later than that near the cathode. As a contrast, the temporal divergence of radiation among different plasma zones of single-wire-array Z-pinch along Z-axis is more than 6ns. Measurements of the x-ray emissions from small segments of pinch (2mm length along axis) indicate that local radiation power profiles almost do not vary for the two loads. Photographs taken by X-ray framing camera give a same description about the radiation process of pinch. One may expect that, as a result of this study, if the single-wire-array can be redesigned so ingeniously that the x-rays are emitted at the same time all over the pinch zone, the radiation power of single wire array Z-pinch may be much greater than what have been achieved.

Dimensionality of Data Matrices with Applications to Gene Expression Profiles

ERIC Educational Resources Information Center

Feng, Xingdong

2009-01-01

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

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

PubMed

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

2014-06-07

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

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.

Identification of the Corn Pathogen Pantoea stewartii by Mass Spectrometry of Whole-Cell Extracts and Its Detection with Novel PCR Primers ▿

PubMed Central

Wensing, Annette; Zimmermann, Stefan; Geider, Klaus

2010-01-01

Pantoea stewartii subsp. stewartii is the causative agent of Stewart's wilt, a bacterial disease transmitted by the corn flea beetle mainly to sweet corn (Zea mays). In many countries, it is classified as a quarantine organism and must be differentiated from other yellow enteric bacteria frequently occurring with corn. We have created novel primers from the pstS-glmS region of P. stewartii for use in conventional PCR (cPCR) and quantitative PCR (qPCR). To facilitate rapid diagnosis, we applied matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Using whole-cell protein extracts, profiles were generated with a Bruker microflex machine, and the bacteria classified. P. stewartii strains were clearly distinguished from strains of Pantoea agglomerans, Pantoea dispersa, and Pantoea ananatis. Dendrogram analysis of the protein profiles confirmed the score values and showed the formation of separate clades for each species. The identification achieved by MALDI-TOF MS analysis agrees with the diagnosis by specific PCR primers. The combination of both methods allows a rapid and simple identification of the corn pathogen. P. stewartii subsp. stewartii and P. stewartii subsp. indologenes are highly related and can be distinguished not only by virulence assays and indole tests but also by a characteristic pattern in the nucleotide sequence of recA. PMID:20656863

Complementary techniques: validation of gene expression data by quantitative real time PCR.

PubMed

Provenzano, Maurizio; Mocellin, Simone

2007-01-01

Microarray technology can be considered the most powerful tool for screening gene expression profiles of biological samples. After data mining, results need to be validated with highly reliable biotechniques allowing for precise quantitation of transcriptional abundance of identified genes. Quantitative real time PCR (qrt-PCR) technology has recently reached a level of sensitivity, accuracy and practical ease that support its use as a routine bioinstrumentation for gene level measurement. Currently, qrt-PCR is considered by most experts the most appropriate method to confirm or confute microarray-generated data. The knowledge of the biochemical principles underlying qrt-PCR as well as some related technical issues must be beard in mind when using this biotechnology.

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.

Antibody profiling sensitivity through increased reporter antibody layering

DOEpatents

Apel, William A.; Thompson, Vicki S.

2013-02-26

A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

Antibody profiling sensitivity through increased reporter antibody layering

DOEpatents

Apel, William A.; Thompson, Vicki S.

2017-03-28

A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

LDL oxidation by THP-1 monocytes: implication of HNP-1, SgIII and DMT-1.

PubMed

He, Chunyan; Huang, Rui; Du, Fen; Zheng, Fang; Wei, Lei; Wu, Junzhu

2009-04-01

Oxidized low-density lipoprotein (oxLDL) plays an important role in the pathogenesis of atherosclerosis. However, the mechanisms of the initiation and progression of LDL oxidation by cells are still unknown. We investigated the molecular mechanism underlying THP-1 cell-mediated LDL oxidation. LDL oxidation was monitored at 234 nm by detecting the formation of conjugated dienes. cDNA array analysis was applied to profile changes in gene expression of human THP-1 monocytes in response to LDL stimulation. The mRNA and protein levels of secretogranin III (SgIII), divalent metal transporter (DMT-1) and human alpha-defensin 1 (HNP-1) were determined by real-time RT-PCR and Western blotting respectively. Eukaryotic expression vectors containing full-length cDNA sequence of HNP-1 (pEGFP-C1/HNP-1) SgIII (pEGFP-C1/SgIII) or DMT-1 (pEGFP-C1/DMT-1) were constructed and transfected to THP-1 cells. The effects of overexpression of these three genes on THP-1 cell-mediated LDL oxidation were observed. LDL oxidation was most pronounced after LDL was incubated with THP-1 cells for 9 h. 1651 genes in total were detected by cDNA array analysis in THP-1 cells with or without LDL treatment for 9 h. Thirteen genes with >2-fold relative expression difference were identified, including nine genes whose expression was up-regulated and four genes whose expression was down-regulated. Among the up-regulated genes, SgIII, DMT-1 and HNP-1 were reported to be associated with atherosclerosis. The increased mRNA expressions of these three genes were confirmed by real-time RT-PCR. Western blotting analysis demonstrated that protein expressions of SgIII and DMT-1 were also enhanced in THP-1 cells in response to LDL. Furthermore, transient overexpression of HNP-1, SgIII or DMT-1 in THP-1 cells significantly increased THP-1 cell-mediated LDL oxidation. Our data suggest that SgIII, DMT-1 and HNP-1 are implicated in cell-mediated LDL oxidation.

Verification of intensity modulated profiles using a pixel segmented liquid-filled linear array.

PubMed

Pardo, J; Roselló, J V; Sánchez-Doblado, F; Gómez, F

2006-06-07

A liquid isooctane (C8H18) filled ionization chamber linear array developed for radiotherapy quality assurance, consisting of 128 pixels (each of them with a 1.7 mm pitch), has been used to acquire profiles of several intensity modulated fields. The results were compared with film measurements using the gamma test. The comparisons show a very good matching, even in high gradient dose regions. The volume-averaging effect of the pixels is negligible and the spatial resolution is enough to verify these regions. However, some mismatches between the detectors have been found in regions where low-energy scattered photons significantly contribute to the total dose. These differences are not very important (in fact, the measurements of both detectors are in agreement using the gamma test with tolerances of 3% and 3 mm in most of those regions), and may be associated with the film energy dependence. In addition, the linear array repeatability (0.27% one standard deviation) is much better than the film one ( approximately 3%). The good repeatability, small pixel size and high spatial resolution make the detector ideal for the real time profile verification of high gradient beam profiles like those present in intensity modulated radiation therapy and radiosurgery.

Prediction of tolerance in children with IgE mediated cow's milk allergy by microarray profiling and chemometric approach.

PubMed

Wulfert, F; Sanyasi, G; Tongen, L; Watanabe, L A; Wang, X; Renault, N K; Falcone, F H; Jacob, C M A; Alcocer, M J C

2012-08-31

The sera of a retrospective cohort (n=41) composed of children with well characterized cow's milk allergy collected from multiple visits were analyzed using a protein microarray system measuring four classes of immunoglobulins. The frequency of the visits, age and gender distribution reflected real situation faced by the clinicians at a pediatric reference center for food allergy in São Paulo, Brazil. The profiling array results have shown that total IgG and IgA share similar specificity whilst IgM and in particular IgE are distantly related. The correlation of specificity of IgE and IgA is variable amongst the patients and this relationship cannot be used to predict atopy or the onset of tolerance to milk. The array profiling technique has corroborated the clinical selection criteria for this cohort albeit it clearly suggested that 4 out of the 41 patients might have allergies other than milk origin. There was also a good correlation between the array data and ImmunoCAP results, casein in particular. By using qualitative and quantitative multivariate analysis routines it was possible to produce validated statistical models to predict with reasonable accuracy the onset of tolerance to milk proteins. If expanded to larger study groups, the array profiling in combination with the multivariate techniques show potential to improve the prognostic of milk allergic patients. Copyright © 2012 Elsevier B.V. All rights reserved.

Investigation of anodic TiO2 nanotube composition with high spatial resolution AES and ToF SIMS

NASA Astrophysics Data System (ADS)

Dronov, Alexey; Gavrilin, Ilya; Kirilenko, Elena; Dronova, Daria; Gavrilov, Sergey

2018-03-01

High resolution Scanning Auger Electron Spectroscopy (AES) and Time-of-Flight Secondary Ion Mass-Spectrometry (ToF SIMS) were used to investigate structure and elemental composition variation of both across an array of TiO2 nanotubes (NTs) and single tube of an array. The TiO2 NT array was grown by anodic oxidation of Ti foil in fluorine-containing ethylene glycol electrolyte. It was found that the studied anodic TiO2 nanotubes have a layered structure with rather sharp interfaces. The differences in AES depth profiling results of a single tube with the focused primary electron beam (point analysis) and over an area of 75 μm in diameter of a nanotube array with the defocused primary electron beam are discussed. Depth profiling by ToF SIMS was carried out over approximately the same size of a nanotube array to determine possible ionic fragments in the structure. The analysis results show that the combination of both mentioned methods is useful for a detailed analysis of nanostructures with complex morphology and multi-layered nature.

A comprehensive assessment of RNA-seq accuracy, reproducibility and information content by the Sequencing Quality Control consortium

PubMed Central

2014-01-01

We present primary results from the Sequencing Quality Control (SEQC) project, coordinated by the United States Food and Drug Administration. Examining Illumina HiSeq, Life Technologies SOLiD and Roche 454 platforms at multiple laboratory sites using reference RNA samples with built-in controls, we assess RNA sequencing (RNA-seq) performance for junction discovery and differential expression profiling and compare it to microarray and quantitative PCR (qPCR) data using complementary metrics. At all sequencing depths, we discover unannotated exon-exon junctions, with >80% validated by qPCR. We find that measurements of relative expression are accurate and reproducible across sites and platforms if specific filters are used. In contrast, RNA-seq and microarrays do not provide accurate absolute measurements, and gene-specific biases are observed, for these and qPCR. Measurement performance depends on the platform and data analysis pipeline, and variation is large for transcript-level profiling. The complete SEQC data sets, comprising >100 billion reads (10Tb), provide unique resources for evaluating RNA-seq analyses for clinical and regulatory settings. PMID:25150838

Global preamplification simplifies targeted mRNA quantification

PubMed Central

Kroneis, Thomas; Jonasson, Emma; Andersson, Daniel; Dolatabadi, Soheila; Ståhlberg, Anders

2017-01-01

The need to perform gene expression profiling using next generation sequencing and quantitative real-time PCR (qPCR) on small sample sizes and single cells is rapidly expanding. However, to analyse few molecules, preamplification is required. Here, we studied global and target-specific preamplification using 96 optimised qPCR assays. To evaluate the preamplification strategies, we monitored the reactions in real-time using SYBR Green I detection chemistry followed by melting curve analysis. Next, we compared yield and reproducibility of global preamplification to that of target-specific preamplification by qPCR using the same amount of total RNA. Global preamplification generated 9.3-fold lower yield and 1.6-fold lower reproducibility than target-specific preamplification. However, the performance of global preamplification is sufficient for most downstream applications and offers several advantages over target-specific preamplification. To demonstrate the potential of global preamplification we analysed the expression of 15 genes in 60 single cells. In conclusion, we show that global preamplification simplifies targeted gene expression profiling of small sample sizes by a flexible workflow. We outline the pros and cons for global preamplification compared to target-specific preamplification. PMID:28332609

Digital RNA sequencing minimizes sequence-dependent bias and amplification noise with optimized single-molecule barcodes

PubMed Central

Shiroguchi, Katsuyuki; Jia, Tony Z.; Sims, Peter A.; Xie, X. Sunney

2012-01-01

RNA sequencing (RNA-Seq) is a powerful tool for transcriptome profiling, but is hampered by sequence-dependent bias and inaccuracy at low copy numbers intrinsic to exponential PCR amplification. We developed a simple strategy for mitigating these complications, allowing truly digital RNA-Seq. Following reverse transcription, a large set of barcode sequences is added in excess, and nearly every cDNA molecule is uniquely labeled by random attachment of barcode sequences to both ends. After PCR, we applied paired-end deep sequencing to read the two barcodes and cDNA sequences. Rather than counting the number of reads, RNA abundance is measured based on the number of unique barcode sequences observed for a given cDNA sequence. We optimized the barcodes to be unambiguously identifiable, even in the presence of multiple sequencing errors. This method allows counting with single-copy resolution despite sequence-dependent bias and PCR-amplification noise, and is analogous to digital PCR but amendable to quantifying a whole transcriptome. We demonstrated transcriptome profiling of Escherichia coli with more accurate and reproducible quantification than conventional RNA-Seq. PMID:22232676

Toll like receptors gene expression of human keratinocytes cultured of severe burn injury.

PubMed

Cornick, Sarita Mac; Noronha, Silvana Aparecida Alves Corrêa de; Noronha, Samuel Marcos Ribeiro de; Cezillo, Marcus V B; Ferreira, Lydia Masako; Gragnani, Alfredo

2014-01-01

To evaluate the expression profile of genes related to Toll Like Receptors (TLR) pathways of human Primary Epidermal keratinocytes of patients with severe burns. After obtaining viable fragments of skin with and without burning, culture hKEP was initiated by the enzymatic method using Dispase (Sigma-Aldrich). These cells were treated with Trizol(r) (Life Technologies) for extraction of total RNA. This was quantified and analyzed for purity for obtaining cDNA for the analysis of gene expression using specific TLR pathways PCR Arrays plates (SA Biosciences). After the analysis of gene expression we found that 21% of these genes were differentially expressed, of which 100% were repressed or hyporegulated. Among these, the following genes (fold decrease): HSPA1A (-58), HRAS (-36), MAP2K3 (-23), TOLLIP (-23), RELA (-18), FOS (-16), and TLR1 (-6.0). This study contributes to the understanding of the molecular mechanisms related to TLR pathways and underlying wound infection caused by the burn. Furthermore, it may provide new strategies to restore normal expression of these genes and thereby change the healing process and improve clinical outcome.

Estrogen effects on cognition and hippocampal transcription in middle-aged mice.

PubMed

Aenlle, Kristina K; Kumar, Ashok; Cui, Li; Jackson, Travis C; Foster, Thomas C

2009-06-01

Young and middle-aged female mice were ovariectomized and given cyclic injections of either estradiol or vehicle treatments. During the fifth week after surgery the Morris water maze was used to assess cognitive function. Age and treatment effects emerged over the course of spatial training such that middle-aged vehicle treated mice exhibited deficits in acquiring a spatial search strategy compared to younger vehicle treated mice and middle-age estradiol treated mice. Following behavioral characterization, mice were maintained on their injection schedule until week seven and hippocampi were collected 24h after the last injection. Hippocampal RNA was extracted and genes responsive to age and estrogen were identified using cDNA microarrays. Estradiol treatment in middle-aged mice altered the expression of genes related to transcriptional regulation, biosynthesis, growth, neuroprotection, and elements of cell signaling pathways. Expression profiles for representative genes were confirmed in a separate set of animals using oligonucleotide arrays and RT-PCR. Our results indicate that estrogen treatment in middle-aged animals may promote hippocampal health during the aging process.

