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Sample records for sensitive multiplex rna

  1. CEL-Seq2: sensitive highly-multiplexed single-cell RNA-Seq.

    PubMed

    Hashimshony, Tamar; Senderovich, Naftalie; Avital, Gal; Klochendler, Agnes; de Leeuw, Yaron; Anavy, Leon; Gennert, Dave; Li, Shuqiang; Livak, Kenneth J; Rozenblatt-Rosen, Orit; Dor, Yuval; Regev, Aviv; Yanai, Itai

    2016-01-01

    Single-cell transcriptomics requires a method that is sensitive, accurate, and reproducible. Here, we present CEL-Seq2, a modified version of our CEL-Seq method, with threefold higher sensitivity, lower costs, and less hands-on time. We implemented CEL-Seq2 on Fluidigm's C1 system, providing its first single-cell, on-chip barcoding method, and we detected gene expression changes accompanying the progression through the cell cycle in mouse fibroblast cells. We also compare with Smart-Seq to demonstrate CEL-Seq2's increased sensitivity relative to other available methods. Collectively, the improvements make CEL-Seq2 uniquely suited to single-cell RNA-Seq analysis in terms of economics, resolution, and ease of use. PMID:27121950

  2. Highly Sensitive Multiplex Assay for Detection of Human Immunodeficiency Virus Type 1 and Hepatitis C Virus RNA

    PubMed Central

    Giachetti, C.; Linnen, J. M.; Kolk, D. P.; Dockter, J.; Gillotte-Taylor, K.; Park, M.; Ho-Sing-Loy, M.; McCormick, M. K.; Mimms, L. T.; McDonough, S. H.

    2002-01-01

    Various nucleic acid assays have been developed and implemented for diagnostics and therapeutic monitoring of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infections. The high-throughput, semiautomated assays described here were developed to provide a method suitable for screening plasma specimens for the presence of HIV-1 and HCV RNAs. Three assays were developed: a multiplex HIV-1/HCV assay for simultaneous detection of HIV-1 and HCV, and discriminatory assays for specific detection of HIV-1 and HCV. The assay systems utilize three proprietary technologies: (i) target capture-based sample preparation, (ii) transcription-mediated amplification (TMA), and (iii) hybridization protection assay (HPA). An internal control is incorporated into each reaction to control for every step of the assay and identify random false-negative reactions. The assays demonstrated a sensitivity of at least 100 copies/ml for each target, and they detected with similar sensitivity all major variants of HCV and HIV-1, including HIV-1 group O strains. Assay sensitivity for one virus was not affected by the presence of the other. The specificity of these TMA-driven assays was ≥99.5% in both normal donor specimens and plasma containing potentially interfering substances or other blood-borne pathogens. Statistical receiver operating characteristic plots of 1 − specificity versus sensitivity data determined very wide analyte cutoff values for each assay at the point at which the assay specificity and sensitivity were both ≥99.5%. The sensitivity, specificity, and throughput capability predict that these assays will be valuable for large-volume plasma screening, either in a blood bank setting or in other diagnostic applications. PMID:12089255

  3. A highly multiplexed and sensitive RNA-seq protocol for simultaneous analysis of host and pathogen transcriptomes.

    PubMed

    Avraham, Roi; Haseley, Nathan; Fan, Amy; Bloom-Ackermann, Zohar; Livny, Jonathan; Hung, Deborah T

    2016-08-01

    The ability to simultaneously characterize the bacterial and host expression programs during infection would facilitate a comprehensive understanding of pathogen-host interactions. Although RNA sequencing (RNA-seq) has greatly advanced our ability to study the transcriptomes of prokaryotes and eukaryotes separately, limitations in existing protocols for the generation and analysis of RNA-seq data have hindered simultaneous profiling of host and bacterial pathogen transcripts from the same sample. Here we provide a detailed protocol for simultaneous analysis of host and bacterial transcripts by RNA-seq. Importantly, this protocol details the steps required for efficient host and bacteria lysis, barcoding of samples, technical advances in sample preparation for low-yield sample inputs and a computational pipeline for analysis of both mammalian and microbial reads from mixed host-pathogen RNA-seq data. Sample preparation takes 3 d from cultured cells to pooled libraries. Data analysis takes an additional day. Compared with previous methods, the protocol detailed here provides a sensitive, facile and generalizable approach that is suitable for large-scale studies and will enable the field to obtain in-depth analysis of host-pathogen interactions in infection models. PMID:27442864

  4. Development of highly sensitive and specific mRNA multiplex system (XCYR1) for forensic human body fluids and tissues identification.

    PubMed

    Xu, Yan; Xie, Jianhui; Cao, Yu; Zhou, Huaigu; Ping, Yuan; Chen, Liankang; Gu, Lihua; Hu, Wei; Bi, Gang; Ge, Jianye; Chen, Xin; Zhao, Ziqin

    2014-01-01

    The identification of human body fluids or tissues through mRNA-based profiling is very useful for forensic investigations. Previous studies have shown mRNA biomarkers are effective to identify the origin of biological samples. In this study, we selected 16 tissue specific biomarkers to evaluate their specificities and sensitivities for human body fluids and tissues identification, including porphobilinogen deaminase (PBGD), hemoglobin beta (HBB) and Glycophorin A (GLY) for circulatory blood, protamine 2 (PRM2) and transglutaminase 4 (TGM4) for semen, mucin 4 (MUC4) and human beta defensin 1(HBD1) for vaginal secretion, matrix metalloproteinases 7 and 11 (MMP7 and MMP11) for menstrual blood, keratin 4(KRT4) for oral mucosa, loricrin (LOR) and cystatin 6 (CST6) for skin, histatin 3(HTN3) for saliva, statherin (STATH) for nasal secretion, dermcidin (DCD) for sweat and uromodulin (UMOD) for urine. The above mentioned ten common forensic body fluids or tissues were used in the evaluation. Based on the evaluation, a reverse transcription (RT) PCR multiplex assay, XCYR1, which includes 12 biomarkers (i.e., HBB, GLY, HTN3, PRM2, KRT4, MMP11, MUC4, DCD, UMOD, MMP7, TGM4, and STATH) and 2 housekeeping genes [i.e., glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18SrRNA], was developed. This assay was further validated with real casework samples and mock samples (with both single source and mixture) and it was approved that XCYR1 is effective to identify common body fluids or tissues (i.e., circulatory blood, saliva, semen, vaginal secretion, menstrual blood, oral mucosa, nasal secretion, sweat and urine) in forensic casework samples. PMID:24991806

  5. Multiplexed miRNA northern blots via hybridization chain reaction.

    PubMed

    Schwarzkopf, Maayan; Pierce, Niles A

    2016-09-01

    Northern blots enable detection of a target RNA of interest in a biological sample using standard benchtop equipment. miRNAs are the most challenging targets as they must be detected with a single short nucleic acid probe. With existing approaches, it is cumbersome to perform multiplexed blots in which several RNAs are detected simultaneously, impeding the study of interacting regulatory elements. Here, we address this shortcoming by demonstrating multiplexed northern blotting based on the mechanism of hybridization chain reaction (HCR). With this approach, nucleic acid probes complementary to RNA targets trigger chain reactions in which fluorophore-labeled DNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability of HCR allows multiple amplifiers to operate simultaneously and independently within a blot, enabling straightforward multiplexing. We demonstrate simultaneous detection of three endogenous miRNAs in total RNA extracted from 293T and HeLa cells. For a given target, HCR signal scales linearly with target abundance, enabling relative and absolute quantitation. Using non-radioactive HCR, sensitive and selective miRNA detection is achieved using 2'OMe-RNA probes. The HCR northern blot protocol takes ∼1.5 days independent of the number of target RNAs. PMID:27270083

  6. Multiplexed miRNA northern blots via hybridization chain reaction

    PubMed Central

    Schwarzkopf, Maayan; Pierce, Niles A.

    2016-01-01

    Northern blots enable detection of a target RNA of interest in a biological sample using standard benchtop equipment. miRNAs are the most challenging targets as they must be detected with a single short nucleic acid probe. With existing approaches, it is cumbersome to perform multiplexed blots in which several RNAs are detected simultaneously, impeding the study of interacting regulatory elements. Here, we address this shortcoming by demonstrating multiplexed northern blotting based on the mechanism of hybridization chain reaction (HCR). With this approach, nucleic acid probes complementary to RNA targets trigger chain reactions in which fluorophore-labeled DNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability of HCR allows multiple amplifiers to operate simultaneously and independently within a blot, enabling straightforward multiplexing. We demonstrate simultaneous detection of three endogenous miRNAs in total RNA extracted from 293T and HeLa cells. For a given target, HCR signal scales linearly with target abundance, enabling relative and absolute quantitation. Using non-radioactive HCR, sensitive and selective miRNA detection is achieved using 2′OMe-RNA probes. The HCR northern blot protocol takes ∼1.5 days independent of the number of target RNAs. PMID:27270083

  7. Simultaneous Multiplexed Measurement of RNA and Proteins in Single Cells

    PubMed Central

    Darmanis, Spyros; Gallant, Caroline Julie; Marinescu, Voichita Dana; Niklasson, Mia; Segerman, Anna; Flamourakis, Georgios; Fredriksson, Simon; Assarsson, Erika; Lundberg, Martin; Nelander, Sven; Westermark, Bengt; Landegren, Ulf

    2015-01-01

    Summary Significant advances have been made in methods to analyze genomes and transcriptomes of single cells, but to fully define cell states, proteins must also be accessed as central actors defining a cell’s phenotype. Methods currently used to analyze endogenous protein expression in single cells are limited in specificity, throughput, or multiplex capability. Here, we present an approach to simultaneously and specifically interrogate large sets of protein and RNA targets in lysates from individual cells, enabling investigations of cell functions and responses. We applied our method to investigate the effects of BMP4, an experimental therapeutic agent, on early-passage glioblastoma cell cultures. We uncovered significant heterogeneity in responses to treatment at levels of RNA and protein, with a subset of cells reacting in a distinct manner to BMP4. Moreover, we found overall poor correlation between protein and RNA at the level of single cells, with proteins more accurately defining responses to treatment. PMID:26748716

  8. Spatially resolved, highly multiplexed RNA profiling in single cells

    PubMed Central

    Chen, Kok Hao; Boettiger, Alistair N.; Moffitt, Jeffrey R.; Wang, Siyuan; Zhuang, Xiaowei

    2015-01-01

    Knowledge of the expression profile and spatial landscape of the transcriptome in individual cells is essential for understanding the rich repertoire of cellular behaviors. Here we report multiplexed error-robust fluorescence in situ hybridization (MERFISH), a single-molecule imaging approach that allows the copy numbers and spatial localizations of thousands of RNA species to be determined in single cells. Using error-robust encoding schemes to combat single-molecule labeling and detection errors, we demonstrated the imaging of 100 – 1000 unique RNA species in hundreds of individual cells. Correlation analysis of the ~104 – 106 pairs of genes allowed us to constrain gene regulatory networks, predict novel functions for many unannotated genes, and identify distinct spatial distribution patterns of RNAs that correlate with properties of the encoded proteins. PMID:25858977

  9. Simultaneous detection of RNA and DNA targets based on multiplex isothermal amplification.

    PubMed

    Dobnik, David; Morisset, Dany; Lenarčič, Rok; Ravnikar, Maja

    2014-04-01

    The detection of pathogenic microorganisms present in food, feed, plant, and other samples is important for providing safe food as well as for preventing the spread of microbes. The genome of pathogens is made of DNA or RNA, therefore a multiplex diagnostics tool would ideally be able to amplify and detect both RNA and DNA targets in parallel. With this goal we have developed an isothermal nucleic acid sequence based amplification [NASBA] implemented microarray analysis (NAIMA) procedure, suitable for the simultaneous multiplex amplification of RNA and DNA targets, coupled with the detection on ArrayTubes. The method is demonstrated to be very sensitive and specific for the detection of two economically important quarantine plant pathogens of potato, the potato spindle tuber viroid (RNA target) and Ralstonia solanacearum (DNA target). Because of its isothermal amplification and simple detection equipment, the method is also applicable for on-site analyses. NAIMA can be used in any domain where there is the need to detect RNA and DNA targets simultaneously. PMID:24625323

  10. A protein multiplex microarray substrate with high sensitivity and specificity

    PubMed Central

    Fici, Dolores A.; McCormick, William; Brown, David W.; Herrmann, John E.; Kumar, Vikram; Awdeh, Zuheir L.

    2010-01-01

    The problems that have been associated with protein multiplex microarray immunoassay substrates and existing technology platforms include: binding, sensitivity, a low signal to noise ratio, target immobilization and the optimal simultaneous detection of diverse protein targets. Current commercial substrates for planar multiplex microarrays rely on protein attachment chemistries that range from covalent attachment to affinity ligand capture, to simple adsorption. In this pilot study, experimental performance parameters for direct monoclonal mouse IgG detection were compared for available two and three dimensional slide surface coatings with a new colloidal nitrocellulose substrate. New technology multiplex microarrays were also developed and evaluated for the detection of pathogen specific antibodies in human serum and the direct detection of enteric viral antigens. Data supports the nitrocellulose colloid as an effective reagent with the capacity to immobilize sufficient diverse protein target quantities for increased specificory signal without compromising authentic protein structure. The nitrocellulose colloid reagent is compatible with the array spotters and scanners routinely used for microarray preparation and processing. More importantly, as an alternate to fluorescence, colorimetric chemistries may be used for specific and sensitive protein target detection. The advantages of the nitrocellulose colloid platform indicate that this technology may be a valuable tool for the further development and expansion of multiplex microarray immunoassays in both the clinical and research laborat environment. PMID:20974147

  11. Rapid and Multiplexed MicroRNA Diagnostic Assay Using Quantum Dot-Based Förster Resonance Energy Transfer.

    PubMed

    Qiu, Xue; Hildebrandt, Niko

    2015-08-25

    The detection of next generation microRNA (miRNA) biomarkers has become a highly important aspect for clinical diagnostics. We use multiplexed Förster resonance energy transfer (FRET) between a luminescent Tb complex and three different semiconductor quantum dots (QDs) to sensitively detect three different miRNAs from a single 150 μL sample with ca. 1 nM (subpicomol) detection limits. The rapid and amplification-free mix-and-measure assay format is based on careful design of miRNA base pairing and stacking to selectively detect different miRNAs with very strong sequence homologies. Clinical applicability is demonstrated by sensitive multiplexed quantification of three miRNAs at low (2 to 10 nM) and varying concentrations in samples that contained up to 10% serum. PMID:26192765

  12. Sensitive giant magnetoresistive-based immunoassay for multiplex mycotoxin detection.

    PubMed

    Mak, Andy C; Osterfeld, Sebastian J; Yu, Heng; Wang, Shan X; Davis, Ronald W; Jejelowo, Olufisayo A; Pourmand, Nader

    2010-03-15

    Rapid and multiplexed measurement is vital in the detection of food-borne pathogens. While highly specific and sensitive, traditional immunochemical assays such as enzyme-linked immunosorbent assays (ELISAs) often require expensive read-out equipment (e.g. fluorescent labels) and lack the capability of multiplex detection. By combining the superior specificity of immunoassays with the sensitivity and simplicity of magnetic detection, we have developed a novel multiplex magnetic nanotag-based detection platform for mycotoxins that functions on a sub-picomolar concentration level. Unlike fluorescent labels, magnetic nanotags (MNTs) can be detected with inexpensive giant magnetoresistive (GMR) sensors such as spin-valve sensors. In the system presented here, each spin-valve sensor has an active area of 90 microm x 90 microm, arranged in an 8 x 8 array. Sample is added to the antibody-immobilized sensor array prior to the addition of the biotinylated detection antibody. The sensor response is recorded in real time upon the addition of streptavidin-linked MNTs on the chip. Here we demonstrate the simultaneous detection of multiple mycotoxins (aflatoxins B(1), zearalenone and HT-2) and show that a detection limit of 50 pg/mL can be achieved. PMID:20047828

  13. RNA Imaging with Multiplexed Error Robust Fluorescence in situ Hybridization

    PubMed Central

    Moffitt, Jeffrey R.; Zhuang, Xiaowei

    2016-01-01

    Quantitative measurements of both the copy number and spatial distribution of large fractions of the transcriptome in single-cells could revolutionize our understanding of a variety of cellular and tissue behaviors in both healthy and diseased states. Single-molecule Fluorescence In Situ Hybridization (smFISH)—an approach where individual RNAs are labeled with fluorescent probes and imaged in their native cellular and tissue context—provides both the copy number and spatial context of RNAs but has been limited in the number of RNA species that can be measured simultaneously. Here we describe Multiplexed Error Robust Fluorescence In Situ Hybridization (MERFISH), a massively parallelized form of smFISH that can image and identify hundreds to thousands of different RNA species simultaneously with high accuracy in individual cells in their native spatial context. We provide detailed protocols on all aspects of MERFISH, including probe design, data collection, and data analysis to allow interested laboratories to perform MERFISH measurements themselves. PMID:27241748

  14. Use of mitochondrial RNA genes for the differentiation of four Trichinella species by multiplex PCR amplification.

    PubMed

    Blaga, R; Fu, BaoQuan; Le Rhun, D; Le Naour, E; Heckman, A; Zocevic, A; Liu, MingYuan; Boireau, P

    2009-06-01

    Until now, four species of the Trichinella genus have been identified in Europe: Trichinella spiralis, T. nativa, T. britovi and T. pseudospiralis. The aim of this work was to establish a sound polymerase chain reaction (PCR)-based method to differentiate these four species using mitochondrial rDNA as a reliable genetic marker and to evaluate the sensitivity of this method. Full-length DNA sequences coding for the small and large mitochondrial rRNA (mt-rrnS and mt-rrnL) of the four species are described. A multiplex PCR was designed and successfully tested on 24 European isolates. As few as one larva, or 100 pg of genomic DNA was detected, providing equivalent sensitivity to previously described PCR methods. The PCR-based method of mitochondrial rDNA amplification was thereby established as a sensitive and reproductive diagnostic method for the four European Trichinella species. PMID:19389269

  15. Wavelength-multiplexing phase-sensitive surface plasmon imaging sensor.

    PubMed

    Shao, Yonghong; Li, Yan; Gu, Dayong; Zhang, Kai; Qu, Junle; He, Jianan; Li, Xuejin; Wu, Shu-Yuen; Ho, Ho-Pui; Somekh, Michael G; Niu, Hanben

    2013-05-01

    A wavelength-multiplexing phase-sensitive surface plasmon resonance (SPR) imaging sensor offering wide dynamic detection range and microarray capability is reported. Phase detection is accomplished by performing self-interference between the s- and p- polarizations within the signal beam. A liquid crystal tunable filter is used to sequentially select the SPR excitation wavelength from a white light source. This wavelength-multiplexing approach enables fast detection of the sensor's SPR phase response over a wide range of wavelengths, thereby covering literally any regions of interest within the SPR dip and thus maintaining the highest sensitivity point at all times. The phase-sensitive approach is particularly important for imaging SPR sensing applications because of its less stringent requirements for intensity signal-to-noise ratio, which also means the possibility of using uncooled modest resolution analog-to-digital conversion imaging devices. Experimental results demonstrate a resolution of 2.7×10(-7) RIU with a dynamic range of 0.0138 RIU. PMID:23632487

  16. Rapid and multiplex microRNA detection on graphically encoded silica suspension array.

    PubMed

    Jiang, Li; Shen, Ye; Zheng, Kexiao; Li, Jiong

    2014-11-15

    MicroRNA (miRNA), an 18-24-nucleotide noncoding RNA molecule, has become an ideal class of biomarker candidates for clinical diagnosis of cancers. By now, a number of detection methods for miRNAs have been developed on planar arrays and suspension arrays. In this work, we describe a hybridization-triggered fluorescence strategy for label-free and multiplex miRNA detection on graphically encoded silica suspension array. The total RNA is directly applied for analysis with an 8-mer Universal Tag which can be selectively captured by the capture probe via base-stacking effects. Benefiting from base-stacking effects, this novel method exhibits superb discrimination ability toward the 5' and 3' end single-nucleotide alteration. Mature miRNAs can be distinguished from their corresponding pre-miRNAs easily. Moreover, the estimated detection limit of 5 amol is comparable to some of the most sensitive methods. All these mentioned characteristics offer exciting possibilities for discovery and clinical applications. PMID:24892784

  17. Developed and evaluated a multiplex mRNA profiling system for body fluid identification in Chinese Han population.

    PubMed

    Song, Feng; Luo, Haibo; Hou, Yiping

    2015-10-01

    In forensic casework, identification the cellular origin from a biological sample is crucial to the case investigation and reconstruction in crime scene. DNA/RNA co-extraction for STR typing and human body fluids identification has been proposed as an efficient and comprehensive assay for forensic analysis. Several cell-specific messenger RNA (mRNA) markers for identification of the body fluids have been proposed by previous studies. In this study, a novel multiplex mRNA profiling system included 19 markers was developed and performed by reverse transcription endpoint polymerase chain reaction (RT-PCR). The multiplex combined 3 housekeeping gene markers and 16 cell-specific markers that have been used to identify five types of human body fluids: peripheral blood, semen, saliva, vaginal secretions and menstrual blood. The specificity, sensitivity, stability and detectability of the mixture were explored in our study. Majority of the cell-specific mRNA markers showed high specificity, although cross-reactivity was observed sporadically. Specific profiling for per body fluid was obtained. Moreover, the interpretation guidelines for inference of body fluid types were performed according to the A. Lindenbergh et al. The scoring guidelines can be applied to any RNA multiplex, which was based on six different scoring categories (observed, observed and fits, sporadically observed and fits, not observed, sporadically observed, not reliable, and non-specific due to high input). The simultaneous extraction of DNA showed positive full or partial profiling results of all samples. It demonstrated that the approach of combined STR-profiling and RNA profiling was suitable and reliable to detect the donor and origin of human body fluids in Chinese Han population. PMID:26311108

  18. A nucleic acid strand displacement system for the multiplexed detection of tuberculosis-specific mRNA using quantum dots

    NASA Astrophysics Data System (ADS)

    Gliddon, H. D.; Howes, P. D.; Kaforou, M.; Levin, M.; Stevens, M. M.

    2016-05-01

    The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar range using only a standard laboratory plate reader. We demonstrate the utility of our QD-based system for the detection of two genes selected from a microarray-derived tuberculosis-specific gene expression signature. Levels of up- and downregulated gene transcripts comprising this signature can be combined to give a disease risk score, making the signature more amenable for use as a diagnostic marker. Our QD-based approach to detect these transcripts could pave the way for novel diagnostic assays for tuberculosis.The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar

  19. Multiplexed miRNA Fluorescence In Situ Hybridization for Formalin-Fixed Paraffin-Embedded Tissues

    PubMed Central

    Renwick, Neil; Cekan, Pavol; Bognanni, Claudia; Tuschl, Thomas

    2015-01-01

    Multiplexed miRNA fluorescence in situ hybridization (miRNA FISH) is an advanced method for visualizing differentially expressed miRNAs, together with other reference RNAs, in archival tissues. Some miRNAs are excellent disease biomarkers due to their abundance and cell-type specificity. However, these short RNA molecules are difficult to visualize due to loss by diffusion, probe mishybridization, and signal detection and signal amplification issues. Here, we describe a reliable and adjustable method for visualizing and normalizing miRNA signals in formalin-fixed paraffin-embedded (FFPE) tissue sections. PMID:25218385

  20. Photocleavable DNA barcode-antibody conjugates allow sensitive and multiplexed protein analysis in single cell

    PubMed Central

    Agasti, Sarit S.; Liong, Monty; Peterson, Vanessa M.; Lee, Hakho; Weissleder, Ralph

    2012-01-01

    DNA barcoding is an attractive technology as it allows sensitive and multiplexed target analysis. However, DNA barcoding of cellular proteins remains challenging, primarily because barcode amplification and readout techniques are often incompatible with the cellular microenvironment. Here, we describe the development and validation of a photocleavable DNA barcode-antibody conjugate method for rapid, quantitative and multiplexed detection of proteins in single live cells. Following target binding, this method allows DNA barcodes to be photoreleased in solution, enabling easy isolation, amplification and readout. As a proof of principle, we demonstrate sensitive and multiplexed detection of protein biomarkers in a variety of cancer cells. PMID:23092113

  1. Highly sensitive and multiplexed platforms for allergy diagnostics

    NASA Astrophysics Data System (ADS)

    Monroe, Margo R.

    Allergy is a disorder of the immune system caused by an immune response to otherwise harmless environmental allergens. Currently 20% of the US population is allergic and 90% of pediatric patients and 60% of adult patients with asthma have allergies. These percentages have increased by 18.5% in the past decade, with predicted similar trends for the future. Here we design sensitive, multiplexed platforms to detect allergen-specific IgE using the Interferometric Reflectance Imaging Sensor (IRIS) for various clinical settings. A microarray platform for allergy diagnosis allows for testing of specific IgE sensitivity to a multitude of allergens, while requiring only small volumes of patient blood sample. However, conventional fluorescent microarray technology is limited by i) the variation of probe immobilization, which hinders the ability to make quantitative, assertive, and statistically relevant conclusions necessary in immunodiagnostics and ii) the use of fluorophore labels, which is not suitable for some clinical applications due to the tendency of fluorophores to stick to blood particulates and require daily calibration methods. This calibrated fluorescence enhancement (CaFE) method integrates the low magnification modality of IRIS with enhanced fluorescence sensing in order to directly correlate immobilized probe (major allergens) density to allergen-specific IgE in patient serum. However, this platform only operates in processed serum samples, which is not ideal for point of care testing. Thus, a high magnification modality of IRIS was adapted as an alternative allergy diagnostic platform to automatically discriminate and size single nanoparticles bound to specific IgE in unprocessed, characterized human blood and serum samples. These features make IRIS an ideal candidate for clinical and diagnostic applications, such a POC testing. The high magnification (nanoparticle counting) modality in conjunction with low magnification of IRIS in a combined instrument

  2. Automated RNA Extraction and Purification for Multiplexed Pathogen Detection

    SciTech Connect

    Bruzek, Amy K.; Bruckner-Lea, Cindy J.

    2005-01-01

    Pathogen detection has become an extremely important part of our nation?s defense in this post 9/11 world where the threat of bioterrorist attacks are a grim reality. When a biological attack takes place, response time is critical. The faster the biothreat is assessed, the faster countermeasures can be put in place to protect the health of the general public. Today some of the most widely used methods for detecting pathogens are either time consuming or not reliable [1]. Therefore, a method that can detect multiple pathogens that is inherently reliable, rapid, automated and field portable is needed. To that end, we are developing automated fluidics systems for the recovery, cleanup, and direct labeling of community RNA from suspect environmental samples. The advantage of using RNA for detection is that there are multiple copies of mRNA in a cell, whereas there are normally only one or two copies of DNA [2]. Because there are multiple copies of mRNA in a cell for highly expressed genes, no amplification of the genetic material may be necessary, and thus rapid and direct detection of only a few cells may be possible [3]. This report outlines the development of both manual and automated methods for the extraction and purification of mRNA. The methods were evaluated using cell lysates from Escherichia coli 25922 (nonpathogenic), Salmonella typhimurium (pathogenic), and Shigella spp (pathogenic). Automated RNA purification was achieved using a custom sequential injection fluidics system consisting of a syringe pump, a multi-port valve and a magnetic capture cell. mRNA was captured using silica coated superparamagnetic beads that were trapped in the tubing by a rare earth magnet. RNA was detected by gel electrophoresis and/or by hybridization of the RNA to microarrays. The versatility of the fluidics systems and the ability to automate these systems allows for quick and easy processing of samples and eliminates the need for an experienced operator.

  3. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system.

    PubMed

    Xie, Kabin; Minkenberg, Bastian; Yang, Yinong

    2015-03-17

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of the tRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplex editing capability of the CRISPR/Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA-gRNA architecture were efficiently and precisely processed into gRNAs with desired 5' targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability and editing efficiency of CRISPR/Cas9 toolkits. PMID:25733849

  4. A nucleic acid strand displacement system for the multiplexed detection of tuberculosis-specific mRNA using quantum dots.

    PubMed

    Gliddon, H D; Howes, P D; Kaforou, M; Levin, M; Stevens, M M

    2016-05-21

    The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar range using only a standard laboratory plate reader. We demonstrate the utility of our QD-based system for the detection of two genes selected from a microarray-derived tuberculosis-specific gene expression signature. Levels of up- and downregulated gene transcripts comprising this signature can be combined to give a disease risk score, making the signature more amenable for use as a diagnostic marker. Our QD-based approach to detect these transcripts could pave the way for novel diagnostic assays for tuberculosis. PMID:27088427

  5. miRge - A Multiplexed Method of Processing Small RNA-Seq Data to Determine MicroRNA Entropy

    PubMed Central

    Myers, Jason R.; Gupta, Simone; Weng, Lien-Chun; Ashton, John M.; Cornish, Toby C.; Pandey, Akhilesh; Halushka, Marc K.

    2015-01-01

    Small RNA RNA-seq for microRNAs (miRNAs) is a rapidly developing field where opportunities still exist to create better bioinformatics tools to process these large datasets and generate new, useful analyses. We built miRge to be a fast, smart small RNA-seq solution to process samples in a highly multiplexed fashion. miRge employs a Bayesian alignment approach, whereby reads are sequentially aligned against customized mature miRNA, hairpin miRNA, noncoding RNA and mRNA sequence libraries. miRNAs are summarized at the level of raw reads in addition to reads per million (RPM). Reads for all other RNA species (tRNA, rRNA, snoRNA, mRNA) are provided, which is useful for identifying potential contaminants and optimizing small RNA purification strategies. miRge was designed to optimally identify miRNA isomiRs and employs an entropy based statistical measurement to identify differential production of isomiRs. This allowed us to identify decreasing entropy in isomiRs as stem cells mature into retinal pigment epithelial cells. Conversely, we show that pancreatic tumor miRNAs have similar entropy to matched normal pancreatic tissues. In a head-to-head comparison with other miRNA analysis tools (miRExpress 2.0, sRNAbench, omiRAs, miRDeep2, Chimira, UEA small RNA Workbench), miRge was faster (4 to 32-fold) and was among the top-two methods in maximally aligning miRNAs reads per sample. Moreover, miRge has no inherent limits to its multiplexing. miRge was capable of simultaneously analyzing 100 small RNA-Seq samples in 52 minutes, providing an integrated analysis of miRNA expression across all samples. As miRge was designed for analysis of single as well as multiple samples, miRge is an ideal tool for high and low-throughput users. miRge is freely available at http://atlas.pathology.jhu.edu/baras/miRge.html. PMID:26571139

  6. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system

    PubMed Central

    Xie, Kabin; Minkenberg, Bastian

    2015-01-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of the tRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplex editing capability of the CRISPR/Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA–gRNA architecture were efficiently and precisely processed into gRNAs with desired 5′ targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability and editing efficiency of CRISPR/Cas9 toolkits. PMID:25733849

  7. Specific multiplex analysis of pathogens using a direct 16S rRNA hybridization in microarray system.

    PubMed

    Hwang, Byeong Hee; Shin, Hwa Hui; Seo, Jeong Hyun; Cha, Hyung Joon

    2012-06-01

    For the rapid multiplex analysis of pathogens, 16S rRNAs from cell lysates were directly applied onto a DNA microarray at room temperature (RT) for RNA-DNA hybridization. To eliminate the labeling step, seven fluorescent-labeled detector probes were cohybridized with 16S rRNA targets and adjacent specific capture probes. We found that eight pathogens were successfully discriminated by the 16S rRNA-based direct method, which showed greater specificity than the polymerase chain reaction (PCR)-labeled method due to chaperone and distance effects. A new specificity criterion for a perfect match between RNA and DNA was suggested to be 21-41% dissimilarity using correlation analysis between the mismatch and the sequence according to the guanine-cytosine (GC) percentage or the distribution of mismatches. Six categories of food matrix (egg, meat, milk, rice, vegetable, and mixed) were also tested, and the target pathogen was successfully discriminated within statistically significant levels. Finally, we found that the intrinsic abundance of 16S rRNA molecules successfully substituted PCR-based amplification with a low limit of detection of 10-10(3) cells mL(-1) and a high quantitative linear correlation. Collectively, our suggested 16S rRNA-based direct method enables the highly sensitive, specific, and quantitative analysis of selected pathogens at RT within 2 h, even in food samples. PMID:22551354

  8. Terbium complex to quantum dot Förster resonance energy transfer for homogeneous and multiplexed microRNA assay (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Qiu, Xue; Hildebrandt, Niko

    2016-03-01

    The importance of microRNA (miRNA) dysregulation in the development and progression of diseases has made these short-length nucleic acids to next generation biomarkers. Tb-to-QD Förster resonance energy transfer (FRET) has several unique advantages over organic dye-based FRET systems for biomolecular sensing. Large Förster distances (6-11 nm) offer much high FRET efficiencies, exceptionally long Tb excited-state lifetimes (ms) enable time-gated detection void of autofluorecence background, and the narrow, symmetric, and tunable emission bands of QDs provide unrivaled potential for multiplexing. Here we report a rapid and homogeneous method to sensitively detect three different miRNAs (hsa-miR-20a-5p, hsa-miR-20b-5p, and hsa-miR-21-5p) from a single 150 µL sample based on multiplexed FRET between a luminescent Lumi4-Tb complex and three different QDs. The biosensing approach exploits both base pairing and stacking. Careful design and optimization of sequence lengths and orientations of the QD and Tb-DNA conjugates was performed to provide maximum selectivity and sensitivity for all three miRNA biomarkers. The assays work at room temperature and were designed for their application on a KRYPTOR diagnostic plate reader system.Only 30 min of sample incubation and 7.5 s of measurement are required to obtain ca. 1 nM (subpicomol) detection limits. We also demonstrate precise multiplexed measurements of these miRNAs at different and varying concentrations and the feasibility of adapting the technology to point-of-care testing (POCT) in buffer containing 10% serum. Our assay does not only demonstrate an important milestone for the integration of quantum dots to multiplexed clinical diagnostics but also a unique rapid miRNA detection technology that is complimentary to the rather complicated high-throughput and high-sensitivity approaches that are established today.

  9. Sensitivity of mRNA Translation

    PubMed Central

    Poker, Gilad; Margaliot, Michael; Tuller, Tamir

    2015-01-01

    Using the dynamic mean-field approximation of the totally asymmetric simple exclusion process (TASEP), we investigate the effect of small changes in the initiation, elongation, and termination rates along the mRNA strand on the steady-state protein translation rate. We show that the sensitivity of mRNA translation is equal to the sensitivity of the maximal eigenvalue of a symmetric, nonnegative, tridiagonal, and irreducible matrix. This leads to new analytical results as well as efficient numerical schemes that are applicable for large-scale models. Our results show that in the usual endogenous case, when initiation is more rate-limiting than elongation, the sensitivity of the translation rate to small mutations rapidly increases towards the 5′ end of the ORF. When the initiation rate is high, as may be the case for highly expressed and/or heterologous optimized genes, the maximal sensitivity is with respect to the elongation rates at the middle of the mRNA strand. We also show that the maximal possible effect of a small increase/decrease in any of the rates along the mRNA is an increase/decrease of the same magnitude in the translation rate. These results are in agreement with previous molecular evolutionary and synthetic biology experimental studies. PMID:26238363

  10. In-solution multiplex miRNA detection using DNA-templated silver nanocluster probes.

    PubMed

    Shah, Pratik; Thulstrup, Peter Waaben; Cho, Seok Keun; Bhang, Yong-Joo; Ahn, Jong Cheol; Choi, Suk Won; Bjerrum, Morten Jannik; Yang, Seong Wook

    2014-05-01

    MicroRNAs (miRNAs) are small regulatory RNAs (size ∼21nt to ∼25nt) that can be used as biomarkers of disease diagnosis, and efforts have been directed towards the invention of a rapid, simple and sequence-selective detection method for miRNAs. We recently developed a DNA/silver nanoclusters (AgNCs)-based turn-off fluorescence method in the presence of target miRNA. To further advance our method toward multiplex miRNA detection in solution, the design of various fluorescent DNA/AgNCs probes was essential. Therefore, tethering of DNA-12nt scaffolds with 9 different AgNCs emitters to target-sensing DNA sequences was investigated. Interestingly, for the creation of spectrally different DNA/AgNCs probes, not only were the emitters encapsulated in 9 different DNA-12nt scaffolds necessary but the tethered target-sensing DNA sequences are also crucial to tune the fluorescence across the visible to infra-red region. In this study, we obtained three spectrally distinctive emitters of each DNA/AgNCs probes such as green, red, and near-infrared (NIR) fluorescence. Using these DNA/AgNCs probes, we here show a proof of concept for a rapid, one-step, in-solution multiplex miRNA detection method. PMID:24616905

  11. Multiplex qRT-PCR for the Detection of Western Equine Encephalomyelitis, St. Louis Encephalitis, and West Nile Viral RNA in Mosquito Pools (Diptera: Culicidae).

    PubMed

    Brault, Aaron C; Fang, Ying; Reisen, William K

    2015-05-01

    Following the introduction of West Nile virus into California during the summer of 2003, public health and vector control programs expanded surveillance efforts and were in need of diagnostics capable of rapid, sensitive, and specific detection of arbovirus infections of mosquitoes to inform decision support for intervention. Development of a multiplex TaqMan or real-time semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay in which three virus specific primer-probe sets were used in the same reaction is described herein for the detection of western equine encephalomyelitis, St. Louis encephalitis and West Nile viral RNA. Laboratory validation and field data from 10 transmission seasons are reported. The comparative sensitivity and specificity of this multiplex assay to singleplex RT-PCR as well as an antigen detection (rapid analyte measurement platform) and standard plaque assays indicate this assay to be rapid and useful in providing mosquito infection data to estimate outbreak risk. PMID:26334826

  12. A HaloTag-Based Small Molecule Microarray Screening Methodology with Increased Sensitivity and Multiplex Capabilities

    PubMed Central

    Noblin, Devin J.; Page, Charlotte M.; Tae, Hyun Seop; Gareiss, Peter C.; Schneekloth, John S.; Crews, Craig M.

    2013-01-01

    Small Molecule Microarrays (SMMs) represent a general platform for screening small molecule–protein interactions independent of functional inhibition of target proteins. In an effort to increase the scope and utility of SMMs, we have modified the SMM screening methodology to increase assay sensitivity and facilitate multiplex screening. Fusing target proteins to the HaloTag protein allows us to covalently prelabel fusion proteins with fluorophores, leading to increased assay sensitivity and an ability to conduct multiplex screens. We use the interaction between FKBP12 and two ligands, rapamycin and ARIAD’s “bumpxs” ligand, to show that the HaloTag-based SMM screening methodology significantly increases assay sensitivity. Additionally, using wild type FKBP12 and the FKBP12 F36V mutant, we show that prelabeling various protein isoforms with different fluorophores allows us to conduct multiplex screens and identify ligands to a specific isoform. Finally, we show this multiplex screening technique is capable of identifying ligands selective for a specific PTP1B isoform using a 20,000 compound screening deck. PMID:23013033

  13. Sensitive multiplex detection of serological liver cancer biomarkers using SERS-active photonic crystal fiber probe.

    PubMed

    Dinish, U S; Balasundaram, Ghayathri; Chang, Young Tae; Olivo, Malini

    2014-11-01

    Surface-enhanced Raman scattering (SERS) spectroscopy possesses the most promising advantage of multiplex detection for biosensing applications, which is achieved due to the narrow 'fingerprint' Raman spectra from the analyte molecules. We developed an ultrasensitive platform for the multiplex detection of cancer biomarkers by combining the SERS technique with a hollow-core photonic crystal fiber (HCPCF). Axially aligned air channels inside the HCPCF provide an excellent platform for optical sensing using SERS. In addition to the flexibility of optical fibers, HCPCF provides better light confinement and a larger interaction length for the guided light and the analyte, resulting in an improvement in sensitivity to detect low concentrations of bioanalytes in extremely low sample volumes. Herein, for the first time, we demonstrate the sensitive multiplex detection of biomarkers immobilized inside the HCPCF using antibody-conjugated SERS-active nanoparticles (SERS nanotags). As a proof-of-concept for targeted multiplex detection, initially we carried out the sensing of epidermal growth factor receptor (EGFR) biomarker in oral squamous carcinoma cell lysate using three different SERS nanotags. Subsequently, we also achieved simultaneous detection of hepatocellular carcinoma (HCC) biomarkers-alpha fetoprotein (AFP) and alpha-1-antitrypsin (A1AT) secreted in the supernatant from Hep3b cancer cell line. Using a SERS-HCPCF sensing platform, we could successfully demonstrate the multiplex detection in an extremely low sample volume of ∼20 nL. In future, this study may lead to sensitive biosensing platform for the low concentration detection of biomarkers in an extremely low sample volume of body fluids to achieve early diagnosis of multiple diseases. (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim). PMID:23963680

  14. Extensible Multiplex Real-time PCR of MicroRNA Using Microparticles.

    PubMed

    Jung, Seungwon; Kim, Junsun; Lee, Dong Jin; Oh, Eun Hae; Lim, Hwasup; Kim, Kwang Pyo; Choi, Nakwon; Kim, Tae Song; Kim, Sang Kyung

    2016-01-01

    Multiplex quantitative real-time PCR (qPCR), which measures multiple DNAs in a given sample, has received significant attention as a mean of verifying the rapidly increasing genetic targets of interest in single phenotype. Here we suggest a readily extensible qPCR for the expression analysis of multiple microRNA (miRNA) targets using microparticles of primer-immobilized networks as discrete reactors. Individual particles, 200~500 μm in diameter, are identified by two-dimensional codes engraved into the particles and the non-fluorescent encoding allows high-fidelity acquisition of signal in real-time PCR. During the course of PCR, the amplicons accumulate in the volume of the particles with high reliability and amplification efficiency over 95%. In a quick assay comprising of tens of particles holding different primers, each particle brings the independent real-time amplification curve representing the quantitative information of each target. Limited amount of sample was analyzed simultaneously in single chamber through this highly multiplexed qPCR; 10 kinds of miRNAs from purified extracellular vesicles (EVs). PMID:26964639

  15. Extensible Multiplex Real-time PCR of MicroRNA Using Microparticles

    PubMed Central

    Jung, Seungwon; Kim, Junsun; Lee, Dong Jin; Oh, Eun Hae; Lim, Hwasup; Kim, Kwang Pyo; Choi, Nakwon; Kim, Tae Song; Kim, Sang Kyung

    2016-01-01

    Multiplex quantitative real-time PCR (qPCR), which measures multiple DNAs in a given sample, has received significant attention as a mean of verifying the rapidly increasing genetic targets of interest in single phenotype. Here we suggest a readily extensible qPCR for the expression analysis of multiple microRNA (miRNA) targets using microparticles of primer-immobilized networks as discrete reactors. Individual particles, 200~500 μm in diameter, are identified by two-dimensional codes engraved into the particles and the non-fluorescent encoding allows high-fidelity acquisition of signal in real-time PCR. During the course of PCR, the amplicons accumulate in the volume of the particles with high reliability and amplification efficiency over 95%. In a quick assay comprising of tens of particles holding different primers, each particle brings the independent real-time amplification curve representing the quantitative information of each target. Limited amount of sample was analyzed simultaneously in single chamber through this highly multiplexed qPCR; 10 kinds of miRNAs from purified extracellular vesicles (EVs). PMID:26964639

  16. Multiplexed analysis of chromosome conformation at vastly improved sensitivity

    PubMed Central

    Davies, James O.J.; Telenius, Jelena M.; McGowan, Simon; Roberts, Nigel A.; Taylor, Stephen; Higgs, Douglas R.; Hughes, Jim R.

    2015-01-01

    Since methods for analysing chromosome conformation in mammalian cells are either low resolution or low throughput and are technically challenging they are not widely used outside of specialised laboratories. We have re-designed the Capture-C method producing a new approach, called next generation (NG) Capture-C. This produces unprecedented levels of sensitivity and reproducibility and can be used to analyse many genetic loci and samples simultaneously. Importantly, high-resolution data can be produced on as few as 100,000 cells and SNPs can be used to generate allele specific tracks. The method is straightforward to perform and should therefore greatly facilitate the task of linking SNPs identified by genome wide association studies with the genes they influence. The complete and detailed protocol presented here, with new publicly available tools for library design and data analysis, will allow most laboratories to analyse chromatin conformation at levels of sensitivity and throughput that were previously impossible. PMID:26595209

  17. Nanogold bioconjugates for direct and sensitive multiplexed immunosensing.

    PubMed

    Dobosz, P; Morais, S; Puchades, R; Maquieira, A

    2015-07-15

    The use of nanogold bioconjugates for direct detection of the antibody-antigen immunoreaction is addressed. The integration of gold nanoparticles tracers as signal generators in microarray immunosensing and compact disc detection technique show important advantages to reach sensitive, selective, high throughput, reliable and cost-effective assays. For that, a thorough study of the performances of the size of spherical nanogold particles and coating density was developed. The size of the nanoparticle determines the optimal antibody dilution, being the smaller particles the best performing ones. Enhancement effect of lower size is also studied. The gold labeling method do not affects the recognition capability of the labeled proteins. As a proof of concept, the nanoconjugates were used for the simultaneous and direct determination of small molecules. Employing nanogold bioconjugates as recognition labels resulted in robust and reliable assays, reaching a sensitivity of 0.03 and 1.3μg/L for sulfasalazine and atrazine, respectively. This shows that the use of nanogold bioconjugates for direct immunosensing is very competitive, achieving highly sensitive and reproducible assays (RSD<10%). This approach would simultaneously determine both small and large molecular size targets, in different formats, using the same detection mode what paves the way for many other applications in different scenarios. PMID:25771301

  18. Validation of an internally controlled one-step real-time multiplex RT-PCR assay for the detection and quantitation of dengue virus RNA in plasma.

    PubMed

    Hue, Kien Duong Thi; Tuan, Trung Vu; Thi, Hanh Tien Nguyen; Bich, Chau Tran Nguyen; Anh, Huy Huynh Le; Wills, Bridget A; Simmons, Cameron P

    2011-11-01

    Dengue is mosquito-borne virus infection that annually causes ~50 million clinically apparent cases worldwide. An internally controlled one-step real-time multiplex RT-PCR assay was developed for detection and quantitation of DENV RNA in plasma sample by using specific primers and fluorogenic TaqMan probes. All primers and probes targeted sequences near the 3' end of the NS5 gene. The method comprised two multiplex assays and was validated for sensitivity, specificity, linearity, reproducibility and precision. An internal control template was spiked into each clinical specimen to provide quality assurance for each experimental step. The assay allowed for detection of between 0.5 and 3 infectious particles per mL, is rapid and has been operationally characterized in 287 Vietnamese dengue patients from two therapeutic intervention trials at the Hospital for Tropical Diseases, Ho Chi Minh City, Viet Nam. PMID:21843553

  19. Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters.

    PubMed

    Lai, Charles P; Kim, Edward Y; Badr, Christian E; Weissleder, Ralph; Mempel, Thorsten R; Tannous, Bakhos A; Breakefield, Xandra O

    2015-01-01

    Accurate spatiotemporal assessment of extracellular vesicle (EV) delivery and cargo RNA translation requires specific and robust live-cell imaging technologies. Here we engineer optical reporters to label multiple EV populations for visualization and tracking of tumour EV release, uptake and exchange between cell populations both in culture and in vivo. Enhanced green fluorescence protein (EGFP) and tandem dimer Tomato (tdTomato) were fused at NH2-termini with a palmitoylation signal (PalmGFP, PalmtdTomato) for EV membrane labelling. To monitor EV-RNA cargo, transcripts encoding PalmtdTomato were tagged with MS2 RNA binding sequences and detected by co-expression of bacteriophage MS2 coat protein fused with EGFP. By multiplexing fluorescent and bioluminescent EV membrane reporters, we reveal the rapid dynamics of both EV uptake and translation of EV-delivered cargo mRNAs in cancer cells that occurred within 1-hour post-horizontal transfer between cells. These studies confirm that EV-mediated communication is dynamic and multidirectional between cells with delivery of functional mRNA. PMID:25967391

  20. Application of multiplex PCR for Rapid and sensitive detection of human papillomaviruses in cervical cancer

    PubMed Central

    Zandnia, Fateme; Doosti, Abbas; Mokhtari-Farsani, Abbas; Kardi, Mohammad Taghi; Movafagh, Abolfazl

    2016-01-01

    Objectives: Reffering to an increase in cervical cancer in the recent years, rapid, sensitive and economical detection of human papillomaviruses (HPVs) as causative agents of cervical cancer is important. The traditional methods for the detection of HPVs in cervical cancer, such as pap smear, suffer from limitation and PCR has a potential to overcome the limitaitons. The purpose of present research work was to identify the five important strains of HPV (16, 18, 31, 33 and 45) simultaneously by Multiplex PCR application. Methods: Study was done on 100 cervical lesions of women. DNA was extracted from specimens by a genomic DNA purification kit. A 5-plex PCR was developed for the simultaneous detection of major HPV. Five pair of new primers was designed for detection of HPV 16, 18, 31, 33 and 45 by Multiplex PCR. Results: Among the 100 evaluated samples, 82 were found positive to HPVs. In the meantime the highest rate of infection was for HPV 16. Also 30 of HPV positive samples had infections with two or more HPV types. Conclusion: Multiplex PCR assay used in present study can provide a rapid, sensitive and economical method for detection of viral infections and is applicable to small volumes of vaginal samples. PMID:27182258

  1. Giant magnetoresistive sensor array for sensitive and specific multiplexed food allergen detection.

    PubMed

    Ng, Elaine; Nadeau, Kari C; Wang, Shan X

    2016-06-15

    Current common allergen detection methods, including enzyme-linked immunosorbent assays (ELISAs) and dip-stick methods, do not provide adequate levels of sensitivity and specificity for at-risk allergic patients. A method for performing highly sensitive and specific detection of multiple food allergens is thus imperative as food allergies are becoming increasingly recognized as a major healthcare concern, affecting an estimated 4% of the total population. We demonstrate first instance of sensitive and specific multiplexed detection of major peanut allergens Ara h 1 and Ara h 2, and wheat allergen Gliadin using giant magnetoresistive (GMR) sensor arrays. Commercialized ELISA kits for Ara h 1 and Ara h 2 report limits of detection (LODs) at 31.5 ng/mL and 0.2 ng/mL, respectively. In addition, the 96-well-based ELISA developed in-house for Gliadin was found to have a LOD of 40 ng/mL. Our multiplexed GMR-based assay demonstrates the ability to perform all three assays on the same chip specifically and with sensitivities at LODs about an order of magnitude lower than those of 96-well-based ELISAs. LODs of GMR-based assays developed for Ara h 1, Ara h 2, and Gliadin were 7.0 ng/mL, 0.2 ng/mL, and 1.5 ng/mL, respectively, with little to no cross-reactivity. These LODs are clinically important as some patients could react strongly against such low allergen levels. Given the limitations of current industrial detection technology, multiplexed GMR-based assays provide a method for highly sensitive and specific simultaneous detection of any combination of food-product allergens, thus protecting allergic patients from life-threatening events, including anaphylaxis, by unintentional consumption. PMID:26859787

  2. Multisite Comparison of High-Sensitivity Multiplex Cytokine Assays▿†

    PubMed Central

    Breen, Elizabeth Crabb; Reynolds, Sandra M.; Cox, Christopher; Jacobson, Lisa P.; Magpantay, Larry; Mulder, Candice B.; Dibben, Oliver; Margolick, Joseph B.; Bream, Jay H.; Sambrano, Elise; Martínez-Maza, Otoniel; Sinclair, Elizabeth; Borrow, Persephone; Landay, Alan L.; Rinaldo, Charles R.; Norris, Philip J.

    2011-01-01

    The concentrations of cytokines in human serum and plasma can provide valuable information about in vivo immune status, but low concentrations often require high-sensitivity assays to permit detection. The recent development of multiplex assays, which can measure multiple cytokines in one small sample, holds great promise, especially for studies in which limited volumes of stored serum or plasma are available. Four high-sensitivity cytokine multiplex assays on a Luminex (Bio-Rad, BioSource, Linco) or electrochemiluminescence (Meso Scale Discovery) platform were evaluated for their ability to detect circulating concentrations of 13 cytokines, as well as for laboratory and lot variability. Assays were performed in six different laboratories utilizing archived serum from HIV-uninfected and -infected subjects from the Multicenter AIDS Cohort Study (MACS) and the Women's Interagency HIV Study (WIHS) and commercial plasma samples spanning initial HIV viremia. In a majority of serum samples, interleukin-6 (IL-6), IL-8, IL-10, and tumor necrosis factor alpha were detectable with at least three kits, while IL-1β was clearly detected with only one kit. No single multiplex panel detected all cytokines, and there were highly significant differences (P < 0.001) between laboratories and/or lots with all kits. Nevertheless, the kits generally detected similar patterns of cytokine perturbation during primary HIV viremia. This multisite comparison suggests that current multiplex assays vary in their ability to measure serum and/or plasma concentrations of cytokines and may not be sufficiently reproducible for repeated determinations over a long-term study or in multiple laboratories but may be useful for longitudinal studies in which relative, rather than absolute, changes in cytokines are important. PMID:21697338

  3. Multiplexed detection of viral infections using rapid in-situ RNA analysis on a chip

    PubMed Central

    Shaffer, Sydney M.; Joshi, Rohan P.; Chambers, Benjamin S.; Sterken, David; Biaesch, Andrew G.; Gabrieli, David J.; Li, Yang; Feemster, Kristen A.; Hensley, Scott E.; Issadore, David; Raj, Arjun

    2015-01-01

    Viral infections are a major cause of human disease, but many require molecular assays for conclusive diagnosis. Current assays typically rely on RT-PCR or ELISA; however, these tests often have limited speed, sensitivity or specificity. Here, we demonstrate that rapid RNA FISH is a viable alternative method that could improve upon these limitations. We describe a platform beginning with software to generate RNA FISH probes both for distinguishing related strains of virus (even those different by a single base) and for capturing large numbers of strains simultaneously. Next, we present a simple fluidic device for reliably performing RNA FISH assays in an automated fashion. Finally, we describe an automated image processing pipeline to robustly identify uninfected and infected samples. Together, our results establish RNA FISH as a methodology with potential for viral point-of-care diagnostics. PMID:26113495

  4. Sensitive and specific miRNA detection method using SplintR Ligase.

    PubMed

    Jin, Jingmin; Vaud, Sophie; Zhelkovsky, Alexander M; Posfai, Janos; McReynolds, Larry A

    2016-07-27

    We describe a simple, specific and sensitive microRNA (miRNA) detection method that utilizes Chlorella virus DNA ligase (SplintR(®) Ligase). This two-step method involves ligation of adjacent DNA oligonucleotides hybridized to a miRNA followed by real-time quantitative PCR (qPCR). SplintR Ligase is 100X faster than either T4 DNA Ligase or T4 RNA Ligase 2 for RNA splinted DNA ligation. Only a 4-6 bp overlap between a DNA probe and miRNA was required for efficient ligation by SplintR Ligase. This property allows more flexibility in designing miRNA-specific ligation probes than methods that use reverse transcriptase for cDNA synthesis of miRNA. The qPCR SplintR ligation assay is sensitive; it can detect a few thousand molecules of miR-122. For miR-122 detection the SplintR qPCR assay, using a FAM labeled double quenched DNA probe, was at least 40× more sensitive than the TaqMan assay. The SplintR method, when coupled with NextGen sequencing, allowed multiplex detection of miRNAs from brain, kidney, testis and liver. The SplintR qPCR assay is specific; individual let-7 miRNAs that differ by one nucleotide are detected. The rapid kinetics and ability to ligate DNA probes hybridized to RNA with short complementary sequences makes SplintR Ligase a useful enzyme for miRNA detection. PMID:27154271

  5. Advancing forensic RNA typing: On non-target secretions, a nasal mucosa marker, a differential co-extraction protocol and the sensitivity of DNA and RNA profiling.

    PubMed

    van den Berge, Margreet; Bhoelai, Bryan; Harteveld, Joyce; Matai, Anuska; Sijen, Titia

    2016-01-01

    The forensic identification of human body fluids and tissues by means of messenger RNA (mRNA) profiling is a long studied methodology that is increasingly applied to casework samples. Previously, we have described an mRNA multiplex system that targets blood, saliva, semen, menstrual secretion, vaginal mucosa and skin (Lindenbergh et al. and van den Berge et al.). In this study we consider various topics to improve this mRNA profiling system or its use and adapt the method accordingly. Bodily secretions that may be encountered at a crime scene whilst not targeted by the multiplex-id est nasal mucosa, sweat, tears, faeces and urine-were examined for false positive signals. The results prompted us to identify a nasal mucosa marker that allows the discrimination of nasal mucosa from saliva or vaginal mucosa and nosebleed blood from peripheral blood. An updated version of the multiplex was prepared to which the nasal mucosa marker was added and in which markers for semen, vaginal mucosa and blood were replaced. Lactobacillus markers were regarded unsuitable as replacement for vaginal mucosa mRNA markers because of background signals on penile swabs that appeared devoid of female DNA. Furthermore, we provide approaches to deal with highly unbalanced mixtures. First, a differential extraction protocol was incorporated into a co-extraction protocol to allow DNA and RNA analysis of separated non-sperm and sperm fractions. In a second approach, besides the standard multiplex, a customized multiplex is used which excludes markers for prevailing cell types. This allows the use of lower cDNA inputs for the prevailing cell types and higher inputs for cell types that appear masked. Additionally, we assessed the relation between the percentage of alleles or markers detected in DNA or RNA profiles when decreasing sample amounts are analysed. While blood, saliva, semen and menstrual secretion show the trend that DNA profiling is more sensitive than RNA profiling, the reverse is seen

  6. Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations

    PubMed Central

    Zonta, Eleonora; Garlan, Fanny; Pécuchet, Nicolas; Perez-Toralla, Karla; Caen, Ouriel; Milbury, Coren; Didelot, Audrey; Fabre, Elizabeth; Blons, Hélène; Laurent-Puig, Pierre; Taly, Valérie

    2016-01-01

    In cancer research, the accuracy of the technology used for biomarkers detection is remarkably important. In this context, digital PCR represents a highly sensitive and reproducible method that could serve as an appropriate tool for tumor mutational status analysis. In particular, droplet-based digital PCR approaches have been developed for detection of tumor-specific mutated alleles within plasmatic circulating DNA. Such an approach calls for the development and validation of a very significant quantity of assays, which can be extremely costly and time consuming. Herein, we evaluated assays for the detection and quantification of various mutations occurring in three genes often misregulated in cancers: the epidermal growth factor receptor (EGFR), the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and the Tumoral Protein p53 (TP53) genes. In particular, commercial competitive allele-specific TaqMan® PCR (castPCR™) technology, as well as TaqMan® and ZEN™ assays, have been evaluated for EGFR p.L858R, p.T790M, p.L861Q point mutations and in-frame deletions Del19. Specificity and sensitivity have been determined on cell lines DNA, plasmatic circulating DNA of lung cancer patients or Horizon Diagnostics Reference Standards. To show the multiplexing capabilities of this technology, several multiplex panels for EGFR (several three- and four-plexes) have been developed, offering new "ready-to-use" tests for lung cancer patients. PMID:27416070

  7. A multiplex readout method for position sensitive boron coated straw neutron detector

    NASA Astrophysics Data System (ADS)

    Yu, Hao; Gong, Hui; Li, Jianmin; Wang, Yongqiang; Wang, Xuewu; Li, Yuanjing; Kang, Kejun

    2015-10-01

    A 1 m×1 m boron coated straw neutron detector is expected to be used to build the small-angle neutron scattering (SANS) instrument of the Compact Pulsed Hadron Source (CPHS) in Tsinghua University. A multiplex readout method based on summing circuits in columns and rows is studied for this large area position sensitive detector. In this method, the outputs of charge sensitive preamplifiers are combined by columns and rows at two ends of the detector, and then the shaped signals are sampled by flash ADCs. With the position reconstructed algorithm implemented in FPGA which analyzes the charge division and column and row number of signals, the 3-D position information of neutron events can be obtained. The position resolution and counting rate performance of this method are analyzed, and the comparison to the delay-line readout method is also given. With the multiplex readout method, the scale of readout electronics can be greatly reduced and a good position resolution can be reached. A readout electronics system for a detector module which consists 4 × 10 straw tubes is designed based on this method, and the test with neutron beam shows an average 3-D spatial resolution of 4 × 4 × 6.8mm3.

  8. Highly Multiplexed RNA Aptamer Selection using a Microplate-based Microcolumn Device

    PubMed Central

    Reinholt, Sarah J.; Ozer, Abdullah; Lis, John T.; Craighead, Harold G.

    2016-01-01

    We describe a multiplexed RNA aptamer selection to 19 different targets simultaneously using a microcolumn-based device, MEDUSA (Microplate-based Enrichment Device Used for the Selection of Aptamers), as well as a modified selection process, that significantly reduce the time and reagents needed for selections. We exploited MEDUSA’s reconfigurable design between parallel and serially-connected microcolumns to enable the use of just 2 aliquots of starting library, and its 96-well microplate compatibility to enable the continued use of high-throughput techniques in downstream processes. Our modified selection protocol allowed us to perform the equivalent of a 10-cycle selection in the time it takes for 4 traditional selection cycles. Several aptamers were discovered with nanomolar dissociation constants. Furthermore, aptamers were identified that not only bound with high affinity, but also acted as inhibitors to significantly reduce the activity of their target protein, mouse decapping exoribonuclease (DXO). The aptamers resisted DXO’s exoribonuclease activity, and in studies monitoring DXO’s degradation of a 30-nucleotide substrate, less than 1 μM of aptamer demonstrated significant inhibition of DXO activity. This aptamer selection method using MEDUSA helps to overcome some of the major challenges with traditional aptamer selections, and provides a platform for high-throughput selections that lends itself to process automation. PMID:27432610

  9. Highly Multiplexed RNA Aptamer Selection using a Microplate-based Microcolumn Device.

    PubMed

    Reinholt, Sarah J; Ozer, Abdullah; Lis, John T; Craighead, Harold G

    2016-01-01

    We describe a multiplexed RNA aptamer selection to 19 different targets simultaneously using a microcolumn-based device, MEDUSA (Microplate-based Enrichment Device Used for the Selection of Aptamers), as well as a modified selection process, that significantly reduce the time and reagents needed for selections. We exploited MEDUSA's reconfigurable design between parallel and serially-connected microcolumns to enable the use of just 2 aliquots of starting library, and its 96-well microplate compatibility to enable the continued use of high-throughput techniques in downstream processes. Our modified selection protocol allowed us to perform the equivalent of a 10-cycle selection in the time it takes for 4 traditional selection cycles. Several aptamers were discovered with nanomolar dissociation constants. Furthermore, aptamers were identified that not only bound with high affinity, but also acted as inhibitors to significantly reduce the activity of their target protein, mouse decapping exoribonuclease (DXO). The aptamers resisted DXO's exoribonuclease activity, and in studies monitoring DXO's degradation of a 30-nucleotide substrate, less than 1 μM of aptamer demonstrated significant inhibition of DXO activity. This aptamer selection method using MEDUSA helps to overcome some of the major challenges with traditional aptamer selections, and provides a platform for high-throughput selections that lends itself to process automation. PMID:27432610

  10. Large area CMOS bio-pixel array for compact high sensitive multiplex biosensing.

    PubMed

    Sandeau, Laure; Vuillaume, Cassandre; Contié, Sylvain; Grinenval, Eva; Belloni, Federico; Rigneault, Hervé; Owens, Roisin M; Fournet, Margaret Brennan

    2015-02-01

    A novel CMOS bio-pixel array which integrates assay substrate and assay readout is demonstrated for multiplex and multireplicate detection of a triplicate of cytokines with single digit pg ml(-1) sensitivities. Uniquely designed large area bio-pixels enable individual assays to be dedicated to and addressed by single pixels. A capability to simultaneously measure a large number of targets is provided by the 128 available pixels. Chemiluminescent assays are carried out directly on the pixel surface which also detects the emitted chemiluminescent photons, facilitating a highly compact sensor and reader format. The high sensitivity of the bio-pixel array is enabled by the high refractive index of silicon based pixels. This in turn generates a strong supercritical angle luminescence response significantly increasing the efficiency of the photon collection over conventional farfield modalities. PMID:25490928

  11. Plasmonic substrates for multiplexed protein microarrays with femtomolar sensitivity and broad dynamic range

    PubMed Central

    Tabakman, Scott M.; Lau, Lana; Robinson, Joshua T.; Price, Jordan; Sherlock, Sarah P.; Wang, Hailiang; Zhang, Bo; Chen, Zhuo; Tangsombatvisit, Stephanie; Jarrell, Justin A.; Utz, Paul J.; Dai, Hongjie

    2012-01-01

    Protein chips are widely used for high-throughput proteomic analysis, but to date, the low sensitivity and narrow dynamic range have limited their capabilities in diagnostics and proteomics. Here we present protein microarrays on a novel nanostructured, plasmonic gold film with near-infrared fluorescence enhancement of up to 100-fold, extending the dynamic range of protein detection by three orders of magnitude towards the fM regime. We employ plasmonic protein microarrays for the early detection of a cancer biomarker, carcinoembryonic antigen, in the sera of mice bearing a xenograft tumour model. Further, we demonstrate a multiplexed autoantigen array for human autoantibodies implicated in a range of autoimmune diseases with superior signal-to-noise ratios and broader dynamic range compared with commercial nitrocellulose and glass substrates. The high sensitivity, broad dynamic range and easy adaptability of plasmonic protein chips presents new opportunities in proteomic research and diagnostics applications. PMID:21915108

  12. A multiplexed miRNA and transgene expression platform for simultaneous repression and expression of protein coding sequences.

    PubMed

    Seyhan, Attila A

    2016-01-01

    Knockdown of single or multiple gene targets by RNA interference (RNAi) is necessary to overcome escape mutants or isoform redundancy. It is also necessary to use multiple RNAi reagents to knockdown multiple targets. It is also desirable to express a transgene or positive regulatory elements and inhibit a target gene in a coordinated fashion. This study reports a flexible multiplexed RNAi and transgene platform using endogenous intronic primary microRNAs (pri-miRNAs) as a scaffold located in the green fluorescent protein (GFP) as a model for any functional transgene. The multiplexed intronic miRNA - GFP transgene platform was designed to co-express multiple small RNAs within the polycistronic cluster from a Pol II promoter at more moderate levels to reduce potential vector toxicity. The native intronic miRNAs are co-transcribed with a precursor GFP mRNA as a single transcript and presumably cleaved out of the precursor-(pre) mRNA by the RNA splicing machinery, spliceosome. The spliced intron with miRNA hairpins will be further processed into mature miRNAs or small interfering RNAs (siRNAs) capable of triggering RNAi effects, while the ligated exons become a mature messenger RNA for the translation of the functional GFP protein. Data show that this approach led to robust RNAi-mediated silencing of multiple Renilla Luciferase (R-Luc)-tagged target genes and coordinated expression of functional GFP from a single transcript in transiently transfected HeLa cells. The results demonstrated that this design facilitates the coordinated expression of all mature miRNAs either as individual miRNAs or as multiple miRNAs and the associated protein. The data suggest that, it is possible to simultaneously deliver multiple negative (miRNA or shRNA) and positive (transgene) regulatory elements. Because many cellular processes require simultaneous repression and activation of downstream pathways, this approach offers a platform technology to achieve that dual manipulation efficiently

  13. Simple, Sensitive and Accurate Multiplex Detection of Clinically Important Melanoma DNA Mutations in Circulating Tumour DNA with SERS Nanotags

    PubMed Central

    Wee, Eugene J.H.; Wang, Yuling; Tsao, Simon Chang-Hao; Trau, Matt

    2016-01-01

    Sensitive and accurate identification of specific DNA mutations can influence clinical decisions. However accurate diagnosis from limiting samples such as circulating tumour DNA (ctDNA) is challenging. Current approaches based on fluorescence such as quantitative PCR (qPCR) and more recently, droplet digital PCR (ddPCR) have limitations in multiplex detection, sensitivity and the need for expensive specialized equipment. Herein we describe an assay capitalizing on the multiplexing and sensitivity benefits of surface-enhanced Raman spectroscopy (SERS) with the simplicity of standard PCR to address the limitations of current approaches. This proof-of-concept method could reproducibly detect as few as 0.1% (10 copies, CV < 9%) of target sequences thus demonstrating the high sensitivity of the method. The method was then applied to specifically detect three important melanoma mutations in multiplex. Finally, the PCR/SERS assay was used to genotype cell lines and ctDNA from serum samples where results subsequently validated with ddPCR. With ddPCR-like sensitivity and accuracy yet at the convenience of standard PCR, we believe this multiplex PCR/SERS method could find wide applications in both diagnostics and research. PMID:27446486

  14. Rapid and sensitive PCR-dipstick DNA chromatography for multiplex analysis of the oral microbiota.

    PubMed

    Tian, Lingyang; Sato, Takuichi; Niwa, Kousuke; Kawase, Mitsuo; Tanner, Anne C R; Takahashi, Nobuhiro

    2014-01-01

    A complex of species has been associated with dental caries under the ecological hypothesis. This study aimed to develop a rapid, sensitive PCR-dipstick DNA chromatography assay that could be read by eye for multiplex and semiquantitative analysis of plaque bacteria. Parallel oligonucleotides were immobilized on a dipstick strip for multiplex analysis of target DNA sequences of the caries-associated bacteria, Streptococcus mutans, Streptococcus sobrinus, Scardovia wiggsiae, Actinomyces species, and Veillonella parvula. Streptavidin-coated blue-colored latex microspheres were to generate signal. Target DNA amplicons with an oligonucleotide-tagged terminus and a biotinylated terminus were coupled with latex beads through a streptavidin-biotin interaction and then hybridized with complementary oligonucleotides on the strip. The accumulation of captured latex beads on the test and control lines produced blue bands, enabling visual detection with the naked eye. The PCR-dipstick DNA chromatography detected quantities as low as 100 pg of DNA amplicons and demonstrated 10- to 1000-fold higher sensitivity than PCR-agarose gel electrophoresis, depending on the target bacterial species. Semiquantification of bacteria was performed by obtaining a series of chromatograms using serial 10-fold dilution of PCR-amplified DNA extracted from dental plaque samples. The assay time was less than 3 h. The semiquantification procedure revealed the relative amounts of each test species in dental plaque samples, indicating that this disposable device has great potential in analysis of microbial composition in the oral cavity and intestinal tract, as well as in point-of-care diagnosis of microbiota-associated diseases. PMID:25485279

  15. Analysis of sensitivity and rapid hybridization of a multiplexed Microbial Detection Microarray

    SciTech Connect

    Thissen, James B.; McLoughlin, Kevin; Gardner, Shea; Gu, Pauline; Mabery, Shalini; Slezak, Tom; Jaing, Crystal

    2014-06-01

    Microarrays have proven to be useful in rapid detection of many viruses and bacteria. Pathogen detection microarrays have been used to diagnose viral and bacterial infections in clinical samples and to evaluate the safety of biological drug materials. A multiplexed version of the Lawrence Livermore Microbial Detection Array (LLMDA) was developed and evaluated with minimum detectable concentrations for pure unamplified DNA viruses, along with mixtures of viral and bacterial DNA subjected to different whole genome amplification protocols. In addition the performance of the array was tested when hybridization time was reduced from 17 h to 1 h. The LLMDA was able to detect unamplified vaccinia virus DNA at a concentration of 14 fM, or 100,000 genome copies in 12 μL of sample. With amplification, positive identification was made with only 100 genome copies of input material. When tested against human stool samples from patients with acute gastroenteritis, the microarray detected common gastroenteritis viral and bacterial infections such as rotavirus and E. coli. Accurate detection was found but with a 4-fold drop in sensitivity for a 1 h compared to a 17 h hybridization. The array detected 2 ng (equivalent concentration of 15.6 fM) of labeled DNA from a virus with 1 h hybridization without any amplification, and was able to identify the components of a mixture of viruses and bacteria at species and in some cases strain level resolution. Sensitivity improved by three orders of magnitude with random whole genome amplification prior to hybridization; for instance, the array detected a DNA virus with only 20 fg or 100 genome copies as input. This multiplexed microarray is an efficient tool to analyze clinical and environmental samples for the presence of multiple viral and bacterial pathogens rapidly.

  16. Rapid and Sensitive PCR-Dipstick DNA Chromatography for Multiplex Analysis of the Oral Microbiota

    PubMed Central

    Niwa, Kousuke; Kawase, Mitsuo; Tanner, Anne C. R.; Takahashi, Nobuhiro

    2014-01-01

    A complex of species has been associated with dental caries under the ecological hypothesis. This study aimed to develop a rapid, sensitive PCR-dipstick DNA chromatography assay that could be read by eye for multiplex and semiquantitative analysis of plaque bacteria. Parallel oligonucleotides were immobilized on a dipstick strip for multiplex analysis of target DNA sequences of the caries-associated bacteria, Streptococcus mutans, Streptococcus sobrinus, Scardovia wiggsiae, Actinomyces species, and Veillonella parvula. Streptavidin-coated blue-colored latex microspheres were to generate signal. Target DNA amplicons with an oligonucleotide-tagged terminus and a biotinylated terminus were coupled with latex beads through a streptavidin-biotin interaction and then hybridized with complementary oligonucleotides on the strip. The accumulation of captured latex beads on the test and control lines produced blue bands, enabling visual detection with the naked eye. The PCR-dipstick DNA chromatography detected quantities as low as 100 pg of DNA amplicons and demonstrated 10- to 1000-fold higher sensitivity than PCR-agarose gel electrophoresis, depending on the target bacterial species. Semiquantification of bacteria was performed by obtaining a series of chromatograms using serial 10-fold dilution of PCR-amplified DNA extracted from dental plaque samples. The assay time was less than 3 h. The semiquantification procedure revealed the relative amounts of each test species in dental plaque samples, indicating that this disposable device has great potential in analysis of microbial composition in the oral cavity and intestinal tract, as well as in point-of-care diagnosis of microbiota-associated diseases. PMID:25485279

  17. Analysis of sensitivity and rapid hybridization of a multiplexed Microbial Detection Microarray

    DOE PAGESBeta

    Thissen, James B.; McLoughlin, Kevin; Gardner, Shea; Gu, Pauline; Mabery, Shalini; Slezak, Tom; Jaing, Crystal

    2014-06-01

    Microarrays have proven to be useful in rapid detection of many viruses and bacteria. Pathogen detection microarrays have been used to diagnose viral and bacterial infections in clinical samples and to evaluate the safety of biological drug materials. A multiplexed version of the Lawrence Livermore Microbial Detection Array (LLMDA) was developed and evaluated with minimum detectable concentrations for pure unamplified DNA viruses, along with mixtures of viral and bacterial DNA subjected to different whole genome amplification protocols. In addition the performance of the array was tested when hybridization time was reduced from 17 h to 1 h. The LLMDA wasmore » able to detect unamplified vaccinia virus DNA at a concentration of 14 fM, or 100,000 genome copies in 12 μL of sample. With amplification, positive identification was made with only 100 genome copies of input material. When tested against human stool samples from patients with acute gastroenteritis, the microarray detected common gastroenteritis viral and bacterial infections such as rotavirus and E. coli. Accurate detection was found but with a 4-fold drop in sensitivity for a 1 h compared to a 17 h hybridization. The array detected 2 ng (equivalent concentration of 15.6 fM) of labeled DNA from a virus with 1 h hybridization without any amplification, and was able to identify the components of a mixture of viruses and bacteria at species and in some cases strain level resolution. Sensitivity improved by three orders of magnitude with random whole genome amplification prior to hybridization; for instance, the array detected a DNA virus with only 20 fg or 100 genome copies as input. This multiplexed microarray is an efficient tool to analyze clinical and environmental samples for the presence of multiple viral and bacterial pathogens rapidly.« less

  18. Engineered nanoconstructs for the multiplexed and sensitive detection of high-risk pathogens.

    PubMed

    Seo, Youngmin; Kim, Ji-eun; Jeong, Yoon; Lee, Kwan Hong; Hwang, Jangsun; Hong, Jongwook; Park, Hansoo; Choi, Jonghoon

    2016-01-28

    Many countries categorize the causative agents of severe infectious diseases as high-risk pathogens. Given their extreme infectivity and potential to be used as biological weapons, a rapid and sensitive method for detection of high-risk pathogens (e.g., Bacillus anthracis, Francisella tularensis, Yersinia pestis, and Vaccinia virus) is highly desirable. Here, we report the construction of a novel detection platform comprising two units: (1) magnetic beads separately conjugated with multiple capturing antibodies against four different high-risk pathogens for simple and rapid isolation, and (2) genetically engineered apoferritin nanoparticles conjugated with multiple quantum dots and detection antibodies against four different high-risk pathogens for signal amplification. For each high-risk pathogen, we demonstrated at least 10-fold increase in sensitivity compared to traditional lateral flow devices that utilize enzyme-based detection methods. Multiplexed detection of high-risk pathogens in a sample was also successful by using the nanoconstructs harboring the dye molecules with fluorescence at different wavelengths. We ultimately envision the use of this novel nanoprobe detection platform in future applications that require highly sensitive on-site detection of high-risk pathogens. PMID:26462853

  19. Engineered nanoconstructs for the multiplexed and sensitive detection of high-risk pathogens

    NASA Astrophysics Data System (ADS)

    Seo, Youngmin; Kim, Ji-Eun; Jeong, Yoon; Lee, Kwan Hong; Hwang, Jangsun; Hong, Jongwook; Park, Hansoo; Choi, Jonghoon

    2016-01-01

    Many countries categorize the causative agents of severe infectious diseases as high-risk pathogens. Given their extreme infectivity and potential to be used as biological weapons, a rapid and sensitive method for detection of high-risk pathogens (e.g., Bacillus anthracis, Francisella tularensis, Yersinia pestis, and Vaccinia virus) is highly desirable. Here, we report the construction of a novel detection platform comprising two units: (1) magnetic beads separately conjugated with multiple capturing antibodies against four different high-risk pathogens for simple and rapid isolation, and (2) genetically engineered apoferritin nanoparticles conjugated with multiple quantum dots and detection antibodies against four different high-risk pathogens for signal amplification. For each high-risk pathogen, we demonstrated at least 10-fold increase in sensitivity compared to traditional lateral flow devices that utilize enzyme-based detection methods. Multiplexed detection of high-risk pathogens in a sample was also successful by using the nanoconstructs harboring the dye molecules with fluorescence at different wavelengths. We ultimately envision the use of this novel nanoprobe detection platform in future applications that require highly sensitive on-site detection of high-risk pathogens.