Triheptanoin improves brain energy metabolism in patients with Huntington disease

PubMed Central

Adanyeguh, Isaac Mawusi; Rinaldi, Daisy; Henry, Pierre-Gilles; Caillet, Samantha; Valabregue, Romain; Durr, Alexandra

2015-01-01

Objective: Based on our previous work in Huntington disease (HD) showing improved energy metabolism in muscle by providing substrates to the Krebs cycle, we wished to obtain a proof-of-concept of the therapeutic benefit of triheptanoin using a functional biomarker of brain energy metabolism validated in HD. Methods: We performed an open-label study using 31P brain magnetic resonance spectroscopy (MRS) to measure the levels of phosphocreatine (PCr) and inorganic phosphate (Pi) before (rest), during (activation), and after (recovery) a visual stimulus. We performed 31P brain MRS in 10 patients at an early stage of HD and 13 controls. Patients with HD were then treated for 1 month with triheptanoin after which they returned for follow-up including 31P brain MRS scan. Results: At baseline, we confirmed an increase in Pi/PCr ratio during brain activation in controls—reflecting increased adenosine triphosphate synthesis—followed by a return to baseline levels during recovery (p = 0.013). In patients with HD, we validated the existence of an abnormal brain energy profile as previously reported. After 1 month, this profile remained abnormal in patients with HD who did not receive treatment. Conversely, the MRS profile was improved in patients with HD treated with triheptanoin for 1 month with the restoration of an increased Pi/PCr ratio during visual stimulation (p = 0.005). Conclusion: This study suggests that triheptanoin is able to correct the bioenergetic profile in the brain of patients with HD at an early stage of the disease. Classification of evidence: This study provides Class III evidence that, for patients with HD, treatment with triheptanoin for 1 month restores an increased MRS Pi/PCr ratio during visual stimulation. PMID:25568297

Transcript and metabolite analysis in Trincadeira cultivar reveals novel information regarding the dynamics of grape ripening.

PubMed

Fortes, Ana M; Agudelo-Romero, Patricia; Silva, Marta S; Ali, Kashif; Sousa, Lisete; Maltese, Federica; Choi, Young H; Grimplet, Jerome; Martinez-Zapater, José M; Verpoorte, Robert; Pais, Maria S

2011-11-02

Grapes (Vitis vinifera L.) are economically the most important fruit crop worldwide. However, the complexity of molecular and biochemical events that lead to the onset of ripening of nonclimacteric fruits is not fully understood which is further complicated in grapes due to seasonal and cultivar specific variation. The Portuguese wine variety Trincadeira gives rise to high quality wines but presents extremely irregular berry ripening among seasons probably due to high susceptibility to abiotic and biotic stresses. Ripening of Trincadeira grapes was studied taking into account the transcriptional and metabolic profilings complemented with biochemical data. The mRNA expression profiles of four time points spanning developmental stages from pea size green berries, through véraison and mature berries (EL 32, EL 34, EL 35 and EL 36) and in two seasons (2007 and 2008) were compared using the Affymetrix GrapeGen® genome array containing 23096 probesets corresponding to 18726 unique sequences. Over 50% of these probesets were significantly differentially expressed (1.5 fold) between at least two developmental stages. A common set of modulated transcripts corresponding to 5877 unigenes indicates the activation of common pathways between years despite the irregular development of Trincadeira grapes. These unigenes were assigned to the functional categories of "metabolism", "development", "cellular process", "diverse/miscellanenous functions", "regulation overview", "response to stimulus, stress", "signaling", "transport overview", "xenoprotein, transposable element" and "unknown". Quantitative RT-PCR validated microarrays results being carried out for eight selected genes and five developmental stages (EL 32, EL 34, EL 35, EL 36 and EL 38). Metabolic profiling using 1H NMR spectroscopy associated to two-dimensional techniques showed the importance of metabolites related to oxidative stress response, amino acid and sugar metabolism as well as secondary metabolism. These results were integrated with transcriptional profiling obtained using genome array to provide new information regarding the network of events leading to grape ripening. Altogether the data obtained provides the most extensive survey obtained so far for gene expression and metabolites accumulated during grape ripening. Moreover, it highlighted information obtained in a poorly known variety exhibiting particular characteristics that may be cultivar specific or dependent upon climatic conditions. Several genes were identified that had not been previously reported in the context of grape ripening namely genes involved in carbohydrate and amino acid metabolisms as well as in growth regulators; metabolism, epigenetic factors and signaling pathways. Some of these genes were annotated as receptors, transcription factors, and kinases and constitute good candidates for functional analysis in order to establish a model for ripening control of a non-climacteric fruit.

Multiplex PCR Tests for Detection of Pathogens Associated with Gastroenteritis

PubMed Central

Zhang, Hongwei; Morrison, Scott; Tang, Yi-Wei

2016-01-01

Synopsis A wide range of enteric pathogens can cause infectious gastroenteritis. Conventional diagnostic algorithms including culture, biochemical identification, immunoassay and microscopic examination are time consuming and often lack sensitivity and specificity. Advances in molecular technology have as allowed its use as clinical diagnostic tools. Multiplex PCR based testing has made its way to gastroenterology diagnostic arena in recent years. In this article we present a review of recent laboratory developed multiplex PCR tests and current commercial multiplex gastrointestinal pathogen tests. We will focus on two FDA cleared commercial syndromic multiplex tests: Luminex xTAG GPP and Biofire FimArray GI test. These multiplex tests can detect and identify multiple enteric pathogens in one test and provide results within hours. Multiplex PCR tests have shown superior sensitivity to conventional methods for detection of most pathogens. The high negative predictive value of these multiplex tests has led to the suggestion that they be used as screening tools especially in outbreaks. Although the clinical utility and benefit of multiplex PCR test are to be further investigated, implementing these multiplex PCR tests in gastroenterology diagnostic algorithm has the potential to improve diagnosis of infectious gastroenteritis. PMID:26004652

New phenolic components and chromatographic profiles of green and fermented teas

USDA-ARS?s Scientific Manuscript database

A standardized profiling method based on liquid chromatography with diode array and electrospray ionization/mass spectrometric detection (LC-DAD-ESI/MS) was applied to establish the phenolic profiles of 41 green teas and 25 fermented teas. More than 80 phenolic compounds were either identified that ...

A Droplet Digital PCR Method for Severe Combined Immunodeficiency Newborn Screening.

PubMed

Vidal-Folch, Noemi; Milosevic, Dragana; Majumdar, Ramanath; Gavrilov, Dimitar; Matern, Dietrich; Raymond, Kimiyo; Rinaldo, Piero; Tortorelli, Silvia; Abraham, Roshini S; Oglesbee, Devin

2017-09-01

Severe combined immunodeficiency (SCID) benefits from early intervention via hematopoietic cell transplantation to reverse T-cell lymphopenia (TCL). Newborn screening (NBS) programs use T-cell receptor excision circle (TREC) levels to detect SCID. Real-time quantitative PCR is often performed to quantify TRECs in dried blood spots (DBSs) for NBS. Yet, real-time quantitative PCR has inefficiencies necessitating normalization, repeat analyses, or standard curves. To address these issues, we developed a multiplex, droplet digital PCR (ddPCR) method for measuring absolute TREC amounts in one DBS punch. TREC and RPP30 levels were simultaneously measured with a Bio-Rad AutoDG and QX200 ddPCR system. DBSs from 610 presumed-normal, 29 lymphocyte-profiled, and 10 clinically diagnosed infants (1 X-linked SCID, 1 RAG1 Omenn syndrome, and other conditions) were tested. Control infants showed 14 to 474 TREC copies/μL blood. SCID infants, and other TCL conditions, had ≤15 TREC copies/μL. The ddPCR lower limit of quantitation was 14 TREC copies/μL, and the limit of detection was 4 TREC copies/μL. Intra-assay and interassay imprecision was <20% CV for DBSs at 54 to 60 TREC copies/μL. Testing 29 infants with known lymphocyte profiles resulted in a sensitivity of 88.9% and a specificity of 100% at TRECs <20 copies/μL. We developed a multiplex ddPCR method for the absolute quantitation of DBS TRECs that can detect SCID and other TCL conditions associated with absent or low TRECs and validated this method for NBS. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

High-throughput deep screening and identification of four peripheral leucocyte microRNAs as novel potential combination biomarkers for preeclampsia

PubMed Central

Wang, Yonghong; Yang, Xukui; Yang, Yuanyuan; Wang, Wenjun; Zhao, Meiling; Liu, Huiqiang; Li, Dongyan; Hao, Min

2016-01-01

Objective: To identify the specific microRNA (miRNA) biomarkers of preeclampsia (PE), the miRNA profiles analysis were performed. Study Design: The blood samples were obtained from five PE patients and five normal healthy pregnant women. The small RNA profiles were analyzed to identify miRNA expression levels and find out miRNAs that may associate with PE. The quantitative reverse transcriptase–PCR (qRT-PCR) assay was used to validate differentially expressed peripheral leucocyte miRNAs in a new cohort. Result: The data analysis showed that 10 peripheral leucocyte miRNAs were significantly differently expressed in severe PE patients. Four differently expressed miRNAs were successfully validated using qRT-PCR method. Conclusion: We successfully constructed a model with high accuracy to predict PE. A combination of four peripheral leucocyte miRNAs has great potential to serve as diagnostic biomarkers of PE. PMID:26675000

Wavelength shift in vertical cavity laser arrays on a patterned substrate

NASA Astrophysics Data System (ADS)

Eng, L. E.; Bacher, K.; Yuen, W.; Larson, M.; Ding, G.; Harris, J. S., Jr.; Chang-Hasnain, C. J.

1995-03-01

The authors demonstrate a spatially chirped emission wavelength in vertical cavity surface emitting laser (VCSEL) arrays grown by molecular beam epitaxy. The wavelength shift is due to a lateral thickness variation in the Al(0.2)Ga(0.8)As cavity, which is induced by a substrate temperature profile during growth. A 20 nm shift in lasing wavelength is obtained in a VCSEL array.

Comparison of STR profiling from low template DNA extracts with and without the consensus profiling method

PubMed Central

2012-01-01

Background The consensus profiling method was introduced to overcome the exaggerated stochastic effects associated with low copy number DNA typing. However, little empirical evidence has been provided which shows that a consensus profile, derived from dividing a sample into separate aliquots and including only alleles seen at least twice, gives the most informative profile, compared to a profile obtained by amplifying the entire low template DNA extract in one reaction. Therefore, this study aimed to investigate the quality of consensus profiles compared to profiles obtained using the whole low template extract for amplification. Methods A total of 100 pg and 25 pg DNA samples were amplified with the PowerPlex® ESI 16 Kits using 30 or 34 PCR cycles. A total of 100 pg and 25 pg DNA samples were then divided into three aliquots for a 34-cycle PCR and a consensus profile derived that included alleles that appeared in at least two of the replicates. Profiles from the non-split samples were compared to the consensus profiles focusing on peak heights, allele drop out, locus drop out and allele drop in. Results Performing DNA profiling on non-split extracts produced profiles with a higher percentage of correct loci compared to the consensus profiling technique. Consensus profiling did eliminate any spurious alleles from the final profile. However, there was a notable increase in allele and locus drop out when a LTDNA sample was divided prior to amplification. Conclusions The loss of information that occurs when a sample is split for amplification indicates that consensus profiling may not be producing the most informative DNA profile for samples where the template amount is limited. PMID:22748106

Variant Profiling of Candidate Genes in Pancreatic Ductal Adenocarcinoma.

PubMed

Huang, Jiaqi; Löhr, Johannes-Matthias; Nilsson, Magnus; Segersvärd, Ralf; Matsson, Hans; Verbeke, Caroline; Heuchel, Rainer; Kere, Juha; Iafrate, A John; Zheng, Zongli; Ye, Weimin

2015-11-01

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis. Variant profiling is crucial for developing personalized treatment and elucidating the etiology of this disease. Patients with PDAC undergoing surgery from 2007 to 2012 (n = 73) were followed from diagnosis until death or the end of the study. We applied an anchored multiplex PCR (AMP)-based next-generation sequencing (NGS) method to a panel of 65 selected genes and assessed analytical performance by sequencing a quantitative multiplex DNA reference standard. In clinical PDAC samples, detection of low-level KRAS (Kirsten rat sarcoma viral oncogene homolog) mutations was validated by allele-specific PCR and digital PCR. We compared overall survival of patients according to KRAS mutation status by log-rank test and applied logistic regression to evaluate the association between smoking and tumor variant types. The AMP-based NGS method could detect variants with allele frequencies as low as 1% given sufficient sequencing depth (>1500×). Low-frequency KRAS G12 mutations (allele frequency 1%-5%) were all confirmed by allele-specific PCR and digital PCR. The most prevalent genetic alterations were in KRAS (78% of patients), TP53 (tumor protein p53) (25%), and SMAD4 (SMAD family member 4) (8%). Overall survival in T3-stage PDAC patients differed among KRAS mutation subtypes (P = 0.019). Transversion variants were more common in ever-smokers than in never-smokers (odds ratio 5.7; 95% CI 1.2-27.8). The AMP-based NGS method is applicable for profiling tumor variants. Using this approach, we demonstrated that in PDAC patients, KRAS mutant subtype G12V is associated with poorer survival, and that transversion variants are more common among smokers. © 2015 American Association for Clinical Chemistry.

Clonal spread and accumulation of β-lactam resistance determinants in Enterobacter aerogenes and Enterobacter cloacae complex isolates from infection and colonization in patients at a public hospital in Recife, Pernambuco, Brazil.

PubMed

Cabral, Adriane Borges; Maciel, Maria Amélia Vieira; Barros, Josineide Ferreira; Antunes, Marcelo Maranhão; Barbosa de Castro, Célia Maria Machado; Lopes, Ana Catarina Souza

2017-01-01

Enterobacter aerogenes and Enterobacter cloacae complex are the two species of this genus most involved in healthcare-associated infections that are ESBL and carbapenemase producers. This study characterized, phenotypically and genotypically, 51 isolates of E. aerogenes and E. cloacae complex originating from infection or colonization in patients admitted to a public hospital in Recife, Pernambuco, Brazil, by antimicrobial susceptibility profile, analysis of β-lactamase genes (blaTEM, blaSHV, blaCTX-M, blaKPC, blaVIM, blaIMP and blaSPM), PCR and DNA sequencing, plasmid profile and ERIC-PCR. In both species, the genes blaTEM, blaCTX-M and blaKPC were detected. The DNA sequencing confirmed the variants blaTEM-1, blaCTX-M-15 and blaKPC-2 in isolates. More than one gene conferring resistance in the isolates, including the detection of the three previously cited genes in strains isolated from infection sites, was observed. The detection of blaCTX-M was more frequent in isolates from infection sites than from colonization. The gene blaKPC predominated in E. cloacae complex isolates obtained from infections; however, in E. aerogenes isolates, it predominated in samples obtained from colonization. A clonal relationship among all of E. aerogenes isolates was detected by ERIC-PCR. The majority of E. cloacae complex isolates presented the same ERIC-PCR pattern. Despite the clonal relation presented by the isolates using ERIC-PCR, different plasmid and resistance profiles and several resistance genes were observed. The clonal dissemination and the accumulation of β-lactam resistance determinants presented by the isolates demonstrated the ability of E. aerogenes and E. cloacae complex, obtained from colonization and infection, to acquire and maintain different resistance genes.