  20. In vivo mutagenesis of miRNA gene families using a scalable multiplexed CRISPR/Cas9 nuclease system

    PubMed Central

    Narayanan, Anand; Hill-Teran, Guillermina; Moro, Albertomaria; Ristori, Emma; Kasper, Dionna M.; A. Roden, Christine; Lu, Jun; Nicoli, Stefania

    2016-01-01

    A large number of microRNAs (miRNAs) are grouped into families derived from the same phylogenetic ancestors. miRNAs within a family often share the same physiological functions despite differences in their primary sequences, secondary structures, or chromosomal locations. Consequently, the generation of animal models to analyze the activity of miRNA families is extremely challenging. Using zebrafish as a model system, we successfully provide experimental evidence that a large number of miRNAs can be simultaneously mutated to abrogate the activity of an entire miRNA family. We show that injection of the Cas9 nuclease and two, four, ten, and up to twenty-four multiplexed single guide RNAs (sgRNAs) can induce mutations in 90% of the miRNA genomic sequences analyzed. We performed a survey of these 45 mutations in 10 miRNA genes, analyzing the impact of our mutagenesis strategy on the processing of each miRNA both computationally and in vivo. Our results offer an effective approach to mutate and study the activity of miRNA families and pave the way for further analysis on the function of complex miRNA families in higher multicellular organisms. PMID:27572667

  1. High sensitive immunoassay for multiplex mycotoxin detection with photonic crystal microsphere suspension array.

    PubMed

    Deng, Guozhe; Xu, Kun; Sun, Yue; Chen, Yu; Zheng, Tiesong; Li, Jianlin

    2013-03-01

    A novel, sensitive, and high throughput competitive immunoassay for multiplex mycotoxins was established by immobilizing the artificial antigens (Ags) of mycotoxins on the surfaces of three kinds of silica photonic crystal microsphere (SPCM) suspension arrays. The SPCMs were encoded by their reflectance peak positions. Aflatoxin B1 (AFB1), fumonisin B1 (FB1), and citrinin (CIT) spiked in the cereals were extracted, and the fluorescein isothiocyanate (FITC) labeled antibodies (Abs) of these mycotoxins were added into the centrifuge tube which contained the SPCMs of the modified artificial antigens (Ags). The fluorescence signal was collected by an array fluorescent scanner. The limit of detection (LOD) was as low as 0.5, 1, and 0.8 pg/mL for AFB1, FB1, and CIT, respectively. The new method provided a wide linear detection range from 0.001 to 10, 0.001 to 10, and 0.001 to 1 ng/mL for AFB1, FB1, and CIT, respectively. The mean recovery rates are in range of 74.7 ± 4.0% to 127.9 ± 4.4% for the three mycotoxins in corn, peanuts, and wheat. The developed method for mycotoxins was used to assay the AFB1, FB1, and CIT level in 10 naturally contaminated cereal samples, and the results of detection were in agreement with that of a classic enzyme-linked immunosorbent assay (ELISA) method. This method saves a large amount of reagents (10 μL volume) and detection time (<3 h) for multiplex mycotoxin assay. PMID:23350906

  2. Sensitive multiplex PCR assay to differentiate Lyme spirochetes and emerging pathogens Anaplasma phagocytophilum and Babesia microti

    PubMed Central

    2013-01-01

    Background The infection with Borrelia burgdorferi can result in acute to chronic Lyme disease. In addition, coinfection with tick-borne pathogens, Babesia species and Anaplasma phagocytophilum has been increasing in endemic regions of the USA and Europe. The currently used serological diagnostic tests are often difficult to interpret and, moreover, antibodies against the pathogens persist for a long time making it difficult to confirm the cure of the disease. In addition, these tests cannot be used for diagnosis of early disease state before the adaptive immune response is established. Since nucleic acids of the pathogens do not persist after the cure, DNA-based diagnostic tests are becoming highly useful for detecting infectious diseases. Results In this study, we describe a real-time multiplex PCR assay to detect the presence of B. burgdorferi, B. microti and A. phagocytophilum simultaneously even when they are present in very low copy numbers. Interestingly, this quantitative PCR technique is also able to differentiate all three major Lyme spirochete species, B. burgdorferi, B. afzelii, and B. garinii by utilizing a post-PCR denaturation profile analysis and a single molecular beacon probe. This could be very useful for diagnosis and discrimination of various Lyme spirochetes in European countries where all three Lyme spirochete species are prevalent. As proof of the principle for patient samples, we detected the presence of low number of Lyme spirochetes spiked in the human blood using our assay. Finally, our multiplex assay can detect all three tick-borne pathogens in a sensitive and specific manner irrespective of the level of each pathogen present in the sample. We anticipate that this novel diagnostic method will be able to simultaneously diagnose early to chronic stages of Lyme disease, babesiosis and anaplasmosis using the patients’ blood samples. Conclusion Real-time quantitative PCR using specific primers and molecular beacon probes for the selected

  3. Development of a Multiplex PCR Method for Detection of the Genes Encoding 16S rRNA, Coagulase, Methicillin Resistance and Enterotoxins in Staphylococcus aureus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A multiplex PCR method was developed for simultaneous detection of the genes encoding methicillin resistance (mecA), staphylococcal enterotoxins A, B and C (sea, seb and sec), coagulase (coa) and 16S rRNA. The primers for amplification of the 16S rRNA gene were specific for Staphylococcus spp., and ...

  4. Rapid and sensitive detection of major uropathogens in a single-pot multiplex PCR assay.

    PubMed

    Padmavathy, B; Vinoth Kumar, R; Patel, Amee; Deepika Swarnam, S; Vaidehi, T; Jaffar Ali, B M

    2012-07-01

    Urinary tract infection (UTI) is among the most common bacterial infections and poses a significant healthcare burden. Escherichia coli is the most common cause of UTI accounting for up to 70 % and a variable contribution from Proteus mirabilis, Pseudomonas aeruginosa and Klebsiella pneumoniae. To establish a complete diagnostic system, we have developed a single-tube multiplex PCR assay (mPCR) for the detection of the above-mentioned four major uropathogens. The sensitivity of the assay was found to be as low as 10(2) cfu/ml of cells. The mPCR evaluated on 280 clinical isolates detected 100 % of E. coli, P. aeruginosa, P. mirabilis and 95 % of K. pneumonia. The assay was performed on 50 urine samples and found to be specific and sensitive for clinical diagnosis. In addition, the mPCR was also validated on spiked urine samples using 40 clinical isolates to demonstrate its application under different strain used in this assay. In total, mPCR reported here is a rapid and simple screening tool that can compete with conventional biochemical-based screening assays that may require 2-3 days for detection. PMID:22526571

  5. High-speed polarization sensitive optical frequency domain imaging with frequency multiplexing

    PubMed Central

    Yun, S.H.; Vakoc, B.J.; Shishkov, M.; Desjardins, A.E.; Park, B.H.; de Boer, J.F.; Tearney, G.J.; Bouma, B.E.

    2009-01-01

    Polarization sensitive optical coherence tomography (PS-OCT) provides a cross-sectional image of birefringence in biological samples that is complementary in many applications to the standard reflectance-based image. Recent ex vivo studies have demonstrated that birefringence mapping enables the characterization of collagen and smooth muscle concentration and distribution in vascular tissues. Instruments capable of applying these measurements percutaneously in vivo may provide new insights into coronary atherosclerosis and acute myocardial infarction. We have developed a polarization sensitive optical frequency domain imaging (PS-OFDI) system that enables high-speed intravascular birefringence imaging through a fiber-optic catheter. The novel design of this system utilizes frequency multiplexing to simultaneously measure reflectance of two incident polarization states, overcoming concerns regarding temporal variations of the catheter fiber birefringence and spatial variations in the birefringence of the sample. We demonstrate circular cross-sectional birefringence imaging of a human coronary artery ex vivo through a flexible fiber-optic catheter with an A-line rate of 62 kHz and a ranging depth of 6.2 mm. PMID:18542183

  6. Quantitative Evaluation of Sensitivity and Selectivity of Multiplex NanoSPR Biosensor Assays

    PubMed Central

    Yu, Chenxu; Irudayaraj, Joseph

    2007-01-01

    A new functionalization procedure was developed to replace cyltrimethylammoniumbromide coating on gold nanorods (GNRs) fabricated through seed-mediated growth with chemically active alkanethiols; antibodies were then attached to the GNRs to yield gold nanorod molecular probes (GNrMPs). The functionalization procedure was shown to minimize nonspecific binding. Multiplex sensing was demonstrated for three targets (goat anti-human IgG, goat anti-rabbit IgG, and goat anti-mouse IgG) through the distinct response of the plasmon spectra of GNrMPs to binding events. Quantification of the plasmonic binding events and estimation of ligand binding kinetics tethered to these nanoscale structures was also demonstrated through a mathematical approach. Evaluation of the experimental and theoretical data yields an affinity constant Ka = 1.34 × 107 M−1, which was in agreement with the IgG-antiIgG binding affinity reported in the literature. The GNrMP sensors were found to be highly specific and sensitive with the dynamic response in the range between 10−9 M and 10−6 M. The limit of detection of GNrMPs was found to be in the low nanomolar range, and is a function of the binding affinity: for a higher probe-target affinity pair, the limit of detection can be expected to reach femto molar levels. This technique can play a key role in developing tunable sensors for sensitive and precise monitoring of biological interactions. PMID:17660314

  7. Simultaneous Detection of CDC Category “A” DNA and RNA Bioterrorism Agents by Use of Multiplex PCR & RT-PCR Enzyme Hybridization Assays

    PubMed Central

    He, Jie; Kraft, Andrea J.; Fan, Jiang; Van Dyke, Meredith; Wang, Lihua; Bose, Michael E.; Khanna, Marilyn; Metallo, Jacob A.; Henrickson, Kelly J.

    2009-01-01

    Assays to simultaneously detect multiple potential agents of bioterrorism are limited. Two multiplex PCR and RT-PCR enzyme hybridization assays (mPCR-EHA, mRT-PCR-EHA) were developed to simultaneously detect many of the CDC category “A” bioterrorism agents. The “Bio T” DNA assay was developed to detect: Variola major (VM), Bacillus anthracis (BA), Yersinia pestis (YP), Francisella tularensis (FT) and Varicella zoster virus (VZV). The “Bio T” RNA assay (mRT-PCR-EHA) was developed to detect: Ebola virus (Ebola), Lassa fever virus (Lassa), Rift Valley fever (RVF), Hantavirus Sin Nombre species (HSN) and dengue virus (serotypes 1–4). Sensitivity and specificity of the 2 assays were tested by using genomic DNA, recombinant plasmid positive controls, RNA transcripts controls, surrogate (spiked) clinical samples and common respiratory pathogens. The analytical sensitivity (limit of detection (LOD)) of the DNA asssay for genomic DNA was 1×100∼1×102 copies/mL for BA, FT and YP. The LOD for VZV whole organism was 1×10−2 TCID50/mL. The LOD for recombinant controls ranged from 1×102∼1×103copies/mL for BA, FT, YP and VM. The RNA assay demonstrated LOD for RNA transcript controls of 1×104∼1×106 copies/mL without extraction and 1×105∼1×106 copies/mL with extraction for Ebola, RVF, Lassa and HSN. The LOD for dengue whole organisms was ∼1×10−4 dilution for dengue 1 and 2, 1×104 LD50/mL and 1×102 LD50/mL for dengue 3 and 4. The LOD without extraction for recombinant plasmid DNA controls was ∼1×103 copies/mL (1.5 input copies/reaction) for Ebola, RVF, Lassa and HSN. No cross-reactivity of primers and probes used in both assays was detected with common respiratory pathogens or between targeted analytes. Clinical sensitivity was estimated using 264 surrogate clinical samples tested with the BioT DNA assay and 549 samples tested with the BioT RNA assay. The clinical specificity is 99.6% and 99.8% for BioT DNA assay and BioT RNA assay

  8. Evaluating Robustness and Sensitivity of the NanoString Technologies nCounter Platform to Enable Multiplexed Gene Expression Analysis of Clinical Samples.

    PubMed

    Veldman-Jones, Margaret H; Brant, Roz; Rooney, Claire; Geh, Catherine; Emery, Hollie; Harbron, Chris G; Wappett, Mark; Sharpe, Alan; Dymond, Michael; Barrett, J Carl; Harrington, Elizabeth A; Marshall, Gayle

    2015-07-01

    Analysis of clinical trial specimens such as formalin-fixed paraffin-embedded (FFPE) tissue for molecular mechanisms of disease progression or drug response is often challenging and limited to a few markers at a time. This has led to the increasing importance of highly multiplexed assays that enable profiling of many biomarkers within a single assay. Methods for gene expression analysis have undergone major advances in biomedical research, but obtaining a robust dataset from low-quality RNA samples, such as those isolated from FFPE tissue, remains a challenge. Here, we provide a detailed evaluation of the NanoString Technologies nCounter platform, which provides a direct digital readout of up to 800 mRNA targets simultaneously. We tested this system by examining a broad set of human clinical tissues for a range of technical variables, including sensitivity and limit of detection to varying RNA quantity and quality, reagent performance over time, variability between instruments, the impact of the number of fields of view sampled, and differences between probe sequence locations and overlapping genes across CodeSets. This study demonstrates that Nanostring offers several key advantages, including sensitivity, reproducibility, technical robustness, and utility for clinical application. PMID:26069246

  9. Sensitive Detection of Thirteen Bacterial Vaginosis-Associated Agents Using Multiplex Polymerase Chain Reaction

    PubMed Central

    Malaguti, Natália; Bahls, Larissa Danielle; Uchimura, Nelson Shozo; Gimenes, Fabrícia; Consolaro, Marcia Edilaine Lopes

    2015-01-01

    Bacterial vaginosis (BV) is characterized by a polymicrobial proliferation of anaerobic bacteria and depletion of lactobacilli, which are components of natural vaginal microbiota. Currently, there are limited conventional methods for BV diagnosis, and these methods are time-consuming, expensive, and rarely allow for the detection of more than one agent simultaneously. Therefore, we conceived and validated a multiplex polymerase chain reaction (M-PCR) assay for the simultaneous screening of thirteen bacterial vaginosis-associated agents (BV-AAs) related to symptomatic BV: Gardnerella vaginalis, Mobiluncus curtisii, Mobiluncus mulieris, Bacteroides fragilis, Mycoplasma hominis, Atopobium vaginae, Ureaplasma urealyticum, Megasphaera type I, Clostridia-like bacteria vaginosis-associated bacteria (BVABs) 1, 2, and 3, Sneathia sanguinegens, and Mycoplasma genitalium. The overall validation parameters of M-PCR compared to single PCR (sPCR) were extremely high, including agreement of 99.1% and sensitivity, specificity, and positive predictive values of 100.0%, negative predictive value of 97.0%, accuracy of 99.3%, and agreement with Nugent results of 100.0%. The prevalence of BV-AAs was very high (72.6%), and simultaneous agents were detected in 53.0%, which demonstrates the effectiveness of the M-PCR assay. Therefore, the M-PCR assay has great potential to impact BV diagnostic methods in vaginal samples and diminish associated complications in the near future. PMID:26078959

  10. A sensitive multiplex real-time PCR panel for rapid diagnosis of viruses associated with porcine respiratory and reproductive disorders.

    PubMed

    Wu, Haigang; Rao, Pinbin; Jiang, Yonghou; Opriessnig, Tanja; Yang, Zongqi

    2014-01-01

    The objective of this study was to develop a multiplex real-time PCR panel using TaqMan probes for the detection and differentiation of porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus North American type (PRRSV-NA), pseudorabies virus (PRV), classical swine fever virus (CSFV), porcine parvovirus type 1 (PPV1) and Japanese encephalitis virus (JEV). Specific primer and probe combinations for PCV2, PRRSV, PRV, CSFV, PPV1 and JEV were selected within the conserved region of each viral genome. The multiplex real-time PCR panel which was run in two separate tubes was capable of specific detection of the six selected pig viruses, without cross-reactions with other non-targeted pig viruses. The detection limit of the assays was 10 copies/μL for PCV2, PRV, CSFV and PRRSV and 100 copies/μL for PPV and JEV. The two-tube multiplex real-time PCR panel showed 99.2% concordance with conventional PCR assays on 118 field samples. Overall, the multiplex real-time PCR panel provides a fast, specific, and sensitive diagnostic tool for detection of multiple viral pathogens in pigs and will be useful not only for diagnostics, or ecological, epidemiological and pathogenesis studies, but also for investigating host/virus or virus/virus interactions, particularly during coinfections. PMID:25044282

  11. Improved Multiplex PCR Using Conserved and Species-Specific 16S rRNA Gene Primers for Simultaneous Detection of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis

    PubMed Central

    Tran, Simon Dangtuan; Rudney, Joel D.

    1999-01-01

    Among putative periodontal pathogens, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis are most convincingly implicated as etiological agents in periodontitis. Therefore, techniques for detection of those three species would be of value. We previously published a description of a multiplex PCR that detects A. actinomycetemcomitans and P. gingivalis. The present paper presents an improvement on that technique, which now allows more sensitive detection of all three periodontal pathogens. Sensitivity was determined by testing serial dilutions of A. actinomycetemcomitans, B. forsythus, and P. gingivalis cells. Primer specificity was tested against (i) all gene sequences from the GenBank-EMBL database, (ii) six A. actinomycetemcomitans, one B. forsythus, and four P. gingivalis strains, (iii) eight different species of oral bacteria, and (iv) supra- and subgingival plaque samples from 20 healthy subjects and subgingival plaque samples from 10 patients with periodontitis. The multiplex PCR had a detection limit of 10 A. actinomycetemcomitans, 10 P. gingivalis, and 100 B. forsythus cells. Specificity was confirmed by the fact that (i) none of our forward primers were homologous to the 16S rRNA genes of other oral species, (ii) amplicons of predicted size were detected for all A. actinomycetemcomitans, B. forsythus, and P. gingivalis strains tested, and (iii) no amplicons were detected for the eight other bacterial species. A. actinomycetemcomitans, B. forsythus, and P. gingivalis were detected in 6 of 20, 1 of 20, and 11 of 20 of supragingival plaque samples, respectively, and 4 of 20, 7 of 20, and 13 of 20 of subgingival plaque samples, respectively, from periodontally healthy subjects. Among patients with periodontitis, the organisms were detected in 7 of 10, 10 of 10, and 7 of 10 samples, respectively. The simultaneous detection of three periodontal pathogens is an advantage of this technique over conventional PCR assays. PMID

  12. Evaluation of Multiplexed 16S rRNA Microbial Population Surveys Using Illumina MiSeq Platform (Seventh Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting 2012)

    SciTech Connect

    Tremblay, Julien

    2012-06-01

    Julien Tremblay from DOE JGI presents "Evaluation of Multiplexed 16S rRNA Microbial Population Surveys Using Illumina MiSeq Platorm" at the 7th Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting held in June, 2012 in Santa Fe, NM.

  13. Evaluation of Multiplexed 16S rRNA Microbial Population Surveys Using Illumina MiSeq Platform (Seventh Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting 2012)

    ScienceCinema

    Tremblay, Julien [DOE JGI

    2013-01-25

    Julien Tremblay from DOE JGI presents "Evaluation of Multiplexed 16S rRNA Microbial Population Surveys Using Illumina MiSeq Platorm" at the 7th Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting held in June, 2012 in Santa Fe, NM.

  14. siRNA Delivery by Stimuli-Sensitive Nanocarriers

    PubMed Central

    Salzano, Giuseppina; Costa, Daniel F.; Torchilin, Vladimir P.

    2016-01-01

    Since its discovery in late 1990s, small interfering RNA (siRNA) has become a significant biopharmaceutical research tool and a powerful option for the treatment of different human diseases based on altered gene-expression. Despite promising data from many pre-clinical studies, concrete hurdles still need to be overcome to bring therapeutic siRNAs in clinic. The design of stimuli-sensitive nanopreparations for gene therapy is a lively area of the current research. Compared to conventional systems for siRNA delivery, this type of platform can respond to local stimuli that are characteristics of the pathological area of interest, allowing the release of nucleic acids at the desired site. Acidic pH, abnormal levels of enzymes, altered redox potential and magnetic field are examples of stimuli exploited in the design of stimuli-sensitive nanoparticles. In this review, we discuss on recent stimuli-sensitive strategies for siRNA delivery and we highlight on the potential of combining multiple stimuli-sensitive strategies in the same nano-platform for a better therapeutic outcome. PMID:26486143

  15. Automated Methods for Multiplexed Pathogen Detection

    SciTech Connect

    Straub, Tim M.; Dockendorff, Brian P.; Quinonez-Diaz, Maria D.; Valdez, Catherine O.; Shutthanandan, Janani I.; Tarasevich, Barbara J.; Grate, Jay W.; Bruckner-Lea, Cindy 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

  16. The Simoa HD-1 Analyzer: A Novel Fully Automated Digital Immunoassay Analyzer with Single-Molecule Sensitivity and Multiplexing.

    PubMed

    Wilson, David H; Rissin, David M; Kan, Cheuk W; Fournier, David R; Piech, Tomasz; Campbell, Todd G; Meyer, Raymond E; Fishburn, Matthew W; Cabrera, Carlos; Patel, Purvish P; Frew, Erica; Chen, Yao; Chang, Lei; Ferrell, Evan P; von Einem, Volker; McGuigan, William; Reinhardt, Marcus; Sayer, Heiko; Vielsack, Claus; Duffy, David C

    2016-08-01

    Disease detection at the molecular level is driving the emerging revolution of early diagnosis and treatment. A challenge facing the field is that protein biomarkers for early diagnosis can be present in very low abundance. The lower limit of detection with conventional immunoassay technology is the upper femtomolar range (10(-13) M). Digital immunoassay technology has improved detection sensitivity three logs, to the attomolar range (10(-16) M). This capability has the potential to open new advances in diagnostics and therapeutics, but such technologies have been relegated to manual procedures that are not well suited for efficient routine use. We describe a new laboratory instrument that provides full automation of single-molecule array (Simoa) technology for digital immunoassays. The instrument is capable of single-molecule sensitivity and multiplexing with short turnaround times and a throughput of 66 samples/h. Singleplex and multiplexed digital immunoassays were developed for 16 proteins of interest in cardiovascular, cancer, infectious disease, neurology, and inflammation research. The average sensitivity improvement of the Simoa immunoassays versus conventional ELISA was >1200-fold, with coefficients of variation of <10%. The potential of digital immunoassays to advance human diagnostics was illustrated in two clinical areas: traumatic brain injury and early detection of infectious disease. PMID:26077162

  17. Multiplex bioimaging of piRNA molecular pathway-regulated theragnostic effects in a single breast cancer cell using a piRNA molecular beacon.

    PubMed

    Lee, Youn Jung; Moon, Sung Ung; Park, Min Geun; Jung, Woon Yong; Park, Yong Keun; Song, Sung Kyu; Ryu, Je Gyu; Lee, Yong Seung; Heo, Hye Jung; Gu, Ha Na; Cho, Su Jeong; Ali, Bahy A; Al-Khedhairy, Abdulaziz A; Lee, Ilkyun; Kim, Soonhag

    2016-09-01

    Recently, PIWI-interacting small non-coding RNAs (piRNAs) have emerged as novel cancer biomarkers candidate because of their high expression level in various cancer types and role in the control of tumor suppressor genes. In this study, a novel breast cancer theragnostics probe based on a single system targeting the piRNA-36026 (piR-36026) molecular pathway was developed using a piR-36026 molecular beacon (MB). The piR-36026 MB successfully visualized endogenous piR-36026 biogenesis, which is highly expressed in MCF7 cells (a human breast cancer cell line), and simultaneously inhibited piR-36026-mediated cancer progression in vitro and in vivo. We discovered two tumor suppressor proteins, SERPINA1 and LRAT, that were directly regulated as endogenous piR-36026 target genes in MCF7 cells. Furthermore, multiplex bioimaging of a single MCF7 cell following treatment with piR-36026 MB clearly visualized the direct molecular interaction of piRNA-36026 with SERPINA1 or LRAT and subsequent molecular therapeutic responses including caspase-3 and PI in the nucleus. PMID:27289065

  18. Analytical Sensitivity Comparison between Singleplex Real-Time PCR and a Multiplex PCR Platform for Detecting Respiratory Viruses.

    PubMed

    Parker, Jayme; Fowler, Nisha; Walmsley, Mary Louise; Schmidt, Terri; Scharrer, Jason; Kowaleski, James; Grimes, Teresa; Hoyos, Shanann; Chen, Jack

    2015-01-01

    Multiplex PCR methods are attractive to clinical laboratories wanting to broaden their detection of respiratory viral pathogens in clinical specimens. However, multiplexed assays must be well optimized to retain or improve upon the analytic sensitivity of their singleplex counterparts. In this experiment, the lower limit of detection (LOD) of singleplex real-time PCR assays targeting respiratory viruses is compared to an equivalent panel on a multiplex PCR platform, the GenMark eSensor RVP. LODs were measured for each singleplex real-time PCR assay and expressed as the lowest copy number detected 95-100% of the time, depending on the assay. The GenMark eSensor RVP LODs were obtained by converting the TCID50/mL concentrations reported in the package insert to copies/μL using qPCR. Analytical sensitivity between the two methods varied from 1.2-1280.8 copies/μL (0.08-3.11 log differences) for all 12 assays compared. Assays targeting influenza A/H3N2, influenza A/H1N1pdm09, influenza B, and human parainfluenza 1 and 2 were most comparable (1.2-8.4 copies/μL, <1 log difference). Largest differences in LOD were demonstrated for assays targeting adenovirus group E, respiratory syncytial virus subtype A, and a generic assay for all influenza A viruses regardless of subtype (319.4-1280.8 copies/μL, 2.50-3.11 log difference). The multiplex PCR platform, the GenMark eSensor RVP, demonstrated improved analytical sensitivity for detecting influenza A/H3 viruses, influenza B virus, human parainfluenza virus 2, and human rhinovirus (1.6-94.8 copies/μL, 0.20-1.98 logs). Broader detection of influenza A/H3 viruses was demonstrated by the GenMark eSensor RVP. The relationship between TCID50/mL concentrations and the corresponding copy number related to various ATCC cultures is also reported. PMID:26569120

  19. Analytical Sensitivity Comparison between Singleplex Real-Time PCR and a Multiplex PCR Platform for Detecting Respiratory Viruses

    PubMed Central

    Parker, Jayme; Fowler, Nisha; Walmsley, Mary Louise; Schmidt, Terri; Scharrer, Jason; Kowaleski, James; Grimes, Teresa; Hoyos, Shanann; Chen, Jack

    2015-01-01

    Multiplex PCR methods are attractive to clinical laboratories wanting to broaden their detection of respiratory viral pathogens in clinical specimens. However, multiplexed assays must be well optimized to retain or improve upon the analytic sensitivity of their singleplex counterparts. In this experiment, the lower limit of detection (LOD) of singleplex real-time PCR assays targeting respiratory viruses is compared to an equivalent panel on a multiplex PCR platform, the GenMark eSensor RVP. LODs were measured for each singleplex real-time PCR assay and expressed as the lowest copy number detected 95–100% of the time, depending on the assay. The GenMark eSensor RVP LODs were obtained by converting the TCID50/mL concentrations reported in the package insert to copies/μL using qPCR. Analytical sensitivity between the two methods varied from 1.2–1280.8 copies/μL (0.08–3.11 log differences) for all 12 assays compared. Assays targeting influenza A/H3N2, influenza A/H1N1pdm09, influenza B, and human parainfluenza 1 and 2 were most comparable (1.2–8.4 copies/μL, <1 log difference). Largest differences in LOD were demonstrated for assays targeting adenovirus group E, respiratory syncytial virus subtype A, and a generic assay for all influenza A viruses regardless of subtype (319.4–1280.8 copies/μL, 2.50–3.11 log difference). The multiplex PCR platform, the GenMark eSensor RVP, demonstrated improved analytical sensitivity for detecting influenza A/H3 viruses, influenza B virus, human parainfluenza virus 2, and human rhinovirus (1.6–94.8 copies/μL, 0.20–1.98 logs). Broader detection of influenza A/H3 viruses was demonstrated by the GenMark eSensor RVP. The relationship between TCID50/mL concentrations and the corresponding copy number related to various ATCC cultures is also reported. PMID:26569120

  20. Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors

    PubMed Central

    Ikten, Cengiz; Ustun, Rustem; Catal, Mursel; Yol, Engin; Uzun, Bulent

    2016-01-01

    Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame (Sesamum indicum L.). Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan® chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 102 and 1.6 x 102 DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease. PMID:27195795

  1. Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors.

    PubMed

    Ikten, Cengiz; Ustun, Rustem; Catal, Mursel; Yol, Engin; Uzun, Bulent

    2016-01-01

    Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame (Sesamum indicum L.). Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan® chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 102 and 1.6 x 102 DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease. PMID:27195795

  2. Multiplexed Identification of Blood-Borne Bacterial Pathogens by Use of a Novel 16S rRNA Gene PCR-Ligase Detection Reaction-Capillary Electrophoresis Assay▿ †

    PubMed Central

    Pingle, Maneesh R.; Granger, Kathleen; Feinberg, Philip; Shatsky, Rebecca; Sterling, Bram; Rundell, Mark; Spitzer, Eric; Larone, Davise; Golightly, Linnie; Barany, Francis

    2007-01-01

    We have developed a novel high-throughput PCR-ligase detection reaction-capillary electrophoresis (PCR-LDR-CE) assay for the multiplexed identification of 20 blood-borne pathogens (Staphylococcus epidermidis, Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Acinetobacter baumannii, Neisseria meningitidis, Bacteroides fragilis, Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Brucella abortus), the last four of which are biothreat agents. The method relies on the amplification of two regions within the bacterial 16S rRNA gene, using universal PCR primers and querying the identity of specific single-nucleotide polymorphisms within the amplified regions in a subsequent LDR. The ligation products vary in color and size and are separated by CE. Each organism generates a specific pattern of ligation products, which can be used to distinguish the pathogens using an automated software program we developed for that purpose. The assay has been verified on 315 clinical isolates and demonstrated a detection sensitivity of 98%. Additionally, 484 seeded blood cultures were tested, with a detection sensitivity of 97.7%. The ability to identify geographically variant strains of the organisms was determined by testing 132 isolates obtained from across the United States. In summary, the PCR-LDR-CE assay can successfully identify, in a multiplexed fashion, a panel of 20 blood-borne pathogens with high sensitivity and specificity. PMID:17428930

  3. miRNA sensitivity to Drosha levels correlates with pre-miRNA secondary structure

    PubMed Central

    Sperber, Henrik; Beem, Alan; Shannon, Sandra; Jones, Ross; Banik, Pratyusha; Chen, Yu; Ku, Sherman; Varani, Gabriele; Yao, Shuyuan; Ruohola-Baker, Hannele

    2014-01-01

    microRNAs (miRNAs) are crucial for cellular development and homeostasis. In order to better understand regulation of miRNA biosynthesis, we studied cleavage of primary miRNAs by Drosha. While Drosha knockdown triggers an expected decrease of many mature miRNAs in human embryonic stem cells (hESC), a subset of miRNAs are not reduced. Statistical analysis of miRNA secondary structure and fold change of expression in response to Drosha knockdown showed that absence of mismatches in the central region of the hairpin, 5 and 9–12 nt from the Drosha cutting site conferred decreased sensitivity to Drosha knockdown. This suggests that, when limiting, Drosha processes miRNAs without mismatches more efficiently than mismatched miRNAs. This is important because Drosha expression changes over cellular development and the fold change of expression for miRNAs with mismatches in the central region correlates with Drosha levels. To examine the biochemical relationship directly, we overexpressed structural variants of miRNA-145, miRNA-137, miRNA-9, and miRNA-200b in HeLa cells with and without Drosha knockdown; for these miRNAs, elimination of mismatches in the central region increased, and addition of mismatches decreased their expression in an in vitro assay and in cells with low Drosha expression. Change in Drosha expression can be a biologically relevant mechanism by which eukaryotic cells control miRNA profiles. This phenomenon may explain the impact of point mutations outside the seed region of certain miRNAs. PMID:24677349

  4. Quantitative and sensitive RNA based detection of Bacillus spores

    PubMed Central

    Osmekhina, Ekaterina; Shvetsova, Antonina; Ruottinen, Maria; Neubauer, Peter

    2014-01-01

    The fast and reliable detection of bacterial spores is of great importance and still remains a challenge. Here we describe a direct RNA-based diagnostic method for the specific detection of viable bacterial spores which does not depends on an enzymatic amplification step and therefore is directly appropriate for quantification. The procedure includes the following steps: (i) heat activation of spores, (ii) germination and enrichment cultivation, (iii) cell lysis, and (iv) analysis of 16S rRNA in crude cell lysates using a sandwich hybridization assay. The sensitivity of the method is dependent on the cultivation time and the detection limit; it is possible to detect 10 spores per ml when the RNA analysis is performed after 6 h of enrichment cultivation. At spore concentrations above 106 spores per ml the cultivation time can be shortened to 30 min. Total analysis times are in the range of 2–8 h depending on the spore concentration in samples. The developed procedure is optimized at the example of Bacillus subtilis spores but should be applicable to other organisms. The new method can easily be modified for other target RNAs and is suitable for specific detection of spores from known groups of organisms. PMID:24653718

  5. Nano metal-organic framework (NMOF)-based strategies for multiplexed microRNA detection in solution and living cancer cells

    NASA Astrophysics Data System (ADS)

    Wu, Yafeng; Han, Jianyu; Xue, Peng; Xu, Rong; Kang, Yuejun

    2015-01-01

    MiRNAs are an emerging type of biomarker for diagnostics and prognostics. A reliable sensing strategy that can monitor miRNA expression in living cancer cells would be critical in view of its extensive advantages for fundamental research related to miRNA-associated bioprocesses and biomedical applications. Conventional miRNA sensing methods include northern blot, microarrays and real-time quantitative PCR. However, none of them is able to monitor miRNA levels expressed in living cancer cells in a real-time fashion. Some fluorescennt biosensors developed recently from carbon nanomaterials, such as single-walled carbon nanotubes (SWNTs), graphene oxide (GO), and carbon nanoparticles, have been successfully used for assaying miRNA in vitro; however the preparation processes are often expensive, complicated and time-consuming, which have motivated the research on other substitute and novel materials. Herein we present a novel sensing strategy based on peptide nucleic acid (PNA) probes labeled with fluorophores and conjugated with an NMOF vehicle to monitor multiplexed miRNAs in living cancer cells. The NMOF works as a fluorescence quencher of the labelled PNA that is firmly bound with the metal center. In the presence of a target miRNA, PNA is hybridized and released from the NMOF leading to the recovery of fluorescence. This miRNA sensor not only enables the quantitative and highly specific detection of multiplexed miRNAs in living cancer cells, but it also allows the precise and in situ monitoring of the spatiotemporal changes of miRNA expression.MiRNAs are an emerging type of biomarker for diagnostics and prognostics. A reliable sensing strategy that can monitor miRNA expression in living cancer cells would be critical in view of its extensive advantages for fundamental research related to miRNA-associated bioprocesses and biomedical applications. Conventional miRNA sensing methods include northern blot, microarrays and real-time quantitative PCR. However, none of

  6. Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing.

    PubMed

    Ståhlberg, Anders; Krzyzanowski, Paul M; Jackson, Jennifer B; Egyud, Matthew; Stein, Lincoln; Godfrey, Tony E

    2016-06-20

    Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing library construction with flexible library content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants. PMID:27060140

  7. Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing

    PubMed Central

    Ståhlberg, Anders; Krzyzanowski, Paul M.; Jackson, Jennifer B.; Egyud, Matthew; Stein, Lincoln; Godfrey, Tony E.

    2016-01-01

    Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing library construction with flexible library content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants. PMID:27060140

  8. Identification of Sensitive Serum microRNA Biomarkers for Radiation Biodosimetry

    PubMed Central

    Jacob, Naduparambil Korah; Cooley, James V.; Yee, Tamara N.; Jacob, Jidhin; Alder, Hansjuerg; Wickramasinghe, Priyankara; Maclean, Kirsteen H.; Chakravarti, Arnab

    2013-01-01

    Exposure to ionizing radiation through environmental, occupational or a nuclear reactor accident such as the recent Fukushima Daiichi incident often results in major consequences to human health. The injury caused by radiation can manifest as acute radiation syndromes within weeks in organs with proliferating cells such as hematopoietic and gastrointestinal systems. Cancers, fibrosis and degenerative diseases are also reported in organs with differentiated cells, months or years later. Studies conducted on atom bomb survivors, nuclear reactor workers and animal models have shown a direct correlation of these effects with the absorbed dose. Physical dosimeters and the available radio-responsive biologics in body fluids, whose responses are rather indirect, have limitations to accurately evaluate the extent of post exposure damage. We have used an amplification-free, hybridization based quantitative assay utilizing the nCounter multiplex platform developed by nanoString Technologies to compare the levels of over 600 miRNAs in serum from mice irradiated at a range of 1 to 12 Gy at 24 and 48 hr time points. Development of a novel normalization strategy using multiple spike-in oligonucleotides allowed accurate measurement of radiation dose and time dependent changes in serum miRNAs. The response of several evolutionarily conserved miRNAs abundant in serum, were found to be robust and sensitive in the dose range relevant for medical triage and in patients who receive total body radiation as preparative regimen for bone marrow transplantation. Notably, miRNA-150, abundant in lymphocytes, exhibited a dose and time dependent decrease in serum, which we propose as a sensitive marker indicative of lymphocyte depletion and bone marrow damage. Our study has identified several markers useful for evaluation of an individual’s response by minimally invasive methods, relevant to triage in case of a radiation accident and evaluation of toxicity and response during and after

  9. Multiplexed, Quantitative Workflow for Sensitive Biomarker Discovery in Plasma Yields Novel Candidates for Early Myocardial Injury*

    PubMed Central

    Keshishian, Hasmik; Burgess, Michael W.; Gillette, Michael A.; Mertins, Philipp; Clauser, Karl R.; Mani, D. R.; Kuhn, Eric W.; Farrell, Laurie A.; Gerszten, Robert E.; Carr, Steven A.

    2015-01-01

    We have developed a novel plasma protein analysis platform with optimized sample preparation, chromatography, and MS analysis protocols. The workflow, which utilizes chemical isobaric mass tag labeling for relative quantification of plasma proteins, achieves far greater depth of proteome detection and quantification while simultaneously having increased sample throughput than prior methods. We applied the new workflow to a time series of plasma samples from patients undergoing a therapeutic, “planned” myocardial infarction for hypertrophic cardiomyopathy, a unique human model in which each person serves as their own biologic control. Over 5300 proteins were confidently identified in our experiments with an average of 4600 proteins identified per sample (with two or more distinct peptides identified per protein) using iTRAQ four-plex labeling. Nearly 3400 proteins were quantified in common across all 16 patient samples. Compared with a previously published label-free approach, the new method quantified almost fivefold more proteins/sample and provided a six- to nine-fold increase in sample analysis throughput. Moreover, this study provides the largest high-confidence plasma proteome dataset available to date. The reliability of relative quantification was also greatly improved relative to the label-free approach, with measured iTRAQ ratios and temporal trends correlating well with results from a 23-plex immunoMRM (iMRM) assay containing a subset of the candidate proteins applied to the same patient samples. The functional importance of improved detection and quantification was reflected in a markedly expanded list of significantly regulated proteins that provided many new candidate biomarker proteins. Preliminary evaluation of plasma sample labeling with TMT six-plex and ten-plex reagents suggests that even further increases in multiplexing of plasma analysis are practically achievable without significant losses in depth of detection relative to iTRAQ four

  10. Multiplexed immunoglobulin E sensitization in relation to exhaled nitric oxide in a population sample of children.

    PubMed

    Yao, T-C; Tsai, H-J; Tu, Y-L; Chang, S-W; Hua, M-C; Liao, S-L; Tsai, M-H; Chiu, C-Y; Lai, S-H; Yeh, K-W; Huang, J-L

    2014-05-01

    This study investigated the relationship between the specific immunoglobulin E (IgE) profile for 40 allergens using a novel microarray technique (BioIC) and fraction of exhaled nitric oxide (FeNO) in a population sample of 1321 children. Significant positive associations were found between FeNO and sensitization to mites (P < 0.001), animals (P = 0.001), cockroaches (P < 0.001), and foods (P = 0.042), and furthermore, between FeNO and the number of sensitizations (all P < 0.05) or the sum of specific IgE (all P ≤ 0.01) against the aforementioned allergen categories. Specifically, sensitization to the following allergens was significantly related to higher FeNO: Dermatophagoides pteronyssinus, Dermatophagoides farina, Blomia tropicalis, cat, German cockroach, Oriental cockroach, codfish, crab, shrimp, and cheese (all P ≤ 0.01). In conclusion, IgE sensitization to mites, pets, cockroaches, seafood, and cheese, respectively, is significantly associated with elevated FeNO levels in a dose-dependent fashion in children. Our results provide new evidence that sensitization to certain food allergens may contribute to prompt inflammation in the airways. PMID:24576320

  11. Multiplexed Digital mRNA Profiling of the Inflammatory Response in the West Nile Swiss Webster Mouse Model

    PubMed Central

    Peña, José; Plante, Jessica A.; Carillo, Alda Celena; Roberts, Kimberly K.; Smith, Jennifer K.; Juelich, Terry L.; Beasley, David W. C.; Freiberg, Alexander N.; Labute, Montiago X.; Naraghi-Arani, Pejman

    2014-01-01

    Background and purpose The ability to track changes in gene expression following viral infection is paramount to understanding viral pathogenesis. This study was undertaken to evaluate the nCounter, a high throughput digital gene expression system, as a means to better understand West Nile virus (WNV) dissemination and the inflammatory response against WNV in the outbred Swiss Webster (SW) mouse model over the course of infection. Methodology The nCounter Mouse Inflammation gene expression kit containing 179 inflammation related genes was used to analyze gene expression changes in multiple tissues over a nine day course of infection in SW mice following intraperitoneal injection with WNV. Protein expression levels for a subset of these cytokine/chemokine genes were determined using a multiplex protein detection system (BioPlex) and comparisons of protein/RNA expression levels made. Results Expression analysis of spleen, lung, liver, kidney and brain of SW mice infected with WNV revealed that Cxcl10 and Il12b are differentially expressed in all tissues tested except kidney. Data stratification of positively confirmed infected (WNV (+)) versus non-infected (WNV (−) tissues allowed differentiation of the systemic inflammatory gene response from tissue-specific responses arising from WNV infection. Significant (p<0.05) decrease in C3ar1 was found in WNV (−) spleen. Il23a was significantly upregulated, while Il10rb was down-regulated in WNV (−) lung. Il3 and Mbl2 were down-regulated in WNV (−) liver. In WNV (+) livers, Stat1, Tlr2, chemokines Cxcl1, Cxcl3, Cxcl9, Cxcl10, cytokines Il6, Il18, cytokine-related gene Il1r and cytokine agonist Ilrn were significantly upregulated. In WNV (−) brain tissues, Csf2 and Cxcl10 were significantly upregulated. Similar gene and protein expression kinetics were found for Ccl2, Ccl3, Ccl4 and Ccl5 and correlated with the presence of infectious virus. In summary, the utility of the nCounter platform for rapid identification of

  12. Sets of RNA Repeated Tags and Hybridization-Sensitive Fluorescent Probes for Distinct Images of RNA in a Living Cell

    PubMed Central

    Kubota, Takeshi; Ikeda, Shuji; Yanagisawa, Hiroyuki; Yuki, Mizue; Okamoto, Akimitsu

    2010-01-01

    Background Imaging the behavior of RNA in a living cell is a powerful means for understanding RNA functions and acquiring spatiotemporal information in a single cell. For more distinct RNA imaging in a living cell, a more effective chemical method to fluorescently label RNA is now required. In addition, development of the technology labeling with different colors for different RNA would make it easier to analyze plural RNA strands expressing in a cell. Methodology/Principal Findings Tag technology for RNA imaging in a living cell has been developed based on the unique chemical functions of exciton-controlled hybridization-sensitive oligonucleotide (ECHO) probes. Repetitions of selected 18-nucleotide RNA tags were incorporated into the mRNA 3′-UTR. Pairs with complementary ECHO probes exhibited hybridization-sensitive fluorescence emission for the mRNA expressed in a living cell. The mRNA in a nucleus was detected clearly as fluorescent puncta, and the images of the expression of two mRNAs were obtained independently and simultaneously with two orthogonal tag–probe pairs. Conclusions/Significance A compact and repeated label has been developed for RNA imaging in a living cell, based on the photochemistry of ECHO probes. The pairs of an 18-nt RNA tag and the complementary ECHO probes are highly thermostable, sequence-specifically emissive, and orthogonal to each other. The nucleotide length necessary for one tag sequence is much shorter compared with conventional tag technologies, resulting in easy preparation of the tag sequences with a larger number of repeats for more distinct RNA imaging. PMID:20885944

  13. Quenching of Unincorporated Amplification Signal Reporters in Reverse-Transcription Loop-Mediated Isothermal Amplification Enabling Bright, Single-Step, Closed-Tube, and Multiplexed Detection of RNA Viruses.

    PubMed

    Ball, Cameron S; Light, Yooli K; Koh, Chung-Yan; Wheeler, Sarah S; Coffey, Lark L; Meagher, Robert J

    2016-04-01

    Reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes postamplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of the reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for quenching of unincorporated amplification signal reporters, read "quasar"), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). Furthermore, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA. PMID:26980448

  14. Silicon on-chip bandpass filters for the multiplexing of high sensitivity photonic crystal microcavity biosensors

    SciTech Connect

    Yan, Hai Zou, Yi; Yang, Chun-Ju; Chakravarty, Swapnajit; Wang, Zheng; Tang, Naimei; Chen, Ray T.; Fan, Donglei

    2015-03-23

    A method for the dense integration of high sensitivity photonic crystal (PC) waveguide based biosensors is proposed and experimentally demonstrated on a silicon platform. By connecting an additional PC waveguide filter to a PC microcavity sensor in series, a transmission passband is created, containing the resonances of the PC microcavity for sensing purpose. With proper engineering of the passband, multiple high sensitivity PC microcavity sensors can be integrated into microarrays and be interrogated simultaneously between a single input and a single output port. The concept was demonstrated with a 2-channel L55 PC biosensor array containing PC waveguide filters. The experiment showed that the sensors on both channels can be monitored simultaneously from a single output spectrum. Less than 3 dB extra loss for the additional PC waveguide filter is observed.

  15. Fluorescent Protein Nanowire-Mediated Protein Microarrays for Multiplexed and Highly Sensitive Pathogen Detection.

    PubMed

    Men, Dong; Zhou, Juan; Li, Wei; Leng, Yan; Chen, Xinwen; Tao, Shengce; Zhang, Xian-En

    2016-07-13

    Protein microarrays are powerful tools for high-throughput and simultaneous detection of different target molecules in complex biological samples. However, the sensitivity of conventional fluorescence-labeling protein detection methods is limited by the availability of signal molecules for binding to the target molecule. Here, we built a multifunctional fluorescent protein nanowire (FNw) by harnessing self-assembly of yeast amyloid protein. The FNw integrated a large number of fluorescent molecules, thereby enhancing the fluorescent signal output in target detection. The FNw was then combined with protein microarray technology to detect proteins derived from two pathogens, including influenza virus (hemagglutinin 1, HA1) and human immunodeficiency virus (p24 and gp120). The resulting detection sensitivity achieved a 100-fold improvement over a commercially available detection reagent. PMID:27315221

  16. High-sensitivity high-throughput chip based biosensor array for multiplexed detection of heavy metals

    NASA Astrophysics Data System (ADS)

    Yan, Hai; Tang, Naimei; Jairo, Grace A.; Chakravarty, Swapnajit; Blake, Diane A.; Chen, Ray T.

    2016-03-01

    Heavy metal ions released into the environment from industrial processes lead to various health hazards. We propose an on-chip label-free detection approach that allows high-sensitivity and high-throughput detection of heavy metals. The sensing device consists of 2-dimensional photonic crystal microcavities that are combined by multimode interferometer to form a sensor array. We experimentally demonstrate the detection of cadmium-chelate conjugate with concentration as low as 5 parts-per-billion (ppb).

  17. Sensitive Simultaneous Detection of Seven Sexually Transmitted Agents in Semen by Multiplex-PCR and of HPV by Single PCR

    PubMed Central

    de Abreu, André Luelsdorf Pimenta; Irie, Mary Mayumi Taguti; Esquiçati, Isis Baroni; Malagutti, Natália; Vasconcellos, Vinícius Rodrigo Bulla; Discacciati, Michele Garcia; Bonini, Marcelo Gialluisi; Maria-Engler, Silvya Stuchi; Consolaro, Marcia Edilaine Lopes

    2014-01-01

    Sexually transmitted diseases (STDs) may impair sperm parameters and functions thereby promoting male infertility. To date limited molecular studies were conducted to evaluate the frequency and type of such infections in semen Thus, we aimed at conceiving and validating a multiplex PCR (M-PCR) assay for the simultaneous detection of the following STD pathogens in semen: Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, Herpes virus simplex (HSV) −1 and −2, and Treponema pallidum; We also investigated the potential usefulness of this M-PCR assay in screening programs for semen pathogens. In addition, we aimed: to detect human Papillomavirus (HPV) and genotypes by single PCR (sPCR) in the same semen samples; to determine the prevalence of the seven STDs, HPV and co-infections; to assess the possibility that these infections affect semen parameters and thus fertility. The overall validation parameters of M-PCR were extremely high including agreement (99.2%), sensitivity (100.00%), specificity (99.70%), positive (96.40%) and negative predictive values (100.00%) and accuracy (99.80%). The prevalence of STDs was very high (55.3%). Furthermore, associations were observed between STDs and changes in semen parameters, highlighting the importance of STD detection in semen. Thus, this M-PCR assay has great potential for application in semen screening programs for pathogens in infertility and STD clinics and in sperm banks. PMID:24921247

  18. Design of a universal biointerface for sensitive, selective, and multiplex detection of biomarkers using surface plasmon resonance imaging.

    PubMed

    Shabani, Arghavan; Tabrizian, Maryam

    2013-10-21

    This paper reports on the sensitive, selective, and simultaneous detection of four protein biomarkers involved in metastasis of various cancers, namely Fas, angiopoietin-2 (Ang-2), human epidermal growth factor receptor 2 (HER2), and matrix metallopeptidase-9 (MMP-9) using an antibody-conjugated quantum dot (QD) chip and surface plasmon resonance imaging (SPRi) biosensors. Initially, a self-assembled monolayer film of l-cysteine, using glutaraldehyde as a linker and QDs as signal enhancement moieties, was employed to immobilize anti-Fas for the detection of Fas as a model protein in buffer. The biointerface was characterized using confocal microscopy, atomic force microscopy, and scanning electron microscopy to provide evidence of uniform surface coverage by the QDs. The SPRi signal was enhanced 100-fold to achieve a detection limit of 25 pg mL(-1) after applying biotinylated detection antibody-conjugate streptavidin-modified QDs. Secondly, this signal amplification strategy was applied to sequentially detect Fas, HER2, MMP-9, and Ang-2 at low concentrations on a protein-microprinted/gold-coated SPRi chip. The results showed the absence of cross-reactivity among these proteins and the feasibility of the approach for multiplex detection of biomarkers as required for the accurate diagnosis of various diseases. PMID:23954940

  19. Phase regeneration for polarization-division multiplexed signals based on vector dual-pump nondegenerate phase sensitive amplification.

    PubMed

    Yang, Weili; Yu, Yu; Ye, Mengyuan; Chen, Guanyu; Zhang, Chi; Zhang, Xinliang

    2015-02-01

    The polarization-division multiplexing (PDM) technology is a practical method to double the transmission capacity, and the corresponding phase regeneration (PR) for PDM signals is meaningful and necessary to extend the transmission distance and increase the transparency for the phase-encoded PDM system. Those reported PDM PR schemes either utilized polarization-diversity technique or required special PDM format. In order to overcome these issues, the PR for the PDM phase-modulated signals is proposed and theoretically demonstrated in this paper, based on the vector dual-pump nondegenerate phase sensitive amplification (PSA). The theoretical model is established and the detailed characteristics are investigated to optimize the PR performance. Results show an obvious phase squeezing for the degraded 80 Gbit/s PDM differential phase-shift keying (DPSK) signals, and the error vector magnitude (EVM) of the regenerated signals on dual polarization states can be improved from 22.58% and 21.39% to 4.57% and 4.63%, respectively. Furthermore, the applicability of the proposed scheme for PDM quaternary-phase shift keying (QPSK) signals is investigated. The proposed scheme can be useful and promising in current PDM based coherent fiber-optic communication. PMID:25836072

  20. Microfluidic based multiplex qRT-PCR identifies diagnostic and prognostic microRNA signatures in sera of prostate cancer patients

    PubMed Central

    Moltzahn, Felix; Olshen, Adam B.; Baehner, Lauren; Peek, Andrew; Fong, Lawrence; Stöppler, Hubert; Simko, Jeffry; Hilton, Joan F.; Carroll, Peter; Blelloch, Robert

    2010-01-01

    Recent prostate specific antigen (PSA) based screening trials indicate an urgent need for novel and non-invasive biomarker identification strategies to improve the prediction of prostate cancer behavior. Non-coding microRNAs (miRNAs) in the serum and plasma have been shown to have potential as non-invasive markers for physiological and pathological conditions. To identify serum miRNAs that diagnose and correlate with prognosis of prostate cancer, we developed a multiplex quantitative reverse transcription PCR (qRT-PCR) method involving purification of multiplex PCR products followed by uniplex analysis on a microfluidics chip to evaluate 384 human miRNAs. Using Dgcr8 and Dicer knockout (small RNA - deficient) mouse ES cells (mESC) as the benchmark, we confirmed the validity of our technique, while uncovering a significant lack of accuracy in previously published methods. Profiling 48 sera from healthy men and untreated prostate cancer patients with differing CAPRA (Cancer of the Prostate Risk Assessment) scores, we identified miRNA signatures that allow to diagnose cancer patients and correlate with prognosis. These serum signatures include oncogenic and tumor suppressive miRNAs suggesting functional roles in prostate cancer progression. PMID:21098088

  1. Use of an internal positive control in a multiplex reverse transcription-PCR to detect West Nile virus RNA in mosquito pools.

    PubMed

    Eisler, Diane L; McNabb, Alan; Jorgensen, Danielle R; Isaac-Renton, Judith L

    2004-02-01

    We report on the use of West Nile virus Armored RNA as an internal positive control (IPC) for the extraction and reverse transcription-PCR (RT-PCR) of RNA extracted from field-collected mosquitoes and on a multiplex real-time Taqman RT-PCR to simultaneously detect the 3' noncoding region of West Nile virus and the West Nile virus NS5-2 region comprising the IPC. Mosquito pools from the province of British Columbia, Canada (n = 635), were tested in duplicate and found to be negative for West Nile virus and positive for the IPC. Known West Nile virus-positive supernatants from mosquito pools from the provinces of Alberta and Manitoba were tested in duplicate and found to be positive for both regions of the West Nile virus genome. The mean cycle threshold (Ct) value for the IPC in batch extraction controls +/- 2 standard deviations was found to be 36.43 +/- 1.78 cycles. IPCs of 98.4% (624) of West Nile virus-negative pools fell within this range, indicating the reproducibility of RNA extraction and RT-PCR for pools varying in mosquito genus and number. A comparison of mosquito pool genera revealed no significant genus effect on the Ct value of the IPC. The incorporation of West Nile virus Armored RNA as an IPC allows monitoring of RNA extraction and RT-PCR and detection of false-negative results due to failures in these processes or to PCR inhibition, respectively. PMID:14766868

  2. Antagonistic functions between the RNA chaperone Hfq and an sRNA regulate sensitivity to the antibiotic colicin

    PubMed Central

    Salvail, Hubert; Caron, Marie-Pier; Bélanger, Justine; Massé, Eric

    2013-01-01

    The RNA chaperone Hfq is a key regulator of the function of small RNAs (sRNAs). Hfq has been shown to facilitate sRNAs binding to target mRNAs and to directly regulate translation through the action of sRNAs. Here, we present evidence that Hfq acts as the repressor of cirA mRNA translation in the absence of sRNA. Hfq binding to cirA prevents translation initiation, which correlates with cirA mRNA instability. In contrast, RyhB pairing to cirA mRNA promotes changes in RNA structure that displace Hfq, thereby allowing efficient translation as well as mRNA stabilization. Because CirA is a receptor for the antibiotic colicin Ia, in addition to acting as an Fur (Ferric Uptake Regulator)-regulated siderophore transporter, translational activation of cirA mRNA by RyhB promotes colicin sensitivity under conditions of iron starvation. Altogether, these results indicate that Fur and RyhB modulate an unexpected feed-forward loop mechanism related to iron physiology and colicin sensitivity. PMID:24065131

  3. Mononeuritis multiplex

    MedlinePlus

    Mononeuropathy multiplex; Multifocal neuropathy; Peripheral neuropathy - mononeuritis multiplex ... Shy ME. Peripheral neuropathies. In: Goldman L, Ausiello D, eds. Goldman's Cecil Medicine . 23rd ed. Philadelphia, PA: Elsevier Saunders; 2007:chap 446.

  4. mCAL: A New Approach for Versatile Multiplex Action of Cas9 Using One sgRNA and Loci Flanked by a Programmed Target Sequence

    PubMed Central

    Finnigan, Gregory C.; Thorner, Jeremy

    2016-01-01

    Genome editing exploiting CRISPR/Cas9 has been adopted widely in academia and in the biotechnology industry to manipulate DNA sequences in diverse organisms. Molecular engineering of Cas9 itself and its guide RNA, and the strategies for using them, have increased efficiency, optimized specificity, reduced inappropriate off-target effects, and introduced modifications for performing other functions (transcriptional regulation, high-resolution imaging, protein recruitment, and high-throughput screening). Moreover, Cas9 has the ability to multiplex, i.e., to act at different genomic targets within the same nucleus. Currently, however, introducing concurrent changes at multiple loci involves: (i) identification of appropriate genomic sites, especially the availability of suitable PAM sequences; (ii) the design, construction, and expression of multiple sgRNA directed against those sites; (iii) potential difficulties in altering essential genes; and (iv) lingering concerns about “off-target” effects. We have devised a new approach that circumvents these drawbacks, as we demonstrate here using the yeast Saccharomyces cerevisiae. First, any gene(s) of interest are flanked upstream and downstream with a single unique target sequence that does not normally exist in the genome. Thereafter, expression of one sgRNA and cotransformation with appropriate PCR fragments permits concomitant Cas9-mediated alteration of multiple genes (both essential and nonessential). The system we developed also allows for maintenance of the integrated, inducible Cas9-expression cassette or its simultaneous scarless excision. Our scheme—dubbed mCAL for “Multiplexing of Cas9 at Artificial Loci”—can be applied to any organism in which the CRISPR/Cas9 methodology is currently being utilized. In principle, it can be applied to install synthetic sequences into the genome, to generate genomic libraries, and to program strains or cell lines so that they can be conveniently (and repeatedly

  5. Rapid and Sensitive Multiplex Detection of Burkholderia pseudomallei-Specific Antibodies in Melioidosis Patients Based on a Protein Microarray Approach

    PubMed Central

    Kohler, Christian; Dunachie, Susanna J.; Müller, Elke; Kohler, Anne; Jenjaroen, Kemajittra; Teparrukkul, Prapit; Baier, Vico; Ehricht, Ralf; Steinmetz, Ivo

    2016-01-01

    Background The environmental bacterium Burkholderia pseudomallei causes the infectious disease melioidosis with a high case-fatality rate in tropical and subtropical regions. Direct pathogen detection can be difficult, and therefore an indirect serological test which might aid early diagnosis is desirable. However, current tests for antibodies against B. pseudomallei, including the reference indirect haemagglutination assay (IHA), lack sensitivity, specificity and standardization. Consequently, serological tests currently do not play a role in the diagnosis of melioidosis in endemic areas. Recently, a number of promising diagnostic antigens have been identified, but a standardized, easy-to-perform clinical laboratory test for sensitive multiplex detection of antibodies against B. pseudomallei is still lacking. Methods and Principal Findings In this study, we developed and validated a protein microarray which can be used in a standard 96-well format. Our array contains 20 recombinant and purified B. pseudomallei proteins, previously identified as serodiagnostic candidates in melioidosis. In total, we analyzed 196 sera and plasmas from melioidosis patients from northeast Thailand and 210 negative controls from melioidosis-endemic and non-endemic regions. Our protein array clearly discriminated between sera from melioidosis patients and controls with a specificity of 97%. Importantly, the array showed a higher sensitivity than did the IHA in melioidosis patients upon admission (cut-off IHA titer ≥1:160: IHA 57.3%, protein array: 86.7%; p = 0.0001). Testing of sera from single patients at 0, 12 and 52 weeks post-admission revealed that protein antigens induce either a short- or long-term antibody response. Conclusions Our protein array provides a standardized, rapid, easy-to-perform test for the detection of B. pseudomallei-specific antibody patterns. Thus, this system has the potential to improve the serodiagnosis of melioidosis in clinical settings. Moreover, our

  6. A target-triggered dual amplification strategy for sensitive detection of microRNA.

    PubMed

    Lv, Weifeng; Zhao, Jiamin; Situ, Bo; Li, Bo; Ma, Wen; Liu, Jumei; Wu, Zixian; Wang, Wen; Yan, Xiaohui; Zheng, Lei

    2016-09-15

    The accurate and quantitative analysis of microRNA (miRNA) expression is critical for biomedical research and clinical theranostics. In this study, we report a novel sensor for the sensitive detection of miRNA based on a duplex-specific nuclease (DSN)-assisted dual signal amplification strategy. A chimeric probe (DNA/2-OMe-RNA) that consists of a miRNA recognition DNA sequence and a Taqman probe hybridization RNA sequence (2'-O-methyl RNA) was designed and synthesized. One molecule of target miRNA can trigger cyclical cleavage of the chimeric probes to produce 2'-O-methyl RNA by DSN in the first round of amplification. The 2'-O-methyl RNA molecules can subsequently hybridize with Taqman probes and initiate the second round of cyclical amplification to generate detectable fluorescence by DSN. The proposed strategy exhibits high specificity in discriminating cognate miRNAs, and the dual signal transduction process enables the detection of miRNA concentrations as low as 7.3fM. We further applied this assay to miRNA quantification in cancer cells to confirm its applicability. The present study provides a sensitive, specific and simple method for miRNA detection and holds great potential for further application in biomedical research and in the clinical laboratory. PMID:27131998

  7. MicroRNA-21 Increases Proliferation and Cisplatin Sensitivity of Osteosarcoma-Derived Cells.

    PubMed

    Vanas, Vanita; Haigl, Barbara; Stockhammer, Verena; Sutterlüty-Fall, Hedwig

    2016-01-01

    Osteosarcoma is the most common primary bone tumor and poor prognosis for osteosarcoma patients is mainly due to chemotherapy resistance. MicroRNAs are important to maintain pathophysiological mechanisms of cancer and influence cell sensitivity to chemotherapy. In this study, we tested the functions of microRNA-21 for malignant features as well as for drug resistance of osteosarcoma. We used Northern blot to measure microRNA-21 levels in osteosarcoma-derived cell lines. MicroRNA-21 activity was modulated by either expressing a sponge to decrease its activity in an osteosarcoma-derived cell line expressing high levels of microRNA-21 or by introducing pri-microRNA-21 in a cell line with low endogenous levels. Cell migration was determined in a scratch assay and cell proliferation was measured by performing growth curve analysis. Sensitivity of the cells towards chemotherapeutics was investigated by performing cell viability assays and calculating the IC50 values. While cell migration was unaffected by modulated microRNA-21 levels, microRNA-21 inhibition slowed proliferation and exogenously expressed microRNA-21 promoted this process. Modulated microRNA-21 activity failed to effect sensitivity of osteosarcoma-derived cell lines to doxorubicin or methotrexate. Contrarily, reduction of microRNA-21 activity resulted in enhanced resistance towards cisplatin while ectopic expression of microRNA-21 showed the opposite effect. Increased microRNA-21 levels repressed the expression of Sprouty2 and ectopic expression of Sprouty2 was able to largely rescue the observed effects of microRNA-21 in osteosarcoma. In summary, our data indicate that in osteosarcoma microRNA-21 expression is an important component for regulation of cell proliferation and for determining sensitivity to cisplatin. PMID:27513462

  8. MicroRNA-21 Increases Proliferation and Cisplatin Sensitivity of Osteosarcoma-Derived Cells

    PubMed Central

    Vanas, Vanita; Haigl, Barbara; Stockhammer, Verena; Sutterlüty-Fall, Hedwig

    2016-01-01

    Osteosarcoma is the most common primary bone tumor and poor prognosis for osteosarcoma patients is mainly due to chemotherapy resistance. MicroRNAs are important to maintain pathophysiological mechanisms of cancer and influence cell sensitivity to chemotherapy. In this study, we tested the functions of microRNA-21 for malignant features as well as for drug resistance of osteosarcoma. We used Northern blot to measure microRNA-21 levels in osteosarcoma-derived cell lines. MicroRNA-21 activity was modulated by either expressing a sponge to decrease its activity in an osteosarcoma-derived cell line expressing high levels of microRNA-21 or by introducing pri-microRNA-21 in a cell line with low endogenous levels. Cell migration was determined in a scratch assay and cell proliferation was measured by performing growth curve analysis. Sensitivity of the cells towards chemotherapeutics was investigated by performing cell viability assays and calculating the IC50 values. While cell migration was unaffected by modulated microRNA-21 levels, microRNA-21 inhibition slowed proliferation and exogenously expressed microRNA-21 promoted this process. Modulated microRNA-21 activity failed to effect sensitivity of osteosarcoma-derived cell lines to doxorubicin or methotrexate. Contrarily, reduction of microRNA-21 activity resulted in enhanced resistance towards cisplatin while ectopic expression of microRNA-21 showed the opposite effect. Increased microRNA-21 levels repressed the expression of Sprouty2 and ectopic expression of Sprouty2 was able to largely rescue the observed effects of microRNA-21 in osteosarcoma. In summary, our data indicate that in osteosarcoma microRNA-21 expression is an important component for regulation of cell proliferation and for determining sensitivity to cisplatin. PMID:27513462

  9. Primers with 5' flaps improve the efficiency and sensitivity of multiplex PCR assays for the detection of Salmonella and Escherichia coli O157:H7.

    PubMed

    Timmons, Chris; Dobhal, Shefali; Fletcher, Jacqueline; Ma, Li Maria

    2013-04-01

    Foodborne illnesses caused by Salmonella enterica and Escherichia coli O157:H7 are worldwide health concerns. Rapid, sensitive, and robust detection of these pathogens in foods and in clinical and environmental samples is essential for routine food quality testing, effective surveillance, and outbreak investigations. The aim of this study was to evaluate the effect on PCR sensitivity of adding a short, AT-rich overhanging nucleotide sequence (flap) to the 5' end of PCR primers specific for the detection of Salmonella and E. coli O157:H7. Primers targeting the invA gene of Salmonella and the rfbE gene of E. coli O157:H7 were synthesized with or without a 12-bp, AT-rich 5' flap (5'-AATAAATCATAA-3'). Singleplex PCR, multiplex PCR, and real-time PCR sensitivity assays were conducted using purified bacterial genomic DNA and crude cell lysates of bacterial cells. The effect of background flora on detection was evaluated by spiking tomato and jalapeno pepper surface washes with E. coli O157:H7 and Salmonella Saintpaul. When targeting individual pathogens, end-point PCR assays using flap-amended primers were more efficient than nonamended primers, with 20.4 and 23.5% increases in amplicon yield for Salmonella and E. coli O157:H7, respectively. In multiplex PCR assays, a 10- to 100-fold increase in detection sensitivity was observed when the primer flap sequence was incorporated. This improvement in both singleplex and multiplex PCR efficiency and sensitivity can lead to improved Salmonella and E. coli O157:H7 detection. PMID:23575131

  10. Interleaved EPI based fMRI improved by multiplexed sensitivity encoding (MUSE) and simultaneous multi-band imaging.

    PubMed

    Chang, Hing-Chiu; Gaur, Pooja; Chou, Ying-hui; Chu, Mei-Lan; Chen, Nan-kuei

    2014-01-01

    Functional magnetic resonance imaging (fMRI) is a non-invasive and powerful imaging tool for detecting brain activities. The majority of fMRI studies are performed with single-shot echo-planar imaging (EPI) due to its high temporal resolution. Recent studies have demonstrated that, by increasing the spatial-resolution of fMRI, previously unidentified neuronal networks can be measured. However, it is challenging to improve the spatial resolution of conventional single-shot EPI based fMRI. Although multi-shot interleaved EPI is superior to single-shot EPI in terms of the improved spatial-resolution, reduced geometric distortions, and sharper point spread function (PSF), interleaved EPI based fMRI has two main limitations: 1) the imaging throughput is lower in interleaved EPI; 2) the magnitude and phase signal variations among EPI segments (due to physiological noise, subject motion, and B0 drift) are translated to significant in-plane aliasing artifact across the field of view (FOV). Here we report a method that integrates multiple approaches to address the technical limitations of interleaved EPI-based fMRI. Firstly, the multiplexed sensitivity-encoding (MUSE) post-processing algorithm is used to suppress in-plane aliasing artifacts resulting from time-domain signal instabilities during dynamic scans. Secondly, a simultaneous multi-band interleaved EPI pulse sequence, with a controlled aliasing scheme incorporated, is implemented to increase the imaging throughput. Thirdly, the MUSE algorithm is then generalized to accommodate fMRI data obtained with our multi-band interleaved EPI pulse sequence, suppressing both in-plane and through-plane aliasing artifacts. The blood-oxygenation-level-dependent (BOLD) signal detectability and the scan throughput can be significantly improved for interleaved EPI-based fMRI. Our human fMRI data obtained from 3 Tesla systems demonstrate the effectiveness of the developed methods. It is expected that future fMRI studies requiring high

  11. Interleaved EPI Based fMRI Improved by Multiplexed Sensitivity Encoding (MUSE) and Simultaneous Multi-Band Imaging

    PubMed Central

    Chang, Hing-Chiu; Gaur, Pooja; Chou, Ying-hui; Chu, Mei-Lan; Chen, Nan-kuei

    2014-01-01

    Functional magnetic resonance imaging (fMRI) is a non-invasive and powerful imaging tool for detecting brain activities. The majority of fMRI studies are performed with single-shot echo-planar imaging (EPI) due to its high temporal resolution. Recent studies have demonstrated that, by increasing the spatial-resolution of fMRI, previously unidentified neuronal networks can be measured. However, it is challenging to improve the spatial resolution of conventional single-shot EPI based fMRI. Although multi-shot interleaved EPI is superior to single-shot EPI in terms of the improved spatial-resolution, reduced geometric distortions, and sharper point spread function (PSF), interleaved EPI based fMRI has two main limitations: 1) the imaging throughput is lower in interleaved EPI; 2) the magnitude and phase signal variations among EPI segments (due to physiological noise, subject motion, and B0 drift) are translated to significant in-plane aliasing artifact across the field of view (FOV). Here we report a method that integrates multiple approaches to address the technical limitations of interleaved EPI-based fMRI. Firstly, the multiplexed sensitivity-encoding (MUSE) post-processing algorithm is used to suppress in-plane aliasing artifacts resulting from time-domain signal instabilities during dynamic scans. Secondly, a simultaneous multi-band interleaved EPI pulse sequence, with a controlled aliasing scheme incorporated, is implemented to increase the imaging throughput. Thirdly, the MUSE algorithm is then generalized to accommodate fMRI data obtained with our multi-band interleaved EPI pulse sequence, suppressing both in-plane and through-plane aliasing artifacts. The blood-oxygenation-level-dependent (BOLD) signal detectability and the scan throughput can be significantly improved for interleaved EPI-based fMRI. Our human fMRI data obtained from 3 Tesla systems demonstrate the effectiveness of the developed methods. It is expected that future fMRI studies requiring high

  12. A combinatorial approach to the repertoire of RNA kissing motifs; towards multiplex detection by switching hairpin aptamers.

    PubMed

    Durand, Guillaume; Dausse, Eric; Goux, Emma; Fiore, Emmanuelle; Peyrin, Eric; Ravelet, Corinne; Toulmé, Jean-Jacques

    2016-05-19

    Loop-loop (also known as kissing) interactions between RNA hairpins are involved in several mechanisms in both prokaryotes and eukaryotes such as the regulation of the plasmid copy number or the dimerization of retroviral genomes. The stability of kissing complexes relies on loop parameters (base composition, sequence and size) and base combination at the loop-loop helix - stem junctions. In order to identify kissing partners that could be used as regulatory elements or building blocks of RNA scaffolds, we analysed a pool of 5.2 × 10(6) RNA hairpins with randomized loops. We identified more than 50 pairs of kissing RNA hairpins. Two kissing motifs, 5'CCNY and 5'RYRY, generate highly stable complexes with KDs in the low nanomolar range. Such motifs were introduced in the apical loop of hairpin aptamers that switch between unfolded and folded state upon binding to their cognate target molecule, hence their name aptaswitch. The aptaswitch-ligand complex is specifically recognized by a second RNA hairpin named aptakiss through loop-loop interaction. Taking advantage of our kissing motif repertoire we engineered aptaswitch-aptakiss modules for purine derivatives, namely adenosine, GTP and theophylline and demonstrated that these molecules can be specifically and simultaneously detected by surface plasmon resonance or by fluorescence anisotropy. PMID:27067541

  13. A combinatorial approach to the repertoire of RNA kissing motifs; towards multiplex detection by switching hairpin aptamers

    PubMed Central

    Durand, Guillaume; Dausse, Eric; Goux, Emma; Fiore, Emmanuelle; Peyrin, Eric; Ravelet, Corinne; Toulmé, Jean-Jacques

    2016-01-01

    Loop–loop (also known as kissing) interactions between RNA hairpins are involved in several mechanisms in both prokaryotes and eukaryotes such as the regulation of the plasmid copy number or the dimerization of retroviral genomes. The stability of kissing complexes relies on loop parameters (base composition, sequence and size) and base combination at the loop–loop helix - stem junctions. In order to identify kissing partners that could be used as regulatory elements or building blocks of RNA scaffolds, we analysed a pool of 5.2 × 106 RNA hairpins with randomized loops. We identified more than 50 pairs of kissing RNA hairpins. Two kissing motifs, 5′CCNY and 5′RYRY, generate highly stable complexes with KDs in the low nanomolar range. Such motifs were introduced in the apical loop of hairpin aptamers that switch between unfolded and folded state upon binding to their cognate target molecule, hence their name aptaswitch. The aptaswitch–ligand complex is specifically recognized by a second RNA hairpin named aptakiss through loop–loop interaction. Taking advantage of our kissing motif repertoire we engineered aptaswitch–aptakiss modules for purine derivatives, namely adenosine, GTP and theophylline and demonstrated that these molecules can be specifically and simultaneously detected by surface plasmon resonance or by fluorescence anisotropy. PMID:27067541

  14. Microgels for multiplex and direct fluorescence detection

    NASA Astrophysics Data System (ADS)

    Causa, Filippo; Aliberti, Anna; Cusano, Angela M.; Battista, Edmondo; Netti, Paolo A.

    2015-05-01

    Blood borne oligonucleotides fragments contain useful clinical information whose detection and monitoring represent the new frontier in liquid biopsy as they can transform the current diagnosis procedure. For instance, recent studies have identified a new class of circulating biomarkers such as s miRNAs, and demonstrated that changes in their concentration are closely associated with the development of cancer and other pathologies. However, direct detection of miRNAs in body fluids is particularly challenging and demands high sensitivity -concentration range between atto to femtomolarspecificity, and multiplexing Here we report on engineered multifunctional microgels and innovative probe design for a direct and multiplex detection of relevant clinical miRNAs in fluorescence by single particle assay. Polyethyleneglycol-based microgels have a coreshell architecture with two spectrally encoded fluorescent dyes for multiplex analyses and are endowed with fluorescent probes for miRNA detection. Encoding and detection fluorescence signals are distinguishable by not overlapping emission spectra. Tuneable fluorescence probe conjugation and corresponding emission confinement on single microgel allows for enhanced target detection. Such suspension array has indeed high selectivity and sensitivity with a detection limit of 10-15 M and a dynamic range from 10-9 to 10-15 M. We believe that sensitivity in the fM concentration range, signal background minimization, multiplexed capability and direct measurement of such microgels will translate into diagnostic benefits opening up new roots toward liquid biopsy in the context of point-of-care testing through an easy and fast detection of sensitive diagnostic biomarkers directly in serum.

  15. mCAL: A New Approach for Versatile Multiplex Action of Cas9 Using One sgRNA and Loci Flanked by a Programmed Target Sequence.

    PubMed

    Finnigan, Gregory C; Thorner, Jeremy

    2016-01-01

    Genome editing exploiting CRISPR/Cas9 has been adopted widely in academia and in the biotechnology industry to manipulate DNA sequences in diverse organisms. Molecular engineering of Cas9 itself and its guide RNA, and the strategies for using them, have increased efficiency, optimized specificity, reduced inappropriate off-target effects, and introduced modifications for performing other functions (transcriptional regulation, high-resolution imaging, protein recruitment, and high-throughput screening). Moreover, Cas9 has the ability to multiplex, i.e., to act at different genomic targets within the same nucleus. Currently, however, introducing concurrent changes at multiple loci involves: (i) identification of appropriate genomic sites, especially the availability of suitable PAM sequences; (ii) the design, construction, and expression of multiple sgRNA directed against those sites; (iii) potential difficulties in altering essential genes; and (iv) lingering concerns about "off-target" effects. We have devised a new approach that circumvents these drawbacks, as we demonstrate here using the yeast Saccharomyces cerevisiae First, any gene(s) of interest are flanked upstream and downstream with a single unique target sequence that does not normally exist in the genome. Thereafter, expression of one sgRNA and cotransformation with appropriate PCR fragments permits concomitant Cas9-mediated alteration of multiple genes (both essential and nonessential). The system we developed also allows for maintenance of the integrated, inducible Cas9-expression cassette or its simultaneous scarless excision. Our scheme-dubbed mCAL for " M: ultiplexing of C: as9 at A: rtificial L: oci"-can be applied to any organism in which the CRISPR/Cas9 methodology is currently being utilized. In principle, it can be applied to install synthetic sequences into the genome, to generate genomic libraries, and to program strains or cell lines so that they can be conveniently (and repeatedly

  16. Rapid, sensitive, and simultaneous detection of three foodborne pathogens using magnetic nanobead-based immunoseparation and quantum dot-based multiplex immunoassay.

    PubMed

    Wang, Hong; Li, Yanbin; Wang, Andrew; Slavik, Michael

    2011-12-01

    Losses caused by foodborne diseases are enormous in terms of human life, illness, medical costs, and food product recalls. Rapid detection of multiple bacterial pathogens in foods is extremely important to ensure food safety. The objective of this research was to develop a multiplex immunoassay by integrating magnetic nanobeads (MNBs) for immunoseparation with quantum dots (QDs) as fluorescent labels for rapid, sensitive, and simultaneous detection of three major pathogenic bacteria, Salmonella Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes, in food products. In this research, both streptavidin-conjugated MNBs (30- and 150-nm diameter) and QDs (530-, 580-, and 620-nm emission wavelength) were separately coated with biotinylated anti-Salmonella, anti-E. coli, and anti-Listeria antibodies. The immuno-MNBs were mixed with a food sample to capture the three target bacteria. After being magnetically separated from the sample, the MNB-cell conjugates were mixed with the immuno-QDs to form the MNB-cell-QD complexes, and unattached QDs were removed. The fluorescence intensity of the MNB-cell-QD complexes was measured at wavelengths of 530, 580, and 620 nm to determine the populations of Salmonella Typhimurium, E. coli O157:H7, and L. monocytogenes, respectively. This multiplex immunoassay simultaneously detected Salmonella Typhimurium, E. coli O157:H7, and L. monocytogenes at levels as low as 20 to 50 CFU/ml in food samples in less than 2 h without enrichment. The change in fluorescence intensity was linearly correlated (R(2) > 0.96) with the logarithmic value of bacterial level in the range of 10 to 10(3) CFU/ml. More than 85% of the three target pathogens could be simultaneously separated from food samples. The multiplex immunoassay could be expanded to detect more target pathogens, depending on the availability of specific antibodies and QDs with different emission wavelengths. PMID:22186043

  17. Steatocystoma multiplex.

    PubMed

    Chu, David H

    2003-10-01

    A 25-year-old man with a 20-year history of asymptomatic nodules on his arms and trunk, which histopathological analysis showed to be consistent with steatocystoma multiplex, is presented. Steatocystoma multiplex is a disorder characterized by multiple, asymptomatic, dermal cysts that usually occur on the trunk and proximal aspects of the extremities. Steatocystoma multiplex with acral predominance has only recently been described. Development of steatocystomas has been hypothesized to be due to alterations in the structure of keratin 17. Treatment for lesions has included surgical excision or drainage, oral retinoids, and liquid nitrogen cryotherapy. PMID:14594591

  18. Sensitive and Selective Detection of HIV-1 RRE RNA Using Vertical Silicon Nanowire Electrode Array.

    PubMed

    Lee, Jaehyung; Hong, Min-Ho; Han, Sanghun; Na, Jukwan; Kim, Ilsoo; Kwon, Yong-Joon; Lim, Yong-Beom; Choi, Heon-Jin

    2016-12-01

    In this study, HIV-1 Rev response element (RRE) RNA was detected via an Au-coated vertical silicon nanowire electrode array (VSNEA). The VSNEA was fabricated by combining bottom-up and top-down approaches and then immobilized by artificial peptides for the recognition of HIV-1 RRE. Differential pulse voltammetry (DPV) analysis was used to measure the electrochemical response of the peptide-immobilized VSNEA to the concentration and types of HIV-1 RRE RNA. DPV peaks showed linearity to the concentration of RNA with a detection limit down to 1.513 fM. It also showed the clear different peaks to the mutated HIV-1 RRE RNA. The high sensitivity and selectivity of VSNEA for the detection of HIV-1 RRE RNA may be attributed to the high surface-to-volume ratio and total overlap diffusion mode of ions of the one-dimensional nanowire electrodes. PMID:27448026

  19. Sensitive and Selective Detection of HIV-1 RRE RNA Using Vertical Silicon Nanowire Electrode Array

    NASA Astrophysics Data System (ADS)

    Lee, Jaehyung; Hong, Min-Ho; Han, Sanghun; Na, Jukwan; Kim, Ilsoo; Kwon, Yong-Joon; Lim, Yong-beom; Choi, Heon-Jin

    2016-07-01

    In this study, HIV-1 Rev response element (RRE) RNA was detected via an Au-coated vertical silicon nanowire electrode array (VSNEA). The VSNEA was fabricated by combining bottom-up and top-down approaches and then immobilized by artificial peptides for the recognition of HIV-1 RRE. Differential pulse voltammetry (DPV) analysis was used to measure the electrochemical response of the peptide-immobilized VSNEA to the concentration and types of HIV-1 RRE RNA. DPV peaks showed linearity to the concentration of RNA with a detection limit down to 1.513 fM. It also showed the clear different peaks to the mutated HIV-1 RRE RNA. The high sensitivity and selectivity of VSNEA for the detection of HIV-1 RRE RNA may be attributed to the high surface-to-volume ratio and total overlap diffusion mode of ions of the one-dimensional nanowire electrodes.