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

PubMed

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

2008-10-06

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

Hydrogen sulphide increases hepatic differentiation of human tooth pulp stem cells compared with human bone marrow stem cells.

PubMed

Okada, M; Ishkitiev, N; Yaegaki, K; Imai, T; Tanaka, T; Fukuda, M; Ono, S; Haapasalo, M

2014-12-01

To determine the differences in stem cell properties, in hepatic differentiation and in the effects of hydrogen sulphide (H2 S) on hepatic differentiation between human bone marrow stem cells (hBMC) and stem cells from human exfoliated primary tooth pulp (SHED). CD117(+) cells were magnetically separated and subjected to hepatic differentiation. CD117(+) cell lineages were characterized for transcription factors indicative of stem cells by qRT-PCR. For the last 9 days of the differentiation, the test cells were exposed to 0.1 ng mL(-1) H2 S. Immunocytochemistry and flow cytometry of albumin, alpha-fetoprotein and carbamoyl phosphate synthetase were carried out after differentiation. Urea concentration and glycogen synthesis were also determined. Genes expressed in SHED were also expressed in BMC. No difference in expression level of hepatic markers was shown by immunofluorescence. SHED showed more positive cells than hBMC (P < 0.01). H2 S increased the number of positive cells in both cultures (P < 0.01). Urea concentration and glycogen synthesis increased significantly after H2 S exposure (P < 0.001 and P < 0.05, respectively). Real-time PCR data were analysed by RT(2) profiler RT-PCR Array Data Analysis version 3.5 (Qiagen), and ELISA data were analysed by Bonferroni's multiple comparison using Windows spss version 16 (SPSS Inc, Chicago, IL, USA). Bonferroni's multiple comparison test was also carried out after angle transformation for the percentage data of flow cytometer using Windows spss(®) version 16 (SPSS Inc). Statistical significance was accepted at P < 0.05. Stem cells from human exfoliated primary tooth pulp and BMC have similar properties. The level of hepatic differentiation in SHED compared with BMC was the same or higher. H2 S increased the level of hepatic differentiation. © 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Transcriptome Analysis of the Dihydrotestosterone-Exposed Fetal Rat Gubernaculum Identifies Common Androgen and Insulin-Like 3 Targets1

PubMed Central

Barthold, Julia S.; Wang, Yanping; Robbins, Alan; Pike, Jack; McDowell, Erin; Johnson, Kamin J.; McCahan, Suzanne M.

2013-01-01

ABSTRACT Androgens and insulin-like 3 (INSL3) are required for development of the fetal gubernaculum and testicular descent. Previous studies suggested that the INSL3-exposed fetal gubernacular transcriptome is enriched for genes involved in neural pathways. In the present study, we profiled the transcriptome of fetal gubernaculum explants exposed to dihydrotestosterone (DHT) and compared this response to that with INSL3. We exposed fetal (Embryonic Day 17) rat gubernacula to DHT for 24 h (10 and 30 nM) or 6 h (1 and 10 nM) in organ culture and analyzed gene expression relative to that of vehicle-treated controls using Affymetrix arrays. Results were annotated using functional, pathway, and promoter analyses and independently validated for selected transcripts using quantitative RT-PCR (qRT-PCR). Transcripts were differentially expressed after 24 h but not 6 h. Most highly overrepresented functional categories included those related to gene expression, skeletal and muscular development and function, and Wnt signaling. Promoter response elements enriched in the DHT-specific transcriptome included consensus sequences for c-ETS1, ELK1, CREB, CRE-BP1/c-June, NRF2, and USF. We observed that 55% of DHT probe sets were also differentially expressed after INSL3 exposure and that the direction of change was the same in 96%. The qRT-PCR results confirmed that DHT increased expression of the INSL3-responsive genes Crlf1 and Chrdl2 but reduced expression of Wnt4. We also validated reduced Tgfb2 and Cxcl12 and increased Slit3 expression following DHT exposure. These data suggest a robust overlap in the DHT- and INSL3-regulated transcriptome that may be mediated in part by CREB signaling and a common Wnt pathway response for both hormones in the fetal gubernaculum. PMID:24174575

Detection of qnrVC6, within a new genetic context, in a NDM-1-producing Citrobacter freundii clinical isolate from Uruguay.

PubMed

Bado, Inés; Ezdra, Romina Papa; Cordeiro, Nicolás; Outeda, Matilde; Caiata, Leticia; García-Fulgueiras, Virginia; Seija, Verónica; Vignoli, Rafael

2018-03-08

The objective of the present study was to characterise the mechanisms underlying quinolone and oxyimino-cephalosporin resistance in a Citrobacter freundii clinical isolate obtained from the ICU in Uruguay's University Hospital. Citrobacter freundii strain CF638 was isolated from a urine culture. Identification and susceptibility testing were performed using the VITEK ® 2 system, and MIC determination and disc diffusion assay, respectively. Resistance genes and mobile genetic elements were identified, by PCR and sequencing. Plasmid transfer was assessed by conjugation; plasmid, size was estimated by treatment with S1 and PFGE. Plasmid incompatibility, group and toxin-antitoxin systems were sought by PCR. Strain CF638 showed a multi-drug resistant profile, including, resistance to carbapenemes and quinolones. Transconjugant TcCF638, harbouring a ∼200 kb IncA/C plasmid, also showed resistance to all β-lactams, (except for aztreonam), and diminished susceptibility to ciprofloxacin. PCR, assays were positive for bla NDM-1 and qnrVC in CF638 and TcCF638. Two different class 1 integrons were detected, In127 and In907. In127, featured the genetic array: aadA2-ltr2. Conversely, complex In907 featured, two variable regions (VR); VR-1 consisted of aadB-bla OXA-10 -aadA1cc, whereas, VR-2, featured a gene qnrVC6 108 bp downstream from the ISCR1 and 45 bp, upstream from the qacEΔ1. Expression of qnrVC6 would be on account of a, putative promoter region, detected using the Neural Network Promoter, Prediction program. To the best of our knowledge this constitutes the first report of a qnrVC gene within a complex class 1 integron, and the first report as well of the occurrence of such gene in an NDM-1-producing enterobacterial clinical isolate. Copyright © 2018. Published by Elsevier Ltd.

Minimizing inhibition of PCR-STR typing using digital agarose droplet microfluidics.

PubMed

Geng, Tao; Mathies, Richard A

2015-01-01

The presence of PCR inhibitors in forensic and other biological samples reduces the amplification efficiency, sometimes resulting in complete PCR failure. Here we demonstrate a high-performance digital agarose droplet microfluidics technique for single-cell and single-molecule forensic short tandem repeat (STR) typing of samples contaminated with high concentrations of PCR inhibitors. In our multifaceted strategy, the mitigation of inhibitory effects is achieved by the efficient removal of inhibitors from the porous agarose microgel droplets carrying the DNA template through washing and by the significant dilution of targets and remaining inhibitors to the stochastic limit within the ultralow nL volume droplet reactors. Compared to conventional tube-based bulk PCR, our technique shows enhanced (20 ×, 10 ×, and 16 ×) tolerance of urea, tannic acid, and humic acid, respectively, in STR typing of GM09948 human lymphoid cells. STR profiling of single cells is not affected by small soluble molecules like urea and tannic acid because of their effective elimination from the agarose droplets; however, higher molecular weight humic acid still partially inhibits single-cell PCR when the concentration is higher than 200 ng/μL. Nevertheless, the full STR profile of 9948 male genomic DNA contaminated with 500 ng/μL humic acid was generated by pooling and amplifying beads carrying single-molecule 9948 DNA PCR products in a single secondary reaction. This superior performance suggests that our digital agarose droplet microfluidics technology is a promising approach for analyzing low-abundance DNA targets in the presence of inhibitors. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Polymerase chain reaction-based denaturing gradient gel electrophoresis in the evaluation of oral microbiota.

PubMed

Li, Y; Saxena, D; Barnes, V M; Trivedi, H M; Ge, Y; Xu, T

2006-10-01

Clinical evaluation of oral microbial reduction after a standard prophylactic treatment has traditionally been based on bacterial cultivation methods. However, not all microbes in saliva or dental plaque can be cultivated. Polymerase chain reaction-based denaturing gradient gel electrophoresis (PCR-DGGE) is a cultivation-independent molecular fingerprinting technique that allows the assessment of the predominant bacterial species present in the oral cavity. This study sought to evaluate the oral microbial changes that occurred after a standard prophylactic treatment with a conventional oral care product using PCR-DGGE. Twelve healthy adults participated in the study. Pooled plaque samples were collected at baseline, 24 h after prophylaxis (T1), and 4 days after toothbrushing with fluoride toothpaste (T4). The total microbial genomic DNA of the plaque was isolated. PCR was performed with a set of universal bacterial 16S rDNA primers. The PCR-amplified 16S rDNA fragments were separated by DGGE. The effects of the treatment and of dental brushing were assessed by comparing the PCR-DGGE fingerprinting profiles. The mean numbers of detected PCR amplicons were 22.3 +/- 6.1 for the baseline group, 13.0 +/- 3.1 for the T1 group, and 13.5 +/- 4.3 for the T4 group; the differences among the three groups were statistically significant (P < 0.01). The study also found a significant difference in the mean similarities of microbial profiles between the baseline and the treatment groups (P < 0.001). PCR-based DGGE has been shown to be an excellent means of rapidly and accurately assessing oral microbial changes in this clinical study.

APOPTOSIS GENE EXPRESSION IN HUMAN EPDERMAL KERATINOCYTES TREATED WITH SODIUM ARSENITE USING REAL TIME PCR ARRAY

EPA Science Inventory

Arsenic exposure via contaminated drinking water is a great public health concern worldwide. Chronic arsenic exposure has been associated with human skin, lung and bladder cancer and other chronic effects. We have previous reported that sodium arsenite stimulated cell proliferati...

Assessment of Phthalate-induced Changes in Fetal Rat Testis Gene Expression using an rt-PCR Drug Metabolism Array.

EPA Science Inventory

Phthalate esters are a large family of compounds used in many industrial and commercial products. Based on numerous studies, phthalates such as diethyl hexyl phthalate (DEHP) produce reproductive malformations in male rodents through reduction of testosterone production and gene ...

PCR-Restriction Fragment Length Polymorphism Analysis of the Phospholipase B (PLB1) Gene for Subtyping of Cryptococcus neoformans Isolates

PubMed Central

Latouche, G. Nicolas; Huynh, Matthew; Sorrell, Tania C.; Meyer, Wieland

2003-01-01

Cryptococcus neoformans is a pathogenic yeast that is currently divided into three varieties, five serotypes, and eight molecular types. The following report describes the use of PCR-restriction fragment length polymorphism (RFLP) analysis of the phospholipase B gene (PLB1) as a simple tool to differentiate between C. neoformans subgroups. A PLB1 fragment, 1,970 bp, was amplified and digested with either AvaI or HindIII. Both sets of profiles grouped the isolates into their respective varieties, but only the AvaI profiles allowed for the identification of the eight molecular types via the corresponding RFLP profiles A1 to A8. Digestion of the same fragments with HindIII resulted in RFLP profiles H1 to H5, which distinguished only between serotype A, AD, D, and B/C. Neither enzyme distinguished serotype B from serotype C. The serotype AD profile was a composite of the serotype A and D profiles. Further investigation showed that the serotype AD isolates used in this study are heterozygous, with one allele of PLB1 originating from a serotype A parent and the other from a serotype D parent. PMID:12676686

An evaluation of direct PCR amplification

PubMed Central

Hall, Daniel E.; Roy, Reena

2014-01-01

Aim To generate complete DNA profiles from blood and saliva samples deposited on FTA® and non-FTA® paper substrates following a direct amplification protocol. Methods Saliva samples from living donors and blood samples from deceased individuals were deposited on ten different FTA® and non-FTA® substrates. These ten paper substrates containing body fluids were kept at room temperature for varying lengths of time ranging from one day to approximately one year. For all assays in this research, 1.2 mm punches were collected from each substrate containing one type of body fluid and amplified with reagents provided in the nine commercial polymerase chain reaction (PCR) amplification kits. The substrates were not subjected to purification reagent or extraction buffer prior to amplification. Results Success rates were calculated for all nine amplification kits and all ten substrates based on their ability to yield complete DNA profiles following a direct amplification protocol. Six out of the nine amplification kits, and four out of the ten paper substrates had the highest success rates overall. Conclusion The data show that it is possible to generate complete DNA profiles following a direct amplification protocol using both standard (non-direct) and direct PCR amplification kits. The generation of complete DNA profiles appears to depend more on the success of the amplification kit rather than the than the FTA®- or non-FTA®-based substrates. PMID:25559837

An ANSERLIN array for mobile satellite applications

NASA Technical Reports Server (NTRS)

Colomb, F. Y.; Kunkee, D. B.; Mayes, P. E.; Smith, D. W.; Jamnejad, V.

1990-01-01

Design, analysis, construction, and test of linear arrays of ANSERLIN (annular sector, radiating line) elements are reported and discussed. Due to feeding simplicity and easy construction as well as good CP performance, a planar array composed of a number of such linear arrays each producing a shaped beam tilted in elevation, is a good candidate as a vehicle-mounted mechanically steered antenna for mobile satellite applications. A single level construction technique was developed that makes this type of array very cost competitive with other low-profile arrays. An asymmetric 19.5 inch long four-element array was fabricated and tested with reasonable performance. A smaller five-element symmetric array (16 inch long) was also designed and tested capable of operating in either sense of circular polarization. Efforts were made to successfully reduce this effect.

In-phased second harmonic wave array generation with intra-Talbot-cavity frequency-doubling.

PubMed

Hirosawa, Kenichi; Shohda, Fumio; Yanagisawa, Takayuki; Kannari, Fumihiko

2015-03-23

The Talbot cavity is one promising method to synchronize the phase of a laser array. However, it does not achieve the lowest array mode with the same phase but the highest array mode with the anti-phase between every two adjacent lasers, which is called out-phase locking. Consequently, their far-field images exhibit 2-peak profiles. We propose intra-Talbot-cavity frequency-doubling. By placing a nonlinear crystal in a Talbot cavity, the Talbot cavity generates an out-phased fundamental wave array, which is converted into an in-phase-locked second harmonic wave array at the nonlinear crystal. We demonstrate numerical calculations and experiments on intra-Talbot-cavity frequency-doubling and obtain an in-phase-locked second harmonic wave array for a Nd:YVO₄ array laser.