  20. Facile semi-automated forensic body fluid identification by multiplex solution hybridization of NanoString® barcode probes to specific mRNA targets.

    PubMed

    Danaher, Patrick; White, Robin Lynn; Hanson, Erin K; Ballantyne, Jack

    2015-01-01

    A DNA profile from the perpetrator does not reveal, per se, the circumstances by which it was transferred. Body fluid identification by mRNA profiling may allow extraction of contextual 'activity level' information from forensic samples. Here we describe the development of a prototype multiplex digital gene expression (DGE) method for forensic body fluid/tissue identification based upon solution hybridization of color-coded NanoString(®) probes to 23 mRNA targets. The method identifies peripheral blood, semen, saliva, vaginal secretions, menstrual blood and skin. We showed that a simple 5 min room temperature cellular lysis protocol gave equivalent results to standard RNA isolation from the same source material, greatly enhancing the ease-of-use of this method in forensic sample processing. We first describe a model for gene expression in a sample from a single body fluid and then extend that model to mixtures of body fluids. We then describe calculation of maximum likelihood estimates (MLEs) of body fluid quantities in a sample, and we describe the use of likelihood ratios to test for the presence of each body fluid in a sample. Known single source samples of blood, semen, vaginal secretions, menstrual blood and skin all demonstrated the expected tissue-specific gene expression for at least two of the chosen biomarkers. Saliva samples were more problematic, with their previously identified characteristic genes exhibiting poor specificity. Nonetheless the most specific saliva biomarker, HTN3, was expressed at a higher level in saliva than in any of the other tissues. Crucially, our algorithm produced zero false positives across this study's 89 unique samples. As a preliminary indication of the ability of the method to discern admixtures of body fluids, five mixtures were prepared. The identities of the component fluids were evident from the gene expression profiles of four of the five mixtures. Further optimization of the biomarker 'CodeSet' will be required

  1. Highly sensitive and ultrafast read mapping for RNA-seq analysis.

    PubMed

    Medina, I; Tárraga, J; Martínez, H; Barrachina, S; Castillo, M I; Paschall, J; Salavert-Torres, J; Blanquer-Espert, I; Hernández-García, V; Quintana-Ortí, E S; Dopazo, J

    2016-04-01

    As sequencing technologies progress, the amount of data produced grows exponentially, shifting the bottleneck of discovery towards the data analysis phase. In particular, currently available mapping solutions for RNA-seq leave room for improvement in terms of sensitivity and performance, hindering an efficient analysis of transcriptomes by massive sequencing. Here, we present an innovative approach that combines re-engineering, optimization and parallelization. This solution results in a significant increase of mapping sensitivity over a wide range of read lengths and substantial shorter runtimes when compared with current RNA-seq mapping methods available. PMID:26740642

  2. Highly sensitive and ultrafast read mapping for RNA-seq analysis

    PubMed Central

    Medina, I.; Tárraga, J.; Martínez, H.; Barrachina, S.; Castillo, M. I.; Paschall, J.; Salavert-Torres, J.; Blanquer-Espert, I.; Hernández-García, V.; Quintana-Ortí, E. S.; Dopazo, J.

    2016-01-01

    As sequencing technologies progress, the amount of data produced grows exponentially, shifting the bottleneck of discovery towards the data analysis phase. In particular, currently available mapping solutions for RNA-seq leave room for improvement in terms of sensitivity and performance, hindering an efficient analysis of transcriptomes by massive sequencing. Here, we present an innovative approach that combines re-engineering, optimization and parallelization. This solution results in a significant increase of mapping sensitivity over a wide range of read lengths and substantial shorter runtimes when compared with current RNA-seq mapping methods available. PMID:26740642

  3. Comparison of rpoB gene sequencing, 16S rRNA gene sequencing, gyrB multiplex PCR, and the VITEK2 system for identification of Acinetobacter clinical isolates.

    PubMed

    Lee, Min Jung; Jang, Sook Jin; Li, Xue Min; Park, Geon; Kook, Joong-Ki; Kim, Min Jung; Chang, Young-Hyo; Shin, Jong Hee; Kim, Soo Hyun; Kim, Dong-Min; Kang, Seong-Ho; Moon, Dae-Soo

    2014-01-01

    Since accurate identification of species is necessary for proper treatment of Acinetobacter infections, we compared the performances of 4 bacterial identification methods using 167 Acinetobacter clinical isolates to identify the best identification method. To secure more non-baumannii Acinetobacter (NBA) strains as target strains, we first identified Acinetobacter baumannii in a total of 495 Acinetobacter clinical isolates identified using the VITEK 2 system. Because 371 of 495 strains were identified as A. baumannii using gyrB multiplex 1 PCR and blaOXA51-like PCR, we performed rpoB gene sequencing and 16S rRNA gene sequencing on remaining 124 strains belonging to NBA and 52 strains of A. baumannii. For identification of Acinetobacter at the species level, the accuracy rates of rpoB gene sequencing, 16S rRNA gene sequencing, gyrB multiplex PCR, and the VITEK 2 were 98.2%, 93.4%, 77.2%, and 35.9%, respectively. The gyrB multiplex PCR seems to be very useful for the detection of ACB complex because its concordance rates to the final identification of strains of ACB complex were 100%. Both the rpoB gene sequencing and the 16S rRNA gene sequencing may be useful in identifying Acinetobacter. PMID:24157058

  4. mRAP, a sensitive method for determination of microRNA expression profiles.

    PubMed

    Mano, Hiroyuki; Takada, Shuji

    2007-10-01

    MicroRNAs (miRNAs) are noncoding RNA molecules of 21-24 nucleotides that regulate the expression of target genes in a posttranscriptional manner. Although evidence indicates that miRNAs play essential roles in embryogenesis, cell differentiation, and pathogenesis of human diseases, extensive miRNA profiling in cells or tissues has been hampered by the lack of sensitive cloning methods. Here we describe a highly efficient profiling strategy, termed miRNA amplification profiling (mRAP), that relies on the use of a long, optimized 5' adaptor, the SMART (switching mechanism at the 5' end of RNA templates of reverse transcriptase) method, the polymerase chain reaction, and cDNA concatamerization after BanI digestion. This approach is highly sensitive, readily allowing the isolation of > 1 x 10(4) independent miRNA-derived cDNAs from < or = 1 x 10(4) cells. The mRAP method thus makes it possible to analyze miRNA expression profiles for small quantities of tissue or cells such as fresh clinical specimens. PMID:17889798

  5. Sensitive SERS detection of miRNA via enzyme-free DNA machine signal amplification.

    PubMed

    Li, Xiaoxiao; Ye, Sujuan; Luo, Xiliang

    2016-08-11

    In this work, an enzyme-free signal amplified detection platform is described for miRNA detection with a DNA fueled molecular machine. Coupling SERS technology with multiple amplification modes, this flexible biosensing system exhibits high sensitivity and specificity. PMID:27469084

  6. Multiplexed digital quantification of binge-like alcohol-mediated alterations in maternal uterine angiogenic mRNA transcriptome.

    PubMed

    Ramadoss, Jayanth; Magness, Ronald R

    2012-06-01

    Genomic studies on fetal alcohol spectrum disorders (FASD) have utilized either genome-wide microarrays/bioinformatics or targeted real-time PCR (RT-PCR). We utilized herein for the first time a novel digital approach with high throughput as well as the capability to focus on one physiological system. The aim of the present study was to investigate alcohol-induced alterations in uterine angiogenesis-related mRNA abundance using digital mRNA technology. Four biological and three technical replicates of uterine arterial endothelial cells from third-trimester ewes were fluorescence-activated cell sorted, validated, and treated without or with binge-like alcohol. A capture probe covalently bound to an oligonucleotide containing biotin and a color-coded reporter probe were designed for 85 angiogenesis-related genes and analyzed with the Nanostring nCounter system. Twenty genes were downregulated (↓) and two upregulated (↑), including angiogenic growth factors/receptors (↓placental growth factor), adhesion molecules (↓angiopoietin-like-3; ↓collagen-18A1; ↓endoglin), proteases/matrix proteins/inhibitors (↓alanyl aminopeptidase; ↓collagen-4A3; ↓heparanase; ↓plasminogen, ↑plasminogen activator urokinase; ↓platelet factor-4; ↓plexin domain containing-1; ↓tissue inhibitor of metalloproteinases-3), transcription/signaling molecules (↓heart and neural crest derivatives-2; ↓DNA-binding protein inhibitor; ↓NOTCH-4; ↓ribosomal protein-L13a1; ↓ribosomal protein large-P1), cytokines/chemokines (↓interleukin-1B), and miscellaneous growth factors (↓leptin; ↓platelet-derived growth factor-α); ↓transforming growth factor (TGF-α; ↑TGF-β receptor-1). These novel data show significant detrimental alcohol effects on genes controlling angiogenesis supporting a mechanistic role for abnormal uteroplacental vascular development in FASD. The tripartite digital gene expression system is therefore a valuable tool to answer many additional

  7. MicroRNA-221 and -222 Regulate Radiation Sensitivity by Targeting the PTEN Pathway

    SciTech Connect

    Zhang Chunzhi; Kang Chunsheng; Wang Ping; Cao Yongzhen; Lv Zhonghong; Yu Shizhu; Wang Guangxiu; Zhang Anling; Jia Zhifan; Han Lei; Yang Chunying; Ishiyama, Hiromichi; Teh, Bin S.; Xu Bo; Pu Peiyu

    2011-05-01

    Purpose: MicroRNAs (miRNAs) are noncoding RNAs inhibiting expression of numerous target genes by posttranscriptional regulation. miRNA-221 and miRNA-222 (miRNA-221/-222) expression is elevated in radioresistant tumor cell lines; however, it is not known whether and how miRNAs control cellular responses to ionizing irradiation. Methods and Materials: We used bioinformatic analyses, luciferase reporter assay, and genetic knockdown and biochemical assays to characterize the regulation pathways of miRNA-221/-222 in response to radiation treatment. Results: We identified the PTEN gene as a target of miRNA-221/-222. Furthermore, we found that knocking down miRNA-221/-222 by antisense oligonucleotides upregulated PTEN expression. Upregulated PTEN expression suppressed AKT activity and increased radiation-induced apoptosis, resulting in enhancement of radiosensitivity in tumor cells. Conclusions: miRNA-221/-222 control radiation sensitivity by regulating the PTEN/AKT pathway and can be explored as novel targets for radiosensitization.

  8. Fast Hepatitis C Virus RNA Elimination and NS5A Redistribution by NS5A Inhibitors Studied by a Multiplex Assay Approach

    PubMed Central

    Liu, Dandan; Ji, Juan; Ndongwe, Tanya P.; Michailidis, Eleftherios; Rice, Charles M.; Ralston, Robert

    2015-01-01

    While earlier therapeutic strategies for the treatment of hepatitis C virus (HCV) infection relied exclusively on interferon (IFN) and ribavirin (RBV), four direct-acting antiviral agents (DAAs) have now been approved, aiming for an interferon-free strategy with a short treatment duration and fewer side effects. To facilitate studies on the mechanism of action (MOA) and efficacy of DAAs, we established a multiplex assay approach, which employs flow cytometry, a Gaussia luciferase reporter system, Western blot analysis, reverse transcription-quantitative PCR (RT-qPCR), a limited dilution assay (50% tissue culture infectious dose [TCID50]), and an image profiling assay that follows the NS5A redistribution in response to drug treatment. We used this approach to compare the relative potency of various DAAs and the kinetics of their antiviral effects as a potential preclinical measure of their potential clinical utility. We evaluated the NS5A inhibitors ledipasvir (LDV) and daclatasvir (DCV), the NS3/4A inhibitor danoprevir (DNV), and the NS5B inhibitor sofosbuvir (SOF). In terms of kinetics, our data demonstrate that the NS5A inhibitor LDV, followed closely by DCV, has the fastest effect on suppression of viral proteins and RNA and on redistribution of NS5A. In terms of MOA, LDV has a more pronounced effect than DCV on the viral replication, assembly, and infectivity of released virus. Our approach can be used to facilitate the study of the biological processes involved in HCV replication and help identify optimal drug combinations. PMID:25845863

  9. Prefrontal microRNA-221 Mediates Environmental Enrichment-Induced Increase of Locomotor Sensitivity to Nicotine

    PubMed Central

    Gomez, Adrian M.; Altomare, Diego; Sun, Wei-Lun; Midde, Narasimha M.; Ji, Hao; Shtutman, Michael; Turner, Jill R.; Creek, Kim E.

    2016-01-01

    Background: Environmental enrichment alters susceptibility in developing drug addiction. We have demonstrated that rats raised in an enriched condition are more sensitive than rats raised in an impoverished condition to nicotine-induced locomotor activity, and this is associated with alterations of phosphorylated extracellular signal-regulated kinase 1/2 within the prefrontal cortex. This study determined the impact of microRNA-221 in the prefrontal cortex on phosphorylated extracellular signal-regulated kinase 1/2 and the enriched environment-dependent behavioral changes in response to nicotine. Methods: A microRNA array was conducted to profile microRNA expression in the prefrontal cortex of enriched condition and impoverished condition rats in response to repeated nicotine (0.35mg/kg, s.c.) administration. microRNA-221 in the prefrontal cortex, nucleus accumbens, and striatum was further verified by quantitative real-time PCR. Lentiviral-mediated overexpression of microRNA-221 in PC12 cells and the medial prefrontal cortex was performed to determine the effects of microRNA-221 on nicotine-mediated phosphorylated extracellular signal-regulated kinase 1/2, phosphorylated cAMP-response element-binding protein, and locomotor activity. Results: microRNA-221 was profoundly upregulated in the prefrontal cortex but not in nucleus accumbens and striatum of enriched condition rats relative to impoverished condition rats following repeated administration of nicotine. Overexpression of lentiviral-microRNA-221 attenuated nicotine-induced increase in phosphorylated extracellular signal-regulated kinase 1/2 in PC12 cells. Lentiviral-microRNA-221 overexpression in the medial prefrontal cortex further increased locomotor activity in impoverished condition but not in enriched condition rats in response to repeated nicotine administration. Accordingly, lentiviral-microRNA-221 attenuated nicotine-induced increases in phosphorylated extracellular signal-regulated kinase 1/2 and

  10. Multiplex Pyrosequencing.

    PubMed

    Pourmand, Nader; Elahi, Elahe; Davis, Ronald W; Ronaghi, Mostafa

    2002-04-01

    We describe here the development of a new and simple single-tube multiplex Pyrosequencing assay. Genomic DNA or cDNA was employed to PCR amplify region(s) using biotinylated and normal primer(s). Subsequent to capture of PCR products on streptavidin-coated beads, single-stranded DNA separation and hybridization of multiple sequencing primers, Pyrosequencing was performed. The obtained pyrogram resulted in a unique pattern in which the intensity of the signal determined the number of incorporated nucleotide(s). Here, we demonstrate the use of this multiplex Pyrosequencing for single nucleotide polymorphisms genotyping and microbial typing. PMID:11917037

  11. Characterizing a novel and sensitive method to measure dsRNA in soil.

    PubMed

    Fischer, Joshua R; Zapata, Fatima; Dubelman, Samuel; Mueller, Geoffrey M; Jensen, Peter D; Levine, Steven L

    2016-10-01

    Performing environmental assessments for double-stranded RNA-based agricultural products require the development of sensitive and selective methods to measure biodegradation rates of dsRNAs. We developed and characterized a novel analytical procedure that uses a molecular hybridization assay (QuantiGene(®)) to accurately measure dsRNA extracted from diverse soils. In this report, we utilize this method to demonstrate that two dsRNAs with distinct size, structure, and sequence degrade rapidly in soil with indistinguishable kinetics. PMID:27441991

  12. Acid Sensitive Polymeric Micelles Combining Folate and Bioreducible Conjugate for Specific Intracellular siRNA Delivery.

    PubMed

    Yang, Yanfang; Xia, Xuejun; Dong, Wujun; Wang, Hongliang; Li, Lin; Ma, Panpan; Sheng, Wei; Xu, Xueqing; Liu, Yuling

    2016-05-01

    An efficiently siRNA transporting nanocarrier still remains to be developed. In this study, utilizing the dual stimulus of acid tumor extracellular environment and redox effect of glutathione in the cytosol, a new siRNA transporting system combining triple effects of folate targeting, acid sensitive polymer micelles, and bio-reducible disulfide bond linked siRNA-cell penetrating peptides (CPPs) conjugate is developed to suppress c-myc gene expression of breast cancer (MCF-7 cells) both in vitro and in vivo. Subsequent research demonstrates that the vesicle has particle size of about 100 nm and siRNA entrapment efficiency of approximately 80%. In vitro studies verified over 90% of encapsulated siRNA-CPPs can be released and the vesicle shows higher cellular uptake in response to the tumorous zone. Determination of gene expression at both mRNA and protein levels indicates the constructed vesicle exhibited enhanced cancer cell apoptosis and improved therapeutic efficacy in vitro and in vivo. PMID:26822264

  13. A handheld flow genetic analysis system (FGAS): towards rapid, sensitive, quantitative and multiplex molecular diagnosis at the point-of-care level.

    PubMed

    Shu, Bowen; Zhang, Chunsun; Xing, Da

    2015-06-21

    A handheld flow genetic analysis system (FGAS) is proposed for rapid, sensitive, multiplex and real-time quantification of nucleic acids at the point-of-care (POC) level. The FGAS includes a helical thermal-gradient microreactor and a microflow actuator, as well as control circuitry for temperature, fluid and power management, and smartphone fluorescence imaging. All of these features are integrated into a field-portable and easy-to-use molecular diagnostic platform powered by lithium batteries. Due to the unique design of the microreactor, not only steady temperatures for denaturation and annealing/extension but also a linear thermal gradient for spatial high-resolution melting can be achieved through simply maintaining a single heater at constant temperature. The smartphone fluorescence imaging system has a wide field of view that captures all PCR channels of the microreactor in a single snapshot without the need for any mechanical scanning. By these designs, the FGAS enables real-time monitoring of the temporal and spatial fluorescence signatures of amplicons during continuous-flow amplification. On the current FGAS, visual detection of as little as 10 copies per μL of genomic DNA of Salmonella enterica was achieved in 15 min, with real-time quantitative detection of the DNA over 6 orders of magnitude concentration from 10(6) to 10(1) copies per μL also completed in 7.5-15 min. In addition, multiple pathogenic DNA targets could be simultaneously discriminated with direct bar-chart readout or multiplex spatial melting in serial flow. We anticipate that the FGAS has great potential to become a next-generation gene analyzer for POC molecular diagnostics. PMID:25953325

  14. A Genome-Wide siRNA Screen to Identify Modulators of Insulin Sensitivity and Gluconeogenesis

    PubMed Central

    Yang, Ruojing; Lacson, Raul G.; Castriota, Gino; Zhang, Xiaohua D.; Liu, Yaping; Zhao, Wenqing; Einstein, Monica; Camargo, Luiz Miguel; Qureshi, Sajjad; Wong, Kenny K.; Zhang, Bei B.; Ferrer, Marc; Berger, Joel P.

    2012-01-01

    Background Hepatic insulin resistance impairs insulin’s ability to suppress hepatic glucose production (HGP) and contributes to the development of type 2 diabetes (T2D). Although the interests to discover novel genes that modulate insulin sensitivity and HGP are high, it remains challenging to have a human cell based system to identify novel genes. Methodology/Principal Findings To identify genes that modulate hepatic insulin signaling and HGP, we generated a human cell line stably expressing beta-lactamase under the control of the human glucose-6-phosphatase (G6PC) promoter (AH-G6PC cells). Both beta-lactamase activity and endogenous G6PC mRNA were increased in AH-G6PC cells by a combination of dexamethasone and pCPT-cAMP, and reduced by insulin. A 4-gene High-Throughput-Genomics assay was developed to concomitantly measure G6PC and pyruvate-dehydrogenase-kinase-4 (PDK4) mRNA levels. Using this assay, we screened an siRNA library containing pooled siRNA targeting 6650 druggable genes and identified 614 hits that lowered G6PC expression without increasing PDK4 mRNA levels. Pathway analysis indicated that siRNA-mediated knockdown (KD) of genes known to positively or negatively affect insulin signaling increased or decreased G6PC mRNA expression, respectively, thus validating our screening platform. A subset of 270 primary screen hits was selected and 149 hits were confirmed by target gene KD by pooled siRNA and 7 single siRNA for each gene to reduce G6PC expression in 4-gene HTG assay. Subsequently, pooled siRNA KD of 113 genes decreased PEPCK and/or PGC1alpha mRNA expression thereby demonstrating their role in regulating key gluconeogenic genes in addition to G6PC. Last, KD of 61 of the above 113 genes potentiated insulin-stimulated Akt phosphorylation, suggesting that they suppress gluconeogenic gene by enhancing insulin signaling. Conclusions/Significance These results support the proposition that the proteins encoded by the genes identified in our cell

  15. Gene and MicroRNA Expression Responses to Exercise; Relationship with Insulin Sensitivity

    PubMed Central

    McLean, Carrie S.; Mielke, Clinton; Cordova, Jeanine M.; Langlais, Paul R.; Bowen, Benjamin; Miranda, Danielle; Coletta, Dawn K.; Mandarino, Lawrence J.

    2015-01-01

    Background Healthy individuals on the lower end of the insulin sensitivity spectrum also have a reduced gene expression response to exercise for specific genes. The goal of this study was to determine the relationship between insulin sensitivity and exercise-induced gene expression in an unbiased, global manner. Methods and Findings Euglycemic clamps were used to measure insulin sensitivity and muscle biopsies were done at rest and 30 minutes after a single acute exercise bout in 14 healthy participants. Changes in mRNA expression were assessed using microarrays, and miRNA analysis was performed in a subset of 6 of the participants using sequencing techniques. Following exercise, 215 mRNAs were changed at the probe level (Bonferroni-corrected P<0.00000115). Pathway and Gene Ontology analysis showed enrichment in MAP kinase signaling, transcriptional regulation and DNA binding. Changes in several transcription factor mRNAs were correlated with insulin sensitivity, including MYC, r=0.71; SNF1LK, r=0.69; and ATF3, r= 0.61 (5 corrected for false discovery rate). Enrichment in the 5’-UTRs of exercise-responsive genes suggested regulation by common transcription factors, especially EGR1. miRNA species of interest that changed after exercise included miR-378, which is located in an intron of the PPARGC1B gene. Conclusions These results indicate that transcription factor gene expression responses to exercise depend highly on insulin sensitivity in healthy people. The overall pattern suggests a coordinated cycle by which exercise and insulin sensitivity regulate gene expression in muscle. PMID:25984722

  16. The Microwave SQUID Multiplexer

    NASA Astrophysics Data System (ADS)

    Mates, John Arthur Benson

    2011-12-01

    This thesis describes a multiplexer of Superconducting Quantum Interference Devices (SQUIDs) with low-noise, ultra-low power dissipation, and great scalability. The multiplexer circuit measures the magnetic flux in a large number of unshunted rf SQUIDs by coupling each SQUID to a superconducting microwave resonator tuned to a unique resonance frequency and driving the resonators from a common feedline. A superposition of microwave tones measures each SQUID simultaneously using only two coaxial cables between the cryogenic device and room temperature. This multiplexer will enable the instrumentation of arrays with hundreds of thousands of low-temperature detectors for new applications in cosmology, materials analysis, and nuclear non-proliferation. The driving application of the Microwave SQUID Multiplexer is the readout of large arrays of superconducting transition-edge sensors, by some figures of merit the most sensitive detectors of electromagnetic signals over a span of more than nine orders of magnitude in energy, from 40 GHz microwaves to 200 keV gamma rays. Modern transition-edge sensors have noise-equivalent power as low as 10-20 W / Hz1/2 and energy resolution as good as 2 eV at 6 keV. These per-pixel sensitivities approach theoretical limits set by the underlying signals, motivating a rapid increase in pixel count to access new science. Compelling applications, like the non-destructive assay of nuclear material for treaty verification or the search for primordial gravity waves from inflation use arrays of these detectors to increase collection area or tile a focal plane. We developed three generations of SQUID multiplexers, optimizing the first for flux noise 0.17 muPhi0 / Hz1/2, the second for input current noise 19 pA / Hz1/2, and the last for practical multiplexing of large arrays of cosmic microwave background polarimeters based on transition-edge sensors. Using the last design we demonstrated multiplexed readout of prototype polarimeters with the

  17. Differential sensitivity of Arabidopsis siRNA biogenesis mutants to genotoxic stress.

    PubMed

    Yao, Youli; Bilichak, Andriy; Golubov, Andrey; Blevins, Todd; Kovalchuk, Igor

    2010-12-01

    Plant response to stress has been linked to different RNA-silencing processes and epigenetic mechanisms. Our recent results showed that Arabidopsis thaliana Dicer-like (DCL) mutants were impaired in transgenerational changes, recombination frequency and stress tolerance. We also found that transgenerational changes were dependent on changes in DNA methylation. Here, we hypothesized that plants deficient in the production of small RNAs would show an impaired abiotic stress response. To test this, we exposed A. thaliana dcl2, dcl3, dcl4, dcl2 dcl3 (d2d3), dcl2 dcl4 (d2d4), dcl2 dcl3 dcl4 (d2d3d4), nrpd1a, rdr2 and rdr6 mutants to methyl methane sulfonate (MMS). We found dcl4 and rdr6 to be more sensitive and dcl2, dcl3, d2d3 and rdr2 plants more resistant to MMS, as shown by fresh weight, root length and survival rate. The in vitro repair assay showed the lower ability of dcl2 and dcl3 to repair UV-damaged DNA. To summarize, we found that whereas mutants impaired in transactivating siRNA biogenesis were more sensitive to MMS, mutants impaired in natural antisense siRNA and heterochromatic siRNA biogeneses were more tolerant. Our data suggest that plant response to MMS is in part regulated through biogenesis of various siRNAs. PMID:20953786

  18. Stem-loop DNA-assisted silicon nanowires-based biochemical sensors with ultra-high sensitivity, specificity, and multiplexing capability

    NASA Astrophysics Data System (ADS)

    Xie, Juan; Jiang, Xiangxu; Zhong, Yiling; Lu, Yimei; Wang, Siyi; Wei, Xinpan; Su, Yuanyuan; He, Yao

    2014-07-01

    A class of stem-loop DNA-assisted silicon nanowires (SiNWs)-based fluorescent biosensor is presented in this report. Significantly, the sensor enables rapid and sensitive detection of DNA targets with a concentration as low as 1 pM. Moreover, the large planar surface of SiNWs facilitates simultaneous assembly with different DNA strands, which is favorable for multiplexed DNA detection. On the other hand, the SiNWs-based sensor is highly efficacious for detecting heavy metal ions. Mercury ions (Hg2+) of low concentrations (e.g., 5 pM) are readily identified from its mixture with over 10 kinds of interfering metal ions, even in real water samples. Given that SiNWs can be fabricated in a facile, reproducible and low-cost manner, this kind of SiNWs-based high-performance sensor is expected to be a practical analytical tool for a variety of biological and environment-protection applications.A class of stem-loop DNA-assisted silicon nanowires (SiNWs)-based fluorescent biosensor is presented in this report. Significantly, the sensor enables rapid and sensitive detection of DNA targets with a concentration as low as 1 pM. Moreover, the large planar surface of SiNWs facilitates simultaneous assembly with different DNA strands, which is favorable for multiplexed DNA detection. On the other hand, the SiNWs-based sensor is highly efficacious for detecting heavy metal ions. Mercury ions (Hg2+) of low concentrations (e.g., 5 pM) are readily identified from its mixture with over 10 kinds of interfering metal ions, even in real water samples. Given that SiNWs can be fabricated in a facile, reproducible and low-cost manner, this kind of SiNWs-based high-performance sensor is expected to be a practical analytical tool for a variety of biological and environment-protection applications. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr01097c

  19. [RNA responsible for conferring a DNase I sensitive structure on albumin gene in assembled chromatin].

    PubMed

    Lv, Zhan-Jun; Wang, Xiu-Fang; Zhai, Yu; Song, Shu-Xia

    2003-01-01

    Although the set of genes is virtually the same in all tissues,differential gene expression is appeared in cells of different kinds. Differentiation and ageing are associated with regulation of gene expression that is a fundamental mechanism in eukaryotic development and survival. The sensitivity to DNase I of actively transcribed genes seems to be a general phenomenon. The purpose of the study is to test whether RNAs obtained from different organs or cells can enhance susceptibility of albumin gene to DNase I digestion in BALB/c mouse brain chromatin assembled.RNAs extracted from rat liver, lung, kidney, brain, tRNA from yeast and synthesized RNAs (23 nt completed with mouse alb gene) were added to a system of chromatin reconstitution that was achieved by dialysis from high ionic strength solution. Assembled chromatin was digested with DNase I (12.5 microg/mL) at 20 degrees for 1 min, then PCR assay was used to detect the level of albumin gene digested. PCR products (1200 bp) were run on a 6% polyacylamide gel and analyzed by silver stain assay. RNAs from different organs and synthesized RNAs all increased the sensitivity of albumin gene to DNase I attack in mouse assembled chromatin. The effect was more obvious in liver and lung RNAs than in kidney and brain ones. tRNA from yeast did not enhance the sensitivity of albumin gene to DNase I digestion. RNA increased albumin gene sensitivity to DNase I in a dose-dependent manner. We report here for the first time that RNAs can enhance susceptibility of albumin gene to DNase I digestion. The effect is associated with RNA sources or sequences. It is generally agreed that the formation of gene sensitivity to DNase I, by unfolding of a tightly packed chromatin fiber, is the first step in gene activation, then RNAs that recognize complementary DNA sequences may be the specific factors that affect DNA supercoiling and determine the sensitivity of gene to DNase I digestion. Here we describes "RNA Population Gene Activating

  20. Influenza A virus drift variants reduced the detection sensitivity of a commercial multiplex nucleic acid amplification assay in the season 2014/15.

    PubMed

    Huzly, Daniela; Korn, Klaus; Bierbaum, Sibylle; Eberle, Björn; Falcone, Valeria; Knöll, Antje; Steininger, Philipp; Panning, Marcus

    2016-09-01

    The influenza season 2014/15 was dominated by drift variants of influenza A(H3N2), which resulted in a reduced vaccine effectiveness. It was not clear if the performance of commercial nucleic-acid-based amplification (NAT) assays for the detection of influenza was affected. The purpose of this study was to perform a real-life evaluation of two commercial NAT assays. During January-April 2015, we tested a total of 665 samples from patients with influenza-like illness using the Fast Track Diagnostics Respiratory pathogens 21, a commercial multiplex kit, (cohorts 1 and 2, n = 563 patients) and the Xpert Flu/RSV XC assay (cohort 3, n = 102 patients), a single-use cartridge system. An in-house influenza real-time RT-PCR (cohort 1) and the RealStar Influenza RT-PCR 1.0 Kit (cohort 2 and 3) served as reference tests. Compared to the reference assay, an overall agreement of 95.9 % (cohort 1), 95 % (cohort 2), and 98 % (cohort 3) was achieved. A total of 24 false-negative results were observed using the Fast Track Diagnostics Respiratory pathogens 21 kit. No false-negative results occurred using the Xpert Flu/RSV XC assay. The Fast Track Diagnostics Respiratory pathogens 21 kit and the Xpert Flu/RSV XC assay had sensitivities of 90.7 % and 100 % and specificities of 100 % and 94.1 %, respectively, compared to the RealStar 1.0 kit. Upon modification of the Fast Track Diagnostics Respiratory pathogens 21 kit, the sensitivity increased to 97.3 %. Influenza virus strains circulating during the 2014/15 season reduced the detection sensitivity of a commercial NAT assay, and continuous monitoring of test performance is therefore necessary. PMID:27316440

  1. Silicon on-chip side-coupled high-Q micro-cavities for the multiplexing of high sensitivity photonic crystal integrated sensors array

    NASA Astrophysics Data System (ADS)

    Yang, Daquan; Wang, Chunhong; Yuan, Wei; Wang, Bo; Yang, Yujie; Ji, Yuefeng

    2016-09-01

    A novel two-dimensional (2D) silicon (Si) photonic crystal (PC) α-H0-slot micro-cavity with high Q-factor and high sensitivity (S) is presented. Based on the proposed α-H0-Slot micro-cavities, an optimal design of photonic crystal integrated sensors array (PC-ISA) on monolithic silicon on insulator (SOI) is displayed. By using finite-difference time-domain (FDTD) method, the simulation results demonstrate that both large S of 200 nm/RIU (RIU=refractive index unit) and high Q-factor >104 at telecom wavelength range can be achieved simultaneously. And the sensor figure of merit (FOM)>7000 is featured, an order of magnitude improvement over previous 2D PC sensors array. In addition, for the proposed 2D PC-ISA device, each sensor unit is shown to independently shift its resonance wavelength in response to the changes in refractive index (RI) and does not perturb the others. Thus, it is potentially an ideal platform for realizing ultra-compact lab-on-a-chip applications with dense arrays of functionalized spots for multiplexed sensing, and also can be used as an opto-fluidic architecture for performing highly parallel detection of biochemical interactions in aqueous environments.

  2. Towards pain-free diagnosis of skin diseases through multiplexed microneedles: biomarker extraction and detection using a highly sensitive blotting method.

    PubMed

    Ng, Keng Wooi; Lau, Wing Man; Williams, Adrian C

    2015-08-01

    Immunodiagnostic microneedles provide a novel way to extract protein biomarkers from the skin in a minimally invasive manner for analysis in vitro. The technology could overcome challenges in biomarker analysis specifically in solid tissue, which currently often involves invasive biopsies. This study describes the development of a multiplex immunodiagnostic device incorporating mechanisms to detect multiple antigens simultaneously, as well as internal assay controls for result validation. A novel detection method is also proposed. It enables signal detection specifically at microneedle tips and therefore may aid the construction of depth profiles of skin biomarkers. The detection method can be coupled with computerised densitometry for signal quantitation. The antigen specificity, sensitivity and functional stability of the device were assessed against a number of model biomarkers. Detection and analysis of endogenous antigens (interleukins 1α and 6) from the skin using the device was demonstrated. The results were verified using conventional enzyme-linked immunosorbent assays. The detection limit of the microneedle device, at ≤10 pg/mL, was at least comparable to conventional plate-based solid-phase enzyme immunoassays. PMID:25939431

  3. Multiplex, Rapid, and Sensitive Isothermal Detection of Nucleic-Acid Sequence by Endonuclease Restriction-Mediated Real-Time Multiple Cross Displacement Amplification.

    PubMed

    Wang, Yi; Wang, Yan; Zhang, Lu; Liu, Dongxin; Luo, Lijuan; Li, Hua; Cao, Xiaolong; Liu, Kai; Xu, Jianguo; Ye, Changyun

    2016-01-01

    We have devised a novel isothermal amplification technology, termed endonuclease restriction-mediated real-time multiple cross displacement amplification (ET-MCDA), which facilitated multiplex, rapid, specific and sensitive detection of nucleic-acid sequences at a constant temperature. The ET-MCDA integrated multiple cross displacement amplification strategy, restriction endonuclease cleavage and real-time fluorescence detection technique. In the ET-MCDA system, the functional cross primer E-CP1 or E-CP2 was constructed by adding a short sequence at the 5' end of CP1 or CP2, respectively, and the new E-CP1 or E-CP2 primer was labeled at the 5' end with a fluorophore and in the middle with a dark quencher. The restriction endonuclease Nb.BsrDI specifically recognized the short sequence and digested the newly synthesized double-stranded terminal sequences (5' end short sequences and their complementary sequences), which released the quenching, resulting on a gain of fluorescence signal. Thus, the ET-MCDA allowed real-time detection of single or multiple targets in only a single reaction, and the positive results were observed in as short as 12 min, detecting down to 3.125 fg of genomic DNA per tube. Moreover, the analytical specificity and the practical application of the ET-MCDA were also successfully evaluated in this study. Here, we provided the details on the novel ET-MCDA technique and expounded the basic ET-MCDA amplification mechanism. PMID:27242766

  4. Highly sensitive and simultaneous detection of melamine and aflatoxin M1 in milk products by multiplexed planar waveguide fluorescence immunosensor (MPWFI).

    PubMed

    Guo, Hongli; Zhou, Xiaohong; Zhang, Yan; Song, Baodong; Zhang, Jingxuan; Shi, Hanchang

    2016-04-15

    Mycotoxins and industrial chemicals, such as aflatoxin M1 and melamine, now commonly exist in milk and cause potential health risks. This study presents an indirect competitive immunoassay through multiplex planar waveguide fluorescence immunosensor (MPWFI) for rapid, sensitive, and simultaneous detection and quantification of aflatoxin M1 and melamine by applying the principle of immunoreaction and total internal reflect fluorescent. Double-channel standard curves with appropriate logistic correlation (R(2)>0.99) were plotted, respectively. The working ranges (0.073-0.400 ng/mL and 26.38-270.00 ng/mL, respectively) were calculated, as well as the limit of detection (0.045 and 13.37 ng/mL, respectively), when two analytes were simultaneously detected. Both results satisfied the requirements for the maximum amount set by the WHO, which illustrated that the current method was better than some other standard methods. The recovery rates in the actual samples ranged from 85% to 103%, with relative standard deviations between 1.3% and 6.5%, which indicated high accuracy and repeatability. PMID:26616961

  5. Stem-loop DNA-assisted silicon nanowires-based biochemical sensors with ultra-high sensitivity, specificity, and multiplexing capability.

    PubMed

    Xie, Juan; Jiang, Xiangxu; Zhong, Yiling; Lu, Yimei; Wang, Siyi; Wei, Xinpan; Su, Yuanyuan; He, Yao

    2014-08-01

    A class of stem-loop DNA-assisted silicon nanowires (SiNWs)-based fluorescent biosensor is presented in this report. Significantly, the sensor enables rapid and sensitive detection of DNA targets with a concentration as low as 1 pM. Moreover, the large planar surface of SiNWs facilitates simultaneous assembly with different DNA strands, which is favorable for multiplexed DNA detection. On the other hand, the SiNWs-based sensor is highly efficacious for detecting heavy metal ions. Mercury ions (Hg(2+)) of low concentrations (e.g., 5 pM) are readily identified from its mixture with over 10 kinds of interfering metal ions, even in real water samples. Given that SiNWs can be fabricated in a facile, reproducible and low-cost manner, this kind of SiNWs-based high-performance sensor is expected to be a practical analytical tool for a variety of biological and environment-protection applications. PMID:24981573

  6. RNase non-sensitive and endocytosis independent siRNA delivery system: delivery of siRNA into tumor cells and high efficiency induction of apoptosis

    NASA Astrophysics Data System (ADS)

    Jiang, Xinglu; Wang, Guobao; Liu, Ru; Wang, Yaling; Wang, Yongkui; Qiu, Xiaozhong; Gao, Xueyun

    2013-07-01

    To date, RNase degradation and endosome/lysosome trapping are still serious problems for siRNA-based molecular therapy, although different kinds of delivery formulations have been tried. In this report, a cell penetrating peptide (CPP, including a positively charged segment, a linear segment, and a hydrophobic segment) and a single wall carbon nanotube (SWCNT) are applied together by a simple method to act as a siRNA delivery system. The siRNAs first form a complex with the positively charged segment of CPP via electrostatic forces, and the siRNA-CPP further coats the surface of the SWCNT via hydrophobic interactions. This siRNA delivery system is non-sensitive to RNase and can avoid endosome/lysosome trapping in vitro. When this siRNA delivery system is studied in Hela cells, siRNA uptake was observed in 98% Hela cells, and over 70% mRNA of mammalian target of rapamycin (mTOR) is knocked down, triggering cell apoptosis on a significant scale. Our siRNA delivery system is easy to handle and benign to cultured cells, providing a very efficient approach for the delivery of siRNA into the cell cytosol and cleaving the target mRNA therein.

  7. Live-cell visualization of pre-mRNA splicing with single-molecule sensitivity

    PubMed Central

    Martin, Robert M.; Rino, José; Carvalho, Célia; Kirchhausen, Tomas; Carmo-Fonseca, Maria

    2013-01-01

    SUMMARY Removal of introns from pre-mRNAs via splicing provides a versatile means of genetic regulation that is often disrupted in human diseases. To decipher how splicing occurs in real time, we have directly examined with single-molecule sensitivity the kinetics of intron excision from pre-mRNA in the nucleus of living human cells. By using two different RNA labeling methods, MS2 and λN, we show that beta-globin introns are transcribed and excised in 20-30 s. We further show that replacing the weak polypyrimidine (Py) tract in mouse immunoglobulin μ (IgM) pre-mRNA by a U-rich Py decreases the intron lifetime, thus providing direct evidence that splice site strength influences splicing kinetics. We also found that RNA polymerase II transcribes at elongation rates ranging between 3 and 6 kb min-1, and that transcription can be rate limiting for splicing. These results have important implications for mechanistic understanding of co-transcriptional splicing regulation in the live-cell context. PMID:24035393

  8. Asymmetric mRNA localization contributes to fidelity and sensitivity of spatially localized systems

    PubMed Central

    Weatheritt, Robert J.; Gibson, Toby J; Babu, M. Madan

    2014-01-01

    While many proteins are localized after translation, asymmetric-protein distribution is also achieved by translation after mRNA localization. Why are certain mRNA transported to a distal location and translated on-site? Here we undertake a systematic, genome-scale study of asymmetrically distributed protein and mRNA in mammalian cells. Our findings suggest that asymmetric-protein distribution by mRNA localization enhances interaction fidelity and signaling sensitivity. Proteins synthesized at distal locations frequently contain intrinsically disordered segments. These regions are generally rich in assembly-promoting modules and are often regulated by post-translational modification sites. Such proteins are tightly regulated but display distinct temporal dynamics upon stimulation with growth factors. Thus proteins synthesized on-site may rapidly alter proteome composition and act as dynamically regulated scaffolds to promote the formation of reversible cellular assemblies. Our observations are consistent across multiple mammalian species, cell types, and developmental stages suggesting that localized translation is a recurring feature of cell signaling and regulation. PMID:25150862

  9. A sensitive electrochemical aptasensor for multiplex antibiotics detection based on high-capacity magnetic hollow porous nanotracers coupling exonuclease-assisted cascade target recycling.

    PubMed

    Yan, Zhongdan; Gan, Ning; Li, Tianhua; Cao, Yuting; Chen, Yinji

    2016-04-15

    A multiplex electrochemical aptasensor was developed for simultaneous detection of two antibiotics such as chloramphenicol (CAP) and oxytetracycline (OTC), and high-capacity magnetic hollow porous nanotracers coupling exonuclease-assisted target recycling was used to improve sensitivity. The cascade amplification process consists of the exonuclease-assisted target recycling amplification and metal ions encoded magnetic hollow porous nanoparticles (MHPs) to produce voltammetry signals. Upon the specific recognition of aptamers to targets (CAP and OTC), exonuclease I (Exo I) selectively digested the aptamers which were bound with CAP and OTC, then the released CAP and OTC participated new cycling to produce more single DNA, which can act as trigger strands to hybrid with nanotracers to generate further signal amplification. MHPs were used as carriers to load more amounts of metal ions and coupling with Exo I assisted cascade target recycling can amplify the signal for about 12 folds compared with silica based nanotracers. Owing to the dual signal amplification, the linear range between signals and the concentrations of CAP and OTC were obtained in the range of 0.0005-50 ng mL(-1). The detection limits of CAP and OTC were 0.15 and 0.10 ng mL(-1) (S/N=3) which is more than 2 orders lower than commercial enzyme-linked immunosorbent immunoassay (ELISA) method, respectively. The proposed method was successfully applied to simultaneously detection of CAP and OTC in milk samples. Besides, this aptasensor can be applied to other antibiotics detection by changing the corresponding aptamer. The whole scheme is facile, selective and sensitive enough for antibiotics screening in food safety. PMID:26594886

  10. Comparison of Conventional PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Arcobacter Species

    PubMed Central

    Wang, Xiaoyu; Seo, Dong Joo; Lee, Min Hwa

    2014-01-01

    This study aimed to develop a loop-mediated isothermal amplification (LAMP) method for the rapid detection of Arcobacter species. Specific primers targeting the 23S ribosomal RNA gene were used to detect Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii. The specificity of the LAMP primer set was assessed using DNA samples from a panel of Arcobacter and Campylobacter species, and the sensitivity was determined using serial dilutions of Arcobacter species cultures. LAMP showed a 10- to 1,000-fold-higher sensitivity than multiplex PCR, with a detection limit of 2 to 20 CFU per reaction in vitro. Whereas multiplex PCR showed cross-reactivity with Campylobacter species, the LAMP method developed in this study was more sensitive and reliable than conventional PCR or multiplex PCR for the detection of Arcobacter species. PMID:24478488

  11. Clinical Impact of a Novel MicroRNA Chemo-Sensitivity Predictor in Gastrooesophageal Cancer

    PubMed Central

    Winther, Mette; Knudsen, Steen; Dahlgaard, Jesper; Jensen, Thomas; Hansen, Anker; Jensen, Peter Buhl; Tramm, Trine; Alsner, Jan; Nordsmark, Marianne

    2016-01-01

    Background miRNAs might be potentially useful biomarkers for prediction of response to chemotherapeutic agents, radiotherapy and survival. The aim of this retrospective study was to validate miRNA response predictors in a cohort of patients with gastrooesophageal cancer in order to predict overall survival (OS) and disease-specific survival (DSS). Material and Methods The study population encompassed 53 patients treated with curative intend for loco-regional gastrooesophageal cancer. miRNA expression was quantified from pre-therapeutic and diagnostic, formalin-fixed, paraffin embedded tumour specimens using Affymetrix GeneChip miRNA 1.0 Array. Based on growth inhibition of the NCI60 panel in the presence of cisplatin, epirubicine and capecitabine, a miRNA based response predictor was developed. The Cox proportional hazards model was applied to assess the correlations of the response predictor with OS and DSS. Results A univariate analysis demonstrated a statistical significant improvement of OS for patients who had undergone surgical resection with prediction scores above the median prediction score (HR: 0.41 (95% CI: 0.17–0.96). Adjusting for surgery and stage, this predictor was identified to be independently associated with both OS (HR: 0.37 (95% CI: 0.16–0.87)) and DSS (HR: 0.32 (0.12–0.87)). Conclusion The miRNA profile predictive for sensitivity to cisplatin, epirubicine and capecitabine was shown to be independently associated with OS and DSS in patients with gastrooesophageal cancer. PMID:26885979

  12. Rapid discrimination of rabies viruses isolated from various host species in Brazil by multiplex reverse transcription-polymerase chain reaction.

    PubMed

    Sato, Go; Tanabe, Hitomi; Shoji, Youko; Itou, Takuya; Ito, Fumio H; Sato, Tetsuo; Sakai, Takeo

    2005-08-01

    Rabies is carried mainly by mammalian carnivores and vampire bats in Latin America. However, rabies virus (RV) has been isolated in recent years from not only vampire bats in rural areas but also from several non-vampire bat species in urban areas, respectively. Therefore, rapid molecular screening is necessary for efficient epidemiology of these RVs. In this study, we investigated the usefulness of multiplex reverse transcription-polymerase chain reaction (RT-PCR) for determining the origins of 54 RV isolates from various host species in Brazil. And to evaluate the multiplex RT-PCR as a potential diagnostic tool, we investigated the sensitivity of this method. In addition, we compared the results with a phylogenetic tree developed from sequences of the RV glycoprotein (G protein) gene. Multiplex RT-PCR products showed five different sizes of products, whereas the phylogenic tree showed six groups. Of these six groups, four corresponded with the four sizes of the multiplex RT-PCR products. The other two groups showed correspondance with another one size of the multiplex RT-PCR products, indicating that multiplex RT-PCR results reflected the lineage of the 54 isolates. This study also showed that this method can detect trace amounts of RNA. In conclusion, this multiplex RT-PCR method allows the rapid, specific, and simultaneous detection of RVs isolated from various host species in Brazil. PMID:16036175

  13. RNA.

    ERIC Educational Resources Information Center

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  14. Antibody array in a multiwell plate format for the sensitive and multiplexed detection of important plant pathogens.

    PubMed

    Charlermroj, Ratthaphol; Himananto, Orawan; Seepiban, Channarong; Kumpoosiri, Mallika; Warin, Nuchnard; Gajanandana, Oraprapai; Elliott, Christopher T; Karoonuthaisiri, Nitsara

    2014-07-15

    The global seed market is considered to be an important industry with a total value of $10,543 million US dollars in 2012. Because plant pathogens such as bacteria and viruses cause a significant economic loss to both producers and exporters, the seed export industry urgently requires rapid, sensitive, and inexpensive testing for the pathogens to prevent disease spreading worldwide. This study developed an antibody array in a multiwell plate format to simultaneously detect four crucial plant pathogens, namely, a bacterial fruit blotch bacterium Acidovorax avenae subsp. citrulli (Aac), Chilli veinal mottle virus (ChiVMV, potyvirus), Watermelon silver mottle virus (WSMoV, tospovirus serogroup IV), and Melon yellow spot virus (MYSV, tospovirus). The capture antibodies specific to the pathogens were immobilized on each well at preassigned positions by an automatic microarrayer. The antibodies on the arrays specifically captured the corresponding pathogens present in the sample extracts. The presence of pathogens bound on the capture antibodies was subsequently detected by a cocktail of fluorescently conjugated secondary antibodies. The limits of detection of the developed antibody array for the detection of Aac, ChiVMV, WSMoV, and MYSV were 5 × 10(5) CFU/mL, 30 ng/mL, 1000 ng/mL, and 160 ng/mL, respectively, which were very similar to those of the conventional ELISA method. The antibody array in a multiwell plate format accurately detected plant pathogens in single and multiple detections. Moreover, this format enables easy handling of the assay at a higher speed of operation. PMID:24945525

  15. Gene and microRNA expression reveals sensitivity to paclitaxel in laryngeal cancer cell line

    PubMed Central

    Xu, Cheng-Zhi; Xie, Jin; Jin, Bin; Chen, Xin-Wei; Sun, Zhen-Feng; Wang, Bao-Xing; Dong, Pin

    2013-01-01

    Paclitaxel is a widely used chemotherapy drug for advanced laryngeal cancer patients. However, the fact that there are 20-40% of advanced laryngeal cancer patients do not response to paclitaxel makes it necessary to figure out potential biomarkers for paclitaxel sensitivity prediction. In this work, Hep2, a laryngeal cancer cell line, untreated or treated with lower dose of paclitaxel for 24 h, was applied to DNA microarray chips for gene and miR expression profile analysis. Expression of eight genes altered significantly following paclitaxel treatment, which was further validated by quantitative real-time PCR. Four up-regulated genes were ID2, BMP4, CCL4 and ACTG2, in which ID2 and BMP4 were implicated to be involved in several drugs sensitivity. While the down-regulated four genes, MAPK4, FASN, INSIG1 and SCD, were mainly linked to the endoplasmic reticulum and fatty acid biosynthesis, these two cell processes that are associated with drug sensitivity by increasing evidences. After paclitaxel treatment, expression of 49 miRs was significantly altered. Within these miRs, the most markedly expression-changed were miR-31-star, miR-1264, miR-3150b-5p and miR-210. While the miRs putatively modulated the mRNA expression of the most significantly expression-altered genes were miR-1264, miR-130a, miR-27b, miR-195, miR-1291, miR-214, miR-1277 and miR-1265, which were obtained by miR target prediction and miRNA target correlation. Collectively, our study might provide potential biomarkers for paclitaxel sensitivity prediction and drug resistance targets in laryngeal cancer patients. PMID:23826416

  16. Knockdown of long noncoding RNA H19 sensitizes human glioma cells to temozolomide therapy

    PubMed Central

    Jiang, Pengfei; Wang, Ping; Sun, Xiaoling; Yuan, Zhongshun; Zhan, Rucai; Ma, Xiangyu; Li, Weiguo

    2016-01-01

    Temozolomide (TMZ) is commonly used in glioma chemotherapy. However, a great clinical challenge for TMZ is chemoresistance. H19 transcripts are recognized as long noncoding RNAs, which potentially interact with chromatin-modifying complexes to regulate gene expression via epigenetic changes. Our data based on glioma patients showed that the expression of H19 was significantly upregulated in TMZ-resistant tumors compared with the TMZ-sensitive tumors. To determine the function of H19 in glioma, cell lines U87 and U251 were exposed to TMZ to establish TMZ-resistant clones U87TMZ and U251TMZ. In U87TMZ and U251TMZ, the expression level of H19 transcripts was increased compared to wild-type or nonresistant clones, as determined by real-time quantitative reverse transcription polymerase chain reaction. Concomitant treatment with small interfering RNA specifically targeting H19 and TMZ in resistant glioma clones resulted in decreased IC50 values for TMZ, and increased apoptotic rates than control small interfering RNA-treated cells. This was also evident by the increased PARP cleavage in resistant cells exposed to TMZ + si-H19. Furthermore, the reduced expression of H19 altered major drug resistance genes, such as MDR, MRP, and ABCG2, both at the mRNA and protein levels. Taken together, these findings suggest that H19 plays an important role in the development of TMZ resistance, and may represent a novel therapeutic target for TMZ-resistant gliomas. PMID:27366087

  17. Multiplex, Rapid, and Sensitive Isothermal Detection of Nucleic-Acid Sequence by Endonuclease Restriction-Mediated Real-Time Multiple Cross Displacement Amplification

    PubMed Central

    Wang, Yi; Wang, Yan; Zhang, Lu; Liu, Dongxin; Luo, Lijuan; Li, Hua; Cao, Xiaolong; Liu, Kai; Xu, Jianguo; Ye, Changyun

    2016-01-01

    We have devised a novel isothermal amplification technology, termed endonuclease restriction-mediated real-time multiple cross displacement amplification (ET-MCDA), which facilitated multiplex, rapid, specific and sensitive detection of nucleic-acid sequences at a constant temperature. The ET-MCDA integrated multiple cross displacement amplification strategy, restriction endonuclease cleavage and real-time fluorescence detection technique. In the ET-MCDA system, the functional cross primer E-CP1 or E-CP2 was constructed by adding a short sequence at the 5′ end of CP1 or CP2, respectively, and the new E-CP1 or E-CP2 primer was labeled at the 5′ end with a fluorophore and in the middle with a dark quencher. The restriction endonuclease Nb.BsrDI specifically recognized the short sequence and digested the newly synthesized double-stranded terminal sequences (5′ end short sequences and their complementary sequences), which released the quenching, resulting on a gain of fluorescence signal. Thus, the ET-MCDA allowed real-time detection of single or multiple targets in only a single reaction, and the positive results were observed in as short as 12 min, detecting down to 3.125 fg of genomic DNA per tube. Moreover, the analytical specificity and the practical application of the ET-MCDA were also successfully evaluated in this study. Here, we provided the details on the novel ET-MCDA technique and expounded the basic ET-MCDA amplification mechanism. PMID:27242766

  18. The supercoiling sensitivity of a bacterial tRNA promoter parallels its responsiveness to stringent control.

    PubMed Central

    Figueroa-Bossi, N; Guérin, M; Rahmouni, R; Leng, M; Bossi, L

    1998-01-01

    In Salmonella typhimurium, expression of the hisR locus, a tRNA operon, decreases upon inhibiting DNA gyrase. Here, the hisR promoter dependence on negative DNA supercoiling was examined in vivo and in vitro. Mutant analysis showed the sequence determinants of this dependence to lie in the region between the -10 box and the transcription start site. As with most promoters subject to stringent control, this portion of the hisR promoter is C-G-rich. Replacing a C/G bp with T/A at position -7 partially relieves the supercoiling response while changing the sequence between -5 and + 1 (-CCCCCG-) for -GTTAA- abolishes the response in vitro and in vivo. The relief of the supercoiling dependence closely correlates with increased promoter susceptibility to melting in vivo and a lesser requirement for initiating nucleotides in the formation of stable initiation complexes in vitro. Studies in isoleucine-starved cells showed that such sequence changes mitigate and abolish the hisR promoter response to stringent control, respectively. The data presented suggest that the hisR promoter's sensitivity to stringent regulation arises from the same physical property that confers supercoiling sensitivity, i.e. resistance to melting. We propose that the stringent control mechanism acts by hampering the ability of RNA polymerase to melt the DNA helix. PMID:9550733

  19. The supercoiling sensitivity of a bacterial tRNA promoter parallels its responsiveness to stringent control.

    PubMed

    Figueroa-Bossi, N; Guérin, M; Rahmouni, R; Leng, M; Bossi, L

    1998-04-15

    In Salmonella typhimurium, expression of the hisR locus, a tRNA operon, decreases upon inhibiting DNA gyrase. Here, the hisR promoter dependence on negative DNA supercoiling was examined in vivo and in vitro. Mutant analysis showed the sequence determinants of this dependence to lie in the region between the -10 box and the transcription start site. As with most promoters subject to stringent control, this portion of the hisR promoter is C-G-rich. Replacing a C/G bp with T/A at position -7 partially relieves the supercoiling response while changing the sequence between -5 and + 1 (-CCCCCG-) for -GTTAA- abolishes the response in vitro and in vivo. The relief of the supercoiling dependence closely correlates with increased promoter susceptibility to melting in vivo and a lesser requirement for initiating nucleotides in the formation of stable initiation complexes in vitro. Studies in isoleucine-starved cells showed that such sequence changes mitigate and abolish the hisR promoter response to stringent control, respectively. The data presented suggest that the hisR promoter's sensitivity to stringent regulation arises from the same physical property that confers supercoiling sensitivity, i.e. resistance to melting. We propose that the stringent control mechanism acts by hampering the ability of RNA polymerase to melt the DNA helix. PMID:9550733

  20. Second Harmonic Super-resolution Microscopy for Quantification of mRNA at Single Copy Sensitivity

    PubMed Central

    2015-01-01

    Cell-specific information on the quantity and localization of key mRNAs at single copy sensitivity in single cells is critical for evaluating basic cellular process, disease risk, and efficacy of therapy. Quantification of overexpressed mRNAs beyond the diffraction limit is constrained by the optical property of the probes and microscopy techniques. In this report, nanosized barium titanium oxide (BaTiO3, BTO) crystals were utilized as probes for mRNA quantification by a second harmonic super-resolution microscopy (SHaSM). The SHaSM was able to detect a single copy of the human epidermal growth factor receptor 2 (Her2) mRNA at a resolution of 55.6 nm with the ability to resolve multiple mRNA copies in a diffraction-limited spot. Her2 mRNA per cell was counted in SK-BR-3, MCF-7, and HeLa cell lines as 595 ± 79.1, 38.9 ± 8.26, and 1.5 ± 2.8, respectively. Our single-cell quantification results were validated with the fluorescence in situ hybridization studies and quantitative PCR, showing better specificity and selectivity over current single-molecule approaches for transcript detection. The SHaSM is expected to have an upper limit of resolving ∼104 transcripts in a single cell with the ability to monitor intracellular transcriptional dynamics at video rate. The developed approach has strong potential in clinical research and in the early diagnosis of life-threatening diseases such as cancer. PMID:25494326

  1. Cold-Sensitive Pseudomonas RNA Polymerase I. Characterization of the Host Dependent Cold-Sensitive Restriction of Phage CB3

    PubMed Central

    Sobieski, Rodney J.; Olsen, Ronald H.

    1973-01-01

    Analysis of bacteriophage CB3 infection of Pseudomonas aeruginosa strain PAT2 establishes that phage induced changes in net macromolecular synthesis are absent at nonpermissive phage growth temperatures (32 C). Alterations which are evident in the PAT2 strain at 37 C or in the fully permissive strain, PAO1C, at either warm or cold temperatures do not occur in PAT2 at low temperatures. CB3 DNA synthesis and the degradation of host DNA to approximately 78S components occur at 37 C, but are absent in PAT2 at 20 C. Nevertheless, attachment of phage DNA to host cytoplasmic material occurs under permissive and nonpermissive conditions. This binding of phage DNA at 20 C is identical in nature to phage DNA bound at 37 C. Thus, the conditional cold-sensitive PAT2 host function in the growth of CB3 is expressed subsequent to membrane binding of the infecting genomes but prior to the onset of the initiation of CB3 DNA synthesis, the inhibition of host DNA synthesis, and the transient depression in RNA synthesis which occurs in permissive cells. PMID:4202617

  2. Multiplex Real-Time PCR Assay for Rapid Detection of Methicillin-Resistant Staphylococci Directly from Positive Blood Cultures

    PubMed Central

    Wang, Hye-young; Kim, Sunghyun; Kim, Jungho; Park, Soon-Deok

    2014-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent cause of bloodstream infections (BSIs) and is recognized as a major nosocomial pathogen. This study aimed to evaluate a newly designed multiplex real-time PCR assay capable of the simultaneous detection of mecA, S. aureus, and coagulase-negative staphylococci (CoNS) in blood culture specimens. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays (M&D, Republic of Korea) use the TaqMan probes 16S rRNA for Staphylococcus spp., the nuc gene for S. aureus, and the mecA gene for methicillin resistance. The detection limit of the multiplex real-time PCR assay was 103 CFU/ml per PCR for each gene target. The multiplex real-time PCR assay was evaluated using 118 clinical isolates from various specimen types and a total of 350 positive blood cultures from a continuous monitoring blood culture system. The results obtained with the multiplex real-time PCR assay for the three targets were in agreement with those of conventional identification and susceptibility testing methods except for one organism. Of 350 positive bottle cultures, the sensitivities of the multiplex real-time PCR kit were 100% (166/166 cultures), 97.2% (35/36 cultures), and 99.2% (117/118 cultures) for the 16S rRNA, nuc, and mecA genes, respectively, and the specificities for all three targets were 100%. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays are very useful for the rapid accurate diagnosis of staphylococcal BSIs. In addition, the Real-MRSA and Real-MRCoNS multiplex real-time PCR assays could have an important impact on the choice of appropriate antimicrobial therapy, based on detection of the mecA gene. PMID:24648566

  3. RNA-seq reveals determinants for irinotecan sensitivity/resistance in colorectal cancer cell lines

    PubMed Central

    Li, Xin-Xiang; Zheng, Hong-Tu; Peng, Jun-Jie; Huang, Li-Yong; Shi, De-Bing; Liang, Lei; Cai, San-Jun

    2014-01-01

    Irinotecan is a topoisomerase I inhibitor approved worldwide as a first- and second-line chemotherapy for advanced or recurrent colorectal cancer (CRC). Although irinotecan showed significant survival advantage for patients, a relatively low response rate and severe adverse effects demonstrated the urgent need for biomarkers searching to select the suitable patients who can benefit from irinotecan-based therapy and avoid the adverse effects. In present work, the irinotecan response (IC50 doses) of 20 CRC cell lines were correlated with the basal expression profiles investigated by RNA-seq to figure out genes responsible for irinotecan sensitivity/resistance. Genes negatively or positively correlated to irinotecan sensitivity were given after biocomputation, and 7 (CDC20, CTNNAL1, FZD7, CITED2, ABR, ARHGEF7, and RNMT) of them were validated in two CRC cell lines by quantitative real-time PCR, several of these 7 genes has been proposed to promote cancer cells proliferation and hence may confer CRC cells resistance to irinotecan. Our work might provide potential biomarkers and therapeutic targets for irinotecan sensitivity in CRC cells. PMID:24966994

  4. Sensitive Detection of Protein and miRNA Cancer Biomarkers using Silicon-Based Photonic Crystals and A Resonance Coupling Laser Scanning Platform

    PubMed Central

    George, Sherine; Chaudhery, Vikram; Lu, Meng; Takagi, Miki; Amro, Nabil; Pokhriyal, Anusha; Tan, Yafang; Ferreira, Placid; Cunningham, Brian T.

    2013-01-01

    Enhancement of the fluorescent output of surface-based fluorescence assays by performing them upon nanostructured photonic crystal (PC) surfaces has been demonstrated to increase signal intensities by >8000×. Using the multiplicative effects of optical resonant coupling to the PC in increasing the electric field intensity experienced by fluorescent labels (“enhanced excitation”) and the spatially biased funneling of fluorophore emissions through coupling to PC resonances (“enhanced extraction”), PC enhanced fluorescence (PCEF) can be adapted to reduce the limits of detection of disease biomarker assays, and to reduce the size and cost of high sensitivity detection instrumentation. In this work, we demonstrate the first silicon-based PCEF detection platform for multiplexed biomarker assay. The sensor in this platform is a silicon-based PC structure, comprised of a SiO2 grating that is overcoated with a thin film of high refractive index TiO2 and is produced in a semiconductor foundry for low cost, uniform, and reproducible manufacturing. The compact detection instrument that completes this platform was designed to efficiently couples fluorescence excitation from a semiconductor laser to the resonant optical modes of the PC, resulting in elevated electric field strength that is highly concentrated within the region <100 nm from the PC surface. This instrument utilizes a cylindrically focused line to scan a microarray in <1 minute. To demonstrate the capabilities of this sensor-detector platform, microspot fluorescent sandwich immunoassays using secondary antibodies labeled with Cy5 for two cancer biomarkers (TNF-α and IL-3) were performed. Biomarkers were detected at concentrations as low as 0.1 pM. In a fluorescent microarray for detection of a breast cancer miRNA biomarker miR-21, the miRNA was detectable at a concentration of 0.6 pM. PMID:23963502

  5. Effects of stathmin 1 silencing by siRNA on sensitivity of esophageal cancer cells Eca-109 to paclitaxel.

    PubMed

    Zhu, H W; Jiang, D; Xie, Z Y; Zhou, M H; Sun, D Y; Zhao, Y G

    2015-01-01

    We investigated the effects of stathmin 1 (STMN1) silencing by small interfering (siRNA) on the sensitivity of esophageal cancer cells Eca-109 to paclitaxel. STMN1 siRNA was transiently transfected into Eca-109 cells. The effects of transfection were detected by quantitative polymerase chain reaction and western blotting. The effects of STMN1 silencing by siRNA on the sensitivity of esophageal cancer cells Eca-109 to paclitaxel was tested by MTT and colony formation assays. Hoechst 33258 nuclear staining was used to investigate the differences in Eca-109 cell apoptosis induced by paclitaxel. STMN1 siRNA was successfully transfected and the expression of STMN1 was inhibited. The sensitivity of STMN1 siRNA-transfected Eca-109 cells to paclitaxel was significantly increased (P < 0.01). The apoptosis of Eca-109 cells significantly increased following treatment with paclitaxel (P < 0.01). STMN1 silencing by siRNA may enhance the sensitivity of esophageal cancer cells Eca-109 to paclitaxel and induce apoptosis. PMID:26782519

  6. pH-Sensitive carboxymethyl chitosan-modified cationic liposomes for sorafenib and siRNA co-delivery

    PubMed Central

    Yao, Yao; Su, Zhihui; Liang, Yanchao; Zhang, Na

    2015-01-01

    Combination of chemotherapeutic drug and small interfering RNA (siRNA) can affect multiple disease pathways and has been proven effective in suppressing tumor progression. Co-delivery of drug and siRNA within a same nanocarrier is a vital means in this field. The present study aimed at the development of a pH-sensitive liposome to co-deliver drug and siRNA to tumor region. Driven by the electrostatic interaction, the pH-sensitive material, carboxymethyl chitosan (CMCS), was coated onto the surface of the cationic liposome (CL) preloaded with sorafenib (Sf) and siRNA (Si). To evaluate whether the resulting CMCS-modified Sf and siRNA co-delivery cationic liposome (CMCS-SiSf-CL) enhanced antitumor efficiency after systematic administration, in vitro and in vivo experiments were evaluated in HepG2 cells and the H22 cells-bearing Kunming mice model. The experimental results demonstrated that CMCS-SiSf-CL was able to condense siRNA efficiently and protect siRNA from being degraded by serum and RNase. The release rate of Sf from CMCS-modified liposome exhibited pH-sensitive release behavior. Furthermore, in vitro cellular uptake results showed that CMCS-SiSf-CL yielded higher fluorescence intensity at pH 6.5 than at pH 7.4, and that siRNA could be delivered to tumor site by CMCS-SiSf-CL in vivo. The in vivo antitumor efficacy showed that CMCS-Sf-CL inhibits tumor growth effectively when compared with free Sf solution. In current experimental conditions, this liposomal formulation did not show significant toxicity both in vitro and in vivo. Therefore, co-delivering Sf with siRNA by CMCS-SiSf-CL might provide a promising approach for tumor therapy. PMID:26491291

  7. pH-Sensitive carboxymethyl chitosan-modified cationic liposomes for sorafenib and siRNA co-delivery.

    PubMed

    Yao, Yao; Su, Zhihui; Liang, Yanchao; Zhang, Na

    2015-01-01

    Combination of chemotherapeutic drug and small interfering RNA (siRNA) can affect multiple disease pathways and has been proven effective in suppressing tumor progression. Co-delivery of drug and siRNA within a same nanocarrier is a vital means in this field. The present study aimed at the development of a pH-sensitive liposome to co-deliver drug and siRNA to tumor region. Driven by the electrostatic interaction, the pH-sensitive material, carboxymethyl chitosan (CMCS), was coated onto the surface of the cationic liposome (CL) preloaded with sorafenib (Sf) and siRNA (Si). To evaluate whether the resulting CMCS-modified Sf and siRNA co-delivery cationic liposome (CMCS-SiSf-CL) enhanced antitumor efficiency after systematic administration, in vitro and in vivo experiments were evaluated in HepG2 cells and the H22 cells-bearing Kunming mice model. The experimental results demonstrated that CMCS-SiSf-CL was able to condense siRNA efficiently and protect siRNA from being degraded by serum and RNase. The release rate of Sf from CMCS-modified liposome exhibited pH-sensitive release behavior. Furthermore, in vitro cellular uptake results showed that CMCS-SiSf-CL yielded higher fluorescence intensity at pH 6.5 than at pH 7.4, and that siRNA could be delivered to tumor site by CMCS-SiSf-CL in vivo. The in vivo antitumor efficacy showed that CMCS-Sf-CL inhibits tumor growth effectively when compared with free Sf solution. In current experimental conditions, this liposomal formulation did not show significant toxicity both in vitro and in vivo. Therefore, co-delivering Sf with siRNA by CMCS-SiSf-CL might provide a promising approach for tumor therapy. PMID:26491291

  8. Exosomes derived from alcohol-treated hepatocytes horizontally transfer liver specific miRNA-122 and sensitize monocytes to LPS.

    PubMed

    Momen-Heravi, Fatemeh; Bala, Shashi; Kodys, Karen; Szabo, Gyongyi

    2015-01-01

    Hepatocyte damage and inflammation in monocytes/macrophages are central to the pathogenesis of alcoholic hepatitis (AH). MicroRNAs (miRNAs) regulate all of these processes. MiRNA-122 is abundantly expressed in hepatocytes while monocytes/macrophages have low levels. The role of exosomes in AH and possible cross talk between hepatocyte-derived exosomes and immune cells is not explored yet. Here, we show that the number of exosomes significantly increases in the sera of healthy individuals after alcohol binge drinking and in mice after binge or chronic alcohol consumption. Exosomes isolated from sera after alcohol consumption or from in vitro ethanol-treated hepatocytes contained miRNA-122. Exosomes derived from ethanol-treated Huh7.5 cells were taken up by the recipients THP1 monocytes and horizontally transferred a mature form of liver-specific miRNA-122. In vivo, liver mononuclear cells and Kupffer cells from alcohol-fed mice had increased miRNA-122 levels. In monocytes, miRNA-122 transferred via exosomes inhibited the HO-1 pathway and sensitized to LPS stimulation and increased levels of pro-inflammatory cytokines. Finally, inflammatory effects of exosomes from ethanol-treated hepatocytes were prevented by using RNA interference via exosome-mediated delivery of a miRNA-122 inhibitor. These results demonstrate that first, exosomes mediate communication between hepatocytes and monocytes/macrophages and second, hepatocyte-derived miRNA-122 can reprogram monocytes inducing sensitization to LPS. PMID:25973575

  9. Exosomes derived from alcohol-treated hepatocytes horizontally transfer liver specific miRNA-122 and sensitize monocytes to LPS

    PubMed Central

    Momen-Heravi, Fatemeh; Bala, Shashi; Kodys, Karen; Szabo, Gyongyi

    2015-01-01

    Hepatocyte damage and inflammation in monocytes/macrophages are central to the pathogenesis of alcoholic hepatitis (AH). MicroRNAs (miRNAs) regulate all of these processes. MiRNA-122 is abundantly expressed in hepatocytes while monocytes/macrophages have low levels. The role of exosomes in AH and possible cross talk between hepatocyte-derived exosomes and immune cells is not explored yet. Here, we show that the number of exosomes significantly increases in the sera of healthy individuals after alcohol binge drinking and in mice after binge or chronic alcohol consumption. Exosomes isolated from sera after alcohol consumption or from in vitro ethanol-treated hepatocytes contained miRNA-122. Exosomes derived from ethanol-treated Huh7.5 cells were taken up by the recipients THP1 monocytes and horizontally transferred a mature form of liver-specific miRNA-122. In vivo, liver mononuclear cells and Kupffer cells from alcohol-fed mice had increased miRNA-122 levels. In monocytes, miRNA-122 transferred via exosomes inhibited the HO-1 pathway and sensitized to LPS stimulation and increased levels of pro-inflammatory cytokines. Finally, inflammatory effects of exosomes from ethanol-treated hepatocytes were prevented by using RNA interference via exosome-mediated delivery of a miRNA-122 inhibitor. These results demonstrate that first, exosomes mediate communication between hepatocytes and monocytes/macrophages and second, hepatocyte-derived miRNA-122 can reprogram monocytes inducing sensitization to LPS. PMID:25973575

  10. CCAT1 is an enhancer-templated RNA that predicts BET sensitivity in colorectal cancer

    PubMed Central

    McCleland, Mark L.; Mesh, Kathryn; Lorenzana, Edward; Chopra, Vivek S.; Segal, Ehud; Watanabe, Colin; Haley, Benjamin; Mayba, Oleg; Yaylaoglu, Murat; Gnad, Florian; Firestein, Ron

    2016-01-01

    Colon tumors arise in a stepwise fashion from either discrete genetic perturbations or epigenetic dysregulation. To uncover the key epigenetic regulators that drive colon cancer growth, we used a CRISPR loss-of-function screen and identified a number of essential genes, including the bromodomain and extraterminal (BET) protein BRD4. We found that BRD4 is critical for colon cancer proliferation, and its knockdown led to differentiation effects in vivo. JQ1, a BET inhibitor, preferentially reduced growth in a subset of epigenetically dysregulated colon cancers characterized by the CpG island methylator phenotype (CIMP). Integrated transcriptomic and genomic analyses defined a distinct superenhancer in CIMP+ colon cancers that regulates cMYC transcription. We found that the long noncoding RNA colon cancer–associated transcript 1 (CCAT1) is transcribed from this superenhancer and is exquisitely sensitive to BET inhibition. Concordantly, cMYC transcription and cell growth were tightly correlated with the presence of CCAT1 RNA in a variety of tumor types. Taken together, we propose that CCAT1 is a clinically tractable biomarker for identifying patients who are likely to benefit from BET inhibitors. PMID:26752646

  11. Single molecule fluorescence microscopy for ultra-sensitive RNA expression profiling

    NASA Astrophysics Data System (ADS)

    Hesse, Jan; Jacak, Jaroslaw; Regl, Gerhard; Eichberger, Thomas; Aberger, Fritz; Schlapak, Robert; Howorka, Stefan; Muresan, Leila; Frischauf, Anna-Maria; Schütz, Gerhard J.

    2007-02-01

    We developed a microarray analysis platform for ultra-sensitive RNA expression profiling of minute samples. It utilizes a novel scanning system for single molecule fluorescence detection on cm2 size samples in combination with specialized biochips, optimized for low autofluorescence and weak unspecific adsorption. 20 μg total RNA was extracted from 10 6 cells of a human keratinocyte cell line (HaCaT) and reversely transcribed in the presence of Alexa647-aha-dUTP. 1% of the resulting labeled cDNA was used for complex hybridization to a custom-made oligonucleotide microarray representing a set of 125 different genes. For low abundant genes, individual cDNA molecules hybridized to the microarray spots could be resolved. Single cDNA molecules hybridized to the chip surface appeared as diffraction limited features in the fluorescence images. The à trous wavelet method was utilized for localization and counting of the separated cDNA signals. Subsequently, the degree of labeling of the localized cDNA molecules was determined by brightness analysis for the different genes. Variations by factors up to 6 were found, which in conventional microarray analysis would result in a misrepresentation of the relative abundance of mRNAs.

  12. Isolation and characterization of temperature-sensitive RNA polymerase II mutants of Saccharomyces cerevisiae.

    PubMed Central

    Himmelfarb, H J; Simpson, E M; Friesen, J D

    1987-01-01

    Three independent, recessive, temperature-sensitive (Ts-) conditional lethal mutations in the largest subunit of Saccharomyces cerevisiae RNA polymerase II (RNAP II) have been isolated after replacement of a portion of the wild-type gene (RPO21) by a mutagenized fragment of the cloned gene. Measurements of cell growth, viability, and total RNA and protein synthesis showed that rpo21-1, rpo21-2, and rpo21-3 mutations caused a slow shutoff of RNAP II activity in cells shifted to the nonpermissive temperature (39 degrees C). Each mutant displayed a distinct phenotype, and one of the mutant enzymes (rpo21-1) was completely deficient in RNAP II activity in vitro. RNAP I and RNAP III in vitro activities were not affected. These results were consistent with the notion that the genetic lesions affect RNAP II assembly or holoenzyme stability. DNA sequencing revealed that in each case the mutations involved nonconservative amino acid substitutions, resulting in charge changes. The lesions harbored by all three rpo21 Ts- alleles lie in DNA sequence domains that are highly conserved among genes that encode the largest subunits of RNAP from a variety of eucaryotes; one mutation lies in a possible Zn2+ binding domain. PMID:3299061

  13. RNA Interference in Schistosoma mansoni Schistosomula: Selectivity, Sensitivity and Operation for Larger-Scale Screening

    PubMed Central

    Horn, Martin; Braschi, Simon; Sojka, Daniel; Ruelas, Debbie S.; Suzuki, Brian; Lim, Kee-Chong; Hopkins, Stephanie D.; McKerrow, James H.; Caffrey, Conor R.

    2010-01-01

    Background The possible emergence of resistance to the only available drug for schistosomiasis spurs drug discovery that has been recently incentivized by the availability of improved transcriptome and genome sequence information. Transient RNAi has emerged as a straightforward and important technique to interrogate that information through decreased or loss of gene function and identify potential drug targets. To date, RNAi studies in schistosome stages infecting humans have focused on single (or up to 3) genes of interest. Therefore, in the context of standardizing larger RNAi screens, data are limited on the extent of possible off-targeting effects, gene-to-gene variability in RNAi efficiency and the operational capabilities and limits of RNAi. Methodology/Principal Findings We investigated in vitro the sensitivity and selectivity of RNAi using double-stranded (ds)RNA (approximately 500 bp) designed to target 11 Schistosoma mansoni genes that are expressed in different tissues; the gut, tegument and otherwise. Among the genes investigated were 5 that had been previously predicted to be essential for parasite survival. We employed mechanically transformed schistosomula that are relevant to parasitism in humans, amenable to screen automation and easier to obtain in greater numbers than adult parasites. The operational parameters investigated included defined culture media for optimal parasite maintenance, transfection strategy, time- and dose- dependency of RNAi, and dosing limits. Of 7 defined culture media tested, Basch Medium 169 was optimal for parasite maintenance. RNAi was best achieved by co-incubating parasites and dsRNA (standardized to 30 µg/ml for 6 days); electroporation provided no added benefit. RNAi, including interference of more than one transcript, was selective to the gene target(s) within the pools of transcripts representative of each tissue. Concentrations of dsRNA above 90 µg/ml were directly toxic. RNAi efficiency was transcript

  14. Label-free fluorescence strategy for sensitive microRNA detection based on isothermal exponential amplification and graphene oxide.

    PubMed

    Li, Wei; Hou, Ting; Wu, Min; Li, Feng

    2016-02-01

    MicroRNAs (miRNAs) play an important role in many biological processes, and have been regarded as potential targets and biomarkers in cancer diagnosis and therapy. Also, to meet the big challenge imposed by the characteristics of miRNAs, such as small size and vulnerability to enzymatic digestion, it is of great importance to develop accurate, sensitive and simple miRNA assays. Herein, we developed a label-free fluorescence strategy for sensitive miRNA detection by combining isothermal exponential amplification and the unique features of SYBR Green I (SG) and graphene oxide (GO), in which SG gives significantly enhanced fluorescence upon intercalation into double-stranded DNAs (dsDNAs), and GO selectively adsorbs miRNA, single-stranded DNA and SG, to protect miRNA from enzymatic digestion, and to quench the fluorescence of the adsorbed SG. In the presence of the target miRNA, the ingeniously designed hairpin probe (HP) is unfolded and the subsequent polymerization and strand displacement reaction takes place to initiate the target recycling process. The newly formed dsDNAs are then recognized and cleaved by the nicking enzyme, generating new DNA triggers with the same sequence as the target miRNA, which hybridize with intact HPs to initiate new extension reactions. As a result, the circular exponential amplification for target miRNA is achieved and large amount of dsDNAs are formed to generate significantly enhanced fluorescence upon the intercalation of SG. Thus sensitive and selective fluorescence miRNA detection is realized, and the detection limit of 3 fM is obtained. Besides, this method exhibits additional advantages of simplicity and low cost, since expensive and tedious labeling process is avoided. Therefore, the as-proposed label-free fluorescence strategy has great potential in the applications in miRNA-related clinical practices and biochemical researches. PMID:26653431

  15. Guanine nucleotide metabolism in a mutant strain of Escherichia coli with a temperature sensitive lesion in rRNA synthesis.

    PubMed

    Harris, J S; Chaney, S G

    1978-12-21

    We have described a mutant of Escherichia coli (designated 2S142) which shows specific inhibition of rRNA synthesis at 42 degrees C. ppGpp levels increase at the restrictive temperature, as expected. However, when the cells are returned to 30 degrees C, rRNA synthesis resumes before ppGpp levels have returned to normal. Furthermore, when ppGpp levels are decreased by the addition of tetracycline or choramphenicol, rRNA synthesis does not resume at 42 degrees C. Also, a derivative of 2S142 with a temperature-sensitive G factor (which cannot synthesize either protein or ppGpp at 42 degrees C) shows identical kinetics of rRNA shut-off at 42 degrees C as 2S142. Thus, the elevated ppGpp levels in this mutant do not appear to be directly responsible for the cessation of rRNA synthesis at 42 degrees C. PMID:367439

  16. N-ethylmaleimide‑sensitive factor siRNA inhibits the release of Weibel-Palade bodies in endothelial cells.

    PubMed

    Zhou, Yong; Yang, Shui-Xiang; Yue, Yu-Nan; Wei, Xiao-Fei; Liu, Yan

    2016-08-01

    The aim of the present study was to examine the effect of small interfering RNA (siRNA) methods on the expression of N‑ethylmaleimide sensitive factor (NSF) and Weibel‑Palade body (WPB) release in endothelial cells. A small hairpin RNA (shRNA), mediated with an adenovirus vector, was designed to target the N‑terminal functional area of NSF. Subsequently, viruses were transfected into human aortic endothelial cells. The mRNA and protein expression levels of NSF were detected using reverse transcription‑quantitative polymerase chain reaction and Western blot analyses, respectively, and the release of WPBs in the endothelial cells was examined using immunofluorescence. The mRNA expression of NSF in the endothelial cells, which were transfected with the adenoviruses carrying the NSF‑shRNA was significantly decreased, compared with the negative control group (P=0.035) and blank control group (P=0.02). In addition, the mRNA expression of NSF was gradually decreased as duration increased; there were marked differences between the 24, 48 and 72 h groups (P<0.05). The protein expression of NSF was significantly decreased in the experimental group, compared with the negative control group (P=0.004) and blank control group (P=0.031), however, no difference was observed between the negative control and blank control groups (P=0.249). The immunofluorescence staining showed that the release of WPBs in the endothelial cells induced with thrombin was inhibited markedly following transfection with the virus carrying the NSF‑shRNA. Therefore NSF‑siRNA inhibited the mRNA and protein expression levels of NSF, and inhibited the release of WPBs in endothelial cells induced with thrombin. These results suggested that NSF-siRNA may be valuable for preventing and treating atherosclerosis and acute coronary syndrome. PMID:27277949

  17. N-ethylmaleimide-sensitive factor siRNA inhibits the release of Weibel-Palade bodies in endothelial cells

    PubMed Central

    Zhou, Yong; Yang, Shui-Xiang; Yue, Yu-Nan; Wei, Xiao-Fei; Liu, Yan

    2016-01-01

    The aim of the present study was to examine the effect of small interfering RNA (siRNA) methods on the expression of N-ethylmaleimide sensitive factor (NSF) and Weibel-Palade body (WPB) release in endothelial cells. A small hairpin RNA (shRNA), mediated with an adenovirus vector, was designed to target the N-terminal functional area of NSF. Subsequently, viruses were transfected into human aortic endothelial cells. The mRNA and protein expression levels of NSF were detected using reverse transcription-quantitative polymerase chain reaction and Western blot analyses, respectively, and the release of WPBs in the endothelial cells was examined using immunofluorescence. The mRNA expression of NSF in the endothelial cells, which were transfected with the adenoviruses carrying the NSF-shRNA was significantly decreased, compared with the negative control group (P=0.035) and blank control group (P=0.02). In addition, the mRNA expression of NSF was gradually decreased as duration increased; there were marked differences between the 24, 48 and 72 h groups (P<0.05). The protein expression of NSF was significantly decreased in the experimental group, compared with the negative control group (P=0.004) and blank control group (P=0.031), however, no difference was observed between the negative control and blank control groups (P=0.249). The immunofluorescence staining showed that the release of WPBs in the endothelial cells induced with thrombin was inhibited markedly following transfection with the virus carrying the NSF-shRNA. Therefore NSF-siRNA inhibited the mRNA and protein expression levels of NSF, and inhibited the release of WPBs in endothelial cells induced with thrombin. These results suggested that NSF-siRNA may be valuable for preventing and treating atherosclerosis and acute coronary syndrome. PMID:27277949

  18. High sensitive method detection of plant RNA viruses by electrochemiluminescence reverse transcription PCR

    NASA Astrophysics Data System (ADS)

    Tang, Ya-bing; Xing, Da; Zhu, De-bin; Zhou, Xiao-ming

    2007-05-01

    It is well known that plant and animal viruses had widely spread the whole of world, and made a big loss in farming and husbandry. It is necessary that a highly efficient and accurate virus's detection method was developed. This research combines reverse transcription polymerase chain reaction (RT-PCR) technique with electrochemiluminescence method, to detect plant RNA viruses for the first time. Biotin-probe hybridizes with PCR product to specific select the target for detection, thus can avoid pseudo-positive result. TBR-probe hybridizes with PCR product to emit light for ECL detection. Specific nucleic acid sequences (20bp) were added to 5' terminal all of the primers, which can improve the chance of hybridization between TBR-probe and PCR product. At the same time, one of the PCR product chain can hybridize two Ru-probes, the ECL signal is intensified. The method was used to detect Odntoglossum ringspot virus ORSV, Sugarcane mosaic virus ScMV, Sorghum mosaic virus SrMV, and Maize dwarf mosaic virus MDMV, the experiment results show that this method could reliably identity virus infected plant samples. In a word, this method has higher sensitivity and lower cost than others. It can effectively detect the plant viruses with simplicity, stability, and high sensitivity.

  19. Low-cost, multiplexed biosensor for disease diagnosis

    NASA Astrophysics Data System (ADS)

    Myatt, Christopher J.; Delaney, Marie; Todorof, Kathryn; Heil, James; Givens, Monique; Schooley, Robert T.; Lochhead, Michael J.

    2009-02-01

    Cost-effective disease diagnosis in resource-limited settings remains a critical global health challenge. Qualitative rapid tests based on lateral flow technology provide valuable screening information, but require relatively expensive confirmatory tests and generally lack quantitation. We report on a fluorescence technology that combines low cost instrumented readout with passive pumping in a disposable cartridge. The detection system utilizes a novel waveguide illumination approach in conjunction with commercial CMOS imagers. Total instrument cost in production are projected to be around $100 This cost structure and instrument ease of use will enable use in point-of-care settings, outside of centralized laboratories. The system has been used for detection and analysis of proteins, antibodies, nucleic acids, and cells. Here we will report first on our development of a multiplexed, array-based serology assay for HIV and common AIDS co-infections. Data will be presented for HIV/HCV antibody testing in human serum samples. In addition, we will present data on the use of the system for sensitive detection of bacterial RNA. Current detection limit for the model multiplexed RNA sandwich assay is 1 femtomolar target RNA. Finally, a high magnification version of the system is used to image immunostained human T cells.

  20. The Effect of MicroRNA-124 Overexpression on Anti-Tumor Drug Sensitivity

    PubMed Central

    Chen, Shiau-Mei; Chou, Wen-Cheng; Hu, Ling-Yueh; Hsiung, Chia-Ni; Chu, Hou-Wei; Huang, Yuan-Ling; Hsu, Huan-Ming; Yu, Jyh-Cherng; Shen, Chen-Yang

    2015-01-01

    MicroRNAs play critical roles in regulating various physiological processes, including growth and development. Previous studies have shown that microRNA-124 (miR-124) participates not only in regulation of early neurogenesis but also in suppression of tumorigenesis. In the present study, we found that overexpression of miR-124 was associated with reduced DNA repair capacity in cultured cancer cells and increased sensitivity of cells to DNA-damaging anti-tumor drugs, specifically those that cause the formation of DNA strand-breaks (SBs). We then examined which DNA repair–related genes, particularly the genes of SB repair, were regulated by miR-124. Two SB repair–related genes, encoding ATM interactor (ATMIN) and poly (ADP-ribose) polymerase 1 (PARP1), were strongly affected by miR-124 overexpression, by binding of miR-124 to the 3¢-untranslated region of their mRNAs. As a result, the capacity of cells to repair DNA SBs, such as those resulting from homologous recombination, was significantly reduced upon miR-124 overexpression. A particularly important therapeutic implication of this finding is that overexpression of miR-124 enhanced cell sensitivity to multiple DNA-damaging agents via ATMIN- and PARP1-mediated mechanisms. The translational relevance of this role of miR-124 in anti-tumor drug sensitivity is suggested by the finding that increased miR-124 expression correlates with better breast cancer prognosis, specifically in patients receiving chemotherapy. These findings suggest that miR-124 could potentially be used as a therapeutic agent to improve the efficacy of chemotherapy with DNA-damaging agents. PMID:26115122

  1. MicroRNA-181a enhances the chemotherapeutic sensitivity of chronic myeloid leukemia to imatinib

    PubMed Central

    WANG, GUANGYU; ZHAO, RAN; ZHAO, XINGSHENG; CHEN, XI; WANG, DONG; JIN, YINJI; LIU, XI; ZHAO, CI; ZHU, YUANYUAN; REN, CHENGCHENG; LI, MINGHUI; JIN, XIAOMING; ZHANG, FENGMIN; ZHONG, ZHAOHUA; WANG, TIANZHEN; LI, XIAOBO

    2015-01-01

    MicroRNA-181 (miR-181) has been recently demonstrated to participate in the differentiation and development of immune cells, including natural killer cells and B and T lymphocytes, and myeloid linages, including erythroid and megakaryocytic cells. The aberrant expression of miR-181, particularly low expression levels, has been observed in a number of leukemia types, including B-cell chronic lymphocytic leukemia and cytogenetically abnormal acute myeloid leukemia. However, the expression and function of miR-181 in chronic myeloid leukemia (CML) remains unknown. In the present study, the aberrant expression of miR-181a was analyzed in a patient with CML and in the CML K562 cell line. In addition, the function and potential mechanisms of miR-181a in K562 cells with regard to their chemotherapeutic sensitivity to imatinib were investigated. The expression levels of miR-181a were significantly reduced in the patient with CML and in the CML K562 cell line. Furthermore, the overexpression of miR-181a in the K562 cells enhanced the chemotherapeutic sensitivity of these cells to imatinib. The potential mechanism mediating these effects may be associated with the capacity of miR-181a to inhibit cell growth and/or to induce cells apoptosis and differentiation in K562 cells. The results of the present study suggested that miR-181a may be a target for the treatment of CML and a useful indicator of the therapeutic sensitivity of CML to imatinib. PMID:26722250

  2. 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; Davies, Peter F.

    2003-01-01

    Although mRNA amplification is necessary for microarray analyses from limited amounts of cells and tissues, the accuracy of transcription profiles following amplification has not been well characterized. We tested the fidelity of differential gene expression following linear amplification by T7-mediated transcription in a well-established in vitro model of cytokine [tumor necrosis factor alpha (TNFalpha)]-stimulated human endothelial cells using filter arrays of 13,824 human cDNAs. Transcriptional profiles generated from amplified antisense RNA (aRNA) (from 100 ng total RNA, approximately 1 ng mRNA) were compared with profiles generated from unamplified RNA originating from the same homogeneous pool. Amplification accurately identified TNFalpha-induced differential expression in 94% of the genes detected using unamplified samples. Furthermore, an additional 1,150 genes were identified as putatively differentially expressed using amplified RNA which remained undetected using unamplified RNA. Of genes sampled from this set, 67% were validated by quantitative real-time PCR as truly differentially expressed. Thus, in addition to demonstrating fidelity in gene expression relative to unamplified samples, linear amplification results in improved sensitivity of detection and enhances the discovery potential of high-throughput screening by microarrays.

  3. Multiplexed Simultaneous High Sensitivity Sensors with High-Order Mode Based on the Integration of Photonic Crystal 1 × 3 Beam Splitter and Three Different Single-Slot PCNCs

    PubMed Central

    Zhou, Jian; Huang, Lijun; Fu, Zhongyuan; Sun, Fujun; Tian, Huiping

    2016-01-01

    We simulated an efficient method for the sensor array of high-sensitivity single-slot photonic crystal nanobeam cavities (PCNCs) on a silicon platform. With the combination of a well-designed photonic crystal waveguide (PhCW) filter and an elaborate single-slot PCNC, a specific high-order resonant mode was filtered for sensing. A 1 × 3 beam splitter carefully established was implemented to split channels and integrate three sensors to realize microarrays. By applying the three-dimensional finite-difference-time-domain (3D-FDTD) method, the sensitivities calculated were S1 = 492 nm/RIU, S2 = 244 nm/RIU, and S3 = 552 nm/RIU, respectively. To the best of our knowledge, this is the first multiplexing design in which each sensor cite features such a high sensitivity simultaneously. PMID:27399712

  4. Multiplexed Simultaneous High Sensitivity Sensors with High-Order Mode Based on the Integration of Photonic Crystal 1 × 3 Beam Splitter and Three Different Single-Slot PCNCs.

    PubMed

    Zhou, Jian; Huang, Lijun; Fu, Zhongyuan; Sun, Fujun; Tian, Huiping

    2016-01-01

    We simulated an efficient method for the sensor array of high-sensitivity single-slot photonic crystal nanobeam cavities (PCNCs) on a silicon platform. With the combination of a well-designed photonic crystal waveguide (PhCW) filter and an elaborate single-slot PCNC, a specific high-order resonant mode was filtered for sensing. A 1 × 3 beam splitter carefully established was implemented to split channels and integrate three sensors to realize microarrays. By applying the three-dimensional finite-difference-time-domain (3D-FDTD) method, the sensitivities calculated were S₁ = 492 nm/RIU, S₂ = 244 nm/RIU, and S₃ = 552 nm/RIU, respectively. To the best of our knowledge, this is the first multiplexing design in which each sensor cite features such a high sensitivity simultaneously. PMID:27399712

  5. MicroRNA-mediated signal amplification coupled with GNP/dendrimers on a mass-sensitive biosensor and its applications in intracellular microRNA quantification.

    PubMed

    Guo, Yingshu; Wang, Yujie; Yang, Guangxu; Xu, Jing-Juan; Chen, Hong-Yuan

    2016-11-15

    Here, a mass-sensitive microRNA sensing surface is developed by utilizing a probe DNA/gold nanoparticles (GNP)/dendrimer composite coupling with an enzymatic amplification process. The probe DNA/GNP/dendrimer composite is prepared via the covalent coupling between the NH2 groups in PAMAM or DNA and the COOH group on GNP. Target microRNA binds to a stem-loop-structured DNA on maganatic NPs, forming a heteroduplex. By enzyme recycling amplification, a large number of linker sequences are produced on MNPs. Via the combination of probe DNA, the linker DNA on MNPs and a capture DNA on gold chip, the DNA/GNP/dendrimer probe could be assembled on the surface of gold chip inducing a sensitive frequency response. It presents an excellent performance in microRNA-203 quantification with a detection linear range of 1.0×10(-11)-1.0×10(-9)M. Both the success in discriminating expressing miroRNA-203 in MCF-7 cell and the well agreement with the commercial qRT-PCR detection method implied its potential application in early cancer diagnosis for future. PMID:27311115

  6. Sensitivity of Ribosomal RNA Character Sampling in the Phylogeny of Rhabditida

    PubMed Central

    Holovachov, Oleksandr; Camp, Lauren; Nadler, Steven A.

    2015-01-01

    Near-full-length 18S and 28S rRNA gene sequences were obtained for 33 nematode species. Datasets were constructed based on secondary structure and progressive multiple alignments, and clades were compared for phylogenies inferred by Bayesian and maximum likelihood methods. Clade comparisons were also made following removal of ambiguously aligned sites as determined using the program ProAlign. Different alignments of these data produced tree topologies that differed, sometimes markedly, when analyzed by the same inference method. With one exception, the same alignment produced an identical tree topology when analyzed by different methods. Removal of ambiguously aligned sites altered the tree topology and also reduced resolution. Nematode clades were sensitive to differences in multiple alignments, and more than doubling the amount of sequence data by addition of 28S rRNA did not fully mitigate this result. Although some individual clades showed substantially higher support when 28S data were combined with 18S data, the combined analysis yielded no statistically significant increases in the number of clades receiving higher support when compared to the 18S data alone. Secondary structure alignment increased accuracy in positional homology assignment and, when used in combination with paired-site substitution models, these structural hypotheses of characters and improved models of character state change yielded high levels of phylogenetic resolution. Phylogenetic results included strong support for inclusion of Daubaylia potomaca within Cephalobidae, whereas the position of Fescia grossa within Tylenchina varied depending on the alignment, and the relationships among Rhabditidae, Diplogastridae, and Bunonematidae were not resolved. PMID:26941463

  7. A sensitive and semi-quantitative method for determination of multi-drug residues in animal body fluids using multiplex dipstick immunoassay.

    PubMed

    Han, Shuaijuan; Zhou, Tianjiao; Yin, Bingjie; He, Pingli

    2016-07-13

    The objective of this research was to develop a multiplex dipstick immunoassay method for the simultaneous determination of multi-veterinary drug residues, such as β-agonists, sulfonamides, and tetracyclines in milk, urine, and serum. The multiplex dipstick assay format was based on an indirect competitive approach: Three test lines (different antigens) and one control line (goat anti-mouse IgG) were located on the strip membrane. Labeled antibodies were freeze-dried in microwells. Samples did not require pretreatment and could be directly analyzed within 10 min. Threshold levels in different sample matrices were visually estimated at 0.3-0.45 ng mL(-1) for clenbuterol; 3-4 ng mL(-1) for sulfadiazine; and 4.5-6 ng mL(-1) for tetracycline, respectively. The linear relationship between the concentrations of veterinary drug residues and the Au nanoparticles plasmon absorbance allowed quantitative determination of these veterinary drug residues. The recoveries of clenbuterol, sulfadiazine and tetracycline in spiked samples ranged from 78.4% to 112.6%, and the relative standard deviations were below 11.2%. Analysis of animal samples suggested that the proposed multiplex dipstick assay method was consistent with the LC-MS/MS method. The percentage of false results was less than or equal to 5%. Thus, the proposed multiplex dipstick assay is inexpensive, easy-to-use, and suitable for the purposes of rapid and comprehensive screening of 3 families of β-agonists, sulfonamides and tetracyclines including 26 drugs in animal body fluids. PMID:27237838

  8. Gold Nanoparticle Coated Silica Nanorods for Sensitive Visual Detection of microRNA on a Lateral Flow Strip Biosensor.

    PubMed

    Takalkar, Sunitha; Xu, Hui; Chen, Jiao; Baryeh, Kwaku; Qiu, Wanwei; Zhao, Julia X; Liu, And Guodong

    2016-01-01

    We present a rapid and highly sensitive approach for visual detection of microRNA (miRNA) using a gold nanoparticles coated silica nanorod label and lateral flow strip biosensor. Gold nanoparticles were decorated on the silica nanorod surface by a seeding and growth procedure. A single strand DNA probe was immobilized on the gold nanoparticles-silica nanorod surface by a self-assembling process, and the formed DNA-gold nanoparticles-silica nanorod conjugate was used to construct the lateral flow nucleic acid biosensor for detecting miRNA. The captured gold nanoparticles-silica nanorods by sandwich-type hybridization reactions (DNA-RNA-DNA) on the test zone of the lateral flow nucleic acid biosensor produced the characteristic color bands, enabling visual detection of miRNA. After systematic optimization, the new lateral flow nucleic acid biosensor was capable of detecting 10 pM of the miRNA target without instrumentation, which is six times lower than that obtained with the gold nanoparticle-based lateral flow nucleic acid biosensor. The gold nanoparticles coated silica nanorod thus provides a new and sensitive nanolabel for visual detection of biological molecules on the lateral flow biosensor. PMID:27302581

  9. Endogenous MCM7 MicroRNA Cluster as a Novel Platform to Multiplex Small Interfering and Nucleolar RNAs for Combinational HIV-1 Gene Therapy

    PubMed Central

    Chung, Janet; Zhang, Jane; Li, Haitang; Ouellet, Dominique L.; DiGiusto, David L.

    2012-01-01

    Abstract Combinational therapy with small RNA inhibitory agents against multiple viral targets allows efficient inhibition of viral production by controlling gene expression at critical time points. Here we explore combinations of different classes of therapeutic anti-HIV-1 RNAs expressed from within the context of an intronic MCM7 (minichromosome maintenance complex component-7) platform that naturally harbors 3 microRNAs (miRNAs). We replaced the endogenous miRNAs with anti-HIV small RNAs, including small interfering RNAs (siRNAs) targeting HIV-1 tat and rev messages that function to induce post-transcriptional gene silencing by the RNA interference pathway, a nucleolar-localizing RNA ribozyme that targets the conserved U5 region of HIV-1 transcripts for degradation, and finally nucleolar trans-activation response (TAR) and Rev-binding element (RBE) RNA decoys designed to sequester HIV-1 Tat and Rev proteins inside the nucleolus. We demonstrate the versatility of the MCM7 platform in expressing and efficiently processing the siRNAs as miRNA mimics along with nucleolar small RNAs. Furthermore, three of the combinatorial constructs tested potently suppressed viral replication during a 1-month HIV challenge, with greater than 5-log inhibition compared with untransduced, HIV-1-infected CEM T lymphocytes. One of the most effective constructs contains an anti-HIV siRNA combined with a nucleolar-localizing U5 ribozyme and TAR decoy. This represents the first efficacious example of combining Drosha-processed siRNAs with small nucleolar ribonucleoprotein (snoRNP)-processed nucleolar RNA chimeras from a single intron platform for effective inhibition of viral replication. Moreover, we demonstrated enrichment/selection for cells expressing levels of the antiviral RNAs that provide optimal inhibition under the selective pressure of HIV. The combinations of si/snoRNAs represent a new paradigm for combinatorial RNA-based gene therapy applications. PMID:22834872

  10. Multiplex PCR for rapid diagnosis of tuberculous meningitis.

    PubMed

    Kusum, Sharma; Aman, Sharma; Pallab, Ray; Kumar, Sharma Shiv; Manish, Modi; Sudesh, Prabhakar; Subhash, Varma; Meera, Sharma

    2011-10-01

    Rapid and specific diagnosis of tubercular meningitis is of paramount importance to decrease morbidity and mortality. The aim of the study was to evaluate multiplex PCR using protein b, MPB 64, and IS6110 primers directed against M. tuberculosis complex for the diagnosis of tuberculous meningitis (TBM). Multiplex PCR was performed on 18 TBM confirmed cases (culture was positive), 92 clinically suspected TBM cases and 100 non-TBM (control group) patients. Multiplex PCR had a sensitivity of 94.4% for confirmed cases and specificity of 100% for confirmed TBM cases. In 92 clinically diagnosed but unconfirmed TBM cases, multiplex PCR was positive in 84.78% cases. The overall sensitivity of microscopy, culture and multiplex cases were 1.81, 16.73, and 86.63% and specificity was 100, 100, and 100% respectively. Multiplex PCR using protein b, MPB 64, and IS6110 primers has a high sensitivity and specificity in diagnosis of tubercular meningitis. PMID:21455603

  11. Pulmonary Codelivery of Doxorubicin and siRNA by pH-Sensitive Nanoparticles for Therapy of Metastatic Lung Cancer.

    PubMed

    Xu, Caina; Wang, Ping; Zhang, Jingpeng; Tian, Huayu; Park, Kinam; Chen, Xuesi

    2015-09-01

    A pulmonary codelivery system that can simultaneously deliver doxorubicin (DOX) and Bcl2 siRNA to the lungs provides a promising local treatment strategy for lung cancers. In this study, DOX is conjugated onto polyethylenimine (PEI) by using cis-aconitic anhydride (CA, a pH-sensitive linker) to obtain PEI-CA-DOX conjugates. The PEI-CA-DOX/siRNA complex nanoparticles are formed spontaneously via electrostatic interaction between cationic PEI-CA-DOX and anionic siRNA. The drug release experiment shows that DOX releases faster at acidic pH than at pH 7.4. Moreover, PEI-CA-DOX/Bcl2 siRNA complex nanoparticles show higher cytotoxicity and apoptosis induction in B16F10 cells than those treated with either DOX or Bcl2 siRNA alone. When the codelivery systems are directly sprayed into the lungs of B16F10 melanoma-bearing mice, the PEI-CA-DOX/Bcl2 siRNA complex nanoparticles exhibit enhanced antitumor efficacy compared with the single delivery of DOX or Bcl2 siRNA. Compared with systemic delivery, most drug and siRNA show a long-term retention in the lungs via pulmonary delivery, and a considerable number of the drug and siRNA accumulate in tumor tissues of lungs, but rarely in normal lung tissues. The PEI-CA-DOX/Bcl2 siRNA complex nanoparticles are promising for the treatment of metastatic lung cancer by pulmonary delivery with low side effects on the normal tissues. PMID:26136261

  12. Development of a multiplex real time PCR to differentiate Sarcocystis spp. affecting cattle.

    PubMed

    Moré, Gastón; Schares, Susann; Maksimov, Aline; Conraths, Franz J; Venturini, María C; Schares, Gereon

    2013-10-18

    Cattle are intermediate hosts of Sarcocystis cruzi, Sarcocystis hirsuta and Sarcocystis hominis which use canids, felids or primates as definitive hosts (DH), respectively, and in addition of Sarcocystis sinensis from which the DH is unknown. The aims of the present study were to develop and optimize a multiplex real time PCR for a sensitive and specific differentiation of Sarcocystis spp. affecting cattle and to estimate the prevalence of Sarcocystis spp. in Argentinean cattle. The 18S rRNA genes from individual sarcocysts were amplified and cloned to serve as controls. For the amplification of bovine Sarcocystis spp. a total of 3 primers were used in combination with specific individual probes. Each assay was evaluated and optimized individually and subsequently combined in a multiplex assay (BovSarcoMultiplex real time PCR). The analytical specificity of the multiplex assay was assessed using 5 ng of DNA of heterologous Sarcocystis spp. and other apicomplexan parasites, and no positive reactions were observed other than for the species the PCR targeted. The analytical sensitivity ranged between 0.0125 and 0.125 fg of plasmid DNA (equivalent to the DNA of 2-20 plasmid DNA copies) or resembling DNA of 0.1-0.3 bradyzoites. A total of 380 DNA loin samples from Argentina were tested and 313, 29, 14 and 2 were positive for S. cruzi, S. sinensis, S. hirsuta and S. hominis, respectively. S. sinensis was the most prevalent species among thick walled Sarcocystis spp. in Argentinean cattle. Mixed infections were detected in 8.9% of all samples. Diagnostic sensitivity and specificity for the BovSarcoMultiplex real time PCR relative to previous microscopic examination for thin and thick-walled cyst were 91.5% and 41.7%, 36.3% and 95.9% respectively. Improved DNA extraction methods may allow to further increase the specific and sensitive detection of Sarcocystis spp. in meat samples. PMID:23680541

  13. A Multiplex Polymerase Chain Reaction Microarray Assay to Detect Bioterror Pathogens in Blood

    PubMed Central

    Tomioka, Keiko; Peredelchuk, Michael; Zhu, Xiangyang; Arena, Roberto; Volokhov, Dmitri; Selvapandiyan, Angamuthu; Stabler, Katie; Mellquist-Riemenschneider, Jenny; Chizhikov, Vladimir; Kaplan, Gerardo; Nakhasi, Hira; Duncan, Robert

    2005-01-01

    Heightened concern about the dangers of bioterrorism requires that measures be developed to ensure the safety of the blood supply. Multiplex detection of such agents using a blood-screening DNA microarray is a sensitive and specific method to screen simultaneously for a number of suspected agents. We have developed and optimized a multiplex polymerase chain reaction microarray assay to screen blood for three potential bioterror bacterial pathogens and a human ribosomal RNA gene internal control. The analytical sensitivity of the assay was demonstrated to be 50 colony-forming units/ml for Bacillus anthracis, Francisella tularensis, and Yersinia pseudotuberculosis (surrogate for Yersinia pestis). The absence of any false-positives demonstrated high analytical specificity. Screening B. anthracis-infected mouse blood samples and uninfected controls demonstrated effectiveness and specificity in a preclinical application. This study represents proof of the concept of microarray technology to screen simultaneously for multiple bioterror pathogens in blood samples. PMID:16237218

  14. Effect of α-Hederin on IL-2 and IL-17 mRNA and miRNA-133a Levels in Lungs of Ovalbumin-Sensitized Male Rats.

    PubMed

    Ebrahimi, Hadi; Fallahi, Maryam; Khamaneh, Amir Mahdi; Ebrahimi Saadatlou, Mohammad Ali; Saadat, Saeideh; Keyhanmanesh, Rana

    2016-03-01

    α-hederin, a saponin that is a major constituent of English Ivy (Hedera helix) is effective in the treatment of asthma. In the present study, the effect of α-hederin on lung tissue pathology and the levels of the inflammatory mediators; IL-2 mRNA, IL-17 mRNA, and MicroRNAs (miRNA)-133a was evaluated in a rat ovalbumin (OVA)-sensitized model of asthma. Rats were divided randomly into control (C), OVA-sensitized (S), OVA-sensitized pretreated with the antioxidant, thymoquinone (3 mg/kg, S + TQ) or OVA-sensitized pretreated with α-hederin (0.02 mg/kg, S + AH) groups. Levels of IL-2 and IL-17 mRNA were higher in the OVA-sensitized group than controls while the level of miRNA-133a gene expression was lower. IL-2 mRNA and miRNA-133a gene expression in the S + TQ group was higher than in the control and OVA-sensitized groups while the level of IL-17 mRNA in the S + TQ group was lower than in the OVA-sensitized group. Pretreatment with α-hederin decreased IL-17 mRNA levels and increased miRNA-133a gene expression compared with OVA-sensitized animals. All pathological changes in pretreated groups were lower than the OVA-sensitized group. These results showed a beneficial effect of α-hederin in OVA-sensitized rats, suggesting that α-hederin affects the IL-2 and IL-17 secretion pathways, altering miRNA-133a expression. PMID:26865286

  15. Optimizing stem-loop qPCR assays through multiplexed cDNA synthesis of U6 and miRNAs

    PubMed Central

    Turner, Marie; Adhikari, Sajag; Subramanian, Senthil

    2013-01-01

    We recently reported that hairpin (or stem-loop) priming is better-suited than polyA tailing to generate cDNA for plant microRNA qPCR. One major limitation of this method is the need to perform individual cDNA synthesis reactions for the reference gene and test miRNAs. Here, we report a novel fusion primer that allows multiplexed hairpin cDNA synthesis of the most-commonly used reference gene, nucleolar small RNA U6, together with test miRNAs. We also propose the use of miR1515 as a house keeping control for tropical legumes. We show that multiplexed cDNA synthesis does not result in loss of sensitivity and reduces the amount of RNA required for miRNA gene expression assays. PMID:23673353

  16. Potential RNA Binding Proteins in Saccharomyces Cerevisiae Identified as Suppressors of Temperature-Sensitive Mutations in Npl3

    PubMed Central

    Henry, M.; Borland, C. Z.; Bossie, M.; Silver, P. A.

    1996-01-01

    The NPL3 gene of the yeast Saccharomyces cerevisiae encodes a protein with similarity to heterogeneous nuclear ribonucleoproteins (hnRNPs). Npl3p has been implicated in many nuclear-related events including RNA export, protein import, and rRNA processing. Several temperature-sensitive alleles of NPL3 have been isolated. We now report the sequence of these alleles. For one allele, npl3-1, four complementation groups of suppressors have been isolated. The cognate genes for the two recessive mutants were cloned. One of these is the previously known RNA15, which, like NPL3, also encodes a protein with similarity to the vertebrate hnRNP A/B protein family. The other suppressor corresponds to a newly defined gene we term HRP1, which also encodes a protein with similarity to the hnRNP A/B proteins of vertebrates. Mutations in HRP1 suppress all npl3 temperature-sensitive alleles but do not bypass an npl3 null allele. We show that HRP1 is essential for cell growth and that the corresponding protein is located in the nucleus. The discovery of two hnRNP homologues that can partially suppress the function of Np13p, also an RNA binding protein, will be discussed in terms of the possible roles for Npl3p in RNA metabolism. PMID:8770588

  17. Portable Multiplex Pathogen Detector

    SciTech Connect

    Visuri, S; McBride, M T; Matthews, D; Rao, R

    2002-07-15

    Tumor marker concentrations in serum provide useful information regarding clinical stage and prognosis of cancer and can thus be used for presymptomatic diagnostic purposes. Currently, detection and identification of soluble analytes in biological fluids is conducted by methods including bioassays, ELISA, PCR, DNA chip or strip tests. While these technologies are generally sensitive and specific, they are time consuming, labor intensive and cannot be multiplexed. Our goal is to develop a simple, point-of-care, portable, liquid array-based immunoassay device capable of simultaneous detection of a variety of cancer markers. Here we describe the development of assays for the detection of Serum Prostate Specific Antigen, and Ovalbumin from a single sample. The multiplexed immunoassays utilize polystyrene microbeads. The beads are imbedded with precise ratios of red and orange fluorescent dyes yielding an array of 100 beads, each with a unique spectral address (Figure 1). Each bead can be coated with capture antibodies specific for a given antigen. After antigen capture, secondary antibodies sandwich the bound antigen and are indirectly labeled by the fluorescent reporter phycoerythrin (PE). Each optically encoded and fluorescently-labeled microbead is then individually interrogated. A red laser excites the dye molecules imbedded inside the bead and classifies the bead to its unique bead set, and a green laser quantifies the assay at the bead surface. This technology has been proven to be comparable to the ELISA in terms of sensitivity and specificity. We also describe the laser-based instrumentation used to acquire fluorescent bead images Following the assay, droplets of bead suspension containing a mixture of bead classes were deposited onto filters held in place by a disposable plexiglass device and the resultant arrays viewed under the fluorescent imaging setup. Using the appropriate filter sets to extract the necessary red, orange and green fluorescence from the

  18. Graphene fluorescence switch-based cooperative amplification: a sensitive and accurate method to detection microRNA.

    PubMed

    Liu, Haiyun; Li, Lu; Wang, Qian; Duan, Lili; Tang, Bo

    2014-06-01

    MicroRNAs (miRNAs) play significant roles in a diverse range of biological progress and have been regarded as biomarkers and therapeutic targets in cancer treatment. Sensitive and accurate detection of miRNAs is crucial for better understanding their roles in cancer cells and further validating their function in clinical diagnosis. Here, we developed a stable, sensitive, and specific miRNAs detection method on the basis of cooperative amplification combining with the graphene oxide (GO) fluorescence switch-based circular exponential amplification and the multimolecules labeling of SYBR Green I (SG). First, the target miRNA is adsorbed on the surface of GO, which can protect the miRNA from enzyme digest. Next, the miRNA hybridizes with a partial hairpin probe and then acts as a primer to initiate a strand displacement reaction to form a complete duplex. Finally, under the action of nicking enzyme, universal DNA fragments are released and used as triggers to initiate next reaction cycle, constituting a new circular exponential amplification. In the proposed strategy, a small amount of target miRNA can be converted to a large number of stable DNA triggers, leading to a remarkable amplification for the target. Moreover, compared with labeling with a 1:1 stoichiometric ratio, multimolecules binding of intercalating dye SG to double-stranded DNA (dsDNA) can induce significant enhancement of fluorescence signal and further improve the detection sensitivity. The extraordinary fluorescence quenching of GO used here guarantees the high signal-to-noise ratio. Due to the protection for target miRNA by GO, the cooperative amplification, and low fluorescence background, sensitive and accurate detection of miRNAs has been achieved. The strategy proposed here will offer a new approach for reliable quantification of miRNAs in medical research and early clinical diagnostics. PMID:24823448

  19. siRNA delivered by EGFR-specific scFv sensitizes EGFR-TKI-resistant human lung cancer cells.

    PubMed

    Lu, Yuan; Liu, Li; Wang, Yuan; Li, Fakai; Zhang, Jian; Ye, Mingxiang; Zhao, Hu; Zhang, Xiang; Zhang, Mi; Zhao, Jing; Yan, Bo; Yang, Angang; Feng, Huasong; Zhang, Rui; Ren, Xinling

    2016-01-01

    The overexpression of epidermal growth factor receptor (EGFR) is closely associated with a poor outcome in non-small cell lung cancer (NSCLC), and EGFR is an ideal biomarker for the targeted therapy of NSCLC. Although patients with EGFR-activating mutations respond to EGFR tyrosine kinase inhibitors (EGFR-TKIs), they eventually acquire resistance, which typically results from a secondary EGFR mutation or the activation of other signaling pathways. Novel approaches to overcome or prevent EGFR-TKI resistance are clinically important. In this study, we developed an EGFR-scFv-arginine nonamer peptide fusion protein, s-9R, as an siRNA carrier. Here, we show that s-9R effectively and specifically delivers EGFR-siRNAs, KRAS-siRNA and MET-siRNA into NSCLC cells and silences the expression of target genes. The sensitivity of NSCLC cells to gefitinib was restored after treatment with the s-9R/siRNA complex, and the apoptosis rates of the treated cells were significantly higher than those of the control groups. Furthermore, the co-administration of s-9R/siRNA and gefitinib successfully suppressed the progression of H1975 xenograft tumors and extended the life span of tumor-bearing nude mice. Collectively, the results of this study provide not only a new scFv derivative for delivering siRNA into EGFR-overexpressing, TKI-resistant NSCLC cells but also a novel method for overcoming TKI resistance. PMID:26524539

  20. Intracavity absorption multiplexed sensor network based on dense wavelength division multiplexing filter.

    PubMed

    Zhang, Haiwei; Lu, Ying; Duan, Liangcheng; Zhao, Zhiqiang; Shi, Wei; Yao, Jianquan

    2014-10-01

    We report the system design and experimental verification of an intracavity absorption multiplexed sensor network with hollow core photonic crystal fiber (HCPCF) sensors and dense wavelength division multiplexing (DWDM) filters. Compared with fiber Bragg grating (FBG), it is easier for the DWDM to accomplish a stable output. We realize the concentration detection of three gas cells filled with acetylene. The sensitivity is up to 100 ppmV at 1536.71 nm. Voltage gradient is firstly used to optimize the intracavity sensor network enhancing the detection efficiency up to 6.5 times. To the best of our knowledge, DWDM is firstly used as a wavelength division multiplexing device to realize intracavity absorption multiplexed sensor network. It make it possible to realize high capacity intracavity sensor network via multiplexed technique. PMID:25322029

  1. Sensitive detection of melanoma metastasis using circulating microRNA expression profiles.

    PubMed

    Shiiyama, Rie; Fukushima, Satoshi; Jinnin, Masatoshi; Yamashita, Junji; Miyashita, Azusa; Nakahara, Satoshi; Kogi, Ai; Aoi, Jun; Masuguchi, Shinichi; Inoue, Yuji; Ihn, Hironobu

    2013-10-01

    Numerous studies have indicated that the serum levels of microRNAs are useful for the diagnosis or evaluation of activity in human diseases. However, determining the level of only one of the nearly 2000 microRNAs identified so far may be less significant. Accordingly, we examined the possibility that the expression pattern of multiple microRNAs in each patient may be a more reliable disease marker for melanoma, especially metastatic disease, focusing on the interaction among microRNAs. Six microRNAs (miR-9, miR-145, miR-150, miR-155, miR-203, and miR-205) were evaluated using real-time PCR in 11 patients with metastatic melanoma and in 16 patients without melanoma. The expression of the six microRNAs was significantly different between the patients with metastasis and those without it. MiR-9 and miR-205 and miR-203 and miR-205 showed significant correlations, and the combination of miR-9, miR-145, miR-150, miR-155, and miR-205 was more sensitive than when each miR was used individually to distinguish the patients with metastasis from those without it. This is the first report demonstrating the expression profiles of multiple microRNAs in melanoma patients. Clarifying the involvement of the microRNA network in the pathogenesis of melanoma may contribute to the development of new diagnostic tools and to advancing the understanding of this disease. PMID:23863473

  2. Multiplex Real-Time PCR Diagnostic of Relapsing Fevers in Africa

    PubMed Central

    Elbir, Haitham; Henry, Mireille; Diatta, Georges; Mediannikov, Oleg; Sokhna, Cheikh; Tall, Adama; Socolovschi, Cristina; Cutler, Sally J.; Bilcha, Kassahum D.; Ali, Jemal; Campelo, Dayana; Barker, Steven C.; Raoult, Didier; Drancourt, Michel

    2013-01-01

    Background In Africa, relapsing fever borreliae are neglected arthropod-borne pathogens causing mild to deadly septicemia and miscarriage. The closely related Borrelia crocidurae, Borrelia duttonii, Borrelia recurrentis and Borrelia hispanica are rarely diagnosed at the species level, hampering refined epidemiological and clinical knowledge of the relapsing fevers. It would be hugely beneficial to have simultaneous detection and identification of Borrelia to species level directly from clinical samples. Methodology/Principal Findings We designed a multiplex real-time PCR protocol targeting the 16S rRNA gene detecting all four Borrelia, the glpQ gene specifically detecting B. crocidurae, the recN gene specifically detecting B. duttonii/B. recurrentis and the recC gene specifically detecting B. hispanica. Compared to combined 16S rRNA gene and flaB gene sequencing as the gold standard, multiplex real-time PCR analyses of 171 Borrelia-positive and 101 Borrelia-negative control blood specimens yielded 100% sensitivity and specificity for B. duttonii/B. recurrentis and B. hispanica and 99% sensitivity and specificity for B. crocidurae. Conclusions/Significance The multiplex real-time PCR developed in this study is a rapid technique for both molecular detection and speciation of relapsing fever borreliae from blood in Africa. It could be incorporated in point-of-care laboratory to confirm diagnosis and provide evidence of the burden of infection attributed to different species of known or potentially novel relapsing fever borreliae. PMID:23390560

  3. Translational sensitivity of the Escherichia coli genome to fluctuating tRNA availability

    PubMed Central

    Wohlgemuth, Sibylle E.; Gorochowski, Thomas E.; Roubos, Johannes A.

    2013-01-01

    The synthesis of protein from messenger RNA during translation is a highly dynamic process that plays a key role in controlling the efficiency and fidelity of genome-wide protein expression. The availability of aminoacylated transfer RNA (tRNA) is a major factor influencing the speed of ribosomal movement, which depending on codon choices, varies considerably along a transcript. Furthermore, it has been shown experimentally that tRNA availability can vary significantly under different growth and stress conditions, offering the cell a way to adapt translational dynamics across the genome. Existing models of translation have neglected fluctuations of tRNA pools, instead assuming fixed tRNA availabilities over time. This has lead to an incomplete understanding of this process. Here, we show for the entire Escherichia coli genome how and to what extent translational speed profiles, which capture local aspects of translational elongation, respond to measured shifts in tRNA availability. We find that translational profiles across the genome are affected to differing degrees, with genes that are essential or related to fundamental processes such as translation, being more robust than those linked to regulation. Furthermore, we reveal how fluctuating tRNA availability influences profiles of specific sequences known to play a significant role in translational control of gene expression. PMID:23842674

  4. Autoregulation of Adenovirus Type 5 Early Gene Expression II. Effect of Temperature-Sensitive Early Mutations on Virus RNA Accumulation

    PubMed Central

    Carter, T. H.; Blanton, R. A.

    1978-01-01

    The kinetics of accumulation of early virus RNA in the cytoplasm of KB cells infected at 40.5°C by wild-type (WT) adenovirus type 5 and a temperature-sensitive “early” mutant, H5ts125 (ts125), were compared by hybridization of unlabeled RNA in solution to the 3H-labeled l strand of Ad5 DNA HindIII restriction endonuclease fragment A. In the presence of 1-β-d-arabinofuranosylcytosine, Al RNA accumulated in WT-infected cells for 9 h and then decreased in concentration to 6% of the 9-h concentration by 18 h. In ts125-infected cells, Al RNA accumulated for 12 h and then remained at the same concentration for at least 6 h thereafter. The concentrations of virus RNA from the four early transcription regions of the genome were measured at 15 h in cells infected at 40.5°C in the presence of 1-β-d-arabinofuranosylcytosine by: (i) ts125 and WT; (ii) two other ts early mutants, ts107 and ts149; and (iii) a revertant of ts125. The revertant and ts149, a mutant from a different complementation group than ts125, both accumulated all early virus cytoplasmic RNA species in amounts similar to, or less than, WT. However, both ts125 and ts107, independently isolated mutations in the 72,000-molecular-weight (72K) DNA-binding protein gene, accumulated cytoplasmic early RNA in excess of that found in WT infection. This pattern of RNA accumulation with the mutants and WT virus was the same in the nuclei as in the cytoplasm at 40.5°C. At 32°C, however, the abundance of nuclear virus RNA from all four early regions was the same in cells infected by either ts125 or WT. Differences in the relative abundance of nuclear RNA from the four early regions were observed in cells infected at 40.5 and 32°C, but were not dependent upon the infecting virus genotype. These results are consistent with autoregulation of early gene expression by the 72K protein and support the hypothesis that the 72K protein either decreases the rate of early virus transcription or increases the rate of virus

  5. Intercalation of quantum dots as the new signal acquisition and amplification platform for sensitive electrochemiluminescent detection of microRNA.

    PubMed

    Chen, Ying; Xiang, Yun; Yuan, Ruo; Chai, Yaqin

    2015-09-01

    Herein, we report on the development of a simple and sensitive biosensor for electrochemiluminescent (ECL) detection of microRNAs (miRNA) based on the intercalation of doxorubicin-conjugated quantum dot nanoparticles (Dox-QDs) into the DNA/RNA hybrids as the new signal acquisition and amplification platform. The thiolated DNA capture probes are self-assembled onto gold electrodes via the formation of Au-S bonds. The sensing surface is then incubated in a target miRNA-containing buffer solution to form the double-stranded duplexes. In this case, massive Dox-QDs can intercalate into the base pairs of the hybrid duplexes, resulting in amplified ECL emissions due to their reactions with the coreactant [Formula: see text] and the dissolved oxygen in the detection buffer. The increase in ECL intensity proportional to the amount of target miRNA in the testing samples serves as the quantitative basis. Different from traditional QDs-based methods such as labeling and embedding, our sensor involves the employment of the intercalation of the Dox-QDs as the signal acquisition and amplification platform. The combination of the QDs intercalation amplification with the high sensitivity of the ECL technique enables us to detect miRNA down to the low femtomolar level. Moreover, our method is also coupled with acceptable selectivity in discriminating the target miRNA and against its family members as well as other interference sequence, and can monitor miRNAs from human prostate carcinoma (22Rv1) cell lysates. PMID:26388371

  6. Silencing MRP1-4 genes by RNA interference enhances sensitivity of human hepatoma cells to chemotherapy

    PubMed Central

    Su, Zheng; Liu, Gaojie; Fang, Tingfeng; Wang, Yang; Zhang, Huayao; Yang, Shanglin; Wei, Jinxing; Lv, Zejian; Tan, Langping; Liu, Jianping

    2016-01-01

    Aim: Besides surgical treatment, systematic chemotherapy plays a crucial role in HCC treatment, especially for patients with advanced HCC. However, none of the single-drug-treatment strategies have shown significant survival benefit due to a high incidence rate of chemoresistance. This study was designed to observe the effect of small interfering of RNA (SiRNA) targeting multidrug resistance-related protein 1-4 (MRP1, MRP2, MRP3, and MRP4) in modulating drug resistance of HepG2/ADM and SMMC7721/ADM cells. Methods: HepG2/Adriamycin (ADM) and SMMC7721/ADM cell lines were developed by exposing parental cells to stepwise increasing concentrations of ADM. MTT assay was used to determine drug sensitivity and half inhibitory concentration (IC50) of drugs was calculated. Flow cytometry was employed to analyze cell cycle distribution. MRP1-4 mRNA expression levels were measured by quantitative real-time PCR (QRT-PCR). Expression of proteins was analyzed by Western blot. The growth curve was draw and the cell apoptosis was also observed. Animal experiment was used to compare the cell growth. Results: MTT assay showed that the values of IC50 and RI of HepG2/ADM and SMMC7721/ADM decreased after siRNA treatment in HepG2/ADM cells and SMMC7721/ADM cells. QRT-PCR analysis demonstrated the MRP1-4 mRNA expression decreased significantly in HepG2/ADM cells and SMMC7721/ADM cells after siRNA transfection. In addition, compared with parental cells, MRP1-4 protein expressions apparently decreased in SMMC7721/ADM and HepG2/ADM cells. Flow cytometry showed significantly elevated apoptosis rate following MRP1-4 siRNA transfection. Animal experiment suggested that silencing MRP1-4 gene in vivo inhibited tumor growth. Conclusion: Inhibition of MRP1-4 by small interfering RNA enhanced and selectively restored sensitivity of hepatoma cells to drugs. MRP1-4 siRNA might represent a new therapeutic option for HCC. PMID:27398162

  7. MicroRNA-145 Modulates Tumor Sensitivity to Radiation in Prostate Cancer.

    PubMed

    Gong, Pijun; Zhang, Tingting; He, Dalin; Hsieh, Jer-Tsong

    2015-12-01

    Radiation therapy prior to surgery has increasingly become the standard of care for locally advanced prostate cancer, however tumor radioresistance remains a major clinical problem. While restoration of microRNA-145 (miR-145) expression reduces chemoradioresistance in glioblastoma and suppress prostate cancer proliferation, migration and invasion, the role of miR-145 in response to radiation therapy for prostate cancer is still unknown. The aim of this study was to investigate the role of miR-145 in determining the tumor response to radiation treatment in prostate cancer. Human prostate cancer cells LNCAP and PC3 were transfected with miR-145 mimic. Clonogenic assay was used to determine whether overexpression of miR-145 could alter radiation response in vitro. Immunofluorescence of γ-H2AX and flow cytometric analysis of phosphorylated histone H3 were performed to investigate the potential mechanisms contributing to the enhanced radiation-induced cell killing induced by miR-145. In addition, a qPCR-based array was used to detect the possible miR-145-mediated regulated genes involved. Tumor growth delay assays and survival curves were then analyzed in an animal model to investigate whether miR-145 induced radiosensitivity in vivo. Furthermore, miR-145 expression was assessed in 30 prostate tumor tissue biopsies taken prior to neoadjuvant radiotherapy using miRNA arrays. Our current study suggested that ectopic expression of miR-145 significantly sensitized prostate cancer cells to radiation and we used γ-H2AX phosphorylation as a surrogate marker of radiotherapy response versus miR-145 expression levels. We observed significantly more foci per cell in the group treated with miR-145 and radiation. In addition, mitotic catastrophe was significantly increased in cells receiving miR-145 and radiation. The above results suggest that miR-145 appears to reduced the efficiency of the repair of radiation-induced DNA double-strand breaks in cells. A detailed examination of

  8. rRNA-based method for sensitive detection of Babesia bigemina in bovine blood.

    PubMed Central

    Reddy, G R; Dame, J B

    1992-01-01

    Three synthetic oligonucleotide probes complementary to unique regions of Babesia bigemina small-subunit rRNA were developed for detecting the parasite in bovine blood. These probes specifically detected a parasitemia of 2 x 10(-5)% by autoradiography in less than 24 h by using a 200-microliters sample of bovine blood. These probes did not bind to total RNA or genomic DNA isolated from another closely related species, Babesia bovis, or to bovine leukocyte RNA. This method detected B. bigemina infections in calves inoculated with as few as 1,000 infected erythrocytes from the second day onward for 16 days. Images PMID:1629339

  9. Delivery of chemo-sensitizing siRNAs to HER2+-breast cancer cells using RNA aptamers

    PubMed Central

    Thiel, Kristina W.; Hernandez, Luiza I.; Dassie, Justin P.; Thiel, William H.; Liu, Xiuying; Stockdale, Katie R.; Rothman, Alissa M.; Hernandez, Frank J.; McNamara, James O.; Giangrande, Paloma H.

    2012-01-01

    Human epidermal growth factor receptor 2 (HER2) expression in breast cancer is associated with an aggressive phenotype and poor prognosis, making it an appealing therapeutic target. Trastuzumab, an HER2 antibody-based inhibitor, is currently the leading targeted treatment for HER2+-breast cancers. Unfortunately, many patients inevitably develop resistance to the therapy, highlighting the need for alternative targeted therapeutic options. In this study, we used a novel, cell-based selection approach for isolating ‘cell-type specific’, ‘cell-internalizing RNA ligands (aptamers)’ capable of delivering therapeutic small interfering RNAs (siRNAs) to HER2-expressing breast cancer cells. RNA aptamers with the greatest specificity and internalization potential were covalently linked to siRNAs targeting the anti-apoptotic gene, Bcl-2. We demonstrate that, when applied to cells, the HER2 aptamer-Bcl-2 siRNA conjugates selectively internalize into HER2+-cells and silence Bcl-2 gene expression. Importantly, Bcl-2 silencing sensitizes these cells to chemotherapy (cisplatin) suggesting a potential new therapeutic approach for treating breast cancers with HER2+-status. In summary, we describe a novel cell-based selection methodology that enables the identification of cell-internalizing RNA aptamers for targeting therapeutic siRNAs to HER2-expressing breast cancer cells. The future refinement of this technology may promote the widespread use of RNA-based reagents for targeted therapeutic applications. PMID:22467215

  10. Establishment and application of a multiplex PCR for rapid and simultaneous detection of six viruses in swine.

    PubMed

    Zeng, Zhiyong; Liu, Zhijie; Wang, Weicheng; Tang, Deyuan; Liang, Haiying; Liu, Zhao

    2014-11-01

    A multiplex PCR assay was developed and evaluated subsequently for its effectiveness in simultaneously detecting mixed viral infections of swine. Specific primers were designed and used for testing the six swine viruses: three DNA viruses, including pseudorabies virus (PRV), porcine parvovirus (PPV), and porcine circovirus type 2 (PCV2); three common RNA viruses, including porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), and Japanese encephalitis virus (JEV). This technique has shown to be highly sensitive in that the minimum detection amounts of nucleic acids from PRV, PPV, PCV2, PRRSV, CSFV, and JEV were 6.6, 96, 12.9, 10.5, 51, and 46 pg, respectively. It also was effective for detecting one or multiple viruses in the specimens, such as the lungs, spleens, lymph nodes, and tonsils collected from clinically ill pigs. The multiplex PCR method can detect simultaneously not only infection of the six viruses, but also other swine DNA and RNA viruses. Given its rapidity, specificity, and sensitivity, the multiplex PCR is a useful tool for diagnosing clinically the mixed infections of swine DNA and RNA viruses. PMID:25116201

  11. Sensitive and label-free detection of miRNA-145 by triplex formation.

    PubMed

    Aviñó, Anna; Huertas, César S; Lechuga, Laura M; Eritja, Ramon

    2016-01-01

    The development of new strategies for detecting microRNAs (miRNAs) has become a crucial step in the diagnostic field. miRNA profiles depend greatly on the sample and the analytical platform employed, leading sometimes to contradictory results. In this work, we study the use of modified parallel tail-clamps to detect a miRNA sequence involved in tumor suppression by triplex formation. Thermal denaturing curves and circular dichroism (CD) measurements have been performed to confirm that parallel clamps carrying 8-aminoguanine form the most stable triplex structures with their target miRNA. The modified tail-clamps have been tested as bioreceptors in a surface plasmon resonance (SPR) biosensor for the detection of miRNA-145. The detection limit was improved 2.4 times demonstrating that a stable triplex structure is formed between target miRNA and 8-aminoguanine tail-clamp bioreceptor. This new approach is an essential step toward the label-free and reliable detection of miRNA signatures for diagnostic purposes. PMID:26603177

  12. 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

  13. Insulin regulates enzyme activity, malonyl-CoA sensitivity and mRNA abundance of hepatic carnitine palmitoyltransferase-I.

    PubMed Central

    Park, E A; Mynatt, R L; Cook, G A; Kashfi, K

    1995-01-01

    The regulation of hepatic mitochondrial carnitine palmitoyltransferase-I (CPT-I) was studied in rats during starvation and insulin-dependent diabetes and in rat H4IIE cells. The Vmax. for CPT-I in hepatic mitochondrial outer membranes isolated from starved and diabetic rats increased 2- and 3-fold respectively over fed control values with no change in Km values for substrates. Regulation of malonyl-CoA sensitivity of CPT-I in isolated mitochondrial outer membranes was indicated by an 8-fold increase in Ki during starvation and by a 50-fold increase in Ki in the diabetic state. Peroxisomal and microsomal CPT also had decreased sensitivity to inhibition by malonyl-CoA during starvation. CPT-I mRNA abundance was 7.5 times greater in livers of 48-h-starved rats and 14.6 times greater in livers of insulin-dependent diabetic rats compared with livers of fed rats. In H4IIE cells, insulin increased CPT-I sensitivity to inhibition by malonyl-CoA in 4 h, and sensitivity continued to increase up to 24 h after insulin addition. CPT-I mRNA levels in H4IIE cells were decreased by insulin after 4 h and continued to decrease so that at 24 h there was a 10-fold difference. The half-life of CPT-I mRNA was 4 h in the presence of actinomycin D or with actinomycin D plus insulin. These results suggest that insulin regulates CPT-I by inhibiting transcription of the CPT-I gene. Images Figure 2 Figure 4 PMID:7575418

  14. An electrochemical biosensor for sensitive detection of microRNA-155: combining target recycling with cascade catalysis for signal amplification.

    PubMed

    Wu, Xiaoyan; Chai, Yaqin; Zhang, Pu; Yuan, Ruo

    2015-01-14

    In this work, a new electrochemical biosensor based on catalyzed hairpin assembly target recycling and cascade electrocatalysis (cytochrome c (Cyt c) and alcohol oxidase (AOx)) for signal amplification was constructed for highly sensitive detection of microRNA (miRNA). It is worth pointing out that target recycling was achieved only based on strand displacement process without the help of nuclease. Moreover, porous TiO2 nanosphere was synthesized, which could offer more surface area for Pt nanoparticles (PtNPs) enwrapping and enhance the amount of immobilized DNA strand 1 (S1) and Cyt c accordingly. With the mimicking sandwich-type reaction, the cascade catalysis amplification strategy was carried out by AOx catalyzing ethanol to acetaldehyde with the concomitant formation of high concentration of H2O2, which was further electrocatalyzed by PtNPs and Cyt c. This newly designed biosensor provided a sensitive detection of miRNA-155 from 0.8 fM to 1 nM with a relatively low detection limit of 0.35 fM. PMID:25495913

  15. Rapid and sensitive microRNA detection with laminar flow-assisted dendritic amplification on power-free microfluidic chip.

    PubMed

    Arata, Hideyuki; Komatsu, Hiroshi; Hosokawa, Kazuo; Maeda, Mizuo

    2012-01-01

    Detection of microRNAs, small noncoding single-stranded RNAs, is one of the key topics in the new generation of cancer research because cancer in the human body can be detected or even classified by microRNA detection. This report shows rapid and sensitive microRNA detection using a power-free microfluidic device, which is driven by degassed poly(dimethylsiloxane), thus eliminating the need for an external power supply. MicroRNA is detected by sandwich hybridization, and the signal is amplified by laminar flow-assisted dendritic amplification. This method allows us to detect microRNA of specific sequences at a limit of detection of 0.5 pM from a 0.5 µL sample solution with a detection time of 20 min. Together with the advantages of self-reliance of this device, this method might contribute substantially to future point-of-care early-stage cancer diagnosis. PMID:23144864

  16. pH-Sensitive siRNA nanovector for targeted gene silencing and cytotoxic effect in cancer cells.

    PubMed

    Mok, Hyejung; Veiseh, Omid; Fang, Chen; Kievit, Forrest M; Wang, Freddy Y; Park, James O; Zhang, Miqin

    2010-12-01

    A small interfering RNA (siRNA) nanovector with dual targeting specificity and dual therapeutic effect is developed for targeted cancer imaging and therapy. The nanovector is composed of an iron oxide magnetic nanoparticle core coated with three different functional molecules: polyethyleneimine (PEI), siRNA, and chlorotoxin (CTX). The primary amine group of PEI is blocked with citraconic anhydride that is removable at acidic conditions, not only to increase its biocompatibility at physiological conditions but also to elicit a pH-sensitive cytotoxic effect in the acidic tumor microenvironment. The PEI is covalently immobilized on the nanovector via a disulfide linkage that is cleavable after cellular internalization of the nanovector. CTX as a tumor-specific targeting ligand and siRNA as a therapeutic payload are conjugated on the nanovector via a flexible and hydrophilic PEG linker for targeted gene silencing in cancer cells. With a size of ∼60 nm, the nanovector exhibits long-term stability and good magnetic property for magnetic resonance imaging. The multifunctional nanovector exhibits both significant cytotoxic and gene silencing effects at acidic pH conditions for C6 glioma cells, but not at physiological pH conditions. Our results suggest that this nanovector system could be safely used as a potential therapeutic agent for targeted treatment of glioma as well as other cancers. PMID:20722417

  17. Selective and sensitive detection of MiRNA-21 based on gold-nanorod functionalized polydiacetylene microtube waveguide.

    PubMed

    Zhu, Yu; Qiu, Dong; Yang, Guang; Wang, Mengqiao; Zhang, Qijin; Wang, Pei; Ming, Hai; Zhang, Douguo; Yu, Yue; Zou, Gang; Badugu, Ramachandram; Lakowicz, Joseph R

    2016-11-15

    Development of rapid, highly selective and sensitive miRNA detection in a complex biological environment has attracted considerable attention. Herein, we describe a novel two step method to construct gold-nanorod functionalized polydiacetylene (PDA) microtube for miRNA detection. In PDA microtube, with a one-dimensional (1D) waveguide nature, the excitation position and emission out-coupling position are far apart, thus helpful in reducing contribution of auto-fluorescence from biological sample. The use of specially designed toehold-mediated strand displacement reaction enables the reliable and selective discrimination of miRNA sequences with high sequence homology. Based on the condensing enrichment effect, the detection limit of the proposed PDA microtube system is as low as 0.01nM, and it can be applied directly to detect disease-specific miRNA targets in human serum. This PDA microtube waveguide system can be further integrated into the chip for the potential applications in minimally invasive, portable clinical diagnostic equipment. PMID:27179561

  18. Stranded Whole Transcriptome RNA-Seq for All RNA Types

    PubMed Central

    Yan, Pearlly X.; Fang, Fang; Buechlein, Aaron; Ford, James B.; Tang, Haixu; Huang, Tim H.; Burow, Matthew E.; Liu, Yunlong; Rusch, Douglas B.

    2015-01-01

    Stranded whole transcriptome RNA-Seq described in this unit captures quantitative expression data for all types of RNA including, but not limited to miRNA (microRNA), piRNA (Piwi-interacting RNA), snoRNA (small nucleolar RNA), lincRNA (large non-coding intergenic RNA), SRP RNA (signal recognition particle RNA), tRNA (transfer RNA), mtRNA (mitochondrial RNA) and mRNA (messenger RNA). The size and nature of these types of RNA are irrelevant to the approach described here. Barcoded libraries for multiplexing on the Illumina platform are generated with this approach but it can be applied to other platforms with a few modifications. PMID:25599667

  19. Rapid Multiplexed Flow Cytometric Assay for Botulinum Neurotoxin Detection Using an Automated Fluidic Microbead-Trapping Flow Cell for Enhanced Sensitivity

    SciTech Connect

    Ozanich, Richard M.; Bruckner-Lea, Cindy J.; Warner, Marvin G.; Miller, Keith D.; Antolick, Kathryn C.; Marks, James D.; Lou, Jianlong; Grate, Jay W.

    2009-07-15

    A bead-based sandwich immunoassay for botulinum neurotoxin serotype A (BoNT/A) has been developed and demonstrated using a recombinant 50 kDa fragment (BoNT/A-HC-fragment) of the BoNT/A heavy chain (BoNT/A-HC) as a structurally valid simulant. Three different anti-BoNT/A antibodies were attached to three different fluorescent dye encoded flow cytometry beads for multiplexing. The assay was conducted in two formats: a manual microcentrifuge tube format and an automated fluidic system format. Flow cytometry detection was used for both formats. The fluidic system used a novel microbead-trapping flow cell to capture antibody-coupled beads with subsequent sequential perfusion of sample, wash, dye-labeled reporter antibody, and final wash solutions. After the reaction period, the beads were collected for analysis by flow cytometry. Sandwich assays performed on the fluidic system gave median fluorescence intensity signals on the flow cytometer that were 2-4 times higher than assays performed manually in the same amount of time. Limits of detection were estimated at 1 pM (~50 pg/mL for BoNT/A-HC-fragment) for the 15 minute fluidic assay.

  20. [Application of Cationic Aluminum Phthalocyanine, a Red-Emitting Fluorescent Probe, for Sensitive Quantitative Analysis of RNA at Nanogram Level].

    PubMed

    Guo, Meng-lin; Yang, Hui-qing; Huang, Ping; Chen, Lin; Li, Dong-hui

    2016-03-01

    Tetrasubstituted trimethyl ammonium iodide aluminum phthalocyanine (TTMAAlPc), a positively charged phthalocyanine compound, is an emerging and potentially useful red-emitting fluorescence probe. The study showed that the fluorescence of TTMAAlPc could be quenched by RNA with high efficiency in weak alkaline media, and the degree of quenching has a linear relationship with RNA in a wide concentration range. The mechanism of quenching behavior of RNA on TTMAAlPc was discussed. It was attributed by the static interaction between RNA and TTMAAlPc, and the assembly of TTMAAlPc induced by RNA. Based on this new discovery, a novel method for quantitative determination of RNA at nanogram level has been established. The factors, including the pH of medium, buffer system, reaction time, reaction temperature, the usage of TTMAAlPc as well as the interferences, which affected the determination, were investigated and discussed. Under optimum conditions, the linear range of the calibration curve was 7.71-1 705.57 ng x mL(-1). The detection limit for RNA was 1.55 ng x mL(-1). This method has been applied to the analysis of practical samples with satisfied results. The constructed method is of high sensitivity and has a wide linear range, it also showed strong ability in the tolerance of foreign substances from anions, cations, surfactants and vitamins, all of which are common interferences encountered in the determination of RNA. Besides, it is the first report that the fluorescence quantum yield of TTMAAlPc has been measured at different pH by reference method in this work. The achieved data indicated that the fluorescence quantum yield of TTMAAlPc is larger than 20% and it keeps constant in a wide range of acidity, implying that TTMAAlPc is a high-quality red-emitting fluorescence probe, it has great potential for practical applications, thus is worthy of further study. This work expands the application of phthalocyanine compound in analytical sciences. PMID:27400518

  1. High Sensitivity RT-qPCR Assay of Nonlabeled siRNA in Small Blood Volume for Pharmacokinetic Studies: Application to Survivin siRNA.

    PubMed

    Yeung, Bertrand Z; Lu, Ze; Wientjes, Guillaume M; Au, Jessie L-S

    2015-11-01

    RNAi therapeutics provide an opportunity to correct faulty genes, and several RNAi have entered clinical evaluation. The existing quantification methods typically use radioactivity- or fluorescence-labeled RNAi, require large blood volumes, and/or have a limited dynamic detection range. We established a quantitative reverse transcriptase real-time polymerase chain reaction (RT-qPCR) assay to measure RNAi; the model analyte was survivin siRNA (siSurvivin). A second siRNA was used as the internal standard. The three major steps were (a) extraction of the two siRNAs from blood or water, (b) synthesis of their cDNA by poly-A extension, and (c) qPCR of cDNA. Standard curves were established. Utility of the assay was demonstrated in a pharmacokinetic study where all 12 samples for the blood concentration-time profile were obtained from a single mouse given an intravenous dose of 1 nmole siSurvivin (prepared as lipoplex with pegylated cationic liposomes). The RT-qPCR assay was sensitive (lower detection limit of 100 fM) and had a 5 × 107-fold dynamic range and low sample volume requirement (10 μL). The 16-point standard curves constructed using whole blood samples were linear (R (2) > 0.98). The intraday and interday variations for the slopes were ≤6%, although the variations for accuracy and precision at individual concentrations were substantially higher (58-145%). Standard curves prepared with water in place of blood showed similar results (<6% difference), indicating water may be used when blood is not available. The current RT-qPCR assay enabled the measurement of nonlabeled siRNA in small volume of blood samples. PMID:26286676

  2. Adult rat brain is sensitive to thyroid hormone. Regulation of RC3/neurogranin mRNA.

    PubMed Central

    Iñiguez, M A; Rodriguez-Peña, A; Ibarrola, N; Morreale de Escobar, G; Bernal, J

    1992-01-01

    The mammalian brain is considered to be poorly responsive to thyroid hormone after the so called "critical periods" of brain development, which occur in the rat before postnatal days 15-20. In a previous work (Muñoz, A., A. Rodriguez-Peña, A. Perez-Castillo, B. Ferreiro, J.G. Sutcliffe, and J. Bernal. 1991. Mol. Endocrinol. 5:273-280) we have identified one neuronal gene, RC3, whose expression is influenced by early neonatal hypothyroidism and thyroid hormone treatment. In the present work we show that adult-onset hypothyroidism leads to a reversible decrease of RC3 mRNA. Rats thyroidectomized on postnatal day 40 and killed three months later showed a decreased RC3 mRNA concentration in the cerebral cortex and striatum. The same effect was observed in animals made hypothyroid on postnatal day 32 and killed on postnatal day 52. RC3 expression was normal when hypothyroid animals were treated with T4 five days before being killed. In contrast, the mRNA encoding myelin proteolipid protein showed no changes in either experimental situation. RC3 mRNA levels were not affected by food restriction demonstrating that the effect of hypothyroidism was not related to the lack of weight gain. The control of RC3 mRNA is so far the only molecular event known to be regulated by thyroid hormone once the critical periods of brain development are over and could represent a molecular correlate for the age-independent, reversible alterations induced by hypothyroidism in the adult brain. Images PMID:1379612

  3. [Development of multiplex PCR for fast detection of Paenibacillus larvae in putrid masses and in isolated bacterial colonies].

    PubMed

    ruseinova, N V; Parvanov, P; Stanilova, S

    2013-01-01

    The present study was performed to develop a fast and sensitive multiplex polymerase chain reaction protocol for routine diagnostics of American foulbrood. A new approach for detection of Paenibacillus larvae in putrid masses was described. Forty five samples of putrid masses obtained from bee combs suspicious for American foulbrood, a reference strain Paenibacillus larvae (NBIMCC 8478), clinical isolates and 4 strains of closely related bacterial species were included in experiments. Bacterial colonies' DNA was isolated by heat and centrifugation method (standard procedure) and with prepGem commercial kit. DNA from putrid masses was isolated by standard and modified procedure. Three pairs of primers specific for 16S rRNA and one pair specific for 35 kDa metalloproteinase genes of Paenibacillus larvae were tested as single pair and in different combinations as multiplex PCR. The sensitivity of the multiplex PCR protocol for putrid masses, developed in study was 100%, versus 45.2% for the standard protocol. The developed multiplex PCR protocol could be successfully used for rapid and specific detection of Paenibacillus larvae in both putrid masses and isolated bacterial colonies. PMID:23662456

  4. p53-Dependent DNA damage response sensitive to editing-defective tRNA synthetase in zebrafish.

    PubMed

    Song, Youngzee; Shi, Yi; Carland, Tristan M; Lian, Shanshan; Sasaki, Tomoyuki; Schork, Nicholas J; Head, Steven R; Kishi, Shuji; Schimmel, Paul

    2016-07-26

    Brain and heart pathologies are caused by editing defects of transfer RNA (tRNA) synthetases, which preserve genetic code fidelity by removing incorrect amino acids misattached to tRNAs. To extend understanding of the broader impact of synthetase editing reactions on organismal homeostasis, and based on effects in bacteria ostensibly from small amounts of mistranslation of components of the replication apparatus, we investigated the sensitivity to editing of the vertebrate genome. We show here that in zebrafish embryos, transient overexpression of editing-defective valyl-tRNA synthetase (ValRS(ED)) activated DNA break-responsive H2AX and p53-responsive downstream proteins, such as cyclin-dependent kinase (CDK) inhibitor p21, which promotes cell-cycle arrest at DNA damage checkpoints, and Gadd45 and p53R2, with pivotal roles in DNA repair. In contrast, the response of these proteins to expression of ValRS(ED) was abolished in p53-deficient fish. The p53-activated downstream signaling events correlated with suppression of abnormal morphological changes caused by the editing defect and, in adults, reversed a shortened life span (followed for 2 y). Conversely, with normal editing activities, p53-deficient fish have a normal life span and few morphological changes. Whole-fish deep sequencing showed genomic mutations associated with the editing defect. We suggest that the sensitivity of p53 to expression of an editing-defective tRNA synthetase has a critical role in promoting genome integrity and organismal homeostasis. PMID:27402763

  5. Chemically modified primers for improved multiplex PCR

    PubMed Central

    Shum, Jonathan; Paul, Natasha

    2009-01-01

    Multiplexed PCR, the amplification of multiple targets in a single reaction, presents a new set of challenges that further complicate more traditional PCR set-ups. These complications include a greater probability for non-specific amplicon formation and for imbalanced amplification of different targets, each of which can compromise quantification and detection of multiple targets. Despite these difficulties, multiplex PCR is frequently used in such applications as pathogen detection, RNA quantification, mutation analysis and now, next generation DNA sequencing. Herein, we investigate the utility of primers with one or two thermolabile 4-oxo-1-pentyl phosphotriester modifications in improving multiplex PCR performance. Initial endpoint and real-time analyses reveal a decrease in off-target amplification and subsequent increase in amplicon yield. Furthermore, the use of modified primers in multiplex set-ups revealed a greater limit of detection and more uniform amplification of each target as compared to unmodified primers. Overall, the thermolabile modified primers present a novel and exciting avenue in improving multiplex PCR performance. PMID:19258004

  6. microRNA160 dictates stage-specific auxin and cytokinin sensitivities and directs soybean nodule development.

    PubMed

    Nizampatnam, Narasimha Rao; Schreier, Spencer John; Damodaran, Suresh; Adhikari, Sajag; Subramanian, Senthil

    2015-10-01

    Legume nodules result from coordinated interactions between the plant and nitrogen-fixing rhizobia. The phytohormone cytokinin promotes nodule formation, and recent findings suggest that the phytohormone auxin inhibits nodule formation. Here we show that microRNA160 (miR160) is a key signaling element that determines the auxin/cytokinin balance during nodule development in soybean (Glycine max). miR160 appears to promote auxin activity by suppressing the levels of the ARF10/16/17 family of repressor ARF transcription factors. Using quantitative PCR assays and a fluorescence miRNA sensor, we show that miR160 levels are relatively low early during nodule formation and high in mature nodules. We had previously shown that ectopic expression of miR160 in soybean roots led to a severe reduction in nodule formation, coupled with enhanced sensitivity to auxin and reduced sensitivity to cytokinin. Here we show that exogenous cytokinin restores nodule formation in miR160 over-expressing roots. Therefore, low miR160 levels early during nodule development favor cytokinin activity required for nodule formation. Suppression of miR160 levels using a short tandem target mimic (STTM160) resulted in reduced sensitivity to auxin and enhanced sensitivity to cytokinin. In contrast to miR160 over-expressing roots, STTM160 roots had increased nodule formation, but nodule maturation was significantly delayed. Exogenous auxin partially restored proper nodule formation and maturation in STTM160 roots, suggesting that high miR160 activity later during nodule development favors auxin activity and promotes nodule maturation. Therefore, miR160 dictates developmental stage-specific sensitivities to auxin and cytokinin to direct proper nodule formation and maturation in soybean. PMID:26287653

  7. Multiplex PageRank.

    PubMed

    Halu, Arda; Mondragón, Raúl J; Panzarasa, Pietro; Bianconi, Ginestra

    2013-01-01

    Many complex systems can be described as multiplex networks in which the same nodes can interact with one another in different layers, thus forming a set of interacting and co-evolving networks. Examples of such multiplex systems are social networks where people are involved in different types of relationships and interact through various forms of communication media. The ranking of nodes in multiplex networks is one of the most pressing and challenging tasks that research on complex networks is currently facing. When pairs of nodes can be connected through multiple links and in multiple layers, the ranking of nodes should necessarily reflect the importance of nodes in one layer as well as their importance in other interdependent layers. In this paper, we draw on the idea of biased random walks to define the Multiplex PageRank centrality measure in which the effects of the interplay between networks on the centrality of nodes are directly taken into account. In particular, depending on the intensity of the interaction between layers, we define the Additive, Multiplicative, Combined, and Neutral versions of Multiplex PageRank, and show how each version reflects the extent to which the importance of a node in one layer affects the importance the node can gain in another layer. We discuss these measures and apply them to an online multiplex social network. Findings indicate that taking the multiplex nature of the network into account helps uncover the emergence of rankings of nodes that differ from the rankings obtained from one single layer. Results provide support in favor of the salience of multiplex centrality measures, like Multiplex PageRank, for assessing the prominence of nodes embedded in multiple interacting networks, and for shedding a new light on structural properties that would otherwise remain undetected if each of the interacting networks were analyzed in isolation. PMID:24205186

  8. The RNA exosome affects iron response and sensitivity to oxidative stress.

    PubMed

    Tsanova, Borislava; Spatrick, Phyllis; Jacobson, Allan; van Hoof, Ambro

    2014-07-01

    RNA degradation plays important roles for maintaining temporal control and fidelity of gene expression, as well as processing of transcripts. In Saccharomyces cerevisiae the RNA exosome is a major 3'-to-5' exoribonuclease and also has an endonuclease domain of unknown function. Here we report a physiological role for the exosome in response to a stimulus. We show that inactivating the exoribonuclease active site of Rrp44 up-regulates the iron uptake regulon. This up-regulation is caused by increased levels of reactive oxygen species (ROS) in the mutant. Elevated ROS also causes hypersensitivity to H2O2, which can be reduced by the addition of iron to H2O2 stressed cells. Finally, we show that the previously characterized slow growth phenotype of rrp44-exo(-) is largely ameliorated during fermentative growth. While the molecular functions of Rrp44 and the RNA exosome have been extensively characterized, our studies characterize how this molecular function affects the physiology of the organism. PMID:24860016

  9. Luciferase mRNA Transfection of Antigen Presenting Cells Permits Sensitive Nonradioactive Measurement of Cellular and Humoral Cytotoxicity.