SU-F-T-179: Fast and Accurate Profile Acquisition for Proton Beam Using Multi-Ion Chamber Arrays

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

Wang, X; Zou, J; Chen, T

2016-06-15

Purpose: Proton beam profile measurement is more time-consuming than photon beam. Due to the energy modulation during proton delivery, chambers have to move step-by-step instead of continuously. Multi-ion chamber arrays are appealing to this task since multiple measurements can be performed at once. However, their utilization suffers from sparse spatial resolution and potential intrinsic volume-averaging effect of the disk-shaped ion chambers. We proposed an approach to measure proton beam profiles accurately and efficiently. Methods: Mevion S250 proton system and IBA Matrixx ion chamber arrays were used in this study. Matrixx has interchamber distance of 7.62 mm, and chamber diameter ofmore » 4.5 mm. We measured the same beam profile by moving the Matrixx seven times with 1 mm each time along y axis. All 7 measurements were superimposed to get a “finer” profile with 1 mm spatial resolution. Coarser resolution profiles of 2 mm and 3 mm were also generated by using subsets of measurements. Those profiles were compared to the TPS calculated beam profile. Gamma analysis was performed for 2D dose maps to evaluate the difference to TPS dose plane. Results: Preliminary results showed a large discrepancy between the TPS calculated profile and the single measurement profile with 7.6 mm resolution. A good match could be achieved when the resolution reduced to 3 mm by adding one extra measurement. Gamma analysis for 2D dose map of a 10×10 field showed a passing rate (γ ≤ 1) of 90.6% using a 3% and 3mm criterion for single measurement, which increased to 92.3% for 2-measurement superimposition, and slightly further increased to 92.9% for 7-measurement superimposition. Conclusion: The results indicated that 2 measurements shifted by 3mm using Matrixx generated a smooth proton beam profile with good matching to Eclipse beam profile. We suggest using this 2-measurement approach in clinic for double scattering proton beam profile measurement.« less

Fabrication of spherical microlens array by combining lapping on silicon wafer and rapid surface molding

NASA Astrophysics Data System (ADS)

Liu, Xiaohua; Zhou, Tianfeng; Zhang, Lin; Zhou, Wenchen; Yu, Jianfeng; Lee, L. James; Yi, Allen Y.

2018-07-01

Silicon is a promising mold material for compression molding because of its properties of hardness and abrasion resistance. Silicon wafers with carbide-bonded graphene coating and micro-patterns were evaluated as molds for the fabrication of microlens arrays. This study presents an efficient but flexible manufacturing method for microlens arrays that combines a lapping method and a rapid molding procedure. Unlike conventional processes for microstructures on silicon wafers, such as diamond machining and photolithography, this research demonstrates a unique approach by employing precision steel balls and diamond slurries to create microlenses with accurate geometry. The feasibility of this method was demonstrated by the fabrication of several microlens arrays with different aperture sizes and pitches on silicon molds. The geometrical accuracy and surface roughness of the microlens arrays were measured using an optical profiler. The measurement results indicated good agreement with the optical profile of the design. The silicon molds were then used to copy the microstructures onto polymer substrates. The uniformity and quality of the samples molded through rapid surface molding were also assessed and statistically quantified. To further evaluate the optical functionality of the molded microlens arrays, the focal lengths of the microlens arrays were measured using a simple optical setup. The measurements showed that the microlens arrays molded in this research were compatible with conventional manufacturing methods. This research demonstrated an alternative low-cost and efficient method for microstructure fabrication on silicon wafers, together with the follow-up optical molding processes.

Measuring the hydraulic fracture-induced deformation of reservoirs and adjacent rocks employing a deeply buried inclinometer array: GRI/DOE multi-site project

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

Branagan, P.T.; Warpinski, N.R.; Engler, B.

A vertical inclinometer array consisting of six biaxial tiltmeters was cemented behind pipe at depths between 4,273 and 4,628 ft. This wide-aperture array provided real-time tilt profiles corresponding to a series of seven hydraulic fractures being conducted in a nearby offset well in a fluvial sandstone reservoir. Array profiles for three KCl water fracs indicated that height growth was confined to the gross thickness of the reservoir despite extensive fracture length extension. Long-term monitoring of the array suggests that a substantial residual frac: width remained long after fracture closure occurred. For two 400-bbl linear gel minifracs, some height growth wasmore » observed but it was not extensive. Tilt amplitudes related to expanded frac widths were found to increase as would be expected with these thicker frac fluids. When cross-linker and proppant were included in the last fracture, tilt-derived heights were seen to grow rapidly extending into the bounding layers as the more complex fluids entered the fracture system. This inclinometer array was one of several independent, yet complimentary, fracture diagnostics tools that included crosswell multilevel microseismic arrays, FRACPRO{reg_sign} and a remote fracture intersection well. Their purpose was to provide integrated field-scale data regarding hydraulic fracture dynamics and geometry that would be used to construct accurate fracture mapping and diagnostic techniques.« less

Discrimination of probiotic Lactobacillus strains for poultry by repetitive sequenced-based PCR fingerprinting.

PubMed

Lee, Chin Mei; Sieo, Chin Chin; Cheah, Yoke-Kqueen; Abdullah, Norhani; Ho, Yin Wan

2012-02-01

Four repetitive element sequence-based polymerase chain reaction (rep-PCR) methods, namely repetitive extragenic palindromic PCR (REP-PCR), enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), polytrinucleotide (GTG)₅ -PCR and BOX-PCR, were evaluated for the molecular differentiation of 12 probiotic Lactobacillus strains previously isolated from the gastrointestinal tract of chickens and used as a multistrain probiotic. This study represents the first analysis of the comparative efficacy of these four rep-PCR methods and their combination (composite rep-PCR) in the molecular typing of Lactobacillus strains based on a discriminatory index (D). Species-specific and strain-specific profiles were observed from rep-PCR. From the numerical analysis of composite rep-PCR, BOX-PCR, (GTG)₅ -PCR, REP-PCR and ERIC-PCR, D values of 0.9118, 0.9044, 0.8897, 0.8750 and 0.8529 respectively were obtained. Composite rep-PCR analysis was the most discriminative method, with eight Lactobacillus strains, namely L. brevis ATCC 14869(T) , L. reuteri C 10, L. reuteri ATCC 23272(T) , L. gallinarum ATCC 33199(T) , L. salivarius ATCC 11741(T) , L. salivarius I 24, L. panis JCM 11053(T) and L. panis C 17, being differentiated at the strain level. Composite rep-PCR analysis is potentially a useful fingerprinting method to discriminate probiotic Lactobacillus strains isolated from the gastrointestinal tract of chickens. Copyright © 2011 Society of Chemical Industry.

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

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.

Apollo lunar surface experiments package. Apollo 17 ALSEP (array E) familiarization course handout

NASA Technical Reports Server (NTRS)

1972-01-01

The familiarization course for the Apollo 17 ALSEP (ARRAY E) is presented. The subjects discussed are: (1) power and data subsystems, (2) lunar surface gravimeter, (3) lunar mass spectrometer, (4) lunar seismic profiling experiment, and (5) heat flow experiment.

GENE EXPRESSION PATTERNS ASSOCIATED WITH INFERTILITY IN HUMAN AND RODENT MODELS

EPA Science Inventory

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

Microfluidics-based digital quantitative PCR for single-cell small RNA quantification.

PubMed

Yu, Tian; Tang, Chong; Zhang, Ying; Zhang, Ruirui; Yan, Wei

2017-09-01

Quantitative analyses of small RNAs at the single-cell level have been challenging because of limited sensitivity and specificity of conventional real-time quantitative PCR methods. A digital quantitative PCR (dqPCR) method for miRNA quantification has been developed, but it requires the use of proprietary stem-loop primers and only applies to miRNA quantification. Here, we report a microfluidics-based dqPCR (mdqPCR) method, which takes advantage of the Fluidigm BioMark HD system for both template partition and the subsequent high-throughput dqPCR. Our mdqPCR method demonstrated excellent sensitivity and reproducibility suitable for quantitative analyses of not only miRNAs but also all other small RNA species at the single-cell level. Using this method, we discovered that each sperm has a unique miRNA profile. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Method and system for homogenizing diode laser pump arrays

DOEpatents

Bayramian, Andrew James

2016-05-03

An optical amplifier system includes a diode pump array including a plurality of semiconductor diode laser bars disposed in an array configuration and characterized by a periodic distance between adjacent semiconductor diode laser bars. The periodic distance is measured in a first direction perpendicular to each of the plurality of semiconductor diode laser bars. The diode pump array provides a pump output propagating along an optical path and characterized by a first intensity profile measured as a function of the first direction and having a variation greater than 10%. The optical amplifier system also includes a diffractive optic disposed along the optical path. The diffractive optic includes a photo-thermo-refractive glass member. The optical amplifier system further includes an amplifier slab having an input face and position along the optical path and separated from the diffractive optic by a predetermined distance. A second intensity profile measured at the input face of the amplifier slab as a function of the first direction has a variation less than 10%.

Method and system for homogenizing diode laser pump arrays

DOEpatents

Bayramian, Andy J

2013-10-01

An optical amplifier system includes a diode pump array including a plurality of semiconductor diode laser bars disposed in an array configuration and characterized by a periodic distance between adjacent semiconductor diode laser bars. The periodic distance is measured in a first direction perpendicular to each of the plurality of semiconductor diode laser bars. The diode pump array provides a pump output propagating along an optical path and characterized by a first intensity profile measured as a function of the first direction and having a variation greater than 10%. The optical amplifier system also includes a diffractive optic disposed along the optical path. The diffractive optic includes a photo-thermo-refractive glass member. The optical amplifier system further includes an amplifier slab having an input face and position along the optical path and separated from the diffractive optic by a predetermined distance. A second intensity profile measured at the input face of the amplifier slab as a function of the first direction has a variation less than 10%.

Genomic analysis of lung cell lines exposures to space radiation and the effect of lunar dust on selected fibrosis gene using RT2 PCR Array

NASA Astrophysics Data System (ADS)

Yeshitla, Samrawit

In the United States (U.S.), lung cancer is the number one cause of cancer death among men and women. Previous studies on human and animal epithelial lung cells showed that ionizing radiation and certain environmental pollutants are carcinogens. The surface area of the lungs and the slow turnover rate of the epithelial cells are suggested to play a role in the vulnerability of the cells, which lead to increase in the progenitor cell of the lung. It has been proposed that these progenitor cells, when exposed to radiation undergo multiple alterations that cause the cells to become cancerous. The current thought is that the lungs contain several facultative progenitor cells that are situated throughout the lung epithelium and are regionally restricted in their regenerative capacity. In this study, normal Human Bronchial Epithelial Cells (HBECs) were immortalized through the expression of Cdk4 and hTERT and evaluated for the effects radiation using in vitro study. The HBECs retained its novel multipotent capacity in vitro and represented unrestricted progenitor cells of the adult lungs, which resemble an embryonic progenitor. Analysis of the transformed clones of human bronchial epithelial cell line, HEBC3KT exposed to Fe ions and gamma rays revealed chromosomal abnormality, which was detected with the Multi-color Fluorescent In Situ Hybridization (mFish). In Part two of this study the F344 rats exposed to lunar dust, for 4 weeks (6h/d; 5d/wk.) in nose-only inhalation chambers at concentrations of 0 (control air), 2.1, 6.8, 20.8, and 61 mg/m3 of lunar dust, were used to determine the lunar dust toxicity on the lung tissues and total RNA were prepared from the tissues and used for gene expression. Analysis of gene expression data using Ingenuity Pathway Analysis tool identified multiple pathways of which fibrosis was one of the pathways. The Rat Fibrosis RT 2 Profile PCR Array was used to profile the expression of 84 genes that are relevant to fibrosis in the lung tissue, after removing the infiltrated of the bronchoalveolar lavage (BAL) cells by lavaging. At a dose of 61mg/m 3, 29 genes showed changes and of 29 genes, nine genes, Bmp7, Cc112, Cc13, Itgb8, Serpinel, Smad6, Tgfbrl, Thbs2, and Tnf, showed significant fold change difference of up-regulation or down-regulation. The data in this study showed that iron ions and gamma rays promote chromosomal abnormality in HBEC3KT, and for the first time, the lunar dust altered gene expression of 29 genes that are relevant to fibrosis in the lung tissue.

Ordered and Ultra-High Aspect Ratio Nanocapillary Arrays as a Model System

DTIC Science & Technology

2015-10-13

formation and deep pore growth of anodized aluminum oxide ( AAO )-based nanocapillary arrays as the basis for high density, safe and high rate gas... anodized aluminum oxide , nanocapillary arrays 16. SECURITY CLASSIFICATION OF: Unclassified 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME... Aluminum Page 7 Copyright © 2015 Mainstream Engineering Corporation CPE Mitigation Schemes  Control thermal and flow profile -> even anodization

Simulated and Real Sheet-of-Light 3D Object Scanning Using a-Si:H Thin Film PSD Arrays.

PubMed

Contreras, Javier; Tornero, Josep; Ferreira, Isabel; Martins, Rodrigo; Gomes, Luis; Fortunato, Elvira

2015-11-30

A MATLAB/SIMULINK software simulation model (structure and component blocks) has been constructed in order to view and analyze the potential of the PSD (Position Sensitive Detector) array concept technology before it is further expanded or developed. This simulation allows changing most of its parameters, such as the number of elements in the PSD array, the direction of vision, the viewing/scanning angle, the object rotation, translation, sample/scan/simulation time, etc. In addition, results show for the first time the possibility of scanning an object in 3D when using an a-Si:H thin film 128 PSD array sensor and hardware/software system. Moreover, this sensor technology is able to perform these scans and render 3D objects at high speeds and high resolutions when using a sheet-of-light laser within a triangulation platform. As shown by the simulation, a substantial enhancement in 3D object profile image quality and realism can be achieved by increasing the number of elements of the PSD array sensor as well as by achieving an optimal position response from the sensor since clearly the definition of the 3D object profile depends on the correct and accurate position response of each detector as well as on the size of the PSD array.

Direct Application of Rep-PCR on Type I Sourdough Matrix to Monitor the Dominance and Persistence of a Lactobacillus plantarum Starter Throughout Back-Slopping.

PubMed

Dolci, Paola; Cocolin, Luca

2017-08-01

This study describes the optimization and application of repetitive element-PCR (rep-PCR) technique directly on microbial DNA extracted from type I sourdoughs for fast monitoring of a Lb. plantarum starter strain (P1FMC) throughout daily back-slopping. The challenge was to follow and study the performance of a starter culture directly in sourdoughs without cultivation on selective media. The extraction of good quality microbial DNA suitable for amplification from a complex matrix such as dough was the first target. In addition, the objective to obtain a clear rep-PCR profile referable to a specific starter strain among a microbial community was pursued. Co-inoculum trials, in flour matrix, with Lb. plantarum P1FMC and L. lactis LC71 strains and, subsequently, type I sourdough back-slopping trials were performed. The rep-PCR amplification profiles obtained were clearly referable to that of Lb. plantarum P1FMC starter in both co-inoculum trials (also when it was present with one order of magnitude less with respect to L. lactis LC71) and back-slopping trials where it dominated the fermentation process with loads of 10 8 cfu g -1 and prevailed on the autochthonous microbiota. Thus, the approach proposed in this paper could be considered a methodological advancement, based on a culture-independent one-step rep-PCR, suitable for fast monitoring of starter performance. © 2017 Institute of Food Technologists®.