    PubMed

    Omokoko, Tana A; Luxemburger, Uli; Bardissi, Shaheer; Simon, Petra; Utsch, Magdalena; Breitkreuz, Andrea; Türeci, Özlem; Sahin, Ugur

    2016-01-01

    Immunotherapy is rapidly evolving as an effective treatment option for many cancers. With the emerging fields of cancer vaccines and adoptive cell transfer therapies, there is an increasing demand for high-throughput in vitro cytotoxicity assays that efficiently analyze immune effector functions. The gold standard (51)Cr-release assay is very accurate but has the major disadvantage of being radioactive. We reveal the development of a versatile and nonradioactive firefly luciferase in vitro transcribed (IVT) RNA-based assay. Demonstrating high efficiency, consistency, and excellent target cell viability, our optimized luciferase IVT RNA is used to transfect dividing and nondividing primary antigen presenting cells. Together with the long-lasting expression and minimal background, the direct measurement of intracellular luciferase activity of living cells allows for the monitoring of killing kinetics and displays paramount sensitivity. The ability to cotransfect the IVT RNA of the luciferase reporter and the antigen of interest into the antigen presenting cells and its simple read-out procedure render the assay high-throughput in nature. Results generated were comparable to the (51)Cr release and further confirmed the assay's ability to measure antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The assay's combined simplicity, practicality, and efficiency tailor it for the analysis of antigen-specific cellular and humoral effector functions during the development of novel immunotherapies. PMID:27057556

  10. Luciferase mRNA Transfection of Antigen Presenting Cells Permits Sensitive Nonradioactive Measurement of Cellular and Humoral Cytotoxicity

    PubMed Central

    Omokoko, Tana A.; Luxemburger, Uli; Bardissi, Shaheer; Simon, Petra; Utsch, Magdalena; Breitkreuz, Andrea; Türeci, Özlem; Sahin, Ugur

    2016-01-01

    Immunotherapy is rapidly evolving as an effective treatment option for many cancers. With the emerging fields of cancer vaccines and adoptive cell transfer therapies, there is an increasing demand for high-throughput in vitro cytotoxicity assays that efficiently analyze immune effector functions. The gold standard 51Cr-release assay is very accurate but has the major disadvantage of being radioactive. We reveal the development of a versatile and nonradioactive firefly luciferase in vitro transcribed (IVT) RNA-based assay. Demonstrating high efficiency, consistency, and excellent target cell viability, our optimized luciferase IVT RNA is used to transfect dividing and nondividing primary antigen presenting cells. Together with the long-lasting expression and minimal background, the direct measurement of intracellular luciferase activity of living cells allows for the monitoring of killing kinetics and displays paramount sensitivity. The ability to cotransfect the IVT RNA of the luciferase reporter and the antigen of interest into the antigen presenting cells and its simple read-out procedure render the assay high-throughput in nature. Results generated were comparable to the 51Cr release and further confirmed the assay's ability to measure antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The assay's combined simplicity, practicality, and efficiency tailor it for the analysis of antigen-specific cellular and humoral effector functions during the development of novel immunotherapies. PMID:27057556

  11. MicroRNA-320a sensitizes tamoxifen-resistant breast cancer cells to tamoxifen by targeting ARPP-19 and ERRγ.

    PubMed

    Lü, Mingrong; Ding, Keshuo; Zhang, Guofeng; Yin, Mianmian; Yao, Guidong; Tian, Hui; Lian, Jie; Liu, Lin; Liang, Meng; Zhu, Tao; Sun, Fei

    2015-01-01

    Tamoxifen represents a major adjuvant therapy to those patients with estrogen receptor-alpha positive breast cancer. However, tamoxifen resistance occurs quite often, either de novo or acquired during treatment. To investigate the role of miR-320a in the development of resistance to tamoxifen, we established tamoxifen-resistant (TamR) models by continually exposing MCF-7 or T47D breast cancer cells to tamoxifen, and identified microRNA(miRNA)-320a as a down-regulated miRNA in tamoxifen resistant cells. Re-expression of miR-320a was sufficient to sensitize TamR cells to tamoxifen by targeting cAMP-regulated phosphoprotein (ARPP-19) and estrogen-related receptor gamma (ERRγ) as well as their downstream effectors, c-Myc and Cyclin D1. Furthermore, progesterone (P4) promoted the expression of miR-320a by repressing c-Myc expression, while estrogen (E2) exerted the opposite effect. These results suggest the potential therapeutic approach for tamoxifen-resistant breast cancer by restorating miR-320a expression or depleting ARPP-19/ERRγ expression. PMID:25736597

  12. MicroRNA-320a sensitizes tamoxifen-resistant breast cancer cells to tamoxifen by targeting ARPP-19 and ERRγ*

    PubMed Central

    Lü, Mingrong; Ding, Keshuo; Zhang, Guofeng; Yin, Mianmian; Yao, Guidong; Tian, Hui; Lian, Jie; Liu, Lin; Liang, Meng; Zhu, Tao; Sun, Fei

    2015-01-01

    Tamoxifen represents a major adjuvant therapy to those patients with estrogen receptor-alpha positive breast cancer. However, tamoxifen resistance occurs quite often, either de novo or acquired during treatment. To investigate the role of miR-320a in the development of resistance to tamoxifen, we established tamoxifen-resistant (TamR) models by continually exposing MCF-7 or T47D breast cancer cells to tamoxifen, and identified microRNA(miRNA)-320a as a down-regulated miRNA in tamoxifen resistant cells. Re-expression of miR-320a was sufficient to sensitize TamR cells to tamoxifen by targeting cAMP-regulated phosphoprotein (ARPP-19) and estrogen-related receptor gamma (ERRγ) as well as their downstream effectors, c-Myc and Cyclin D1. Furthermore, progesterone (P4) promoted the expression of miR-320a by repressing c-Myc expression, while estrogen (E2) exerted the opposite effect. These results suggest the potential therapeutic approach for tamoxifen-resistant breast cancer by restorating miR-320a expression or depleting ARPP-19/ERRγ expression. PMID:25736597

  13. Multicopy suppressors of temperature-sensitive mutations of yeast mRNA capping enzyme.

    PubMed

    Schwer, B; Shuman, S

    1996-01-01

    We have isolated three Saccharomyces cerevisiae genes-CES1, CES2, and CES3-- that, when present in high copy, suppress the ts growth defect caused by mutations in the CEG1 gene encoding mRNA guanylyltransferase (capping enzyme). Molecular characterization of the capping enzyme suppressor genes reveals the following. CES2 is identical to ESP1, a gene required for proper nuclear division. We show by deletion analysis that the 1573-amino acid ESP1 polypeptide is composed of distinct functional domains. The C-terminal portion of ESP1 is essential for cell growth, but dispensable for CES2 activity. The N-terminal half of ESP1, which is sufficient for CES2 function, displays local sequence similarity to the small subunit of the vaccinia virus RNA capping enzyme. This suggests a basis for suppression by physical or functional interaction between the CES2 domain of ESP1 and the yeast guanylyltransferase. CES1 encodes a novel hydrophilic 915-amino acid protein. The amino acid sequence of CES1 is uninformative, except for its extensive similarity to another yeast gene product of unknown function. The CES1 homologue (designated CES4) is also a multicopy suppressor of capping enzyme ts mutations. Neither CES1 nor CES4 is essential for cell growth, and a double deletion mutant is viable. CES3 corresponds to BUD5, which encodes a putative guanine nucleotide exchange factor. We hypothesize that CES1, CES4, and BUD5 may impact on RNA transactions downstream of cap synthesis that are cap dependent in vivo. PMID:8836740

  14. Multiplex gas chromatography

    NASA Technical Reports Server (NTRS)

    Valentin, Jose R.

    1990-01-01

    The principles of the multiplex gas chromatography (GC) technique, which is a possible candidate for chemical analysis of planetary atmospheres, are discussed. Particular attention is given to the chemical modulators developed by present investigators for multiplex GC, namely, the thermal-desorption, thermal-decomposition, and catalytic modulators, as well as to mechanical modulators. The basic technique of multiplex GC using chemical modulators and a mechanical modulator is demonstrated. It is shown that, with the chemical modulators, only one gas stream consisting of the carrier in combination with the components is being analyzed, resulting in a simplified instrument that requires relatively few consumables. The mechanical modulator demonstrated a direct application of multiplex GC for the analysis of gases in atmosphere of Titan at very low pressures.

  15. Notch3-specific inhibition using siRNA knockdown or GSI sensitizes paclitaxel-resistant ovarian cancer cells.

    PubMed

    Kang, Haeyoun; Jeong, Ju-Yeon; Song, Ji-Ye; Kim, Tae Heon; Kim, Gwangil; Huh, Jin Hyung; Kwon, Ah-Young; Jung, Sang Geun; An, Hee Jung

    2016-07-01

    Notch signaling plays an important role in ovarian cancer chemoresistance, which is responsible for recurrence. Gamma-secretase inhibitor (GSI) is a broad-spectrum Notch inhibitor, but it has serious side effects. The efficacy of Notch3-specific inhibition in paclitaxel-resistant ovarian cancers was assessed in this study, which has not yet been evaluated relative to GSI. To analyze the effect of Notch3-specific inhibition on paclitaxel-resistant ovarian cancers, we compared cell viability, apoptosis, cell migration, angiogenesis, cell cycle, and spheroid formation after treatment with either Notch3 siRNA or GSI in paclitaxel-resistant SKpac cells and parental SKOV3 cells. Expression levels of survival, cell cycle, and apoptosis-related proteins were measured and compared between groups. Notch3 was significantly overexpressed in chemoresistant cancer tissues and cell lines relative to chemosensitive group. In paclitaxel-resistant cancer cells, Notch inhibition significantly reduced viability, migration, and angiogenesis and increased apoptosis, thereby boosting sensitivity to paclitaxel. Spheroid formation was also significantly reduced. Both Notch3 siRNA-treated cells and GSI-treated cells arrested in the G2/M phase of the cell cycle. Proteins of cell survival, cyclin D1 and cyclin D3 were reduced, whereas p21 and p27 were elevated. Both GSI and Notch3 siRNA treatment reduced expression of anti-apoptotic proteins (BCL-W, BCL2, and BCL-XL) and increased expression of pro-apoptotic proteins (Bad, Bak, Bim, Bid, and Bax). These results indicate that Notch3-specific inhibition sensitizes paclitaxel-resistant cancer cells to paclitaxel treatment, with an efficacy comparable to that of GSI. This approach would be likely to avoid the side effects of broad-spectrum GSI treatment. © 2015 Wiley Periodicals, Inc. PMID:26207830

  16. Knockdown of microRNA-127 reverses adriamycin resistance via cell cycle arrest and apoptosis sensitization in adriamycin-resistant human glioma cells

    PubMed Central

    Feng, Ren; Dong, Lei

    2015-01-01

    The aim of this study was to investigate signaling pathways for reversal of microRNA-127-mediated multi-drug resistance (MDR) in gliomas cells. Adriamycin-resistant glioma cell lines U251/adr and U87-MG/adr were established and we found that anti-microRNA-127 markedly reduced microRNA-127 expression levels in a time-dependent manner, leading to distinct inhibition of cell proliferation and increased apoptosis and the content of intracellular Rh123. Silencing of microRNA-127 significantly increased the sensitivity of U251/ADR and U87-MG/adr cells to adriamycin, compared to cells transfected with negative control siRNA. Silencing of microRNA-127 also significantly reduced the mRNA and protein expression levels of MDR1 and MRP1, which are major ATP-binding cassette (ABC) transporter linked to multi-drug resistance in cancer cells. And Runx2, p53, bcl-2 and survivin, which are important role in cell apoptosis, also markedly changed after microRNA-127 silencing. In addition, down-regulating microRNA-127 decreased the level of phosphorylated-Akt. Our data indicate that down-regulation of micorRNA-127 can trigger apoptosis and overcome drug resistance of gliomas cells. Therefore, this resistance of adriamycin in gliomas can be cancelled by silencing expression of microRNA-127. PMID:26261488

  17. Knockdown of microRNA-127 reverses adriamycin resistance via cell cycle arrest and apoptosis sensitization in adriamycin-resistant human glioma cells.

    PubMed

    Feng, Ren; Dong, Lei

    2015-01-01

    The aim of this study was to investigate signaling pathways for reversal of microRNA-127-mediated multi-drug resistance (MDR) in gliomas cells. Adriamycin-resistant glioma cell lines U251/adr and U87-MG/adr were established and we found that anti-microRNA-127 markedly reduced microRNA-127 expression levels in a time-dependent manner, leading to distinct inhibition of cell proliferation and increased apoptosis and the content of intracellular Rh123. Silencing of microRNA-127 significantly increased the sensitivity of U251/ADR and U87-MG/adr cells to adriamycin, compared to cells transfected with negative control siRNA. Silencing of microRNA-127 also significantly reduced the mRNA and protein expression levels of MDR1 and MRP1, which are major ATP-binding cassette (ABC) transporter linked to multi-drug resistance in cancer cells. And Runx2, p53, bcl-2 and survivin, which are important role in cell apoptosis, also markedly changed after microRNA-127 silencing. In addition, down-regulating microRNA-127 decreased the level of phosphorylated-Akt. Our data indicate that down-regulation of micorRNA-127 can trigger apoptosis and overcome drug resistance of gliomas cells. Therefore, this resistance of adriamycin in gliomas can be cancelled by silencing expression of microRNA-127. PMID:26261488

  18. Apollo Multiplexer operations manual

    SciTech Connect

    Miller, M.M.

    1985-04-01

    This report describes the operation of the the Apollo Multiplexer, a microprocessor based communications device designed to process data between an Apollo computer and up to four Gandalf PACXIV data switches. Details are given on overall operation, hardware, and troubleshooting. The reader should gain sufficient knowledge from this report to understand the operation of the multiplexer and effectively analyze and correct any problems that might occur.

  19. Downlink data multiplexer

    NASA Technical Reports Server (NTRS)

    Holland, S. Douglas (Inventor); Steele, Glen F. (Inventor); Romero, Denise M. (Inventor); Koudelka, Robert David (Inventor)

    2008-01-01

    A data multiplexer that accommodates both industry standard CCSDS data packets and bits streams and standard IEEE 1394 data is described. The multiplexer provides a statistical allotment of bandwidth to the channels in turn, preferably four, but expandable in increments of four up to sixteen. A microcontroller determines bandwidth requested by the plurality of channels, as well as the bandwidth available, and meters out the available bandwidth on a statistical basis employing flow control to the input channels.

  20. Multiplexed chirp waveform synthesizer

    DOEpatents

    Dudley, Peter A.; Tise, Bert L.

    2003-09-02

    A synthesizer for generating a desired chirp signal has M parallel channels, where M is an integer greater than 1, each channel including a chirp waveform synthesizer generating at an output a portion of a digital representation of the desired chirp signal; and a multiplexer for multiplexing the M outputs to create a digital representation of the desired chirp signal. Preferably, each channel receives input information that is a function of information representing the desired chirp signal.

  1. RNA interference screening identifies lenalidomide sensitizers in multiple myeloma, including RSK2

    PubMed Central

    Zhu, Yuan Xiao; Yin, Hongwei; Bruins, Laura A.; Shi, Chang-Xin; Jedlowski, Patrick; Aziz, Meraj; Sereduk, Chris; Kortuem, Klaus Martin; Schmidt, Jessica E.; Champion, Mia; Braggio, Esteban

    2015-01-01

    To identify molecular targets that modify sensitivity to lenalidomide, we measured proliferation in multiple myeloma (MM) cells transfected with 27 968 small interfering RNAs in the presence of increasing concentrations of drug and identified 63 genes that enhance activity of lenalidomide upon silencing. Ribosomal protein S6 kinase (RPS6KA3 or RSK2) was the most potent sensitizer. Other notable gene targets included 5 RAB family members, 3 potassium channel proteins, and 2 peroxisome family members. Single genes of interest included I-κ-B kinase-α (CHUK), and a phosphorylation dependent transcription factor (CREB1), which associate with RSK2 to regulate several signaling pathways. RSK2 knockdown induced cytotoxicity across a panel of MM cell lines and consistently increased sensitivity to lenalidomide. Accordingly, 3 small molecular inhibitors of RSK2 demonstrated synergy with lenalidomide cytotoxicity in MM cells even in the presence of stromal contact. Both RSK2 knockdown and small molecule inhibition downregulate interferon regulatory factor 4 and MYC, and provides an explanation for the synergy between lenalidomide and RSK2 inhibition. Interestingly, RSK2 inhibition also sensitized MM cells to bortezomib, melphalan, and dexamethasone, but did not downregulate Ikaros or influence lenalidomide-mediated downregulation of tumor necrosis factor-α or increase lenalidomide-induced IL-2 upregulation. In summary, inhibition of RSK2 may prove a broadly useful adjunct to MM therapy. PMID:25395420

  2. Coevolution and Correlated Multiplexity in Multiplex Networks

    NASA Astrophysics Data System (ADS)

    Kim, Jung Yeol; Goh, K.-I.

    2013-08-01

    Distinct channels of interaction in a complex networked system define network layers, which coexist and cooperate for the system’s function. Towards understanding such multiplex systems, we propose a modeling framework based on coevolution of network layers, with a class of minimalistic growing network models as working examples. We examine how the entangled growth of coevolving layers can shape the network structure and show analytically and numerically that the coevolution can induce strong degree correlations across layers, as well as modulate degree distributions. We further show that such a coevolution-induced correlated multiplexity can alter the system’s response to the dynamical process, exemplified by the suppressed susceptibility to a social cascade process.

  3. New sensitive one-step real-time duplex PCR method for group A and B HIV-2 RNA load.

    PubMed

    Avettand-Fenoel, Véronique; Damond, Florence; Gueudin, Marie; Matheron, Sophie; Mélard, Adeline; Collin, Gilles; Descamps, Diane; Chaix, Marie-Laure; Rouzioux, Christine; Plantier, Jean-Christophe

    2014-08-01

    The Agence Nationale de Recherche sur le Sida et les hépatites virales (ANRS) previously developed a widely used method for HIV-1 RNA quantification (Biocentric). Here, we report the development of a new specific and sensitive method for HIV-2 RNA quantification, based on an adaptation of the existing HIV-1 protocol. The new test is based on TaqMan one-step reverse transcription-quantitative PCR (qRT-PCR) targeting two conserved consensus regions of HIV-2 (long terminal repeat [LTR] and gag). Analytic performances were determined in three laboratories. Clinical performances were evaluated on 100 plasma samples from HIV-2-infected patients (groups A, B, and H) by comparison with the assay currently used for the ANRS HIV-2 cohort. The specificity was 100%. Sensitivity was 50 copies/ml (cp/ml) and was optimized to 10 cp/ml. The within-run coefficients of variation in the three laboratories varied from 0.54% to 1.61% at 4 log10 copies/ml and from 7.24% to 14.32% at 2 log10 cp/ml. The between-run coefficients of variation varied from 2.28% to 6.43%. Of the 39 clinical samples below 2 log10 in the current assay, the new test improved the detection or quantification of 17 samples, including eight group B samples. For quantifiable samples, similar loads were obtained with the two assays for group A samples. The median difference between the two assays for group B samples was +0.18 but with greater heterogeneity than for group A. The HIV-2 group H sample had similar results with the two assays. This new assay is highly sensitive and accurately quantifies the most prevalent HIV-2 groups. This test will be useful for monitoring low viral loads in HIV-2-infected patients. PMID:24920771

  4. Compact spatial multiplexers for mode division multiplexing.

    PubMed

    Chen, Haoshuo; van Uden, Roy; Okonkwo, Chigo; Koonen, Ton

    2014-12-29

    Spatial multiplexer (SMUX) for mode division multiplexing (MDM) has evolved from mode-selective excitation, multiple-spot and photonic-lantern based solutions in order to minimize both mode-dependent loss (MDL) and coupler insertion loss (CIL). This paper discusses the implementation of all the three solutions by compact components in a small footprint. Moreover, the compact SMUX can be manufactured in mass production and packaged to assure high reliability. First, push-pull scheme and center launch based SMUXes are demonstrated on two mostly-popular photonic integration platforms: Silicon-on-insulator (SOI) and Indium Phosphide (InP) for selectively exciting LP01 and LP11 modes. 2-dimensional (2D) top-coupling by using vertical emitters is explored to provide a coupling interface between a few-mode fiber (FMF) and the photonic integrated SMUX. SOI-based grating couplers and InP-based 45° vertical mirrors are proposed and researched as vertical emitters in each platform. Second, a 3-spot SMUX is realized on an InP-based circuit through employing 45° vertical mirrors. Third, as a newly-emerging photonic integration platform, laser-inscribed 3D waveguide (3DW) technology is applied for a fully-packaged dual-channel 6-mode SMUX including two 6-core photonic lantern structures as mode multiplexer and demultiplexer, respectively. PMID:25607130

  5. An MRPS12 mutation modifies aminoglycoside sensitivity caused by 12S rRNA mutations

    PubMed Central

    Emperador, Sonia; Pacheu-Grau, David; Bayona-Bafaluy, M. Pilar; Garrido-Pérez, Nuria; Martín-Navarro, Antonio; López-Pérez, Manuel J.; Montoya, Julio; Ruiz-Pesini, Eduardo

    2015-01-01

    Several homoplasmic pathologic mutations in mitochondrial DNA, such as those causing Leber hereditary optic neuropathy or non-syndromic hearing loss, show incomplete penetrance. Therefore, other elements must modify their pathogenicity. Discovery of these modifying factors is not an easy task because in multifactorial diseases conventional genetic approaches may not always be informative. Here, we have taken an evolutionary approach to unmask putative modifying factors for a particular homoplasmic pathologic mutation causing aminoglycoside-induced and non-syndromic hearing loss, the m.1494C>T transition in the mitochondrial DNA. The mutation is located in the decoding site of the mitochondrial ribosomal RNA. We first looked at mammalian species that had fixed the human pathologic mutation. These mutations are called compensated pathogenic deviations because an organism carrying one must also have another that suppresses the deleterious effect of the first. We found that species from the primate family Cercopithecidae (old world monkeys) harbor the m.1494T allele even if their auditory function is normal. In humans the m.1494T allele increases the susceptibility to aminoglycosides. However, in primary fibroblasts from a Cercopithecidae species, aminoglycosides do not impair cell growth, respiratory complex IV activity and quantity or the mitochondrial protein synthesis. Interestingly, this species also carries a fixed mutation in the mitochondrial ribosomal protein S12. We show that the expression of this variant in a human m.1494T cell line reduces its susceptibility to aminoglycosides. Because several mutations in this human protein have been described, they may possibly explain the absence of pathologic phenotype in some pedigree members with the most frequent pathologic mutations in mitochondrial ribosomal RNA. PMID:25642242

  6. MicroRNA-224 promotes the sensitivity of osteosarcoma cells to cisplatin by targeting Rac1.

    PubMed

    Geng, Shuo; Gu, Lina; Ju, Fang; Zhang, Hepeng; Wang, Yiwen; Tang, Han; Bi, ZhengGang; Yang, Chenglin

    2016-09-01

    Osteosarcoma is the most common primary bone tumour in children and adolescents. Accumulating evidence has shown that microRNAs (miRNAs) participate in the development of almost all types of cancer. Here, we investigated the role of miR-224 in the development and progression of osteosarcoma. We demonstrated that miR-224 was down-regulated in osteosarcoma cell lines and tissues. Lower miR-224 levels were correlated with shorter survivalin osteosarcoma patients. Furthermore, overexpression of miR-224 suppressed osteosarcoma cell proliferation, migration and invasion and contributed to the increased sensitivity of MG-63 cells to cisplatin. We identified Rac1 as a direct target gene of miR-224 in osteosarcoma. Rac1 expression was up-regulated in the osteosarcoma cell lines and tissues, and there was an inverse correlation between Rac1 and miR-224 expression in osteosarcoma tissues. Furthermore, rescuing Rac1 expression decreased the sensitivity of miR-224-overexpressing MG-63 cells to cisplatin. We also demonstrated that ectopic expression of Rac1 promoted the proliferation, migration and invasion of miR-224-overexpressing MG-63 cells. These data suggest that miR-224 plays a tumour suppressor role in the development of osteosarcoma and is related to the sensitivity of osteosarcoma to cisplatin. PMID:27222381

  7. Flexible wavelength de-multiplexer for elastic optical networking.

    PubMed

    Zhou, Rui; Gutierrez Pascual, M Deseada; Anandarajah, Prince M; Shao, Tong; Smyth, Frank; Barry, Liam P

    2016-05-15

    We report an injection locked flexible wavelength de-multiplexer (de-mux) that shows 24-h frequency stability of 1 kHz for optical comb-based elastic optical networking applications. We demonstrate 50 GHz, 87.5 GHz equal spacing and 6.25G-25G-50 GHz, 75G-50G-100 GHz unequal spacing for the de-multiplexer outputs. We also implement an unequally spaced (75G-50G-100 GHz), mixed symbol rate (12.5 GBaud and 40 GBaud) and modulation format (polarization division multiplexed quadrature phase shift keying and on-off keying) wavelength division multiplexed transmission system using the de-multiplexer outputs. The results show 0.6 dB receiver sensitivity penalty, at 7% hard decision forward error correction coding limit, of the 100 km transmitted de-mux outputs when compared to comb source seeding laser back-to-back. PMID:27176972

  8. Specific temperature-induced perturbations of secondary mRNA structures are associated with the cold-adapted temperature-sensitive phenotype of influenza A virus

    PubMed Central

    Chursov, Andrey; Kopetzky, Sebastian J.; Leshchiner, Ignaty; Kondofersky, Ivan; Theis, Fabian J.; Frishman, Dmitrij; Shneider, Alexander

    2012-01-01

    For decades, cold-adapted, temperature-sensitive (ca/ts) strains of influenza A virus have been used as live attenuated vaccines. Due to their great public health importance it is crucial to understand the molecular mechanism(s) of cold adaptation and temperature sensitivity that are currently unknown. For instance, secondary RNA structures play important roles in influenza biology. Thus, we hypothesized that a relatively minor change in temperature (32–39°C) can lead to perturbations in influenza RNA structures and, that these structural perturbations may be different for mRNAs of the wild type (wt) and ca/ts strains. To test this hypothesis, we developed a novel in silico method that enables assessing whether two related RNA molecules would undergo (dis)similar structural perturbations upon temperature change. The proposed method allows identifying those areas within an RNA chain where dissimilarities of RNA secondary structures at two different temperatures are particularly pronounced, without knowing particular RNA shapes at either temperature. We identified such areas in the NS2, PA, PB2 and NP mRNAs. However, these areas are not identical for the wt and ca/ts mutants. Differences in temperature-induced structural changes of wt and ca/ts mRNA structures may constitute a yet unappreciated molecular mechanism of the cold adaptation/temperature sensitivity phenomena. PMID:22995831

  9. A novel multiplex PCR coupled with Luminex assay for the simultaneous detection of Cryptosporidium spp., Cryptosporidium parvum and Giardia duodenalis.

    PubMed

    Li, Wei; Zhang, Nan; Gong, Pengtao; Cao, Lili; Li, Jianhua; Su, Libo; Li, Shuhong; Diao, Yumei; Wu, Kang; Li, He; Zhang, Xichen

    2010-10-11

    Cryptosporidium parvum and Giardia duodenalis are the most frequently identified enteric parasites associated with diarrhea-causing disease outbreaks, and many non-parvum species of Cryptosporidium also can replicate and cause illness in mammals including humans. In this study, we describe a novel multiplex PCR coupled with Luminex assay for the identification of Cryptosporidium spp., C. parvum and G. duodenalis in a rapid manner. The multiplex PCR for the simultaneous detection of Cryptosporidium and Giardia was developed using three pairs of biotinylated primers which amplify 424, 223 and 267 bp products from the U1 small nuclear ribonucleoprotein (U1 snr) gene, 18S rRNA gene of Cryptosporidium and the beta-giardin gene of Giardia, respectively. The genus and species-specific capture probes linked to carboxylated Luminex microspheres hybridized to the multiplex PCR amplicons to enhance sensitivity and specificity. The conditions of multiplex PCR and Luminex hybridization reaction were optimized to enable the minimum detection limits of 5x10(-6), 5x10(-6), and 5x10(-6) ng DNAs (corresponding approximately to 0.1 oocyst/cyst). The Luminex approach proved to be 100% specific and accurate by testing a total of 240 fecal samples compared with microscopic examination of fecal smears and further modified acid-fast staining or iodine-staining observation. The established assay offers the potential for rapid detection of Cryptosporidium spp., C. parvum and G. duodenalis in fecal and environmental samples. PMID:20594647

  10. FGFR1 mRNA and Protein Expression, not Gene Copy Number, Predict FGFR TKI Sensitivity Across All Lung Cancer Histologies

    PubMed Central

    Wynes, Murry W.; Hinz, Trista K.; Gao, Dexiang; Martini, Michael; Marek, Lindsay A.; Ware, Kathryn E.; Edwards, Michael G.; Böhm, Diana; Perner, Sven; Helfrich, Barbara A.; Dziadziuszko, Rafal; Jassem, Jacek; Wojtylak, Szymon; Sejda, Aleksandra; Gozgit, Joseph M.; Bunn, Paul A.; Camidge, D. Ross; Tan, Aik-Choon; Hirsch, Fred R.; Heasley, Lynn E.

    2014-01-01

    Purpose FGFR1 gene copy number (GCN) is being evaluated as a biomarker for FGFR tyrosine kinase inhibitor (TKI) response in squamous-cell lung cancers (SCC). The exclusive use of FGFR1 GCN for predicting FGFR TKI sensitivity assumes increased GCN is the only mechanism for biologically-relevant increases in FGFR1 signaling. Herein, we tested whether FGFR1 mRNA and protein expression may serve as better biomarkers of FGFR TKI sensitivity in lung cancer. Experimental Design Histologically diverse lung cancer cell lines were submitted to assays for ponatinib sensitivity, a potent FGFR TKI. A tissue microarray comprised of resected lung tumors was submitted to FGFR1 GCN and mRNA analyses and the results were validated with TCGA lung cancer data. Results 14/58 cell lines exhibited ponatinib sensitivity (IC50 values ≤ 50 nM) that correlated with FGFR1 mRNA and protein expression, but not with FGFR1 GCN or histology. Moreover, ponatinib sensitivity associated with mRNA expression of the ligands, FGF2 and FGF9. In resected tumors, 22% of adenocarcinomas and 28% of SCCs expressed high FGFR1 mRNA. Importantly, only 46% of SCCs with increased FGFR1 GCN expressed high mRNA. Lung cancer TCGA data validated these findings and unveiled overlap of FGFR1 mRNA positivity with KRAS and PIK3CA mutations. Conclusions FGFR1 dependency is frequent across various lung cancer histologies and FGFR1 mRNA may serve as a better biomarker of FGFR TKI response in lung cancer than FGFR1 GCN. The study provides important and timely insight into clinical testing of FGFR TKIs in lung cancer and other solid tumor types. PMID:24771645

  11. Effect of Alpha-Hederin, the active constituent of Nigella sativa, on miRNA-126, IL-13 mRNA levels and inflammation of lungs in ovalbumin-sensitized male rats

    PubMed Central

    Fallahi, Maryam; Keyhanmanesh, Rana; Khamaneh, Amir Mahdi; Ebrahimi Saadatlou, Mohammad Ali; Saadat, Saeideh; Ebrahimi, Hadi

    2016-01-01

    Objective: In previous studies the therapeutic effects of Nigella sativa have been demonstrated on asthmatic animals. In the present study, the preventive effect of single dose of alpha-hederin, its active constituent, has been evaluated on lung inflammation and some inflammatory mediators in lungs of ovalbumin sensitized rat in order to elicit its mechanism. Materials and Methods: Forty rats were randomly grouped in 4 groups; control (C), sensitized (S), sensitized pretreated groups with thymoquinone (3 mg/kg i.p., S+TQ) and alpha-hederin (0.02 mg/kg i.p., S+AH). Levels of IL-13 mRNA and miRNA-126 in lung tissue and its pathological changes in each group were assessed. Results: Elevated levels of miRNA-126, IL-13 mRNA and pathological changes were observed in the sensitized group compared to the control group (p<0.001 to p<0.05). All of these factors were significantly reduced in S+TQ and S+AH groups in comparison to S group (p<0.001 to p<0.05). Although alpha-hederin decreased the levels of miRNA-126, IL-13 mRNA and pathological changes in comparison with thymoquinone, the results were statistically not significant. Conclusion: The results suggested that alpha-hederin had preventive effect on sensitized rats like thymoquinone. It may intervene in miRNA-126 expression, which consequently could interfere with IL-13 secretion pathway leading to a reduction in inflammatory responses. PMID:27247924

  12. The intranuclear mobility of messenger RNA binding proteins is ATP dependent and temperature sensitive

    PubMed Central

    Calapez, Alexandre; Pereira, Henrique M.; Calado, Angelo; Braga, José; Rino, José; Carvalho, Célia; Tavanez, João Paulo; Wahle, Elmar; Rosa, Agostinho C.; Carmo-Fonseca, Maria

    2002-01-01

    fAter being released from transcription sites, messenger ribonucleoprotein particles (mRNPs) must reach the nuclear pore complexes in order to be translocated to the cytoplasm. Whether the intranuclear movement of mRNPs results largely from Brownian motion or involves molecular motors remains unknown. Here we have used quantitative photobleaching techniques to monitor the intranuclear mobility of protein components of mRNPs tagged with GFP. The results show that the diffusion coefficients of the poly(A)-binding protein II (PABP2) and the export factor TAP are significantly reduced when these proteins are bound to mRNP complexes, as compared with nonbound proteins. The data further show that the mobility of wild-type PABP2 and TAP, but not of a point mutant variant of PABP2 that fails to bind to RNA, is significantly reduced when cells are ATP depleted or incubated at 22°C. Energy depletion has only minor effects on the intranuclear mobility of a 2,000-kD dextran (which corresponds approximately in size to 40S mRNP particles), suggesting that the reduced mobility of PABP2 and TAP is not caused by a general alteration of the nuclear environment. Taken together, the data suggest that the mobility of mRNPs in the living cell nucleus involves a combination of passive diffusion and ATP-dependent processes. PMID:12473688

  13. MicroRNA-26a regulates insulin sensitivity and metabolism of glucose and lipids

    PubMed Central

    Fu, Xianghui; Dong, Bingning; Tian, Yan; Lefebvre, Philippe; Meng, Zhipeng; Wang, Xichun; Pattou, François; Han, Weidong; Wang, Xiaoqiong; Lou, Fang; Jove, Richard; Staels, Bart; Moore, David D.; Huang, Wendong

    2015-01-01

    Type 2 diabetes (T2D) is characterized by insulin resistance and increased hepatic glucose production, yet the molecular mechanisms underlying these abnormalities are poorly understood. MicroRNAs (miRs) are a class of small, noncoding RNAs that have been implicated in the regulation of human diseases, including T2D. miR-26a is known to play a critical role in tumorigenesis; however, its function in cellular metabolism remains unknown. Here, we determined that miR-26a regulates insulin signaling and metabolism of glucose and lipids. Compared with lean individuals, overweight humans had decreased expression of miR-26a in the liver. Moreover, miR-26 was downregulated in 2 obese mouse models compared with control animals. Global or liver-specific overexpression of miR-26a in mice fed a high-fat diet improved insulin sensitivity, decreased hepatic glucose production, and decreased fatty acid synthesis, thereby preventing obesity-induced metabolic complications. Conversely, silencing of endogenous miR-26a in conventional diet–fed mice impaired insulin sensitivity, enhanced glucose production, and increased fatty acid synthesis. miR-26a targeted several key regulators of hepatic metabolism and insulin signaling. These findings reveal miR-26a as a regulator of liver metabolism and suggest miR-26a should be further explored as a potential target for the treatment of T2D. PMID:25961460

  14. Silver Nanoclusters for High-Efficiency Quenching of CdS Nanocrystal Electrochemiluminescence and Sensitive Detection of microRNA.

    PubMed

    Zhang, Yan-Yan; Feng, Qiu-Mei; Xu, Jing-Juan; Chen, Hong-Yuan

    2015-12-01

    In this work, oligonucleotide-encapusulated silver nanoclusters were applied in the electrochemiluminescence (ECL) system of CdS nanocrystals (NCs)/ K2S2O8 based on dual ECL quenching effects. We found that the ECL emission of CdS NCs matched well with the absorption band of oligonucleotide encapsulated Ag nanoclusters, which could act as the energy acceptor of CdS NCs ECL so as to lead to an effective ECL resonance energy transfer (RET). On the other hand, the Ag nanoclusters could also catalyze electrochemical reduction of K2S2O8, resulting in increased consumption of ECL coreactant near the working electrode and decreased ECL intensity from CdS NCs. On the basis of the dual ECL quenching effects, a sensitive ECL biosensor for detection of microRNA was successfully achieved with a wide linear range from 10 fM to 100 pM. PMID:26561442

  15. Multiplexed Biosensors for Mycotoxins.

    PubMed

    Maragos, Chris M

    2016-07-01

    Significant progress has been made in the development of biosensors that can be used to detect low-MW toxins produced by fungi (mycotoxins). The number of formats that have been investigated is impressive and is an indication of the importance attached to finding easy-to-use, accurate, and rapid methods for detecting these toxins in commodities and foods. This review explores the details of multiplexed biosensors based on many formats, including multiplexed immunoassays, suspension arrays, membrane-based devices (flow-through and immunochromatographic), and planar microarrays. Each assay format has its own strengths and areas that need improvement. Certain formats, such as multiplexed immunochromatographic devices, are well developed and relatively easy to use, and in some cases, commercial products are being sold. Others, such as the suspension arrays and microarrays, are laboratory-based assays that, although more complicated, are also more amenable to a larger scale of multiplexing. The diversity of such efforts and the multitude of formats under investigation suggest that multiple solutions will be found to satisfy the need for multiplexed toxin detection. PMID:27455928

  16. Inhibition of microRNA-155 sensitizes lung cancer cells to irradiation via suppression of HK2-modulated glucose metabolism.

    PubMed

    Lv, Xin; Yao, Li; Zhang, Jianli; Han, Ping; Li, Cuiyun

    2016-08-01

    MicroRNAs (miRNAs) are small non-coding regulatory RNAs, which are involved in the post-transcriptional regulation of gene expression. miRNA (miR)-155, which has previously been reported to be overexpressed in lung cancer, is correlated with poor patient prognosis. The present study aimed to investigate the effects of miR‑155 on the radiosensitivity of human non‑small cell lung cancer (NSCLC) cells. To explore the roles of miRNAs in the regulation of irradiation sensitivity of human lung cancer cells, the expressions of miR‑155 in response to irradiation, have been studied by RT‑qPCR, and the putative direct target of miR‑155 was identified by western blot and luciferase assays. The results of the present study revealed that the expression of miR‑155 was induced by irradiation, thus suggesting a positive correlation between miR‑155 and radiosensitivity. Furthermore, overexpression of miR‑155 rendered lung cancer cells resistant to irradiation. In addition, hexokinase 2 (HK2) was identified as an indirect target of miR‑155; exogenous overexpression of miR‑155 upregulated the expression of HK2, whereas inhibition of miR‑155 by antisense miRNA suppressed HK2 expression. In addition, HK2‑modulated glucose metabolism was significantly upregulated by overexpression of miR‑155. Notably, inhibition of miR‑155 sensitized lung cancer cells to irradiation via suppression of glucose metabolism. In conclusion, the present study reported a novel function for miR‑155 in the regulation of NSCLC cell radiosensitivity, thus suggesting that miR‑155 may be considered a therapeutic target for the development of anticancer drugs. PMID:27315591

  17. Microwave multiplex readout for superconducting sensors

    NASA Astrophysics Data System (ADS)

    Ferri, E.; Becker, D.; Bennett, D.; Faverzani, M.; Fowler, J.; Gard, J.; Giachero, A.; Hays-Wehle, J.; Hilton, G.; Maino, M.; Mates, J.; Puiu, A.; Nucciotti, A.; Reintsema, C.; Schmidt, D.; Swetz, D.; Ullom, J.; Vale, L.

    2016-07-01

    The absolute neutrino mass scale is still an outstanding challenge in both particle physics and cosmology. The calorimetric measurement of the energy released in a nuclear beta decay is a powerful tool to determine the effective electron-neutrino mass. In the last years, the progress on low temperature detector technologies has allowed to design large scale experiments aiming at pushing down the sensitivity on the neutrino mass below 1 eV. Even with outstanding performances in both energy (~ eV on keV) and time resolution (~ 1 μs) on the single channel, a large number of detectors working in parallel is required to reach a sub-eV sensitivity. Microwave frequency domain readout is the best available technique to readout large array of low temperature detectors, such as Transition Edge Sensors (TESs) or Microwave Kinetic Inductance Detectors (MKIDs). In this way a multiplex factor of the order of thousands can be reached, limited only by the bandwidth of the available commercial fast digitizers. This microwave multiplexing system will be used to readout the HOLMES detectors, an array of 1000 microcalorimeters based on TES sensors in which the 163Ho will be implanted. HOLMES is a new experiment for measuring the electron neutrino mass by means of the electron capture (EC) decay of 163Ho. We present here the microwave frequency multiplex which will be used in the HOLMES experiment and the microwave frequency multiplex used to readout the MKID detectors developed in Milan as well.

  18. Micro RNA 100 sensitizes luminal A breast cancer cells to paclitaxel treatment in part by targeting mTOR

    PubMed Central

    He, Yuan; Fu, Xing; Fu, Liya; Zhu, Zhengmao; Fu, Li; Dong, Jin-Tang

    2016-01-01

    Luminal A breast cancer usually responds to hormonal therapies but does not benefit from chemotherapies, including microtubule-targeted paclitaxel. MicroRNAs could play a role in mediating this differential response. In this study, we examined the role of micro RNA 100 (miR-100) in the sensitivity of breast cancer to paclitaxel treatment. We found that while miR-100 was downregulated in both human breast cancer primary tumors and cell lines, the degree of downregulation was greater in the luminal A subtype than in other subtypes. The IC50 of paclitaxel was much higher in luminal A than in basal-like breast cancer cell lines. Ectopic miR-100 expression in the MCF-7 luminal A cell line enhanced the effect of paclitaxel on cell cycle arrest, multinucleation, and apoptosis, while knockdown of miR-100 in the MDA-MB-231 basal-like line compromised these effects. Similarly, overexpression of miR-100 enhanced the effects of paclitaxel on tumorigenesis in MCF-7 cells. Rapamycin-mediated inhibition of the mammalian target of rapamycin (mTOR), a target of miR-100, also sensitized MCF-7 cells to paclitaxel. Gene set enrichment analysis showed that genes that are part of the known paclitaxel-sensitive signature had a significant expression correlation with miR-100 in breast cancer samples. In addition, patients with lower levels of miR-100 expression had worse overall survival. These results suggest that miR-100 plays a causal role in determining the sensitivity of breast cancers to paclitaxel treatment. PMID:26744318

  19. Isolation and characterization of temperature-sensitive mutations in RPA190, the gene encoding the largest subunit of RNA polymerase I from Saccharomyces cerevisiae.

    PubMed Central

    Wittekind, M; Dodd, J; Vu, L; Kolb, J M; Buhler, J M; Sentenac, A; Nomura, M

    1988-01-01

    The isolation and characterization of temperature-sensitive mutations in RNA polymerase I from Saccharomyces cerevisiae are described. A plasmid carrying RPA190, the gene encoding the largest subunit of the enzyme, was subjected to in vitro mutagenesis with hydroxylamine. Using a plasmid shuffle screening system, five different plasmids were isolated which conferred a temperature-sensitive phenotype in haploid yeast strains carrying the disrupted chromosomal RPA190 gene. These temperature-sensitive alleles were transferred to the chromosomal RPA190 locus for mapping and physiology experiments. Accumulation of RNA was found to be defective in all mutant strains at the nonpermissive temperature. In addition, analysis of pulse-labeled RNA from two mutant strains at 37 degrees C showed that the transcription of rRNA genes was decreased, while that of 5S RNA was relatively unaffected. RNA polymerase I was partially purified from several of the mutant strains grown at the nonpermissive temperature and was shown to be deficient when assayed in vitro. Fine-structure mapping and sequencing of the mutant alleles demonstrated that all five mutations were unique. The rpa190-1 and rpa190-5 mutations are tightly clustered in region I (S.S. Broyles and B. Moss, Proc. Natl. Acad. Sci. USA 83:3141-3145, 1986), the putative zinc-binding region that is common to all eucaryotic RNA polymerase large subunits. The rpa190-3 mutation is located between regions III and IV, and a strain carrying it behaves as a mutant that is defective in the synthesis of the enzyme. This mutation lies within a previously unidentified segment of highly conserved amino acid sequence homology that is shared among the largest subunits of eucaryotic nuclear RNA polymerases. Another temperature-sensitive mutation, rpa190-2, creates a UGA nonsense codon. Images PMID:3054507

  20. Matrix metalloproteinase 2-sensitive multifunctional polymeric micelles for tumor-specific co-delivery of siRNA and hydrophobic drugs.

    PubMed

    Zhu, Lin; Perche, Federico; Wang, Tao; Torchilin, Vladimir P

    2014-04-01

    Co-delivery of hydrophilic siRNA and hydrophobic drugs is one of the major challenges for nanomaterial-based medicine. Here, we present a simple but multifunctional micellar platform constructed by a matrix metalloproteinase 2 (MMP2)-sensitive copolymer (PEG-pp-PEI-PE) via self-assembly for tumor-targeted siRNA and drug co-delivery. The micellar nanocarrier possesses several key features for siRNA and drug delivery, including (i) excellent stability; (ii) efficient siRNA condensation by PEI; (iii) hydrophobic drug solubilization in the lipid "core"; (iv) passive tumor targeting via the enhanced permeability and retention (EPR) effect; (v) tumor targeting triggered by the up-regulated tumoral MMP2; and (vi) enhanced cell internalization after MMP2-activated exposure of the previously hidden PEI. These cooperative functions ensure the improved tumor targetability, enhanced tumor cell internalization, and synergistic antitumor activity of co-loaded siRNA and drug. PMID:24529391

  1. A multiplexed quantum memory.

    PubMed

    Lan, S-Y; Radnaev, A G; Collins, O A; Matsukevich, D N; Kennedy, T A; Kuzmich, A

    2009-08-01

    A quantum repeater is a system for long-distance quantum communication that employs quantum memory elements to mitigate optical fiber transmission losses. The multiplexed quantum memory (O. A. Collins, S. D. Jenkins, A. Kuzmich, and T. A. B. Kennedy, Phys. Rev. Lett. 98, 060502 (2007)) has been shown theoretically to reduce quantum memory time requirements. We present an initial implementation of a multiplexed quantum memory element in a cold rubidium gas. We show that it is possible to create atomic excitations in arbitrary memory element pairs and demonstrate the violation of Bell's inequality for light fields generated during the write and read processes. PMID:19654771

  2. Acid-Sensitive Sheddable PEGylated PLGA Nanoparticles Increase the Delivery of TNF-α siRNA in Chronic Inflammation Sites.

    PubMed

    Aldayel, Abdulaziz M; Naguib, Youssef W; O'Mary, Hannah L; Li, Xu; Niu, Mengmeng; Ruwona, Tinashe B; Cui, Zhengrong

    2016-01-01

    There has been growing interest in utilizing small interfering RNA (siRNA) specific to pro-inflammatory cytokines, such as tumor necrosis factor-α ( TNF-α), in chronic inflammation therapy. However, delivery systems that can increase the distribution of the siRNA in chronic inflammation sites after intravenous administration are needed. Herein we report that innovative functionalization of the surface of siRNA-incorporated poly (lactic-co-glycolic) acid (PLGA) nanoparticles significantly increases the delivery of the siRNA in the chronic inflammation sites in a mouse model. The TNF-α siRNA incorporated PLGA nanoparticles were prepared by the standard double emulsion method, but using stearoyl-hydrazone-polyethylene glycol 2000, a unique acid-sensitive surface active agent, as the emulsifying agent, which renders (i) the nanoparticles PEGylated and (ii) the PEGylation sheddable in low pH environment such as that in chronic inflammation sites. In a mouse model of lipopolysaccharide-induced chronic inflammation, the acid-sensitive sheddable PEGylated PLGA nanoparticles showed significantly higher accumulation or distribution in chronic inflammation sites than PLGA nanoparticles prepared with an acid-insensitive emulsifying agent (i.e., stearoyl-amide-polyethylene glycol 2000) and significantly increased the distribution of the TNF-α siRNA incorporated into the nanoparticles in inflamed mouse foot. PMID:27434685

  3. Differential Sensitivity of Bat Cells to Infection by Enveloped RNA Viruses: Coronaviruses, Paramyxoviruses, Filoviruses, and Influenza Viruses

    PubMed Central

    Hoffmann, Markus; Müller, Marcel Alexander; Drexler, Jan Felix; Glende, Jörg; Erdt, Meike; Gützkow, Tim; Losemann, Christoph; Binger, Tabea; Deng, Hongkui; Schwegmann-Weßels, Christel; Esser, Karl-Heinz; Drosten, Christian; Herrler, Georg

    2013-01-01

    Bats (Chiroptera) host major human pathogenic viruses including corona-, paramyxo, rhabdo- and filoviruses. We analyzed six different cell lines from either Yinpterochiroptera (including African flying foxes and a rhinolophid bat) or Yangochiroptera (genera Carollia and Tadarida) for susceptibility to infection by different enveloped RNA viruses. None of the cells were sensitive to infection by transmissible gastroenteritis virus (TGEV), a porcine coronavirus, or to infection mediated by the Spike (S) protein of SARS-coronavirus (SARS-CoV) incorporated into pseudotypes based on vesicular stomatitis virus (VSV). The resistance to infection was overcome if cells were transfected to express the respective cellular receptor, porcine aminopeptidase N for TGEV or angiotensin-converting enzyme 2 for SARS-CoV. VSV pseudotypes containing the S proteins of two bat SARS-related CoV (Bg08 and Rp3) were unable to infect any of the six tested bat cell lines. By contrast, viral pseudotypes containing the surface protein GP of Marburg virus from the family Filoviridae infected all six cell lines though at different efficiency. Notably, all cells were sensitive to infection by two paramyxoviruses (Sendai virus and bovine respiratory syncytial virus) and three influenza viruses from different subtypes. These results indicate that bat cells are more resistant to infection by coronaviruses than to infection by paramyxoviruses, filoviruses and influenza viruses. Furthermore, these results show a receptor-dependent restriction of the infection of bat cells by CoV. The implications for the isolation of coronaviruses from bats are discussed. PMID:24023659

  4. Differential sensitivity of bat cells to infection by enveloped RNA viruses: coronaviruses, paramyxoviruses, filoviruses, and influenza viruses.

    PubMed

    Hoffmann, Markus; Müller, Marcel Alexander; Drexler, Jan Felix; Glende, Jörg; Erdt, Meike; Gützkow, Tim; Losemann, Christoph; Binger, Tabea; Deng, Hongkui; Schwegmann-Weßels, Christel; Esser, Karl-Heinz; Drosten, Christian; Herrler, Georg

    2013-01-01

    Bats (Chiroptera) host major human pathogenic viruses including corona-, paramyxo, rhabdo- and filoviruses. We analyzed six different cell lines from either Yinpterochiroptera (including African flying foxes and a rhinolophid bat) or Yangochiroptera (genera Carollia and Tadarida) for susceptibility to infection by different enveloped RNA viruses. None of the cells were sensitive to infection by transmissible gastroenteritis virus (TGEV), a porcine coronavirus, or to infection mediated by the Spike (S) protein of SARS-coronavirus (SARS-CoV) incorporated into pseudotypes based on vesicular stomatitis virus (VSV). The resistance to infection was overcome if cells were transfected to express the respective cellular receptor, porcine aminopeptidase N for TGEV or angiotensin-converting enzyme 2 for SARS-CoV. VSV pseudotypes containing the S proteins of two bat SARS-related CoV (Bg08 and Rp3) were unable to infect any of the six tested bat cell lines. By contrast, viral pseudotypes containing the surface protein GP of Marburg virus from the family Filoviridae infected all six cell lines though at different efficiency. Notably, all cells were sensitive to infection by two paramyxoviruses (Sendai virus and bovine respiratory syncytial virus) and three influenza viruses from different subtypes. These results indicate that bat cells are more resistant to infection by coronaviruses than to infection by paramyxoviruses, filoviruses and influenza viruses. Furthermore, these results show a receptor-dependent restriction of the infection of bat cells by CoV. The implications for the isolation of coronaviruses from bats are discussed. PMID:24023659

  5. Cloning and characterization of SRP1, a suppressor of temperature-sensitive RNA polymerase I mutations, in Saccharomyces cerevisiae.

    PubMed Central

    Yano, R; Oakes, M; Yamaghishi, M; Dodd, J A; Nomura, M

    1992-01-01

    The SRP1-1 mutation is an allele-specific dominant suppressor of temperature-sensitive mutations in the zinc-binding domain of the A190 subunit of Saccharomyces cerevisiae RNA polymerase I (Pol I). We found that it also suppresses temperature-sensitive mutations in the zinc-binding domain of the Pol I A135 subunit. This domain had been suggested to be in physical proximity to the A190 zinc-binding domain. We have cloned the SRP1 gene and determined its nucleotide sequence. The gene encodes a protein of 542 amino acids consisting of three domains: the central domain, which is composed of eight (degenerate) 42-amino-acid contiguous tandem repeats, and the surrounding N-terminal and C-terminal domains, both of which contain clusters of acidic and basic amino acids and are very hydrophilic. The mutational alteration (P219Q) responsible for the suppression was found to be in the central domain. Using antibody against the SRP1 protein, we have found that SRP1 is mainly localized at the periphery of the nucleus, apparently more concentrated in certain regions, as suggested by a punctate pattern in immunofluorescence microscopy. We suggest that SRP1 is a component of a larger macromolecular complex associated with the nuclear envelope and interacts with Pol I either directly or indirectly through other components in the structure containing SRP1. Images PMID:1448093

  6. Underproduction of the Largest Subunit of RNA Polymerase II Causes Temperature Sensitivity, Slow Growth, and Inositol Auxotrophy in Saccharomyces Cerevisiae

    PubMed Central

    Archambault, J.; Jansma, D. B.; Friesen, J. D.

    1996-01-01

    In the yeast Saccharomyces cerevisiae, mutations in genes encoding subunits of RNA polymerase II (RNAPII) often give rise to a set of pleiotropic phenotypes that includes temperature sensitivity, slow growth and inositol auxotrophy. In this study, we show that these phenotypes can be brought about by a reduction in the intracellular concentration of RNAPII. Underproduction of RNAPII was achieved by expressing the gene (RPO21), encoding the largest subunit of the enzyme, from the LEU2 promoter or a weaker derivative of it, two promoters that can be repressed by the addition of leucine to the growth medium. We found that cells that underproduced RPO21 were unable to derepress fully the expression of a reporter gene under the control of the INO1 UAS. Our results indicate that temperature sensitivity, slow growth and inositol auxotrophy is a set of phenotypes that can be caused by lowering the steadystate amount of RNAPII; these results also lead to the prediction that some of the previously identified RNAPII mutations that confer this same set of phenotypes affect the assembly/stability of the enzyme. We propose a model to explain the hypersensitivity of INO1 transcription to mutations that affect components of the RNAPII transcriptional machinery. PMID:8849884

  7. Study on the Association Between miRNA-202 Expression and Drug Sensitivity in Multiple Myeloma Cells.

    PubMed

    Shen, Xianjuan; Guo, Yuehua; Qi, Jing; Shi, Wei; Wu, Xinhua; Ni, Hongbing; Ju, Shaoqing

    2016-07-01

    An increasing amount of experimental evidence has shown that miRNAs play a causal role in hematologic tumorigenesis. In this study, we characterized the role of miR-202 in multiple myeloma (MM) drug sensitivity. The potential binding site of miR-202 and B cell-activating factor (BAFF) was confirmed by luciferase reporter assay. MM cells were transfected with miR-202 mimics and inhibitor. Cells growth was measured by WST-1 cell proliferation assay and Annexin V-FLUOS apoptosis assay. BAFF and miR-202 mRNA levels were measured by real-time PCR. Meanwhile, BAFF, Bcl-2 family survival proteins and MAPK pathway proteins were measured by Western blot. It was found that miR-202 was functioned as a modulator of BAFF expression. miR-202 over-expression sensitized MM cells to bortezomib (Bort) but less to Thalidomide (Thal) and dexamethasone (Dex). miR-202 mimics in combination with Bort inhibited MM cell survival more effectively as compared with Bort treatment alone. Our study also provided experimental evidence that JNK/SAPK signaling pathway was involved in the regulatory effect of miR-202 on drug resistance of MM cells. These results suggest that the regulatory mechanism of miR-202 expression may be a promising target for fine-tuning anti-myeloma therapy. PMID:26689580

  8. Development of multiplex loop-mediated isothermal amplification assays to detect medically important yeasts in dairy products.

    PubMed

    Kasahara, Kohei; Ishikawa, Hiroshi; Sato, Sumie; Shimakawa, Yasuhisa; Watanabe, Koichi

    2014-08-01

    Rapid detection of yeast contamination is important in the food industry. We have developed loop-mediated isothermal amplification (LAMP) assays to detect the emerging opportunistic pathogenic yeasts: Candida albicans, Candida glabrata, Candida tropicalis, the Candida parapsilosis group, Trichosporon asahii, and Trichosporon mucoides. These yeasts may cause deep-seated candidiasis or trichosporonosis. Four LAMP primer sets specific for Candida were designed to target the internal transcribed spacer 2 (ITS2) region between the 5.8S and 26S rRNA genes, and two LAMP primer sets specific for Trichosporon were designed to target the intergenic spacer 1 (IGS1) region between the 26S and 5S rRNA genes. The LAMP assays could detect these yeasts in a range between 10(0) and 10(3)  cells mL(-1) in a contaminated dairy product within 1 h. We also developed multiplex LAMP assays to detect these Candida or Trichosporon species in a single reaction. Multiplex LAMP assays can detect contamination if at least one of the target species is present; they are more time- and cost-efficient than conventional methods and could detect target yeasts with sensitivity close to that of the LAMP assays. Multiplex LAMP assays established in this study can be used as a primary screening method for yeast contamination in food products. PMID:24965944

  9. Detecting miRNA by producing RNA: a sensitive assay that combines rolling-circle DNA polymerization and rolling circle transcription.

    PubMed

    Li, Xuemei; Zheng, Fuwei; Ren, Rui

    2015-08-01

    Target miRNA was detected by producing RNA: rolling circle polymerization (RCP) and rolling circle transcription (RCT) were interlinked to provide dual amplification, producing multiplied malachite green (MG) aptamers, and a signal was generated by the SERS (surface-enhanced Raman scattering) quantification of the MG molecules that were bound to the transcripts. PMID:26120604

  10. Color-Multiplexing-Based Fluorescent Test Paper: Dosage-Sensitive Visualization of Arsenic(III) with Discernable Scale as Low as 5 ppb.

    PubMed

    Zhou, Yujie; Huang, Xiaoyan; Liu, Cui; Zhang, Ruilong; Gu, Xiaoling; Guan, Guijian; Jiang, Changlong; Zhang, Liying; Du, Shuhu; Liu, Bianhua; Han, Ming-Yong; Zhang, Zhongping

    2016-06-21

    Fluorescent colorimetry test papers are promising for the assays of environments, medicines, and foods by the observation of the naked eye on the variations of fluorescence brightness and color. Unlike dye-absorption-based pH test paper, however, the fluorescent test papers with wide color-emissive variations with target dosages for accurate quantification remain unsuccessful even if the multicolorful fluorescent probes are used. Here, we report the dosage-sensitive fluorescent colorimetry test paper with a very wide/consecutive "from red to cyan" response to the presence and amount of arsenic ions, As(III). Red quantum dots (QDs) were modified with glutathione and dithiothreitol to obtain the supersensitivity to As(III) by the quenching of red fluorescence through the formation of dispersive QDs aggregates. A small amount of cyan carbon dots (CDs) with spectral blue-green components as the photostable internal standard were mixed into the QDs solution to produce a composited red fluorescence. Upon the addition of As(III) into the sensory solution, the fluorescence color could gradually be reversed from red to cyan with a detection limit of 1.7 ppb As(III). When the sensory solution was printed onto a piece of filter paper, surprisingly a serial of color evolution from peach to pink to orange to khaki to yellowish to yellow-green to final cyan with the addition of As(III) was displayed and clearly discerned the dosage scale as low as 5 ppb. The methodology reported here opens a novel pathway toward the real applications of fluorescent test papers. PMID:27230307

  11. MicroRNA-183 correlates cancer prognosis, regulates cancer proliferation and bufalin sensitivity in epithelial ovarian caner.

    PubMed

    Chen, Huixiao; Zhang, Ling; Zhang, Lili; Du, Jing; Wang, Hongying; Wang, Bin

    2016-01-01

    Background we intended to explore the functional implication of microRNA-183 (miR-183) in predicting clinical prognosis and regulating cancer proliferation and bufalin sensitivity in epithelial ovarian cancer (EOC). Methods In 75 EOC patients, miR-183 expression was examined, by quantitative RT-PCR (qRT-PCR), between paired EOC tumors and adjacent normal tissues, and between tumor samples from patients at early clinical stages and those at advanced clinical stages. The association of serum miR-183 and patients' clinicopathological variables were examined. The overall survival (OS) was estimated by Kaplan-Meier model. And the possibility of miR-183 as a prognostic biomarker for EOC was examined by cox proportional hazard regression model. In EOC cell lines SKOV3 and ES-2 cells, lentiviral transduction was conducted to genetically suppress miR-183. The effect of miR-183 downregulation on EOC in vitro growth, bufalin sensitivity and in vivo tumorigenicity were examined. Results MiR-183 was highly expressed in EOC tumors, as well ass in patients at advanced clinical stages. Serum miR-183 was significantly associated with major clinicopathological variables in EOC patients, such as clinical stage and lymph node metastases. High level of serum miR-183 was associated with poor OS in EOC patients, and proved to be a potential biomarker for EOC. In EOC cell lines, functional assays demonstrated that miR-183 downregulation inhibited cancer proliferation, enhanced bufalin sensitivity and reduced tumorigenicity in vivo. Conclusion MiR-183 may be a prognostic biomarker for EOC, and inhibiting miR-183 may have therapeutic effect to inhibit tumor growth in EOC. PMID:27186298

  12. MicroRNA-183 correlates cancer prognosis, regulates cancer proliferation and bufalin sensitivity in epithelial ovarian caner

    PubMed Central

    Chen, Huixiao; Zhang, Ling; Zhang, Lili; Du, Jing; Wang, Hongying; Wang, Bin

    2016-01-01

    Background we intended to explore the functional implication of microRNA-183 (miR-183) in predicting clinical prognosis and regulating cancer proliferation and bufalin sensitivity in epithelial ovarian cancer (EOC). Methods In 75 EOC patients, miR-183 expression was examined, by quantitative RT-PCR (qRT-PCR), between paired EOC tumors and adjacent normal tissues, and between tumor samples from patients at early clinical stages and those at advanced clinical stages. The association of serum miR-183 and patients’ clinicopathological variables were examined. The overall survival (OS) was estimated by Kaplan-Meier model. And the possibility of miR-183 as a prognostic biomarker for EOC was examined by cox proportional hazard regression model. In EOC cell lines SKOV3 and ES-2 cells, lentiviral transduction was conducted to genetically suppress miR-183. The effect of miR-183 downregulation on EOC in vitro growth, bufalin sensitivity and in vivo tumorigenicity were examined. Results MiR-183 was highly expressed in EOC tumors, as well ass in patients at advanced clinical stages. Serum miR-183 was significantly associated with major clinicopathological variables in EOC patients, such as clinical stage and lymph node metastases. High level of serum miR-183 was associated with poor OS in EOC patients, and proved to be a potential biomarker for EOC. In EOC cell lines, functional assays demonstrated that miR-183 downregulation inhibited cancer proliferation, enhanced bufalin sensitivity and reduced tumorigenicity in vivo. Conclusion MiR-183 may be a prognostic biomarker for EOC, and inhibiting miR-183 may have therapeutic effect to inhibit tumor growth in EOC. PMID:27186298

  13. Development and validation of a multiplex reverse transcription quantitative PCR (RT-qPCR) assay for the rapid detection of Citrus tristeza virus, Citrus psorosis virus, and Citrus leaf blotch virus.

    PubMed

    Osman, Fatima; Hodzic, Emir; Kwon, Sun-Jung; Wang, Jinbo; Vidalakis, Georgios

    2015-08-01

    A single real-time multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay for the simultaneous detection of Citrus tristeza virus (CTV), Citrus psorosis virus (CPsV), and Citrus leaf blotch virus (CLBV) was developed and validated using three different fluorescently labeled minor groove binding qPCR probes. To increase the detection reliability, coat protein (CP) genes from large number of different isolates of CTV, CPsV and CLBV were sequenced and a multiple sequence alignment was generated with corresponding CP sequences from the GenBank and a robust multiplex RT-qPCR assay was designed. The capacity of the multiplex RT-qPCR assay in detecting the viruses was compared to singleplex RT-qPCR designed specifically for each virus and was assessed using multiple virus isolates from diverse geographical regions and citrus species as well as graft-inoculated citrus plants infected with various combination of the three viruses. No significant difference in detection limits was found and specificity was not affected by the inclusion of the three assays in a multiplex RT-qPCR reaction. Comparison of the viral load for each virus using singleplex and multiplex RT-qPCR assays, revealed no significant differences between the two assays in virus detection. No significant difference in Cq values was detected when using one-step and two-step multiplex RT-qPCR detection formats. Optimizing the RNA extraction technique for citrus tissues and testing the quality of the extracted RNA using RT-qPCR targeting the cytochrome oxidase citrus gene as an RNA specific internal control proved to generate better diagnostic assays. Results showed that the developed multiplex RT-qPCR can streamline viruses testing of citrus nursery stock by replacing three separate singleplex assays, thus reducing time and labor while retaining the same sensitivity and specificity. The three targeted RNA viruses are regulated pathogens for California's mandatory "Section 3701

  14. 3D multiplexed immunoplasmonics microscopy

    NASA Astrophysics Data System (ADS)

    Bergeron, Éric; Patskovsky, Sergiy; Rioux, David; Meunier, Michel

    2016-07-01

    Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K+ channel subunit KV1.1) on human cancer CD44+ EGFR+ KV1.1+ MDA-MB-231 cells and reference CD44- EGFR- KV1.1+ 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third, the developed

  15. Combining Single RNA Sensitive Probes with Subdiffraction-Limited and Live-Cell Imaging Enables the Characterization of Virus Dynamics in Cells

    PubMed Central

    2013-01-01

    The creation of fluorescently labeled viruses is currently limited by the length of imaging observation time (e.g., labeling an envelope protein) and the rescue of viral infectivity (e.g., encoding a GFP protein). Using single molecule sensitive RNA hybridization probes delivered to the cytoplasm of infected cells, we were able to isolate individual, infectious, fluorescently labeled human respiratory syncytial virus virions. This was achieved without affecting viral mRNA expression, viral protein expression, or infectivity. Measurements included the characterization of viral proteins and genomic RNA in a single virion using dSTORM, the development of a GFP fusion assay, and the development of a pulse-chase assay for viral RNA production that allowed for the detection of both initial viral RNA and nascent RNA production at designated times postinfection. Live-cell measurements included imaging and characterization of filamentous virion fusion and the quantification of virus replication within the same cell over an eight-hour period. Using probe-labeled viruses, individual viral particles can be characterized at subdiffraction-limited resolution, and viral infections can be quantified in single cells over an entire cycle of replication. The implication of this development is that MTRIP labeling of viral RNA during virus assembly has the potential to become a general methodology for the labeling and study of many important RNA viruses. PMID:24351207

  16. pH-responsive polymeric siRNA carriers sensitize multidrug resistant ovarian cancer cells to doxorubicin via knockdown of polo-like kinase 1

    PubMed Central

    Benoit, Danielle S.W.; Henry, Scott M.; Shubin, Andrew D.; Hoffman, Allan S.; Stayton, Patrick S.

    2010-01-01

    Small interfering RNA (siRNA)-based therapies have great potential for the treatment of debilitating diseases such as cancer, but an effective delivery strategy for siRNA is elusive. Here, pH-responsive complexes were developed for the delivery of siRNA in order to sensitize drug-resistant ovarian cancer cells (NCI/ADR-RES) to doxorubicin. The electrostatic complexes consisted of a cationic micelle used as a nucleating core, siRNA, and a pH-responsive endosomolytic polymer. Cationic micelles were formed from diblock copolymers of dimethylaminoethyl methacrylate (pDMAEMA) and butyl methacrylate (pDbB). The hydrophobic butyl core mediated micelle formation while the positively-charged pDMAEMA corona enabled siRNA condensation. To enhance cytosolic delivery through endosomal release, a pH-responsive copolymer of poly(styrene-alt-maleic anhydride) (pSMA) was electrostatically complexed with the positively-charged siRNA/micelle to form a ternary complex. Complexes exhibited size (30–105 nm) and charge (slightly positive) properties important for endocytosis and were found to be non-cytotoxic and mediate uptake in >70% of ovarian cancer cells after 1 hour of incubation. The pH-responsive ternary complexes were used to deliver siRNA against polo-like kinase 1 (plk1), a gene upregulated in many cancers and responsible for cell cycle progression, to ovarian cancer cell lines. Treatment resulted in ∼50% reduction of plk1 gene expression in the drug-resistant NCI/ADR-RES ovarian cancer cell model and in the drug-sensitive parental cell line, OVCAR8. This knockdown functionally sensitized NCI/ADR-RES cells to doxorubicin at levels similar to OVCAR8. Sensitization occurred through a p53 signaling pathway, as indicated by caspase 3/7 upregulation following plk1 knockdown and doxorubicin treatment, and this effect could be abrogated using a p53 inhibitor. To demonstrate the potential for dual delivery from this polymer system, micelle cores were subsequently loaded with

  17. Time-division SQUID multiplexers

    NASA Astrophysics Data System (ADS)

    Irwin, K. D.; Vale, L. R.; Bergren, N. E.; Deiker, S.; Grossman, E. N.; Hilton, G. C.; Nam, S. W.; Reintsema, C. D.; Rudman, D. A.; Huber, M. E.

    2002-02-01

    SQUID multiplexers make it possible to build arrays of thousands of low-temperature bolometers and microcalorimeters based on superconducting transition-edge sensors with a manageable number of readout channels. We discuss the technical tradeoffs between proposed time-division multiplexer and frequency-division multiplexer schemes and motivate our choice of time division. Our first-generation SQUID multiplexer is now in use in an astronomical instrument. We describe our second-generation SQUID multiplexer, which is based on a new architecture that significantly reduces the dissipation of power at the first stage, allowing thousands of SQUIDs to be operated at the base temperature of a cryostat. .

  18. Extracting information from multiplex networks.

    PubMed

    Iacovacci, Jacopo; Bianconi, Ginestra

    2016-06-01

    Multiplex networks are generalized network structures that are able to describe networks in which the same set of nodes are connected by links that have different connotations. Multiplex networks are ubiquitous since they describe social, financial, engineering, and biological networks as well. Extending our ability to analyze complex networks to multiplex network structures increases greatly the level of information that is possible to extract from big data. For these reasons, characterizing the centrality of nodes in multiplex networks and finding new ways to solve challenging inference problems defined on multiplex networks are fundamental questions of network science. In this paper, we discuss the relevance of the Multiplex PageRank algorithm for measuring the centrality of nodes in multilayer networks and we characterize the utility of the recently introduced indicator function Θ̃(S) for describing their mesoscale organization and community structure. As working examples for studying these measures, we consider three multiplex network datasets coming for social science. PMID:27368796

  19. Extracting information from multiplex networks

    NASA Astrophysics Data System (ADS)

    Iacovacci, Jacopo; Bianconi, Ginestra

    2016-06-01

    Multiplex networks are generalized network structures that are able to describe networks in which the same set of nodes are connected by links that have different connotations. Multiplex networks are ubiquitous since they describe social, financial, engineering, and biological networks as well. Extending our ability to analyze complex networks to multiplex network structures increases greatly the level of information that is possible to extract from big data. For these reasons, characterizing the centrality of nodes in multiplex networks and finding new ways to solve challenging inference problems defined on multiplex networks are fundamental questions of network science. In this paper, we discuss the relevance of the Multiplex PageRank algorithm for measuring the centrality of nodes in multilayer networks and we characterize the utility of the recently introduced indicator function Θ ˜ S for describing their mesoscale organization and community structure. As working examples for studying these measures, we consider three multiplex network datasets coming for social science.

  20. Pre-Clinical Validation of a Novel, Highly Sensitive Assay to Detect PML-RARα mRNA Using Real-Time Reverse-Transcription Polymerase Chain Reaction

    PubMed Central

    Slack, James L.; Bi, WanLi; Livak, Kenneth J.; Beaubier, Nike; Yu, Min; Clark, Michelle; Kim, Soon H.; Gallagher, Robert E.; Willman, Cheryl L.

    2001-01-01

    We have developed a sensitive and quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay for detection of PML-RARα, the fusion oncogene present as a specific marker in >99% of cases of acute promyelocytic leukemia (APL). The assay is linear over at least 5 orders of magnitude of input DNA or RNA, and detects as few as 4 copies of PML-RARα plasmid DNA. PML-RARα transcripts could be detected in mixtures containing 2 to 5 pg of RNA from fusion-containing cells in a background of 1 μg of RNA from PML-RARα-negative cells. Using 1.0 to 2.5 μg of input RNA, the sensitivity of the assay was between 10−5 and 10−6. Furthermore, determination of GAPDH copy number in each reaction allowed an accurate assessment of sample-to-sample variation in RNA quality and reaction efficiency, with consequent definition of a detection limit for each sample assayed. Using an internal calibrator, assay precision was high, with coefficients of variation between 10 and 20%. An interlaboratory study using coded samples demonstrated excellent reproducibility and high concordance between laboratories. This assay will be used to test the hypothesis that sensitive and quantitative measurement of leukemic burden, during or after therapy of APL, can stratify patients into discrete risk groups, and thereby serve as a basis for risk-adapted therapy in APL. PMID:11687597

  1. A rapid and sensitive nonradioactive method applicable for genome-wide analysis of Saccharomyces cerevisiae genes involved in small RNA biology

    PubMed Central

    Wu, Jingyan; Huang, Hsiao-Yun; Hopper, Anita K.

    2013-01-01

    The conventional small RNA isolation and detection methods for yeast cells have been designed for a small number of samples. In order to conduct a genome-wide assessment of how each gene product impacts upon small non-coding RNAs, we developed a rapid method for analyzing small RNAs from Saccharomyces cerevisiae wild-type and mutants cells in the deletion and temperature-sensitive (ts) collections. Our method implements three optimized techniques: a procedure for growing small yeast cultures in 96-deepwell plates, a fast procedure for small RNA isolation from the plates, and a sensitive nonradioactive Northern method for RNA detection. The RNA isolation procedure is highly reproducible and requires only 4 hours for processing 96 samples, and yields RNA of good quality and quantity. The nonradioactive Northern method employs digoxigenin (DIG)-labeled DNA probes and chemiluminescence. It detects femtomole-level small RNAs within 1-minute exposure time. We minimized the processing time for large-scale analysis and optimized the stripping and re-probing procedures for analysis of multiple RNAs from a single membrane. The method described is rapid, sensitive, safe, and cost-effective for genome-wide screens of novel genes involved in the biogenesis, subcellular trafficking, and stability of small RNAs. Moreover, it will be useful to educational laboratory class venues and to research institutions with limited access to radioisotopes or robots. PMID:23417998

  2. Optimized Ultrasound Conditions for Enhanced Sensitivity of Molecular Beacons in the Detection of MDR1 mRNA in Living Cells.

    PubMed

    Zhou, Qiumei; Ma, Yi; Wang, Zhaohui; Wang, Ke; Liu, Ruonan; Han, Zhihao; Zhang, Min; Li, Siwen; Gu, Yueqing

    2016-03-01

    P-glycoprotein (P-gp), aprognostic indicator for chemotherapy failure, is encoded by multidrug resistance gene (MDR1). MDR1 mRNA expression could serve as a guidance for personalized medicine. However, the traditional PCR process for mRNA measurement is complicated and cannot realize the real-time detection of mRNA in living single cells. In this work, optimized gold nanoparticle-based molecular beacons were employed to determine MDR1 mRNA levels in living cancer cells. To improve detection sensitivity, ultrasound (US) irradiation was applied to facilitate and enhance cellular uptake of hairpin DNA-coated gold nanoparticle (hDAuNP). The US conditions including irradiation power, exposure time, duty cycle, and incubation time were optimized. The slight difference in MDR1 expression manipulated by siRNA silence could be recognized by US assisted hDAuNP beacons; a 10-fold increase of detection sensitivity was achieved compared with the nonultrasound assistance. Meanwhile, the detection cycle could be shortened from 12 to 2 h. Furthermore, this hDAuNP beacon can serve as an antisense agent to down-regulate P-gp expression and to reverse drug resistance of MCF-7/Adr cells to doxorubicin. Our results demonstrated that the MDR1 hDAuNP beacon assisted by US irradiation had great potential to predict chemotherapy sensitivity and to overcome multidrug resistance in cancer cells and was thus a promising tool for individualized medicine. PMID:26821347

  3. Small interfering RNA targeting S100A4 sensitizes non-small-cell lung cancer cells (A549) to radiation treatment

    PubMed Central

    Qi, Ruixue; Qiao, Tiankui; Zhuang, Xibing

    2016-01-01

    Objective This study aimed to investigate the impact of S100A4-small interfering RNA (S100A4-siRNA) on apoptosis and enhanced radiosensitivity in non-small-cell lung cancer (A549) cells. We also explored the mechanisms of radiosensitization and identified a new target to enhance radiosensitivity and gene therapy for non-small-cell lung cancer. Methods RNA interference is a powerful tool for gene silencing. In this study, we constructed an effective siRNA to knock down S100A4. A549 cells were randomly divided into three groups: blank, negative control, and S100A4-siRNA. To investigate the effect of S100A4-siRNA, the expression of S100A4, E-cadherin, and p53 proteins and their messenger RNA (mRNA) was detected by Western blot and quantitative real-time polymerase chain reaction. Transwell chambers were used to assess cell invasion. Cell cycle and apoptosis were analyzed by flow cytometry. Radiosensitivity was determined by colony formation ability. Results Our results demonstrate that S100A4-siRNA effectively silenced the S100A4 gene. When siRNA against S100A4 was used, S100A4 protein expression was downregulated, whereas the expressions of E-cadherin and p53 were upregulated. In addition, a clear reduction in S100A4 mRNA levels was noted compared with the blank and negative control groups, whereas E-cadherin and p53 mRNA levels increased. Transfection with S100A4-siRNA significantly reduced the invasiveness of A549 cells. S100A4 silencing induced immediate G2/M arrest in cell cycle studies and increased apoptosis rates in A549 cells. In clonogenic assays, we used a multitarget, single-hit model to detect radiosensitivity after S100A4 knockdown. All parameters (D0, Dq, α, β) indicated that the downregulation of S100A4 enhanced radiosensitivity in A549 cells. Furthermore, S100A4-siRNA upregulated p53 expression, suggesting that S100A4 may promote A549 cell proliferation, invasion, and metastasis by regulating the expression of other proteins. Therefore, siRNA

  4. Co-delivery of hydrophobic paclitaxel and hydrophilic AURKA specific siRNA by redox-sensitive micelles for effective treatment of breast cancer.

    PubMed

    Yin, Tingjie; Wang, Lei; Yin, Lifang; Zhou, Jianping; Huo, Meirong

    2015-08-01

    In this study, a novel redox-sensitive micellar system constructed from a hyaluronic acid-based amphiphilic conjugate (HA-ss-(OA-g-bPEI), HSOP) was successfully developed for tumor-targeted co-delivery of paclitaxel (PTX) and AURKA specific siRNA (si-AURKA). HSOP exhibited excellent loading capacities for both PTX and siRNA with adjustable dosing ratios and desirable redox-sensitivity independently verified by morphological changes of micelles alongside in vitro release of both drugs in different reducing environments. Moreover, flow cytometry and confocal microscopy analysis confirmed that HSOP micelles were capable of simultaneously delivering PTX and siRNA into MDA-MB-231 breast cancer cells via HA-receptor mediated endocytosis followed by rapid transport of cargoes into the cytosol. Successful delivery and transport amplified the synergistic effects between the drugs while leading to substantially greater antitumor efficacy when compared with single drug-loaded micelles and non-sensitive co-loaded micelles. In vivo investigation demonstrated that HSOP micelles could effectively accumulate in tumor sites and possessed the greatest antitumor efficacy over non-sensitive co-delivery control and redox-sensitive single-drug controls. These findings indicated that redox-sensitive HSOP co-delivery system holds great promise for combined drug/gene treatment for targeted cancer therapy. PMID:25996409

  5. Quartz-Seq: a highly reproducible and sensitive single-cell RNA sequencing method, reveals non-genetic gene-expression heterogeneity

    PubMed Central

    2013-01-01

    Development of a highly reproducible and sensitive single-cell RNA sequencing (RNA-seq) method would facilitate the understanding of the biological roles and underlying mechanisms of non-genetic cellular heterogeneity. In this study, we report a novel single-cell RNA-seq method called Quartz-Seq that has a simpler protocol and higher reproducibility and sensitivity than existing methods. We show that single-cell Quartz-Seq can quantitatively detect various kinds of non-genetic cellular heterogeneity, and can detect different cell types and different cell-cycle phases of a single cell type. Moreover, this method can comprehensively reveal gene-expression heterogeneity between single cells of the same cell type in the same cell-cycle phase. PMID:23594475

  6. Fluorescence behavior of a unique two-photon fluorescent probe in aggregate and solution states and highly sensitive detection of RNA in water solution and living systems.

    PubMed

    Liu, Yong; Meng, Fangfang; He, Longwei; Yu, Xiaoqiang; Lin, Weiying

    2016-07-01

    It is found that 2,7-substituted carbazole derivative possesses distinct luminescence features in both aggregate and solution states. In view of this, probe realizes highly sensitive detection of RNA in pure water systems by an aggregation-disaggregation method for the first time. PMID:27346863

  7. Downlink Data Multiplexer

    NASA Technical Reports Server (NTRS)

    Holland, Douglas; Steele, Glen F.; Romero, Denise M.; Koudelka, Robert David

    2004-01-01

    A multiplexer/demultiplexer system has been developed to enable the transmission, over a single channel, of four data streams generated by a variety of sources at different (including variable) bit rates. In the original intended application, replicas of this multiplexer/demultiplexer system would be incorporated into the spacecraft-to-ground communication systems of the space shuttles. The multiplexer of each system would be installed in the spacecraft, where it would acquire and process data from such sources as commercial digital camcorders, video tape recorders, and the spacecraft telemetry system. The demultiplexer of each system would be installed in a ground station. Purely terrestrial systems of similar design could be attractive for use in situations in which there are requirements to transmit multiple streams of high-quality video data and possibly other data over single channels. The figure is a block diagram of the multiplexer as configured to process data received via three fiber-optic channels like those of the International Space Station and one electrical-cable channel that conforms to the Institute of Electrical and Electronic Engineers (IEEE) 1394 standard. (This standard consists of specifications of a high-speed serial data interface, the physical layer of which includes a cable known in the art as "FireWire." An IEEE 1394 interface can also transfer power between the components to which it is connected.) The fiber-optic channels carry packet and/or bit-stream signals that conform to the standards of the Consultative Committee for Space Data Systems (CCSDS). The IEEE 1394 interface accepts an isochronous signal like that from a digital camcorder or a video tape recorder. The processing of the four input data streams to combine them into one output stream is governed by a statistical multiplexing algorithm that features a flow-control capability and makes it possible to utilize the transmission channel with nearly 100-percent efficiency. This

  8. Identification and characterization of a double-stranded RNA- reovirus temperature-sensitive mutant defective in minor core protein mu2.

    PubMed Central

    Coombs, K M

    1996-01-01

    A newly identified temperature-sensitive mutant whose defect was mapped to the reovirus M1 gene (minor core protein mu2) was studied to better understand the functions of this virion protein. Sequence determination of the Ml gene of this mutant (tsH11.2) revealed a predicted methionine-to-threonine alteration at amino acid 399 and a change from proline to histidine at amino acid 414. The mutant made normal amounts of single-stranded RNA, both in in vitro transcriptase assays and in infected cells, and normal amounts of progeny viral protein at early times in a restrictive infection. However, tsH11.2 produced neither detectable progeny protein nor double-stranded RNA at late times in a restrictive infection. These studies indicate that mu2 plays a role in the conversion of reovirus mRNA to progeny double-stranded RNA. PMID:8676444

  9. Co-delivery of doxorubicin and siRNA with reduction and pH dually sensitive nanocarrier for synergistic cancer therapy.

    PubMed

    Chen, Weicai; Yuan, Yuanyuan; Cheng, Du; Chen, Jifeng; Wang, Lu; Shuai, Xintao

    2014-07-01

    Drug resistance is the greatest challenge in clinical cancer chemotherapy. Co-delivery of chemotherapeutic drugs and siRNA to tumor cells is a vital means to silence drug resistant genes during the course of cancer chemotherapy for an improved chemotherapeutic effect. This study aims at effective co-delivery of siRNA and anticancer drugs to tumor cells. A ternary block copolymer PEG-PAsp(AED)-PDPA consisting of pH-sensitive poly(2-(diisopropyl amino)ethyl methacrylate) (PDPA), reduction-sensitive poly(N-(2,2'-dithiobis(ethylamine)) aspartamide) PAsp(AED), and poly(ethylene glycol) (PEG) is synthesized and assembled into a core-shell structural micelle which encapsulated doxorubicin (DOX) in its pH-sensitive core and the siRNA-targeting anti-apoptosis BCL-2 gene (BCL-2 siRNA) in a reduction-sensitive interlayer. At the optimized size and zeta potential, the nanocarriers loaded with DOX and BCL-2 siRNA may effectively accumulate in the tumor site via blood circulation. Moreover, the dual stimuli-responsive design of micellar carriers allows microenviroment-specific rapid release of both DOX and BCL-2 siRNA inside acidic lysosomes with enriched reducing agent, glutathione (GSH, up to 10 mM). Consequently, the expression of anti-apoptotic BCL-2 protein induced by DOX treatment is significantly down-regulated, which results in synergistically enhanced apoptosis of human ovarian cancer SKOV-3 cells and thus dramatically inhibited tumor growth. PMID:24668891

  10. Highly selective and sensitive detection of miRNA based on toehold-mediated strand displacement reaction and DNA tetrahedron substrate.

    PubMed

    Li, Wei; Jiang, Wei; Ding, Yongshun; Wang, Lei

    2015-09-15

    MicroRNAs (miRNAs) play important roles in a variety of biological processes and have been regarded as tumor biomarkers in cancer diagnosis and prognosis. In this work, a single-molecule counting method for miRNA analysis was proposed based on toehold-mediated strand displacement reaction (SDR) and DNA tetrahedron substrate. Firstly, a specially designed DNA tetrahedron was assembled with a hairpin at one of the vertex, which has an overhanging toehold domain. Then, the DNA tetrahedron was immobilized on the epoxy-functional glass slide by epoxy-amine reaction, forming a DNA tetrahedron substrate. Next, the target miRNA perhybridized with the toehold domain and initiated a strand displacement reaction along with the unfolding of the hairpin, realizing the selective recognization of miRNA. Finally, a biotin labeled detection DNA was hybridized with the new emerging single strand and the streptavidin coated QDs were used as fluorescent probes. Fluorescent images were acquired via epi-fluorescence microscopy, the numbers of fluorescence dots were counted one by one for quantification. The detection limit is 5 fM, which displayed an excellent sensitivity. Moreover, the proposed method which can accurately be identified the target miRNA among its family members, demonstrated an admirable selectivity. Furthermore, miRNA analysis in total RNA samples from human lung tissues was performed, suggesting the feasibility of this method for quantitative detection of miRNA in biomedical research and early clinical diagnostics. PMID:25950935

  11. The use of pH-sensitive functional selenium nanoparticles shows enhanced in vivo VEGF-siRNA silencing and fluorescence imaging.

    PubMed

    Yu, Qianqian; Liu, Yanan; Cao, Chengwen; Le, Fangling; Qin, Xiuying; Sun, Dongdong; Liu, Jie

    2014-08-01

    The utility of small interfering RNAs (siRNAs) has shown great promise in treating a variety of diseases including many types of cancer. While their ability to silence a wide range of target genes underlies their effectiveness, the application of therapies remains hindered by a lack of an effective delivery system. In this study, we sought to develop an siRNA-delivery system for VEGF, a known signaling molecule involved in cancer, that consists of two selenium nanoparticles SeNPs and G2/PAH-Cit/SeNPs. A G2/PAH-Cit/SeNP is a pH-sensitive delivery system that is capable of enhancing siRNA loading, thus increasing siRNA release efficiency and subsequent target gene silencing both in vitro and in vivo. In vivo experiments using G2/PAH-Cit/SeNPs@siRNA led to significantly higher accumulation of siRNA within the tumor itself, VEGF gene silencing, and reduced angiogenesis in the tumor. Furthermore, the G2/PAH-Cit/SeNP delivery system not only enhanced anti-tumor effects on tumor-bearing nude mice as compared to SeNPs@siRNA, but also resulted in weak occurrence of lesions in major target organs. In sum, this study provides a new class of siRNA delivery system, thereby providing an alternative therapeutic route for cancer treatment. PMID:24986368

  12. Temperature-sensitive mutations for live-attenuated Rift Valley fever vaccines: implications from other RNA viruses

    PubMed Central

    Nishiyama, Shoko; Ikegami, Tetsuro

    2015-01-01

    Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to the African continent. RVF is characterized by high rate of abortions in ruminants and hemorrhagic fever, encephalitis, or blindness in humans. RVF is caused by the Rift Valley fever virus (RVFV: genus Phlebovirus, family Bunyaviridae). Vaccination is the only known effective strategy to prevent the disease, but there are no licensed RVF vaccines available for humans. A live-attenuated vaccine candidate derived from the wild-type pathogenic Egyptian ZH548 strain, MP-12, has been conditionally licensed for veterinary use in the U.S. MP-12 displays a temperature-sensitive (ts) phenotype and does not replicate at 41°C. The ts mutation limits viral replication at a specific body temperature and may lead to an attenuation of the virus. Here we will review well-characterized ts mutations for RNA viruses, and further discuss the potential in designing novel live-attenuated vaccines for RVF. PMID:26322023

  13. A novel nested multiplex polymerase chain reaction (PCR) assay for differential detection of Entamoeba histolytica, E. moshkovskii and E. dispar DNA in stool samples

    PubMed Central

    Khairnar, Krishna; Parija, Subhash C

    2007-01-01

    Background E. histolytica, a pathogenic amoeba, is indistinguishable in its cyst and trophozoite stages from those of non-pathogenic E. moshkovskii and E. dispar by light microscopy. We have developed a nested multiplex PCR targeting a 16S-like rRNA gene for differential detection of all the three morphologically similar forms of E. histolytica, E. moshkovskii and E. dispar simultaneously in stool samples. Results The species specific product size for E. histolytica, E. moshkovskii and E. dispar was 439, 553 and 174 bp respectively, which was clearly different for all the three Entamoeba species. The nested multiplex PCR showed a sensitivity of 94% and specificity of 100% for the demonstration of E. histolytica, E. moshkovskii and E. dispar DNA in stool samples. The PCR was positive for E. histolytica, E. moshkovskii and E. dispar in a total of 190 out of 202 stool specimens (94% sensitive) that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture. All the 35 negative control stool samples that were negative for E. histolytica/E. dispar/E. moshkovskii by microscopy and culture were also found negative by the nested multiplex PCR (100% specific). The result from the study shows that only 34.6% of the patient stool samples that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture, were actually positive for pathogenic E. histolytica and the remaining majority of the stool samples were positive for non-pathogenic E. dispar or E. moshkovskii as demonstrated by the use of nested multiplex PCR. Conclusion The present study reports a new nested multiplex PCR strategy for species specific detection and differentiation of E. histolytica, E. dispar and E. moshkovskii DNA in stool specimens. The test is highly specific, sensitive and also rapid, providing the results within 12 hours of receiving stool specimens. PMID:17524135

  14. Multiplexing oscillatory biochemical signals.

    PubMed

    de Ronde, Wiet; ten Wolde, Pieter Rein

    2014-04-01

    In recent years it has been increasingly recognized that biochemical signals are not necessarily constant in time and that the temporal dynamics of a signal can be the information carrier. Moreover, it is now well established that the protein signaling network of living cells has a bow-tie structure and that components are often shared between different signaling pathways. Here we show by mathematical modeling that living cells can multiplex a constant and an oscillatory signal: they can transmit these two signals simultaneously through a common signaling pathway, and yet respond to them specifically and reliably. We find that information transmission is reduced not only by noise arising from the intrinsic stochasticity of biochemical reactions, but also by crosstalk between the different channels. Yet, under biologically relevant conditions more than 2 bits of information can be transmitted per channel, even when the two signals are transmitted simultaneously. These observations suggest that oscillatory signals are ideal for multiplexing signals. PMID:24685537

  15. Multiplex data bus simulator

    SciTech Connect

    Garbo, D.L.

    1983-01-01

    A multiplex data-bus simulator for analyzing multiprocessor designs is presented. The simulator was designed to be user-friendly, thus allowing a multiprocessor designer to enter various configuration inputs in a concise and orderly fashion through the use of menus. The designer is also provided a method of visualizing a message traffic flow through the use of graphical representation of events. 3 references.

  16. Simultaneous multiplex PCR detection of seven cucurbit-infecting viruses.

    PubMed

    Kwon, Ji Yeon; Hong, Jin Sung; Kim, Min Jea; Choi, Sun Hee; Min, Byeong Eun; Song, Eun Gyeong; Kim, Hyun Hee; Ryu, Ki Hyun

    2014-09-01

    Two multiplex polymerase chain reaction (PCR) systems using dual priming oligonucleotide (DPO) primers were developed for the simultaneous detection of seven cucurbit-infecting viruses. One system allows for the detection of papaya ringspot virus, watermelon mosaic virus, and zucchini yellow mosaic virus, whereas the other permits the detection of cucumber green mottle mosaic virus, cucumber fruit mottle mosaic virus, kyuri green mottle mosaic virus, and zucchini green mottle mosaic virus. Viral species-specific DPO primers developed in this study detected as little as 10 fg/μl of viral RNA under monoplex conditions and 10 pg/μl of viral RNA under multiplex conditions. Multiplex PCR using the DPO primer sets was capable of amplifying viral genes at annealing temperatures ranging from 53 °C to 63 °C. Whereas the use of conventional primers gave rise to non-specific bands, the DPO primers detected target viral genes in the absence of non-specific amplification. When these DPO multiplex primer sets were applied to virus-infected cucurbit samples obtained in the field, multiple infection as well as single infection was accurately identified. This novel approach could also detect multiple viruses in infected seeds. The reliability of multiplex PCR systems using DPO primers for plant virus detection is discussed. PMID:24937806

  17. On-chip noninterference angular momentum multiplexing of broadband light

    NASA Astrophysics Data System (ADS)

    Ren, Haoran; Li, Xiangping; Zhang, Qiming; Gu, Min

    2016-05-01

    Angular momentum division has emerged as a physically orthogonal multiplexing method in high-capacity optical information technologies. However, the typical bulky elements used for information retrieval from the overall diffracted field, based on the interference method, impose a fundamental limit toward realizing on-chip multiplexing. We demonstrate noninterference angular momentum multiplexing by using a mode-sorting nanoring aperture with a chip-scale footprint as small as 4.2 micrometers by 4.2 micrometers, where nanoring slits exhibit a distinctive outcoupling efficiency on tightly confined plasmonic modes. The nonresonant mode-sorting sensitivity and scalability of our approach enable on-chip parallel multiplexing over a bandwidth of 150 nanometers in the visible wavelength range. The results offer the possibility of ultrahigh-capacity and miniaturized nanophotonic devices harnessing angular momentum division.

  18. Burst noise in the HAWAII-1RG multiplexer

    NASA Astrophysics Data System (ADS)

    Bacon, Candice M.; McMurtry, Craig W.; Pipher, Judith L.; Forrest, William J.; Garnett, James D.

    2005-08-01

    Burst noise (also known as popcorn noise and random telegraph signal/noise) is a phenomenon that is understood to be a result of defects in the vicinity of a p-n junction. It is characterized by rapid level shifts in both positive and negative directions and can have varying magnitudes. This noise has been seen in both HAWAII-1RG and HAWAII-2RG multiplexers and is under investigation. We have done extensive burst noise testing on a HAWAII-1RG multiplexer, where we have determined a significant percentage of pixels exhibit the phenomenon. In addition, the prevalence of small magnitude transitions make sensitivity of detection the main limiting factor. Since this is a noise source for the HAWAII-1RG multiplexer, its elimination would make the HAWAII-1RG and the HAWAII-2RG even lower noise multiplexers.

  19. On-chip noninterference angular momentum multiplexing of broadband light.

    PubMed

    Ren, Haoran; Li, Xiangping; Zhang, Qiming; Gu, Min

    2016-05-13

    Angular momentum division has emerged as a physically orthogonal multiplexing method in high-capacity optical information technologies. However, the typical bulky elements used for information retrieval from the overall diffracted field, based on the interference method, impose a fundamental limit toward realizing on-chip multiplexing. We demonstrate noninterference angular momentum multiplexing by using a mode-sorting nanoring aperture with a chip-scale footprint as small as 4.2 micrometers by 4.2 micrometers, where nanoring slits exhibit a distinctive outcoupling efficiency on tightly confined plasmonic modes. The nonresonant mode-sorting sensitivity and scalability of our approach enable on-chip parallel multiplexing over a bandwidth of 150 nanometers in the visible wavelength range. The results offer the possibility of ultrahigh-capacity and miniaturized nanophotonic devices harnessing angular momentum division. PMID:27056843

  20. 3D multiplexed immunoplasmonics microscopy.

    PubMed

    Bergeron, Éric; Patskovsky, Sergiy; Rioux, David; Meunier, Michel

    2016-07-21

    Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K(+) channel subunit KV1.1) on human cancer CD44(+) EGFR(+) KV1.1(+) MDA-MB-231 cells and reference CD44(-) EGFR(-) KV1.1(+) 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third

  1. Anti-EphA10 antibody-conjugated pH-sensitive liposomes for specific intracellular delivery of siRNA.

    PubMed

    Zang, Xinlong; Ding, Huaiwei; Zhao, Xiufeng; Li, Xiaowei; Du, Zhouqi; Hu, Haiyang; Qiao, Mingxi; Chen, Dawei; Deng, Yuihui; Zhao, Xiuli

    2016-01-01

    Therapeutic delivery of small interfering RNA (siRNA) is a major challenge that limits its potential clinical application. Here, a pH-sensitive cholesterol-Schiff base-polyethylene glycol (Chol-SIB-PEG)-modified cationic liposome-siRNA complex, conjugated with the recombinant humanized anti-EphA10 antibody (Eph), was developed as an efficient nonviral siRNA delivery system. Chol-SIB-PEG was successfully synthesized and confirmed with FTIR and (1)H-NMR. An Eph-PEG-SIB-Chol-modified liposome-siRNA complex (EPSLR) was prepared and characterized by size, zeta potential, gel retardation, and encapsulation efficiency. Electrophoresis results showed that EPSLR was resistant to heparin replacement and protected siRNA from fetal bovine serum digestion. EPSLR exhibited only minor cytotoxicity in MCF-7/ADR cells. The results of flow cytometry and confocal laser scanning microscopy suggested that EPSLR enhanced siRNA transfection in MCF-7/ADR cells. Intracellular distribution experiment revealed that EPSLR could escape from the endo-lysosomal organelle and release siRNA into cytoplasm at 4 hours posttransfection. Western blot experiment demonstrated that EPSLR was able to significantly reduce the levels of MDR1 protein in MCF-7/ADR cells. The in vivo study of DIR-labeled complexes in mice bearing MCF-7/ADR tumor indicated that EPSLR could reach the tumor site rather than other organs more effectively. All these results demonstrate that EPSLR has much potential for effective siRNA delivery and may facilitate its therapeutic application. PMID:27574425

  2. Anti-EphA10 antibody-conjugated pH-sensitive liposomes for specific intracellular delivery of siRNA

    PubMed Central

    Zang, Xinlong; Ding, Huaiwei; Zhao, Xiufeng; Li, Xiaowei; Du, Zhouqi; Hu, Haiyang; Qiao, Mingxi; Chen, Dawei; Deng, Yuihui; Zhao, Xiuli

    2016-01-01

    Therapeutic delivery of small interfering RNA (siRNA) is a major challenge that limits its potential clinical application. Here, a pH-sensitive cholesterol–Schiff base–polyethylene glycol (Chol–SIB–PEG)-modified cationic liposome–siRNA complex, conjugated with the recombinant humanized anti-EphA10 antibody (Eph), was developed as an efficient nonviral siRNA delivery system. Chol–SIB–PEG was successfully synthesized and confirmed with FTIR and 1H-NMR. An Eph–PEG–SIB–Chol-modified liposome–siRNA complex (EPSLR) was prepared and characterized by size, zeta potential, gel retardation, and encapsulation efficiency. Electrophoresis results showed that EPSLR was resistant to heparin replacement and protected siRNA from fetal bovine serum digestion. EPSLR exhibited only minor cytotoxicity in MCF-7/ADR cells. The results of flow cytometry and confocal laser scanning microscopy suggested that EPSLR enhanced siRNA transfection in MCF-7/ADR cells. Intracellular distribution experiment revealed that EPSLR could escape from the endo-lysosomal organelle and release siRNA into cytoplasm at 4 hours posttransfection. Western blot experiment demonstrated that EPSLR was able to significantly reduce the levels of MDR1 protein in MCF-7/ADR cells. The in vivo study of DIR-labeled complexes in mice bearing MCF-7/ADR tumor indicated that EPSLR could reach the tumor site rather than other organs more effectively. All these results demonstrate that EPSLR has much potential for effective siRNA delivery and may facilitate its therapeutic application. PMID:27574425

  3. Self-calibrating multiplexer circuit

    DOEpatents

    Wahl, Chris P.

    1997-01-01

    A time domain multiplexer system with automatic determination of acceptable multiplexer output limits, error determination, or correction is comprised of a time domain multiplexer, a computer, a constant current source capable of at least three distinct current levels, and two series resistances employed for calibration and testing. A two point linear calibration curve defining acceptable multiplexer voltage limits may be defined by the computer by determining the voltage output of the multiplexer to very accurately known input signals developed from predetermined current levels across the series resistances. Drift in the multiplexer may be detected by the computer when the output voltage limits, expected during normal operation, are exceeded, or the relationship defined by the calibration curve is invalidated.

  4. Self-calibrating multiplexer circuit

    SciTech Connect

    Wahl, C.P.

    1995-12-31

    A time domain multiplexer system with automatic determination of acceptable multiplexer output limits, error determination, or correction is comprised of a time domain multiplexer, a computer, a constant current source capable of at least three distinct current levels, and two series resistances employed for calibration and testing. A two point linear calibration curve defining acceptable multiplexer voltage limits may be defined by the computer by determining the voltage output of the multiplexer to very accurately known input signals developed from predetermined current levels across the series resistances. Drift in the multiplexer may be detected by the computer when the output voltage limits, expected during normal operation, are exceeded, or the relationship defined by the calibration curve is invalidated.

  5. Microenvironmental Effect of 2'-O-(1-Pyrenylmethyl)uridine Modified Fluorescent Oligonucleotide Probes on Sensitive and Selective Detection of Target RNA.

    PubMed

    Imincan, Gülnur; Pei, Fen; Yu, Lijia; Jin, Hongwei; Zhang, Liangren; Yang, Xiaoda; Zhang, Lihe; Tang, XinJing

    2016-04-19

    2'-O-(1-Pyrenylmethyl)uridine modified oligoribonucleotides provide highly sensitive pyrene fluorescent probes for detecting specific nucleotide mutation of RNA targets. To develop more stable and cost-effective oligonucleotide probes, we investigated the local microenvironmental effects of nearby nucleobases on pyrene fluorescence in duplexes of RNAs and 2'-O-(1-pyrenylmethyl)uridine modified oligonucleotides. By incorporation of deoxyribonucleotides, ribonucleotides, 2'-MeO-nucleotides and 2'-F-nucleotides at both sides of 2'-O-(1-pyrenylmethyl)uridine (Up) in oligodeoxynucleotide probes, we synthesized a series of pyrene modified oligonucleotide probes. Their pyrene fluorescence emission spectra indicated that only two proximal nucleotides have a substantial effect on the pyrene fluorescence properties of these oligonucleotide probes hybridized with target RNA with an order of fluorescence sensitivity of 2'-F-nucleotides > 2'-MeO-nucleotides > ribonucleotides ≫ deoxyribonucleotides. While based on circular dichroism spectra, overall helix conformations (either A- or B-form) of the duplexes have marginal effects on the sensitivity of the probes. Instead, the local substitution reflected the propensity of the nucleotide sugar ring to adopt North type conformation and, accordingly, shifted their helix geometry toward a more A-type like conformation in local microenvironments. Thus, higher enhancement of pyrene fluorescence emission favored local A-type helix structures and more polar and hydrophobic environments (F > MeO > OH at 2' substitution) of duplex minor grooves of probes with the target RNA. Further dynamic simulation revealed that local microenvironmental effect of 2'-F-nucleotides or ribonucleotides was enough for pyrene moiety to move out of nucleobases to the minor groove of duplexes; in addition, 2'-F-nucleotide had less effect on π-stack of pyrene-modified uridine with upstream and downstream nucleobases. The present oligonucleotide probes

  6. Dual phase multiplex polymerase chain reaction

    DOEpatents

    Pemov, Alexander; Bavykin, Sergei

    2008-10-07

    Highly specific and sensitive methods were developed for multiplex amplification of nucleic acids on supports such as microarrays. Based on a specific primer design, methods include five types of amplification that proceed in a reaction chamber simultaneously. These relate to four types of multiplex amplification of a target DNA on a solid support, directed by forward and reverse complex primers immobilized to the support and a fifth type--pseudo-monoplex polymerase chain reaction (PCR) of multiple targets in solution, directed by a single pair of unbound universal primers. The addition of the universal primers in the reaction mixture increases the yield over the traditional "bridge" amplification on a solid support by approximately ten times. Methods that provide multitarget amplification and detection of as little as 0.45-4.5.times.10.sup.-12 g (equivalent to 10.sup.2-10.sup.3 genomes) of a bacterial genomic DNA are disclosed.

  7. Development of a multiplex assay to measure the effects of shipping and storage conditions on the quality of RNA used in molecular assays for detection of viral haemorrhagic septicemia virus.

    PubMed

    Siah, A; Duesund, H; Frisch, K; Nylund, A; McKenzie, P; Saksida, S

    2014-09-01

    Abstract In routine diagnostics, real-time reverse transcriptase quantitative PCR (RT-qPCR) has become a powerful method for fish health screening. Collection, transportation, and storage conditions of specimens could dramatically affect their integrity and could consequently affect RT-qPCR test results. In this study, to assess the expression profile of elongation factor 1 alpha (ELF-1α) gene, head kidney (HK) tissues from Atlantic Salmon Salmo salar were exposed at room temperature, 4°C, -20°C, and -80°C as well as in 70% ethanol for 6, 12, 24, 48, and 72 h. Data showed a significant increase of RT-qPCR cycle threshold (Ct) values for ELF-1α ranging from 14.7 to 26.5 cycles for tissues exposed to room temperature. In order to mimic the sample transportation conditions, different temperatures of storage were used and tissue quality was evaluated using ELF-1α gene expression. Data showed that Ct values for ELF-1α increased significantly when the tissues were transported on ice for 2 h, stored at -20°C, thawed on ice for 6 h, and stored again at -80°C. The HK tissues collected from Atlantic Salmon challenged with viral hemorrhagic septicemia virus (VHSV) through intraperitoneal injection were exposed at room temperature for 0, 6, 12, 24, 48, 72, and 96 h. Data showed a good correlation of values for ELF-1α and VHSV Ct although the ELF-1α mRNA of the host degraded faster than the RNA of VHSV. Based on these data, HK tissues could be transported on ice or ice packs without the quality of the tissue being affected when stored at -80°C upon arrival at the laboratory. In addition, 70% ethanol could be used as a preservative for long-distance transportation. For an efficient diagnostic test, a duplex VHSV-ELF-1α was developed and optimized. Data showed that the sensitivity of the duplex assay for VHSV was similar to the singleplex. Received November 25, 2013; accepted February 14, 2014. PMID:25229489

  8. Detecting respiratory viral RNA using expanded genetic alphabets and self-avoiding DNA.

    PubMed

    Glushakova, Lyudmyla G; Sharma, Nidhi; Hoshika, Shuichi; Bradley, Andrea C; Bradley, Kevin M; Yang, Zunyi; Benner, Steven A

    2015-11-15

    Nucleic acid (NA)-targeted tests detect and quantify viral DNA and RNA (collectively xNA) to support epidemiological surveillance and, in individual patients, to guide therapy. They commonly use polymerase chain reaction (PCR) and reverse transcription PCR. Although these all have rapid turnaround, they are expensive to run. Multiplexing would allow their cost to be spread over multiple targets, but often only with lower sensitivity and accuracy, noise, false positives, and false negatives; these arise by interactions between the multiple nucleic acid primers and probes in a multiplexed kit. Here we offer a multiplexed assay for a panel of respiratory viruses that mitigates these problems by combining several nucleic acid analogs from the emerging field of synthetic biology: (i) self-avoiding molecular recognition systems (SAMRSs), which facilitate multiplexing, and (ii) artificially expanded genetic information systems (AEGISs), which enable low-noise PCR. These are supplemented by "transliteration" technology, which converts standard nucleotides in a target to AEGIS nucleotides in a product, improving hybridization. The combination supports a multiplexed Luminex-based respiratory panel that potentially differentiates influenza viruses A and B, respiratory syncytial virus, severe acute respiratory syndrome coronavirus (SARS), and Middle East respiratory syndrome (MERS) coronavirus, detecting as few as 10 MERS virions in a 20-μl sample. PMID:26299645

  9. Characterization of a temperature-sensitive mutant of vaccinia virus reveals a novel function that prevents virus-induced breakdown of RNA.

    PubMed Central

    Pacha, R F; Condit, R C

    1985-01-01

    We have attempted to characterize the molecular defect in a temperature-sensitive mutant of vaccinia virus, ts22, which has an abortive late phenotype. At the nonpermissive temperature, ts22 displays normal viral protein synthesis until 8 h postinfection. Between 8 and 10 h after infection all viral protein synthesis ceases abruptly. Characterization of ts22 revealed that (i) primary transcription of late viral genes was not grossly impaired, (ii) late viral mRNA was biologically inactive since it could not stimulate in vitro protein synthesis, and (iii) extensive cleavage of rRNA and late viral mRNA occurred at the time that viral protein synthesis aborted in vivo. These data suggest that ts22 is defective in a function which prevents host rRNA and viral mRNA from being degraded. Inhibitor studies with cytosine arabinoside and cycloheximide showed that induction of and protection from rRNA breakdown occurred at approximately the same time during infection and required late viral gene expression. The viral protein synthesis pattern observed in vaccinia virus-infected cells treated with the drug isatin-beta-thiosemicarbazone was strikingly similar to that observed in ts22-infected cells at the nonpermissive temperature (J. Cooper, B. Moss, and E. Katz, Virology 96:381-392, 1979). Analysis of rRNA integrity in isatin-beta-thiosemicarbazone-treated, vaccinia virus-infected cells revealed extensive cleavage of rRNA, suggesting that the ts22 and drug inhibitor may function in the same pathway. Images PMID:4057355

  10. 3D multiplexed immunoplasmonics microscopy

    NASA Astrophysics Data System (ADS)

    Bergeron, Éric; Patskovsky, Sergiy; Rioux, David; Meunier, Michel

    2016-07-01

    Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K+ channel subunit KV1.1) on human cancer CD44+ EGFR+ KV1.1+ MDA-MB-231 cells and reference CD44- EGFR- KV1.1+ 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third, the developed

  11. Coupling Specificity of NOP Opioid Receptors to Pertussis-Toxin-Sensitive Gα Proteins in Adult Rat Stellate Ganglion Neurons Using Small Interference RNA

    PubMed Central

    Margas, Wojciech; Sedeek, Khaled; Ruiz-Velasco, Victor

    2008-01-01

    The opioid receptor-like 1 (NOP or ORL1) receptor is a G-protein-coupled receptor the endogenous ligand of which is the heptadecapeptide, nociceptin (Noc). NOP receptors are known to modulate pain processing at spinal, supraspinal, and peripheral levels. Previous work has demonstrated that NOP receptors inhibit N-type Ca2+ channel currents in rat sympathetic stellate ganglion (SG) neurons via pertussis toxin (PTX)-sensitive Gαi/o subunits. However, the identification of the specific Gα subunit that mediates the Ca2+ current modulation is unknown. The purpose of the present study was to examine coupling specificity of Noc-activated NOP receptors to N-type Ca2+ channels in SG neurons. Small interference RNA (siRNA) transfection was employed to block the expression of PTX-sensitive Gα subunits. RT-PCR results showed that siRNA specifically decreased the expression of the intended Gα subunit. Evaluation of cell surface protein expression and Ca2+ channel modulation were assessed by immunofluorescence staining and electrophysiological recordings, respectively. Furthermore, the presence of mRNA of the intended siRNA target Gα protein was examined by RT-PCR experiments. Fluorescence imaging showed that Gαi1, Gαi3, and Gαo were expressed in SG neurons. The transfection of Gαi1-specific siRNA resulted in a significant decrease in Noc-mediated Ca2+ current inhibition, while silencing of either Gαi3 or Gαo was without effect. Taken together, these results suggest that in SG neurons Gαi1 subunits selectively couple NOP receptors to N-type Ca2+ channels. PMID:18562551

  12. Expression of microRNA-30a-5p in drug-resistant and drug-sensitive ovarian cancer cell lines

    PubMed Central

    Liu, Jin; Wu, Xiaohua; Liu, Hongmei; Liang, Yijuan; Gao, Xinping; Cai, Zhihui; Wang, Weiming; Zhang, Hui

    2016-01-01

    The present study aimed to explore the expression of microRNA (miRNA or miR) in drug-resistant and drug-sensitive ovarian cancer cell lines, and to seek the potential therapeutic target of ovarian cancer drug-resistant mechanism in order to improve drug resistance by altering miRNA levels. The drug-resistant characteristics of SKOV3/DDP, SKOV3, COC1/DDP and COC1 cell lines were studied. The miRNAs that were differentially expressed between cisplatin-resistant cells and its parental cells in ovarian cancer were screened with a miRNA chip. The effect of miRNAs was detected, and their drug-resistant mechanism was investigated by transfection and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide methods. Among the expression screening of miRNAs, 41 mRNAs, including Homo sapiens (hsa)-miR-30a-5p and hsa-miR-34c-5p, were highly expressed in the drug-resistant cells, whereas 44 miRNAs, including hsa-miR-96-5p and hsa-miR-200c-3p, were lowly expressed. The expression levels of hsa-miR-30a-5p in two types of ovarian cancer chemotherapy-resistant cell lines were significantly higher than those in chemotherapy-sensitive cell lines, which was associated with ovarian cancer chemotherapy resistance. In conclusion, high expression of miRNA-30a-5p was able to promote cell growth and colony forming ability, and enhance cell migration and invasion. Thus, miRNA-30a-5p is expected to become a meaningful novel target for ovarian cancer resistant treatment. PMID:27602140

  13. A multiplex real-time PCR panel assay for simultaneous detection and differentiation of 12 common swine viruses.

    PubMed

    Shi, Xiju; Liu, Xuming; Wang, Qin; Das, Amaresh; Ma, Guiping; Xu, Lu; Sun, Qing; Peddireddi, Lalitha; Jia, Wei; Liu, Yanhua; Anderson, Gary; Bai, Jianfa; Shi, Jishu

    2016-10-01

    Mixed infection with different pathogens is common in swine production systems especially under intensive production conditions. Quick and accurate detection and differentiation of different pathogens are necessary for epidemiological surveillance, disease management and import and export controls. In this study, we developed and validated a panel of multiplex real-time PCR/RT-PCR assays composed of four subpanels, each detects three common swine pathogens. The panel detects 12 viruses or viral serotypes, namely, VSV-IN, VSV-NJ, SVDV, CSFV, ASFV, FMDV, PCV2, PPV, PRV, PRRSV-NA, PRRSV-EU and SIV. Correlation coefficients (R(2)) and PCR amplification efficiencies of all singular and triplex real-time PCR reactions are within the acceptable range. Comparison between singular and triplex real-time PCR assays of each subpanel indicates that there is no significant interference on assay sensitivities caused by multiplexing. Specificity tests on 226 target clinical samples or 4 viral strains and 91 non-target clinical samples revealed that the real-time PCR panel is 100% specific, and there is no cross amplification observed. The limit of detection of each triplex real-time PCR is less than 10 copies per reaction for DNA, and less than 16 copies per reaction for RNA viruses. The newly developed multiplex real-time PCR panel also detected different combinations of co-infections as confirmed by other means of detections. PMID:27506582

  14. Multiplexed capillary electrophoresis system

    DOEpatents

    Yeung, Edward S.; Li, Qingbo; Lu, Xiandan

    1998-04-21

    The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification ("base calling") is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations.

  15. Multiplexed capillary electrophoresis system

    DOEpatents

    Yeung, Edward S.; Chang, Huan-Tsang; Fung, Eliza N.; Li, Qingbo; Lu, Xiandan

    1996-12-10

    The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification ("base calling") is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations.

  16. Multiplexed capillary electrophoresis system

    DOEpatents

    Yeung, E.S.; Li, Q.; Lu, X.

    1998-04-21

    The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification (``base calling``) is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations. 19 figs.

  17. Multiplexed capillary electrophoresis system

    DOEpatents

    Yeung, E.S.; Chang, H.T.; Fung, E.N.; Li, Q.; Lu, X.

    1996-12-10

    The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification (``base calling``) is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations. 19 figs.

  18. Hardware Counter Multiplexing

    Energy Science and Technology Software Center (ESTSC)

    2000-10-13

    The Hardware Counter Multiplexer works with the built-in counter registers on computer processors. These counters record various low-level events as software runs, but they can not record all possible events at the same time. This software helps work around that limitation by counting a series of different events in sequence over a period of time. This in turn allows programmers to measure interesting combinations of events, rather than single events. The software is designed tomore » work with multithreaded or single-threaded programs.« less

  19. Multiplex Detection and SNP Genotyping in a Single Fluorescence Channel

    PubMed Central

    Fu, Guoliang; Miles, Andrea; Alphey, Luke

    2012-01-01

    Probe-based PCR is widely used for SNP (single nucleotide polymorphism) genotyping and pathogen nucleic acid detection due to its simplicity, sensitivity and cost-effectiveness. However, the multiplex capability of hydrolysis probe-based PCR is normally limited to one target (pathogen or allele) per fluorescence channel. Current fluorescence PCR machines typically have 4–6 channels. We present a strategy permitting the multiplex detection of multiple targets in a single detection channel. The technique is named Multiplex Probe Amplification (MPA). Polymorphisms of the CYP2C9 gene (cytochrome P450, family 2, subfamily C, polypeptide 9, CYP2C9*2) and human papillomavirus sequences HPV16, 18, 31, 52 and 59 were chosen as model targets for testing MPA. The allele status of the CYP2C9*2 determined by MPA was entirely concordant with the reference TaqMan® SNP Genotyping Assays. The four HPV strain sequences could be independently detected in a single fluorescence detection channel. The results validate the multiplex capacity, the simplicity and accuracy of MPA for SNP genotyping and multiplex detection using different probes labeled with the same fluorophore. The technique offers a new way to multiplex in a single detection channel of a closed-tube PCR. PMID:22272339

  20. Multiplex detection and SNP genotyping in a single fluorescence channel.

    PubMed

    Fu, Guoliang; Miles, Andrea; Alphey, Luke

    2012-01-01

    Probe-based PCR is widely used for SNP (single nucleotide polymorphism) genotyping and pathogen nucleic acid detection due to its simplicity, sensitivity and cost-effectiveness. However, the multiplex capability of hydrolysis probe-based PCR is normally limited to one target (pathogen or allele) per fluorescence channel. Current fluorescence PCR machines typically have 4-6 channels. We present a strategy permitting the multiplex detection of multiple targets in a single detection channel. The technique is named Multiplex Probe Amplification (MPA). Polymorphisms of the CYP2C9 gene (cytochrome P450, family 2, subfamily C, polypeptide 9, CYP2C9*2) and human papillomavirus sequences HPV16, 18, 31, 52 and 59 were chosen as model targets for testing MPA. The allele status of the CYP2C9*2 determined by MPA was entirely concordant with the reference TaqMan® SNP Genotyping Assays. The four HPV strain sequences could be independently detected in a single fluorescence detection channel. The results validate the multiplex capacity, the simplicity and accuracy of MPA for SNP genotyping and multiplex detection using different probes labeled with the same fluorophore. The technique offers a new way to multiplex in a single detection channel of a closed-tube PCR. PMID:22272339

  1. Shift-multiplexing complex spectral-domain optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Huang, Haochong; Jiang, Zhuqing; Wang, Dayong; Cai, Wenyuan; Man, Tianlong; Wang, Zhe; Panezai, Spozmai

    2014-01-01

    We propose and experimentally demonstrate a shift-multiplexing complex spectral-domain optical coherence tomography (shift-multiplexing CSD-OCT) method, in which the maximum detection depth of SD-OCT can be greatly extended by incorporating the shift-multiplexing of detection positions with CSD-OCT. The tomographic imaging with twofold or threefold microscopic slides as the target sample is performed. The experimental results show that the tomographic imaging with more uniform brightness and clarity for the different depth regions in a thick sample can be achieved by the shift-multiplexing CSD-OCT system. In particular, even while the sample's depth is beyond the maximum imaging depth of CSD-OCT system, the tomographic imaging of this sample can still be realized by using the shift-multiplexing CSD-OCT method without the need for any replacement of the equipment, such as high spectral capacity grating or high resolution of CCD. The shift-multiplexing CSD-OCT system can perform the imaging with the optimization and less reduction of sensitivity for the deeper detection position in the sample.

  2. The use of pH-sensitive functional selenium nanoparticles shows enhanced in vivo VEGF-siRNA silencing and fluorescence imaging

    NASA Astrophysics Data System (ADS)

    Yu, Qianqian; Liu, Yanan; Cao, Chengwen; Le, Fangling; Qin, Xiuying; Sun, Dongdong; Liu, Jie

    2014-07-01

    The utility of small interfering RNAs (siRNAs) has shown great promise in treating a variety of diseases including many types of cancer. While their ability to silence a wide range of target genes underlies their effectiveness, the application of therapies remains hindered by a lack of an effective delivery system. In this study, we sought to develop an siRNA-delivery system for VEGF, a known signaling molecule involved in cancer, that consists of two selenium nanoparticles SeNPs and G2/PAH-Cit/SeNPs. A G2/PAH-Cit/SeNP is a pH-sensitive delivery system that is capable of enhancing siRNA loading, thus increasing siRNA release efficiency and subsequent target gene silencing both in vitro and in vivo. In vivo experiments using G2/PAH-Cit/SeNPs@siRNA led to significantly higher accumulation of siRNA within the tumor itself, VEGF gene silencing, and reduced angiogenesis in the tumor. Furthermore, the G2/PAH-Cit/SeNP delivery system not only enhanced anti-tumor effects on tumor-bearing nude mice as compared to SeNPs@siRNA, but also resulted in weak occurrence of lesions in major target organs. In sum, this study provides a new class of siRNA delivery system, thereby providing an alternative therapeutic route for cancer treatment.The utility of small interfering RNAs (siRNAs) has shown great promise in treating a variety of diseases including many types of cancer. While their ability to silence a wide range of target genes underlies their effectiveness, the application of therapies remains hindered by a lack of an effective delivery system. In this study, we sought to develop an siRNA-delivery system for VEGF, a known signaling molecule involved in cancer, that consists of two selenium nanoparticles SeNPs and G2/PAH-Cit/SeNPs. A G2/PAH-Cit/SeNP is a pH-sensitive delivery system that is capable of enhancing siRNA loading, thus increasing siRNA release efficiency and subsequent target gene silencing both in vitro and in vivo. In vivo experiments using G2/PAH-Cit/SeNPs@siRNA

  3. microRNA-7 Suppresses the Invasive Potential of Breast Cancer Cells and Sensitizes Cells to DNA Damages by Targeting Histone Methyltransferase SET8*

    PubMed Central

    Yu, Na; Huangyang, Peiwei; Yang, Xiaohan; Han, Xiao; Yan, Ruorong; Jia, Hongti; Shang, Yongfeng; Sun, Luyang

    2013-01-01

    SET8 (SET domain containing 8) is a histone H4 lysine 20 (H4K20)-specific monomethyltransferase in higher eukaryotes that exerts diverse functions in transcription regulation, DNA repair, tumor metastasis, and genome integrity. The activity of SET8 is tightly controlled during cell cycle through post-translational modifications, including ubiquitination, phosphorylation, and sumoylation. However, how the expression of SET8 is regulated is not fully understood. Here, we report that microRNA-7 is a negative regulator of SET8. We demonstrated that microRNA-7 inhibits H4K20 monomethylation and suppresses epithelial-mesenchymal transition and the invasive potential of breast cancer cells. We showed that microRNA-7 promotes spontaneous DNA damages and sensitizes cells to induced DNA damages. Our experiments provide a molecular mechanism for the regulation of SET8 and extend the biological function of microRNA-7 to DNA damage response, supporting the pursuit of microRNA-7 as a potential target for breast cancer intervention. PMID:23720754

  4. Matrix Metalloproteinase 2-sensitive Multifunctional Polymeric Micelles for Tumor-specific Co-delivery of siRNA and Hydrophobic Drugs

    PubMed Central

    Zhu, Lin; Perche, Federico; Wang, Tao; Torchilin, Vladimir P

    2014-01-01

    Co-delivery of hydrophilic siRNA and hydrophobic drugs is one of the major challenges for nanomaterial-based medicine. Here, we present a simple but multifunctional micellar platform constructed by a matrix metalloproteinase 2 (MMP2)-sensitive copolymer (PEG-pp-PEI-PE) via self-assembly for tumor-targeted siRNA and drug co-delivery. The micellar nanocarrier possesses several key features for siRNA and drug delivery, including (i) excellent stability; (ii) efficient siRNA condensation by PEI; (iii) hydrophobic drug solubilization in the lipid “core”; (iv) passive tumor targeting via the enhanced permeability and retention (EPR) effect; (v) tumor targeting triggered by the up-regulated tumoral MMP2; and (vi) enhanced cell internalization after MMP2-activated exposure of the previously hidden PEI. These cooperative functions ensure the improved tumor targetability, enhanced tumor cell internalization, and synergistic antitumor activity of co-loaded siRNA and drug. PMID:24529391

  5. SBP2 binding affinity is a major determinant in differential selenoprotein mRNA translation and sensitivity to nonsense-mediated decay.

    PubMed

    Squires, Jeffrey E; Stoytchev, Ilko; Forry, Erin P; Berry, Marla J

    2007-11-01

    Selenoprotein mRNAs are potential targets for degradation via nonsense-mediated decay due to the presence of in-frame UGA codons that can be decoded as either selenocysteine or termination codons. When UGA decoding is inefficient, as occurs when selenium is limiting, termination occurs at these positions. Based on the predicted exon-intron structure, 14 of the 25 human selenoprotein mRNAs are predicted to be sensitive to nonsense-mediated decay. Among these, sensitivity varies widely, resulting in a hierarchy of preservation or degradation of selenoprotein mRNAs and, thus, of selenoprotein synthesis. Potential factors in dictating the hierarchy of selenoprotein synthesis are the Sec insertion sequence RNA-binding proteins, SBP2 and nucleolin. To investigate the mechanistic basis for this hierarchy and the role of these two proteins, we carried out knockdowns of SBP2 expression and assessed the effects on selenoprotein mRNA levels. We also investigated in vivo binding of selenoprotein mRNAs by SBP2 and nucleolin via immunoprecipitation of the proteins and quantitation of bound mRNAs. We report that SBP2 exhibits strong preferential binding to some selenoprotein mRNAs over others, whereas nucleolin exhibits minimal differences in binding. Thus, SBP2 is a major determinant in dictating the hierarchy of selenoprotein synthesis via differential selenoprotein mRNA translation and sensitivity to nonsense-mediated decay. PMID:17846120

  6. Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses.

    PubMed

    Simmons, Monika; Myers, Todd; Guevara, Carolina; Jungkind, Donald; Williams, Maya; Houng, Huo-Shu

    2016-07-01

    Dengue virus (DENV) and chikungunya virus (CHIKV) are important human pathogens with common transmission vectors and similar clinical presentations. Patient care may be impacted by the misdiagnosis of DENV and CHIKV in areas where both viruses cocirculate. In this study, we have developed and validated a one-step multiplex reverse transcriptase PCR (RT-PCR) to simultaneously detect, quantify, and differentiate between four DENV serotypes (pan-DENV) and chikungunya virus. The assay uses TaqMan technology, employing two forward primers, three reverse primers, and four fluorophore-labeled probes in a single-reaction format. Coextracted and coamplified RNA was used as an internal control (IC), and in vitro-transcribed DENV and CHIKV RNAs were used to generate standard curves for absolute quantification. The diagnostic 95% limits of detection (LOD) within the linear range were 50 and 60 RNA copies/reaction for DENV (serotypes 1 to 4) and CHIKV, respectively. Our assay was able to detect 53 different strains of DENV, representing four serotypes, and six strains of CHIKV. No cross-reactivity was observed with related flaviviruses and alphaviruses, To evaluate diagnostic sensitivity and specificity, 89 clinical samples positive or negative for DENV (serotypes 1 to 4) and CHIKV by the standard virus isolation method were tested in our assay. The multiplex RT-PCR assay showed 95% sensitivity and 100% specificity for DENV and 100% sensitivity and specificity for CHIKV. With an assay turnaround time of less than 2 h, including extraction of RNA, the multiplex quantitative RT-PCR assay provides rapid diagnosis for the differential detection of two clinically indistinguishable diseases, whose geographical occurrence is increasingly overlapping. PMID:27098955

  7. Quantitative multiplexed quantum dot immunohistochemistry

    SciTech Connect

    Sweeney, E.; Ward, T.H.; Gray, N.; Womack, C.; Jayson, G.; Hughes, A.; Dive, C.; Byers, R.

    2008-09-19

    Quantum dots are photostable fluorescent semiconductor nanocrystals possessing wide excitation and bright narrow, symmetrical, emission spectra. These characteristics have engendered considerable interest in their application in multiplex immunohistochemistry for biomarker quantification and co-localisation in clinical samples. Robust quantitation allows biomarker validation, and there is growing need for multiplex staining due to limited quantity of clinical samples. Most reported multiplexed quantum dot staining used sequential methods that are laborious and impractical in a high-throughput setting. Problems associated with sequential multiplex staining have been investigated and a method developed using QDs conjugated to biotinylated primary antibodies, enabling simultaneous multiplex staining with three antibodies. CD34, Cytokeratin 18 and cleaved Caspase 3 were triplexed in tonsillar tissue using an 8 h protocol, each localised to separate cellular compartments. This demonstrates utility of the method for biomarker measurement enabling rapid measurement of multiple co-localised biomarkers on single paraffin tissue sections, of importance for clinical trial studies.

  8. Suppressor analysis of temperature-sensitive mutations of the largest subunit of RNA polymerase I in Saccharomyces cerevisiae: a suppressor gene encodes the second-largest subunit of RNA polymerase I.

    PubMed Central

    Yano, R; Nomura, M

    1991-01-01

    The SRP3-1 mutation is an allele-specific suppressor of temperature-sensitive mutations in the largest subunit (A190) of RNA polymerase I from Saccharomyces cerevisiae. Two mutations known to be suppressed by SRP3-1 are in the putative zinc-binding domain of A190. We have cloned the SRP3 gene by using its suppressor activity and determined its complete nucleotide sequence. We conclude from the following evidence that the SRP3 gene encodes the second-largest subunit (A135) of RNA polymerase I. First, the deduced amino acid sequence of the gene product contains several regions with high homology to the corresponding regions of the second-largest subunits of RNA polymerases of various origins, including those of RNA polymerase II and III from S. cerevisiae. Second, the deduced amino acid sequence contains known amino acid sequences of two tryptic peptides from the A135 subunit of RNA polymerase I purified from S. cerevisiae. Finally, a strain was constructed in which transcription of the SRP3 gene was controlled by the inducible GAL7 promoter. When this strain, which can grow on galactose but not on glucose, was shifted from galactose medium to glucose medium, a large decrease in the cellular concentration of A135 was observed by Western blot analysis. We have also identified the specific amino acid alteration responsible for suppression by SRP3-1 and found that it is located within the putative zinc-binding domain conserved among the second-largest subunits of eucaryotic RNA polymerases. From these results, it is suggested that this putative zinc-binding domain is in physical proximity to and interacts with the putative zinc-binding domain of the A190 subunit. Images PMID:1990281

  9. Highspeed multiplexed heterodyne interferometry.

    PubMed

    Isleif, Katharina-S; Gerberding, Oliver; Köhlenbeck, Sina; Sutton, Andrew; Sheard, Benjamin; Goßler, Stefan; Shaddock, Daniel; Heinzel, Gerhard; Danzmann, Karsten

    2014-10-01

    Digitally enhanced heterodyne interferometry is a metrology technique that uses pseudo-random noise codes for modulating the phase of the laser light. Multiple interferometric signals from the same beam path can thereby be isolated based on their propagation delay, allowing one to use advantageous optical layouts in comparison to classic laser interferometers. We present here a high speed version of this technique for measuring multiple targets spatially separated by only a few centimetres. This allows measurements of multiplexed signals using free beams, making the technique attractive for several applications requiring compact optical set-ups like for example space-based interferometers. In an experiment using a modulation and sampling rate of 1.25 GHz we are able to demonstrate multiplexing between targets only separated by 36 cm and we achieve a displacement measurement noise floor of <3 pm/√Hz at 10 Hz between them. We identify a limiting excess noise at low frequencies which is unique to this technique and is probably caused by the finite bandwidth in our measurement set-up. Utilising an active clock jitter correction scheme we are also able to reduce this noise in a null measurement configuration by one order of magnitude. PMID:25322043

  10. Scalable multiplexing for parallel imaging with interleaved optical coherence tomography.

    PubMed

    Lee, Hee Yoon; Marvdashti, Tahereh; Duan, Lian; Khan, Saara A; Ellerbee, Audrey K

    2014-09-01

    We demonstrate highly parallel imaging with interleaved optical coherence tomography (iOCT) using an in-house-fabricated, air-spaced virtually-imaged phased array (VIPA). The air-spaced VIPA performs spectral encoding of the interferograms from multiple lateral points within a single sweep of the source and allows us to tune and balance several imaging parameters: number of multiplexed points, ranging depth, and sensitivity. In addition to a thorough discussion of the parameters and operating principles of the VIPA, we experimentally demonstrate the effect of different VIPA designs on the multiplexing potential of iOCT. Using a 200-kHz light source, we achieve an effective A-scan rate of 3.2-MHz by multiplexing 16 lateral points onto a single wavelength sweep. The improved sensitivity of this system is demonstrated for 3D imaging of biological samples such as a human finger and a fruit fly. PMID:25401031

  11. Rare Earth core/shell nanobarcodes for multiplexed trace biodetection.

    PubMed

    Chen, Lei; Li, Xiaomin; Shen, Dengke; Zhou, Lei; Zhu, Dan; Fan, Chunhai; Zhang, Fan

    2015-06-01

    Multiplexed detection technology has been attractive for its simultaneous assay of several analytes, which play significant roles in applications such as screening for combinatorial chemistry, genetic analysis, and clinical diagnostics. This work reports a novel and potentially powerful encoding system based upon dispersible suspension arrays of multilayer rare earth core/shell nanoparticles that are capable of multiplexed, high-sensitivity reporting for biomolecule detection by the Z-contrast imaging. These nanobarcode arrays are encoded by nanostructure design based on different atomic numbers. With the well-resolved high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) decoding technique, many thousands of unique nanobarcodes can be identified by multilayer core/shell nanostructure. Their applications to multiplexed biodetection of DNA demonstrated the highly sensitive (picomole) features of this novel nanobarcode system. PMID:25951110

  12. Scalable multiplexing for parallel imaging with interleaved optical coherence tomography

    PubMed Central

    Lee, Hee Yoon; Marvdashti, Tahereh; Duan, Lian; Khan, Saara A.; Ellerbee, Audrey K.

    2014-01-01

    We demonstrate highly parallel imaging with interleaved optical coherence tomography (iOCT) using an in-house-fabricated, air-spaced virtually-imaged phased array (VIPA). The air-spaced VIPA performs spectral encoding of the interferograms from multiple lateral points within a single sweep of the source and allows us to tune and balance several imaging parameters: number of multiplexed points, ranging depth, and sensitivity. In addition to a thorough discussion of the parameters and operating principles of the VIPA, we experimentally demonstrate the effect of different VIPA designs on the multiplexing potential of iOCT. Using a 200-kHz light source, we achieve an effective A-scan rate of 3.2-MHz by multiplexing 16 lateral points onto a single wavelength sweep. The improved sensitivity of this system is demonstrated for 3D imaging of biological samples such as a human finger and a fruit fly. PMID:25401031

  13. shRNA Depletion of cIAP1 Sensitizes Human Ovarian Cancer Cells to Anticancer Agent-Induced Apoptosis.

    PubMed

    Jin, Hong; Dong, You-Yuan; Zhang, Hong; Cui, Ying; Xie, Kai; Lou, Ge

    2014-01-01

    Emerging evidence suggests a potential role of cellular inhibitor of apoptosis protein 1 (cIAP1) in the development of human ovarian cancer. However, its function in the progression of ovarian cancer has not been clearly determined. Our study aimed to investigate the effect of cIAP1 gene depletion on the chemosensitivity of ovarian cancer cells. We developed a novel short hairpin RNA (shRNA) plasmid specifically targeting cIAP1. Cell proliferation, invasion, and apoptosis of the shRNA-transfected cells were evaluated using MTT, Transwell chamber, and flow cytometric assays, respectively. The concentration of MMP-9 in the supernatant was detected by ELISA. Targeted depletion of cIAP1 by shRNA significantly reduced expression levels of cIAP1 mRNA and protein, leading to inhibition of cell proliferation and invasion capability in SKOV3 cells. At the same time, cIAP1 downregulation decreased the secretion of MMP-9. shRNA depletion of cIAP1 enhanced chemosensitivity of ovarian cancer cells to Taxol and carboplatin-induced apoptosis. cIAP1 is associated with tumor progression in human ovarian cancer. Therefore, cIAP1 might be a potential target for therapeutic anticancer drugs. PMID:26168135

  14. Multiplex PCR: Optimization and Application in Diagnostic Virology

    PubMed Central

    Elnifro, Elfath M.; Ashshi, Ahmed M.; Cooper, Robert J.; Klapper, Paul E.

    2000-01-01

    PCR has revolutionized the field of infectious disease diagnosis. To overcome the inherent disadvantage of cost and to improve the diagnostic capacity of the test, multiplex PCR, a variant of the test in which more than one target sequence is amplified using more than one pair of primers, has been developed. Multiplex PCRs to detect viral, bacterial, and/or other infectious agents in one reaction tube have been described. Early studies highlighted the obstacles that can jeopardize the production of sensitive and specific multiplex assays, but more recent studies have provided systematic protocols and technical improvements for simple test design. The most useful of these are the empirical choice of oligonucleotide primers and the use of hot start-based PCR methodology. These advances along with others to enhance sensitivity and specificity and to facilitate automation have resulted in the appearance of numerous publications regarding the application of multiplex PCR in the diagnosis of infectious agents, especially those which target viral nucleic acids. This article reviews the principles, optimization, and application of multiplex PCR for the detection of viruses of clinical and epidemiological importance. PMID:11023957

  15. A novel electrically magnetic-controllable electrochemical biosensor for the ultra sensitive and specific detection of attomolar level oral cancer-related microRNA.

    PubMed

    Wang, ZongWen; Zhang, Jing; Guo, Ying; Wu, XiaoYan; Yang, WeiJuan; Xu, LiangJun; Chen, JingHua; Fu, FengFu

    2013-07-15

    Non-invasive early diagnosis of oral cancer is the most effective means to reduce mortality rate from this disease. In this paper, we described a novel magnetic-controllable electrochemical RNA biosensor for the ultra sensitive and specific detection of oral cancer-related microRNA (miRNA) based on a home-made electrically magnetic-controllable gold electrode. The electrically magnetic-controllable gold electrode combined the merits of heated electrode and magnetic electrode, has notable advantage such as that the strength and direction of the magnetic field and the temperature of the electrode's surface can be easily regulated. The advantage of electrically magnetic-controllable gold electrode, as well as the utilization of "junction-probe" strategy and magnetic beads (MBs)-based enzymatic catalysis amplification, make the biosensor has ultra-high sensitivity and discrimination ability even for the detection of similar miRNAs. It can be used to detect as low as 0.22 aM (2.2 × 10(-19)M) of oral cancer-related miRNA with a recovery of 93-108% and a RSD<6 (n=5). The high sensitivity and selectivity, as well as the easiness of fabrication, operational convenience, short analysis time, good stability and re-usability, make the biosensor a promising alternative for the early point-of-care diagnosis of oral cancer. The success of the biosensor also leads to a great potential in the development of biosensor for the early diagnosis of other diseases. PMID:23455049

  16. Site-specific fluorescence dynamics in an RNA ‘thermometer’ reveals the role of ribosome binding in its temperature-sensitive switch function

    PubMed Central

    Narayan, Satya; Kombrabail, Mamta H.; Das, Sudipta; Singh, Himanshu; Chary, Kandala V. R.; Rao, Basuthkar J.; Krishnamoorthy, Guruswamy

    2015-01-01

    RNA thermometers control the translation of several heat shock and virulence genes by their temperature-sensitive structural transitions. Changes in the structure and dynamics of MiniROSE RNA, which regulates translation in the temperature range of 20–45°C, were studied by site specifically replacing seven adenine residues with the fluorescent analog, 2-aminopurine (2-AP), one at a time. Dynamic fluorescence observables of 2-AP-labeled RNAs were compared in their free versus ribosome-bound states for the first time. Noticeably, position dependence of fluorescence observables, which was prominent at 20°C, was persistent even at 45ºC, suggesting the persistence of structural integrity up to 45ºC. Interestingly, position-dependent dispersion of fluorescence lifetime and quenching constant at 45°C was ablated in ribosome-bound state, when compared to those at 20°C, underscoring loss of structural integrity at 45°C, in ribosome-bound RNA. Significant increase in the value of mean lifetime for 2-AP corresponding to Shine–Dalgarno sequences, when the temperature was raised from 20 to 45°C, to values seen in the presence of urea at 45°C was a strong indicator of melting of the 3D structure of MiniROSE RNA at 45°C, only when it was ribosome bound. Taken all together, we propose a model where we invoke that ribosome binding of the RNA thermometer critically regulates temperature sensing functions in MiniROSE RNA. PMID:25477380

  17. Delayed Correlation of mRNA and Protein Expression in Rapamycin-treated Cells and a Role for Ggc1 in Cellular Sensitivity to Rapamycin*

    PubMed Central

    Fournier, Marjorie L.; Paulson, Ariel; Pavelka, Norman; Mosley, Amber L.; Gaudenz, Karin; Bradford, William D.; Glynn, Earl; Li, Hua; Sardiu, Mihaela E.; Fleharty, Brian; Seidel, Christopher; Florens, Laurence; Washburn, Michael P.

    2010-01-01

    To identify new molecular targets of rapamycin, an anticancer and immunosuppressive drug, we analyzed temporal changes in yeast over 6 h in response to rapamycin at the transcriptome and proteome levels and integrated the expression patterns with functional profiling. We show that the integration of transcriptomics, proteomics, and functional data sets provides novel insights into the molecular mechanisms of rapamycin action. We first observed a temporal delay in the correlation of mRNA and protein expression where mRNA expression at 1 and 2 h correlated best with protein expression changes after 6 h of rapamycin treatment. This was especially the case for the inhibition of ribosome biogenesis and induction of heat shock and autophagy essential to promote the cellular sensitivity to rapamycin. However, increased levels of vacuolar protease could enhance resistance to rapamycin. Of the 85 proteins identified as statistically significantly changing in abundance, most of the proteins that decreased in abundance were correlated with a decrease in mRNA expression. However, of the 56 proteins increasing in abundance, 26 were not correlated with an increase in mRNA expression. These protein changes were correlated with unchanged or down-regulated mRNA expression. These proteins, involved in mitochondrial genome maintenance, endocytosis, or drug export, represent new candidates effecting rapamycin action whose expression might be post-transcriptionally or post-translationally regulated. We identified GGC1, a mitochondrial GTP/GDP carrier, as a new component of the rapamycin/target of rapamycin (TOR) signaling pathway. We determined that the protein product of GGC1 was stabilized in the presence of rapamycin, and the deletion of the GGC1 enhanced growth fitness in the presence of rapamycin. A dynamic mRNA expression analysis of Δggc1 and wild-type cells treated with rapamycin revealed a key role for Ggc1p in the regulation of ribosome biogenesis and cell cycle progression

  18. Information multiplexing in ptychography.

    PubMed

    Batey, Darren J; Claus, Daniel; Rodenburg, John M

    2014-03-01

    We show for the first time that ptychography (a form of lensless diffractive imaging) can recover the spectral response of an object through simultaneous reconstruction of multiple images that represent the object's response to a particular mode present in the illumination. We solve the phase problem for each mode independently, even though the intensity arriving at every detector pixel is an incoherent superposition of several uncorrelated diffracted waves. Until recently, the addition of incoherent modes has been seen as a nuisance in diffractive imaging: here we show that not only can the difficulties they pose be removed, but that they can also be used to discover much more information about the object. If the illumination function is also mode-specific, we show that we can also solve simultaneously for a multiplicity of such illumination modes. The work opens exciting possibilities for information multiplexing in ptychography over all visible, X-ray and electron wavelengths. PMID:24413077

  19. Detection of carbapenemases in Enterobacteriaceae by a commercial multiplex PCR.

    PubMed

    Kaase, Martin; Szabados, Florian; Wassill, Lars; Gatermann, Sören G

    2012-09-01

    A commercial multiplex PCR (hyplex SuperBug ID) was tested with a collection of 132 clinical Enterobacteriaceae strains producing different carbapenemases. The sensitivity for the detection of KPC-, VIM-, NDM-, and OXA-48-encoding genes was 100%, whereas two IMP variants were missed. PMID:22785190

  20. Efficient detection of Alport syndrome COL4A5 mutations with multiplex genomic PCR-SSCP.

    PubMed

    Barker, D F; Denison, J C; Atkin, C L; Gregory, M C

    2001-01-15

    We have performed effective mutation screening of COL4A5 with a new method of direct, multiplex genomic amplification that employs a single buffer condition and PCR profile. Application of the method to a consecutive series of 46 United States patients with diverse indications of Alport syndrome resulted in detection of mutations in 31 cases and of five previously unreported polymorphisms. With a correction for the presence of cases that are not likely to be due to changes at the COL4A5 locus, the mutation detection sensitivity is greater than 79%. The test examines 52 segments, including the COL4A6/COL4A5 intergenic promoter region, all 51 of the previously recognized exons and two newly detected exons between exons 41 and 42 that encode an alternatively spliced mRNA segment. New genomic sequence information was generated and used to design primer pairs that span substantial intron sequences on each side of all 53 exons. For SSCP screening, 16 multiplex PCR combinations (15 4-plex and 1 3-plex) were used to provide complete, partially redundant coverage of the gene. The selected combinations allow clear resolution of products from each segment using various SSCP gel formulations. One of the 29 different mutations detected initially seemed to be a missense change in exon 32 but was found to cause exon skipping. Another missense variant may mark a novel functional site located in the collagenous domain. PMID:11223851

  1. Specific identification of chicken and soybean fraud in premium burgers using multiplex-PCR method.

    PubMed

    Tafvizi, Farzaneh; Hashemzadegan, Masumeh

    2016-01-01

    The increased consumption of meat products, such as hamburger in large cities such as Tehran, has highlighted the importance of quality control for these products. Due to the escalating cost of red meat, and the difficulty of detecting adulteration in ground meat, the replacement of red meat with cheaper animal and plant proteins in these products is clearly possible. As a result, the aim of this study was to investigate the validity of labeling in premium hamburgers made of beef. In addition, the presence of soybean and chicken meat, which constitutes commercial fraud in premium hamburgers, was detected using a sensitive and quick multiplex-PCR method. In total, 10 specified brands of premium hamburgers purported to consist of beef were collected from markets in Tehran City, Iran. DNA was extracted from the premium hamburgers, then, simplex-PCR and multiplex-PCR fwere optimized using specific beef, chicken and soybean primers. The 118, 183, and 274 bp fragments, were amplified in all samples from soybean lectin, 12 s rRNA, and mitochondrial cytochrome b genes, respectively. The results indicated the addition of chicken meat and soybean in the premium hamburgers which were not indicated on their labels. PMID:26788003

  2. Multiplex Touchdown PCR for Rapid Typing of the Opportunistic Pathogen Propionibacterium acnes

    PubMed Central

    Barnard, Emma; Nagy, István; Hunyadkürti, Judit; Patrick, Sheila

    2015-01-01

    The opportunistic human pathogen Propionibacterium acnes is composed of a number of distinct phylogroups, designated types IA1, IA2, IB, IC, II, and III, which vary in their production of putative virulence factors, their inflammatory potential, and their biochemical, aggregative, and morphological characteristics. Although multilocus sequence typing (MLST) currently represents the gold standard for unambiguous phylogroup classification and individual strain identification, it is a labor-intensive and time-consuming technique. As a consequence, we developed a multiplex touchdown PCR assay that in a single reaction can confirm the species identity and phylogeny of an isolate based on its pattern of reaction with six primer sets that target the 16S rRNA gene (all isolates), ATPase (types IA1, IA2, and IC), sodA (types IA2 and IB), atpD (type II), and recA (type III) housekeeping genes, as well as a Fic family toxin gene (type IC). When applied to 312 P. acnes isolates previously characterized by MLST and representing types IA1 (n = 145), IA2 (n = 20), IB (n = 65), IC (n = 7), II (n = 45), and III (n = 30), the multiplex displayed 100% sensitivity and 100% specificity for detecting isolates within each targeted phylogroup. No cross-reactivity with isolates from other bacterial species was observed. This multiplex assay will provide researchers with a rapid, high-throughput, and technically undemanding typing method for epidemiological and phylogenetic investigations. It will facilitate studies investigating the association of lineages with various infections and clinical conditions, and it will serve as a prescreening tool to maximize the number of genetically diverse isolates selected for downstream higher-resolution sequence-based analyses. PMID:25631794

  3. miR-33-5p, a novel mechano-sensitive microRNA promotes osteoblast differentiation by targeting Hmga2

    PubMed Central

    Wang, Han; Sun, Zhongyang; Wang, Yixuan; Hu, Zebing; Zhou, Hua; Zhang, Lianchang; Hong, Bo; Zhang, Shu; Cao, Xinsheng

    2016-01-01

    MicroRNAs (miRNAs) interfere with the translation of specific target mRNAs and are thought to thereby regulate many cellular processes. However, the role of miRNAs in osteoblast mechanotransduction remains to be defined. In this study, we investigated the ability of a miRNA to respond to different mechanical environments and regulate mechano-induced osteoblast differentiation. First, we demonstrated that miR-33-5p expressed by osteoblasts is sensitive to multiple mechanical environments, microgravity and fluid shear stress. We then confirmed the ability of miR-33-5p to promote osteoblast differentiation. Microgravity or fluid shear stress influences osteoblast differentiation partially via miR-33-5p. Through bioinformatics analysis and a luciferase assay, we subsequently confirmed that Hmga2 is a target gene of miR-33-5p that negatively regulates osteoblast differentiation. Moreover, miR-33-5p regulates osteoblast differentiation partially via Hmga2. In summary, our findings demonstrate that miR-33-5p is a novel mechano-sensitive miRNA that can promote osteoblast differentiation and participate in the regulation of differentiation induced by changes in the mechanical environment, suggesting this miRNA as a potential target for the treatment of pathological bone loss. PMID:26980276

  4. On-Electrode Synthesis of Shape-Controlled Hierarchical Flower-Like Gold Nanostructures for Efficient Interfacial DNA Assembly and Sensitive Electrochemical Sensing of MicroRNA.

    PubMed

    Su, Shao; Wu, Yan; Zhu, Dan; Chao, Jie; Liu, Xingfen; Wan, Ying; Su, Yan; Zuo, Xiaolei; Fan, Chunhai; Wang, Lianhui

    2016-07-01

    The performance for biomolecular detection is closely associated with the interfacial structure of a biosensor, which profoundly affects both thermodynamics and kinetics of the assembly, binding and signal transduction of biomolecules. Herein, it is reported on a one-step and template-free on-electrode synthesis method for making shape-controlled gold nanostructures on indium tin oxide substrates, which provide an electrochemical sensing platform for ultrasensitive detection of nucleic acids. Thus-prepared hierarchical flower-like gold nanostructures (HFGNs) possess large surface area that can readily accommodate the assembly of DNA probes for subsequent hybridization detection. It is found that the sensitivity for electrochemical DNA sensing is critically dependent on the morphology of HFGNs. By using this new strategy, a highly sensitive electrochemical biosensor is developed for label-free detection of microRNA-21 (miRNA-21), a biomarker for lung cancers. Importantly, it is demonstrated that this biosensor can be employed to measure the miRNA-21 expression level from human lung cancer cell (A549) lysates and worked well in 100% serum, suggesting its potential for applications in clinical diagnosis and a wide range of bioanalysis. PMID:27305644

  5. RNA-ID, a highly sensitive and robust method to identify cis-regulatory sequences using superfolder GFP and a fluorescence-based assay

    PubMed Central

    Dean, Kimberly M.; Grayhack, Elizabeth J.

    2012-01-01

    We have developed a robust and sensitive method, called RNA-ID, to screen for cis-regulatory sequences in RNA using fluorescence-activated cell sorting (FACS) of yeast cells bearing a reporter in which expression of both superfolder green fluorescent protein (GFP) and yeast codon-optimized mCherry red fluorescent protein (RFP) is driven by the bidirectional GAL1,10 promoter. This method recapitulates previously reported progressive inhibition of translation mediated by increasing numbers of CGA codon pairs, and restoration of expression by introduction of a tRNA with an anticodon that base pairs exactly with the CGA codon. This method also reproduces effects of paromomycin and context on stop codon read-through. Five key features of this method contribute to its effectiveness as a selection for regulatory sequences: The system exhibits greater than a 250-fold dynamic range, a quantitative and dose-dependent response to known inhibitory sequences, exquisite resolution that allows nearly complete physical separation of distinct populations, and a reproducible signal between different cells transformed with the identical reporter, all of which are coupled with simple methods involving ligation-independent cloning, to create large libraries. Moreover, we provide evidence that there are sequences within a 9-nt library that cause reduced GFP fluorescence, suggesting that there are novel cis-regulatory sequences to be found even in this short sequence space. This method is widely applicable to the study of both RNA-mediated and codon-mediated effects on expression. PMID:23097427

  6. Simultaneous detection of four causal agents of tobacco bushy top disease by a multiplex one-step RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tobacco bushy top disease is a complex disease caused by mixed infection of Tobacco bushy top virus (TBTV), Tobacco vein distorting virus (TVDV), satellite RNA of TBTV (Sat-TBTV) and Tobacco vein distorting virus associate RNA (TVDVaRNA). A one-tube multiplex reverse transcription-PCR (RT-PCR) assay...

  7. A Novel Multiplex PCR Discriminates Bacillus anthracis and Its Genetically Related Strains from Other Bacillus cereus Group Species

    PubMed Central

    Ogawa, Hirohito; Fujikura, Daisuke; Ohnuma, Miyuki; Ohnishi, Naomi; Hang'ombe, Bernard M.; Mimuro, Hitomi; Ezaki, Takayuki; Mweene, Aaron S.; Higashi, Hideaki

    2015-01-01

    Anthrax is an important zoonotic disease worldwide that is caused by Bacillus anthracis, a spore-forming pathogenic bacterium. A rapid and sensitive method to detect B. anthracis is important for anthrax risk management and control in animal cases to address public health issues. However, it has recently become difficult to identify B. anthracis by using previously reported molecular-based methods because of the emergence of B. cereus, which causes severe extra-intestinal infection, as well as the human pathogenic B. thuringiensis, both of which are genetically related to B. anthracis. The close genetic relation of chromosomal backgrounds has led to complexity of molecular-based diagnosis. In this study, we established a B. anthracis multiplex PCR that can screen for the presence of B. anthracis virulent plasmids and differentiate B. anthracis and its genetically related strains from other B. cereus group species. Six sets of primers targeting a chromosome of B. anthracis and B. anthracis-like strains, two virulent plasmids, pXO1 and pXO2, a bacterial gene, 16S rRNA gene, and a mammalian gene, actin-beta gene, were designed. The multiplex PCR detected approximately 3.0 CFU of B. anthracis DNA per PCR reaction and was sensitive to B. anthracis. The internal control primers also detected all bacterial and mammalian DNAs examined, indicating the practical applicability of this assay as it enables monitoring of appropriate amplification. The assay was also applied for detection of clinical strains genetically related to B. anthracis, which were B. cereus strains isolated from outbreaks of hospital infections in Japan, and field strains isolated in Zambia, and the assay differentiated B. anthracis and its genetically related strains from other B. cereus group strains. Taken together, the results indicate that the newly developed multiplex PCR is a sensitive and practical method for detecting B. anthracis. PMID:25774512

  8. Multiplexer and time duration measuring circuit

    SciTech Connect

    Gray, Jr., James

    1980-01-01

    A multiplexer device is provided for multiplexing data in the form of randomly developed, variable width pulses from a plurality of pulse sources to a master storage. The device includes a first multiplexer unit which includes a plurality of input circuits each coupled to one of the pulse sources, with all input circuits being disabled when one input circuit receives an input pulse so that only one input pulse is multiplexed by the multiplexer unit at any one time.

  9. A 128 Multiplexing Factor Time-Domain SQUID Multiplexer

    NASA Astrophysics Data System (ADS)

    Prêle, D.; Voisin, F.; Piat, M.; Decourcelle, T.; Perbost, C.; Chapron, C.; Rambaud, D.; Maestre, S.; Marty, W.; Montier, L.