COMPARISON BETWEEN AUTOMATED SYSTEM AND PCR-BASED METHOD FOR IDENTIFICATION AND ANTIMICROBIAL SUSCEPTIBILITY PROFILE OF CLINICAL Enterococcus spp

PubMed Central

Furlaneto-Maia, Luciana; Rocha, Kátia Real; Siqueira, Vera Lúcia Dias; Furlaneto, Márcia Cristina

2014-01-01

Enterococci are increasingly responsible for nosocomial infections worldwide. This study was undertaken to compare the identification and susceptibility profile using an automated MicrosScan system, PCR-based assay and disk diffusion assay of Enterococcus spp. We evaluated 30 clinical isolates of Enterococcus spp. Isolates were identified by MicrosScan system and PCR-based assay. The detection of antibiotic resistance genes (vancomycin, gentamicin, tetracycline and erythromycin) was also determined by PCR. Antimicrobial susceptibilities to vancomycin (30 µg), gentamicin (120 µg), tetracycline (30 µg) and erythromycin (15 µg) were tested by the automated system and disk diffusion method, and were interpreted according to the criteria recommended in CLSI guidelines. Concerning Enterococcus identification the general agreement between data obtained by the PCR method and by the automatic system was 90.0% (27/30). For all isolates of E. faecium and E. faecalis we observed 100% agreement. Resistance frequencies were higher in E. faecium than E. faecalis. The resistance rates obtained were higher for erythromycin (86.7%), vancomycin (80.0%), tetracycline (43.35) and gentamicin (33.3%). The correlation between disk diffusion and automation revealed an agreement for the majority of the antibiotics with category agreement rates of > 80%. The PCR-based assay, the van(A) gene was detected in 100% of vancomycin resistant enterococci. This assay is simple to conduct and reliable in the identification of clinically relevant enterococci. The data obtained reinforced the need for an improvement of the automated system to identify some enterococci. PMID:24626409

Quantitative PCR Profiling of Escherichia coli in Livestock Feces Reveals Increased Population Resilience Relative to Culturable Counts under Temperature Extremes.

PubMed

Oliver, David M; Bird, Clare; Burd, Emmy; Wyman, Michael

2016-09-06

The relationship between culturable counts (CFU) and quantitative PCR (qPCR) cell equivalent counts of Escherichia coli in dairy feces exposed to different environmental conditions and temperature extremes was investigated. Fecal samples were collected in summer and winter from dairy cowpats held under two treatments: field-exposed versus polytunnel-protected. A significant correlation in quantified E. coli was recorded between the qPCR and culture-based methods (r = 0.82). Evaluation of the persistence profiles of E. coli over time revealed no significant difference in the E. coli numbers determined as either CFU or gene copies during the summer for the field-exposed cowpats, whereas significantly higher counts were observed by qPCR for the polytunnel-protected cowpats, which were exposed to higher ambient temperatures. In winter, the qPCR returned significantly higher counts of E. coli for the field-exposed cowpats, thus representing a reversal of the findings from the summer sampling campaign. Results from this study suggest that with increasing time post-defecation and with the onset of challenging environmental conditions, such as extremes in temperature, culture-based counts begin to underestimate the true resilience of viable E. coli populations in livestock feces. This is important not only in the long term as the Earth changes in response to climate-change drivers but also in the short term during spells of extremely cold or hot weather.

A comparison of PCR assays for beak and feather disease virus and high resolution melt (HRM) curve analysis of replicase associated protein and capsid genes.

PubMed

Das, Shubhagata; Sarker, Subir; Ghorashi, Seyed Ali; Forwood, Jade K; Raidal, Shane R

2016-11-01

Beak and feather disease virus (BFDV) threatens a wide range of endangered psittacine birds worldwide. In this study, we assessed a novel PCR assay and genetic screening method using high-resolution melt (HRM) curve analysis for BFDV targeting the capsid (Cap) gene (HRM-Cap) alongside conventional PCR detection as well as a PCR method that targets a much smaller fragment of the virus genome in the replicase initiator protein (Rep) gene (HRM-Rep). Limits of detection, sensitivity, specificity and discriminatory power for differentiating BFDV sequences were compared. HRM-Cap had a high positive predictive value and could readily differentiate between a reference genotype and 17 other diverse BFDV genomes with more discriminatory power (genotype confidence percentage) than HRM-Rep. Melt curve profiles generated by HRM-Cap correlated with unique DNA sequence profiles for each individual test genome. The limit of detection of HRM-Cap was lower (2×10 -5 ng/reaction or 48 viral copies) than that for both HRM-Rep and conventional BFDV PCR which had similar sensitivity (2×10 -6 ng or 13 viral copies/reaction). However, when used in a diagnostic setting with 348 clinical samples there was strong agreement between HRM-Cap and conventional PCR (kappa=0.87, P<0.01, 98% specificity) and HRM-Cap demonstrated higher specificity (99.9%) than HRM-Rep (80.3%). Copyright © 2016 Elsevier B.V. All rights reserved.

KDNA Genetic Signatures Obtained by LSSP-PCR Analysis of Leishmania (Leishmania) infantum Isolated from the New and the Old World

PubMed Central

Alvarenga, Janaína Sousa Campos; Ligeiro, Carla Maia; Gontijo, Célia Maria Ferreira; Cortes, Sofia; Campino, Lenea; Vago, Annamaria Ravara; Melo, Maria Norma

2012-01-01

Background Visceral Leishmaniasis (VL) caused by species from the Leishmania donovani complex is the most severe form of the disease, lethal if untreated. VL caused by Leishmania infantum is a zoonosis with an increasing number of human cases and millions of dogs infected in the Old and the New World. In this study, L. infantum (syn. L.chagasi) strains were isolated from human and canine VL cases. The strains were obtained from endemic areas from Brazil and Portugal and their genetic polymorphism was ascertained using the LSSP-PCR (Low-Stringency Single Specific Primer PCR) technique for analyzing the kinetoplastid DNA (kDNA) minicircles hypervariable region. Principal Findings KDNA genetic signatures obtained by minicircle LSSP-PCR analysis of forty L. infantum strains allowed the grouping of strains in several clades. Furthermore, LSSP-PCR profiles of L. infantum subpopulations were closely related to the host origin (human or canine). To our knowledge this is the first study which used this technique to compare genetic polymorphisms among strains of L. infantum originated from both the Old and the New World. Conclusions LSSP-PCR profiles obtained by analysis of L. infantum kDNA hypervariable region of parasites isolated from human cases and infected dogs from Brazil and Portugal exhibited a genetic correlation among isolates originated from the same reservoir, human or canine. However, no association has been detected among the kDNA signatures and the geographical origin of L. infantum strains. PMID:22912862

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

Riot, Vincent J.

The present disclosure provides a system and a method for measuring fluorescence of a sample. The sample may be a polymerase-chain-reaction (PCR) array, a loop-mediated-isothermal amplification array, etc. LEDs are used to excite the sample, and a photodiode is used to collect the sample's fluorescence. An electronic offset signal is used to reduce the effects of background fluorescence and the noises from the measurement system. An integrator integrates the difference between the output of the photodiode and the electronic offset signal over a given period of time. The resulting integral is then converted into digital domain for further processing andmore » storage.« less

Gene expression profile of patients with Mayer-Rokitansky-Küster-Hauser syndrome: new insights into the potential role of developmental pathways.

PubMed

Nodale, Cristina; Ceccarelli, Simona; Giuliano, Mariateresa; Cammarota, Marcella; D'Amici, Sirio; Vescarelli, Enrica; Maffucci, Diana; Bellati, Filippo; Panici, Pierluigi Benedetti; Romano, Ferdinando; Angeloni, Antonio; Marchese, Cinzia

2014-01-01

Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is a rare disease characterized by congenital aplasia of uterus and vagina. Although many studies have investigated several candidate genes, up to now none of them seem to be responsible for the aetiology of the syndrome. In our study, we identified differences in gene expression profile of in vitro cultured vaginal tissue of MRHKS patients using whole-genome microarray analysis. A group of eight out of sixteen MRKHS patients that underwent reconstruction of neovagina with an autologous in vitro cultured vaginal tissue were subjected to microarray analysis and compared with five healthy controls. Results obtained by array were confirmed by qRT-PCR and further extended to other eight MRKHS patients. Gene profiling of MRKHS patients delineated 275 differentially expressed genes, of which 133 downregulated and 142 upregulated. We selected six deregulated genes (MUC1, HOXC8, HOXB2, HOXB5, JAG1 and DLL1) on the basis of their fold change, their differential expression in most patients and their relevant role in embryological development. All patients showed upregulation of MUC1, while HOXB2 and HOXB5 were downregulated, as well as Notch ligands JAG1 and DLL1 in the majority of them. Interestingly, HOXC8 was significantly upregulated in 47% of patients, with a differential expression only in MRKHS type I patients. Taken together, our results highlighted the dysregulation of developmental genes, thus suggesting a potential alteration of networks involved in the formation of the female reproductive tract and providing a useful clue for understanding the pathophysiology of MRKHS.

Gene Expression Profile of Patients with Mayer-Rokitansky-Küster-Hauser Syndrome: New Insights into the Potential Role of Developmental Pathways

PubMed Central

Giuliano, Mariateresa; Cammarota, Marcella; D’Amici, Sirio; Vescarelli, Enrica; Maffucci, Diana; Bellati, Filippo; Panici, Pierluigi Benedetti; Romano, Ferdinando; Angeloni, Antonio; Marchese, Cinzia

2014-01-01

Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is a rare disease characterized by congenital aplasia of uterus and vagina. Although many studies have investigated several candidate genes, up to now none of them seem to be responsible for the aetiology of the syndrome. In our study, we identified differences in gene expression profile of in vitro cultured vaginal tissue of MRHKS patients using whole-genome microarray analysis. A group of eight out of sixteen MRKHS patients that underwent reconstruction of neovagina with an autologous in vitro cultured vaginal tissue were subjected to microarray analysis and compared with five healthy controls. Results obtained by array were confirmed by qRT-PCR and further extended to other eight MRKHS patients. Gene profiling of MRKHS patients delineated 275 differentially expressed genes, of which 133 downregulated and 142 upregulated. We selected six deregulated genes (MUC1, HOXC8, HOXB2, HOXB5, JAG1 and DLL1) on the basis of their fold change, their differential expression in most patients and their relevant role in embryological development. All patients showed upregulation of MUC1, while HOXB2 and HOXB5 were downregulated, as well as Notch ligands JAG1 and DLL1 in the majority of them. Interestingly, HOXC8 was significantly upregulated in 47% of patients, with a differential expression only in MRKHS type I patients. Taken together, our results highlighted the dysregulation of developmental genes, thus suggesting a potential alteration of networks involved in the formation of the female reproductive tract and providing a useful clue for understanding the pathophysiology of MRKHS. PMID:24608967

Dynamics of cytokines and immunoglobulins serum profiles in primary and secondary Cryptosporidium parvum infection: usefulness of Luminex® xMAP technology.

PubMed

Codices, Vera; Martins, Catarina; Novo, Carlos; de Sousa, Bruno; Lopes, Ângela; Borrego, Miguel; Matos, Olga

2013-01-01

Infection by Cryptosporidium parvum triggers a complex array of innate and adaptive cell mediated immune response, playing an important role in controlling the infection. To date, there are no studies applying the Luminex® xMAP technology to determine profiles of cytokines and immunoglobulins in the context of an infection by C. parvum. In this study, we analyzed these immune mediators in the serum of immunocompetent mice inoculated with C. parvum oocysts, using Luminex, to understand how the immune system responds to an infection by this parasite. Animal sera were also analyzed by ELISA to determine the expressed immunoglobulin isotype profile, and compare the obtained trend with data obtained by Luminex. Specific-pathogen-free BALB/C mice were inoculated with oocysts of C. parvum at days 0 and 22. Peripheral blood was aseptically collected from sacrificed mice on several time points, and immune mediators were evaluated in serum samples. Infection was confirmed by the presence of C. parvum DNA in feces by a nested-PCR assay (60-kDa glycoprotein). Luminex results showed predominance in the secretion of IgG1 and IgG2a, confirmed by ELISA, which also showed that IgG1 is the major immunoglobulin isotype produced during the infection. The analysis of cytokines suggests a preferential Th(1) over the Th(2) response, with increased production of TNF-α, IFN-γ and GM-CSF. This work contributed to a better understanding of the immune response to the infection by C. parvum, as well as demonstrated the advantage of Luminex® xMAP technology to study immune mediators, using small sample volumes. Copyright © 2012 Elsevier Inc. All rights reserved.

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

PubMed

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

2014-01-01

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

Using microRNA profiling in urine samples to develop a non-invasive test for bladder cancer.

PubMed

Mengual, Lourdes; Lozano, Juan José; Ingelmo-Torres, Mercedes; Gazquez, Cristina; Ribal, María José; Alcaraz, Antonio

2013-12-01

Current standard methods used to detect and monitor bladder urothelial cell carcinoma (UCC) are invasive or have low sensitivity. The incorporation into clinical practice of a non-invasive tool for UCC assessment would enormously improve patients' quality of life and outcome. This study aimed to examine the microRNA (miRNA) expression profiles in urines of UCC patients in order to develop a non-invasive accurate and reliable tool to diagnose and provide information on the aggressiveness of the tumor. We performed a global miRNA expression profiling analysis of the urinary cells from 40 UCC patients and controls using TaqMan Human MicroRNA Array followed by validation of 22 selected potentially diagnostic and prognostic miRNAs in a separate cohort of 277 samples using a miRCURY LNA qPCR system. miRNA-based signatures were developed by multivariate logistic regression analysis and internally cross-validated. In the initial cohort of patients, we identified 40 and 30 aberrantly expressed miRNA in UCC compared with control urines and in high compared with low grade tumors, respectively. Quantification of 22 key miRNAs in an independent cohort resulted in the identification of a six miRNA diagnostic signature with a sensitivity of 84.8% and specificity of 86.5% (AUC = 0.92) and a two miRNA prognostic model with a sensitivity of 84.95% and a specificity of 74.14% (AUC = 0.83). Internal cross-validation analysis confirmed the accuracy rates of both models, reinforcing the strength of our findings. Although the data needs to be externally validated, miRNA analysis in urine appears to be a valuable tool for the non-invasive assessment of UCC. Copyright © 2013 UICC.

Global microRNA expression profiling of microdissected tissues identifies miR-135b as a novel biomarker for pancreatic ductal adenocarcinoma.

PubMed

Munding, Johanna B; Adai, Alex T; Maghnouj, Abdelouahid; Urbanik, Aleksandra; Zöllner, Hannah; Liffers, Sven T; Chromik, Ansgar M; Uhl, Waldemar; Szafranska-Schwarzbach, Anna E; Tannapfel, Andrea; Hahn, Stephan A

2012-07-15

Pancreatic ductal adenocarcinoma (PDAC) is known for its poor prognosis resulting from being diagnosed at an advanced stage. Accurate early diagnosis and new therapeutic modalities are therefore urgently needed. MicroRNAs (miRNAs), considered a new class of biomarkers and therapeutic targets, may be able to fulfill those needs. Combining tissue microdissection with global miRNA array analyses, cell type-specific miRNA expression profiles were generated for normal pancreatic ductal cells, acinar cells, PDAC cells derived from xenografts and also from macrodissected chronic pancreatitis (CP) tissues. We identified 78 miRNAs differentially expressed between ND and PDAC cells providing new insights into the miRNA-driven pathophysiological mechanisms involved in PDAC development. Having filtered miRNAs which are upregulated in the three pairwise comparisons of PDAC vs. ND, PDAC vs. AZ and PDAC vs. CP, we identified 15 miRNA biomarker candidates including miR-135b. Using relative qRT-PCR to measure miR-135b normalized to miR-24 in 75 FFPE specimens (42 PDAC and 33 CP) covering a broad range of tumor content, we discriminated CP from PDAC with a sensitivity and specificity of 92.9% [95% CI=(80.5, 98.5)] and 93.4% [95% CI=(79.8, 99.3)], respectively. Furthermore, the area under the curve (AUC) value reached of 0.97 was accompanied by positive and negative predictive values of 95% and 91%, respectively. In conclusion, we report pancreatic cell-specific global miRNA profiles, which offer new candidate miRNAs to be exploited for functional studies in PDAC. Furthermore, we provide evidence that miRNAs are well-suited analytes for development of sensitive and specific aid-in-diagnosis tests for PDAC. Copyright © 2011 UICC.