    2016-01-01

    A cryogenic 128:1 Time-Domain Multiplexer (TDM) has been developed for the readout of kilo-pixel Transition Edge Sensor (TES) arrays dedicated to the Q&U Bolometric Interferometer for Cosmology (QUBIC) instrument which aims to measure the B-mode polarization of the Cosmic Microwave Background. Superconducting QUantum Interference Devices (SQUIDs) are usually used to read out TESs. Moreover, SQUIDs are used to build TDM by biasing sequentially the SQUIDs connected together—one for each TES. In addition to this common technique which allows a typical 32 multiplexing factor, a cryogenic integrated circuit provides a 4:1 second multiplexing stage. This cryogenic integrated circuit is one of the original part of our TDM achieving an unprecedented 128 multiplexing factor. We present these two dimension TDM stages: topology of the SQUID multiplexer, operation of the cryogenic integrated circuit, and integration of the full system to read out a TES array dedicated to the QUBIC instrument. Flux-locked loop operation in multiplexed mode is also discussed.

  10. A 128 Multiplexing Factor Time-Domain SQUID Multiplexer

    NASA Astrophysics Data System (ADS)

    Prêle, D.; Voisin, F.; Piat, M.; Decourcelle, T.; Perbost, C.; Chapron, C.; Rambaud, D.; Maestre, S.; Marty, W.; Montier, L.

    2016-07-01

    A cryogenic 128:1 Time-Domain Multiplexer (TDM) has been developed for the readout of kilo-pixel Transition Edge Sensor (TES) arrays dedicated to the Q&U Bolometric Interferometer for Cosmology (QUBIC) instrument which aims to measure the B-mode polarization of the Cosmic Microwave Background. Superconducting QUantum Interference Devices (SQUIDs) are usually used to read out TESs. Moreover, SQUIDs are used to build TDM by biasing sequentially the SQUIDs connected together—one for each TES. In addition to this common technique which allows a typical 32 multiplexing factor, a cryogenic integrated circuit provides a 4:1 second multiplexing stage. This cryogenic integrated circuit is one of the original part of our TDM achieving an unprecedented 128 multiplexing factor. We present these two dimension TDM stages: topology of the SQUID multiplexer, operation of the cryogenic integrated circuit, and integration of the full system to read out a TES array dedicated to the QUBIC instrument. Flux-locked loop operation in multiplexed mode is also discussed.

  11. Differential Gene and MicroRNA Expression between Etoposide Resistant and Etoposide Sensitive MCF7 Breast Cancer Cell Lines

    PubMed Central

    Moitra, Karobi; Im, Kate; Limpert, Katy; Borsa, Alexander; Sawitzke, Julie; Robey, Rob; Yuhki, Naoya; Savan, Ram; Huang, Da Wei; Lempicki, Richard A.; Bates, Susan; Dean, Michael

    2012-01-01

    In order to develop targeted strategies for combating drug resistance it is essential to understand it’s basic molecular mechanisms. In an exploratory study we have found several possible indicators of etoposide resistance operating in MCF7VP cells, including up-regulation of ABC transporter genes, modulation of miRNA, and alteration in copy numbers of genes. PMID:23028896

  12. Expression of the RNA-binding protein RBM3 is associated with a favourable prognosis and cisplatin sensitivity in epithelial ovarian cancer

    PubMed Central

    2010-01-01

    Background We recently demonstrated that increased expression of the RNA-binding protein RBM3 is associated with a favourable prognosis in breast cancer. The aim of this study was to examine the prognostic value of RBM3 mRNA and protein expression in epithelial ovarian cancer (EOC) and the cisplatin response upon RBM3 depletion in a cisplatin-sensitive ovarian cancer cell line. Methods RBM3 mRNA expression was analysed in tumors from a cohort of 267 EOC cases (Cohort I) and RBM3 protein expression was analysed using immunohistochemistry (IHC) in an independent cohort of 154 prospectively collected EOC cases (Cohort II). Kaplan Meier analysis and Cox proportional hazards modelling were applied to assess the relationship between RBM3 and recurrence free survival (RFS) and overall survival (OS). Immunoblotting and IHC were used to examine the expression of RBM3 in a cisplatin-resistant ovarian cancer cell line A2780-Cp70 and its cisplatin-responsive parental cell line A2780. The impact of RBM3 on cisplatin response in EOC was assessed using siRNA-mediated silencing of RBM3 in A2780 cells followed by cell viability assay and cell cycle analysis. Results Increased RBM3 mRNA expression was associated with a prolonged RFS (HR = 0.64, 95% CI = 0.47-0.86, p = 0.003) and OS (HR = 0.64, 95% CI = 0.44-0.95, p = 0.024) in Cohort I. Multivariate analysis confirmed that RBM3 mRNA expression was an independent predictor of a prolonged RFS, (HR = 0.61, 95% CI = 0.44-0.84, p = 0.003) and OS (HR = 0.62, 95% CI = 0.41-0.95; p = 0.028) in Cohort I. In Cohort II, RBM3 protein expression was associated with a prolonged OS (HR = 0.53, 95% CI = 0.35-0.79, p = 0.002) confirmed by multivariate analysis (HR = 0.61, 95% CI = 0.40-0.92, p = 0.017). RBM3 mRNA and protein expression levels were significantly higher in the cisplatin sensitive A2780 cell line compared to the cisplatin resistant A2780-Cp70 derivative. siRNA-mediated silencing of RBM3 expression in the A2780 cells resulted in a

  13. Tumor specific lung cancer diagnostics with multiplexed FRET immunoassays

    NASA Astrophysics Data System (ADS)

    Geißler, D.; Hill, D.; Löhmannsröben, H.-G.; Thomas, E.; Lavigne, A.; Darbouret, B.; Bois, E.; Charbonnière, L. J.; Ziessel, R. F.; Hildebrandt, N.

    2010-02-01

    An optical multiplexed homogeneous (liquid phase) immunoassay based on FRET from a terbium complex to eight different fluorescent dyes is presented. We achieved highly sensitive parallel detection of four different lung cancer specific tumor markers (CEA, NSE, SCC and CYFRA21-1) within a single assay and show a proof-of-principle for 5- fold multiplexing. The method is well suited for fast and low-cost miniaturized point-of-care testing as well as for highthroughput screening in a broad range of in-vitro diagnostic applications.

  14. Ribosomal pausing at a frameshifter RNA pseudoknot is sensitive to reading phase but shows little correlation with frameshift efficiency.

    PubMed

    Kontos, H; Napthine, S; Brierley, I

    2001-12-01

    Here we investigated ribosomal pausing at sites of programmed -1 ribosomal frameshifting, using translational elongation and ribosome heelprint assays. The site of pausing at the frameshift signal of infectious bronchitis virus (IBV) was determined and was consistent with an RNA pseudoknot-induced pause that placed the ribosomal P- and A-sites over the slippery sequence. Similarly, pausing at the simian retrovirus 1 gag/pol signal, which contains a different kind of frameshifter pseudoknot, also placed the ribosome over the slippery sequence, supporting a role for pausing in frameshifting. However, a simple correlation between pausing and frameshifting was lacking. Firstly, a stem-loop structure closely related to the IBV pseudoknot, although unable to stimulate efficient frameshifting, paused ribosomes to a similar extent and at the same place on the mRNA as a parental pseudoknot. Secondly, an identical pausing pattern was induced by two pseudoknots differing only by a single loop 2 nucleotide yet with different functionalities in frameshifting. The final observation arose from an assessment of the impact of reading phase on pausing. Given that ribosomes advance in triplet fashion, we tested whether the reading frame in which ribosomes encounter an RNA structure (the reading phase) would influence pausing. We found that the reading phase did influence pausing but unexpectedly, the mRNA with the pseudoknot in the phase which gave the least pausing was found to promote frameshifting more efficiently than the other variants. Overall, these experiments support the view that pausing alone is insufficient to mediate frameshifting and additional events are required. The phase dependence of pausing may be indicative of an activity in the ribosome that requires an optimal contact with mRNA secondary structures for efficient unwinding. PMID:11713298

  15. MicroRNA-triggered, cascaded and catalytic self-assembly of functional ``DNAzyme ferris wheel'' nanostructures for highly sensitive colorimetric detection of cancer cells

    NASA Astrophysics Data System (ADS)

    Zhou, Wenjiao; Liang, Wenbin; Li, Xin; Chai, Yaqin; Yuan, Ruo; Xiang, Yun

    2015-05-01

    The construction of DNA nanostructures with various sizes and shapes has significantly advanced during the past three decades, yet the application of these DNA nanostructures for solving real problems is still in the early stage. On the basis of microRNA-triggered, catalytic self-assembly formation of the functional ``DNAzyme ferris wheel'' nanostructures, we show here a new signal amplification platform for highly sensitive, label-free and non-enzyme colorimetric detection of a small number of human prostate cancer cells. The microRNA (miR-141), which is catalytically recycled and reused, triggers isothermal self-assembly of a pre-designed, G-quadruplex sequence containing hairpin DNAs into ``DNAzyme ferris wheel''-like nanostructures (in association with hemin) with horseradish peroxidase mimicking activity. These DNAzyme nanostructures catalyze an intensified color transition of the probe solution for highly sensitive detection of miR-141 down to 0.5 pM with the naked eye, and the monitoring of as low as 283 human prostate cancer cells can also, theoretically, be achieved in a colorimetric approach. The work demonstrated here thus offers new opportunities for the construction of functional DNA nanostructures and for the application of these DNA nanostructures as an effective signal amplification means in the sensitive detection of nucleic acid biomarkers.

  16. Evaluation of 16S rRNA Gene PCR Sensitivity and Specificity for Diagnosis of Prosthetic Joint Infection: a Prospective Multicenter Cross-Sectional Study

    PubMed Central

    Plouzeau, Chloé; Tande, Didier; Léger, Julie; Giraudeau, Bruno; Valentin, Anne Sophie; Jolivet-Gougeon, Anne; Vincent, Pascal; Corvec, Stéphane; Gibaud, Sophie; Juvin, Marie Emmanuelle; Héry-Arnaud, Genevieve; Lemarié, Carole; Kempf, Marie; Bret, Laurent; Quentin, Roland; Coffre, Carine; de Pinieux, Gonzague; Bernard, Louis; Burucoa, Christophe

    2014-01-01

    There is no standard method for the diagnosis of prosthetic joint infection (PJI). The contribution of 16S rRNA gene PCR sequencing on a routine basis remains to be defined. We performed a prospective multicenter study to assess the contributions of 16S rRNA gene assays in PJI diagnosis. Over a 2-year period, all patients suspected to have PJIs and a few uninfected patients undergoing primary arthroplasty (control group) were included. Five perioperative samples per patient were collected for culture and 16S rRNA gene PCR sequencing and one for histological examination. Three multicenter quality control assays were performed with both DNA extracts and crushed samples. The diagnosis of PJI was based on clinical, bacteriological, and histological criteria, according to Infectious Diseases Society of America guidelines. A molecular diagnosis was modeled on the bacteriological criterion (≥1 positive sample for strict pathogens and ≥2 for commensal skin flora). Molecular data were analyzed according to the diagnosis of PJI. Between December 2010 and March 2012, 264 suspected cases of PJI and 35 control cases were included. PJI was confirmed in 215/264 suspected cases, 192 (89%) with a bacteriological criterion. The PJIs were monomicrobial (163 cases [85%]; staphylococci, n = 108; streptococci, n = 22; Gram-negative bacilli, n = 16; anaerobes, n = 13; others, n = 4) or polymicrobial (29 cases [15%]). The molecular diagnosis was positive in 151/215 confirmed cases of PJI (143 cases with bacteriological PJI documentation and 8 treated cases without bacteriological documentation) and in 2/49 cases without confirmed PJI (sensitivity, 73.3%; specificity, 95.5%). The 16S rRNA gene PCR assay showed a lack of sensitivity in the diagnosis of PJI on a multicenter routine basis. PMID:25056331

  17. Evaluation of 16S rRNA gene PCR sensitivity and specificity for diagnosis of prosthetic joint infection: a prospective multicenter cross-sectional study.

    PubMed

    Bémer, Pascale; Plouzeau, Chloé; Tande, Didier; Léger, Julie; Giraudeau, Bruno; Valentin, Anne Sophie; Jolivet-Gougeon, Anne; Vincent, Pascal; Corvec, Stéphane; Gibaud, Sophie; Juvin, Marie Emmanuelle; Héry-Arnaud, Genevieve; Lemarié, Carole; Kempf, Marie; Bret, Laurent; Quentin, Roland; Coffre, Carine; de Pinieux, Gonzague; Bernard, Louis; Burucoa, Christophe

    2014-10-01

    There is no standard method for the diagnosis of prosthetic joint infection (PJI). The contribution of 16S rRNA gene PCR sequencing on a routine basis remains to be defined. We performed a prospective multicenter study to assess the contributions of 16S rRNA gene assays in PJI diagnosis. Over a 2-year period, all patients suspected to have PJIs and a few uninfected patients undergoing primary arthroplasty (control group) were included. Five perioperative samples per patient were collected for culture and 16S rRNA gene PCR sequencing and one for histological examination. Three multicenter quality control assays were performed with both DNA extracts and crushed samples. The diagnosis of PJI was based on clinical, bacteriological, and histological criteria, according to Infectious Diseases Society of America guidelines. A molecular diagnosis was modeled on the bacteriological criterion (≥ 1 positive sample for strict pathogens and ≥ 2 for commensal skin flora). Molecular data were analyzed according to the diagnosis of PJI. Between December 2010 and March 2012, 264 suspected cases of PJI and 35 control cases were included. PJI was confirmed in 215/264 suspected cases, 192 (89%) with a bacteriological criterion. The PJIs were monomicrobial (163 cases [85%]; staphylococci, n = 108; streptococci, n = 22; Gram-negative bacilli, n = 16; anaerobes, n = 13; others, n = 4) or polymicrobial (29 cases [15%]). The molecular diagnosis was positive in 151/215 confirmed cases of PJI (143 cases with bacteriological PJI documentation and 8 treated cases without bacteriological documentation) and in 2/49 cases without confirmed PJI (sensitivity, 73.3%; specificity, 95.5%). The 16S rRNA gene PCR assay showed a lack of sensitivity in the diagnosis of PJI on a multicenter routine basis. PMID:25056331

  18. High thickness acrylamide photopolymer for peristrophic multiplexing

    NASA Astrophysics Data System (ADS)

    Ortuño, M.; Fernández, E.; Márquez, A.; Gallego, S.; Neipp, C.; Pascual, I.

    2006-05-01

    The acrylamide photolymers are considered interesting materials for holographic media. They have high diffraction efficiency (ratio of the intensities of the diffracted and the incident beams), an intermediate energetic sensitivity among other materials and post-processing steps are not necessary, therefore the media is not altered. The layers of these materials, about 1 mm thick, are a suitable media for recording many diffraction gratings in the same volume of photopolymer using peristrophic multiplexing technique, with great practical importance in the field of holographic memories type WORM (write once read many). In this work we study the recording of diffraction gratings by peristrophic multiplexing with axis of rotation perpendicular to the recording media. The photopolymer is composed of acrylamide as the polymerizable monomer, triethanolamine as radical generator, yellowish eosin as sensitizer and a binder of polyvinyl alcohol. We analyze the holographic behaviour of the material during recording and reconstruction of diffraction gratings using a continuous Nd:YAG laser (532 nm) at an intensity of 5 mW/cm2 as recording laser. The response of the material is monitored after recording with an He-Ne laser. We study the recording process of unslanted diffraction gratings of 1125 lines/mm. The diffraction efficiency of each hologram is seen to decrease as the number of holograms recorded increases, due to consumption of the available dynamic range, in a constant exposure scheduling. It can be seen that the photopolymer works well with high energy levels, without excessive dispersion of light by noise gratings. In order to homogenize the diffraction efficiency of each hologram we use the method proposed by Pu. This method is designed to share all or part of the avaliable dynamic range of the recording material among the holograms to be multiplexed. Using exposure schedules derived from this method we have used 3 scheduling recordings from the algorithm used

  19. Variable Sensitivity to Bacterial Methionyl-tRNA Synthetase Inhibitors Reveals Subpopulations of Streptococcus pneumoniae with Two Distinct Methionyl-tRNA Synthetase Genes

    PubMed Central

    Gentry, Daniel R.; Ingraham, Karen A.; Stanhope, Michael J.; Rittenhouse, Stephen; Jarvest, Richard L.; O'Hanlon, Peter J.; Brown, James R.; Holmes, David J.

    2003-01-01

    As reported previously (J. R. Jarvest et al., J. Med. Chem. 45:1952-1962, 2002), potent inhibitors (at nanomolar concentrations) of Staphylococcus aureus methionyl-tRNA synthetase (MetS; encoded by metS1) have been derived from a high-throughput screening assay hit. Optimized compounds showed excellent activities against staphylococcal and enterococcal pathogens. We report on the bimodal susceptibilities of S. pneumoniae strains, a significant fraction of which was found to be resistant (MIC, ≥8 mg/liter) to these inhibitors. Using molecular genetic techniques, we have found that the mechanism of resistance is the presence of a second, distantly related MetS enzyme, MetS2, encoded by metS2. We present evidence that the metS2 gene is necessary and sufficient for resistance to MetS inhibitors. PCR analysis for the presence of metS2 among a large sample (n = 315) of S. pneumoniae isolates revealed that it is widespread geographically and chronologically, occurring at a frequency of about 46%. All isolates tested also contained the metS1 gene. Searches of public sequence databases revealed that S. pneumoniae MetS2 was most similar to MetS in Bacillus anthracis, followed by MetS in various non-gram-positive bacterial, archaeal, and eukaryotic species, with streptococcal MetS being considerably less similar. We propose that the presence of metS2 in specific strains of S. pneumoniae is the result of horizontal gene transfer which has been driven by selection for resistance to some unknown class of naturally occurring antibiotics with similarities to recently reported synthetic MetS inhibitors. PMID:12760849

  20. Developmental changes in the hypothalamic mRNA expression levels of PACAP and its receptor PAC1 and their sensitivity to fasting in male and female rats.

    PubMed

    Iwasa, Takeshi; Matsuzaki, Toshiya; Tungalagsuvd, Altankhuu; Munkhzaya, Munkhsaikhan; Yiliyasi, Maira; Kato, Takeshi; Kuwahara, Akira; Irahara, Minoru

    2016-08-01

    The actions and responses of hypothalamic appetite regulatory and factors change markedly during the neonatal to pre-pubertal period. Pituitary adenylate cyclase-activating polypeptide (PACAP) has been found to play pivotal roles in the regulation of metabolic and nutritional status through its specific receptor PAC1. PACAP/PAC1 have anorectic roles, and their functions are regulated by leptin in adulthood. In the present study, we showed that hypothalamic PACAP mRNA expression decreases during the neonatal to pre-pubertal period (from postnatal day 10-30) in both male and female rats. During this period, hypothalamic PACAP mRNA expression was not affected by 24h fasting in either sex, while the serum leptin levels (leptin is a positive regulator of hypothalamic PACAP expression in adulthood) of both sexes were decreased by fasting. On the other hand, hypothalamic PAC1 mRNA expression did not change during the neonatal to pre-pubertal period in either sex; however, its levels were consistently higher in males than in females. Hypothalamic PAC1 mRNA expression was decreased by 24h fasting in males, but no such changes were observed in females. These results indicate while hypothalamic PACAP expression is sensitive to a negative energy state and the serum leptin level in adulthood, no such relationships are seen in the pre-pubertal period. In addition, we speculate that differences in the gonadal steroidal milieu might induce sexual dimorphism in the basal hypothalamic PAC1 mRNA level and its response to fasting. The mechanisms responsible for and the physiological effects of such changes in hypothalamic PACAP and PAC1 expression during the developmental period remain to be clarified. PMID:27181029

  1. Microbial Contaminants of Cord Blood Units Identified by 16S rRNA Sequencing and by API Test System, and Antibiotic Sensitivity Profiling.

    PubMed

    França, Luís; Simões, Catarina; Taborda, Marco; Diogo, Catarina; da Costa, Milton S

    2015-01-01

    Over a period of ten months a total of 5618 cord blood units (CBU) were screened for microbial contamination under routine conditions. The antibiotic resistance profile for all isolates was also examined using ATB strips. The detection rate for culture positive units was 7.5%, corresponding to 422 samples.16S rRNA sequence analysis and identification with API test system were used to identify the culturable aerobic, microaerophilic and anaerobic bacteria from CBUs. From these samples we recovered 485 isolates (84 operational taxonomic units, OTUs) assigned to the classes Bacteroidia, Actinobacteria, Clostridia, Bacilli, Betaproteobacteria and primarily to the Gammaproteobacteria. Sixty-nine OTUs, corresponding to 447 isolates, showed 16S rRNA sequence similarities above 99.0% with known cultured bacteria. However, 14 OTUs had 16S rRNA sequence similarities between 95 and 99% in support of genus level identification and one OTU with 16S rRNA sequence similarity of 90.3% supporting a family level identification only. The phenotypic identification formed 29 OTUs that could be identified to the species level and 9 OTUs that could be identified to the genus level by API test system. We failed to obtain identification for 14 OTUs, while 32 OTUs comprised organisms producing mixed identifications. Forty-two OTUs covered species not included in the API system databases. The API test system Rapid ID 32 Strep and Rapid ID 32 E showed the highest proportion of identifications to the species level, the lowest ratio of unidentified results and the highest agreement to the results of 16S rRNA assignments. Isolates affiliated to the Bacilli and Bacteroidia showed the highest antibiotic multi-resistance indices and microorganisms of the Clostridia displayed the most antibiotic sensitive phenotypes. PMID:26512991

  2. Microbial Contaminants of Cord Blood Units Identified by 16S rRNA Sequencing and by API Test System, and Antibiotic Sensitivity Profiling

    PubMed Central

    França, Luís; Simões, Catarina; Taborda, Marco; Diogo, Catarina; da Costa, Milton S.

    2015-01-01

    Over a period of ten months a total of 5618 cord blood units (CBU) were screened for microbial contamination under routine conditions. The antibiotic resistance profile for all isolates was also examined using ATB strips. The detection rate for culture positive units was 7.5%, corresponding to 422 samples.16S rRNA sequence analysis and identification with API test system were used to identify the culturable aerobic, microaerophilic and anaerobic bacteria from CBUs. From these samples we recovered 485 isolates (84 operational taxonomic units, OTUs) assigned to the classes Bacteroidia, Actinobacteria, Clostridia, Bacilli, Betaproteobacteria and primarily to the Gammaproteobacteria. Sixty-nine OTUs, corresponding to 447 isolates, showed 16S rRNA sequence similarities above 99.0% with known cultured bacteria. However, 14 OTUs had 16S rRNA sequence similarities between 95 and 99% in support of genus level identification and one OTU with 16S rRNA sequence similarity of 90.3% supporting a family level identification only. The phenotypic identification formed 29 OTUs that could be identified to the species level and 9 OTUs that could be identified to the genus level by API test system. We failed to obtain identification for 14 OTUs, while 32 OTUs comprised organisms producing mixed identifications. Forty-two OTUs covered species not included in the API system databases. The API test system Rapid ID 32 Strep and Rapid ID 32 E showed the highest proportion of identifications to the species level, the lowest ratio of unidentified results and the highest agreement to the results of 16S rRNA assignments. Isolates affiliated to the Bacilli and Bacteroidia showed the highest antibiotic multi-resistance indices and microorganisms of the Clostridia displayed the most antibiotic sensitive phenotypes. PMID:26512991

  3. Patterned Plasmonic Nanoparticle Arrays for Microfluidic and Multiplexed Biological Assays.

    PubMed

    He, Jie; Boegli, Michelle; Bruzas, Ian; Lum, William; Sagle, Laura

    2015-11-17

    For applications ranging from medical diagnostics and drug screening to chemical and biological warfare detection, inexpensive, rapid-readout, portable devices are required. Localized surface plasmon resonance (LSPR) technologies show substantial promise toward meeting these goals, but the generation of portable, multiplexed and/or microfluidic devices incorporating sensitive nanoparticle arrays is only in its infancy. Herein, we have combined photolithography with Hole Mask Colloidal lithography to pattern uniform nanoparticle arrays for both microfluidic and multiplexed devices. The first proof-of-concept study is carried out with 5- and 7-channel microfluidic devices to acquire one-shot binding curves and protein binding kinetic data. The second proof-of-concept study involved the fabrication of a 96-spot plate that can be inserted into a standard plate reader for the multiplexed detection of protein binding. This versatile fabrication technique should prove useful in next generation chips for bioassays and genetic screening. PMID:26494412

  4. RNA Profiles of Porcine Embryos during Genome Activation Reveal Complex Metabolic Switch Sensitive to In Vitro Conditions

    PubMed Central

    Østrup, Olga; Olbricht, Gayla; Østrup, Esben; Hyttel, Poul; Collas, Philippe; Cabot, Ryan

    2013-01-01

    Fertilization is followed by complex changes in cytoplasmic composition and extensive chromatin reprogramming which results in the abundant activation of totipotent embryonic genome at embryonic genome activation (EGA). While chromatin reprogramming has been widely studied in several species, only a handful of reports characterize changing transcriptome profiles and resulting metabolic changes in cleavage stage embryos. The aims of the current study were to investigate RNA profiles of in vivo developed (ivv) and in vitro produced (ivt) porcine embryos before (2-cell stage) and after (late 4-cell stage) EGA and determine major metabolic changes that regulate totipotency. The period before EGA was dominated by transcripts responsible for cell cycle regulation, mitosis, RNA translation and processing (including ribosomal machinery), protein catabolism, and chromatin remodelling. Following EGA an increase in the abundance of transcripts involved in transcription, translation, DNA metabolism, histone and chromatin modification, as well as protein catabolism was detected. The further analysis of members of overlapping GO terms revealed that despite that comparable cellular processes are taking place before and after EGA (RNA splicing, protein catabolism), different metabolic pathways are involved. This strongly suggests that a complex metabolic switch accompanies EGA. In vitro conditions significantly altered RNA profiles before EGA, and the character of these changes indicates that they originate from oocyte and are imposed either before oocyte aspiration or during in vitro maturation. IVT embryos have altered content of apoptotic factors, cell cycle regulation factors and spindle components, and transcription factors, which all may contribute to reduced developmental competence of embryos produced in vitro. Overall, our data are in good accordance with previously published, genome-wide profiling data in other species. Moreover, comparison with mouse and human embryos

  5. Multiplexed protein profiling by sequential affinity capture.

    PubMed

    Ayoglu, Burcu; Birgersson, Elin; Mezger, Anja; Nilsson, Mats; Uhlén, Mathias; Nilsson, Peter; Schwenk, Jochen M

    2016-04-01

    Antibody microarrays enable parallelized and miniaturized analysis of clinical samples, and have proven to provide novel insights for the analysis of different proteomes. However, there are concerns that the performance of such direct labeling and single antibody assays are prone to off-target binding due to the sample context. To improve selectivity and sensitivity while maintaining the possibility to conduct multiplexed protein profiling, we developed a multiplexed and semi-automated sequential capture assay. This novel bead-based procedure encompasses a first antigen capture, labeling of captured protein targets on magnetic particles, combinatorial target elution and a read-out by a secondary capture bead array. We demonstrate in a proof-of-concept setting that target detection via two sequential affinity interactions reduced off-target contribution, while lowered background and noise levels, improved correlation to clinical values compared to single binder assays. We also compared sensitivity levels with single binder and classical sandwich assays, explored the possibility for DNA-based signal amplification, and demonstrate the applicability of the dual capture bead-based antibody microarray for biomarker analysis. Hence, the described concept enhances the possibilities for antibody array assays to be utilized for protein profiling in body fluids and beyond. PMID:26935855

  6. Multiplexing holograms in an acrylamide photopolymer

    NASA Astrophysics Data System (ADS)

    Fernández, Elena; Ortuño, Manuel; Márquez, Andrés; Gallego, Sergi; Pascual, Inmaculada

    2006-04-01

    A peristrophic multiplexing method is used to store various diffraction gratings at the same spot in the material. This material is formed of acrylamide photopolymers which are considered interesting materials for recording holographic memories. They have high diffraction efficiency (ratio between diffracted and incident beams), high energetic sensitivity and optical quality, and developing processes are not necessary. In this work, the photopolymer is composed of acrylamide (AA) as the polymerizable monomer, triethanolamine (TEA) as radical generator, N,N'methylene-bis-acrylamide (BMA) as crosslinker, yellowish eosin (YE) as sensitizer and a binder of polyvinyl alcohol (PVA). The layers of material obtained are approximately 1 mm thick. Using holographic recording schedules, the exposure energy each hologram should receive in order to achieve uniform diffraction efficiency is optimized. The purpose of these recording schedules is to enable full advantage to be taken of the whole dynamic range of the material and to share it between the individual holograms. The Scheduled Exposure Method (SEM) and the Incremental Exposure Method (IEM) are the two multiplexing schedules used to determine the recording times. Having determined these times, the results obtained with both methods are compared to ascertain which method enables the greatest number of holograms to be recorded with the highest, most uniform diffraction efficiencies.

  7. Sensitive detection of miRNA by using hybridization chain reaction coupled with positively charged gold nanoparticles.

    PubMed

    Miao, Xiangmin; Ning, Xue; Li, Zongbing; Cheng, Zhiyuan

    2016-01-01

    Positively charged gold nanoparticles (+)AuNPs can adsorb onto the negatively charged surface of single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA). Herein, long-range dsDNA polymers could form based on the hybridization chain reaction (HCR) of two hairpin probes (H1 and H2) by using miRNA-21 as an initiator. (+)AuNPs could adsorb onto the negatively charged surface of such long-range dsDNA polymers based on the electrostatic adsorption, which directly resulted in the precipitation of (+)AuNPs and the decrease of (+)AuNPs absorption spectra. Under optimal conditions, miRNA-21 detection could be realized in the range of 20 pM-10 nM with a detection limit of 6.8 pM. In addition, (+)AuNPs used here are much more stable than commonly used negatively charged gold nanoparticles ((-)AuNPs) in mixed solution that contained salt, protein or other metal ions. Importantly, the assay could realize the detection of miRNA in human serum samples. PMID:27576601

  8. Sensitive detection of miRNA by using hybridization chain reaction coupled with positively charged gold nanoparticles

    PubMed Central

    Miao, Xiangmin; Ning, Xue; Li, Zongbing; Cheng, Zhiyuan

    2016-01-01

    Positively charged gold nanoparticles (+)AuNPs can adsorb onto the negatively charged surface of single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA). Herein, long-range dsDNA polymers could form based on the hybridization chain reaction (HCR) of two hairpin probes (H1 and H2) by using miRNA-21 as an initiator. (+)AuNPs could adsorb onto the negatively charged surface of such long-range dsDNA polymers based on the electrostatic adsorption, which directly resulted in the precipitation of (+)AuNPs and the decrease of (+)AuNPs absorption spectra. Under optimal conditions, miRNA-21 detection could be realized in the range of 20 pM-10 nM with a detection limit of 6.8 pM. In addition, (+)AuNPs used here are much more stable than commonly used negatively charged gold nanoparticles ((−)AuNPs) in mixed solution that contained salt, protein or other metal ions. Importantly, the assay could realize the detection of miRNA in human serum samples. PMID:27576601

  9. Investigation of riboflavin sensitized degradation of purine and pyrimidine derivatives of DNA and RNA under UVA and UVB.

    PubMed

    Joshi, Prakash C; Keane, Thomas C

    2010-10-01

    DNA and RNA undergo photodegradation in UVC (200-290nm) due to direct absorption by the purine and pyrimidine bases. Limited effects are observed under UVB (290-320nm) or UVA (320-400nm). We have observed that an endogenous photosensitizer, riboflavin (RF), upon exposure to UVB or UVA can extensively damage the DNA and RNA bases. Guanine, uracil, thymine, adenine and cytosine were degraded by 100%, 82%, 60.4%, 46.3% and 10.3% under UVA (12J) and by 100%, 54.1%, 38.9%, 42.2% and <1.0% under UVB (6J), respectively. Guanosine and deoxyguanosine were degraded by 98±1.0% and 80±1.0% under UVA (4J) and UVB (12J), respectively. With an exception of GMP (53-82%), dGMP (51-88%) and to some extent TMP (3-4%) the remaining nucleosides and nucleotides were resistant to RF-induced photodecomposition. The photodegradation of G derivatives by RF was 2-fold higher than a well known photodynamic agent rose bengal. A comparison of the intensities of UVA and UVB sources used in this study with natural sunlight suggests that exposure with the latter along with an endogenous photosensitizer can have similar effects on DNA and RNA depending upon the duration of exposure. PMID:20816939

  10. Rapid and Sensitive Detection of RNA Viruses Based on Reverse Transcription Loop-Mediated Isothermal Amplification, Magnetic Nanoparticles, and Chemiluminescence.

    PubMed

    Wang, Jiuhai; Lu, Peng; Yan, Jieni; Zhang, Yufan; Huang, Lanye; Ali, Zeeshan; Li, Zhiyang; He, Nongyue

    2016-04-01

    RNA viruses, particularly, the highly pathogenic avian influenza (HPAI) virus, pose serious health concerns, and cause huge economic losses worldwide. Diagnostic tools for the early detection of these deadly RNA viruses are urgently needed to implement treatment and disease control strategies. Conventional reverse transcription polymerase chain reaction (RT-PCR)-based chemiluminescent (RT-PCR-CL) detection is frequently used for the diagnosis of viral infections. However, the requirements for expensive PCR machines and longer thermocycling times are significant drawbacks. In this study, we propose a method based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with chemiluminescence (CL) to detect H7N9 virus. The proposed method does not require any expensive instruments, and processing time is remarkably shortened compared to that of RT-PCR-CL. Since several factors including RT-LAMP temperature, probe concentration, hybridization temperature, and hybridization duration might affect the CL signal, each of these parameters was investigated and optimized. One thousand copies/mL of H7N9 RNA were detectable using the optimized RT-LAMP-CL method. The detection time was significantly reduced by using RT-LAMP, in comparison with conventional RT-PCR-CL. This technique holds great promise for viral detection and diagnosis, especially with regard to avian influenza virus. PMID:27301197

  11. Multiplex Real-Time PCR for Detection of Campylobacter, Salmonella, and Shigella

    PubMed Central

    Barletta, F.; Mercado, E. H.; Lluque, A.; Ruiz, J.; Cleary, T. G.

    2013-01-01

    Infectious diarrhea can be classified based on its clinical presentation as noninflammatory or inflammatory disease. In developing countries, among inflammatory diarrhea cases, Shigella is the most common cause, followed by Campylobacter and Salmonella. Because the time frame in which treatment choices must be made is short and conventional stool cultures lack good sensitivity, there is a need for a rapid, sensitive, and inexpensive detection technique. The purpose of our study was to develop a multiplex real-time PCR procedure to simultaneously identify Campylobacter spp., Salmonella spp., and Shigella spp. Primers were designed to amplify the invA, ipaH, and 16S rRNA genes simultaneously in a single reaction to detect Salmonella, Shigella, and Campylobacter, respectively. Using this approach, we correctly identified 102 of 103 strains of the targeted enteropathogens and 34 of 34 other pathogens. The melting temperatures were 82.96 ± 0.05°C for invA, 85.56 ± 0.28°C for ipaH, and 89.21 ± 0.24°C for 16S rRNA. The limit of accurate quantification for the assay in stool samples was 104 CFU g−1; however, the limit of detection was 103 CFU g−1. This assay is a simple, rapid, inexpensive, and reliable system for the practical detection of these three enteropathogens in clinical specimens. PMID:23761159

  12. Viability study of a multiplex diagnostic platform for Chagas disease.

    PubMed

    Foti, Leonardo; Fonseca, Bruna de Paula Fonseca e; Nascimento, Lilian Dias; Marques, Christiane de Fatima Silva; da Silva, Edmilson Domingos; Duarte, Cesar Augusto Barros; Probst, Christian M; Goldenberg, Samuel; Pinto, Antônio Gomes; Krieger, Marco Aurélio

    2009-07-01

    A new multiplex assay platform was evaluated to detect Trypanosoma cruzi infection using the recombinant antigens CRA, FRA, CRAFRA fusion and parasite lysate. The antigens presented different sensitivity and specificity in a singleplex test when compared to a serial dilution of two pools comprising 10 positive serum samples and one pool of 10 negative samples. The recombinant protein CRA presented lower sensitivity (55%) in contrast to the 100% specificity and sensitivity of FRA, CRAFRA and T. cruzi lysate. These antigens also showed good results in a duplex test and the duplex test with CRAFRA/T. cruzi lysate showed better performance with 100% specificity and sensitivity, as well as a lower cut-off value in comparison to the other duplex test, FRA/T. cruzi lysate. Hence, when the antigens were used in duplex format, both tests showed decreased cut-off values and no interference between different bead sets, resulting in increasing sensitivity and specificity. The results of these multiplex tests show that they could be an alternative to singleplex detection for Chagas disease, and also indicate the necessity of using multiplex diagnostic tools to increase the sensitivity and specificity for diagnostic tests. Emerging data from the T. cruzi genome and from its ORFeome project will also allow the identification of new antigens for this disease detection application. PMID:19753468

  13. Multiplex component-based allergen microarray in recent clinical studies.

    PubMed

    Patelis, A; Borres, M P; Kober, A; Berthold, M

    2016-08-01

    During the last decades component-resolved diagnostics either as singleplex or multiplex measurements has been introduced into the field of clinical allergology, providing important information that cannot be obtained from extract-based tests. Here we review recent studies that demonstrate clinical applications of the multiplex microarray technique in the diagnosis and risk assessment of allergic patients, and its usefulness in studies of allergic diseases. The usefulness of ImmunoCAP ISAC has been validated in a wide spectrum of allergic diseases like asthma, allergic rhinoconjunctivitis, atopic dermatitis, eosinophilic esophagitis, food allergy and anaphylaxis. ISAC provides a broad picture of a patient's sensitization profile from a single test, and provides information on specific and cross-reactive sensitizations that facilitate diagnosis, risk assessment, and disease management. Furthermore, it can reveal unexpected sensitizations which may explain anaphylaxis previously categorized as idiopathic and also display for the moment clinically non-relevant sensitizations. ISAC can facilitate a better selection of relevant allergens for immunotherapy compared with extract testing. Microarray technique can visualize the allergic march and molecular spreading in the preclinical stages of allergic diseases, and may indicate that the likelihood of developing symptomatic allergy is associated with specific profiles of sensitization to allergen components. ISAC is shown to be a useful tool in routine allergy diagnostics due to its ability to improve risk assessment, to better select relevant allergens for immunotherapy as well as detecting unknown sensitization. Multiplex component testing is especially suitable for patients with complex symptomatology. PMID:27196983

  14. Evidence for genetic regulation of mRNA expression of the dosage-sensitive gene retinoic acid induced-1 (RAI1) in human brain.

    PubMed

    Chen, Li; Tao, Yu; Song, Fan; Yuan, Xi; Wang, Jian; Saffen, David

    2016-01-01

    RAI1 (retinoic acid induced-1) is a dosage-sensitive gene that causes Smith-Magenis syndrome (SMS) when mutated or deleted and Potocki-Lupski Syndrome (PTLS) when duplicated, with psychiatric features commonly observed in both syndromes. How common genetic variants regulate this gene, however, is unknown. In this study, we found that RAI1 mRNA expression in Chinese prefrontal and temporal cortex correlate with genotypes of common single nucleotide polymorphisms (SNPs) located in the RAI1 5'-upstream region. Using genotype imputation, "R(2)-Δ(2)" analysis, and data from the RegulomeDB database, we identified SNPs rs4925102 and rs9907986 as possible regulatory variants, accounting for approximately 30-40% of the variance in RAI1 mRNA expression in both brain regions. Specifically, rs4925102 and rs9907986 are predicted to disrupt the binding of retinoic acid RXR-RAR receptors and the transcription factor DEAF1 (Deformed epidermal autoregulatory factor-1), respectively. Consistent with these predictions, we observed binding of RXRα and RARα to the predicted RAI1 target in chromatin immunoprecipitation assays. Retinoic acid is crucial for early development of the central neural system, and DEAF1 is associated with intellectual disability. The observation that a significant portion of RAI1 mRNA expression is genetically controlled raises the possibility that common RAI1 5'-region regulatory variants contribute more generally to psychiatric disorders. PMID:26743651

  15. Conformation-sensitive nucleoside analogues as topology-specific fluorescence turn-on probes for DNA and RNA G-quadruplexes

    PubMed Central

    Tanpure, Arun A.; Srivatsan, Seergazhi G.

    2015-01-01

    Development of probes that can discriminate G-quadruplex (GQ) structures and indentify efficient GQ binders on the basis of topology and nucleic acid type is highly desired to advance GQ-directed therapeutic strategies. In this context, we describe the development of minimally perturbing and environment-sensitive pyrimidine nucleoside analogues, based on a 5-(benzofuran-2-yl)uracil core, as topology-specific fluorescence turn-on probes for human telomeric DNA and RNA GQs. The pyrimidine residues of one of the loop regions (TTA) of telomeric DNA and RNA GQ oligonucleotide (ON) sequences were replaced with 5-benzofuran-modified 2′-deoxyuridine and uridine analogues. Depending on the position of modification the fluorescent nucleoside analogues distinguish antiparallel, mixed parallel-antiparallel and parallel stranded DNA and RNA GQ topologies from corresponding duplexes with significant enhancement in fluorescence intensity and quantum yield. Further, these GQ sensors enabled the development of a simple fluorescence binding assay to quantify topology- and nucleic acid-specific binding of small molecule ligands to GQ structures. Together, our results demonstrate that these nucleoside analogues are useful GQ probes, which are anticipated to provide new opportunities to study and discover efficient G-quadruplex binders of therapeutic potential. PMID:26202965

  16. A temperature-sensitive allele of a putative mRNA splicing helicase down-regulates many cell wall genes and causes radial swelling in Arabidopsis thaliana.

    PubMed

    Howles, Paul A; Gebbie, Leigh K; Collings, David A; Varsani, Arvind; Broad, Ronan C; Ohms, Stephen; Birch, Rosemary J; Cork, Ann H; Arioli, Tony; Williamson, Richard E

    2016-05-01

    The putative RNA helicase encoded by the Arabidopsis gene At1g32490 is a homolog of the yeast splicing RNA helicases Prp2 and Prp22. We isolated a temperature-sensitive allele (rsw12) of the gene in a screen for root radial swelling mutants. Plants containing this allele grown at the restrictive temperature showed weak radial swelling, were stunted with reduced root elongation, and contained reduced levels of cellulose. The role of the protein was further explored by microarray analysis. By using both fold change cutoffs and a weighted gene coexpression network analysis (WGCNA) to investigate coexpression of genes, we found that the radial swelling phenotype was not linked to genes usually associated with primary cell wall biosynthesis. Instead, the mutation has strong effects on expression of secondary cell wall related genes. Many genes potentially associated with secondary walls were present in the most significant WGCNA module, as were genes coding for arabinogalactans and proteins with GPI anchors. The proportion of up-regulated genes that possess introns in rsw12 was above that expected if splicing was unrelated to the activity of the RNA helicase, suggesting that the helicase does indeed play a role in splicing in Arabidopsis. The phenotype may be due to a change in the expression of one or more genes coding for cell wall proteins. PMID:27008640

  17. Evidence for genetic regulation of mRNA expression of the dosage-sensitive gene retinoic acid induced-1 (RAI1) in human brain

    PubMed Central

    Chen, Li; Tao, Yu; Song, Fan; Yuan, Xi; Wang, Jian; Saffen, David

    2016-01-01

    RAI1 (retinoic acid induced-1) is a dosage-sensitive gene that causes Smith-Magenis syndrome (SMS) when mutated or deleted and Potocki-Lupski Syndrome (PTLS) when duplicated, with psychiatric features commonly observed in both syndromes. How common genetic variants regulate this gene, however, is unknown. In this study, we found that RAI1 mRNA expression in Chinese prefrontal and temporal cortex correlate with genotypes of common single nucleotide polymorphisms (SNPs) located in the RAI1 5′-upstream region. Using genotype imputation, “R2-Δ2” analysis, and data from the RegulomeDB database, we identified SNPs rs4925102 and rs9907986 as possible regulatory variants, accounting for approximately 30–40% of the variance in RAI1 mRNA expression in both brain regions. Specifically, rs4925102 and rs9907986 are predicted to disrupt the binding of retinoic acid RXR-RAR receptors and the transcription factor DEAF1 (Deformed epidermal autoregulatory factor-1), respectively. Consistent with these predictions, we observed binding of RXRα and RARα to the predicted RAI1 target in chromatin immunoprecipitation assays. Retinoic acid is crucial for early development of the central neural system, and DEAF1 is associated with intellectual disability. The observation that a significant portion of RAI1 mRNA expression is genetically controlled raises the possibility that common RAI1 5′-region regulatory variants contribute more generally to psychiatric disorders. PMID:26743651

  18. Efficient exploration of multiplex networks

    NASA Astrophysics Data System (ADS)

    Battiston, Federico; Nicosia, Vincenzo; Latora, Vito

    2016-04-01

    Efficient techniques to navigate networks with local information are fundamental to sample large-scale online social systems and to retrieve resources in peer-to-peer systems. Biased random walks, i.e. walks whose motion is biased on properties of neighbouring nodes, have been largely exploited to design smart local strategies to explore a network, for instance by constructing maximally mixing trajectories or by allowing an almost uniform sampling of the nodes. Here we introduce and study biased random walks on multiplex networks, graphs where the nodes are related through different types of links organised in distinct and interacting layers, and we provide analytical solutions for their long-time properties, including the stationary occupation probability distribution and the entropy rate. We focus on degree-biased random walks and distinguish between two classes of walks, namely those whose transition probability depends on a number of parameters which is extensive in the number of layers, and those whose motion depends on intrinsically multiplex properties of the neighbouring nodes. We analyse the effect of the structure of the multiplex network on the steady-state behaviour of the walkers, and we find that heterogeneous degree distributions as well as the presence of inter-layer degree correlations and edge overlap determine the extent to which a multiplex can be efficiently explored by a biased walk. Finally we show that, in real-world multiplex transportation networks, the trade-off between efficient navigation and resilience to link failure has resulted into systems whose diffusion properties are qualitatively different from those of appropriately randomised multiplex graphs. This fact suggests that multiplexity is an important ingredient to include in the modelling of real-world systems.

  19. A Multiplex PCR Approach for Detecting Dual Infections and Recombinants Involving Major HIV Variants.

    PubMed

    Cappy, Pierre; De Oliveira, Fabienne; Gueudin, Marie; Alessandri-Gradt, Elodie; Plantier, Jean-Christophe

    2016-05-01

    The cocirculation of different HIV types and groups can lead to dual infections and recombinants, which hinder diagnosis and therapeutic management. We designed two multiplex PCRs (mPCRs) coupled with capillary electrophoresis to facilitate the detection of such infections. The first, MMO2, targets three variants (HIV-1/M, HIV-1/O, and HIV-2), and the second, MMO, targets HIV-1/M and HIV-1/O. These mPCRs were validated on DNA and RNA extracts from 19 HIV-1/M, 12 HIV-1/O, and 13 HIV-2 cultures and from mixtures simulating dual infections. They were then assessed with DNA and RNA extracts from samples of 47 clinical monoinfections and HIV-1/M+O dual infections or infections with HIV-1/MO recombinants. Both mPCRs had excellent specificity. Sensitivities ranged from 80 to 100% for in vitro samples and from 58 to 100% for clinical samples, with the results obtained depending on the material used and the region of the genome concerned. Sensitivity was generally lower for DNA than for RNA and for amplifications of the integrase and matrix regions. In terms of global detection (at least one target gene for each strain), both mPCRs yielded a detection rate of 100% for in vitro samples. MMO2 detected 100% of the clinical strains from DNA and 97% from RNA, whereas MMO detected 100% of the strains from both materials. Thus, for in vitro and clinical samples, MMO2 was a useful tool for detecting dual infections with HIV-1 and HIV-2 (referred to as HIV-1+HIV-2) and HIV-1/M+O, and MMO was useful for detecting both MO dual infections and MO mosaic patterns. PMID:26912747

  20. RapMap: a rapid, sensitive and accurate tool for mapping RNA-seq reads to transcriptomes

    PubMed Central

    Srivastava, Avi; Sarkar, Hirak; Gupta, Nitish; Patro, Rob

    2016-01-01

    Motivation: The alignment of sequencing reads to a transcriptome is a common and important step in many RNA-seq analysis tasks. When aligning RNA-seq reads directly to a transcriptome (as is common in the de novo setting or when a trusted reference annotation is available), care must be taken to report the potentially large number of multi-mapping locations per read. This can pose a substantial computational burden for existing aligners, and can considerably slow downstream analysis. Results: We introduce a novel concept, quasi-mapping, and an efficient algorithm implementing this approach for mapping sequencing reads to a transcriptome. By attempting only to report the potential loci of origin of a sequencing read, and not the base-to-base alignment by which it derives from the reference, RapMap—our tool implementing quasi-mapping—is capable of mapping sequencing reads to a target transcriptome substantially faster than existing alignment tools. The algorithm we use to implement quasi-mapping uses several efficient data structures and takes advantage of the special structure of shared sequence prevalent in transcriptomes to rapidly provide highly-accurate mapping information. We demonstrate how quasi-mapping can be successfully applied to the problems of transcript-level quantification from RNA-seq reads and the clustering of contigs from de novo assembled transcriptomes into biologically meaningful groups. Availability and implementation: RapMap is implemented in C ++11 and is available as open-source software, under GPL v3, at https://github.com/COMBINE-lab/RapMap. Contact: rob.patro@cs.stonybrook.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27307617

  1. High-Resolution Mapping of RNA Polymerases Identifies Mechanisms of Sensitivity and Resistance to BET Inhibitors in t(8;21) AML.

    PubMed

    Zhao, Yue; Liu, Qi; Acharya, Pankaj; Stengel, Kristy R; Sheng, Quanhu; Zhou, Xiaofan; Kwak, Hojoong; Fischer, Melissa A; Bradner, James E; Strickland, Stephen A; Mohan, Sanjay R; Savona, Michael R; Venters, Bryan J; Zhou, Ming-Ming; Lis, John T; Hiebert, Scott W

    2016-08-16

    Bromodomain and extra-terminal domain (BET) family inhibitors offer an approach to treating hematological malignancies. We used precision nuclear run-on transcription sequencing (PRO-seq) to create high-resolution maps of active RNA polymerases across the genome in t(8;21) acute myeloid leukemia (AML), as these polymerases are exceptionally sensitive to BET inhibitors. PRO-seq identified over 1,400 genes showing impaired release of promoter-proximal paused RNA polymerases, including the stem cell factor receptor tyrosine kinase KIT that is mutated in t(8;21) AML. PRO-seq also identified an enhancer 3' to KIT. Chromosome conformation capture confirmed contacts between this enhancer and the KIT promoter, while CRISPRi-mediated repression of this enhancer impaired cell growth. PRO-seq also identified microRNAs, including MIR29C and MIR29B2, that target the anti-apoptotic factor MCL1 and were repressed by BET inhibitors. MCL1 protein was upregulated, and inhibition of BET proteins sensitized t(8:21)-containing cells to MCL1 inhibition, suggesting a potential mechanism of resistance to BET-inhibitor-induced cell death. PMID:27498870

  2. Mutations in UVSSA cause UV-sensitive syndrome and impair RNA polymerase IIo processing in transcription-coupled nucleotide-excision repair.

    PubMed

    Nakazawa, Yuka; Sasaki, Kensaku; Mitsutake, Norisato; Matsuse, Michiko; Shimada, Mayuko; Nardo, Tiziana; Takahashi, Yoshito; Ohyama, Kaname; Ito, Kosei; Mishima, Hiroyuki; Nomura, Masayo; Kinoshita, Akira; Ono, Shinji; Takenaka, Katsuya; Masuyama, Ritsuko; Kudo, Takashi; Slor, Hanoch; Utani, Atsushi; Tateishi, Satoshi; Yamashita, Shunichi; Stefanini, Miria; Lehmann, Alan R; Yoshiura, Koh-ichiro; Ogi, Tomoo

    2012-05-01

    UV-sensitive syndrome (UV(S)S) is a genodermatosis characterized by cutaneous photosensitivity without skin carcinoma. Despite mild clinical features, cells from individuals with UV(S)S, like Cockayne syndrome cells, are very UV sensitive and are deficient in transcription-coupled nucleotide-excision repair (TC-NER), which removes DNA damage in actively transcribed genes. Three of the seven known UV(S)S cases carry mutations in the Cockayne syndrome genes ERCC8 or ERCC6 (also known as CSA and CSB, respectively). The remaining four individuals with UVSS , one of whom is described for the first time here, formed a separate UV(S)S-A complementation group; however, the responsible gene was unknown. Using exome sequencing, we determine that mutations in the UVSSA gene (formerly known as KIAA1530) cause UV(S)S-A. The UVSSA protein interacts with TC-NER machinery and stabilizes the ERCC6 complex; it also facilitates ubiquitination of RNA polymerase IIo stalled at DNA damage sites. Our findings provide mechanistic insights into the processing of stalled RNA polymerase and explain the different clinical features across these TC-NER–deficient disorders. PMID:22466610

  3. Modulating the Growth and Imatinib Sensitivity of Chronic Myeloid Leukemia Stem/Progenitor Cells with Pullulan/MicroRNA Nanoparticles In Vitro.

    PubMed

    Ma, Wenjuan; Liu, Jian; Xie, Jundan; Zhang, Xiuyan; Zhou, Haixia; Yao, Hong; Zhang, Weiqi; Guo, Dawei; Zhu, Lingying; Xiao, Lun; Wu, Depei; Xu, Haiyan; Chen, Suning; Zhao, Yun

    2015-11-01

    Chronic myeloid leukemia (CML) originates from normal hematopoietic stem cells acquiring Philadelphia chromosome (Ph) to generate BCR-ABL fusion gene whose protein product has deregulated tyrosine kinase activity. Specific inhibitors against BCR-ABL, such as Imatinib mesylate (IM), have greatly improved CML management; however, no single agent is a cure yet. Delivery of microRNA (miRNA) using non-viral vectors has been utilized to inhibit various cancer cells; however, the efficacy of this approach to target CML stem/progenitor cells has not been elucidated. In this study, we firstly validated that spermine-introduced pullulan (Ps) was a robust non-viral vector for delivery of miRNA to CML cells, including the CD34+ cells from clinical isolates. We then found that the miR-181a/RALA (V-ral simian leukemia viral oncogene homolog A) axis was aberrantly expressed in the CML CD34+ cells. The delivery of miR-181a specifically inhibited the growth of CML CD34+ cells, possibly via the inhibition of RALA. In contrast, miR-181a did not evidently affect the normal hematopoietic CD34+ cells. In addition, miR-181a increased IM sensitivity of the CD34+ CML cells. Taken together, we have therefore demonstrated that the delivery of miR-181a using Ps to CML stem/progenitor cells leads to their growth inhibition and enhancement of IM sensitivity, which will possibly be beneficial to CML treatment. PMID:26554155

  4. Multiplexed Western Blotting Using Microchip Electrophoresis.

    PubMed

    Jin, Shi; Furtaw, Michael D; Chen, Huaxian; Lamb, Don T; Ferguson, Stephen A; Arvin, Natalie E; Dawod, Mohamed; Kennedy, Robert T

    2016-07-01

    Western blotting is a commonly used protein assay that combines the selectivity of electrophoretic separation and immunoassay. The technique is limited by long time, manual operation with mediocre reproducibility, and large sample consumption, typically 10-20 μg per assay. Western blots are also usually used to measure only one protein per assay with an additional housekeeping protein for normalization. Measurement of multiple proteins is possible; however, it requires stripping membranes of antibody and then reprobing with a second antibody. Miniaturized alternatives to Western blot based on microfluidic or capillary electrophoresis have been developed that enable higher-throughput, automation, and greater mass sensitivity. In one approach, proteins are separated by electrophoresis on a microchip that is dragged along a polyvinylidene fluoride membrane so that as proteins exit the chip they are captured on the membrane for immunoassay. In this work, we improve this method to allow multiplexed protein detection. Multiple injections made from the same sample can be deposited in separate tracks so that each is probed with a different antibody. To further enhance multiplexing capability, the electrophoresis channel dimensions were optimized for resolution while keeping separation and blotting times to less than 8 min. Using a 15 μm deep × 50 μm wide × 8.6 cm long channel, it is possible to achieve baseline resolution of proteins that differ by 5% in molecular weight, e.g., ERK1 (44 kDa) from ERK2 (42 kDa). This resolution allows similar proteins detected by cross-reactive antibodies in a single track. We demonstrate detection of 11 proteins from 9 injections from a single Jurkat cell lysate sample consisting of 400 ng of total protein using this procedure. Thus, multiplexed Western blots are possible without cumbersome stripping and reprobing steps. PMID:27270033

  5. Thermally multiplexed polymerase chain reaction

    PubMed Central

    Phaneuf, Christopher R.; Pak, Nikita; Saunders, D. Curtis; Holst, Gregory L.; Birjiniuk, Joav; Nagpal, Nikita; Culpepper, Stephen; Popler, Emily; Shane, Andi L.; Jerris, Robert; Forest, Craig R.

    2015-01-01

    Amplification of multiple unique genetic targets using the polymerase chain reaction (PCR) is commonly required in molecular biology laboratories. Such reactions are typically performed either serially or by multiplex PCR. Serial reactions are time consuming, and multiplex PCR, while powerful and widely used, can be prone to amplification bias, PCR drift, and primer-primer interactions. We present a new thermocycling method, termed thermal multiplexing, in which a single heat source is uniformly distributed and selectively modulated for independent temperature control of an array of PCR reactions. Thermal multiplexing allows amplification of multiple targets simultaneously—each reaction segregated and performed at optimal conditions. We demonstrate the method using a microfluidic system consisting of an infrared laser thermocycler, a polymer microchip featuring 1 μl, oil-encapsulated reactions, and closed-loop pulse-width modulation control. Heat transfer modeling is used to characterize thermal performance limitations of the system. We validate the model and perform two reactions simultaneously with widely varying annealing temperatures (48 °C and 68 °C), demonstrating excellent amplification. In addition, to demonstrate microfluidic infrared PCR using clinical specimens, we successfully amplified and detected both influenza A and B from human nasopharyngeal swabs. Thermal multiplexing is scalable and applicable to challenges such as pathogen detection where patients presenting non-specific symptoms need to be efficiently screened across a viral or bacterial panel. PMID:26339317

  6. Structural measures for multiplex networks

    NASA Astrophysics Data System (ADS)

    Battiston, Federico; Nicosia, Vincenzo; Latora, Vito

    2014-03-01

    Many real-world complex systems consist of a set of elementary units connected by relationships of different kinds. All such systems are better described in terms of multiplex networks, where the links at each layer represent a different type of interaction between the same set of nodes rather than in terms of (single-layer) networks. In this paper we present a general framework to describe and study multiplex networks, whose links are either unweighted or weighted. In particular, we propose a series of measures to characterize the multiplexicity of the systems in terms of (i) basic node and link properties such as the node degree, and the edge overlap and reinforcement, (ii) local properties such as the clustering coefficient and the transitivity, and (iii) global properties related to the navigability of the multiplex across the different layers. The measures we introduce are validated on a genuinely multiplex data set of Indonesian terrorists, where information among 78 individuals are recorded with respect to mutual trust, common operations, exchanged communications, and business relationships.

  7. Structural measures for multiplex networks.

    PubMed

    Battiston, Federico; Nicosia, Vincenzo; Latora, Vito

    2014-03-01

    Many real-world complex systems consist of a set of elementary units connected by relationships of different kinds. All such systems are better described in terms of multiplex networks, where the links at each layer represent a different type of interaction between the same set of nodes rather than in terms of (single-layer) networks. In this paper we present a general framework to describe and study multiplex networks, whose links are either unweighted or weighted. In particular, we propose a series of measures to characterize the multiplexicity of the systems in terms of (i) basic node and link properties such as the node degree, and the edge overlap and reinforcement, (ii) local properties such as the clustering coefficient and the transitivity, and (iii) global properties related to the navigability of the multiplex across the different layers. The measures we introduce are validated on a genuinely multiplex data set of Indonesian terrorists, where information among 78 individuals are recorded with respect to mutual trust, common operations, exchanged communications, and business relationships. PMID:24730896

  8. Bond Percolation on Multiplex Networks

    NASA Astrophysics Data System (ADS)

    Hackett, A.; Cellai, D.; Gómez, S.; Arenas, A.; Gleeson, J. P.

    2016-04-01

    We present an analytical approach for bond percolation on multiplex networks and use it to determine the expected size of the giant connected component and the value of the critical bond occupation probability in these networks. We advocate the relevance of these tools to the modeling of multilayer robustness and contribute to the debate on whether any benefit is to be yielded from studying a full multiplex structure as opposed to its monoplex projection, especially in the seemingly irrelevant case of a bond occupation probability that does not depend on the layer. Although we find that in many cases the predictions of our theory for multiplex networks coincide with previously derived results for monoplex networks, we also uncover the remarkable result that for a certain class of multiplex networks, well described by our theory, new critical phenomena occur as multiple percolation phase transitions are present. We provide an instance of this phenomenon in a multiplex network constructed from London rail and European air transportation data sets.

  9. High-throughput Multiplexed xMAP Luminex Array Panel for Detection of Twenty TWO Medically Important Mosquito-borne Arboviruses based on Innovations in Synthetic Biology

    PubMed Central

    Glushakova, Lyudmyla G.; Bradley, Andrea; Bradley, Kevin M.; Alto, Barry W.; Hoshika, Shuichi; Hutter, Daniel; Sharma, Nidhi; Yang, Zunyi; Kim, Myong-Jung; Benner, Steven A.

    2015-01-01

    Mosquito-borne arboviruses are emerging world-wide as important human and animal pathogens. This makes assays for their accurate and rapid identification essential for public health, epidemiological, ecological studies. Over the past decade, many mono- and multiplexed assays targeting arboviruses nucleic acids have been reported. None has become established for the routine identification of multiple viruses in a “single tube” setting. With increasing multiplexing, the detection of viral RNAs is complicated by noise, false positives and negatives. In this study, an assay was developed that avoids these problems by combining two new kinds of nucleic acids emerging from the field of synthetic biology. The first is a “self-avoiding molecular recognition system” (SAMRS), which enables high levels of multiplexing. The second is an “artificially expanded genetic information system” (AEGIS), which enables clean PCR amplification in nested PCR formats. A conversion technology was used to place AEGIS component into amplicon, improving their efficiency of hybridization on Luminex beads. When Luminex “liquid microarrays” are exploited for downstream detection, this combination supports single-tube PCR amplification assays that can identify 22 mosquito-borne RNA viruses from the genera Flavivirus, Alphavirus, Orthobunyavirus. The assay differentiates between closely-related viruses, as dengue, West Nile, Japanese encephalitis, and the California serological group. The performance and the sensitivity of the assay were evaluated with dengue viruses and infected mosquitoes; as few as 6–10 dengue virions can be detected in a single mosquito. PMID:25680538

  10. Liquid Hybridization and Solid Phase Detection: A Highly Sensitive and Accurate Strategy for MicroRNA Detection in Plants and Animals.

    PubMed

    Li, Fosheng; Mei, Lanju; Zhan, Cheng; Mao, Qiang; Yao, Min; Wang, Shenghua; Tang, Lin; Chen, Fang

    2016-01-01

    MicroRNAs (miRNAs) play important roles in nearly every aspect of biology, including physiological, biochemical, developmental and pathological processes. Therefore, a highly sensitive and accurate method of detection of miRNAs has great potential in research on theory and application, such as the clinical approach to medicine, animal and plant production, as well as stress response. Here, we report a strategic method to detect miRNAs from multicellular organisms, which mainly includes liquid hybridization and solid phase detection (LHSPD); it has been verified in various species and is much more sensitive than traditional biotin-labeled Northern blots. By using this strategy and chemiluminescent detection with digoxigenin (DIG)-labeled or biotin-labeled oligonucleotide probes, as low as 0.01-0.25 fmol [for DIG-CDP Star (disodium2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate) system], 0.005-0.1 fmol (for biotin-CDP Star system), or 0.05-0.5 fmol (for biotin-luminol system) of miRNA can be detected and one-base difference can be distinguished between miRNA sequences. Moreover, LHSPD performed very well in the quantitative analysis of miRNAs, and the whole process can be completed within about 9 h. The strategy of LHSPD provides an effective solution for rapid, accurate, and sensitive detection and quantitative analysis of miRNAs in plants and animals. PMID:27598139

  11. Enhanced Therapeutic siRNA to Tumor Cells by a pH-Sensitive Agmatine-Chitosan Bioconjugate.

    PubMed

    Li, Yongmao; Yang, Jianhai; Xu, Bing; Gao, Fei; Wang, Wei; Liu, Wenguang

    2015-04-22

    Charge-conversional naturally occurring chitosan-agmatine bioconjugates are prepared by dimethylmaleic anhydride (DMA) modification and the nucleophilic reaction between tosyl of tosylated chitosan and primary amine of agmatine. These bioconjugates (CS-DM-Agm) are shown to condense siRNA into nanocomplexes, which are stable in the presence of serum at physical pH values. Furthermore, the surface charge of complexes can tune from negative to positive while pH is changed to weak acid tumor micromilieu, thus facilitating the target cancer cell internalization in resisting serum adsorption. More importantly, this smart biogenic system shows remarkable gene silencing efficiency and a high apoptotic rate of tumor cells both in vitro and in vivo, indicating its great potential for cancer therapy. PMID:25832629

  12. Helicity multiplexed broadband metasurface holograms

    PubMed Central

    Wen, Dandan; Yue, Fuyong; Li, Guixin; Zheng, Guoxing; Chan, Kinlong; Chen, Shumei; Chen, Ming; Li, King Fai; Wong, Polis Wing Han; Cheah, Kok Wai; Yue Bun Pun, Edwin; Zhang, Shuang; Chen, Xianzhong

    2015-01-01

    Metasurfaces are engineered interfaces that contain a thin layer of plasmonic or dielectric nanostructures capable of manipulating light in a desirable manner. Advances in metasurfaces have led to various practical applications ranging from lensing to holography. Metasurface holograms that can be switched by the polarization state of incident light have been demonstrated for achieving polarization multiplexed functionalities. However, practical application of these devices has been limited by their capability for achieving high efficiency and high image quality. Here we experimentally demonstrate a helicity multiplexed metasurface hologram with high efficiency and good image fidelity over a broad range of frequencies. The metasurface hologram features the combination of two sets of hologram patterns operating with opposite incident helicities. Two symmetrically distributed off-axis images are interchangeable by controlling the helicity of the input light. The demonstrated helicity multiplexed metasurface hologram with its high performance opens avenues for future applications with functionality switchable optical devices. PMID:26354497

  13. Helicity multiplexed broadband metasurface holograms.

    PubMed

    Wen, Dandan; Yue, Fuyong; Li, Guixin; Zheng, Guoxing; Chan, Kinlong; Chen, Shumei; Chen, Ming; Li, King Fai; Wong, Polis Wing Han; Cheah, Kok Wai; Pun, Edwin Yue Bun; Zhang, Shuang; Chen, Xianzhong

    2015-01-01

    Metasurfaces are engineered interfaces that contain a thin layer of plasmonic or dielectric nanostructures capable of manipulating light in a desirable manner. Advances in metasurfaces have led to various practical applications ranging from lensing to holography. Metasurface holograms that can be switched by the polarization state of incident light have been demonstrated for achieving polarization multiplexed functionalities. However, practical application of these devices has been limited by their capability for achieving high efficiency and high image quality. Here we experimentally demonstrate a helicity multiplexed metasurface hologram with high efficiency and good image fidelity over a broad range of frequencies. The metasurface hologram features the combination of two sets of hologram patterns operating with opposite incident helicities. Two symmetrically distributed off-axis images are interchangeable by controlling the helicity of the input light. The demonstrated helicity multiplexed metasurface hologram with its high performance opens avenues for future applications with functionality switchable optical devices. PMID:26354497

  14. Multiplexed quantification for data-independent acquisition.

    PubMed

    Minogue, Catherine E; Hebert, Alexander S; Rensvold, Jarred W; Westphall, Michael S; Pagliarini, David J; Coon, Joshua J

    2015-03-01

    Data-independent acquisition (DIA) strategies provide a sensitive and reproducible alternative to data-dependent acquisition (DDA) methods for large-scale quantitative proteomic analyses. Unfortunately, DIA methods suffer from incompatibility with common multiplexed quantification methods, specifically stable isotope labeling approaches such as isobaric tags and stable isotope labeling of amino acids in cell culture (SILAC). Here we expand the use of neutron-encoded (NeuCode) SILAC to DIA applications (NeuCoDIA), producing a strategy that enables multiplexing within DIA scans without further convoluting the already complex MS(2) spectra. We demonstrate duplex NeuCoDIA analysis of both mixed-ratio (1:1 and 10:1) yeast and mouse embryo myogenesis proteomes. Analysis of the mixed-ratio yeast samples revealed the strong accuracy and precision of our NeuCoDIA method, both of which were comparable to our established MS(1)-based quantification approach. NeuCoDIA also uncovered the dynamic protein changes that occur during myogenic differentiation, demonstrating the feasibility of this methodology for biological applications. We consequently establish DIA quantification of NeuCode SILAC as a useful and practical alternative to DDA-based approaches. PMID:25621425

  15. Multiplexed Quantification for Data-Independent Acquisition

    PubMed Central

    Minogue, Catherine E.; Hebert, Alexander S.; Rensvold, Jarred W.; Westphall, Michael S.; Pagliarini, David J.; Coon, Joshua J.

    2015-01-01

    Data-independent acquisition (DIA) strategies provide a sensitive and reproducible alternative to data-dependent acquisition (DDA) methods for large-scale quantitative proteomic analyses. Unfortunately, DIA methods suffer from incompatibility with common multiplexed quantification methods, specifically stable isotope labeling approaches such as isobaric tags and stable isotope labeling of amino acids in cell culture (SILAC). Here we expand the use of neutron-encoded (NeuCode) SILAC to DIA applications (NeuCoDIA), producing a strategy that enables multiplexing within DIA scans without further convoluting the already complex MS2 spectra. We demonstrate duplex NeuCoDIA analysis of both mixed-ratio (1:1 and 10:1) yeast and mouse embryo myogenesis proteomes. Analysis of the mixed-ratio yeast samples revealed the strong accuracy and precision of our NeuCoDIA method, both of which were comparable to our established MS1-based quantification approach. NeuCoDIA also uncovered the dynamic protein changes that occur during myogenic differentiation, demonstrating the feasibility of this methodology for biological applications. We consequently establish DIA quantification of NeuCode SILAC as a useful and practical alternative to DDA-based approaches. PMID:25621425

  16. Integrated multiplexed capillary electrophoresis system

    SciTech Connect

    Yeung, Edward S.; Tan, Hongdong

    2002-05-14

    The present invention provides an integrated multiplexed capillary electrophoresis system for the analysis of sample analytes. The system integrates and automates multiple components, such as chromatographic columns and separation capillaries, and further provides a detector for the detection of analytes eluting from the separation capillaries. The system employs multiplexed freeze/thaw valves to manage fluid flow and sample movement. The system is computer controlled and is capable of processing samples through reaction, purification, denaturation, pre-concentration, injection, separation and detection in parallel fashion. Methods employing the system of the invention are also provided.

  17. Turing patterns in multiplex networks

    NASA Astrophysics Data System (ADS)

    Asllani, Malbor; Busiello, Daniel M.; Carletti, Timoteo; Fanelli, Duccio; Planchon, Gwendoline

    2014-10-01

    The theory of patterns formation for a reaction-diffusion system defined on a multiplex is developed by means of a perturbative approach. The interlayer diffusion constants act as a small parameter in the expansion and the unperturbed state coincides with the limiting setting where the multiplex layers are decoupled. The interaction between adjacent layers can seed the instability of a homogeneous fixed point, yielding self-organized patterns which are instead impeded in the limit of decoupled layers. Patterns on individual layers can also fade away due to cross-talking between layers. Analytical results are compared to direct simulations.

  18. On-chip multiplexing conversion between wavelength division multiplexing-polarization division multiplexing and wavelength division multiplexing-mode division multiplexing.

    PubMed

    Ye, Mengyuan; Yu, Yu; Zou, Jinghui; Yang, Weili; Zhang, Xinliang

    2014-02-15

    A compact silicon-on-insulator device used for conversions between polarization division multiplexing (PDM) and mode division multiplexing (MDM) signals is proposed and experimentally demonstrated by utilizing a structure combining the improved two-dimensional grating coupler and two-mode multiplexer. The detailed design of the proposed device is presented and the results show the extinction ratio of 16 and 20 dB for X- and Y-pol input, respectively. The processing of 40  Gb/s signal is achieved within the C-band with good performance. The proposed converter is capable of handling multiple wavelengths in wavelength division multiplexing (WDM) networks, enabling the conversions between WDM-PDM and WDM-MDM, which is promising to further increase the throughput at the network interface. PMID:24562199

  19. Comparison of Three Different FDA-Approved Plasma HIV-1 RNA Assay Platforms Confirms the Virologic Failure Endpoint of 200 Copies per Milliliter Despite Improved Assay Sensitivity

    PubMed Central

    Jennings, Cheryl; Johnson, Victoria A.; Coombs, Robert W.; McKinnon, John E.; Bremer, James W.; Cobb, Bryan R.; Cloherty, Gavin A.; Mellors, John W.; Ribaudo, Heather J.

    2015-01-01

    Discrepancies between HIV-1 RNA results assayed by different FDA-approved platforms have been reported. Plasma samples collected from 332 randomly selected clinical trial participants during the second year of antiretroviral treatment were assayed with three FDA-approved platforms: UltraSensitive Roche Amplicor Monitor, v1.5 (Monitor), the Abbott RealTime HIV-1 test on the m2000 system (Abbott), and the Roche TaqMan HIV-1 test, v2.0 (TaqMan). Samples from 61 additional participants with confirmed HIV-1 RNA levels of >50 copies/ml during trial follow-up were also included. Endpoints were HIV-1 RNA quantification of ≤50 copies/ml versus >50 copies/ml at an individual-sample level (primary) and determination of confirmed virologic failure (VF) from longitudinal samples. A total of 389 participants had results obtained from all assays on at least one sample (median = 6). Proportions of results of >50 copies/ml were 19% (Monitor), 22% (TaqMan), and 25% (Abbott). Despite indication of strong agreement (Cohen's kappa, 0.76 to 0.82), Abbott was more likely to detect HIV-1 RNA levels of >50 copies/ml than Monitor (matched-pair odds ratio [mOR] = 4.2; modified Obuchowski P < 0.001) and TaqMan (mOR = 2.1; P < 0.001); TaqMan was more likely than Monitor (mOR = 2.6; P < 0.001). Despite strong agreement in classifying VF across assay comparisons (kappa, 0.75 to 0.92), at a 50-copies/ml threshold, differences in the probability of VF classification (in the same direction as primary) were apparent (all McNemar's P < 0.007). At a 200-copies/ml VF threshold, no differences between assays were apparent (all P > 0.13). Despite strong agreement among assays, significant differences were observed with respect to detecting HIV-1 RNA levels of >50 copies/ml and identifying VF at the 50-copies/ml threshold. This has important implications for the definition of VF in clinical trials and clinical practice. PMID:26063861

  20. Comparison of Three Different FDA-Approved Plasma HIV-1 RNA Assay Platforms Confirms the Virologic Failure Endpoint of 200 Copies per Milliliter Despite Improved Assay Sensitivity.

    PubMed

    Lalama, Christina M; Jennings, Cheryl; Johnson, Victoria A; Coombs, Robert W; McKinnon, John E; Bremer, James W; Cobb, Bryan R; Cloherty, Gavin A; Mellors, John W; Ribaudo, Heather J

    2015-08-01

    Discrepancies between HIV-1 RNA results assayed by different FDA-approved platforms have been reported. Plasma samples collected from 332 randomly selected clinical trial participants during the second year of antiretroviral treatment were assayed with three FDA-approved platforms: UltraSensitive Roche Amplicor Monitor, v1.5 (Monitor), the Abbott RealTime HIV-1 test on the m2000 system (Abbott), and the Roche TaqMan HIV-1 test, v2.0 (TaqMan). Samples from 61 additional participants with confirmed HIV-1 RNA levels of >50 copies/ml during trial follow-up were also included. Endpoints were HIV-1 RNA quantification of ≤50 copies/ml versus >50 copies/ml at an individual-sample level (primary) and determination of confirmed virologic failure (VF) from longitudinal samples. A total of 389 participants had results obtained from all assays on at least one sample (median = 6). Proportions of results of >50 copies/ml were 19% (Monitor), 22% (TaqMan), and 25% (Abbott). Despite indication of strong agreement (Cohen's kappa, 0.76 to 0.82), Abbott was more likely to detect HIV-1 RNA levels of >50 copies/ml than Monitor (matched-pair odds ratio [mOR] = 4.2; modified Obuchowski P < 0.001) and TaqMan (mOR = 2.1; P < 0.001); TaqMan was more likely than Monitor (mOR = 2.6; P < 0.001). Despite strong agreement in classifying VF across assay comparisons (kappa, 0.75 to 0.92), at a 50-copies/ml threshold, differences in the probability of VF classification (in the same direction as primary) were apparent (all McNemar's P < 0.007). At a 200-copies/ml VF threshold, no differences between assays were apparent (all P > 0.13). Despite strong agreement among assays, significant differences were observed with respect to detecting HIV-1 RNA levels of >50 copies/ml and identifying VF at the 50-copies/ml threshold. This has important implications for the definition of VF in clinical trials and clinical practice. PMID:26063861

  1. Holographic data storage system combining shift-multiplexing with peristrophic-multiplexing

    NASA Astrophysics Data System (ADS)

    Yoshikawa, Kengo; Tsukamoto, Yu; Okubo, Kaito; Yamamoto, Manabu

    2014-02-01

    Holographic data storage (HDS) is a next-generation optical storage that uses the principles of holography. The multiplex holographic recording method is an important factor that affects the recording capacity of this storage. Various multiplex recording methods have been proposed so far. In this study, we focus on shift multiplexing with spherical waves and propose a method of shift multiplex recording that combines the peristrophic multiplexed recording. Simulation and experimental verification shows that the proposed method is effective in principle.

  2. Multiplexed label-free optical biosensor for medical diagnostics.

    PubMed

    Bottazzi, Barbara; Fornasari, Lucia; Frangolho, Ana; Giudicatti, Silvia; Mantovani, Alberto; Marabelli, Franco; Marchesini, Gerardo; Pellacani, Paola; Therisod, Rita; Valsesia, Andrea

    2014-01-01

    This paper describes a new multiplexed label-free biosensor. The detection technology is based on nanostructured gold-polymer surfaces. These surfaces support surface plasmon resonance modes that can be probed by a miniaturized optical setup. The optical characterization of the sensing chip shows the sensitivity and the limit-of-detection to refractive index changes. Moreover, by studying the progressive adhesion of molecular monolayers of polyelectrolytes, the decay of the plasmonic mode electric field above the surface has been reconstructed. A multiplexed label-free biosensing device is then described and characterized in terms of sensitivity, lateral resolution, and sensitivity to a model biological assay. The sensitivity in imaging mode of the device is of the order of 10-6 refractive index units, while the measured lateral resolution is 6.25 μm within a field of view of several tenths of mm2, making the instrument unique in terms of multiplexing capability. Finally, the proof-of-concept application of the technology as a point-of-care diagnostic tool for an inflammatory marker is demonstrated. PMID:24474511

  3. MicroRNA-137 inhibits tumor growth and sensitizes chemosensitivity to paclitaxel and cisplatin in lung cancer

    PubMed Central

    Ge, Xin; Jiang, Cheng-Fei; Shi, Zhu-Mei; Li, Dong-Mei; Liu, Wei-Tao; Yu, Xiaobo; Shu, Yong-Qian

    2016-01-01

    Chemotherapy resistance frequently drives tumour progression. However, the underlying molecular mechanisms are poorly characterized. In this study, we explored miR-137's role in the chemosensitivity of lung cancer. We found that the expression level of miR-137 is down-regulated in the human lung cancer tissues and the resistant cells strains: A549/paclitaxel(A549/PTX) and A549/cisplatin (A549/CDDP) when compared with lung cancer A549 cells. Moreover, we found that overe-expression of miR-137 inhibited cell proliferation, migration, cell survival and arrest the cell cycle in G1 phase in A549/PTX and A549/CDDP. Furthermore, Repression of miR-137 significantly promoted cell growth, migration, cell survival and cell cycle G1/S transition in A549 cells. We further demonstrated that the tumor suppressive role of miR-137 was mediated by negatively regulating Nuclear casein kinase and cyclin-dependent kinase substrate1(NUCKS1) protein expression. Importantly, miR-137 inhibits A549/PTX, A549/CDDP growth and angiogenesis in vivo. Our study is the first to identify the tumor suppressive role of over-expressed miR-137 in chemosensitivity. Identification of a novel miRNA-mediated pathway that regulates chemosensitivity in lung cancer will facilitate the development of novel therapeutic strategies in the future. PMID:26989074

  4. Point-of-care multiplexed assays of nucleic acids using microcapillary-based loop-mediated isothermal amplification.

    PubMed

    Zhang, Yi; Zhang, Lu; Sun, Jiashu; Liu, Yulei; Ma, Xingjie; Cui, Shangjin; Ma, Liying; Xi, Jianzhong Jeff; Jiang, Xingyu

    2014-07-15

    This report demonstrates a straightforward, robust, multiplexed and point-of-care microcapillary-based loop-mediated isothermal amplification (cLAMP) for assaying nucleic acids. This assay integrates capillaries (glass or plastic) to introduce and house sample/reagents, segments of water droplets to prevent contamination, pocket warmers to provide heat, and a hand-held flashlight for a visual readout of the fluorescent signal. The cLAMP system allows the simultaneous detection of two RNA targets of human immunodeficiency virus (HIV) from multiple plasma samples, and achieves a high sensitivity of two copies of standard plasmid. As few nucleic acid detection methods can be wholly independent of external power supply and equipment, our cLAMP holds great promise for point-of-care applications in resource-poor settings. PMID:24937125

  5. Application of a novel and automated branched DNA in situ hybridization method for the rapid and sensitive localization of mRNA molecules in plant tissues1

    PubMed Central

    Bowling, Andrew J.; Pence, Heather E.; Church, Jeffrey B.

    2014-01-01

    • Premise of the study: A novel branched DNA detection technology, RNAscope in situ hybridization (ISH), originally developed for use on human clinical and animal tissues, was adapted for use in plant tissue in an attempt to overcome some of the limitations associated with traditional ISH assays. • Methods and Results: Zea mays leaf tissue was formaldehyde fixed and paraffin embedded (FFPE) and then probed with the RNAscope ISH assay for two endogenous genes, phosphoenolpyruvate carboxylase (PEPC) and phosphoenolpyruvate carboxykinase (PEPCK). Results from both manual and automated methods showed tissue- and cell-specific mRNA localization patterns expected from these well-studied genes. • Conclusions: RNAscope ISH is a sensitive method that generates high-quality, easily interpretable results from FFPE plant tissues. Automation of the RNAscope method on the Ventana Discovery Ultra platform allows significant advantages for repeatability, reduction in variability, and flexibility of workflow processes. PMID:25202621

  6. Bixin regulates mRNA expression involved in adipogenesis and enhances insulin sensitivity in 3T3-L1 adipocytes through PPAR{gamma} activation

    SciTech Connect

    Takahashi, Nobuyuki; Goto, Tsuyoshi; Taimatsu, Aki; Egawa, Kahori; Katoh, Sota; Kusudo, Tatsuya; Sakamoto, Tomoya; Ohyane, Chie; Lee, Joo-Young; Kim, Young-il; Uemura, Taku; Hirai, Shizuka; Kawada, Teruo

    2009-12-25

    Insulin resistance is partly due to suppression of insulin-induced glucose uptake into adipocytes. The uptake is dependent on adipocyte differentiation, which is controlled at mRNA transcription level. The peroxisome proliferator-activated receptor (PPAR), a ligand-regulated nuclear receptor, is involved in the differentiation. Many food-derived compounds serve as ligands to activate or inactivate PPAR. In this study, we demonstrated that bixin and norbixin (annatto extracts) activate PPAR{gamma} by luciferase reporter assay using GAL4-PPAR chimera proteins. To examine the effects of bixin on adipocytes, 3T3-L1 adipocytes were treated with bixin or norbixin. The treatment induced mRNA expression of PPAR{gamma} target genes such as adipocyte-specific fatty acid-binding protein (aP2), lipoprotein lipase (LPL), and adiponectin in differentiated 3T3-L1 adipocytes and enhanced insulin-dependent glucose uptake. The observations indicate that bixin acts as an agonist of PPAR{gamma} and enhances insulin sensitivity in 3T3-L1 adipocytes, suggesting that bixin is a valuable food-derived compound as a PPAR ligand to regulate lipid metabolism and to ameliorate metabolic syndrome.

  7. Hormone-sensitive lipase in yellow catfish Pelteobagrus fulvidraco: molecular characterization, mRNA tissue expression and transcriptional regulation by leptin in vivo and in vitro.

    PubMed

    Chen, Qi-Liang; Luo, Zhi; Song, Yu-Feng; Wu, Kun; Huang, Chao; Pan, Ya-Xiong; Zhu, Qing-Ling

    2014-09-15

    Hormone-sensitive lipase (hsl) plays a pivotal role in regulation of lipolysis in mammals, but information is very scarce about its gene structure and function in fish. In this study, two distinct hsl cDNAs, designated hsl1 and hsl2, were firstly isolated and characterized from yellow catfish Pelteobagrus fulvidraco. The validated cDNAs encoding for hsl1 and hsl2 were 2739 and 2629bp in length, encoding peptides of 679 and 813 amino acid residues, respectively, and shared 57.7% amino acid identity. The phylogenetic analysis revealed that hsl1 and hsl2 derived from paralogous genes that might have arisen during a teleost-specific genome duplication event. Both hsl mRNAs were expressed in a wide range of tissues, but the abundance of each hsl mRNA showed the tissue- and developmental stage-dependent expression patterns. Intraperitoneal injection in vivo and incubation in vitro of recombinant human leptin (rb-hLEP) stimulated the mRNA expression of hsl2, but not hsl1, in the liver and hepatocytes of P. fulvidraco, respectively, suggesting that two hsl isoforms might serve different roles in lipid metabolism. To our knowledge, for the first time, the present study provides evidence that two hsl mRNAs are differentially expressed with and among tissues during different developmental stages and also differentially regulated by leptin both in vivo and in vitro, which serves to increase our understanding on hsl physiological function in fish. PMID:25016050

  8. Mechanistic Study on the Nuclear Modifier Gene MSS1 Mutation Suppressing Neomycin Sensitivity of the Mitochondrial 15S rRNA C1477G Mutation in Saccharomyces cerevisiae

    PubMed Central

    Zhou, Qiyin; Wang, Wei; He, Xiangyu; Zhu, Xiaoyu; Shen, Yaoyao; Yu, Zhe; Wang, Xuexiang; Qi, Xuchen; Zhang, Xuan; Fan, Mingjie; Dai, Yu; Yang, Shuxu; Yan, Qingfeng

    2014-01-01

    The phenotypic manifestation of mitochondrial DNA (mtDNA) mutations can be modulated by nuclear genes and environmental factors. However, neither the interaction among these factors nor their underlying mechanisms are well understood. The yeast Saccharomyces cerevisiae mtDNA 15S rRNA C1477G mutation (PR) corresponds to the human 12S rRNA A1555G mutation. Here we report that a nuclear modifier gene mss1 mutation suppresses the neomycin-sensitivity phenotype of a yeast C1477G mutant in fermentable YPD medium. Functional assays show that the mitochondrial function of the yeast C1477G mutant was impaired severely in YPD medium with neomycin. Moreover, the mss1 mutation led to a significant increase in the steady-state level of HAP5 (heme activated protein), which greatly up-regulated the expression of glycolytic transcription factors RAP1, GCR1, and GCR2 and thus stimulated glycolysis. Furthermore, the high expression of the key glycolytic enzyme genes HXK2, PFK1 and PYK1 indicated that enhanced glycolysis not only compensated for the ATP reduction from oxidative phosphorylation (OXPHOS) in mitochondria, but also ensured the growth of the mss1(PR) mutant in YPD medium with neomycin. This study advances our understanding of the phenotypic manifestation of mtDNA mutations. PMID:24595024

  9. Robert Feulgen Prize Lecture 1999. Detection and amplification systems for sensitive, multiple-target DNA and RNA in situ hybridization: looking inside cells with a spectrum of colors.

    PubMed

    Speel, E J

    1999-08-01

    In situ hybridization (ISH) is a powerful technique for localizing specific nucleic acid sequences (DNA, RNA) in microscopic preparations of tissues, cells, chromosomes, and linear DNA fibers. To date, a wide variety of research and diagnostic applications of ISH have been described, making the technique an integral part of studies concerning gene mapping, gene expression, RNA processing and transport, the three-dimensional organization of the nucleus, tumor genetics, microbial infections, and prenatal diagnosis. In this review, I first describe the ISH procedure in short and then focus on the currently available non-radioactive probe-labeling and cytochemical detection methodologies that are utilized to visualize one or multiple different nucleic acid targets in situ with different colors. Special emphasis is placed on the procedures applying fluorescence and brightfield microscopy, the simultaneous detection of nucleic acids and proteins by combined ISH and immunocytochemistry, and, in addition, on the recent progress that has been made with the introduction of signal amplification procedures to increase the detection sensitivity of ISH. Finally, a comparison of fluorescence, enzyme cytochemical, and colloidal gold silver probe detection systems will be presented, and possible future directions of in situ nucleic acid detection will be discussed. PMID:10460463

  10. Use of a Hierarchical Oligonucleotide Primer Extension Approach for Multiplexed Relative Abundance Analysis of Methanogens in Anaerobic Digestion Systems

    PubMed Central

    Chuang, Hui-Ping; Hsu, Mao-Hsuan; Chen, Wei-Yu

    2013-01-01

    In this study, we established a rapid multiplex method to detect the relative abundances of amplified 16S rRNA genes from known cultivatable methanogens at hierarchical specificities in anaerobic digestion systems treating industrial wastewater and sewage sludge. The method was based on the hierarchical oligonucleotide primer extension (HOPE) technique and combined with a set of 27 primers designed to target the total archaeal populations and methanogens from 22 genera within 4 taxonomic orders. After optimization for their specificities and detection sensitivity under the conditions of multiple single-nucleotide primer extension reactions, the HOPE approach was applied to analyze the methanogens in 19 consortium samples from 7 anaerobic treatment systems (i.e., 513 reactions). Among the samples, the methanogen populations detected with order-level primers accounted for >77.2% of the PCR-amplified 16S rRNA genes detected using an Archaea-specific primer. The archaeal communities typically consisted of 2 to 7 known methanogen genera within the Methanobacteriales, Methanomicrobiales, and Methanosarcinales and displayed population dynamic and spatial distributions in anaerobic reactor operations. Principal component analysis of the HOPE data further showed that the methanogen communities could be clustered into 3 distinctive groups, in accordance with the distribution of the Methanosaeta, Methanolinea, and Methanomethylovorans, respectively. This finding suggested that in addition to acetotrophic and hydrogenotrophic methanogens, the methylotrophic methanogens might play a key role in the anaerobic treatment of industrial wastewater. Overall, the results demonstrated that the HOPE approach is a specific, rapid, and multiplexing platform to determine the relative abundances of targeted methanogens in PCR-amplified 16S rRNA gene products. PMID:24077716

  11. Multiplexed Recombinase Polymerase Amplification Assay To Detect Intestinal Protozoa.

    PubMed

    Crannell, Zachary; Castellanos-Gonzalez, Alejandro; Nair, Gayatri; Mejia, Rojelio; White, A Clinton; Richards-Kortum, Rebecca

    2016-02-01

    This work describes a proof-of-concept multiplex recombinase polymerase amplification (RPA) assay with lateral flow readout that is capable of simultaneously detecting and differentiating DNA from any of the diarrhea-causing protozoa Giardia, Cryptosporidium, and Entamoeba. Together, these parasites contribute significantly to the global burden of diarrheal illness. Differential diagnosis of these parasites is traditionally accomplished via stool microscopy. However, microscopy is insensitive and can miss up to half of all cases. DNA-based diagnostics such as polymerase chain reaction (PCR) are far more sensitive; however, they rely on expensive thermal cycling equipment, limiting their availability to centralized reference laboratories. Isothermal DNA amplification platforms, such as the RPA platform used in this study, alleviate the need for thermal cycling equipment and have the potential to broaden access to more sensitive diagnostics. Until now, multiplex RPA assays have not been developed that are capable of simultaneously detecting and differentiating infections caused by different pathogens. We developed a multiplex RPA assay to detect the presence of DNA from Giardia, Cryptosporidium, and Entamoeba. The multiplex assay was characterized using synthetic DNA, where the limits-of-detection were calculated to be 403, 425, and 368 gene copies per reaction of the synthetic Giardia, Cryptosporidium, and Entamoeba targets, respectively (roughly 1.5 orders of magnitude higher than for the same targets in a singleplex RPA assay). The multiplex assay was also characterized using DNA extracted from live parasites spiked into stool samples where the limits-of-detection were calculated to be 444, 6, and 9 parasites per reaction for Giardia, Cryptosporidium, and Entamoeba parasites, respectively. This proof-of-concept assay may be reconfigured to detect a wide variety of targets by re-designing the primer and probe sequences. PMID:26669715

  12. Weak percolation on multiplex networks.

    PubMed

    Baxter, Gareth J; Dorogovtsev, Sergey N; Mendes, José F F; Cellai, Davide

    2014-04-01

    Bootstrap percolation is a simple but nontrivial model. It has applications in many areas of science and has been explored on random networks for several decades. In single-layer (simplex) networks, it has been recently observed that bootstrap percolation, which is defined as an incremental process, can be seen as the opposite of pruning percolation, where nodes are removed according to a connectivity rule. Here we propose models of both bootstrap and pruning percolation for multiplex networks. We collectively refer to these two models with the concept of "weak" percolation, to distinguish them from the somewhat classical concept of ordinary ("strong") percolation. While the two models coincide in simplex networks, we show that they decouple when considering multiplexes, giving rise to a wealth of critical phenomena. Our bootstrap model constitutes the simplest example of a contagion process on a multiplex network and has potential applications in critical infrastructure recovery and information security. Moreover, we show that our pruning percolation model may provide a way to diagnose missing layers in a multiplex network. Finally, our analytical approach allows us to calculate critical behavior and characterize critical clusters. PMID:24827287

  13. Code retrieval via undercover multiplexing

    NASA Astrophysics Data System (ADS)

    Barrera, John Fredy; Henao, Rodrigo; Tebaldi, Myrian; Torroba, Roberto; Bolognini, Nestor

    2008-02-01

    The purpose of this research is to develop an undercover multiplexing technique to give additional protection for optical information encryption. We employ the double random phase mask as our basic optical encryption system. The holographic storage medium of choice is a photorefractive crystal. To achieve the multiplexing we use the aperture size of the pupil in the optical system, as it governs the speckle size. We introduce such variation in order to produce a decorrelation between two consecutively stored speckle patterns. Each stored speckle pattern is associated to an input encrypted image, thus producing a multiplexing of the encrypted information. We implement this operation without altering the setup architecture and the random phase masks. This multiplexing is our undercover operation to encipher a true code behind a fake code. Under this approach, the user can only recover the bulk information stored in the volume hologram. However, he cannot recover the true code without the additional information on the pupil size key, even if accessed in position of the original decoding mask.

  14. Weak percolation on multiplex networks

    NASA Astrophysics Data System (ADS)

    Baxter, Gareth J.; Dorogovtsev, Sergey N.; Mendes, José F. F.; Cellai, Davide

    2014-04-01

    Bootstrap percolation is a simple but nontrivial model. It has applications in many areas of science and has been explored on random networks for several decades. In single-layer (simplex) networks, it has been recently observed that bootstrap percolation, which is defined as an incremental process, can be seen as the opposite of pruning percolation, where nodes are removed according to a connectivity rule. Here we propose models of both bootstrap and pruning percolation for multiplex networks. We collectively refer to these two models with the concept of "weak" percolation, to distinguish them from the somewhat classical concept of ordinary ("strong") percolation. While the two models coincide in simplex networks, we show that they decouple when considering multiplexes, giving rise to a wealth of critical phenomena. Our bootstrap model constitutes the simplest example of a contagion process on a multiplex network and has potential applications in critical infrastructure recovery and information security. Moreover, we show that our pruning percolation model may provide a way to diagnose missing layers in a multiplex network. Finally, our analytical approach allows us to calculate critical behavior and characterize critical clusters.

  15. A Synthetic Lethality Screen Using a Focused siRNA Library to Identify Sensitizers to Dasatinib Therapy for the Treatment of Epithelial Ovarian Cancer

    PubMed Central

    Pathak, Harsh B.; Zhou, Yan; Sethi, Geetika; Hirst, Jeff; Schilder, Russell J.; Golemis, Erica A.; Godwin, Andrew K.

    2015-01-01

    Molecular targeted therapies have been the focus of recent clinical trials for the treatment of patients with recurrent epithelial ovarian cancer (EOC). The majority have not fared well as monotherapies for improving survival of these patients. Poor bioavailability, lack of predictive biomarkers, and the presence of multiple survival pathways can all diminish the success of a targeted agent. Dasatinib is a tyrosine kinase inhibitor of the Src-family kinases (SFK) and in preclinical studies shown to have substantial activity in EOC. However, when evaluated in a phase 2 clinical trial for patients with recurrent or persistent EOC, it was found to have minimal activity. We hypothesized that synthetic lethality screens performed using a cogently designed siRNA library would identify second-site molecular targets that could synergize with SFK inhibition and improve dasatinib efficacy. Using a systematic approach, we performed primary siRNA screening using a library focused on 638 genes corresponding to a network centered on EGFR, HER2, and the SFK-scaffolding proteins BCAR1, NEDD9, and EFS to screen EOC cells in combination with dasatinib. We followed up with validation studies including deconvolution screening, quantitative PCR to confirm effective gene silencing, correlation of gene expression with dasatinib sensitivity, and assessment of the clinical relevance of hits using TCGA ovarian cancer data. A refined list of five candidates (CSNK2A1, DAG1, GRB2, PRKCE, and VAV1) was identified as showing the greatest potential for improving sensitivity to dasatinib in EOC. Of these, CSNK2A1, which codes for the catalytic alpha subunit of protein kinase CK2, was selected for additional evaluation. Synergistic activity of the clinically relevant inhibitor of CK2, CX-4945, with dasatinib in reducing cell proliferation and increasing apoptosis was observed across multiple EOC cell lines. This overall approach to improving drug efficacy can be applied to other targeted agents

  16. Multiplexed Microsphere Suspension Array-Based Immunoassays.

    PubMed

    Lin, Andrew; Salvador, Alexandra; Carter, J Mark

    2015-01-01

    ELISA is an extremely powerful tool to detect analytes because of its sensitivity, selectivity, reproducibility and ease of use. Here we describe sandwich immunoassays performed in suspension on spectrally unique microspheres developed by Luminex. Luminex assays offer the benefit of multiplex analysis of large numbers of analytes in a single reaction. Because the microspheres are spectrally unique, many microspheres, each attached to various antibodies, can be added to a single sample. Luminex instruments can distinguish each microsphere and detect the intensity of a reporter signal for each microsphere. Results are reported in Median Fluorescent Intensities for each analyte. Luminex assays can be used to detect up to 500 analytes in a high-throughput format. Luminex refers to this technology as xMAP(®). Here we describe a routine protocol for a Luminex immunoassay. Other Luminex assays would have to be optimized for specific conditions according to their use. PMID:26160569

  17. The complete chloroplast genome sequence of the Bambusa multiplex (Poaceae: Bambusoideae).

    PubMed

    Gao, Ju; Li, Kui; Gao, Li-zhi

    2016-01-01

    The complete nucleotide sequence of the Bambusa multiplex chloroplast genome (cpDNA) was determined in this study. The cpDNA was 139,394 bp in length, containing a pair of 21,798 bp inverted repeat regions (IR), which were separated by small and large single copy regions (SSC and LSC) of 12,875 and 82,923 bp, respectively. The B. multiplex cp genome encodes 129 predicted functional genes; 110 are unique (77 protein-coding genes, 29 tRNA genes, 4 rRNA), 19 are duplicated in the IR regions and one gene extended into the IR region in the junctions between IR and SSC. 43.20% of the genome sequence encodes proteins. The B. multiplex cp genome is AT-rich (61.08%). In these genes, fourteen genes contained one intron, while one gene had two introns. PMID:24938112

  18. CRISPR-Cas-Assisted Multiplexing (CAM): Simple Same-Day Multi-Locus Engineering in Yeast.

    PubMed

    Walter, Jessica M; Chandran, Sunil S; Horwitz, Andrew A

    2016-12-01

    Demands on the industrial and academic yeast strain engineer have increased significantly in the era of synthetic biology. Installing complex biosynthetic pathways and combining point mutations are tedious and time-consuming using traditional methods. With multiplex engineering tools, these tasks can be completed in a single step, typically achieving up to sixfold compression in strain engineering timelines. To capitalize on this potential, a variety of yeast CRISPR-Cas methods have been developed, differing largely in how the guide RNA (gRNA) reagents that direct the Cas9 nuclease are delivered. However, in nearly all reported protocols, the time savings of multiplexing is offset by multiple days of cloning to prepare the required reagents. Here, we discuss the advantages and opportunities of CRISPR-Cas-assisted multiplexing (CAM), a same-day, cloning-free method for multi-locus engineering in yeast. J. Cell. Physiol. 231: 2563-2569, 2016. © 2016 Wiley Periodicals, Inc. PMID:26991244

  19. MicroRNA-223 Enhances Radiation Sensitivity of U87MG Cells In Vitro and In Vivo by Targeting Ataxia Telangiectasia Mutated

    SciTech Connect

    Liang, Liping; Zhu, Ji; Zaorsky, Nicholas G.; Deng, Yun; Wu, Xingzhong; Liu, Yong; Liu, Fangqi; Cai, Guoxiang; Gu, Weilie; Shen, Lijun; Zhang, Zhen

    2014-03-15

    Purpose: Ataxia telangiectasia mutated (ATM) protein is important in the DNA damage response because it repairs radiation-induced damage in cancers. We examined the effect of microRNA-223 (miR-223), a regulator of ATM expression, on radiation sensitivity of cancer cells. Methods and Materials: Human embryonic kidney 293 T (293T) cells were infected with pLL3.7-miR-223 plasmid to generate the pLL3.7-miR-223 and -empty virus (EV) lentivirus (miR-223 and EV). A dual luciferase assay in which the reporter contained wild-type 3′ untranslated region (UTR) of ATM was performed. U87MG cells were infected with miR-223 or EV to establish the overexpressed stable cell lines (U87-223 or U87-EV, respectively). Cells were irradiated in vitro, and dose enhancement ratios at 2 Gy (DER{sub 2}) were calculated. Hind legs of BALB/c athymic mice were injected with U87-223 or U87-EV cells; after 2 weeks, half of the tumors were irradiated. Tumor volumes were tracked for a total of 5 weeks. Results: The dual luciferase reporter assay showed a significant reduction in luciferase activity of 293T cells cotransfected with miR-223 and the ATM 3′UTR compared to that in EV control. Overexpression of miR-223 in U87MG cells showed that ATM expression was significantly downregulated in the U87-223 cells compared to that in U87-EV (ATM/β-actin mRNA 1.0 vs 1.5, P<.05). U87-223 cells were hypersensitive to radiation compared to U87-EV cells in vitro (DER{sub 2} = 1.32, P<.01). Mice injected with miR-223-expressing tumors had almost the same tumors after 3 weeks (1.5 cm{sup 3} vs 1.7 cm{sup 3}). However, irradiation significantly decreased tumor size in miR-223-expressing tumors compared to those in controls (0.033 cm{sup 3} vs 0.829 cm{sup 3}). Conclusions: miR-223 overexpression downregulates ATM expression and sensitizes U87 cells to radiation in vitro and in vivo. MicroRNA-223 may be a novel cancer-targeting therapy, although its cancer- and patient-specific roles are

  20. Detection of virulence genes of Clostridium difficile by multiplex PCR.

    PubMed

    Antikainen, Jenni; Pasanen, Tanja; Mero, Sointu; Tarkka, Eveliina; Kirveskari, Juha; Kotila, Saara; Mentula, Silja; Könönen, Eija; Virolainen-Julkunen, Anni-Riitta; Vaara, Martti; Tissari, Päivi

    2009-08-01

    Clostridium difficile strains belonging to the PCR ribotype 027, pulse-field gel electrophoresis (PFGE) type NAP1, toxinotype III and restriction endonuclease analysis group BI harbouring mutations in the tcdC gene and possessing binary toxin components A and B have been described to cause epidemics with increased morbidity and mortality. In the present study we developed a conventional multiplex PCR designed to detect selected virulence associated markers of the hypervirulent C. difficile PCR ribotype 027. The multiplex PCR assay detected the major toxins A and B, binary toxin components A and B as well as a possible deletion in the tcdC gene: a characteristic pattern of amplification products for the PCR ribotype 027 strains was detected. This rather simple method was specific for the screening of this hypervirulent C. difficile strain. The correlation between the multiplex PCR and PCR ribotyping methods was excellent. The sensitivity and specificity were 100% in our epidemiological situation. In conclusion, this multiplex PCR was found useful in the preliminary screening for the hypervirulent C. difficile PCR ribotype 027. PMID:19664132

  1. Multiplexed Dip Pen Nanolithography patterning by simple desktop nanolithography platform

    NASA Astrophysics Data System (ADS)

    Jang, Jae-Won; Smetana, Alexander; Stiles, Paul

    2010-02-01

    Multiplexed patterning in the micro-scale has been required in order to accomplish functional bio-materials templating on the subcellular length scale. Multiplexed bio-material patterns can be used in several fields: high sensitivity DNA/protein chip development, cell adhesion/differentiation studies, and biological sensor applications. Especially, two or more materials' patterning in subcellular length scale is highly demanding to develop a multi-functional and highintegrated chip device. The multiplexing patterning of two or more materials is a challenge because of difficulty in an alignment and a precision of patterning. In this work, we demonstrate that multiplexed dip pen nanolithography® (DPN®) patterning up to four different material inks by means of using recently developed new generation nanolithography platform (NLP 2000™, NanoInk, Inc., Skokie, IL). Ink materials were prepared by adding different colored fluorescent dyes to matrix carrier materials, such as poly(ethylene glycol) dimethacrylate (PEG-DMA) and lipid material (1,2- dioleoyl-sn-glycero-3-phosphocholine, DOPC). Finally, dot-array patterns of four different inks were obtained in 50 × 50 μm2 area. This lithography platform is capable of patterning 12 separate materials within micrometer areas by efficient use of the available MEMS accessories. This number can be scaled up further with development of new accessories.

  2. Multiplex suspension array for human anti-carbohydrate antibody profiling.

    PubMed

    Pochechueva, Tatiana; Chinarev, Alexander; Spengler, Marianne; Korchagina, Elena; Heinzelmann-Schwarz, Viola; Bovin, Nicolai; Rieben, Robert

    2011-02-01

    Glycan-binding antibodies form a significant subpopulation of both natural and acquired antibodies and play an important role in various immune processes. They are for example involved in innate immune responses, cancer, autoimmune diseases, and neurological disorders. In the present study, a microsphere-based flow-cytometric immunoassay (suspension array) was applied for multiplexed detection of glycan-binding antibodies in human serum. Several approaches for immobilization of glycoconjugates onto commercially available fluorescent microspheres were compared, and as the result, the design based on coupling of end-biotinylated glycopolymers has been selected. This method requires only minute amounts of glycans, similar to a printed glycan microarray. The resulting glyco-microspheres were used for detection of IgM and IgG antibodies directed against ABO blood group antigens. The possibility of multiplexing this assay was demonstrated with mixtures of microspheres modified with six different ABO related glycans. Multiplexed detection of anti-glycan IgM and IgG correlated well with singleplex assays (Pearson's correlation coefficient r = 0.95-0.99 for sera of different blood groups). The suspension array in singleplex format for A/B trisaccharide, H(di) and Le(x) microspheres corresponded well to the standard ELISA (r > 0.94). Therefore, the described method is promising for rapid, sensitive, and reproducible detection of anti-glycan antibodies in a multiplexed format. PMID:21107457

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

    SciTech Connect

    Hindson, B J; Baker, B R; Bentley Tammero, L F; Lenhoff, R J; Naraghi-Arani, P; Vitalis, E A; Slezak, T R; Hullinger, P J; Reid, S M; Ebert, K; Ferris, N P; King, D P

    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 suspect 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.

  4. A Temperature Sensitive Variant of p53 Drives p53-Dependent MicroRNA Expression without Evidence of Widespread Post-Transcriptional Gene Silencing

    PubMed Central

    Cabrita, Miguel A.; Vanzyl, Erin J.; Hamill, Jeff D.; Pan, Elysia; Marcellus, Kristen A.; Tolls, Victoria J.; Alonzi, Rhea C.; Pastic, Alyssa; Rambo, Teeghan M. E.; Sayed, Hadil; McKay, Bruce C.

    2016-01-01

    The p53 tumour suppressor is a transcription factor that can regulate the expression of numerous genes including many encoding proteins and microRNAs (miRNAs). The predominant outcomes of a typical p53 response are the initiation of apoptotic cascades and the activation of cell cycle checkpoints. HT29-tsp53 cells express a temperature sensitive variant of p53 and in the absence of exogenous DNA damage, these cells preferentially undergo G1 phase cell cycle arrest at the permissive temperature that correlates with increased expression of the cyclin-dependent kinase inhibitor p21WAF1. Recent evidence also suggests that a variety of miRNAs can induce G1 arrest by inhibiting the expression of proteins like CDK4 and CDK6. Here we used oligonucleotide microarrays to identify p53-regulated miRNAs that are induced in these cells undergoing G1 arrest. At the permissive temperature, the expression of several miRNAs was increased through a combination of either transcriptional or post-transcriptional regulation. In particular, miR-34a-5p, miR-143-3p and miR-145-5p were strongly induced and they reached levels comparable to that of reference miRNAs (miR-191 and miR-103). Importantly, miR-34a-5p and miR-145-5p are known to silence the Cdk4 and/or Cdk6 G1 cyclin-dependent kinases (cdks). Surprisingly, there was no p53-dependent decrease in the expression of either of these G1 cdks. To search for other potential targets of p53-regulated miRNAs, p53-downregulated mRNAs were identified through parallel microarray analysis of mRNA expression. Once again, there was no clear effect of p53 on the repression of mRNAs under these conditions despite a remarkable increase in p53-induced mRNA expression. Therefore, despite a strong p53 transcriptional response, there was no clear evidence that p53-responsive miRNA contributed to gene silencing. Taken together, the changes in cell cycle distribution in this cell line at the permissive temperature is likely attributable to transcriptional

  5. Genome-wide methylation profiling and a multiplex construction for the identification of body fluids using epigenetic markers.

    PubMed

    Lee, Hwan Young; An, Ja Hyun; Jung, Sang-Eun; Oh, Yu Na; Lee, Eun Young; Choi, Ajin; Yang, Woo Ick; Shin, Kyoung-Jin

    2015-07-01

    The identification of body fluids found at crime scenes can contribute to solving crimes by providing important insights into crime scene reconstruction. In the present study, body fluid-specific epigenetic marker candidates were identified from genome-wide DNA methylation profiling of 42 body fluid samples including blood, saliva, semen, vaginal fluid and menstrual blood using the Illumina Infinium HumanMethylation450 BeadChip array. A total of 64 CpG sites were selected as body fluid-specific marker candidates by having more than 20% discrepancy in DNA methylation status between a certain type of body fluid and other types of body fluids and to have methylation or unmethylation pattern only in a particular type of body fluid. From further locus-specific methylation analysis in additional samples, 1 to 3 CpG sites were selected for each body fluid. Then, a multiplex methylation SNaPshot reaction was constructed to analyze methylation status of 8 body fluid-specific CpG sites. The developed multiplex reaction positively identifies blood, saliva, semen and the body fluid which originates from female reproductive organ in one reaction, and produces successful DNA methylation profiles in aged or mixed samples. Although it remains to be investigated whether this approach is more sensitive, more practical than RNA- or peptide-based assays and whether it can be successfully applied to forensic casework, the results of the present study will be useful for the forensic investigators dealing with body fluid samples. PMID:25796047

  6. Reducing Multiplexing Artifacts in Multi-Pinhole SPECT with a Stacked Silicon-Germanium System: a Simulation Study

    PubMed Central

    Johnson, Lindsay C.; Shokouhi, Sepideh; Peterson, Todd E

    2015-01-01

    In pinhole SPECT, multi-pinhole collimators can increase sensitivity but may lead to projection overlap, or multiplexing, which can cause image artifacts. In this work we explore whether a stacked-detector configuration with a germanium and a silicon detector, used with 123I (27–32, 159 keV), where little multiplexing occurs in the Si projections, can reduce image artifacts caused by highly-multiplexed Ge projections. Simulations are first used to determine a reconstruction method that combines the Si and Ge projections to maximize image quality. Next, simulations of different pinhole configurations (varying projection multiplexing) in conjunction with digital phantoms are used to examine whether additional Si projections mitigate artifacts from the multiplexing in the Ge projections. Reconstructed images using both Si and Ge data are compared to those using Ge data alone. Normalized mean-square error and normalized standard deviation provide a quantitative evaluation of reconstructed images’ error and noise, respectively, and are used to evaluate the impact of the additional non-multiplexed data on image quality. For a qualitative comparison, the differential point response function is used to examine multiplexing artifacts. Results show that in cases of highly-multiplexed Ge projections, the addition of low-multiplexed Si projections helps to reduce image artifacts both quantitatively and qualitatively. PMID:25055382

  7. Detection of the A2058G and A2059G 23S rRNA Gene Point Mutations Associated with Azithromycin Resistance in Treponema pallidum by Use of a TaqMan Real-Time Multiplex PCR Assay

    PubMed Central

    Chi, Kai-Hua; Pillay, Allan; Nachamkin, Eli; Su, John R.; Ballard, Ronald C.

    2013-01-01

    Macrolide treatment failure in syphilis patients is associated with a single point mutation (either A2058G or A2059G) in both copies of the 23S rRNA gene in Treponema pallidum strains. The conventional method for the detection of both point mutations uses nested PCR combined with restriction enzyme digestions, which is laborious and time-consuming. We initially developed a TaqMan-based real-time duplex PCR assay for detection of the A2058G mutation, and upon discovery of the A2059G mutation, we modified the assay into a triplex format to simultaneously detect both mutations. The point mutations detected by the real-time triplex PCR were confirmed by pyrosequencing. A total of 129 specimens PCR positive for T. pallidum that were obtained from an azithromycin resistance surveillance study conducted in the United States were analyzed. Sixty-six (51.2%) of the 129 samples with the A2058G mutation were identified by both real-time PCR assays. Of the remaining 63 samples that were identified as having a macrolide-susceptible genotype by the duplex PCR assay, 17 (27%) were found to contain the A2059G mutation by the triplex PCR. The proportions of macrolide-susceptible versus -resistant genotypes harboring either the A2058G or the A2059G mutation among the T. pallidum strains were 35.6, 51.2, and 13.2%, respectively. None of the T. pallidum strains examined had both point mutations. The TaqMan-based real-time triplex PCR assay offers an alternative to conventional nested PCR and restriction fragment length polymorphism analyses for the rapid detection of both point mutations associated with macrolide resistance in T. pallidum. PMID:23284026

  8. Effects of the level of mRNA expression on biophysical properties, sensitivity to neurotoxins, and regulation of the brain delayed-rectifier K+ channels Kv1.2.

    PubMed

    Guillemare, E; Honoré, E; Pradier, L; Lesage, F; Schweitz, H; Attali, B; Barhanin, J; Lazdunski, M

    1992-12-15

    Injection of 0.2 ng of cRNA encoding the brain Kv1.2 channel into Xenopus oocytes leads to the expression of a very slowly inactivating K+ current. Inactivation is absent in oocytes injected with 20 ng of cRNA although activation remains unchanged. Low cRNA concentrations generate a channel which is sensitive to dendrotoxin I (IC50 = 2 nM at 0.2 ng of cRNA/oocyte) and to less potent analogs of this toxin from Dendroaspis polylepis venom. A good correlation is found between blockade of the K+ current and binding of the different toxins to rat brain membranes. High cRNA concentrations generate another form of the K+ channel which is largely insensitive to dendrotoxin I (IC50 = 200 nM at 20 ng of cRNA per oocyte). At low cRNA concentrations, the expressed Kv1.2 channel is also blocked by other polypeptide toxins such as MCD peptide (IC50 = 20 nM), charybdotoxin (IC50 = 50 nM), and beta-bungarotoxin (IC50 = 50 nM), which bind to distinct and allosterically related sites on the channel protein. The pharmacologically distinct type of K+ channel expressed at high cRNA concentrations (20 ng of cRNA/oocyte) is nearly totally resistant to 100 nM MCD peptide and hardly altered by charybdotoxin and beta-bungarotoxin at concentrations as high as 1 microM. Both at low and at high cRNA concentrations, the expressed Kv1.2 channel is blocked by an increase in intracellular Ca2+ from the inositol trisphosphate sensitive pools and by the phorbol ester PMA that activates protein kinase C. PMID:1281425

  9. Modulation of K2P2.1 and K2P10.1 K+ channel sensitivity to carvedilol by alternative mRNA translation initiation

    PubMed Central

    Kisselbach, J; Seyler, C; Schweizer, P A; Gerstberger, R; Becker, R; Katus, H A; Thomas, D

    2014-01-01

    Background and Purpose The β-receptor antagonist carvedilol blocks a range of ion channels. K2P2.1 (TREK1) and K2P10.1 (TREK2) channels are expressed in the heart and regulated by alternative translation initiation (ATI) of their mRNA, producing functionally distinct channel variants. The first objective was to investigate acute effects of carvedilol on human K2P2.1 and K2P10.1 channels. Second, we sought to study ATI-dependent modulation of K2P K+ current sensitivity to carvedilol. Experimental Approach Using standard electrophysiological techniques, we recorded currents from wild-type and mutant K2P2.1 and K2P10.1 channels in Xenopus oocytes and HEK 293 cells. Key Results Carvedilol concentration-dependently inhibited K2P2.1 channels (IC50,oocytes = 20.3 μM; IC50,HEK = 1.6 μM) and this inhibition was frequency-independent. When K2P2.1 isoforms generated by ATI were studied separately in oocytes, the IC50 value for carvedilol inhibition of full-length channels (16.5 μM) was almost 5-fold less than that for the truncated channel variant (IC50 = 79.0 μM). Similarly, the related K2P10.1 channels were blocked by carvedilol (IC50,oocytes = 24.0 μM; IC50,HEK = 7.6 μM) and subject to ATI-dependent modulation of drug sensitivity. Conclusions and Implications Carvedilol targets K2P2.1 and K2P10.1 K+ channels. This previously unrecognized mechanism supports a general role of cardiac K2P channels as antiarrhythmic drug targets. Furthermore, the work reveals that the sensitivity of the cardiac ion channels K2P2.1 and K2P10.1 to block was modulated by alternative mRNA translation initiation. PMID:25168769

  10. MiRNA-621 sensitizes breast cancer to chemotherapy by suppressing FBXO11 and enhancing p53 activity.

    PubMed

    Xue, J; Chi, Y; Chen, Y; Huang, S; Ye, X; Niu, J; Wang, W; Pfeffer, L M; Shao, Z-M; Wu, Z-H; Wu, J

    2016-01-28

    MicroRNAs (miRNAs) have been demonstrated to have critical roles in regulating cancer cell proliferation, survival and sensitivity to chemotherapy. The potential application of using miRNAs to predict therapeutic response to cancer treatment holds high promise, but miRNAs with predictive value remain to be identified and underlying mechanisms have not been completely understood. Here, we show a strong correlation between miR-621 expression and chemosensitivity to paclitaxel plus carboplatin (PTX/CBP) regimen, an effective neoadjuvant chemotherapy for breast cancer patients. High level of miR-621 predicts better response to PTX/CBP regimen neoadjuvant chemotherapy in breast cancer patients, who also tend to achieve pathological complete response. Ectopic overexpression of miR-621 promoted apoptosis and increased chemosensitivity to PTX and CBP both in cultured breast cancer cells and in xenograft tumor model. We further show that FBXO11 is a direct functional target of miR-621 and miR-621 level is negatively correlated with FBXO11 expression in breast cancer patients. Ectopic expression of FBXO11 attenuated increased apoptosis in breast cancer cells overexpressing miR-621 upon PTX or CBP treatment. Consistently, high FBXO11 expression significantly correlated with poor survival in breast cancer patients. Mechanistically, we found in breast cancer cells FBXO11 interacts with p53 and promotes its neddylation, which suppressed the p53 transactivity. Accordingly, miR-621-dependent FBXO11 suppression enhanced p53 activity and increased apoptosis in breast cancer cells exposed to chemotherapeutics. Taken together, our data suggest that miR-621 enhances chemosensitivity of breast cancer cells to PTX/CBP chemotherapy by suppressing FBXO11-dependent inhibition of p53. miR-621 may serve as a predictive biomarker and a potential therapeutic target in breast cancer treatment. PMID:25867061

  11. Draft genome sequence of Xylella fastidiosa supsp. multiplex strain Griffin-1 from Quercus rubra in Georgia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The draft genome sequence of Xylella fastidiosa subsp. multiplex Strain Griffin-1 isolated from a red oak tree (Quercus rubra) in Georgia, U.S.A. is reported. The bacterium has a genome size of 2,387,314 bp with 51.7% G+C content and comprises 2,903 predicted open reading frames (ORFs), and 50 RNA g...

  12. Parallel multiplex laser feedback interferometry

    SciTech Connect

    Zhang, Song; Tan, Yidong; Zhang, Shulian

    2013-12-15

    We present a parallel multiplex laser feedback interferometer based on spatial multiplexing which avoids the signal crosstalk in the former feedback interferometer. The interferometer outputs two close parallel laser beams, whose frequencies are shifted by two acousto-optic modulators by 2Ω simultaneously. A static reference mirror is inserted into one of the optical paths as the reference optical path. The other beam impinges on the target as the measurement optical path. Phase variations of the two feedback laser beams are simultaneously measured through heterodyne demodulation with two different detectors. Their subtraction accurately reflects the target displacement. Under typical room conditions, experimental results show a resolution of 1.6 nm and accuracy of 7.8 nm within the range of 100 μm.

  13. Pattern formation in multiplex networks

    PubMed Central

    Kouvaris, Nikos E.; Hata, Shigefumi; Guilera, Albert Díaz-

    2015-01-01

    The advances in understanding complex networks have generated increasing interest in dynamical processes occurring on them. Pattern formation in activator-inhibitor systems has been studied in networks, revealing differences from the classical continuous media. Here we study pattern formation in a new framework, namely multiplex networks. These are systems where activator and inhibitor species occupy separate nodes in different layers. Species react across layers but diffuse only within their own layer of distinct network topology. This multiplicity generates heterogeneous patterns with significant differences from those observed in single-layer networks. Remarkably, diffusion-induced instability can occur even if the two species have the same mobility rates; condition which can never destabilize single-layer networks. The instability condition is revealed using perturbation theory and expressed by a combination of degrees in the different layers. Our theory demonstrates that the existence of such topology-driven instabilities is generic in multiplex networks, providing a new mechanism of pattern formation. PMID:26042606

  14. Optical Fiber Multiplexer For Industrial Nd:YAG Lasers

    NASA Astrophysics Data System (ADS)

    Goethals, Walther A.

    1989-03-01

    A lot of industrial Nd:YAG lasers are now being equipped with fiber optics for application in flexible manufacturing. One laser can be supplied with several fibers so different processing positions can receive laser power according to the time-and/or energy sharing principle. Most of the time-sharing devices (multiplexers) are based on a galvanometer mirror that couples a converging laser beam into different fibers respectively. From the industry several questions have risen to make these multiplexers faster, more reliable and suitable for smaller diameter fibers. The current designs are limited by the positioning accuracy of the galvanometer mirror and by their sensitiveness to variations in the parameters of the laser beam due to thermal lensing effects. In the patented design of the multiplexer presented here, based on a telescopic image relay and a precision fiber positioning unit, these problems were solved. A prototype has been built which addresses five fibers with 200 μm cores at a switching rate higher than 100 times per second. The fibers have special termination connectors and can be exchanged easily without losing the alignment of the optical system. For spot welding applications with pulsed Nd:YAG lasers this means that this type of multiplexer could be used to take advantage of the high laser pulse rates and the pulse shape and energy programming possibilities that manufacturers of these types of lasers offer nowadays. Another application that has been shown now is the use of several remote laser engraving units served by a single CW/Q-Switched Nd:YAG laser and a fiber multiplexer.

  15. Detection of toxigenic strains of Aeromonas species in foods by a multiplex PCR assay.

    PubMed

    Balakrishna, K; Murali, H S; Batra, H V

    2010-06-01

    Aeromonas hydrophila and other aeromonads are ubiquitous organisms found in meat, vegetables, drinking water and various other food items. They cause diarrhea and extra-intestinal infections in normal and immunocompromised patients. The aim of the study was to develop a multiplex PCR assay for the detection of virulence-associated genes of Aeromonas including hemolysin (hlyA), aerolysin (aerA), glycerophospholipid-cholesterol acyl transferase (GCAT) alongwith a 16S rRNA gene. Internal amplification control (IAC), which was coamplified with aerA primers, was also included in this study. The results showed that all cultures of Aeromonas were accurately identified by the assay without showing non-specificity. A. hydrophila could be detected at a range of 10-50 CFU ml(-1) from experimentally spiked fish, chicken and milk samples following overnight enrichment in alkaline peptone water supplemented with 10 μg/ml ampicillin (APW-A) by this multiplex PCR (mPCR). When evaluated on a total of 74 naturally occurring food samples, four samples were identified to contain Aeromonas by mPCR. All these results were compared to the conventional culture, isolation and biochemical identification procedures. The high throughput and cost-effective mPCR method developed in this study could provide a powerful tool for detection of pathogenic Aeromonas spp. from food and environmental samples and in addition, the method has advantages in terms of specificity, sensitivity and ease of use compared to other reported PCR methods and DNA hybridization assays. PMID:23100820

  16. Multiplex detection of agricultural pathogens

    DOEpatents

    Siezak, Thomas R.; Gardner, Shea; Torres, Clinton; Vitalis, Elizabeth; Lenhoff, Raymond J.

    2013-01-15

    Described are kits and methods useful for detection of agricultural pathogens in a sample. Genomic sequence information from agricultural pathogens was analyzed to identify signature sequences, e.g., polynucleotide sequences useful for confirming the presence or absence of a pathogen in a sample. Primer and probe sets were designed and optimized for use in a PCR based, multiplexed Luminex assay and/or an array assay to successfully identify the presence or absence of pathogens in a sample.

  17. Multiplex detection of agricultural pathogens

    DOEpatents

    McBride, Mary Teresa; Slezak, Thomas Richard; Messenger, Sharon Lee

    2010-09-14

    Described are kits and methods useful for detection of seven agricultural pathogens (BPSV; BHV; BVD; FMDV; BTV; SVD; and VESV) in a sample. Genomic sequence information from 7 agricultural pathogens was analyzed to identify signature sequences, e.g., polynucleotide sequences useful for confirming the presence or absence of a pathogen in a sample. Primer and probe sets were designed and optimized for use in a PCR based, multiplexed Luminex assay to successfully identify the presence or absence of pathogens in a sample.

  18. Antibody Colocalization Microarray: A Scalable Technology for Multiplex Protein Analysis in Complex Samples*

    PubMed Central

    Pla-Roca, M.; Leulmi, R. F.; Tourekhanova, S.; Bergeron, S.; Laforte, V.; Moreau, E.; Gosline, S. J. C.; Bertos, N.; Hallett, M.; Park, M.; Juncker, D.

    2012-01-01

    DNA microarrays were rapidly scaled up from 256 to 6.5 million targets, and although antibody microarrays were proposed earlier, sensitive multiplex sandwich assays have only been scaled up to a few tens of targets. Cross-reactivity, arising because detection antibodies are mixed, is a known weakness of multiplex sandwich assays that is mitigated by lengthy optimization. Here, we introduce (1) vulnerability as a metric for assays. The vulnerability of multiplex sandwich assays to cross-reactivity increases quadratically with the number of targets, and together with experimental results, substantiates that scaling up of multiplex sandwich assays is unfeasible. We propose (2) a novel concept for multiplexing without mixing named antibody colocalization microarray (ACM). In ACMs, both capture and detection antibodies are physically colocalized by spotting to the same two-dimensional coordinate. Following spotting of the capture antibodies, the chip is removed from the arrayer, incubated with the sample, placed back onto the arrayer and then spotted with the detection antibodies. ACMs with up to 50 targets were produced, along with a binding curve for each protein. The ACM was validated by comparing it to ELISA and to a small-scale, conventional multiplex sandwich assay (MSA). Using ACMs, proteins in the serum of breast cancer patients and healthy controls were quantified, and six candidate biomarkers identified. Our results indicate that ACMs are sensitive, robust, and scalable. PMID:22171321

  19. Development of multiplex PCR assay for simultaneous detection of five bacterial fish pathogens.

    PubMed

    Altinok, Ilhan; Capkin, Erol; Kayis, Sevki

    2008-10-15

    A multiplex polymerase chain reaction (PCR) method was designed for the simultaneous detection of the five major fish pathogens, Aeromonas hydrophila, Aeromonas salmonicida subsp. salmonicida, Flavobacterium columnare, Renibacterium salmoninarum, and Yersinia ruckeri. Each of the five pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism. The detection limits of the multiplex PCR was in the range of 2, 1, 1, 3, and 1CFU for A. hydrophila, A. salmonicida, F. columnare, R. salmoninarum, and Y. ruckeri, respectively. Multiplex PCR did not produce any nonspecific amplification products when tested against 23 related species of bacteria. The multiplex PCR assay was useful for the detection of the bacteria in naturally infected fish. This assay is a sensitive and specific and reproducible diagnostic tool for the simultaneous detection of five pathogenic bacteria that cause disease in fish. Therefore, it could be a useful alternative to the conventional culture based method. PMID:18499358

  20. Lifetime-based tomographic multiplexing

    NASA Astrophysics Data System (ADS)

    Raymond, Scott B.; Boas, David A.; Bacskai, Brian J.; Kumar, Anand T. N.

    2010-07-01

    Near-infrared (NIR) fluorescence tomography of multiple fluorophores has previously been limited by the bandwidth of the NIR spectral regime and the broad emission spectra of most NIR fluorophores. We describe in vivo tomography of three spectrally overlapping fluorophores using fluorescence lifetime-based separation. Time-domain images are acquired using a voltage-gated, intensified charge-coupled device (CCD) in free-space transmission geometry with 750 nm Ti:sapphire laser excitation. Lifetime components are fit from the asymptotic portion of fluorescence decay curve and reconstructed separately with a lifetime-adjusted forward model. We use this system to test the in vivo lifetime multiplexing suitability of commercially available fluorophores, and demonstrate lifetime multiplexing in solution mixtures and in nude mice. All of the fluorophores tested exhibit nearly monoexponential decays, with narrow in vivo lifetime distributions suitable for lifetime multiplexing. Quantitative separation of two fluorophores with lifetimes of 1.1 and 1.37 ns is demonstrated for relative concentrations of 1:5. Finally, we demonstrate tomographic imaging of two and three fluorophores in nude mice with fluorophores that localize to distinct organ systems. This technique should be widely applicable to imaging multiple NIR fluorophores in 3-D.

  1. Small RNA and degradome deep sequencing reveals drought-and tissue-specific micrornas and their important roles in drought-sensitive and drought-tolerant tomato genotypes.

    PubMed

    Candar-Cakir, Bilgin; Arican, Ercan; Zhang, Baohong

    2016-08-01

    Drought stress has adverse impacts on plant production and productivity. MicroRNAs (miRNAs) are one class of noncoding RNAs regulating gene expression post-transcriptionally. In this study, we employed small RNA and degradome sequencing to systematically investigate the tissue-specific miRNAs responsible to drought stress, which are understudied in tomato. For this purpose, root and upground tissues of two different drought-responsive tomato genotypes (Lycopersicon esculentum as sensitive and L. esculentum var. cerasiforme as tolerant) were subjected to stress with 5% polyethylene glycol for 7 days. A total of 699 conserved miRNAs belonging to 578 families were determined and 688 miRNAs were significantly differentially expressed between different treatments, tissues and genotypes. Using degradome sequencing, 44 target genes were identified associated with 36 miRNA families. Drought-related miRNAs and their targets were enriched functionally by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Totally, 53 miRNAs targeted 23 key drought stress- and tissue development-related genes, including DRP (dehydration-responsive protein), GTs (glycosyltransferases), ERF (ethylene responsive factor), PSII (photosystem II) protein, HD-ZIP (homeodomain-leucine zipper), MYB and NAC-domain transcription factors. miR160, miR165, miR166, miR171, miR398, miR408, miR827, miR9472, miR9476 and miR9552 were the key miRNAs functioning in regulation of these genes and involving in tomato response to drought stress. Additionally, plant hormone signal transduction pathway genes were differentially regulated by miR169, miR172, miR393, miR5641, miR5658 and miR7997 in both tissues of both sensitive and tolerant genotypes. These results provide new insight into the regulatory role of miRNAs in drought response with plant hormone signal transduction and drought-tolerant tomato breeding. PMID:26857916

  2. Downregulation of yidC in Escherichia coli by Antisense RNA Expression Results in Sensitization to Antibacterial Essential Oils Eugenol and Carvacrol

    PubMed Central

    Patil, Supriya Deepak; Sharma, Rajnikant; Srivastava, Santosh; Navani, Naveen Kumar; Pathania, Ranjana

    2013-01-01

    Background The rising drug resistance in pathogenic bacteria and inefficiency of current antibiotics to meet clinical requirements has augmented the need to establish new and innovative approaches for antibacterial drug discovery involving identification of novel antibacterial targets and inhibitors. Being obligatory for bacterial growth, essential gene products are considered vital as drug targets. The bacterial protein YidC is highly conserved among pathogens and is essential for membrane protein insertion due to which it holds immense potential as a promising target for antibacterial therapy. Methods/Principal Findings The aim of this study was to explore the feasibility and efficacy of expressed antisense-mediated gene silencing for specific downregulation of yidC in Escherichia coli. We induced RNA silencing of yidC which resulted in impaired growth of the host cells. This was followed by a search for antibacterial compounds sensitizing the YidC depleted cells as they may act as inhibitors of the essential protein or its products. The present findings affirm that reduction of YidC synthesis results in bacterial growth retardation, which warrants the use of this enzyme as a viable target in search of novel antibacterial agents. Moreover, yidC antisense expression in E. coli resulted in sensitization to antibacterial essential oils eugenol and carvacrol. Fractional Inhibitory Concentration Indices (FICIs) point towards high level of synergy between yidC silencing and eugenol/carvacrol treatment. Finally, as there are no known YidC inhibitors, the RNA silencing approach applied in this study put forward rapid means to screen novel potential YidC inhibitors. Conclusions/Significance The present results suggest that YidC is a promising candidate target for screening antibacterial agents. High level of synergy reported here between yidC silencing and eugenol/carvacrol treatment is indicative of a potential antibacterial therapy. This is the first report indicating

  3. Rotation spacing and multiplexing number in angle-peristrophic multiplexing holographic memory

    NASA Astrophysics Data System (ADS)

    Sawada, Masamitsu; Kinoshita, Nobuhiro; Muroi, Tetsuhiko; Motohashi, Mitsuya; Saito, Nobuo

    2015-09-01

    Holographic memory is expected to be the next-generation optical memory with several advantages including high data transfer rate and high recording density. Holographic memory enables the storage of holograms in the same location in a holographic medium typically using the angle multiplexing method. The multiplexing number is an important factor that determines the recording density when using this method. To increase the multiplexing number, it is known as an effective method to combine peristrophic (or rotation) multiplexing with angle multiplexing. We use the k-sphere to describe that the rotation spacing for peristrophic multiplexing depends on both the numerical aperture in the signal beam path and the angle between the reference and signal beams. We then formulate the rotation spacing and compare the results obtained using the theoretical formula with the measured results. Finally, we estimate the maximum multiplexing number for our experimental system using the angle-peristrophic multiplexing method on the basis of the measured results.

  4. Multiplex detection of food allergens and gluten.

    PubMed

    Cho, Chung Y; Nowatzke, William; Oliver, Kerry; Garber, Eric A E

    2015-05-01

    To help safeguard the food supply and detect the presence of undeclared food allergens and gluten, most producers and regulatory agencies rely on commercial test kits. Most of these are ELISAs with a few being PCR-based. These methods are very sensitive and analyte specific, requiring different assays to detect each of the different food allergens. Mass spectrometry offers an alternative approach whereby multiple allergens may be detected simultaneously. However, mass spectrometry requires expensive equipment, highly trained analysts, and several years before a quantitative approach can be achieved. Using multianalyte profiling (xMAP®) technology, a commercial multiplex test kit based on the use of established antibodies was developed for the simultaneous detection of up to 14 different food allergens plus gluten. The assay simultaneously detects crustacean seafood, egg, gluten, milk, peanut, soy, and nine tree nuts (almond, Brazil nut, cashew, coconut, hazelnut, macadamia, pine nut, pistachio, and walnut). By simultaneously performing multiple tests (typically two) for each analyte, this magnetic bead-based assay offers built-in confirmatory analyses without the need for additional resources. Twenty-five of the assays were performed on buffer extracted samples, while five were conducted on samples extracted using reduced-denatured conditions. Thus, complete analysis for all 14 allergens and gluten requires only two wells of a 96-well microtiter plate. This makes it possible to include in a single analytical run up to 48 samples. All 30 bead sets in this multiplex assay detected 5 ng/mL of food allergen and gluten with responses greater than background. In addition, 26 of the bead sets displayed signal/noise ratios of five or greater. The bead-based design makes this 30-plex assay expandable to incorporate new antibodies and capture/detector methodologies by ascribing these new detectors to any of the unassigned bead sets that are commercially available. PMID

  5. Sensitivity, Specificity, and Clinical Value of Human Papillomavirus (HPV) E6/E7 mRNA Assay as a Triage Test for Cervical Cytology and HPV DNA Test ▿

    PubMed Central

    Benevolo, Maria; Vocaturo, Amina; Caraceni, Donatella; French, Deborah; Rosini, Sandra; Zappacosta, Roberta; Terrenato, Irene; Ciccocioppo, Lucia; Frega, Antonio; Rossi, Paolo Giorgi

    2011-01-01

    There is evidence that testing for human papillomavirus (HPV) E6/E7 mRNA is more specific than testing for HPV DNA. A retrospective study was carried out to evaluate the performance of the PreTect HPV-Proofer E6/E7 mRNA assay (Norchip) as a triage test for cytology and HPV DNA testing. This study analyzed 1,201 women, 688 of whom had a colposcopy follow-up and 195 of whom had histology-confirmed high-grade intraepithelial neoplasia or worse (CIN2+). The proportion of positive results and the sensitivity and specificity for CIN2+ were determined for HPV mRNA in comparison to HPV DNA and cytology. All data were adjusted for follow-up completeness. Stratified by cytological grades, the HPV mRNA sensitivity was 83% (95% confidence interval [CI] = 63 to 94%) in ASC-US (atypical squamous cells of undetermined significance), 62% (95% CI = 47 to 75%) in L-SIL (low-grade squamous intraepithelial lesion), and 67% (95% CI = 57 to 76%) in H-SIL (high-grade squamous intraepithelial lesion). The corresponding figures were 99, 91, and 96%, respectively, for HPV DNA. The specificities were 82, 76, and 45%, respectively, for HPV mRNA and 29, 13, and 4%, respectively, for HPV DNA. Used as a triage test for ASC-US and L-SIL, mRNA reduced colposcopies by 79% (95% CI = 74 to 83%) and 69% (95% CI = 65 to 74%), respectively, while HPV DNA reduced colposcopies by 38% (95% CI = 32 to 44%) and by 15% (95% CI = 12 to 19%), respectively. As a HPV DNA positivity triage test, mRNA reduced colposcopies by 63% (95% CI = 60 to 66%), having 68% sensitivity (95% CI = 61 to 75%), whereas cytology at the ASC-US+ threshold reduced colposcopies by 23% (95% CI = 20 to 26%), showing 92% sensitivity (95% CI = 87 to 95%). In conclusion, PreTect HPV-Proofer mRNA can serve as a better triage test than HPV DNA to reduce colposcopy referral in both ASC-US and L-SIL. It is also more efficient than cytology for the triage of HPV DNA-positive women. Nevertheless, its low sensitivity demands a strict follow-up of

  6. A Localized Surface Plasmon Resonance Imaging Instrument for Multiplexed Biosensing

    PubMed Central

    Ruemmele, Julia A.; Hall, W. Paige; Ruvuna, Laura K.; Van Duyne, Richard P.

    2013-01-01

    Localized surface plasmon resonance (LSPR) spectroscopy has been widely used for label-free, highly-sensitive measurements of interactions at a surface. LSPR imaging (LSPRi) has the full advantages of LSPR, but enables high-throughput, multiplexed measurements by simultaneously probing multiple individually addressable sensors on a single sample surface. Each spatially distinct sensor can be tailored to provide data regarding different surface functionalities or reaction environments. Previously, LSPRi has focused on single particle sensing where the size scale is very small. Here, we create defined macroscale arrays of nanoparticles that are compatible with common patterning methods such as dip-pen nanolithography and multi-channel microfluidic delivery devices. With this new LSPR sensing format, we report the first demonstration of multiplexed LSPR imaging and show that the increased throughput of our instrument enables the collection of a complete Langmuir binding curve on a single sensor surface. In addition, the multiplexed LSPR sensor is highly selective, as demonstrated by the hybridization of single-stranded DNA to complementary sequences immobilized on the sensor surface. The LSPR arrays described in this work exhibit uniform sensitivity and tailorable optical properties, making them an ideal platform for high-throughput, label-free analysis of a variety of molecular binding interactions. PMID:23560643

  7. Multiplex Electrochemical Immunoassay Using Gold Nanoparticle Probes and Immunochromatographic Strips

    SciTech Connect

    Mao, Xun; Baloda, Meenu; Gurung, Anant; Lin, Yuehe; Liu, Guodong

    2008-10-20

    We describe a multiplex electrochemical immunoassay based on the use of gold nanoparticle (Au-NP) probes and immunochromatographic strips (ISs). The approach takes advantage of the speed and low cost of the conventional IS tests and the high sensitivities of the nanoparticle-based electrochemical immunoassays. Rabbit IgG(R-IgG) and human IgM (H-IgM) were used as model targets for the demonstration of the proof of concept. The Au-NPs based sandwich immunoreactions were performed on the IS, and the captured gold nanoparticle labels on the test zones were determined by highly-sensitive stripping voltammetric measurement of the dissolved gold ions (III) with a carbon paste electrode. The detection limits are 1.0 and 1.5 ng/mL with the linear ranges of 2.5-250 ng/mL for quantitative detection of R-IgG and H-IgM, respectively. The total assay time is around 25 minutes. Such multiplex electrochemical immunoassay could be readily highly multiplexed to allow simultaneous parallel detection of numerous proteins and is expected to open new opportunities for protein diagnostics and biosecurity.

  8. A two-tube multiplex reverse transcription PCR assay for simultaneous detection of viral and bacterial pathogens of infectious diarrhea.

    PubMed

    Wang, Ji; Xu, Ziqian; Niu, Peihua; Zhang, Chen; Zhang, Jingyun; Guan, Li; Kan, Biao; Duan, Zhaojun; Ma, Xuejun

    2014-01-01

    Diarrhea caused by viral and bacterial infections is a major health problem in developing countries. The purpose of this study is to develop a two-tube multiplex PCR assay using automatic electrophoresis for simultaneous detection of 13 diarrhea-causative viruses or bacteria, with an intended application in provincial Centers for Diseases Control and Prevention, China. The assay was designed to detect rotavirus A, norovirus genogroups GI and GII, human astrovirus, enteric adenoviruses, and human bocavirus (tube 1), and Salmonella, Vibrio parahaemolyticus, diarrheagenic Escherichia coli, Campylobacter jejuni, Shigella, Yersinia, and Vibrio cholera (tube 2). The analytical specificity was examined with positive controls for each pathogen. The analytical sensitivity was evaluated by performing the assay on serial tenfold dilutions of in vitro transcribed RNA, recombinant plasmids, or bacterial culture. A total of 122 stool samples were tested by this two-tube assay and the results were compared with those obtained from reference methods. The two-tube assay achieved a sensitivity of 20-200 copies for a single virus and 10(2)-10(3) CFU/mL for bacteria. The clinical performance demonstrated that the two-tube assay had comparable sensitivity and specificity to those of reference methods. In conclusion, the two-tube assay is a rapid, cost-effective, sensitive, specific, and high throughput method for the simultaneous detection of enteric bacteria and virus. PMID:24711998

  9. Multiplexing of encrypted data using fractal masks.

    PubMed

    Barrera, John F; Tebaldi, Myrian; Amaya, Dafne; Furlan, Walter D; Monsoriu, Juan A; Bolognini, Néstor; Torroba, Roberto

    2012-07-15

    In this Letter, we present to the best of our knowledge a new all-optical technique for multiple-image encryption and multiplexing, based on fractal encrypting masks. The optical architecture is a joint transform correlator. The multiplexed encrypted data are stored in a photorefractive crystal. The fractal parameters of the key can be easily tuned to lead to a multiplexing operation without cross talk effects. Experimental results that support the potential of the method are presented. PMID:22825170

  10. Multiplex Holograms And Their Applications In Medicine

    NASA Astrophysics Data System (ADS)

    Tsujiuchi, Jumpei

    1988-01-01

    Fundamental properties of reconstructed images from a multiplex hologram are studied, and conditions for compensating distortions and for designing a reconstructing source are proposed. Applications of multiplex hologram to medical objects are reviewed, and a computer-aided hologram synthesizing system is proposed for obtaining better images and wider applications. An example of multiplex holograms synthesized from a series of CT images is also presented.

  11. Multiplexing Bioluminescent and Fluorescent Reporters to Monitor Live Cells

    PubMed Central

    Haugwitz, Michael; Nourzaie, Omar; Garachtchenko, Tatiana; Hu, Lanrong; Gandlur, Suvarna; Olsen, Cathy; Farmer, Andrew; Chaga, Grigoriy; Sagawa, Hiroaki

    2008-01-01

    Reporter proteins are valuable tools to monitor promoter activities and characterize signal transduction pathways. Many of the currently available promoter reporters have drawbacks that compromise their performance. Enzyme-based reporter systems using cytosolic luciferases are highly sensitive, but require a cell lysis step that prevents their use in long-term monitoring. By contrast, secreted bioluminescent reporters like Metridia luciferase and Secreted Alkaline Phosphatase can be assayed repeatedly, using supernatant from the same live cell population to produce many sets of data over time. This is crucial for studies with limited amounts of cells, as in the case of stem cells. The use of secreted bioluminescent reporters also enables broader applications to provide more detailed information using live cells; for example, multiplexing with fluorescent proteins. Here, data is presented describing the characteristics of secreted Metridia luciferase and its use in multiplexing applications with either Secreted Alkaline Phosphatase or a fluorescent protein. PMID:20161823

  12. Multiplexing bioluminescent and fluorescent reporters to monitor live cells.

    PubMed

    Haugwitz, Michael; Nourzaie, Omar; Garachtchenko, Tatiana; Hu, Lanrong; Gandlur, Suvarna; Olsen, Cathy; Farmer, Andrew; Chaga, Grigoriy; Sagawa, Hiroaki

    2008-01-01

    Reporter proteins are valuable tools to monitor promoter activities and characterize signal transduction pathways. Many of the currently available promoter reporters have drawbacks that compromise their performance. Enzyme-based reporter systems using cytosolic luciferases are highly sensitive, but require a cell lysis step that prevents their use in long-term monitoring. By contrast, secreted bioluminescent reporters like Metridia luciferase and Secreted Alkaline Phosphatase can be assayed repeatedly, using supernatant from the same live cell population to produce many sets of data over time. This is crucial for studies with limited amounts of cells, as in the case of stem cells. The use of secreted bioluminescent reporters also enables broader applications to provide more detailed information using live cells; for example, multiplexing with fluorescent proteins. Here, data is presented describing the characteristics of secreted Metridia luciferase and its use in multiplexing applications with either Secreted Alkaline Phosphatase or a fluorescent protein. PMID:20161823

  13. Measuring and modeling correlations in multiplex networks.

    PubMed

    Nicosia, Vincenzo; Latora, Vito

    2015-09-01

    The interactions among the elementary components of many complex systems can be qualitatively different. Such systems are therefore naturally described in terms of multiplex or multilayer networks, i.e., networks where each layer stands for a different type of interaction between the same set of nodes. There is today a growing interest in understanding when and why a description in terms of a multiplex network is necessary and more informative than a single-layer projection. Here we contribute to this debate by presenting a comprehensive study of correlations in multiplex networks. Correlations in node properties, especially degree-degree correlations, have been thoroughly studied in single-layer networks. Here we extend this idea to investigate and characterize correlations between the different layers of a multiplex network. Such correlations are intrinsically multiplex, and we first study them empirically by constructing and analyzing several multiplex networks from the real world. In particular, we introduce various measures to characterize correlations in the activity of the nodes and in their degree at the different layers and between activities and degrees. We show that real-world networks exhibit indeed nontrivial multiplex correlations. For instance, we find cases where two layers of the same multiplex network are positively correlated in terms of node degrees, while other two layers are negatively correlated. We then focus on constructing synthetic multiplex networks, proposing a series of models to reproduce the correlations observed empirically and/or to assess their relevance. PMID:26465526

  14. Measuring and modeling correlations in multiplex networks

    NASA Astrophysics Data System (ADS)

    Nicosia, Vincenzo; Latora, Vito

    2015-09-01

    The interactions among the elementary components of many complex systems can be qualitatively different. Such systems are therefore naturally described in terms of multiplex or multilayer networks, i.e., networks where each layer stands for a different type of interaction between the same set of nodes. There is today a growing interest in understanding when and why a description in terms of a multiplex network is necessary and more informative than a single-layer projection. Here we contribute to this debate by presenting a comprehensive study of correlations in multiplex networks. Correlations in node properties, especially degree-degree correlations, have been thoroughly studied in single-layer networks. Here we extend this idea to investigate and characterize correlations between the different layers of a multiplex network. Such correlations are intrinsically multiplex, and we first study them empirically by constructing and analyzing several multiplex networks from the real world. In particular, we introduce various measures to characterize correlations in the activity of the nodes and in their degree at the different layers and between activities and degrees. We show that real-world networks exhibit indeed nontrivial multiplex correlations. For instance, we find cases where two layers of the same multiplex network are positively correlated in terms of node degrees, while other two layers are negatively correlated. We then focus on constructing synthetic multiplex networks, proposing a series of models to reproduce the correlations observed empirically and/or to assess their relevance.

  15. Application of a multiplex PCR for the detection of protozoan pathogens of the eastern oyster Crassostrea virginica in field samples.

    PubMed

    Russell, Spencer; Frasca, Salvatore; Sunila, Inke; French, Richard A

    2004-04-21

    Populations of eastern oysters Crassostrea virginica along the east coast of North America have repeatedly experienced epizootic mass mortality due to infections by protozoan parasites, and molecular diagnostic methodologies are fast becoming more widely available for the diagnosis of protozoan diseases of oysters. In this study we applied a modified version of an existing multiplex polymerase chain reaction (PCR) for detection of the eastern oyster parasites Haplosporidium nelsoni, H. costale and Perkinsus marinus from field-collected samples. We incorporated primers for DNA quality control based on the large subunit ribosomal RNA (LSU rRNA) gene of C. virginica. The multiplex PCR (MPCR) simultaneously amplified genomic DNA of C. virginica, and cloned DNA of H. nelsoni, P. marinus and H. costale. In field trial applications, we compared the performance of the MPCR to that of the conventional diagnostic techniques of histopathological tissue examination and the Ray/Mackin fluid thioglycollate medium (RMFT) assay. A total of 530 oysters were sampled from 18 sites at 12 locations along the east coast of the United States from the Gulf of Mexico to southern New England. The modified MPCR detected 21% oysters with H. nelsoni, 2% oysters with H. costale, and 40% oysters with P. marinus infections. In comparison, histopathological examination detected H. nelsoni and H. costale infections in 6 and 0.8% oysters, respectively, and the RMFT assay detected P. marinus infection in 31% oysters. The MPCR is a more sensitive diagnostic assay for detection of H. nelsoni, H. costale, and P. marinus, and incorporation of an oyster quality control product limits false negative results. PMID:15212297

  16. Cells on chip for multiplex screening.

    PubMed

    Berthuy, Ophélie I; Blum, Loïc J; Marquette, Christophe A

    2016-02-15

    Microarray technology was developed in the early 1990s to measure the transcription levels of thousands of genes in parallel. The basic premise of high-density arraying has since been expanded to create cells microarrays. Cells on chip are powerful experimental tools for high-throughput and multiplex screening of samples or cellular functions. Miniaturization increases assay throughput while reducing both reagent consumption and cell population heterogeneity effect, making these systems attractive for a wide range of assays, from drug discovery to toxicology, stem cell research and therapy. One form of cell microarrays, the transfected cell microarray, wherein plasmid DNA or siRNA, spotted on the surface of a substrate, is reverse-transfected locally into adherent cells, has become a standard tool for parallel cell-based analysis. With the advent of technologies, cells can also be directly spotted onto functionalized surfaces using robotic fluid-dispensing devices, or printed directly in bio-ink material. We are providing herein an overview of the last developments in optical cell microarrays allowing high-throughput and high-content analysis. PMID:25892543

  17. Computer assisted multiplex sequencing. Performance report, August 1, 1992--July 15, 1993

    SciTech Connect

    Not Available

    1993-07-01

    The objectives of this project are automation for optimization of multiplex sequencing. We have integrated direct transfer electrophoresis, automated multiplex hybridizations and automated film reading and applied this toward sequencing of E. coli and human DNA. Primers for the directed dideoxy sequence walking and sequence confirmation steps are synthesized to include DNA tags complementary to an alkaline phosphatase conjugate. A higher throughput synthesis device is well along in testing as are new automated hybridization devices. We have developed software for automatically annotating ORFs and databases of precise termini of proteins and RNA.

  18. Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector.

    PubMed

    Kabadi, Ami M; Ousterout, David G; Hilton, Isaac B; Gersbach, Charles A

    2014-10-29

    Engineered DNA-binding proteins that manipulate the human genome and transcriptome have enabled rapid advances in biomedical research. In particular, the RNA-guided CRISPR/Cas9 system has recently been engineered to create site-specific double-strand breaks for genome editing or to direct targeted transcriptional regulation. A unique capability of the CRISPR/Cas9 system is multiplex genome engineering by delivering a single Cas9 enzyme and two or more single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can be used to simultaneously create multiple DNA breaks or to target multiple transcriptional activators to a single promoter for synergistic enhancement of gene induction. To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells. This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types. PMID:25122746

  19. Space-division multiplexing optical coherence tomography

    PubMed Central

    Zhou, Chao; Alex, Aneesh; Rasakanthan, Janarthanan; Ma, Yutao

    2013-01-01

    High speed, high resolution and high sensitivity are desirable for optical coherence tomography (OCT). Here, we demonstrate a space-division multiplexing (SDM) technology that translates long coherence length of a commercially available wavelength tunable laser into high OCT imaging speed. We achieved an effective 800,000 A-scans/s imaging speed using a 100,000 Hz tunable vertical cavity surface-emitting laser (VCSEL). A sensitivity of 94.6 dB and a roll-off of < 2 dB over ~30 mm imaging depth were measured from a single channel in the prototype SDM-OCT system. An axial resolution of ~11 μm in air (or ~8.3 μm in tissue) was achieved throughout the entire depth range. An in vivo, 3D SDM-OCT volume of an entire Drosophila larva consisting of 400 x 605 A-scans was acquired in 0.37 seconds. Synchronized cross-sectional OCT imaging of three different segments of a beating Drosophila larva heart is demonstrated. The SDM technology provides a new orthogonal dimension for further speed improvement for OCT with favorable cost scaling. SDM-OCT also preserves image resolution and allows synchronized cross-sectional and three-dimensional (3D) imaging of biological samples, enabling new biomedical applications. PMID:23938839

  20. Detection of RNA viruses: current technologies and future perspectives.

    PubMed

    Cella, Lakshmi N; Blackstock, Daniel; Yates, Marylynn A; Mulchandani, Ashok; Chen, Wilfred

    2013-01-01

    RNA viruses constitute one of the major classes of pathogenic organisms causing human diseases, with varying degrees of severity. This review summarizes the conventional and emerging technologies that are available for the detection of these organisms. Cell culture-based techniques for viral detection have been popular since their inception and continue to be the gold standard against which all other techniques. Over many years, these techniques have undergone some radical changes, reducing the total time needed for detection and improving sensitivity, although even with their reliability and improved features they are being slowly replaced by nucleic acid-based technologies. These molecular detection techniques have revolutionized the area of viral detection by their high sensitivity, selectivity, and short detection time. The majority of nucleic acid-based techniques depend on amplifying viral RNA; however, there are some newer emerging techniques that detect viral RNA in live cells using various configurations of florescent probes. In addition, nucleic acid-based technology has made it possible for multiviral detection with either multiplex polymerase chain reaction assays or microarrays. Every technique described in this review has its own unique abilities, making them indispensable for viral detection. However, we believe that nucleic acid-based technologies will find widespread use after being standardized, limiting other technologies to very specific uses. PMID:23582035

  1. Single-pair fluorescence resonance energy transfer analysis of mRNA transcripts for highly sensitive gene expression profiling in near real time.

    PubMed

    Peng, Zhiyong; Young, Brandon; Baird, Alison E; Soper, Steven A

    2013-08-20

    Expression analysis of mRNAs transcribed from certain genes can be used as important sources of biomarkers for in vitro diagnostics. While the use of reverse transcription quantitative PCR (RT-qPCR) can provide excellent analytical sensitivity for monitoring transcript numbers, more sensitive approaches for expression analysis that can report results in near real-time are needed for many critical applications. We report a novel assay that can provide exquisite limits-of-quantitation and consists of reverse transcription (RT) followed by a ligase detection reaction (LDR) with single-pair fluorescence resonance energy transfer (spFRET) to provide digital readout through molecular counting. For this assay, no PCR was employed, which enabled short assay turnaround times. To facilitate implementation of the assay, a cyclic olefin copolymer (COC) microchip, which was fabricated using hot embossing, was employed to carry out the LDR in a continuous flow format with online single-molecule detection following the LDR. As demonstrators of the assay's utility, MMP-7 mRNA was expression profiled from several colorectal cancer cell lines. It was found that the RT-LDR/spFRET assay produced highly linear calibration plots even in the low copy number regime. Comparison to RT-qPCR indicated a better linearity over the low copy number range investigated (10-10,000 copies) with an R(2) = 0.9995 for RT-LDR/spFRET and R(2) = 0.98 for RT-qPCR. In addition, differentiating between copy numbers of 10 and 50 could be performed with higher confidence using RT-LDR/spFRET. To demonstrate the short assay turnaround times obtainable using the RT-LDR/spFRET assay, a two thermal cycle LDR was carried out on amphiphysin gene transcripts that can serve as important diagnostic markers for ischemic stroke. The ability to supply diagnostic information on possible stroke events in short turnaround times using RT-LDR/spFRET will enable clinicians to treat patients effectively with appropriate time-sensitive

  2. Development of multiplex real-time reverse transcriptase PCR assays for detecting eight medically important flaviviruses in mosquitoes.

    PubMed

    Chao, Day-Yu; Davis, Brent S; Chang, Gwong-Jen J

    2007-02-01

    A multiplex real-time reverse transcriptase PCR has been developed for the rapid detection and identification of eight medically important flaviviruses from laboratory-reared, virus-infected mosquito pools. The method used involves the gene-specific amplification of yellow fever virus (YFV), Japanese encephalitis virus (JEV), West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and dengue virus (DENV) serotypes 1 to 4 (DENV-1 to DENV-4, respectively) by use of the flavivirus consensus amplimers