Differential miRNA expression profiling reveals miR-205-3p to be a potential radiosensitizer for low- dose ionizing radiation in DLD-1 cells.

PubMed

Andaur, Rodrigo; Tapia, Julio C; Moreno, José; Soto, Leopoldo; Armisen, Ricardo; Marcelain, Katherine

2018-05-29

Enhanced radiosensitivity at low doses of ionizing radiation (IR) (0.2 to 0.6 Gy) has been reported in several cell lines. This phenomenon, known as low doses hyper-radiosensitivity (LDHRS), appears as an opportunity to decrease toxicity of radiotherapy and to enhance the effects of chemotherapy. However, the effect of low single doses IR on cell death is subtle and the mechanism underlying LDHRS has not been clearly explained, limiting the utility of LDHRS for clinical applications. To understand the mechanisms responsible for cell death induced by low-dose IR, LDHRS was evaluated in DLD-1 human colorectal cancer cells and the expression of 80 microRNAs (miRNAs) was assessed by qPCR array. Our results show that DLD-1 cells display an early DNA damage response and apoptotic cell death when exposed to 0.6 Gy. miRNA expression profiling identified 3 over-expressed (miR-205-3p, miR-1 and miR-133b) and 2 down-regulated miRNAs (miR-122-5p, and miR-134-5p) upon exposure to 0.6 Gy. This miRNA profile differed from the one in cells exposed to high-dose IR (12 Gy), supporting a distinct low-dose radiation-induced cell death mechanism. Expression of a mimetic miR-205-3p, the most overexpressed miRNA in cells exposed to 0.6 Gy, induced apoptotic cell death and, more importantly, increased LDHRS in DLD-1 cells. Thus, we propose miR-205-3p as a potential radiosensitizer to low-dose IR.

The pulse profile of the Crab pulsar in the energy range 45 keV-1.2 MeV

NASA Technical Reports Server (NTRS)

Wilson, R. B.; Fishman, G. J.

1983-01-01

The Crab Nebula pulsar (PSR 0531+21) is the best studied and most intense of the nontransient X-ray pulsars. However, since its spectrum drops rapidly with energy, a well-resolved pulse profile has not previously been obtained above 200 keV. In the hard X-ray and low-energy gamma-ray region, an accurate pulse profile can be obtained with a balloon-borne detector of sufficient area during a single transit of the source. A new measurement of the pulse profile of PSR 0531+21 in the energy range above 45 keV obtained with a large-area scintillation detector array is reported. The detector array was flown on a balloon launched from Palestine, Texas on 1980 October 6, reaching a float altitude 4.5 g/sq cm at 0230 UTC October 7. The primary objective of the experiment was to detect and study weak gamma-ray bursts.

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.

Adaptation of the Biolog Phenotype MicroArrayTM Technology to Profile the Obligate Anaerobe Geobacter metallireducens

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

Joyner, Dominique; Fortney, Julian; Chakraborty, Romy

2010-05-17

The Biolog OmniLog? Phenotype MicroArray (PM) plate technology was successfully adapted to generate a select phenotypic profile of the strict anaerobe Geobacter metallireducens (G.m.). The profile generated for G.m. provides insight into the chemical sensitivity of the organism as well as some of its metabolic capabilities when grown with a basal medium containing acetate and Fe(III). The PM technology was developed for aerobic organisms. The reduction of a tetrazolium dye by the test organism represents metabolic activity on the array which is detected and measured by the OmniLog(R) system. We have previously adapted the technology for the anaerobic sulfate reducingmore » bacterium Desulfovibrio vulgaris. In this work, we have taken the technology a step further by adapting it for the iron reducing obligate anaerobe Geobacter metallireducens. In an osmotic stress microarray it was determined that the organism has higher sensitivity to impermeable solutes 3-6percent KCl and 2-5percent NaNO3 that result in osmotic stress by osmosis to the cell than to permeable non-ionic solutes represented by 5-20percent ethylene glycol and 2-3percent urea. The osmotic stress microarray also includes an array of osmoprotectants and precursor molecules that were screened to identify substrates that would provide osmotic protection to NaCl stress. None of the substrates tested conferred resistance to elevated concentrations of salt. Verification studies in which G.m. was grown in defined medium amended with 100mM NaCl (MIC) and the common osmoprotectants betaine, glycine and proline supported the PM findings. Further verification was done by analysis of transcriptomic profiles of G.m. grown under 100mM NaCl stress that revealed up-regulation of genes related to degradation rather than accumulation of the above-mentioned osmoprotectants. The phenotypic profile, supported by additional analysis indicates that the accumulation of these osmoprotectants as a response to salt stress does not occur in G.m. and response to stress must occur by other mechanisms. The Phenotype MicroArray technology can be reliably used as a rapid screening tool for characterization in anaerobic microbial ecology.« less

Identification of Brucella genus and eight Brucella species by Luminex bead-based suspension array.

PubMed

Lusk Pfefer, Tina S; Timme, Ruth; Kase, Julie A

2018-04-01

Globally, unpasteurized milk products are vehicles for the transmission of brucellosis, a zoonosis responsible for cases of foodborne illness in the United States and elsewhere. Existing PCR assays to detect Brucella species are restricted by the resolution of band sizes on a gel or the number of fluorescent channels in a single real-time system. The Luminex bead-based suspension array is performed in a 96-well plate allowing for high throughput screening of up to 100 targets in one sample with easily discernible results. We have developed an array using the Bio-Plex 200 to differentiate the most common Brucella species: B. abortus, B. melitensis, B. suis, B. suis bv5, B. canis, B. ovis, B. pinnipedia, and B. neotomae, as well as Brucella genus. All probes showed high specificity, with no cross-reaction with non-Brucella strains. We could detect pure DNA from B. abortus, B. melitensis, and genus-level Brucella at concentrations of ≤5 fg/μL. Pure DNA from all other species tested positive at concentrations well below 500 fg/μL and we positively identified B. neotomae in six artificially contaminated cheese and milk products. An intra-laboratory verification further demonstrated the assay's accuracy and robustness in the rapid screening (3-4 h including PCR) of DNA. Published by Elsevier Ltd.

A novel X-linked disorder with developmental delay and autistic features.

PubMed

Kaya, Namik; Colak, Dilek; Albakheet, Albandary; Al-Owain, Mohammad; Abu-Dheim, Nada; Al-Younes, Banan; Al-Zahrani, Jawaher; Mukaddes, Nahit M; Dervent, Aysin; Al-Dosari, Naji; Al-Odaib, Ali; Kayaalp, Inci V; Al-Sayed, Moeenaladin; Al-Hassnan, Zuhair; Nester, Michael J; Al-Dosari, Mohammad; Al-Dhalaan, Hesham; Chedrawi, Aziza; Gunoz, Hulya; Karakas, Bedri; Sakati, Nadia; Alkuraya, Fowzan S; Gascon, Generaso G; Ozand, Pinar T

2012-04-01

Genomic duplications that lead to autism and other human diseases are interesting pathological lesions since the underlying mechanism almost certainly involves dosage sensitive genes. We aim to understand a novel genomic disorder with profound phenotypic consequences, most notably global developmental delay, autism, psychosis, and anorexia nervosa. We evaluated the affected individuals, all maternally related, using childhood autism rating scale (CARS) and Vineland Adaptive scales, magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) brain, electroencephalography (EEG), electromyography (EMG), muscle biopsy, high-resolution molecular karyotype arrays, Giemsa banding (G-banding) and fluorescent in situ hybridization (FISH) experiments, mitochondrial DNA (mtDNA) sequencing, X-chromosome inactivation study, global gene expression analysis on Epstein-Barr virus (EBV)-transformed lymphoblasts, and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). We have identified a novel Xq12-q13.3 duplication in an extended family. Clinically normal mothers were completely skewed in favor of the normal chromosome X. Global transcriptional profiling of affected individuals and controls revealed significant alterations of genes and pathways in a pattern consistent with previous microarray studies of autism spectrum disorder patients. Moreover, expression analysis revealed copy number-dependent increased messenger RNA (mRNA) levels in affected patients compared to control individuals. A subset of differentially expressed genes was validated using qRT-PCR. Xq12-q13.3 duplication is a novel global developmental delay and autism-predisposing chromosomal aberration; pathogenesis of which may be mediated by increased dosage of genes contained in the duplication, including NLGN3, OPHN1, AR, EFNB1, TAF1, GJB1, and MED12. Copyright © 2011 American Neurological Association.

MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo

PubMed Central

Eskildsen, Tilde; Taipaleenmäki, Hanna; Stenvang, Jan; Abdallah, Basem M.; Ditzel, Nicholas; Nossent, Anne Yael; Bak, Mads; Kauppinen, Sakari; Kassem, Moustapha

2011-01-01

Elucidating the molecular mechanisms that regulate human stromal (mesenchymal) stem cell (hMSC) differentiation into osteogenic lineage is important for the development of anabolic therapies for treatment of osteoporosis. MicroRNAs (miRNAs) are short, noncoding RNAs that act as key regulators of diverse biological processes by mediating translational repression or mRNA degradation of their target genes. Here, we show that miRNA-138 (miR-138) modulates osteogenic differentiation of hMSCs. miRNA array profiling and further validation by quantitative RT-PCR (qRT-PCR) revealed that miR-138 was down-regulated during osteoblast differentiation of hMSCs. Overexpression of miR-138 inhibited osteoblast differentiation of hMSCs in vitro, whereas inhibition of miR-138 function by antimiR-138 promoted expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Furthermore, overexpression of miR-138 reduced ectopic bone formation in vivo by 85%, and conversely, in vivo bone formation was enhanced by 60% when miR-138 was antagonized. Target prediction analysis and experimental validation by luciferase 3′ UTR reporter assay confirmed focal adhesion kinase, a kinase playing a central role in promoting osteoblast differentiation, as a bona fide target of miR-138. We show that miR-138 attenuates bone formation in vivo, at least in part by inhibiting the focal adhesion kinase signaling pathway. Our findings suggest that pharmacological inhibition of miR-138 by antimiR-138 could represent a therapeutic strategy for enhancing bone formation in vivo. PMID:21444814

High-resolution depth profiling using a range-gated CMOS SPAD quanta image sensor.

PubMed

Ren, Ximing; Connolly, Peter W R; Halimi, Abderrahim; Altmann, Yoann; McLaughlin, Stephen; Gyongy, Istvan; Henderson, Robert K; Buller, Gerald S

2018-03-05

A CMOS single-photon avalanche diode (SPAD) quanta image sensor is used to reconstruct depth and intensity profiles when operating in a range-gated mode used in conjunction with pulsed laser illumination. By designing the CMOS SPAD array to acquire photons within a pre-determined temporal gate, the need for timing circuitry was avoided and it was therefore possible to have an enhanced fill factor (61% in this case) and a frame rate (100,000 frames per second) that is more difficult to achieve in a SPAD array which uses time-correlated single-photon counting. When coupled with appropriate image reconstruction algorithms, millimeter resolution depth profiles were achieved by iterating through a sequence of temporal delay steps in synchronization with laser illumination pulses. For photon data with high signal-to-noise ratios, depth images with millimeter scale depth uncertainty can be estimated using a standard cross-correlation approach. To enhance the estimation of depth and intensity images in the sparse photon regime, we used a bespoke clustering-based image restoration strategy, taking into account the binomial statistics of the photon data and non-local spatial correlations within the scene. For sparse photon data with total exposure times of 75 ms or less, the bespoke algorithm can reconstruct depth images with millimeter scale depth uncertainty at a stand-off distance of approximately 2 meters. We demonstrate a new approach to single-photon depth and intensity profiling using different target scenes, taking full advantage of the high fill-factor, high frame rate and large array format of this range-gated CMOS SPAD array.

Measurement and Interpretation of DT Neutron Emission from Tftr.

NASA Astrophysics Data System (ADS)

McCauley, John Scott, Jr.

A fast-ion diffusion coefficient of 0.1 +/- 0.1 m^2s ^{-1} has been deduced from the triton burnup neutron emission profile measured by a collimated array of helium-4 spectrometers. The experiment was performed with high-power deuterium discharges produced by Princeton University's Tokamak Fusion Test Reactor (TFTR). The fast ions monitored were the 1.0 MeV tritons produced from the d(d,t)p triton burnup reaction. These tritons "burn up" with deuterons and emit a 14 MeV neutron by the d(t, alpha)n reaction. The measured radial profiles of DT emission were compared with the predictions of a computer transport code. The ratio of the measured-to -calculated DT yield is typically 70%. The measured DT profile width is typically 5 cm larger than predicted by the transport code. The radial 14 MeV neutron profile was measured by a radial array of helium-4 recoil neutron spectrometers installed in the TFTR Multichannel Neutron Collimator (MCNC). The spectrometers are capable of measuring the primary and secondary neutron fluxes from deuterium discharges. The response to 14 MeV neutrons of the array has been measured by cross calibrating with the MCNC ZnS detector array when the emission from TFTR is predominantly DT neutrons. The response was also checked by comparing a model of the recoil spectrum based on nuclear physics data to the observed spectrum from ^{252 }Cf, ^{238}Pu -Be, and DT neutron sources. Extensions of this diagnostic to deuterium-tritium plasma and the implications for fusion research are discussed.

Antibody profiling sensitivity through increased reporter antibody layering

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

Apel, William A.; Thompson, Vicki S

A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immunemore » complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.« less

Flat dielectric metasurface lens array for three dimensional integral imaging

NASA Astrophysics Data System (ADS)

Zhang, Jianlei; Wang, Xiaorui; Yang, Yi; Yuan, Ying; Wu, Xiongxiong

2018-05-01

In conventional integral imaging, the singlet refractive lens array limits the imaging performance due to its prominent aberrations. Different from the refractive lens array relying on phase modulation via phase change accumulated along the optical paths, metasurfaces composed of nano-scatters can produce phase abrupt over the scale of wavelength. In this letter, we propose a novel lens array consisting of two neighboring flat dielectric metasurfaces for integral imaging system. The aspherical phase profiles of the metasurfaces are optimized to improve imaging performance. The simulation results show that our designed 5 × 5 metasurface-based lens array exhibits high image quality at designed wavelength 865 nm.

Real-time PCR (qPCR) primer design using free online software.

PubMed

Thornton, Brenda; Basu, Chhandak

2011-01-01

Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most commonly used fluorescent chemistries are SYBR® Green dyes and TaqMan®, Molecular Beacon or Scorpion probes. SYBR® Green is very simple to use and cost efficient. As SYBR® Green dye binds to any double-stranded DNA product, its success depends greatly on proper primer design. Many types of online primer design software are available, which can be used free of charge to design desirable SYBR® Green-based qPCR primers. This laboratory exercise is intended for those who have a fundamental background in PCR. It addresses the basic fluorescent chemistries of real-time PCR, the basic rules and pitfalls of primer design, and provides a step-by-step protocol for designing SYBR® Green-based primers with free, online software. Copyright © 2010 Wiley Periodicals, Inc.

Jet array impingement flow distributions and heat transfer characteristics. Effects of initial crossflow and nonuniform array geometry. [gas turbine engine component cooling

NASA Technical Reports Server (NTRS)

Florschuetz, L. W.; Metzger, D. E.; Su, C. C.; Isoda, Y.; Tseng, H. H.

1982-01-01

Two-dimensional arrays of circular air jets impinging on a heat transfer surface parallel to the jet orifice plate are considered. The jet flow, after impingement, is constrained to exit in a single direction along the channel formed by the jet orifice plate and the heat transfer surface. The configurations considered are intended to model those of interest in current and contemplated gas turbine airfoil midchord cooling applications. The effects of an initial crossflow which approaches the array through an upstream extension of the channel are considered. Flow distributions as well as heat transfer coefficients and adiabatic wall temperatures resolved to one streamwise hole spacing were measured as a function of the initial crossflow rate and temperature relative to the jet flow rate and temperature. Both Nusselt number profiles and dimensionless adiabatic wall temperature (effectiveness) profiles are presented and discussed. Special test results which show a significant reduction of jet orifice discharge coefficients owing to the effect of a confined crossflow are also presented, along with a flow distribution model which incorporates those effects. A nonuniform array flow distribution model is developed and validated.

Shear wave velocities in the upper mantle of the Western Alps: new constraints using array analysis of seismic surface waves

NASA Astrophysics Data System (ADS)

Lyu, Chao; Pedersen, Helle A.; Paul, Anne; Zhao, Liang; Solarino, Stefano

2017-07-01

It remains challenging to obtain absolute shear wave velocities of heterogeneities of small lateral extension in the uppermost mantle. This study presents a cross-section of Vs across the strongly heterogeneous 3-D structure of the western European Alps, based on array analysis of data from 92 broad-band seismic stations from the CIFALPS experiment and from permanent networks in France and Italy. Half of the stations were located along a dense sublinear array. Using a combination of these stations and off-profile stations, fundamental-mode Rayleigh wave dispersion curves were calculated using a combined frequency-time beamforming approach. We calculated dispersion curves for seven arrays of approximately 100 km aperture and 14 arrays of approximately 50 km aperture, the latter with the aim of obtaining a 2-D vertical cross-section of Vs beneath the western Alps. The dispersion curves were inverted for Vs(z), with crustal interfaces imposed from a previous receiver function study. The array approach proved feasible, as Vs(z) from independent arrays vary smoothly across the profile length. Results from the seven large arrays show that the shear velocity of the upper mantle beneath the European plate is overall low compared to AK135 with the lowest velocities in the internal part of the western Alps, and higher velocities east of the Alps beneath the Po plain. The 2-D Vs model is coherent with (i) a ∼100 km thick eastward-dipping European lithosphere west of the Alps, (ii) very high velocities beneath the Po plain, coherent with the presence of the Alpine (European) slab and (iii) a narrow low-velocity anomaly beneath the core of the western Alps (from the Briançonnais to the Dora Maira massif), and approximately colocated with a similar anomaly observed in a recent teleseismic P-wave tomography. This intriguing anomaly is also supported by traveltime variations of subvertically propagating body waves from two teleseismic events that are approximately located on the profile great circle.

Identification of molecular pathways affected by pterostilbene, a natural dimethylether analog of resveratrol

PubMed Central

Pan, Zhiqiang; Agarwal, Ameeta K; Xu, Tao; Feng, Qin; Baerson, Scott R; Duke, Stephen O; Rimando, Agnes M

2008-01-01

Background Pterostilbene, a naturally occurring phenolic compound produced by agronomically important plant genera such as Vitis and Vacciunium, is a phytoalexin exhibiting potent antifungal activity. Additionally, recent studies have demonstrated several important pharmacological properties associated with pterostilbene. Despite this, a systematic study of the effects of pterostilbene on eukaryotic cells at the molecular level has not been previously reported. Thus, the aim of the present study was to identify the cellular pathways affected by pterostilbene by performing transcript profiling studies, employing the model yeast Saccharomyces cerevisiae. Methods S. cerevisiae strain S288C was exposed to pterostilbene at the IC50 concentration (70 μM) for one generation (3 h). Transcript profiling experiments were performed on three biological replicate samples using the Affymetrix GeneChip Yeast Genome S98 Array. The data were analyzed using the statistical methods available in the GeneSifter microarray data analysis system. To validate the results, eleven differentially expressed genes were further examined by quantitative real-time RT-PCR, and S. cerevisiae mutant strains with deletions in these genes were analyzed for altered sensitivity to pterostilbene. Results Transcript profiling studies revealed that pterostilbene exposure significantly down-regulated the expression of genes involved in methionine metabolism, while the expression of genes involved in mitochondrial functions, drug detoxification, and transcription factor activity were significantly up-regulated. Additional analyses revealed that a large number of genes involved in lipid metabolism were also affected by pterostilbene treatment. Conclusion Using transcript profiling, we have identified the cellular pathways targeted by pterostilbene, an analog of resveratrol. The observed response in lipid metabolism genes is consistent with its known hypolipidemic properties, and the induction of mitochondrial genes is consistent with its demonstrated role in apoptosis in human cancer cell lines. Furthermore, our data show that pterostilbene has a significant effect on methionine metabolism, a previously unreported effect for this compound. PMID:18366703

SU-F-T-281: Monte Carlo Investigation of Sources of Dosimetric Discrepancies with 2D Arrays

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

Afifi, M; Deiab, N; El-Farrash, A

2016-06-15

Purpose: Intensity modulated radiation therapy (IMRT) poses a number of challenges for properly measuring commissioning data and quality assurance (QA). Understanding the limitations and use of dosimeters to measure these dose distributions is critical to safe IMRT implementation. In this work, we used Monte Carlo simulations to investigate the possible sources of discrepancy between our measurement with 2D array system and our dose calculation using our treatment planning system (TPS). Material and Methods: MCBEAM and MCSIM Monte Carlo codes were used for treatment head simulation and phantom dose calculation. Accurate modeling of a 6MV beam from Varian trilogy machine wasmore » verified by comparing simulated and measured percentage depth doses and profiles. Dose distribution inside the 2D array was calculated using Monte Carlo simulations and our TPS. Then Cross profiles for different field sizes were compared with actual measurements for zero and 90° gantry angle setup. Through the analysis and comparison, we tried to determine the differences and quantify a possible angular calibration factor. Results: Minimum discrepancies was seen in the comparison between the simulated and the measured profiles for the zero gantry angles at all studied field sizes (4×4cm{sup 2}, 10×10cm{sup 2}, 15×15cm{sup 2}, and 20×20cm{sup 2}). Discrepancies between our measurements and calculations increased dramatically for the cross beam profiles at the 90° gantry angle. This could ascribe mainly to the different attenuation caused by the layer of electronics at the base behind the ion chambers in the 2D array. The degree of attenuation will vary depending on the angle of beam incidence. Correction factors were implemented to correct the errors. Conclusion: Monte Carlo modeling of the 2D arrays and the derivation of angular dependence correction factors will allow for improved accuracy of the device for IMRT QA.« less

Altered gene expression by low-dose arsenic exposure in humans and cultured cardiomyocytes: Assessment by real-time PCR array

EPA Science Inventory

Arsenic contamination in drinking water has become a great public health concern worldwide. Chronic arsenic exposure results in higher risk of skin, lung and bladder cancer, as well as cardiovascular disease and diabetes. The purpose of this study was to investigate the effects o...

SEWAGE DECOMPOSITION IN AMBIENT WATER: INFLUENCE OF SOLARRADIATION AND BIOTIC INTERACTIONS ON MICROORGANISM COMMUNITIES AND BACTEROIDALES REAL-TIME QUANTITATIVE PCR MEASUREMENTS - poster

EPA Science Inventory

AIMS: Sewage and ambient water both consist of a highly complex array of bacteria and eukaryotic microbes. When these communities are mixed, solar radiation and biotic interactions (predation and competition) can influence pathogen decay based on experiments targeting indicator ...

Sewage Decomposition in Ambient Water: Influence of Solarradiation and Biotic Interactions on Microorganism Communities and Bacteroidales Real-Time Quantitative PCR Measurements - poster/abstract

EPA Science Inventory

AIMS: Sewage and ambient water both consist of a highly complex array of bacteria and eukaryotic microbes. When these communities are mixed, solar radiation and biotic interactions (predation and competition) can influence pathogen decay based on experiments targeting indicator ...

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

EPA Science Inventory

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

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

EPA Science Inventory

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

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.

Infralimbic EphB2 Modulates Fear Extinction in Adolescent Rats.

PubMed

Cruz, Emmanuel; Soler-Cedeño, Omar; Negrón, Geovanny; Criado-Marrero, Marangelie; Chompré, Gladys; Porter, James T

2015-09-09

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. Copyright © 2015 the authors 0270-6474/15/3512394-10$15.00/0.

Solid State Research

DTIC Science & Technology

1975-04-17

1-3. CO2 laser raster scan sensitivity profile of HgCdTe quadrantal array with two of the four elements connected to 50-ohm load. Fig. 1-4...Response of HgCdTe quadrantal array to CO2 laser beam scanned across center with (a) two opposite photodiodes connected, and (b) all four photodiodes...RESEARCH 1 A. Planar HgCdTe Quadrantal Arrays for Gigahertz Heterodyne Operation at 10.6 (im 1 B. Electrical Properties of Silicon Ion-Implanted

Development and Validation of PCR Primers To Assess the Diversity of Clostridium spp. in Cheese by Temporal Temperature Gradient Gel Electrophoresis

PubMed Central

Le Bourhis, Anne-Gaëlle; Saunier, Katiana; Doré, Joël; Carlier, Jean-Philippe; Chamba, Jean-François; Popoff, Michel-Robert; Tholozan, Jean-Luc

2005-01-01

A nested-PCR temporal temperature gradient gel electrophoresis (TTGE) approach was developed for the detection of bacteria belonging to phylogenetic cluster I of the genus Clostridium (the largest clostridial group, which represents 25% of the currently cultured clostridial species) in cheese suspected of late blowing. Primers were designed based on the 16S rRNA gene sequence, and the specificity was confirmed in PCRs performed with DNAs from cluster I and non-cluster I species as the templates. TTGE profiles of the PCR products, comprising the V5-V6 region of the 16S rRNA gene, allowed us to distinguish the majority of cluster I species. PCR-TTGE was applied to analyze commercial cheeses with defects. All cheeses gave a signal after nested PCR, and on the basis of band comigration with TTGE profiles of reference strains, all the bands could be assigned to a clostridial species. The direct identification of Clostridium spp. was confirmed by sequencing of excised bands. C. tyrobutyricum and C. beijerinckii contaminated 15 and 14 of the 20 cheese samples tested, respectively, and C. butyricum and C. sporogenes were detected in one cheese sample. Most-probable-number counts and volatile fatty acid were determined for comparison purposes. Results obtained were in agreement, but only two species, C. tyrobutyricum and C. sporogenes, could be isolated by the plating method. In all cheeses with a high amount of butyric acid (>100 mg/100 g), the presence of C. tyrobutyricum DNA was confirmed by PCR-TTGE, suggesting the involvement of this species in butyric acid fermentation. These results demonstrated the efficacy of the PCR-TTGE method to identify Clostridium in cheeses. The sensitivity of the method was estimated to be 100 CFU/g. PMID:15640166

Clinical Application of Picodroplet Digital PCR Technology for Rapid Detection of EGFR T790M in Next-Generation Sequencing Libraries and DNA from Limited Tumor Samples.

PubMed

Borsu, Laetitia; Intrieri, Julie; Thampi, Linta; Yu, Helena; Riely, Gregory; Nafa, Khedoudja; Chandramohan, Raghu; Ladanyi, Marc; Arcila, Maria E

2016-11-01

Although next-generation sequencing (NGS) is a robust technology for comprehensive assessment of EGFR-mutant lung adenocarcinomas with acquired resistance to tyrosine kinase inhibitors, it may not provide sufficiently rapid and sensitive detection of the EGFR T790M mutation, the most clinically relevant resistance biomarker. Here, we describe a digital PCR (dPCR) assay for rapid T790M detection on aliquots of NGS libraries prepared for comprehensive profiling, fully maximizing broad genomic analysis on limited samples. Tumor DNAs from patients with EGFR-mutant lung adenocarcinomas and acquired resistance to epidermal growth factor receptor inhibitors were prepared for Memorial Sloan-Kettering-Integrated Mutation Profiling of Actionable Cancer Targets sequencing, a hybrid capture-based assay interrogating 410 cancer-related genes. Precapture library aliquots were used for rapid EGFR T790M testing by dPCR, and results were compared with NGS and locked nucleic acid-PCR Sanger sequencing (reference high sensitivity method). Seventy resistance samples showed 99% concordance with the reference high sensitivity method in accuracy studies. Input as low as 2.5 ng provided a sensitivity of 1% and improved further with increasing DNA input. dPCR on libraries required less DNA and showed better performance than direct genomic DNA. dPCR on NGS libraries is a robust and rapid approach to EGFR T790M testing, allowing most economical utilization of limited material for comprehensive assessment. The same assay can also be performed directly on any limited DNA source and cell-free DNA. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Reference Gene Selection for qPCR Normalization of Kosteletzkya virginica under Salt Stress

PubMed Central

Tang, Xiaoli; Wang, Hongyan; Shao, Chuyang; Shao, Hongbo

2015-01-01

Kosteletzkya virginica (L.) is a newly introduced perennial halophytic plant. Presently, reverse transcription quantitative real-time PCR (qPCR) is regarded as the best choice for analyzing gene expression and its accuracy mainly depends on the reference genes which are used for gene expression normalization. In this study, we employed qPCR to select the most stable reference gene in K. virginica which showed stable expression profiles under our experimental conditions. The candidate reference genes were 18S ribosomal RNA (18SrRNA), β-actin (ACT), α-tubulin (TUA), and elongation factor (EF). We tracked the gene expression profiles of the candidate genes and analyzed their stabilities through BestKeeper, geNorm, and NormFinder software programs. The results of the three programs were identical and 18SrRNA was assessed to be the most stable reference gene in this study. However, TUA was identified to be the most unstable. Our study proved again that the traditional reference genes indeed displayed a certain degree of variations under given experimental conditions. Importantly, our research also provides guidance for selecting most suitable reference genes and lays the foundation for further studies in K. virginica. PMID:26581422

Enterobacterial Repetitive Intergenic Consensus Sequences as Molecular Targets for Typing of Mycobacterium tuberculosis Strains

PubMed Central

Sechi, Leonardo A.; Zanetti, Stefania; Dupré, Ilaria; Delogu, Giovanni; Fadda, Giovanni

1998-01-01

The presence of enterobacterial repetitive intergenic consensus (ERIC) sequences was demonstrated for the first time in the genome of Mycobacterium tuberculosis; these sequences have been found in transcribed regions of the chromosomes of gram-negative bacteria. In this study genetic diversity among clinical isolates of M. tuberculosis was determined by PCR with ERIC primers (ERIC-PCR). The study isolates comprised 71 clinical isolates collected from Sardinia, Italy. ERIC-PCR was able to identify 59 distinct profiles. The results obtained were compared with IS6110 and PCR-GTG fingerprinting. We found that the level of differentiation obtained by ERIC-PCR is greater than that obtained by IS6110 fingerprinting and comparable to that obtained by PCR-GTG. This method of fingerprinting is rapid and sensitive and can be applied to the study of the epidemiology of M. tuberculosis infections, especially when IS6110 fingerprinting is not of any help. PMID:9431935

Gestational form of Selenium in Free-Choice Mineral Mixes Affects Transcriptome Profiles of the Neonatal Calf Testis, Including those of Steroidogenic and Spermatogenic Pathways.

PubMed

Cerny, K L; Garbacik, S; Skees, C; Burris, W R; Matthews, J C; Bridges, P J

2016-01-01

In areas where soils are deficient in Selenium (Se), dietary supplementation of this trace mineral directly to cattle is recommended. Because Se status affects testosterone synthesis and frequency of sperm abnormalities, and the form of Se supplemented to cows affects tissue-specific gene expression, the objective of this study was to determine whether the form of Se consumed by cows during gestation would affect the expression of mRNAs that regulate steroidogenesis and/or spermatogenesis in the neonatal calf testis. Twenty-four predominantly Angus cows were assigned randomly to have individual, ad libitum, access of a mineral mix containing 35 ppm of Se in free-choice vitamin-mineral mixes as either inorganic (ISe), organic (OSe), or a 50/50 mix of ISe and OSe (MIX), starting 4 months prior to breeding and continuing throughout gestation. Thirteen male calves were born over a 3-month period (ISe, n = 5; OSe, n = 4; MIX, n = 4), castrated within 2 days of birth, and extracted testis RNA subjected to transcriptomal analysis by microarray (Affymetrix Bovine 1.0 ST arrays) and targeted gene expression analysis by real-time reverse-transcription PCR (RT-PCR) of mRNAs encoding proteins known to affect steroidogenesis and/or spermatogenesis. The form of dam Se affected (P < 0.05) the expression of 853 annotated genes, including 17 mRNAs putatively regulating steroidogenesis and/or spermatogenesis. Targeted RT-PCR analysis indicated that the expression of mRNA encoding proteins CYP2S1 (cytochrome P450, family 2, subfamily S, polypeptide 1), HSD17B7 (hydroxysteroid (17β) dehydrogenase 7), SULT1E1 (sulfotransferase family 1E, estrogen preferring, member 1), LDHA (lactate dehydrogenase A), CDK5R1 (cyclin-dependent kinase 5, regulatory subunit 1), and LEP (leptin) was affected (P < 0.05) by form of Se consumed by dams of developing bull calves, while AKR1C4 (aldo-keto reductase family 1, member C4) and CCND2 (cyclin D2) tended (P < 0.09) to be affected. Our results indicate that form of Se fed to dams during gestation affected the transcriptome of the neonatal calf testis. If these profiles are maintained throughout maturation, then the form of Se fed to dams may impact bull fertility and the development of Se form-dependent mineral mixes that target gestational development of the testis are warranted.

Apollo lunar surface experiments package

NASA Technical Reports Server (NTRS)

1972-01-01

Developments in the ALSEP program are reported. A summary of the status for the total ALSEP program is included. Other areas discussed include: (1) status of Apollo 16 (array D) and Apollo 17 (array E), (2) lunar seismic profiling experiment, (3) lunar ejecta and meteorites experiment, and (4) lunar mass spectrometer experiments.

Molecular genetic and biochemical responses in human airway epithelial cell cultures exposed to titanium nanoparticles in vitro.

PubMed

Aydın, Elanur; Türkez, Hasan; Hacımüftüoğlu, Fazıl; Tatar, Abdulgani; Geyikoğlu, Fatime

2017-07-01

Titanium nanoparticles (NPs) have very wide application areas such as paint, cosmetics, pharmaceuticals, and biomedical applications. And, to translate these nanomaterials to the clinic and industrial domains, their safety needs to be verified, particularly in terms of genotoxicity and cytotoxicity. Therefore, in this study, we aimed to investigate of cytotoxicity and changes in gene expression profiles influenced by commonly titanium (as titanium carbide, titanium carbo-nitride, titanium (II) oxide, titanium (III) oxide, titanium (IV) oxide, titanium nitride, titanium silicon oxide) NPs in human alveolar epithelial (HPAEpiC) and pharynx (HPPC) cell lines in vitro since inhalation is an important pathway for exposure to these NPs. HPAEpiC and HPPC cells were treated with titanium (0-100 µg/mL), NPs for 24 and 48 h, and then cytotoxicity was detected by, [3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide] (MTT), uptake of neutral red (NR) and lactate dehydrogenase (LDH) release assays, while genotoxicity was also analyzed by cDNA array - RT-PCR assay. According to the results of MTT, NR and LDH assays, all tested NPs induced cytotoxicity on both HPAEpiC and HPPC cells in a time- and dose-dependent manner. Determining and analyzing the gene expression profiles of HPAEpiC and HPPC cells, titanium NPs showed more changes in genes related to DNA damage or repair, oxidative stress, and apoptosis. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2056-2064, 2017. © 2017 Wiley Periodicals, Inc.

Transcriptome Profiling of Lotus japonicus Roots During Arbuscular Mycorrhiza Development and Comparison with that of Nodulation

PubMed Central

Deguchi, Yuichi; Banba, Mari; Shimoda, Yoshikazu; Chechetka, Svetlana A.; Suzuri, Ryota; Okusako, Yasuhiro; Ooki, Yasuhiro; Toyokura, Koichi; Suzuki, Akihiro; Uchiumi, Toshiki; Higashi, Shiro; Abe, Mikiko; Kouchi, Hiroshi; Izui, Katsura; Hata, Shingo

2007-01-01

Abstract To better understand the molecular responses of plants to arbuscular mycorrhizal (AM) fungi, we analyzed the differential gene expression patterns of Lotus japonicus, a model legume, with the aid of a large-scale cDNA macroarray. Experiments were carried out considering the effects of contaminating microorganisms in the soil inoculants. When the colonization by AM fungi, i.e. Glomus mosseae and Gigaspora margarita, was well established, four cysteine protease genes were induced. In situ hybridization revealed that these cysteine protease genes were specifically expressed in arbuscule-containing inner cortical cells of AM roots. On the other hand, phenylpropanoid biosynthesis-related genes for phenylalanine ammonia-lyase (PAL), chalcone synthase, etc. were repressed in the later stage, although they were moderately up-regulated on the initial association with the AM fungus. Real-time RT–PCR experiments supported the array experiments. To further confirm the characteristic expression, a PAL promoter was fused with a reporter gene and introduced into L. japonicus, and then the transformants were grown with a commercial inoculum of G. mosseae. The reporter activity was augmented throughout the roots due to the presence of contaminating microorganisms in the inoculum. Interestingly, G. mosseae only colonized where the reporter activity was low. Comparison of the transcriptome profiles of AM roots and nitrogen-fixing root nodules formed with Mesorhizobium loti indicated that the PAL genes and other phenylpropanoid biosynthesis-related genes were similarly repressed in the two organs. PMID:17634281

Transcriptional profiling of rat skeletal muscle hypertrophy under restriction of blood flow.

PubMed

Xu, Shouyu; Liu, Xueyun; Chen, Zhenhuang; Li, Gaoquan; Chen, Qin; Zhou, Guoqing; Ma, Ruijie; Yao, Xinmiao; Huang, Xiao

2016-12-15

Blood flow restriction (BFR) under low-intensity resistance training (LIRT) can produce similar effects upon muscles to that of high-intensity resistance training (HIRT) while overcoming many of the restrictions to HIRT that occurs in a clinical setting. However, the potential molecular mechanisms of BFR induced muscle hypertrophy remain largely unknown. Here, using a BFR rat model, we aim to better elucidate the mechanisms regulating muscle hypertrophy as induced by BFR and reveal possible clinical therapeutic targets for atrophy cases. We performed genome wide screening with microarray analysis to identify unique differentially expressed genes during rat muscle hypertrophy. We then successfully separated the differentially expressed genes from BRF treated soleus samples by comparing the Affymetrix rat Genome U34 2.0 array with the control. Using qRT-PCR and immunohistochemistry (IHC) we also analyzed other related differentially expressed genes. Results suggested that muscle hypertrophy induced by BFR is essentially regulated by the rate of protein turnover. Specifically, PI3K/AKT and MAPK pathways act as positive regulators in controlling protein synthesis where ubiquitin-proteasome acts as a negative regulator. This represents the first general genome wide level investigation of the gene expression profile in the rat soleus after BFR treatment. This may aid our understanding of the molecular mechanisms regulating and controlling muscle hypertrophy and provide support to the BFR strategies aiming to prevent muscle atrophy in a clinical setting. Copyright © 2016 Elsevier B.V. All rights reserved.

Therapeutic effects of Euphorbia Pekinensis and Glycyrrhiza glabra on Hepatocellular Carcinoma Ascites Partially Via Regulating the Frk-Arhgdib-Inpp5d-Avpr2-Aqp4 Signal Axis

NASA Astrophysics Data System (ADS)

Zhang, Yanqiong; Yan, Chen; Li, Yuting; Mao, Xia; Tao, Weiwei; Tang, Yuping; Lin, Ya; Guo, Qiuyan; Duan, Jingao; Lin, Na

2017-02-01

To clarify unknown rationalities of herbaceous compatibility of Euphorbia Pekinensis (DJ) and Glycyrrhiza glabra (GC) acting on hepatocellular carcinoma (HCC) ascites, peritoneum transcriptomics profiling of 15 subjects, including normal control (Con), HCC ascites mouse model (Mod), DJ-alone, DJ/GC-synergy and DJ/GC-antagonism treatment groups were performed on OneArray platform, followed by differentially expressed genes (DEGs) screening. DEGs between Mod and Con groups were considered as HCC ascites-related genes, and those among different drug treatment and Mod groups were identified as DJ/GC-combination-related genes. Then, an interaction network of HCC ascites-related gene-DJ/GC combination-related gene-known therapeutic target gene for ascites was constructed. Based on nodes’ degree, closeness, betweenness and k-coreness, the Frk-Arhgdib-Inpp5d-Avpr2-Aqp4 axis with highly network topological importance was demonstrated to be a candidate target of DJ/GC combination acting on HCC ascites. Importantly, both qPCR and western blot analyses verified this regulatory effects based on HCC ascites mice in vivo and M-1 collecting duct cells in vitro. Collectively, different combination designs of DJ and GC may lead to synergistic or antagonistic effects on HCC ascites partially via regulating the Frk-Arhgdib-Inpp5d-Avpr2-Aqp4 axis, implying that global gene expression profiling combined with network analysis can offer an effective way to understand pharmacological mechanisms of traditional Chinese medicine prescriptions.

Using Contaminant Transport Simulations to Optimize Electrical Resistivity Tomography Survey Design for Improved Contaminant Detection at Lined Ponds

NASA Astrophysics Data System (ADS)

Herring, T.; Pidlisecky, A.

2015-12-01

The saline flowback water produced during hydraulic fracturing is often stored in lined surface ponds. Leakage from these ponds poses a significant environmental threat and there is a need for a reliable and economical long term monitoring strategy. Electrical resistivity tomography (ERT), being sensitive to changes in groundwater salinity, is therefore well suited to such a problem. The goal of this work is to compare the leak detection capabilities of a surface ERT array and a downhole ERT array. In this study several plausible 3D electrical conductivity models were created that simulated a contaminant plume evolving over time, using realistic contaminant concentrations, plume geometries, water saturation profiles, and seasonal temperature profiles. The forward modeled data were used to identify the advantages and drawbacks of using each ERT array orientation.

Basic research for development of the beam profile monitor based on a Faraday cup array system

NASA Astrophysics Data System (ADS)

Park, Mook-Kwang

2015-10-01

The basic design used to develop a beam profile monitor based on a Faraday cup array (FCA), which has the advantages of high robustness, reliability, and long-term stability, along with the ability to measure the ion current over a wide dynamic range, was developed. The total system is divided into three parts: i.e., a faraday cup, measuring electronics, and a display program part. The FCA was considered to consist of a collimator, suppressor, insulator frame, and 64 (8 × 8 array) tiny Faraday cups (FC). An electronic circuit using a multiplexer was applied to effectively address many signal lines and the printed circuit board (PCB) was designed to be divided into three parts, i.e., an electrode PCB (ELEC PCB), capacitance PCB (CAP PCB), and control PCB (CON PCB).

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.

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.

Role of microbiological culture and polymerase chain reaction (PCR) of actinomyces in medication-related osteonecrosis of the jaw (MRONJ).

PubMed

Panya, Sappasith; Fliefel, Riham; Probst, Florian; Tröltzsch, Matthias; Ehrenfeld, Michael; Schubert, Sören; Otto, Sven

2017-03-01

We hypothesized that local infection plays a critical role in the pathogenesis of medication-related osteonecrosis of the jaw (MRONJ). Recent developments in molecular methods have revolutionized new approaches for the rapid detection of microorganisms including those difficult to culture. The aim of our study is to identify the bacterial profiles in MRONJ by microbiological culture and polymerase chain reactions (PCR). A retrospective analysis was performed on MRONJ patients from 2008 to 2014. The bacterial profile from MRONJ bone samples was determined using microbiological culture and PCR. Ninety five patients fulfilled the inclusion criteria with mean age of 69.85 ± 8.71 years. A female predilection was detected. The mandible was more commonly affected than maxilla. Tooth extraction was the frequent triggering factor. Breast cancer was the primary cause for administration and intravenous bisphosphonates were the most commonly administrated antiresorptive drugs. The majority of patients were classified as stage 2. Posterior teeth were most commonly affected. Based on bone culture results, the most common microorganism were both actinomyces and mixed flora. PCR confirmed the presence of actinomyces in 55 patients. Our data suggest that PCR might be an innovative method for detection of microorganisms difficult to culture using traditional microbiological techniques. Copyright © 2017 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

Detection of clinically relevant copy number alterations in oral cancer progression using multiplexed droplet digital PCR.

PubMed

Hughesman, Curtis B; Lu, X J David; Liu, Kelly Y P; Zhu, Yuqi; Towle, Rebecca M; Haynes, Charles; Poh, Catherine F

2017-09-19

Copy number alterations (CNAs), a common genomic event during carcinogenesis, are known to affect a large fraction of the genome. Common recurrent gains or losses of specific chromosomal regions occur at frequencies that they may be considered distinctive features of tumoral cells. Here we introduce a novel multiplexed droplet digital PCR (ddPCR) assay capable of detecting recurrent CNAs that drive tumorigenesis of oral squamous cell carcinoma. Applied to DNA extracted from oral cell lines and clinical samples of various disease stages, we found good agreement between CNAs detected by our ddPCR assay with those previously reported using comparative genomic hybridization or single nucleotide polymorphism arrays. Furthermore, we demonstrate that the ability to target specific locations of the genome permits detection of clinically relevant oncogenic events such as small, submicroscopic homozygous deletions. Additional capabilities of the multiplexed ddPCR assay include the ability to infer ploidy level, quantify the change in copy number of target loci with high-level gains, and simultaneously assess the status and viral load for high-risk human papillomavirus types 16 and 18. This novel multiplexed ddPCR assay therefore may have clinical value in differentiating between benign oral lesions from those that are at risk of progressing to oral cancer.